Targeted chimeric antigen receptor modified t cells for treatment of il13ralpha2 positive malignancies

Abstract

Immune cells lacking expression of TGFβR2 and harboring nucleic molecule comprising a nucleotide sequence encoding a chimeric antigen receptor targeted to IL13Rα2 (IL13Rα2-CAR), wherein the chimeric antigen receptor comprises: an IL-13 variant having the E13Y mutation; a spacer domain; a transmembrane domain; a costimulatory domain and a CD3zeta domain are described, amongst other things.

Description

[0001] Attorney Docket No.: 40056-0090WO1 TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF IL13Rα2 POSITIVE MALIGNANCIES CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Application Serial No. 63 / 594,121, filed on October 30, 2023. The entire contents of the foregoing are incorporated herein by reference. TECHNICAL FIELD This disclosure concerns T cells that express a chimeric antigen receptor targeted to IL13Rα2 and lack functional TGFβR2 as well as methods for using such T cells for treatment of cancers, including glioblastoma. BACKGROUND There is a need for effective therapy against recurrent high-grade glioma (rHGG), including astrocytoma IDH-mutant (grade III and IV) and glioblastoma (GBM-grade IV). HGG is one of the most common types of primary brain tumor and one of most lethal of human cancers. Despite aggressive standard-of-care (SOC) therapy, including initial tumor resection and post-operative adjuvant radiation plus temozolomide or nitrosourea chemotherapy, tumor recurrence is almost inevitable and uniformly lethal. GBM, the most common and aggressive HGG histology (∼45% of all malignant gliomas), has a 5-year relative survival of ∼5%. Furthermore, there is no SOC for treating recurrent GBM, and the current treatment options include additional surgery, bevacizumab, nitrosourea, lomustine, temozolomide rechallenge, carmustine wafers, or experimental therapies. Median overall survival for GBM following first recurrence is estimated to be 5-8 month. The intractable nature of rHGGs, particularly recurrent GBM (rGBM), provides compelling motivation for developing novel treatment interventions for this devastating disease. SUMMARY Described herein are T cells expressing an IL13Rα2 targeted chimeric antigen receptor (IL13Rα2-CAR) and lacking a functional TGFβR2 gene. The IL13Rα2-CAR includes a targeting domain that includes, for example, the sequence: Attorney Docket No.: 40056-0090WO1 GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 1), a variant of IL-13 having the E13Y mutation; a spacer domain; a transmembrane domain; a co-stimulatory domain; and a CD3zeta domain. A phase 1 clinical trial evaluating locoregional delivery of IL13Rα2-CAR T cells in 65 patients with recurrent high-grade glioma, the majority being rGBM has been completed. In brief, this study evaluated five treatment arms to assess three locoregional delivery routes [intratumoral (ICT), intraventricular (ICT) and dual ICT / ICV] and two manufacturing platforms (Tcm-derived and Tn / mem-derived T cells for CAR engineering).Overall, it was found that weekly delivery of up to 22 cycles of IL13Rα2-CAR T cells were well-tolerated with no dose-limiting toxicities and clinically manageable adverse events at doses up to 200M / cycle. Stable disease or better was achieved in 50% of patients (29 of 58 evaluable patients), with two partial responses, one complete response (CR), and a second CR after additional CAR T cycles were given off-protocol therapy. Patients with rGBM treated with dual ICT / ICV delivery and the optimized Tn / mem manufacturing platform exhibited the best median overall survival of 10.2 months. While this is highly encouraging for this difficult to treat disease, all patients eventually relapsed and clinical responses for GBM patients were transient. Described herein are IL13Rα2-CAR T cells resistant to TGFβ. These cells have been developed in order to address the suppressive effects of TGFβ in glioma microenvironment, one of the key drivers of immune suppression in GBM, which has been linked to T cell dysfunction / suppression as well as glioma invasion / progression. IL13Rα2-CAR A preferred IL13Rα2-CAR CAR (IL13 variant / IgG4 (EQQ) spacer / CD4TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAI EKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNESKY GPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHQAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT Attorney Docket No.: 40056-0090WO1 TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMA LIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR SEQ ID NO: 30 A preferred IL13Rα2-CAR (IL13 variant / IgG4 (EQ) spacer / CD4TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNESK YGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM ALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR SEQ ID NO:31 In some embodiments, an IL13Rα2-CAR (IL13 variant / IgG4 (EQ) spacer / CD8TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNESK YGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYI WAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREYDVLDKRRGRDPEMGGKPR Attorney Docket No.: 40056-0090WO1 RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR SEQ ID NO:32 In some embodiments, an IL13Rα2-CAR (IL13 variant / IgG4 ΔCH2 spacer / CD28TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWVLVVVGG VLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR SEQ ID NO:33 In some embodiments, an IL13Rα2-CAR (IL13 variant / IgG4 ΔCH2 spacer / CD4TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMALIVLGGV AGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGG RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO:34 In some embodiments, an IL13Rα2-CAR (IL13 variant / IgG4 ΔCH2 spacer / CD8TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: Attorney Docket No.: 40056-0090WO1 GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGT CGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRV KFSRSADAPAYQQGQNQLYNELNLGRREYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:35 In some embodiments, an IL13Rα2-CAR (IL13 variant / IgG4 (EQQ) spacer / CD8TM / 41BB / GGG / CD3zeta) comprises the amino acid sequence: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSA IEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNESK YGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHQAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYI WAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR SEQ ID NO:36 In some embodiments, an IL13Rα2-CAR comprises or consists of an amino acid sequence selected from SEQ ID NOs:30-36. In some embodiments, an IL13Rα2-CAR comprises an amino acid sequence selected from SEQ ID NOs:30-36 or a variant thereof having 1-10 (e.g., 1, 2, 3, 4, 5…) amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions). In some embodiments, the amino acid changes (e.g., amino acid substitutions) are not in the IL-13 variant sequence. An IL13Rα2-CAR includes: an targeting domain comprising a variant of IL-13, a spacer domain, a transmembrane domain, a co-stimulatory domain and a CD3zeta domain. A variety of IL13Rα2-CAR are described in WO 2021 / 184960, hereby incorporated by reference. Attorney Docket No.: 40056-0090WO1 Spacer Domain The CAR or polypeptide described herein can include a spacer located between the targeting domain (i.e., IL13 or variant thereof) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can be used in the CARs described herein. Table 1: Examples of Spacers Attorney Docket No.: 40056-0090WO1 Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ΔCH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off- target binding. The spacer region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3). The spacer region can also comprise the hinge sequence ESKYGPPCPPCP (SEQ ID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:12). Thus, the entire spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK (SEQ ID NO:9). Transmembrane Domain A variety of transmembrane domains can be used in the CAR. In some cases, the transmembrane domain is a CD28 transmembrane domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: Attorney Docket No.: 40056-0090WO1 FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:14). In some cases, the CD28 transmembrane domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:14. Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain (TM) is located carboxy terminal to the spacer region. Table 2: Examples of Transmembrane Domains Costimulatory Domain The costimulatory domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the co-signaling domain is a CD28 co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 22). In Attorney Docket No.: 40056-0090WO1 some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:22. The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3ζ signaling domain.

[0002] Attorney Docket No.: 40056-0090WO1 Table 3: CD3ζ Domain and Examples of Costimulatory Domains Attorney Docket No.: 40056-0090WO1 In various embodiments: the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present. In some embodiments there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. The costimulatory domain is amino terminal to the CD3ζ signaling domain and a short linker consisting of 2 – 10, e.g., 3 amino acids (e.g., GGG) is can be positioned between the costimulatory domain and the CD3ζ signaling domain. Signaling Domain The CD3ζ signaling domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the CD3ζ signaling domain includes a sequence that is at least 90%, Attorney Docket No.: 40056-0090WO1 at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO:21). In some cases, the CD3ζ signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21. In some case the CD3ζ signaling domain comprises any of SEQ ID NOs: 50-56. These variant CD3ζ signaling domains have Y to F mutations in one or more ITAM domains. In some cases it is preferable to use a variant with mutations that inactive ITAMs 2 and 3. Truncated EGFR and Truncated CD19 The CD3ζ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 26) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVA FRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHG QFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISN RGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREF VENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVAL GIGLFM (SEQ ID NO: 27). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 27. Alternatively the CD3ζ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 26) and a truncated CD19R having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESP LKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVN VEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGE PPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLS LELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWH WLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 28) An amino acid modification refers to an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. An “amino acid substitution” or "substitution" refers to Attorney Docket No.: 40056-0090WO1 replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine. In some cases, the CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt) or truncated CD19 (CD19t). In this arrangement, co-expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt or CD19t incorporated in the lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity. The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte, and most preferably an autologous T lymphocyte. Attorney Docket No.: 40056-0090WO1 Various T cell subsets isolated from the patient can be transduced with a vector for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+ / CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3 / CD28, transduced with, for example, a lentiviral vector that directs the expression of the CAR as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated / genetically modified CAR T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Additional methods of preparing CAR T cells can be found in PCT / US2016 / 043392. In some embodiments, any of the populations of CAR T cells described herein are genetically modified. In some embodiments, one or more genes are knocked out, down regulated, or upregulated. In some embodiments, of the population of CAR T cells lack expression of TGFβR2. In some embodiments, TGFβR2 is knocked out. In some embodiments, TGFβR2 is down regulated. In some embodiments, genetic modification is achieved by methods described herein and those known in the art. In some embodiments, genetic modification methods comprise gene editing, homologous recombination, nonhomologous recombination, RNA-mediated genetic modification, DNA-mediated genetic modification, zinc finger nucleases, meganucleases, TALEN, or CRISPR / CAS9. In some embodiments, TGFβR2 is knocked out by CRISPR-Cas9 gene editing technology using ribonucleoprotein (RNP) complex delivery (e.g., electroporation of a Cas9 and a TGFβR2-targeting sgRNA). In some embodiments, any of the populations of CAR T cells described herein are administered with a TGFβR1 inhibitor (TGFβR1i), e.g., LY3200882, or a functional variant thereof (such as a blockade of TGFβ signaling pathway or an agent that acts as a TGFβR1 inhibitor). In some embodiments, the TGFβR1i (or functional variant thereof) is administered before the population of CAR T cells. In some embodiments, the TGFβR1i (or functional variant thereof) is administered at the same time as the population of CAR T cells. In some embodiments, the TGFβR1i (or functional variant thereof) is administered before the population of CAR T cells and at the same time as the population of CAR T cells. Also described herein are methods of treating a patient suffering from a glioma (e.g., glioblastoma), pancreatic ductal adenocarcinoma, melanoma, ovarian carcinoma, renal cell Attorney Docket No.: 40056-0090WO1 carcinoma, breast cancer or lung cancer, comprising administering a population of autologous or allogeneic CAR immune cells lacking expression of TGFβR2. In some embodiments, the glioma is recurrent high-grade glioma (rHGG), astrocytoma IDH-mutant (grade III and IV) or glioblastoma (GBM-grade IV). In various embodiments: the cells are administered locally or systemically; the cells are administered intraventricularly; the cells administered by single or repeat dosing. In some embodiments, methods of treating a patient suffering from a glioma (e.g., glioblastoma), pancreatic ductal adenocarcinoma, melanoma, ovarian carcinoma, renal cell carcinoma, breast cancer or lung cancer, comprising administering a population of autologous or allogeneic immune cells and a TGFβR1i (or functional variant thereof). In some embodiments, the glioma is recurrent high-grade glioma (rHGG), astrocytoma IDH-mutant (grade III and IV) or glioblastoma (GBM-grade IV). In various embodiments: the cells are administered locally or systemically; the cells are administered intraventricularly; the cells administered by single or repeat dosing. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety for any and all purposes. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIG 1: Amino acid sequence of an exemplary IL13Rα2-CAR (SEQ ID NO: 30). FIG 2: Schematic diagram of one method for preparing T cells that express a chimeric antigen receptor targeted to IL13Rα2 and lack functional TGFβR2. Attorney Docket No.: 40056-0090WO1 FIG 3: Expansion of Qualification Run Products. Viable cell numbers starting at the day of thaw and Transact™ stimulation are indicated for qualification runs HD562, HD624 and HD680, as well as their wildtype (WT; non-TGFβ2RKO) counterparts. Log 2 scales are depicted for the Y-axes to better depict doubling time of cells in culture. FIG 4: Surface Phenotype of Qualification Run Products. Qualification run TGFβR2KO / IL13Rα2-CAR T cell products were stained with fluorochrome-conjugated antibodies to detect either the T cell marker CD3, or the CD19t transgene marker. Percentages of viable cells (DAPI negative) with immunoreactivity above control staining are indicated in each histogram. FIG 5: Surface Phenotype of Qualification Run Products and their WT (non- TGFβR2KO) Counterparts. Qualification run TGFβR2KO / IL13Rα2-CAR T cell products (KO) and their wildtype IL13Ra2-CAR T cell counterparts (WT) were stained with fluorochrome-conjugated antibodies to detect the indicated cell surface markers. Percentages of viable, CD19t-gated cells with immunoreactivity above control staining are plotted for each cell product. Bars indicate mean and S.E. FIGS 6A-6C: Effector Activity of TGFβR2KO / IL13Rα2-CAR T Cells. A, Relative IL13Ra2 expression levels on human primary brain tumor (PBT) lines that were used in effector activity assays. Percentages of viable cells (DAPI negative) with immunoreactivity above isotype control staining are indicated in each histogram. Parental PBT138 was used as a negative control target / stimulator, PBT138 cells that had been transduced to express either low (PBT138-low) or high (PBT138-high) levels of IL13Rα2, and PBT030 cells that endogenously expressed high levels IL13Rα2 were used as positive targets / stimulators. B, Qualification run TGFβR2KO / IL13Rα2-CAR T cell products (KO) and their wildtype (WT; non-TGFβR2 knockout) IL13Rα2-CAR T cell counterparts were used as effectors in a flow cytometry based 48-hour killing assay at an E:T ratio of 1:4 CAR+ T cells to tumor cells. % Tumor killing was calculated by comparing the viable numbers of the indicated target line co- cultured with CAR+ T cells to that of tumor only; i.e., using the following equation: 100 – (100 x (counts in co-culture / counts in tumor only)). Bars indicate Mean ± S.E.M.. C, Qualification run TGFβR2KO / IL13Rα2-CAR T cell products and their WT IL13Rα2-CAR T cell counterparts were used as effectors in a cytokine production assay in which supernatants were collected after overnight co-culture with the indicated targets at an E:T ratio of 1:2 Attorney Docket No.: 40056-0090WO1 CAR+ T cells to tumor cells. IFN-γ (top) and IL-2 (bottom) levels in the supernatants were then measured by bioplex assay. Bars indicate Mean ± S.E.M.. FIGS 7A-7B: Long-Term Activity of TGFβR2KO / IL13Rα2-CAR T Cells in the Presence of TGF-β. A, Qualification run TGFβR2KO / IL13Rα2-CAR T cell products (KO) and their wildtype (WT; non-TGFβR2 knockout) IL13Rα2-CAR T cell and mock-transduced (Mock) T cell counterparts were used as effectors in a 7-day killing assay at an E:T ratio of 1:50 CAR+ T cells to tumor cells (or 1:50 total Mock T cells to tumor cells). TGF-β was added to the co-cultures as indicated by ‘+TGF-β’. % Tumor killing was calculated by comparing the viable numbers of the indicated target line co-cultured with T cells to that of tumor only; i.e., using the following equation: 100 – (100 x (counts in co-culture / counts in tumor only)). Bars indicate Mean ± S.E.M.. B, Numbers of viable CAR+ T cells (or total Mock T cells) were determined after the 7-day co-cultures by flow cytometry. Mean ± S.E.M. of triplicate wells are depicted. Using a Student’s t-test to compare the WT and KO values for each condition: *, P < 0.02; **, P < 0.005; ***, P < 0.0005; ****, P < 0.000001. FIGS 8A-8C: Control of GBM xenografts after adoptive transfer of TGFβR2KO / IL13Rα2-CAR T cells. A, ffLuc+PBT030-2 cells (0.1x106) were administered IC into NSG mice. On day 8 mice were left untreated or treated IC with 10x103CAR+ T cells – either wildtype (WT) IL13Rα2-CAR T cells or TGFβR2KO / IL13Rα2-CAR T cells, n = 5-8 per group. B, Quantification of ffLuc flux (photons / sec) in each group over time. Each mouse is depicted as a dashed line, with geometric means indicated as a thick solid line. C, Kaplan Meier survival curves. A log-rank (Mantel-Cox) test was used to compare the WT IL13Rα2- CAR T cell and TGFβR2KO / IL13Rα2-CAR T cell treated groups (p = 0.03). FIGS 9A-9C: Control of GBM tumors after treatment with mouse TGFβR2KO / IL13Rα2-CAR T cells in an immunocompetent mouse model. A, K-Luc- Rα2 cells (0.1x106) were administered IC into C57BL / 6 mice. On day 13 mice were left untreated or treated IC with 1 x 106CAR+ mouse T cells – either wildtype (WT) IL13Rα2- CAR T cells or TGFβR2KO / IL13Rα2-CAR T cells, n = 5-10 per group. B, Quantification of ffLuc flux (photons / sec) in each group over time. Each mouse is depicted as a dashed line, with geometric means indicated as a thick solid line. A mixed-effects analysis was used to compare WT IL13Rα2-CAR T cell and TGFβR2KO / IL13Rα2-CAR T cell treated groups (p < 0.0001). C, Kaplan Meier survival curves. A log-rank (Mantel-Cox) test was used to Attorney Docket No.: 40056-0090WO1 compare the WT IL13Rα2-CAR T cell and TGFβR2KO / IL13Rα2-CAR T cell treated groups (p = 0.0005). FIGS 10A-10E: TGFβ levels in patients with glioblastoma. A. Schematic of IL13Rα2- CAR structure. B. Schema of phase I clinical trial (NCT02208362) using IL13Rα2-CAR T cells in patients with recurrent glioblastoma. C. TGFβ levels in tumor fluid (TF) and cerebrospinal fluid (CSF) of patients with GBM prior to CAR T therapy. D. Comparison of TGFβ levels in tumor fluid (TF, left) or cerebrospinal fluid (CSF, right) before and after CAR T treatment. E. Correlation between TGFβ levels in tumor fluid (TF, left) or cerebrospinal fluid (CSF, right) in relation to clinical response to CAR T therapy. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease. FIGS 11A-11E: Inhibition of TGFβ signaling in combination with CAR T therapy changes in the tumor microenvironment A. Schematic of experimental design. B. Graphs quantify flow cytometry analysis of CAR and endogenous T cell phenotype in untreated, CAR T, TGFβRIi and combination CAR T plus TGFβRIi treatments. C. Histograms of mean fluorescence intensity of the indicated markers on the myeloid population, measured by flow cytometry in each treatment groups. D-E. Heatmaps show mRNA expression of tumor microenvironment for each treatment group. Data are presented as means ± SEM and were analyzed by two-tailed, unpaired student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 for the indicated comparison. FIGS 12A-12C: Blockade of TGFβ signaling improves efficacy of CAR T therapy in a large established glioma model A. Schematic of experimental design. Mice bearing glioma tumors (14-day old tumor) were treated with TGFβRI inhibitor daily before and after IL13Rα2-CAR T treatment. B. Tumor growth kinetics demonstrate changes in tumor progression over time. C. Kaplan-Meier survival curve shows overall survival of mice receiving monotherapy (TGFβRI inhibitor alone or CAR T alone) compared to combination therapy (TGFβRI inhibitor plus CAR T therapy). Data are presented as means ± SEM and were analyzed by two-tailed, unpaired student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for indicated comparison. FIG 13: Additional IL13Rα2-CAR (SEQ ID NOs: 31 to 35). DETAILED DESCRIPTION Attorney Docket No.: 40056-0090WO1 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Described below is the preparation of T cells expressing IL13Rα2-CAR and lacking expression of TGFβR2. In the methods described below CRISPR / Cas9 was used to knock out TGFβR2. Example 1: Manufacturing of TGFβR2KO / IL13Rα2-CAR T cells. FIG 2 provides a schematic depiction of an example of a method for preparing TGFβR2KO / IL13Rα2-CAR T cells. The cells were prepared essentially as described below. Recovery, Isolation, and Activation (Days 1 to 2) Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the leukapheresis collection using Ficoll in either a centrifugation or Sepax cell separation system. PBMCs were washed and placed in complete medium (X-VIVO™ 15 + 10% FBS) overnight on a rotator at room temperature. Naïve and memory T cells (Tn / mem) were isolated from the PBMCs using immunomagnetic depletion followed by selection. Up to 3 x 109viable PBMCs were incubated with anti-CD14 and anti-CD25 reagent and depleted on a CliniMACS®Plus device. Negatively selected (CD14 / CD25-negative) cells were incubated with anti-CD62L reagent and cells were enriched using the CliniMACS®Plus device. After CD62L+ cell enrichment, Tn / mem cells were either cryopreserved or proceeded directly to the TransAct activation step. Thawed or fresh Tn / mem, at a concentration of 1 million (M) / mL, were mixed with 200μL TranAct / mL and placed in cell culture flask(s). Transduction (Day 3) Activated Tn / mem cells were transduced with lentiviral vector encoding IL13Rα2-CAR at an MOI of 0.5 in closed culture bags with protamine sulfate (25 ug / mL) as a transduction enhancer. After the transduction period was complete (6 hours minimum), cells were diluted in complete medium with cytokines (i.e., rhIL-2 [final] = 50 U / mL, and rhIL-15 [final] = 0.5 Attorney Docket No.: 40056-0090WO1 ng / mL). The following day (Day 4), there was a scheduled half-media change and cytokines were replenished prior to CRISPR / Cas9 transfection on Day 5. If transfection is delayed to Day 7, then a half-media change and replenishment of cytokines can take place again on Day 6. CRISPR / Cas9 Transfection (Day 5 to Day 7) CAR transduced Tn / mem cells were counted and prepared for transfection. Ribonucleoprotein (RNP) was be prepared by mixing TGFβR2-targeting sgRNA and SpyFi™ Cas9 at a ratio of 3:1 (225pmol sgRNA and 75 pmol Cas9 for every 5M T cells) and incubated at room temperature for a minimum of 15 minutes. The appropriate number of T cells were centrifuged, and washed with electroporation (EP) buffer, and then mixed with the prepared RNP at a cell concentration of 50-100M / mL. The RNP / cell mixture was transferred to an appropriate sized Maxcyte GT™ single-use vials, and the program "Expanded T Cell – 1” was used for electroporation. After transfection, cells were resuspended at 1M / mL in complete medium with cytokines. Culture and Harvest Transfected cells were maintained in complete medium with cytokines to maintain a concentration of approximately 0.3-0.6M / mL. On the day of harvest, cells were washed and concentrated using either a centrifuge or LOVO device. After washing, cells were pelleted by centrifugation and resuspended in CryoStor CS5 cryopreservation medium. Cryovials were filled with cells resuspended in CS5 and frozen using a controlled rate freezer (CRF). Cryopreserved autologous TGFβR2KO / IL13Rα2-CAR T cells (IP) can be stored at LN2 vapor temperatures until preparation for patient infusions. Table 4 summarizes certain steps in the process. Table 4: Example of cell preparation steps Attorney Docket No.: 40056-0090WO1 Example 2: Characterization of expansion and transduction of TGFβR2KO / IL13Rα2- CAR T cells The objective of this study was to evaluate the ex vivo expansion and cell surface phenotype of human T cells that had undergone immunomagnetic enrichment for the CD62L+ naïve and memory T cell populations (Tn / mem), lentiviral transduction with GMP-grade IL13(EQ)BBZ-T2A-CD19t _epHIV7 lentiviral vector, further gene editing with CRISPR / Cas9 to knockout TGFβR2, and expanded ex vivo as is proposed for clinical use. Qualification run CAR T cell products from three healthy donor PBMC were generated using methodologies proposed for clinical use. Briefly, Tn / mem cells were enriched from healthy donor PBMC. Specifically, one day after leukapheresis, CliniMACS® depletion was employed to remove the CD25+ regulatory T cells, and the CD14+ monocytes, followed by the positive selection of the CD62L+ naïve / memory (Tn / mem) population. The selected Tn / mem were activated using T cell TransAct and transduced with IL13(EQ)BBZ-T2A- CD19t _epHIV7 GMP-grade lentivirus in accordance. Cells were then maintained in Attorney Docket No.: 40056-0090WO1 complete X-VIVO 15 media with cytokine supplementation until they were electroporated 2- 4 days later for delivery of the CRISPR / Cas9 protein along with TGFβR2-targeting sgRNA. Expansion in complete media with cytokine supplementation was then carried out, with viable cell numbers being monitored during this expansion process. Final cell products were cryopreserved and then freshly thawed at a later time for flow cytometric evaluation of the T cell marker CD3 and the truncated CD19 (CD19t) transgene marker. Freshly thawed TGFβR2KO / IL13Rα2-CAR T cell products and their wildtype (WT; non-TGFβR2 knockout) IL13Rα2-CAR T cell counterparts were also examined for their surface expression of the markers CD4, CD8, CD27, CD28, CD45RA, CD57, CD62L, CCR7, Lag-3, PD-1, and Tim-3 to evaluate if the knockout of TGFαβR2 affected the overall T cell phenotype of the CAR T cells. Healthy donor blood products were ficolled and the resulting peripheral blood mononuclear cells (PBMC) underwent sequential rounds of CliniMACS® depletion and selection to enrich for the Tn / mem population. The first step involved magnetic depletion to remove the CD25+ regulatory T cells and the CD14+ monocytes. The remaining cells then underwent a positive selection for the CD62L+ population. The resulting Tn / mem cells were cryopreserved. On the day of thaw, the Tn / mem cells were resuspended in complete X-VIVO media in T-75 flasks and activated overnight with T Cell TransAct™ (Miltenyi). The next day the cells were lentivirally transduced with GMP-grade IL13(EQ)BBZ-T2A-CD19t _epHIV7 at a multiplicity of infection (MOI) of 0.5. Cultures were then maintained with addition of complete X-VIVO 15 media and cytokine supplementation (rhIL-2 and rhIL-15) until electroporation 2-4 days later. On the day of electroporation, cells were washed and concentrated to 50-100 x 106cells / mL, mixed with Cas9 / sgRNA mixture, incubated briefly, and the mixture was transferred to the MaxCyte GT system for electroporation. The electroporated cells were resuspended in complete X-VIVO 15 media, cytokines were added after 72 hours, and expansion was carried out with addition of complete X-VIVO 15 media as required based on cell density, and cytokine supplementation every Monday, Wednesday and Friday of culture. Viable cell numbers were monitored during this expansion process by taking culture sample cell counts using the Muse Cell Analyzer. The resulting TGFβR2KO / IL13Rα2-CAR T cell qualification runs were cryopreserved. Freshly thawed cells were washed in FACS Stain Solution (FSS) using a tabletop centrifuge, resuspended in FSS and 100 µL per sample was aliquoted into pre-labeled 12x75 mm FACS Attorney Docket No.: 40056-0090WO1 tubes (1 tube per condition). The required volume of antibody was added to their respective FACS tubes, and tubes were then incubated for 30 minutes in the dark at 4°C. At the end of incubation each tube was washed twice in FSS and resuspended in 200 ^L FSS and 100 ^L working dilution of DAPI. Samples were then run and analyzed on a MACSQuant (Miltenyi) instrument. Percentages of immunoreactive cells were calculated above isotype control staining using FCS Express (De Novo Software, Los Angeles, CA). After lentiviral transduction with IL13(EQ)BBZ-T2A-CD19t _epHIV7 and electroporation with Cas9 and the TGFβR2-targeting sgRNA, the preclinical qualification run T cell products were expanded. Growth curves for each of the TGFβR2KO / IL13Rα2-CAR T cell products and their wildtype (WT; non-TGFβR2 knockout) IL13Rα2-CAR T cell counterparts are depicted in FIG 3. Flow cytometric analyses of each of the qualification run TGFβR2KO / IL13Rα2-CAR T cell products following ex vivo expansion revealed that the final cell products expressed surface the T cell marker CD3 (FIG 4). Furthermore, following transduction at an MOI of 0.5, each of the qualification run products exhibited transgene surface expression, with 34-76% of cells immunoreactive for CD19-specific staining (i.e., to detect the truncated CD19 transduction marker that is expressed as part of the IL13(EQ)BBZ-T2A-CD19t _epHIV7 gene cassette) (FIG 4). Gating on transduced, CD19t+ cells, it was also determined that the qualification run TGFβR2KO / IL13Rα2-CAR T cells expressed a similar surface phenotype as that of their WT (non-TGFβR2 knockout) IL13Rα2-CAR T cell counterparts (FIG 5). Together, these data indicate that enriched Tn / mem that had undergone lentiviral transduction with IL13(EQ)BBZ-T2A-CD19t _epHIV7 and further gene editing with CRISPR / Cas9 to knockout TGFβR2, could be expanded to numbers sufficient for clinical application. Flow cytometric analysis confirmed that the ex vivo expanded qualification run samples expressed the T cell surface marker CD3 and were efficiently transduced using an MOI of 0.5 to express the CD19t transgene. Furthermore, comparison of surface marker expression to that of wildtype IL13Rα2-CAR T cells confirmed that the knockout of TGFβR2 does not significantly alter the surface phenotype of the CAR T cell products. These data obtained from the qualification runs indicate that our protocol for generating genetically-altered Tn / mem-derived cells results in cell products that exhibit a T cell phenotype. Furthermore, transduction with IL13(EQ)BBZ-T2A-CD19t _epHIV7 at an MOI Attorney Docket No.: 40056-0090WO1 of 0.5 is sufficient to produce a cell product that expresses the transgenes in 34% or more of the population as determined by staining for the CD19t marker. Example 3: Effector Activity of TGFβR2KO / IL13Rα2-CAR T Cells. The objective of this study was to evaluate the effector activity of qualification run TGFβR2KO / IL13Rα2-CAR T cells as compared to their wildtype (WT; non-TGFβR2 knockout) IL13Rα2-CAR T cell counterparts. Short-term effector activity against IL13Rα2+ tumor cells was evaluated using a flow cytometry-based 48-hour killing assay, and cytokine production was evaluated by bioplex analysis of supernatants after co-culture with various stimulator cell lines. Long-term activity was evaluated using flow cytometry to measure tumor cell killing and CAR T cell viability after 7 days of co-culture with IL13Rα2+ tumor cells in the presence or absence of TGFβ. Qualification run TGFβR2KO / IL13Rα2-CAR T cell products were generated from the PBMC of three different healthy donors using methodologies proposed for clinical use. Briefly, Tn / mem cells that had been enriched from PBMC by MACS™ selection were lenti- transduced with GMP-grade IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentivirus, further gene edited by CRISPR / Cas9 to knockout TGFβR2, expanded and then cryopreserved. Cell products were then evaluated for effector function by a flow cytometry-based 48-hour kill assay, and by bioplex analysis of harvested supernatants for cytokine production after overnight in vitro co-culture with stimulator cell lines. Long-term activity of the cell products was also evaluated using flow cytometry to measure tumor cell killing and CAR T cell viability after 7 days of co-culture with IL13Rα2+ tumor cells in the presence or absence of TGFβ. Samples of target / stimulator cell lines were washed in FACS Stain Solution (FSS) using a tabletop centrifuge, resuspended in FSS and aliquoted into a pre-labeled 96-well plate. The samples were stained with fluorchrome-conjugated anti-IL13Rα2 antibody for 30 minutes in the dark at 4°C. At the end of incubation each sample was washed twice in FSS and resuspended in 200 ^L FSS and 100 ^L working dilution of DAPI. Samples were then run and analyzed on a MACSQuant (Miltenyi) instrument. Percentages of immunoreactive cells were calculated above isotype control staining using FCS Express (De Novo Software, Los Angeles, CA). Attorney Docket No.: 40056-0090WO1 Samples from each qualification run product were thawed, rested for 3-4 days in the presence of 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15. Each T cell line and target cell line was then counted and seeded at a 1:4 (48-hour assay) or 1:50 (7-day assay) CAR+ effector to total target cell ratio into a 96-well plate in 200uL X-VIVO 15 media with 10% FBS per well. Where indicated, TGFβ was also added to the wells. After 48-hours or 7-days of co-culture, cells were collected with trypsinization, washed in cold FACS buffer (HBSS- / - with 2% FBS), and then stained with anti-human CD45 to detect the T cells, with DAPI as a viability dye. Samples were run and analyzed on a MACSQuant (Miltenyi) instrument, and numbers of viable immunoreactive cells were calculated via FlowJo software (FlowJo, LLC, Ashland, OR). Final results were then graphed as percentages of live (DAPI-negative), CD45-negative, forward scatter high tumor cells remaining in the co-cultures when compared to that of tumor cell alone cultures which had been normalized to 100%. Samples from each qualification run product were thawed, rested for 3 days in X-Vivo15 Complete Media (containing 10% FCS) with 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15, and then plated at a 1:2 CAR+ effector to target cell ratio in a 96-well plate in 200uL X-VIVO 15 media with 10% FBS per well. After overnight culture, supernatants were harvested, diluted 1:2 or 1:10 and were analyzed by cytokine bead array in accordance with the FlexMap 3D System, and the Human Cytokine Magnetic 10-Plex Panel kit instructions (Thermo Scientific). After co-culture in the 7-day killing assay described above, cell samples were stained with anti-human CD45 to detect the mock-transduced, non-TGFβR2 knockout T cells, and anti- human CD19 to detect the CAR T cells (either WT or TGFβR2KO), with DAPI as a viability dye. Samples were run and analyzed on a MACSQuant (Miltenyi) instrument, and numbers of viable immunoreactive cells were calculated via FlowJo software (FlowJo, LLC, Ashland, OR).Qualification run TGFβR2KO / IL13Rα2-CAR T cell products were cultured with tumor lines that expressed high levels of IL13Rα2 (i.e., PBT030, or PBT138-high), low levels of IL13Rα2 (i.e., PBT138-low) or an IL13Rα2-negative tumor line (parental PBT138) as a negative control (FIG 6A). Results are presented below and demonstrate that TGFβR2KO / IL13Rα2-CAR T cells exhibit IL13Rα2-directed cytolytic activity (FIG 6B) and cytokine (e.g., IFN-γ and IL-2) production (FIG 6C) that is significantly higher than that seen with the negative control target. Furthermore, the IL13Rα2-specific activity of each Attorney Docket No.: 40056-0090WO1 qualification run is similar to that of their wildtype (WT; non-TGFβR2 knockout) IL13Rα2- CAR T cell counterpart. Qualification run TGFβR2KO / IL13Rα2-CAR T cell products and their wildtype (WT; non- TGFβR2 knockout) IL13Rα2-CAR T cell or mock-transduced T cell counterparts were cultured for seven days with IL13Rα2-expressing tumor lines (i.e., PBT030, or PBT138- high), in either the presence or absence of TGF-β to see if the knockout of TGFβR2 conferred any advantage. Results are presented below and demonstrate that TGFβR2KO / IL13Rα2-CAR T cells exhibit long-term killing that is similar to that of WT IL13Rα2-CAR T cells (FIG 7A). However, looking at viable CAR T cell numbers after the seven days of co-culture, the TGFβR2KO / IL13Rα2-CAR T cells were seen to have a significantly improved expansion compared to their WT IL13Rα2-CAR T cell counterparts (FIG 7B). This improved persistence of the TGFβR2KO / IL13Rα2-CAR T cells was observed both with and without the addition of TGFβ to the cultures, suggesting that TGFβ is produced in these long-term cultures. These data confirmed that the qualification run TGFβR2KO / IL13Rα2-CAR T cell products exhibited IL13Rα2-specific cytolytic activity and cytokine production upon co-culture with IL13Rα2-expressing tumor cells. Importantly, CRISPR / Cas9-mediated knock out of TGFβR2 does not significantly affect the CAR-mediated function of these T cells, as seen when comparing TGFβR2KO / IL13Rα2-CAR T cells to their wildtype IL13Rα2-CAR T counterparts in both short-term and long-term co-cultures. Furthermore, the long-term assays also revealed that the knockout of TGFβR2 conferred improved expansion of the IL13Rα2- CAR T cells, as expected. These data obtained from three qualification runs indicate that our protocol for generating TGFβR2KO / IL13Rα2-CAR T cells results in cell products that exhibit IL13Rα2-directed effector activity, and improved cell survival after long-term co-culture with tumor targets. Example 4: In Vivo Efficacy of TGFβR2KO / IL13Rα2-CAR T Cells. The objective of this study was to evaluate the in vivo activity of TGFβR2KO / IL13Rα2-CAR T cell products. In vivo anti-tumor efficacy of these cells was examined in both an immunodeficient mouse xenograft model, as well as in an immunocompetent mouse syngeneic model. Attorney Docket No.: 40056-0090WO1 For the human xenograft model, TGFβR2KO / IL13Rα2-CAR T cells were derived from human PBMC that had undergone enrichment for Tn / mem cells using MACS™ selection, lenti-transduction with the IL13(EQ)BBZ-T2A-CD19t_epHIV7 vector, CRISPR / Cas9- mediated knockout of TGFβR2, expansion and then cryopreservation. The human TGFβR2KO / IL13Rα2-CAR T cells were then evaluated for their ability to control the in vivo growth of intracranially (IC) engrafted IL13Rα2+ PBT030-2 cells in immunodeficient NSG mice. For the mouse syngeneic model, T cells expressing a murine IL13Rα2-targeting CAR and gene edited with CRISPR / Cas9 to knockout TGFβR2 were generated from immunocompetent C57BL / 6 mice. These mouse TGFβR2KO / IL13Rα2-CAR T cells were then evaluated for their ability to control the in vivo growth of IC engrafted IL13Rα2+ K-luc cells in C57BL / 6 mice. In both models, tumor burden and survival was examined after treatment with the CAR T cells delivered IC, as described below. For the human xenograft model, NSG mice (8-12 weeks old) were stereotactically injected IC with 0.1 x 106ffLuc+ PBT030-2 glioma cells (endogenously IL13Rα2+) at day 0 and allowed to engraft for 8 days. Mice were then grouped based on tumor size as determined by biophotonic imaging for similar tumor size distributions per group. Groups of mice (n = 5 to 8) were then left untreated, or treated IC (i.e., at the same stereotactic coordinates as the tumor) with 10 x 103CAR+ wild type (WT; non-TGFβR2KO) IL13Rα2-CAR T cells, or TGFβR2KO / IL13Rα2-CAR T cells. For the syngeneic mouse model, C57BL / 6 mice (8-12 weeks old) were stereotactically injected IC with 0.1 x 106ffLuc+ K-Luc cells that had been transduced to express mouse IL13Rα2 (K-Luc-Rα2 cells) at day 0 and allowed to engraft for 8 days. Mice were then grouped based on tumor size as determined by biophotonic imaging for similar tumor size distributions per group. Groups of mice (n = 5 to 10) were then left untreated, or treated IC (i.e., at the same stereotactic coordinates as the tumor) with 1 x 106CAR+ wild type (WT; non-TGFβR2KO) mouse IL13Rα2-CAR T cells, or mouse TGFβR2KO / IL13Rα2-CAR T cells. Attorney Docket No.: 40056-0090WO1 In both models, ffLuc+ tumor growth was monitored over time by LagoX imaging and quantification of ffLuc flux (photons / sec), and mouse survival was monitored by Kaplan Meier curve analysis. We first evaluated the anti-tumor potency of human TGFβR2KO / IL13Rα2-CAR T cells against the IL13Rα2+ primary low-passage glioblastoma tumor sphere line PBT030-2, which has been engineered to express the firefly luciferase (ffLuc) reporter gene. The ffLuc+ PBT030-2 xenograft model has been previously used to evaluate in vivo anti-tumor activity of IL13Rα2-specific CAR expressing T cells in NSG mice (e.g., PMID: 22407828). In the experiment depicted in FIG 8, a single IC injection of TGFβR2KO / IL13Rα2-CAR T cells (10x103CAR+ cells) significantly improved survival as compared to treatment with non- TGFβ resistant, wildtype IL13Rα2-CAR T cells (p = 0.03). While these were not formal toxicity studies, the mice were monitored daily for any obvious signs of distress or general toxicity. Mice treated IC with the CAR T cells in these xenograft experiments did not exhibit any weight loss, and were bright, alert and reactive throughout the experiment, unless they were succumbing to high tumor burden. To evaluate anti-tumor potency in an immunocompetent mouse model, mouse TGFβR2KO / IL13Rα2-CAR T cells were used to treat the C57BL / 6 derived glioma K-Luc- Rα2 line, which has been engineered to express both the ffLuc reporter gene and the mouse IL13Rα2 antigen. The ffLuc+ K-Luc-Rα2 xenograft model has been previously used to evaluate in vivo anti-tumor activity of IL13Rα2-specific CAR expressing mouse T cells in C57BL / 6 mice (PMID: 33837065). In the experiment depicted in FIG 9, a single IC injection of mouse TGFβR2KO / IL13Rα2-CAR T cells (1 x 106CAR+ cells) significantly improved tumor growth control and survival as compared to treatment with non-TGFβ resistant, wildtype mouse IL13Rα2-CAR T cells. While these were not formal toxicity studies, the mice were monitored daily for any obvious signs of distress or general toxicity. Mice treated IC with the CAR T cells in these syngeneic experiments did not exhibit any weight loss, and were bright, alert and reactive throughout the experiment, unless they were succumbing to high tumor burden. Example 5: TGF β in glioblastoma Attorney Docket No.: 40056-0090WO1 We recently completed a phase I trial of IL13Rα2-targeted CAR T cells for recurrent glioblastomas (GBM) and other high-grade gliomas, the largest clinical study completed to date (NCT02208362). Samples from this trial were evaluated for TGFβ, an immunosuppressive cytokine highly upregulated in GBM. We measured TGFβ levels in 781 patient samples collected from the tumor site (tumor fluid; TF) or cerebral spinal fluid (CSF) before and after CAR T treatments. Compared to CSF, TF exhibited a higher concentration of TGFβ indicating a more immunosuppressive environment in the tumor bed. We also found that TGFβ levels decreased in the TF after CAR T therapy, but not the CSF. Furthermore, higher TGFβ levels in TF correlated with poor prognosis and lack of response to CAR T therapy. This patient information strongly indicates that targeting the TGFβ pathway may be of importance for CAR T therapy of GBM. FIG 10 demonstrates these results. To evaluate the importance of targeting TGFβ pathway for CAR T therapy, we used a syngeneic glioma model characterized as highly invasive and nonresponsive to immunotherapies, such as checkpoint blockade. We demonstrated that pretreatment with TGFβR1 inhibitor (TGFβR1i), LY3200882, significantly augmented the efficacy of CAR T therapy and improved overall survival of mice bearing large established tumors. TME characterization revealed upregulation of migratory phenotype in both CAR and endogenous T cells. These results indicate that blockade of TGFβ in the TME augments the antitumor function CAR T cells by potentially enhancing both endogenous and CAR T cells migration and trafficking to the tumor. Next, we assessed the impact of blocking the TGFβ signaling on human CAR T cells. Blockade of TGFβ pathway (using TGFβR1 inhibitor) enhanced CAR T cell antitumor activity and proliferation upon multiple tumor-rechallenge in vitro. Gene expression analyses revealed upregulation of genes associated with cytotoxic and memory-stem-like phenotype and down regulation of genes associated with exhaustion and inhibitory pathways. The results of these studies are presented in FIG 11 and FIG 12. These studies demonstrate that in vivo anti-tumor efficacy can be observed with TGFβR2KO / IL13Rα2-CAR T cells. Indeed, IC administration of TGFβR2KO / IL13Rα2- CAR T cells resulted in significant survival benefit over that of non-knockout IL13Rα2-CAR T cells in both the PBT030-2 murine xenograft tumor model and immunocompetent, synegeneic mouse tumor model without inducing any obvious signs of distress or general toxicity in the mice. Attorney Docket No.: 40056-0090WO1 These data suggest that IC administered TGFβR2KO / IL13Rα2-CAR T cells exhibit in vivo anti-tumor activity against IC IL13Rα2-expressing GBM tumors in mice that is superior to that of WT (i.e., non-TGFβR2 knockout) IL13Rα2-CAR T cells. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. All references are herein incorporated in their entirety for any and all purposes.

Claims

Attorney Docket No.: 40056-0090WO1 WHAT IS CLAIMED IS:

1. A population of immune cells lacking expression of TGFβR2 and harboring nucleic molecule comprising a nucleotide sequence encoding a chimeric antigen receptor targeted to IL13Rα2 (IL13Rα2-CAR), wherein the chimeric antigen receptor comprises: an IL-13 variant having the E13Y mutation; a spacer domain; a transmembrane domain; a costimulatory domain and a CD3zeta domain.

2. The population of immune cells of claim 1, wherein the spacer domain is selected from the group consisting of: and IgG4(EQ) spacer domain, a IgG4(HL-CH3) spacer domain and an IgG4(CH3) spacer domain.

3. The population of immune cells of claim 1, wherein: the IL-13 variant comprises or consists of SEQ ID NO:1; the spacer domain comprises or consists of any one of SEQ ID NOs:2-12 and 64; the transmembrane domain comprises or consists of any one of SEQ ID NOs:13-20 and 66; the costimulatory domain comprises or consists of any one of SEQ ID NOs: 22-25 and 66; and the CD3zeta domain comprises or consists of any one of SEQ ID NO:21 and 50-56.

4. The population of immune cells of claim 1, wherein IL13Rα2-CAR comprises or consist of an amino acid sequence of any one of SEQ ID NOs: 30-36.

5. The population of immune cells of claim 1, wherein IL13Rα2-CAR comprises an amino acid sequence of any one of SEQ ID NOs: 30-36 or a variant thereof having 1-10 amino acid substitutions.

6. The population of immune cells of claim 5, wherein the amino acid substitutions are not in the IL13 variant portion of the CAR.

7. The population of immune cells claim 1, wherein the immune cells are human T cells.

8. The population of human T cells of claim 7, wherein the human T cells comprise central memory T cells, naive memory T cells, pan T cells, or PBMC substantially depleted for CD25+ cells and CD14+ cells.Attorney Docket No.: 40056-0090WO1 9. A method of treating a patient suffering from a glioma (e.g., glioblastoma), pancreatic ductal adenocarcinoma, melanoma, ovarian carcinoma, renal cell carcinoma, breast cancer or lung cancer, comprising administering a population of autologous or allogeneic immune cells of any one of claims 1-8.

10. The method of claim 9, wherein the cells are administered locally or systemically.

11. The method of claim 9, wherein the cells are administered intraventricularly.

12. The method of claim 9, wherein the cells administered by single or repeat dosing.

13. The method of claim 9, wherein in the glioma is recurrent high-grade glioma (rHGG), astrocytoma IDH-mutant (grade III and IV) or glioblastoma (GBM-grade IV).

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