IL-13 ALPHA 2 RECEPTOR CHIMERIC ANTIGEN RECEPTOR (IL13RA2) FOR TUMOR-SPECIFIC T-CELL IMMUNOTHERAPY

Abstract

The present invention relates to certain embodiments of the methods and compositions provided herein, relating to chimeric antigen receptors (CARs) that specifically bind to human extracellular domains of the IL-13 alpha 2 receptor (IL13Ra2), cells containing these CARs, and cell-based immunotherapy methods that target (or take as their target) cancer cells, such as solid tumor cells.

Description

IL-13 ALPHA 2 RECEPTOR CHIMERIC ANTIGEN RECEPTOR (IL13RA2) FOR TUMOR-SPECIFIC T-CELL IMMUNOTHERAPY Cross-reference to Related Applications

[0001] This application claims priority to United States of America Provisional Application No. 62 / 643055 filed on March 14, 2018 entitled “ILL-13 ALPHA 2 RECEPTOR CHIMERIC ANTIGEN RECEPTOR (IL13RA2) FOR TUMOR-SPECIFIC T-CELL IMMUNOTHERAPY” which is expressly incorporated herein by reference in its entirety. Reference to the Sequence List

[0002] This application is being submitted together with a Sequence List in electronic format. The Sequence List is provided as a file named SCRI169WOSEQLIST, created on Tuesday, March 12, 2019, which is approximately 62 KB in size. The information in the electronic Sequence List is expressly incorporated herein by reference in its entirety. Field of Invention

[0003] Some modalities of the methods and compositions provided herein relate to chimeric antigen receptors (CARs) that specifically bind to human extracellular domains of an IL-13 alpha 2 receptor (IL13Ra2), to cells containing these CARs, and to cell-based immunotherapy methods that target (or take as their target) cancer cells, such as solid tumor cells. Background of the Invention

[0004] Despite significant advances in understanding brain cancer, the mortality rate has remained consistent over the past decade, and innovative new therapies are urgently needed. To date, T-cell immunotherapy has emerged as a promising cancer therapy supported by remarkable clinical data reporting complete remission in patients with B-cell malignancies following administration of CAR T-cell therapies targeting CD19. However, the need for additional and improved T-cell immunotherapies remains. Brief Description of the Invention

[0005] Some embodiments of the methods and compositions provided herein include a nucleic acid encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: a ligand-binding domain that binds to / interacts with an IL-13 alpha 2 receptor (IL13Ra2); a polypeptide spacer between the ligand-binding domain and a transmembrane domain; the transmembrane domain; and an intracellular signaling region.

[0006] In some embodiments, the ligand-binding domain comprises: a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 20, or conservative variations thereof; a heavy chain complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 21, or conservative variations thereof; and / or a heavy chain complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 22, or conservative variations thereof.

[0007] In some embodiments, the ligand-binding domain comprises: a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 23, or conservative variations thereof; a light chain complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 24, or conservative variations thereof; and / or a light chain complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 25, or conservative variations thereof.

[0008] In some embodiments, the ligand-binding domain comprises a heavy chain variable domain (HV) comprising a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO:18.

[0009] In some embodiments, the ligand-binding domain comprises a light chain variable domain (LV) comprising a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 19.

[00010] In some embodiments, wherein the ligand-binding domain comprises: a heavy-chain CDR1 having the amino acid sequence of SEQ ID NQ:20; a heavy-chain CDR2 having the amino acid sequence of SEQ ID NO:21; and / or a heavy-chain CDR3 having the amino acid sequence of SEQ ID NO:22.

[00011] In some embodiments, the ligand-binding domain comprises: a light chain CDR1 having the amino acid sequence of SEQ ID NO:23; a light chain CDR2 having the amino acid sequence of SEQ ID NO:24; and / or a light chain CDR3 having the amino acid sequence of SEQ ID NO:25.

[00012] In some embodiments, the ligand-binding domain comprises a VH domain comprising a polypeptide having the amino acid sequence of SEQ ID NO:18.

[00013] In some embodiments, the ligand-binding domain comprises a VL domain having the amino acid sequence of SEQ ID NO:19.

[00014] In some forms, the ligand-binding domain is an antibody fragment, just as an antigen-binding fragment.

[00015] In some embodiments, the ligand-binding domain is a single-strand variable fragment (scFv). In some embodiments, the scFv comprises a VL-VH orientation. In some embodiments, the single-strand variable fragment (scFv) comprises a sequence that has at least 95% identity with the nucleotide sequence of SEQ ID NO:61. In some embodiments, the single-strand variable fragment (scFv) comprises the nucleotide sequence of SEQ ID NO:61.

[00016] In some embodiments, the separator comprises an amino acid sequence of X1PPX2P.

[00017] In some embodiments, the spacer region comprises a portion of a hinge region of a human antibody or a modified variant thereof.

[00018] In some forms, the spacer is 15 amino acids or less but not less than 1 or 2 amino acids.

[00019] In some embodiments, the spacer comprises, consists of, or essentially consists of a sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO:09. In some embodiments, the spacer comprises, consists of, or essentially consists of an amino acid sequence of SEQ ID NO:09, or conservative substitutions thereof.

[00020] In some embodiments, the spacer comprises, consists of, or consists essentially of, an lgG4 (S) hinge spacer.

[00021] In some embodiments, the spacer comprises, consists of, or essentially consists of, an lgG4-CH3 (M) hinge spacer.

[00022] In some embodiments, the spacer comprises, consists of, or essentially consists of, an lgG4-CH2(L234D, N297A)-CHE (L) hinge spacer.

[00023] In some modalities, the intracellular signaling region comprises primary and co-stimulatory signaling domains, optionally comprising all or a portion of a CD3-zeta in combination with a co-stimulatory domain selected from the group consisting of signaling domains of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, and B7-H3 and combinations thereof.

[00024] In some modalities, the intracellular signaling region comprises a CD3-zeta signaling portion and a 4-1 BB signaling portion.

[00025] In some forms, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm).

[00026] Some modality also includes a nucleic acid that encodes a marker sequence. In some modality, the marker sequence is a truncated receptor and is optionally an EGFRt, a HER2t, or a CD19t.

[00027] Some modality also includes a dihydrofolate reductase transgene configured for methotrexate selection. In some modality, the dihydrofolate reductase transgene is a dihydrofolate reductase double mutant (DHFRdm). In some modality, the dihydrofolate reductase double mutant comprises amino acid mutations at L22F and F31S.

[00028] Some embodiments of the methods and compositions provided herein include an expression vector comprising the nucleic acid of any of the embodiments provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral or adenoviral vector.

[00029] Some modalities of the methods and compositions provided herein include a nucleic acid-encoded chimeric antigen receptor (CAR) polypeptide of any of the modalities provided herein.

[00030] Some embodiments of the methods and compositions provided herein include a host cell comprising the nucleic acid of any of the embodiments provided herein.

[00031] In some modalities, the host cell is a CD8+ cytotoxic T lymphocyte selected from the pool consisting of previously untreated CD8+ T cells, CD8+ central memory T cells, CD8+ memory effector T cells, and CD8+ bulk T cells. In some modalities, the CD8+ cytotoxic T lymphocyte is a central memory T cell, wherein the central memory T cell is positive for CD45RO+, CD62L+, and CD8+.

[00032] In some modalities, the host cell is a CD4+ helper T cell selected from the pool consisting of previously untreated CD4+ T cells, CD4+ central memory T cells, CD4+ memory effector T cells, and CD4+ bulk T cells. In some modalities, the CD4+ helper T cell is a previously untreated CD4+ T cell, wherein the previously untreated CD4+ T cell is positive for CD45RA+, CD62L+, and CD4+ and negative for CD45RO.

[00033] In some modalities, the host cell is a precursor T cell.

[00034] In some modalities, the host cell is a hematopoietic stem cell.

[00035] Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising the host cell of any of the embodiments provided herein, and a pharmaceutically acceptable excipient.

[00036] Some embodiments of the methods and compositions provided herein include a method for preparing the host cell of any of the embodiments provided herein, comprising: introducing the nucleic acid of any of the embodiments provided herein into a cell; culturing the cell in the presence of anti-CD3 antibody and / or anti-CD28 antibody, and at least one homeostatic cytokine under conditions sufficient for the cells to expand; and selecting the cell with a selection reagent; wherein the selection reagent is configured to selectively enrich cells transduced with the nucleic acid or vector.

[00037] In some modalities, the cell is a lymphocyte. In some modalities, the lymphocyte has a CD45RA-, CD45RO+, and CD62L+ phenotype. In some modalities, the lymphocyte is CD8+ or CD4+.

[00038] In some modalities, the selection reagent is methotrexate.

[00039] In some modalities, the cytokine is IL-15, II-7 and / or 11-21.

[00040] Some modality also includes introducing a second nucleic acid into the host cell, where the second nucleic acid encodes a marker protein. In some modality, the marker protein is EGFRt.

[00041] Some modalities of the methods and compositions provided herein include a host cell of any of the modalities provided herein for use in a drug or for use in the treatment or inhibition of a cancer or solid tumor expressing an IL-13a2 receptor. In some modalities, the cancer comprises a brain cancer. In some modalities, the cancer is a glioblastoma or glioma tumor. In some modalities, the cancer is glioblastoma multiforme (GBM). In some modalities, the cancer is a glioma.

[00042] Some modalities of the methods and compositions provided herein include a method for treating, inhibiting, or ameliorating cancer in a subject, comprising: administering the host cell of any of the modalities provided herein to the subject in need thereof. In some modalities, the cancer is an IL13Ra-positive malignancy. In some modalities, the cancer is brain cancer. In some modalities, the cancer is a glioblastoma or glioma tumor. In some modalities, the cancer is a glioma. In some modalities, the cancer is glioblastoma multiforme (GBM). In some modalities, the subject is mammalian. In some modalities, the subject is human.

[00043] Some modalities also include administering selected additional chemotherapy and radiation therapy. In some modalities, the chemotherapeutic drug comprises electrochemotherapy, an alkylating agent, antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (XelodaMR), cladribine, clofarabine, cytarabine (Ara-CMR), floxuridine, fludarabine, gemcitabine (GemzarMR), hydroxyurea, methotrexate, pemetrexed (AlimtaMR), pentostatin, and thioguanine), an antitumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, a DNA intercalation agent, or a checkpoint inhibitor (CHK1, CHK2 checkpoint kinase). Brief Description of the Figures

[00044] Figure 1 represents a schematic of certain CAR modalities that target IL-13Ra2, and components used for the construction of CAR modalities that target IL-13Ra2.The IL-13Ra2-specific CARs included: one of a variable number of single-chain fragments (scFvs) that specifically recognize an extracellular epitope of the IL-13 alpha 2 receptor (IL-13Ra2) derived from one of two antibodies 'hu08' (Ab01) or 'hu07' (Ab02) and included VH and VL domains with a linker between them; one of several spacer domains (labeled “S”, “M”, and “L”); a transmembrane domain derived from a human CD28 (CD28tm); a costimulatory domain derived from human 4-1BB; a CD3-derived signaling domain; a T2A ribosomal jump sequence; and a truncated EGFR transduction marker (EG FRt); and optionally an additional T2A ribosomal jump sequence and a dihydrofolate reductase double mutator transgene (DHFRdm) configured for methotrexate selection.

[00045] Figure 2A represents a flow cytometry analysis of EGFRt expression in CD8+ T cells (top panel) or CD4+ T cells (bottom panel) transduced with various CARs represented in Figure 1. From top row to bottom row of each panel: Simulated T cells; T cell with IL-13Ra2 Ab01 S VL-VH spacer CAR; T cell with IL-13Ra2 Ab01 S VH-VL spacer CAR; T cell with IL-13Ra2 Ab02 S VL-VH spacer CAR; and T cell with IL-13Ra2 Ab02 S VH-VL spacer CAR.

[00046] Figure 2B shows cytokine production graphs (IL-2, 1FN-γ, TNF-α) from CD8+ T cells transduced with various CARs and incubated with the indicated target cells. The key corresponds to T cell simulation; T cell with VL-VH Ab01 S-spacer CAR of IL-13Ra2; T cell with VH-VL Ab01 S-spacer CAR of IL-13Ra2; T cell with VL-VH Ab02 S-spacer CAR of IL-13Ra2; and T cell with VHVL Ab02 S-spacer CAR of IL-13Ra2.

[00047] Figure 2C shows cytokine production graphs (IL-2, 1FN-γ, TNF-α) from CD4+ T cells transduced with various CARs and incubated with the indicated target cells. The key corresponds to T cell simulation; T cell with VL-VH Ab01 S-spacer CAR of IL-13Ra2; T cell with VH-VL Ab01 S-spacer CAR of IL-13Ra2; T cell with VL-VH Ab02 S-spacer CAR of IL-13Ra2; and T cell with VHVL Ab02 S-spacer CAR of IL-13Ra2.

[00048] Figure 3A represents a multiparameter flow cytometry analysis of healthy donor-derived T cells transduced with IL-13Ra2 hu08 CARs, with histogram quadrants extracted based on control staining (top and middle panels); and a flow cytometry analysis of EGFRt expression in CD8+ T cells transduced with a CAR (bottom panel). The indicated CARs included: mock-up; IL-13Ra2 Ab01 VL-VH S-spacer CAR; IL-13Ra2 Ab01 VL-VH M-spacer CAR; IL-13Ra2 Ab01 VL-VH L-spacer CAR; IL-13Ra2 Ab01 VH-VL S-spacer CAR; IL-13Ra2 Ab01 VH-VL M-spacer CAR; and CAR of spacer-L VH-VL of Ab01 of IL-13Ra2.

[00049] Figure 3B represents an analysis for cytokine production and cytolytic activity of CD8+ T cells containing a CAR, after co-culture with certain indicated target cells. The indicated CARs included: sham; Ab01 IL-13Ra2 VL-VH S-spacer CAR; Ab01 IL-13Ra2 VL-VH M-spacer CAR; Ab01 IL-13Ra2 VL-VH L-spacer CAR; Ab01 IL-13Ra2 VH-VL S-spacer CAR; Ab01 IL-13Ra2 VH-VL M-spacer CAR; and Ab01 IL-13Ra2 VH-VL L-spacer CAR.

[00050] Figure 4A represents graphs of total flux (photons / sec) as a measure of tumor burden (y-axis) over time (days after tumor inoculation, x-axis), for mice treated with: vehicle, sham cells, 2 x 10⁶ T cells containing an anti-IL-13Ra2 CAR; 1 x 10⁶ T cells containing an anti-IL-13Ra2 CAR; or 0.5 x 10⁶ T cells containing an anti-IL-13Ra2 CAR. The anti-IL-13Ra2 CAR was an IL13Ra2 AbO1 VL-VH spacer-S CAR.

[00051] Figure 4B represents a Kaplan-Meier survival curve for mice treated with increasing doses of T cells expressing an anti-IL-13Ra2 CAR (IL-13Ra2 Ab01 VL-VH spacer-S CAR).

[00052] Figure 5A represents graphs of total flux (photons / sec) as a measure of tumor burden (y-axis) over time (days after tumor inoculation, x-axis), for mice treated with: sham cells; 2 x 106 T cells containing an anti-IL-13Ra2 CAR containing a short spacer; 2 x 106 T cells containing an anti-IL-13Ra2 CAR containing a medium spacer; 2 x 106 T cells containing an anti-IL-13Ra2 CAR containing a long spacer.

[00053] Figure 5B represents a Kaplan-Meier survival curve for T-cell-treated mice expressing an anti-IL-13Ra2 CAR containing a long spacer, a medium spacer, or a short spacer.

[00054] Figure 5C represents an area under the curve (AUC) analysis of bioluminescent data for mice treated with T cells expressing an anti-IL-13Ra2 CAR containing a long, medium, or short spacer. Mean AUCs were compared between the IL13Ra2 Ab01 spacer variants, CAR T cells, and sham T cells. *, P<0.05. **, P<0.01. ****, P<0.0001. Detailed Description of the Invention

[00055] Some of the methods and compositions provided herein include chimeric antigen receptors (CARs) targeting IL13Ra2, such as second-generation IL-13Ra2-specific CARs, cells containing these CARs, nucleic acids encoding the CARs, and related therapeutic methods and uses thereof. Among the CARs provided are those having particular combinations of components or domains, such as those resulting from CAR function optimization. In some respects, IL13Ra2-targeted T-cell therapeutics may also serve as an alternative or supplement to current IL-13Ra2-specific and non-specific cancer treatments, such as combination therapies.

[00056] IL-13Ra2 was previously found to be abundant in metastatic or late-stage BLBC (Papageorgis et al. Breast Cancer Research, 2015; 17(1); expressly incorporated herein by reference in its entirety). Based on publicly available data, correlations were made between progression-free survival probability and high levels of IL-13Ra2. A subtype of BLBC that tended to spread rapidly to the lungs was observed to have high levels of IL-13Ra2. IL-13Ra2 was also found to stimulate human glioma cell growth and metastasis via the Src / PI3K / Akt / mTOR signaling pathway (Tu et al. Tumour Biol. 2016 Nov; 37(11): 14701-14709; expressly incorporated herein by reference in its entirety). Therapies targeting IL13Ra2 have been described, such as chimeric receptor-based therapies (see, for example, Brown et al. Clin Cancer Res 2015; Brown et al. N Engl J Med 2016; Brown et al.Mol Ther 2017; WO 2014072888-Al, which describe anti-IL-13 alpha 2 receptor antibodies (IL-13-Ra2) and antibody-drug conjugates for the treatment of cancer, each expressly incorporated in the presence as a reference in its entirety). Among these therapies are those based on or including IL13-mutant-based antigen recognition or binding domains (e.g., E13Y), such as zetakines.

[00057] In some embodiments, the provided CARs and associated methods and uses are based in part on observations described herein that show particularly advantageous activity and / or anti-cancer effects in vivo in the context of glioblastoma or a glioblastoma model, of CARs containing specific binding domains in combination with other specific components such as specific curative separator domains. In some respects, the provided compositions, methods, and uses are employed in the treatment, inhibition, or prevention of glioblastoma and / or other IL-13Ra2-positive malignancies. In some respects, the provided CARs described herein differ from other CARs in several aspects, such as in one or more features of their molecular structure, such as the IL-13Ra2 epitope specifically recognized by the binding domain and targeted by the provided CARs herein.

[00058] The provided modalities are based in part on observations herein, as well as those demonstrated in preclinical studies described herein, which show in vitro antitumor activity of the provided CAR T cells against glioblastoma cell lines and the ability to reduce tumor growth in xenograft models, in some aspects to a greater extent compared to different CARs that have different zcRRQn / nznz / a / YiAi molecular structures. In view of these findings, the commercial use of compositions comprising these CARs in immunotherapy for brain tumors and other IL-13Ra2-positive cancers is contemplated. Definitions

[00059] As used herein, “nucleic acid” or “nucleic acid molecule” has its plain and ordinary meaning when read in view of the specification and may include, but is not limited to, for example, polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogues of naturally occurring nucleotides (for example, enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides may have alterations in sugar portions and / or purine or pyrimidine base portions.Sugar modifications include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or sugars can be functionalized as ethers or esters. Furthermore, the entire sugar portion can be replaced with spherically shaped and electronically similar structures, such as aza sugars and carbocyclic sugar analogues. Examples of modifications to a base portion include alkylated purines and pyrimidines, adiated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogues thereof. Phosphodiester bond analogues include phosphorothioate, phosphorodithioate, phosphoroselenote, phosphorodiselelenote, phosphoranilothioate, phosphoranilidate, phosphoramidate, and the like.The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise nucleic acid bases that are modified or occur naturally attached to a polyamide structure. Nucleic acids can be either single-stranded or double-stranded. In some alternatives, a nucleic acid sequence that codes for a protein is provided. In some alternatives, the nucleic acid is either RNA or DNA.

[00060] As used herein, “chimeric antigen receptor” has its plain and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, a synthetically designed receptor comprising a ligand-binding domain of an antibody or other protein sequence that binds to a disease- or disorder-associated molecule and links via a spacer domain to one or more intracellular signaling domains of a cell, such as a T cell, or other receptors, such as a costimulatory domain. The chimeric receptor may also be referred to as artificial cell receptors or T cell receptors, chimeric cell receptors or T cell receptors, chimeric immunoreceptors, and / or chimeric antigen receptors (CARs).These receptors can be used to graft the specificity of a monoclonal antibody or its binding fragment into a cell, preferably a T cell, with the transfer of its coding sequence facilitated by viral vectors, such as a retroviral or lentiviral vector. CARs can, in some cases, be genetically modified T cell receptors designed to redirect T cells to target cells that express specific cell surface antigens. T cells can be removed from a subject and modified so that they can express receptors specific to an antigen through a process called adoptive cell transfer. The T cells are then reintroduced into the patient, where they can recognize and target an antigen. CARs are also modified receptors that can graft arbitrary specificity onto an immune receptor cell.The term chimeric antigen receptor (CAR) is considered by some researchers to include the antibody or antibody fragment, preferably an antigen-binding fragment of an antibody, the spacer, signaling domain, and transmembrane region. Due to the surprising effects of modifying the different CAR components or domains described herein, such as the epitope-binding region (e.g., antibody fragment, scFv, or portion thereof), spacer, transmembrane domain, and / or signaling domain, the CAR components are frequently distinguished from the beginning of this description in terms of independent elements. Variation of the different CAR elements can, for example, lead to a stronger binding affinity for a specific epitope or antigen.

[00061] CARs graft the specificity of a monoclonal antibody or antibody-binding fragment or scFv into a T cell, with the transfer of its coding sequence facilitated by vectors. In order to use CARs as therapy for a subject in need, a technique called adoptive cell transfer is used, in which T cells are removed from a subject and modified so that they can express CARs that are specific for an antigen. The T cells, which can then recognize and target an antigen, are reintroduced into the patient.

[00062] In some embodiments, the transmembrane domain is a hydrophobic, membrane-spanning region of a protein that can reside in a cell's bilayer to anchor a protein embedded in the biological membrane. Without limitation, the topology of the transmembrane domain can be a transmembrane alpha helix. In some chimeric antigen receptor (CAR) alternatives, the CAR comprises a sequence encoding a transmembrane domain. In some alternatives, the transmembrane domain comprises a transmembrane sequence of CD28 or a fragment thereof that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids in length, or a length within a range defined by any two of the above lengths. In some alternatives, the transmembrane sequence of CD28 or a fragment thereof comprises 28 amino acids in length.

[00063] In some modalities, signaling domains, such as primary signaling domains or costimulatory domains, include an intracellular or cytoplasmic domain of a protein or receptor protein that interacts with components within cells and is capable of relaying or participating in the relay of a signal. These interactions can, in some respects, occur through the intracellular domain, which communicates via specific protein-protein or protein-ligand interactions with an effector molecule or effector protein, which in turn can transmit the signal along a signaling pathway to its destination. In some modalities, the signaling domain includes a costimulatory domain.In some aspects, the costimulatory domain includes a signaling portion that provides T cells with a signal that, in addition to the primary signal provided by, for example, the CD3 zeta chain of the TCR / CD3 complex, potentiates the response, such as a T cell effector response, including, for example, an immune response, activation, proliferation, differentiation, cytokine secretion, cytolytic activity, perforin and / or granzyme activity, and the like. In some modalities, the intracellular signaling domain and / or the costimulatory domain may include all or a portion of CD27, CD28, 4-1BB, 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-I), CD2, CD7, LIGHT, NKG2C, and / or B7-H3, and / or a ligand that binds specifically to CD83.

[00064] As used herein, an “antibody” has its simple and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, a large Y-shaped protein produced by plasma cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. In some contexts, the term antibody refers to the antigen-binding domains of an antibody. The antibody protein may comprise four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains may contain 70–110 amino acids and are classified into different categories according to their size and function.A chimeric antigen receptor may comprise a ligand-binding domain, which includes an antibody fragment, preferably an antigen-binding fragment. In some alternatives, a nucleic acid encoding a chimeric antigen receptor (CAR) is provided, the nucleic acid comprising: a) a first polynucleotide encoding a ligand-binding domain, wherein the ligand-binding domain binds to or interacts with an IL-13 alpha 2 receptor (IL13Ra2), b) a second polynucleotide encoding a polypeptide spacer of sufficient length to allow the ligand-binding domain to interact with the IL-13 alpha 2 receptor (IL13Ra2), c) a third polynucleotide encoding a transmembrane domain, and d) a fourth polynucleotide encoding an intracellular signaling domain. In some alternatives, the ligand-binding domain is an antibody fragment.

[00065] As used herein, a single-chain variable fragment, or scFv, has its plain and ordinary meaning when read in view of the specification and may include, but is not limited to, for example, a fusion protein comprising the variable regions of the heavy chain (HV) and light chains (LV) of an immunoglobulin, which are linked together by a short-linker peptide. Without limitation, the linker may comprise glycine for flexibility and hydrophilic amino acids, for example, serine or threonine, for solubility. The linker may connect the N-terminus of the HV to the C-terminus of the LV or it may connect the C-terminus of the HV to the N-terminus of the LV. In some alternatives, the ligand-binding domain present in a CAR is a single-chain variable fragment (scFv). In some alternatives, the scFv domain present in a CAR is specific for an IL-13 alpha 2 receptor (IL13Ra2) present on a tumor cell.

[00066] In some embodiments, the compositions, cells, and vectors include marker sequences or nucleic acids encoding them, which may include, for example, a protein that serves as a tag for a cell. In some cell alternatives described herein, the cells co-express a marker protein for a specific chimeric antigen protein that is expressed. In some cell alternatives provided herein, the chimeric antigen receptor is co-expressed with a specific marker protein. In some cell alternatives provided herein, the cells comprise a nucleic acid encoding a chimeric antigen receptor. The markers may include a selectable marker sequence, such as a gene introduced into a vector or cell that confers a trait for artificial selection.A marker sequence or selectable marker sequence can be a screenable marker to allow a researcher to distinguish between desired and undesired cells, or to be enriched for a specific cell type. In some alternatives, a vector is provided where the vector encodes a chimeric antigen receptor comprising a marker sequence, and the marker sequence encodes a selectable cell surface marker. In the alternatives described herein, the provided fusion protein may comprise a marker sequence that can be selected in experiments, such as flow cytometry. In some alternatives, the marker is the Her2tG or EGFRt protein.

[00067] Methotrexate (MTX) may include, but is not limited to, for example, an antimetabolite and antifolate drug. In some respects, it acts by inhibiting folic acid metabolism. In some alternatives, a method is provided for generating multiplexed modified T cells for adoptive T cell immunotherapy. In the broadest sense, the method may comprise providing the gene-delivery polynucleotide of any of the alternatives described herein, selecting cells comprising the gene-delivery polynucleotide, wherein the selection comprises adding a selection reagent. In some alternatives described herein, the selection reagent comprises a selection agent. In some alternatives, the selection reagent is MTX.

[00068] As used herein, “dihydrofolate reductase, or DHFR, as described herein, has its plain and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as an electron donor, which can be converted into the types of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. In some alternatives described herein, a gene-delivery polynucleotide is provided. In some alternatives, the gene-delivery polynucleotide comprises at least one selectable marker cassette encoding a dihydrofolate reductase double mutan (DHFRdm).”

[00069] In some modalities, the provided constructs and sequences are modified or optimized, such as by codon optimization, which may include, but is not limited to, for example, the process of designing codons to those known to increase the efficiency of maximizing protein expression in a desired cell, preferably a human cell. In some alternatives, codon optimization is described, where codon optimization can be performed using algorithms known to those skilled in the art of creating synthetic genetic transcripts optimized for high protein yield. Programs containing algorithms for codon optimization are known to those skilled in the art. The programs may include, for example, algorithms from OptimumGeneMR and GeneGPSMR.Additionally, synthetic codon-optimized sequences can be obtained commercially, for example, from Integrated DNA Technologies and other commercially available DNA sequencing services. In some alternatives, nucleic acids are described where nucleic acid genes for complete gene transcription are codon-optimized for human expression. In some alternatives, genes are optimized to have codons specifically selected for maximum protein expression in human cells, which can increase the concentration of proteins or CARs in a T cell.

[00070] Codon optimization can also be performed to reduce the presentation of secondary structure in a polynucleotide. In some alternatives, codon optimization can also be performed to reduce the total GC / AT ratio. Strict codon optimization can also lead to unwanted secondary structure or undesirable GC content. As such, secondary structures affect transcriptional efficiency. Programs such as GeneOptimizer can be used after codon usage optimization for secondary structure avoidance and GC content optimization. These additional programs can be used for further optimization and troubleshooting after initial codon optimization to limit the secondary structures that may be present after the first round of optimization.Alternative optimization programs are known to those skilled in the technique. In some alternatives, the nucleic acid comprises sequences that are optimized by codon for human expression and / or to remove secondary structure and / or to reduce the total GC / AT ratio. In some alternatives, the sequences are optimized for secondary structure avoidance. In some alternatives, the sequences are optimized to reduce the total GC / AT ratio.

[00071] As used herein, “vector,” “expression vector,” or “construct” has its plain and ordinary meaning when read in view of the specification and may include, but is not limited to, for example, a nucleic acid used to introduce heterologous nucleic acids into a cell that has regulatory elements to provide expression of the heterologous nucleic acids in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some alternatives, the vectors are plasmids, minicircles, or viral genomes. In some alternatives, the vector is a viral vector. In some alternatives, the viral vector is a lentivirus. In some alternatives, the vector is a lentiviral vector. In some alternatives, the vector is a foamy viral vector, adenoviral vector, retroviral vector, or lentiviral vector.

[00072] T cells or T lymphocytes in some modalities may include T cells from any mammalian species, preferably primates, including monkeys, dogs, and humans. In some alternatives, the T cells are allogeneic (from the same species but a different donor) as the recipient subject who receives or is to receive the cells, such as in the form of a therapeutic composition; in some alternatives, the T cells are autologous (the donor and recipient are the same); in some alternatives, the T cells are syngeneic (the donor and recipients are different but are identical twins).

[00073] As used herein, a “ribosome jump sequence” as described herein refers to a sequence that, during translation, forces the ribosome to “jump” the ribosome jump sequence and translate the region after the ribosome jump sequence without the formation of a peptide bond. Several viruses, for example, zcRRQn / nznz / a / YiAi, have ribosome jump sequences that allow the sequential translation of several proteins into a single nucleic acid without the proteins being linked by a peptide bond. As described herein, this is the “linker” sequence. In some alternative nucleic acids provided herein, the nucleic acids comprise a ribosome jump sequence between the sequence for the chimeric antigen receptor and the sequence for the marker protein, such that the proteins are co-expressed and not linked by a peptide bond.In some alternatives, the ribosome jump sequence is a P2A, T2A, E2A, or F2A sequence. In some alternatives, the ribosome jump sequence is a T2A sequence.

[00074] Mature T cells express the surface protein CD4 and are known as CD4+ T cells. CD4+ T cells are generally treated as having a predefined function as helper T cells within the immune system. For example, when an antigen-presenting cell expresses an antigen on MHC class II, a CD4+ cell will assist those cells through a combination of cell-to-cell interactions (e.g., CD40 and CD40L) and through cytokines. However, there are rare exceptions; for example, subsets of regulatory T cells, natural killer cells, and cytotoxic T cells express CD4. All of these latter groups of CD4+-expressing T cells are not considered helper T cells.

[00075] As used herein, “central memory” T cells (or “Tcm”) have their plain and ordinary meaning when read in view of the specification, and may include, but are not limited to, for example, an antigen-experienced CTL that expresses CD62L or CCR7 and CD45RO on its surface, and does not express or has decreased expression of CD45RA compared to untreated cells. In some alternatives, central memory cells are positive for the expression of CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95, and have decreased expression of CD54RA compared to untreated cells.

[00076] As used herein, “effector” T cells “Te” has its plain and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, antigen-experienced cytotoxic T lymphocyte cells that do not express or have decreased expression of CD62L, CCR7, or CD28, or are positive for granzyme B and / or perforin, compared to untreated or central memory T cells.

[00077] As used herein, “pharmaceutical excipient, or pharmaceutical vehicle” has its plain and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, an inert carrier or medium used as a solvent in which the medically active agent or T cells are formulated and / or administered for treatment or therapy. Vehicles may include polymeric micelles, liposomes, lipoprotein-based carriers, nanoparticle carriers, dendrimers, and / or other T-cell vehicles known to a person skilled in the art. An ideal vehicle or excipient may be nontoxic, biocompatible, nonimmunogenic, biodegradable, and may avoid recognition by host defense mechanisms.

[00078] As used herein, “T cell precursors” has its plain and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, lymphoid precursor cells that can migrate to the thymus and become T cell precursors, which do not express a T cell receptor. All cells T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) of hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes do not express CD4 or CD8 and are therefore classified as double-negative cells (CD4_CD8). As they progress through development, they become double-positive thymocytes (CD4+CD8+), and finally mature into single-positive thymocytes (CD4+CD8_ or CD4_CD8+) that are then released from the thymus into peripheral tissues.

[00079] Glioblastoma multiforme (GBM) is generally considered the most aggressive cancer that begins within the brain. The initial signs and symptoms of glioblastoma are generally nonspecific. They may include headaches, personality changes, nausea, and stroke-like symptoms.

[00080] As used herein, a "leader sequence," also known as the 5' untranslated region (5j UTR), is the mRNA region located 5' upstream of the start codon and is important for regulating the translation of an mRNA transcript. In some alternative methods for producing genetically modified T cells that have a chimeric antigen receptor (CAR), the vector encoding the chimeric antigen receptor comprises a sequence encoding a leader sequence.

[00081] As described in the alternatives herein, these are ligands. “Ligand” has its simple and ordinary meaning when read in light of the specification, and may include, but is not limited to, for example, a substance that can form a complex with a biomolecule. By way of example, and not as a limitation, ligands may include substrates, proteins, small molecules, inhibitors, activators, nucleic acids, or neurotransmitters. Binding may occur through intermolecular forces, for example, ionic bonds, hydrogen bonds, or van der Waals interactions. A ligand that binds to a receptor protein may alter its three-dimensional structure and determine its functional state. The binding strength of a ligand is known as its binding affinity and can be determined by direct interactions and solvent effects. A ligand may bind via a ligand-binding domain.A ligand-binding domain, for example, can refer to a conserved sequence in a protein structure that can bind to a specific ligand or epitope. The ligand-binding domain or ligand-binding portion may comprise an antibody or antibody-binding fragment, a receptor ligand or receptor-binding molecules, a peptide, and / or a polypeptide affinity molecule or binding partner. Without limitation, a ligand-binding domain can also be a protein-specific domain or epitope in a protein that is specific for a ligand or ligands.

[00082] As described in the alternatives herein, it is the interleukin-13 receptor alpha-2 subunit (IL-13Ra2). “Interleukin-13 receptor alpha-2 subunit (IL-13Ra2),” also known as CD213A2 (differentiation cluster 213A2), has its simple and ordinary meaning when read in view of the specification, and may include, but is not limited to, for example, a membrane-bound protein that in humans is encoded by the IL-13RA2 gene. IL13Ra2 is closely related to IL-13Ra1, a subunit of the interleukin-13 receptor complex. IL-13Ra2 generally binds to IL-13 with high affinity, but lacks any significant cytoplasmic domain, and does not appear to function as a signal mediator. However, it is able to regulate the effects of both IL-13 and IL-4, despite the fact that it cannot bind directly to the latter. It is also reported to play a role in the internalization of IL-13.

[00083] In some alternatives, the peptide spacer is 15 amino acids or less but not less than 1 or 2 amino acids. In some embodiments, the spacer is a polypeptide chain. In some embodiments, the spacer is a polypeptide chain. In some aspects, the polypeptide chain may vary in length, such as from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, or 240 amino acids, or a length within a range defined by any two of the lengths mentioned above. A spacer may comprise any 20 amino acids, for example, in any order to create a desirable polypeptide chain length in a chimeric antigen receptor, including the amino acids arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and / or tryptophan. A spacer sequence can be a linker between the scFv (or ligand-binding domain) and the transmembrane domain of the chimeric antigen receptor. In some chimeric antigen receptor alternatives, the chimeric antigen receptor also includes a sequence that encodes a spacer.In some alternatives, the separator comprises a sequence with a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, or 240 amino acids, or a length within a range defined by any two of the lengths mentioned above. In some alternatives, the spacer resides between the scFv and the transmembrane region of the chimeric antigen receptor. In some alternatives, the spacer resides between the ligand-binding domain of the chimeric antigen receptor and the transmembrane region of the chimeric antigen receptor.

[00084] A spacer can also be customized, selected, or optimized for a desired length in order to improve scFv domain binding to the target cell, which may increase cytotoxic efficacy. In some alternatives, the ligand or separator between the scFv domain or ligand-binding domain and the transmembrane can be 25 to 55 amino acids in length (e.g., at least equal to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids or a length within a range defined by any two of the lengths mentioned above).

[00085] Some modalities include polypeptide sequences or conservative variations thereof, such as conservative substitutions within a polypeptide sequence. In some modalities, “conservative amino acid substitution” refers to amino acid substitutions that replace functionally equivalent amino acids. Conservative amino acid changes result in imperceptible changes to the amino acid sequence of the resulting peptide. For example, one or more amino acids of similar polarity act as functional equivalents and result in an imperceptible alteration within the amino acid sequence of the peptide. Charge-neutral substitutions that replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacing phenylalanine with the smaller isoleucine).Families of amino acid residues with similar side chains have been defined in the technique. Several families of conservative amino acid substitutions are shown in Table 1. zcRRQn / nznz / a / YiAi Table 1 Nonpolar Amino Acid Family: Trp, Phe, Met, Leu, Ie, Val, Ala, Pro; Polar uncharged: Gly, Ser, Thr, Asn, Gln, Tyr, Cys; Acidic / negatively charged: Asp, Glu; Basic / positively charged: Arg, Lys, His; Branched beta: Thr, Val, Ie; Residues that influence chain orientation: Gly, Pro; Aromatic: Trp, Tyr, Phe, His

[00086] High levels of interleukin-13 alpha 2 receptor (IL-13RA2) are found in a number of cancer cells, including pancreatic, breast, and ovarian cancers, or malignant gliomas such as glioblastoma. IL-13RA2 has also been shown to be overexpressed in a large majority of human patients with high-grade astrocytomas (see PLoS One. 2013 Oct 16; 8(10):e77719; incorporated herein by reference in its entirety). In addition, previous research has shown that reducing the amount of IL-13RA2 expression in cancer cells significantly slowed tumor growth in models (Breast Cancer Research, 2015; 17(1); incorporated herein by reference in its entirety). It is thought that few normal tissue types express IL-13-RA2 and only at low levels.

[00087] In glioblastoma multiforme (GBM), high IL13Ra2 expression may be a prognostic marker of tumor progression and poor patient survival. Chimeric antigen receptors (CARs), and cells and therapies containing or utilizing them, are provided that selectively target IL13Ra2. The CARs provided generally comprise an extracellular domain linked to a transmembrane region and an intracellular signaling region that includes one or more signaling domains, generally including primary and costimulatory signaling domains.

[00088] The extracellular domain includes an antigen-binding motif, which in some respects is or includes an antigen-binding antibody fragment such as scFv. In some respects, the binding domain, such as the scFv, is derived from an antibody specific for human interleukin-13Ra2. In some embodiments, the CAR includes a spacer region that connects the extracellular binding domain to the transmembrane domain. In some respects, one or more properties of the spacer are designed to optimize the CAR's characteristics, such as its function, for example, by providing an appropriate distance between the epitope contact and the binding region and the cell membranes, and / or by providing flexibility.In some aspects, the spacer is less than or no more than 20 amino acids in length, such as less than or no more than 15 amino acids in length, less than or no more than 14 amino acids in length, less than or no more than 13 amino acids in length, or no more than 12 amino acids in length. In some aspects, it is approximately 12 or 15 amino acids in length. In some aspects, the spacer comprises all or a portion of an immunoglobulin hinge region (such as a human IgG hinge region, such as a human IgG4 hinge region) or a modified version thereof.

[00089] In some alternatives, the spacer comprises a hinge region of a human antibody. In some alternatives of the method, the spacer comprises an IgG4 hinge. In some alternatives, the IgG4 hinge region is a modified IgG4 hinge. A modified lgG4 hinge, as described herein, may refer to a hinge region that may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or a sequence identity within a range defined by any two of the above-mentioned percentages, with a hinge region amino acid sequence as set out in SEQ ID NO:01, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, or SEQ ID NO8.

[00090] In some alternatives, the CAR comprises an S spacer, an M spacer, or an L spacer. The S spacer comprises a sequence exposed in SEQ ID NO:09. The M spacer comprises a sequence exposed in SEQ ID NO:10. The L spacer comprises a sequence exposed in SEQ ID NO:11.

[00091] In some embodiments, the transmembrane portion includes or is a CD28 transmembrane domain (CD28tm) such as a human CD28 transmembrane domain. CD28tm is encoded by a sequence listed in SEQ ID NO: 12. CD28tm comprises the amino acids listed in SEQ ID NO: 13. In some embodiments, the transmembrane domain links to an intracellular signaling region containing one or more costimulatory domains, such as a costimulatory domain derived from the intracellular segment of human 4-1BB (CD137) and a CD3-zeta (CD3ζ) signaling domain such as that of human Oϋ3ζ. In some alternatives, the CAR design is based on different combinations of two scFv molecules, targeting different extracellular epitopes of IL13Ra2, with different VH-VL orientations and three possible CAR spacer lengths. The rest of the CAR structure, the transmembrane and costimulatory domains are shared among CARs.Other alternatives include a truncated version of the epidermal growth factor receptor (EGFRt), which is co-expressed with CARs on the surface of T cells. Additional alternatives may include a dihydrofolate reductase double mutant (DHFRdm) transgene, which allows for methotrexate selection from T cell products. As described herein, CD8 and CD4 IL13Ra2 CAR T cells exhibit potent in vitro tumor cytotoxicity and specific efficacy in vivo against glioblastoma tumor models.

[00092] Some alternatives include a CAR containing a scFv that includes VL and VH domains of an antibody known as IL13Ra2 Ab01, in the VL-linker-VH orientation. In some respects, these CARs further include particular combinations of spacer, transmembrane, and / or signaling domains. For example, in some respects, these CARs include a spacer consisting of no more than 15 or no more than 12 amino acids and / or containing or consisting of an immunoglobulin hinge region or a modified variant thereof, such as an IgG4 hinge region or a modified variant thereof. In some respects, the CAR domains further include a costimulatory signaling domain and a primary signaling domain, such as 41BB and CD3zeta signaling domains.In some respects, preferred alternatives may be advantageous in their ability to enhance T cell effector functions, such as by driving increased cytotoxicity and cytokine secretion in vitro and / or improving function in vivo, such as in the context of human glioblastoma, compared to alternative CARs, such as those containing different binding domains (e.g., with different VH and / or VL regions and / or in which the VH and VL regions are present in a different orientation) and / or those containing the same binding domain but with one or more differences in other CAR regions, such as with an alternative spacer, such as a spacer with an increased length.The modalities provided herein are, in some respects, based on observations herein that demonstrate the ability to control tumor growth and / or increase median survival in subjects bearing tumors that express IL-13Ra2, such as in a mouse model involving mice bearing intracranially grafted human GBM, treated with a single injection of IL13Ra2-targeted CAR T cells. Certain nucleic acids

[00093] Some embodiments of the methods and compositions provided herein include nucleic acids encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: a) a ligand-binding domain that binds to or interacts with an IL-13 alpha 2 receptor (IL13Ra2) and / or comprising CDR3, and optionally CDR1 and CDR2, from a VH region of SEQ ID NO:18 and CDR3, and optionally CDR1 and CDR2, from a VL region of SEQ ID NO:19; b) a polypeptide spacer between the ligand-binding domain and a transmembrane domain; c) the transmembrane domain; and d) an intracellular signaling region. In some embodiments, the ligand-binding domain is an antibody fragment, such as an antigen-binding fragment. In some embodiments, the ligand-binding domain is a single-strand variable fragment (scFv).In some alternatives, the spacer is 15 amino acids or less, but not less than 1 or 2 amino acids. In some alternatives, the spacer comprises an amino acid sequence of X1PPX2P. In some alternatives, the spacer region comprises a portion of a hinge region of a human antibody or a modified variant thereof. In some alternatives, the intracellular signaling region comprises primary and costimulatory signaling domains, optionally comprising all or a portion of a CD3-zeta in combination with a costimulatory domain selected from the group consisting of signaling domains of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, and B7-H3, and combinations thereof. In some alternatives, the intracellular signaling region comprises a signaling portion of a CD3-zeta and a signaling portion of a 41BB.In some alternatives, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm). In some alternatives, the spacer comprises, consists of, or essentially consists of, an lgG4 hinge spacer (S). In some alternatives, the spacer comprises, consists of, or essentially consists of, an lgG4-CH3 hinge spacer (M). In some alternatives, the spacer comprises, consists of, or essentially consists of, an lgG4-CH2 (L234D, N297A)-CHE hinge spacer (L). In some alternatives, the scFv comprises a VL-VH orientation and spacer S. In some alternatives, the scFv comprises a VH-VL orientation and a spacer, wherein the spacer is either spacer M or spacer L. In some alternatives, the single-strand variable fragment (scFv) comprises a sequence exposed in SEQ ID NO:61 or SEQ ID NO:62.In some alternatives, the single-strand variable fragment (scFv) comprises a sequence exposed in SEQ ID NO:63 or SEQ ID NO:64. In some alternatives, the nucleic acid further comprises a nucleic acid encoding a marker sequence. In some alternatives, the marker sequence is a truncated receptor and is optionally an EGFRt, a HER2t, or a CD19t. In some alternatives, the nucleic acid further comprises a dihydrofolate reductase transgene configured for methotrexate selection. In some alternatives, the dihydrofolate reductase transgene is a dihydrofolate reductase double mutator (DHFRdm). In some alternatives, the first, second, third, and / or fourth polynucleotides are codon-optimized to reduce the total GC / AT ratio of the nucleic acid. In some alternatives, the first, second, third, and / or fourth polynucleotides are codon-optimized for human expression.In some alternatives, the dihydrofolate reductase double mutant comprises amino acid mutations of L22F and F31S.

[00094] Some embodiments of the methods and compositions provided herein include expression vectors comprising nucleic acid from any of the alternatives herein. In some alternatives, the vector is a viral vector. In some alternatives, the vector is a lentiviral or adenoviral vector. Certain chimeric receptors

[00095] Some modalities of the methods and compositions provided herein include chimeric receptor polypeptides, encoded by the nucleic acid of any of the alternatives herein or by the vector of any of the alternatives herein. Certain host cells

[00096] Some embodiments of the methods and compositions provided herein include host cells comprising the nucleic acid of any of the alternatives herein or the expression vector of any of the alternatives herein or expressing the chimeric receptor of any of the alternatives herein. In some embodiments, a host cell comprises a genetically modified cell. In some embodiments, the chimeric antigen receptor is encoded by the nucleic acid of any of the alternatives herein or by the vector of any of the alternatives herein. In some embodiments, the expression vector comprising the nucleic acid of any of the alternatives herein is provided.In some alternatives, the host cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of previously untreated CD8+ T cells, CD8+ central memory T cells, CD8+ memory effector T cells, and CD8+ bulk T cells. In some alternatives, the CD8+ cytotoxic T lymphocyte is a central memory T cell, wherein the central memory T cell is positive for CD45RO+, CD62L+, and CD8+. In some alternatives, the host cell is a CD4+ helper T lymphocyte selected from the group consisting of previously untreated CD4+ T cells, CD4+ central memory T cells, CD4+ memory effector T cells, and CD4+ bulk T cells. In some alternatives, the CD4+ helper lymphocyte cell is a previously untreated CD4+ T cell, where the previously untreated CD4+ T cell is positive for CD45RA+, CD62L+ and CD4+ and negative for CD45RO.In some alternatives, the host cell is a precursor T cell. In some alternatives, the host cell is a hematopoietic stem cell.

[00097] Some embodiments of the methods and compositions provided herein include a host cell of any of the alternatives herein, or the composition of any of the alternatives herein is provided for use in the treatment or inhibition of a cancer or solid tumor expressing an IL-13α2 receptor. The composition comprises the host cell of any of the alternatives herein and a pharmaceutical excipient. In some embodiments, the cancer is a glioblastoma tumor. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13α-positive malignancy. In some embodiments, the cancer is brain cancer or brain tumors. Certain compositions

[00098] Some embodiments of the methods and compositions provided herein include compositions comprising a host cell of any of the alternatives herein, and a pharmaceutically acceptable excipient. Certain methods for preparing host cells

[00099] Some embodiments of the methods and compositions provided herein include methods for preparing a host cell of any of the alternatives herein comprising: (a) introducing a nucleic acid of any of the alternatives herein or an expression vector of any of the alternatives herein into lymphocytes; (b) culturing the lymphocytes in the presence of anti-CD3 and / or anti-CD28 antibodies, and at least one homeostatic cytokine until the cells expand; and (c) selecting the lymphocytes with a selection reagent; wherein the selection reagent is configured to selectively enrich cells transduced with the nucleic acid or vector. In some embodiments, the selection reagent is methotrexate. In some embodiments, the lymphocytes have a CD45RA-, CD45RO+, and CD62L+ phenotype. In some embodiments, the lymphocytes are CD8+ or CD4+. In some alternatives, the cytokine is IL-15, II-7 and / or 11-21.In some alternatives, the method also involves introducing a second nucleic acid into the host cell, where the second nucleic acid encodes a marker protein. In some alternatives, the marker protein is EGFRt. Certain therapy methods [000100] Some embodiments of the methods and compositions provided herein include methods for performing cellular immunotherapy in a subject having a cancer or tumor comprising: administering the host cell of any of the alternatives herein or the composition of the alternatives herein is provided to the subject. The composition comprising a host cell of any of the alternatives herein is provided, and a pharmaceutically acceptable excipient is provided. In some embodiments, the cancer is a glioblastoma tumor. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13Ra-positive malignancy. In some embodiments, the cancer is brain cancer. In some embodiments, the subject is selected to receive combination therapy. In some embodiments, the combination therapy comprises administering a chemotherapeutic drug.In some alternatives, combination therapy includes administering radiation therapy. In some alternatives, chemotherapy includes electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, and thioguanine), antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalation agents, or checkpoint inhibitors (CHK1, CHK2 checkpoint kinases). In some alternatives, the cancer is a glioma. Development of certain IL13Ra2 CARs [000101] Figure 1 shows schematic representations of the nucleic acids encoding the structure of several IL-13Ra2 CARs that were created, each having one of several binding domains, one of several spacer regions, along with a transmembrane domain and intracellular signaling domains. The nucleic acid sequence of each of these example IL-13Ra2 CARs includes a leader sequence, which can be used, for example, to enable translation of the mRNA transcript encoding the CAR. Each of the example CARs also comprised an IL-13Ra2 scFv, specifically an anti-IL-13Ra2 scFv having VH and VL domains exposed in SEQ ID NO: 14 and 16, respectively, or having VH and VL domains exposed in SEQ ID NO: 15 and 17, respectively. Each of the generated and analyzed CARs was generated with four different scFv binding domains.The four binding domains included each of the VH / VL anti-IL13Ra2 pairs, in each of the VL-VH and VH-VL orientations, individually. [000102] The three different spacers, individually present in the example CARs, are shown in Figure 1, located between the scFv and the transmembrane domain. The spacers tested varied in length and were derived from immunoglobulin constant regions. The spacer with the shortest amino acid sequence length was a modified human lgG4 hinge region; a spacer of intermediate (medium) length included the modified human hinge region and also included a CH3 domain (lgG4-CH3); the longest spacer tested included the modified lgG4 hinge region, a modified CH2 region, and the CH3 region (lgG4-CH2 (L235D, N297Q)-CH3 (L)). Each of the generated example CARs also included a human CD28-derived transmembrane domain (CD28tm domain) and human 4-1BB and CD3zeta signaling domains (4-1BB domain, CD3 zeta domain). [000103] Each of the nucleic acids that encode for, and are used to express, CARs further included a sequence encoding a T2A skip sequence and a nucleic acid encoding a truncated CAR expression surrogate marker. [000104] In some alternatives, the ligand-binding domain of provided CAR modalities comprises a heavy-chain variable region having or consisting of the amino acid sequence of SEQ ID NO: 18, or having or consisting of a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 18, or comprising a CDR1, CDR2, and / or CDR3 of, usually at least a CDR3 of, a VH region having the sequence of SEQ ID NO: 18. [000105] In some alternatives, the ligand-binding domain of the provided CAR modalities comprises a light-chain variable region having or consisting of the amino acid sequence of SEQ ID NO: 19, or having or consisting of a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 19, or comprising CDR1, CDR2, and CDR3 of a VL region having the sequence of SEQ ID NO: 19. Sequences have been described in WO2014072888, included herein by reference in full. [000106] In some alternatives, the ligand-binding region that specifically binds to human IL-13-Ra2 comprises: (a) a heavy chain CDR1 comprising SEQ ID NO:20; (b) a heavy chain CDR2 comprising SEQ ID NO:21; (c) a heavy chain CDR3 comprising SEQ ID NO:22; and / or comprises (d) a light chain CDR1 comprising SEQ ID NO:23; (e) a light chain CDR2 comprising SEQ ID NO:24; and / or (f) a light chain CDR3 comprising SEQ ID NO:25. [000107] In some alternatives, the ligand-binding region comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 18 and the light chain variable region amino acid sequence of SEQ ID NO: 19. [000108] In some alternatives, the ligand-binding domain comprises a heavy chain CDR2 (CDRH2) comprising SEQ ID NO:28; (c) a heavy chain CDR3 (CDRH3) comprising SEQ ID NO:29; (d) a light chain CDR1 comprising SEQ ID NO:30; (e) a light chain CDR2 comprising SEQ ID NO:31; and / or (f) a light chain CDR3 comprising SEQ ID NO:32. [000109] In some alternatives, the ligand-binding domain comprises a heavy-chain CDR1 (CDR-H1) comprising a sequence exposed in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36. In some alternatives, CDR-H2 comprises a sequence exposed in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40. In some alternatives, CDRH3 comprises a sequence exposed in SEQ ID NO:41. [000110] In some embodiments, the CAR comprises a spacer interposed between the binding domain and the transmembrane domain. In some alternatives, the spacer comprises or consists of, or essentially consists of, at least a portion of an immunoglobulin constant region, such as all or a portion of an immunoglobulin hinge region, such as an IgG4 hinge region, or a functional variant thereof. In some embodiments, the immunoglobulin hinge and / or constant region is a human IgG region, such as IgG4 or IgG1, or a variant thereof. The spacer may be of a length that provides increased cellular responsiveness after antigen binding, compared to the absence of the spacer. Example spacers include those described in International Patent Application Publication No. WO2014031687, expressly incorporated herein by reference in its entirety.In some examples, the spacer is either approximately 12 amino acids in length, or no more than 12 amino acids in length, or is approximately 15 amino acids in length, or is approximately no more than 15 amino acids in length. [000111] Example spacers include those that are at least 10 to 229 amino acids, 10 to 200 amino acids, 10 to 175 amino acids, 10 to 150 amino acids, 10 to 125 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 20 amino acids, or 10 to 15 amino acids, and including any whole number between the endpoints of any of the listed ranges. In some forms, a spacer region has 12 amino acids or less but not zero, 119 amino acids or less but not zero, or 229 amino acids or less but not zero.In some forms, the spacer is less than 250 amino acids in length but not zero, less than 200 amino acids in length but not zero, less than 150 amino acids in length but not zero, less than 100 amino acids in length but not zero, less than 75 amino acids in length but not zero, less than 50 amino acids in length but not zero, less than 25 amino acids in length but not zero, less than 20 amino acids in length but not zero, less than 15 amino acids in length but not zero, less than 12 amino acids in length but not zero, or less than 10 amino acids in length but not zero.In some forms, the spacer is either 10 to 250 amino acids in length, 10 to 150 amino acids in length, 10 to 100 amino acids in length, 10 to 50 amino acids in length, 10 to 25 amino acids in length, 10 to 15 amino acids in length, 15 to 250 amino acids in length, 15 to 150 amino acids in length, 15 to 100 amino acids in length, 15 to 50 amino acids in length, 15 to 25 amino acids in length, 25 to 250 amino acids in length, 25 to 100 amino acids in length, 25 to 50 amino acids in length, 50 to 250 amino acids in length, 50 to 150 amino acids in length, 50 to 100 amino acids in length, 100 to 250 amino acids in length, or 150 to 250 amino acids in length. Example spacers include the lgG4 hinge alone, the lgG4 hinge linked to CH2 and CH3 domains, or the lgG4 hinge linked to the CH3 domain. Example spacers include, but are not limited to, those described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013), publication of international patent application No. WO2014031687, United States Patent No. 8,822,647 or published application No. US2014 / 0271635 expressly incorporated herein by reference in their entirety. [000112] In some respects, the CAR spacer consists of or comprises an lgG4 hinge, an lgG4-CH3 hinge, or an lgG4-CH2(L235D, N297Q)-CH3 hinge. [000113] In some alternatives, the ligand-binding domain comprises (a) a heavy-chain variable region comprising a CDR1, a CDR2, and a CDR3 of the VH sequence of SEQ ID NO:18; and, (b) a light-chain variable region comprising a CDR1, a CDR2, and a CDR3 of the VL sequence of SEQ ID NO: 19. Sequences can be found in WO2014 / 072888, which is hereby expressly incorporated by reference in its entirety herein. [000114] In some alternatives, the orientation of the chains is VL-linker-VH. In some aspects, the orientation is VH-linker-VL. [000115] In some alternatives, the ligand-binding region comprises a heavy-chain variable region comprising CDR1, CDR2, and CDR3 of the VH sequence shown in SEQ ID NO: 18; a light-chain variable region comprising CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 19. In some alternatives, a spacer is provided where the spacer comprises an immunoglobulin constant region, such as one including all or part of an Fe region that includes at least one pair of amino acid substitutions selected from the group consisting of the amino acid sequence of SEQ ID NO: 42 and SEQ ID NO: 43; or a modified Fe region and at least one genetically selected modified light chain constant region from the group consisting of L443C (SEQ ID NO:44), Q347C (SEQ ID NO:45), kK183C (SEQ ID NO:46), SEQ ID NO:47, L443C / kK183C (SEQ ID NO:44 and SEQ ID NO:46), Q347C / kA111 C (SEQ ID NO:45 and SEQ ID NO:47), and Q347C / kK183C (SEQ ID NO:45 and SEQ ID NO:46).[000116] In some alternatives, the constant region or portion thereof comprises a sequence exposed in SEQ ID NO:48. [000117] In some alternatives, scFv derived from Ab02 comprises a sequence exposed in SEQ ID NO: 49 (LCDR1), SEQ ID NO: 50 (LCDR2), and / or SEQ ID NO: 51 (LCDR3). In some alternatives, antibody Ab02 comprises a sequence as exposed in SEQ ID NO: 55 (HCDR1), SEQ ID NO: 56 (HCDR2), and / or SEQ ID NO: 57 (HCDR3). [000118] In some alternatives, antibody Ab01 comprises a sequence exposed in SEQ ID NO: 52 (LCDR1), SEQ ID NO: 53 (LCDR2), and / or SEQ ID NO: 54 (LCDR3). In some alternatives, antibody Ab01 comprises a sequence as exposed in SEQ ID NO: 58 (HCDR1), SEQ ID NO: 59 (HCDR2), and / or SEQ ID NO: 60 (HCDR3). [000119] In some embodiments, the CAR includes an antigen-binding region that is or comprises a scFv including VH and VL domains in the VL-binder-VH orientation. In some embodiments, such as in some aspects of these embodiments, the VH domain comprises an amino acid sequence of SEQ ID NO:18, a CDR3 (and, optionally, CDR1 and CDR2) of the VH sequence exposed in SEQ ID NO:18, and / or comprises or consists of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:18, or comprising a CDR1, CDR2, and / or CDR3 of, usually at least one CDR3 of, a VH region having the sequence of SEQ ID NO:18; In some aspects, for example, of these modalities, VH is a VH derived from Ab01.In some aspects of these modalities, the VL domain comprises an amino acid sequence of SEQ ID NO: 19, a CDR3 (and, optionally, CDR1 and CDR2) of the VL sequence exposed in SEQ ID NO: 19, and / or comprises or consists of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 19, or comprising a CDR1, CDR2, and / or CDR3 of, usually at least one CDR3 of, a VL region having the sequence of SEQ ID NO: 19 in some aspects; for example, in these modalities, laVH is a VH derived from Ab01.In some aspects of any of these modalities, such as aspects of CARs having these binding domains, the CAR includes a spacer region between the binding domain and transmembrane domain that contains no more than or approximately 50, 40, 30, or 20 amino acids in length, such as no more than or approximately 15 or no more than or approximately 12 amino acids, and / or contains or is or essentially consists of an immunoglobulin hinge region or variant thereof, such as an lgG4 hinge or variant thereof. [000120] In some respects, CARs and / or cells expressing them exhibit one or more increased or superior functional activities compared to a reference CAR. In some respects, the reference CAR is a CAR that has the same (and optionally otherwise identical) binding domain but has a spacer that differs in length and / or composition, such as one longer than or approximately 50, 40, 30, 20, 15, or 12 amino acids in length, and / or a CAR that has a binding domain with the same or similar variable regions, but in an alternative orientation.In some modalities, the function is as observed in an in vitro assay such as a co-culture assay with cells expressing IL-13Ra2 (such as an assay that measures cytokine production or cytolytic activity) or an in vivo assay using an animal model with an IL-13Ra2-positive tumor, such as one that assesses tumor burden reduction or survival after administration of cells, such as T cells, expressing CAR. [000121] In the alternatives presented herein, eight CARs having various combinations of antigen-binding domains and other domains as depicted in Figure 1 were tested in several assays. Certain sequences [000122] In some alternatives, the VH of Ab01 comprises a sequence exposed in SEQ ID NO:14. In some alternatives, the VL of Ab01 comprises a sequence exposed in SEQ ID NO:16. In some alternatives, the scFvVLVH of Ab01 of IL13Ra2 is encoded by a sequence exposed in SEQ ID NO:61. In some alternatives, the scFv VHVL of Ab01 of IL13Ra2 is encoded by a sequence exposed in SEQ ID NO:62 that includes an Ab01-VL sequence, an Ab01-VH sequence, an ATG start codon, a 5' Nhe\ restriction site (GCTAGC), and a 3' Rsr\I restriction site (CGGACCG). In some alternatives, the Ab01-VH protein sequence comprises SEQ ID NO:65. In some alternatives, the HCDR1 of Ab01 comprises a sequence exposed in SEQ ID NO:66. In some alternatives, HCDR2 of Ab01 comprises a sequence exposed in SEQ ID NO:67. In some alternatives, HCDR3 of Ab01 comprises a sequence exposed in SEQ ID NO:68.In some alternatives, Ab01-VL comprises a sequence exposed in SEQ ID NO:69. In some alternatives, LCDR1 of Ab01 comprises a sequence exposed in SEQ ID zcRRQn / nznz / a / YiAi. N07O. In some alternatives, LCDR2 of Ab01 comprises a sequence exposed in SEQ ID NO:71. In some alternatives, LCDR3 of Ab01 comprises a sequence exposed in SEQ ID NO:72. [000123] In some alternatives, the VH of AbO2 comprises a sequence exposed in SEQ ID NO:15. In some alternatives, the VL of AbO2 comprises a sequence exposed in SEQ ID NO:17. In some alternatives, the scFv VLVH of AbO2 from IL13Ra2 is encoded by a sequence exposed in SEQ ID NO:63. In some alternatives, the scFv VHVL of AbO2 is encoded by a sequence exposed in SEQ ID NO:64, which includes an AbO2-VL sequence, an AbO2-VH sequence, an ATG start codon, a 5' Nhe\ restriction site (GCTAGC), and a 3' Rsril restriction site (CGGACCG). In some alternatives, the AbO2-VH protein sequence comprises a sequence exposed in SEQ ID NO:73. In some alternatives, the HCDR1 of AbO2 comprises a sequence exposed in SEQ ID NO:74. In some alternatives, HCDR2 of Ab02 comprises a sequence exposed in SEQ ID NO:75. In some alternatives, HCDR3 of Ab02 comprises a sequence exposed in SEQ ID NO:76.In some alternatives, the Ab02-VL protein sequence comprises a sequence exposed in SEQ ID NO:77. In some alternatives, the Ab02 LCDR1 comprises a sequence exposed in SEQ ID NO:78. In some alternatives, the Ab02 LCDR2 comprises a sequence exposed in SEQ ID NO:79. In some alternatives, the Ab02 LCDR3 comprises a sequence exposed in SEQ ID NO:80. EXAMPLES Example 1 - Construction of CARs targeting IL-13Ra2 [000124] Several CARs targeting IL-13Ra2 were constructed. As shown in Figure 1, the IL-13Ra2-specific CARs included: one of a variable amount of single-chain fragment (scFv) that specifically recognized an extracellular epitope of the IL-13 alpha 2 receptor (IL-13Ra2); one of several spacer domains (labeled “S”, “M”, and “L”); a transmembrane domain derived from human CD28 (CD28tm); a costimulatory domain derived from human 4-1BB; a signaling domain derived from CD3; a T2A ribosomal skipping sequence; and a truncated EGFR transduction marker (EGFRt); and optionally an additional T2A ribosomal skipping sequence and a dihydrofolate reductase double mutator transgene (DHFRdm) configured for methotrexate selection. [000125] Examples of antibodies or epitope-binding fragments from which single-chain variable fragment (scFv) can be derived include humanized anti-IL-13-Ra2 lgG1 antibody hu08 (Creative Biolabs, NY) and anti-IL13RA2 therapeutic antibody (hu07 v1.1) (Creative Biolabs, NY). [000126] The spacer domain was linked in each CAR to the scFv and connected the extracellular binding element to the transmembrane domain. Each of the CARs was generated to include one of three different spacers (S, M, or L), each with an amino acid sequence of a different length (Spacer S, SEQ ID NO: 9; Spacer M, SEQ ID NO: 10; or Spacer L, SEQ ID NO: 11). Different scFv configurations were used individually in the various IL-13Ra2 CARs, including VH and VL domains derived from one of two different anti-IL-13Ra2 directed antibodies ('HuO8', AbO1; and 'HuOZ', AbO2). The scFv portion of each CAR consisted of a VH region having a sequence as described in SEQ ID NO:14 or SEQ ID NO:15 and a VL region having a sequence as described in SEQ ID NO:16 or zcRRQn / nznz / a / YiAi SEQ ID NO: 17, present in a VL-VH or VH-VL orientation, connected by a flexible peptide ligand. Example 2-Functional comparison of CARs derived from different antibodies [000127] A functional comparison of CARs incorporating scFv-binding domains derived from different antibodies in various orientations was performed. The comparison included an EGFRt surface expression analysis and a cytokine release assay. In this study, each CAR included the S” spacer (having the amino acid sequence SEQ ID NO:9, a modified immunoglobulin hinge region), as well as the primary and costimulatory signaling and transmembrane domains described and shown in Figure 1, with one of the various scFv-binding regions (VH / VL domains and orientations) shown in Figure 1. The CARs included: IL-13Ra2 Ab01 VLVH S spacer; IL-13Ra2 Ab01 VHVL S spacer; IL-13Ra2 Ab02 VLVH S spacer; and IL-13Ra2 Ab02 VHVL S spacer. [000128] CAR expression in cells was examined by determining the expression of the EGFRt marker in CD8+ and CD4+ T cells using flow cytometry (Figure 2A) [000129] CAR T cells were co-cultured individually with DAOY cells (human cerebellum / brain cell line), U251T cells (human giobastoma cell line), and K562 cells (human chronic myelogenous leukemic cells) modified to express IL13Ra2. After 24 h, IL-2, IFN-γ, and TNF-α levels were measured in supernatants using a cytokine assay. Increased cytokine production was observed after incubation with target cells for CAR-expressing CD8+ and CD4+ T cells containing binding domains that have VH and VL domains of the Ab01 antibody, in both VH / VL orientations (Figure 2B and Figure 2C). Example 3 - Functional comparison of CARs that include different spacers [000130] A functional comparison was performed of CARs including scFvs derived from the same antibody (in different orientations) and different spacers of varying lengths. The four CARs represented in Figure 1 were tested for cell surface expression on CD8+ cells by cleavage of the marker sequence, EGFRt (Figure 3A). In addition, CD8+ cells bearing the CARs were also tested for their ability to induce specific lysis, as well as their ability to release cytokines.In this study, each of the example CARs included one of two different binding domains, an scFv having Ab01 VL and VH domains, either in the VL-VH or VH-VL orientation, and one of the various spacers (S” (having the amino acid sequence of SEQ ID NO:9), “M” (having the amino acid sequence of SEQ ID NO:10), and “L” (having the amino acid sequence of SEQ ID NO:11), as well as the primary and co-stimulatory signaling and transmembrane domains described and depicted in Figure 1. [000131] Representative results of multiparameter flow cytometry analysis of healthy donor-derived T cells transduced with the indicated CAR lentivirus are shown in Figure 3A (top and middle panels), in which quadrants were extracted from histograms based on control staining. The expression of the surrogate surface marker (EGFRt) on IL-13Ra2 CD8+ CAR T cells was analyzed by flow cytometry (Figure 3A, bottom panel). [000132] CD8+ T cells expressing CARs targeting IL-13Ra2 were co-cultured with different IL-13Ra2-expressing target cell lines in varying effector-to-target ratios (Figure 3B). A chromium release assay was used to assess cytolytic activity, and cytokine production was evaluated in supernatants. The chromium release assay was performed over a 4-hour co-culture period, while cytokine release was assessed after a 24-hour co-culture (effector-to-target ratio of 2:1). As shown in Figure 3C, co-culture with the various CAR-expressing T cells led to increasingly specific lysis with increasing effector-to-target ratios and cytokine secretion levels. Compared to other CARs evaluated, increased cytolytic activity was observed for cells expressing the CAR with the S” spacer and including scFv with the VL-VH orientation.In each of the evaluated CARs with VL-VH oriented binding domains, target-specific cytolytic activity and cytokine production were observed. Additionally, in the VLVH-oriented CARs, the presence of the S” spacer resulted in superior effects compared to the other spacers tested. In contrast, no cytolytic activity was observed in this assay for the VH-VL oriented CARs with the S” spacer (whereas activity was observed for each of the VH-VL CARs with the “M” and “L” spacers). [000133] Regarding cytokine production, as shown in Figure 3B, T cells with S-spacer CARs of IL-13Ra2 Ab01 VLVH, T cells with M-spacer CARs of IL-13Ra2 Ab01 VLVH, and T cells with L-spacer CARs of IL-13Ra2 Ab01 VLVH led to higher IFN-gamma expression. T cells with M-spacer CARs of IL-13Ra2 Ab01 VHVL and T cells with L-spacer CARs of IL-13Ra2 Ab01 VHVL showed IFN-gamma expression. T cell with S spacer CAR of IL-13Ra2 Ab01 VLVH, T cell with M spacer CAR of IL-13Ra2 Ab01 VLVH, T cell with L spacer CAR of IL-13Ra2 Ab01 VLVH led to higher specific lysis. [000134] Regarding specific lysis, as shown in Figure 3B, at increased concentrations, CD8+ cells bearing the IL-13Ra2 Ab01 S VLVH spacer CAR, the IL-13Ra2 Ab01 medium VLVH CAR, and the IL-13Ra2 Ab01 long VLVH CAR induced a significant amount of specific lysis at high cell concentrations. Furthermore, these cells led to high production of the cytokine IFN-gamma. [000135] In contrast, cells expressing the Ab01 IL-13Ra2 S VHVL spacer CAR, the Ab01 IL-13Ra2 medium VHVL CAR, and the Ab01 IL-13Ra2 long VHVL CAR had reduced amounts of specific lysis compared to the Ab01 IL-13Ra2 S BLVH spacer CAR, the Ab01 IL-13Ra2 medium VLVH CAR, and the Ab01 IL-13Ra2 long VLVH CAR. No specific lysis was observed with the Ab01 IL-13Ra2 S VHVL spacer CAR. Additionally, the expression of the S-spacer VHVL CAR of IL-13Ra2 Ab01 did not lead to IFNgamma expression, which was observed with the medium VHVL CAR of IL-13Ra2 Ab01 and the long VHVL CAR of IL-13Ra2 Ab01. Example 4—In vivo antitumor activity of an anti-IL13Ra2 CAR [000136] The therapeutic outcomes following administration of cells expressing an IL13Ra2-specific CAR containing an Ab01 antibody-derived binding domain, VLVH targeting, and S-spacer were evaluated in a mouse glioblastoma model. The CARs used for the experiments were expressed in ζGLLoη / ηζηζ / α / γίΛΐ cells CD8+. For the experiments, ffLuc+ U87 glioblastoma cells (0.2 x 106) were injected intracranially into the forebrain of NSG mice on day 0. On day 7, mice (n=3 per group) were either untreated (vehicle or sham control) or treated with different doses (2 x 106, 1 x 106 or 0.5 x 106 cells) of IL-13Ra2 Ab01 transduced CD8+ CAR T cells. [000137] Figure 4A and Figure 4B show results from a study demonstrating in vivo antitumor activity of an IL13Ra2-specific CAR containing the Ab01-derived VH and VL domains, in the VL-VH orientation, and the “S” spacer, along with the signaling and transmembrane domains as depicted in Figure 1. Firefly luciferase-expressing human glioblastoma cells (ffLuc+U87 glioblastoma cells) (0.2 x 10⁶) were intracranially injected into the forebrains of NSG mice on day 0. On day 7, mice (n=3 per group) received either vehicle control or sham administration, or were treated with different doses (2 x 10⁶, 1 x 10⁶, or 0.5 x 10⁶ cells) of CD8+ cells expressing the IL-13Ra2-specific CAR (Ab01 VL-VH).Figure 4A shows graphs of total flux (photons / sec) as a measure of tumor burden (y-axis) over time (days after tumor inoculation, x-axis) for vehicle-treated or sham-treated animals (left) and indicated decreasing numbers of CAR+ cells (right; right to left), demonstrating tumor regression after administration of CAR-expressing cells. A Kaplan-Meier survival curve demonstrated progressively improved survival for mice treated with increasing doses of anti-IL-13Ra2 CAR-expressing T cells (Figure 4B). The vehicle control had a 0% survival rate at 25 days. However, mice receiving 2 x 10⁶ CD8+ T cells expressing the IL-13Ra2 Ab01 had -50% survival after 90 days. Example 5—In vivo antitumor activity of an anti-IL13Ra2 CAR [000138] To evaluate the impact of the IL13Ra2 Ab01 CAR VLVH spacer, an experiment substantially similar to Example 3 was performed using U251T tumor-bearing mice as a glioblastoma model. On day 0, U251T GFP:ffluc tumor cells were injected intracranially, followed by intratumoral injection of either CD8+ CAR T cells or CD8+ simulated T cells on day 7. Mice injected with simulated T cells served as a control. [000139] All mice treated with short-spacer VLVH 2G Ab01 IL13Ra2 CAR T cells showed competent tumor regression. Only one mouse in the medium-CAR group exhibited tumor relapse and required euthanasia on day 58 post-tumor injection. Three of the U251T tumor-bearing mice treated with intratumoral injection of long-spacer IL13Ra2 CAR T cells exhibited less therapeutic activity and tumor relapse. [000140] Taken together, all these data suggest that the short extracellular spacer size is the optimal configuration for the IL13Ra2 Ab01 VLVH that confers optimal antitumor potency in vivo, supporting its clinical development. [000141] Table 2 lists certain amino acid and nucleotide sequences for some modalities of the methods and compositions provided herein. Table 2 SEQIDNO: Characteristica(s) Sequence SEQ ID NQ:01 lgG1 human EPKSCDKTHTCPPCP SEQ ID NO:02 lgG2 human ERKCCVECPPCP SEQ ID ΝΟΌ3 lgG3 human ELKTPLGDTHTCPRCPEPPKSCDTPPPCPCPCPCPCPCPCPCPCPCPPDCPCPCPQPDCPCPQPDC NQ:04 lgG4 human ESKYGPPCPSCP SEQ ID ΝΟΌ5 lgG4 human modified ESKYGPPCPPCP SEQ ID ΝΟΌ6 lgG4 human modified YGPPCPPCP SEQ ID ΝΟΌ7 lgG4 human modified KYGPPCPPCP SEQG4 human modified ID ΝΝ EWKYGPPCPPCP SEQ ID ΝΟΌ9 Spacer S ESKYGPPCPPCP SEQ ID NQ:10 Spacer M ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDGSFFLYS MHEALHNHYTQKSLSLSLGK SEQ ID NO:11 Spacer L ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVWWDVSQE DPEVQFNWYVDGVEVHNACTKPREEQFNSTYRWSVLTVLHQDWLNGKE YCKKVSNKGLPSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:12 CD28tm ATGTTCTGGGTGCTGGTGGTGGTCGGAGGC GTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTT SEQIDNO: Característica(s) Secuencia TGGGTG SEQ ID NO:13 CD28tm MFWVLVWGGVLACYSLLVWAFIIFWV SEQ ID NO:14 Ab01-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSS SEQ ID NO:15 AbO2-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTKYGVHWVRQAPGKGLEWV AVKWAGGSTDYNSALMSRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DHRDAMDYWGQGTLVTVSS SEQ ID NO:16 AbO1-VL DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLUYS ASYRSTGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQHHYSAPWTFGGGTKVEIK SEQ ID NO:17 AÓ02-VL DIQMTQSPSSSLSASVGDRVTITCTASLSSVSSTYLHWYQQKPGKAPKLLIYS TSNLASGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCHQYHRSPLTFGGGTKVEIK SEQ ID NO:18 Región VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSS SEQ ID NO:19 Región VL DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYS ASYRSTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSAPWTFGGGTKVEIK SEQ ID NQ:20 CDR1 de cadena pesada SRNGMS SEQ ID NO:21 CDR2 de cadena pesadaTVSSGGSYIYYADSVKG SEQ ID NO:22 CDR3 heavy chain QGTTALATRFFDV 7CRRQn / n7n7 / 3 / YIAI SEQIDNO: Feature(s) Sequence SEQ ID NO:23 Light Chain CDR1 KASQDVGTAVA SEQ ID NO:24 Light Chain CDR2 SASYRST SEQ ID NO:25 Light Chain CDR3 QHHYSAPWT SEQ ID NO:26 Space Marker SEQ ID NO:27 Space Marker SEQ ID NO:28 Heavy Chain CDR2 TVSSGGSYIYYADSVKG SEQ ID NO:29 Heavy Chain CDR3 QGTTALATRFFDV SEQ ID NO:30 Light Chain CDR1 KASQDVGTAVA SEQ ID NO:31 Light Chain CDR2 SASYRST SEQ ID NO:32 Light Chain CDR3 QHHYSAPWT SEQ ID NO:33 Heavy Chain CDR1 GFTFSRN SEQ ID NO:34 Heavy Chain CDR1 GFTFSRNGMS SEQ ID NO:35 Heavy Chain CDR1 RNGMS 7CRRQn / n7n7 / 3 / YIAI SEQIDNO: Characteristica(s) Secuencia SEQ ID NO:36 CDR1 de cadena pesada SRNGMS SEQ ID NO:37 CDR2 de cadena pesada SSGGSY SEQ ID NO:38 CDR2 de cadena pesada TVSSGGSYIY SEQ ID NO:39 CDR2 de cadena pesada TVSSGGSYIY SEQ ID NO:40 CDR2 de cadena pesada TVSSGGSYIYYADSVKG SEQ ID NO:41 CDR3 de cadena pesada ARQGTTALATRFFDV SEQ ID NO:42 Región Fe EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSC SPGK SEQ ID NO:43 Región Fe EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Característica(s) Secuencia EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNCFSCSVMHEALHNHYTQKSLSC SPGK SEQ ID NO:44 Región Fe EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSC SPGK SEQ ID NO:45 Región Fe EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPCVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SEQ ID NO:46 Región Fe DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLUYS ASYRSTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSAPWTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQGL SSPVTKSFNRGEC SEQ ID NO:47 Región Fe DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYS 7CRRQn / n7n7 / 3 / YIAI SEQIDNO: Característica(s) Secuencia ASYRSTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSAPWTFGG GTKVEIKRTVACPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC SEQ ID NO:48 Región Fe TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKAAYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO:49 LCDR1 TASLSVSSTYLH SEQ ID NO:50 LCDR2 STSNLAS SEQ ID NO:51 LCDR3 HQYHRSPLT SEQ ID NO:52 LCDR1 KASQDVGTAVA SEQ ID NO:53 LCDR2 SASYRST SEQ ID NO:54 LCDR3 QHHYSAPWT SEQ ID NO:55 HCDR1 TKYGVH SEQ ID NO:56 HCDR2 VKWAGGSTDYNSALMS SEQ ID NO:57 HCDR3 DHRDAMDY SEQ ID NO:58 HCDR1 SRNGMS SEQ ID NO:59 HCDR2 TVSSGGSYIYYADSVKG SEQ ID NQ:60 HCDR3 QGTTALATRFFDV SEQ ID NO:61 ScFv VLVH de Ab01 de IL13Ra2 GCTAGCCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTG AGTTACCACACCCAGCATTCCTCCTGATCCCAGACATCCAGATGACCC AGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCA CCTGTAAGGCCAGTCAGGATGTAGGTACTGCTGTAGCCTGGTATCAGC AGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTCGGCATCCTACCGGTCCACTGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCG 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Sequence ATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTA CTACTGCCAGCACCATTATAGTGCTCCGTGGACGTTTGGCGGCGGAAC AAAGGTGGAGATCAAGGGTGGTGGTGGTTCTGGCGGCGGCGTCCG GTGGTGGTGGTTCTGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTG GTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTC ACCTTCAGTAGGAATGGCATGTCTTGGGTGAGGCAGGCCCCTGGCAA CTATGCAGACAGTGTGAAGGGGCGGTTCACCATCTCCAGGGACAACG CCAAGAACTCCCTGTACCTCCAGATGAACTCCCTGAGGGCCGAGGATA CCGCCGTGTACTACTGTGCCAGACAAGGGACTACGGCACTAGCTACGA GGTTCTTCGATGTCTGGGGCCAGGCACCTAGCTGAGTTCGTTGGGGCCAG SEQ ID NO:62 scFv VHVL of Ab01 of IL13Ra2: AbO1-VL; Ab01VH; ATG start codon; 5' Nhel restriction site (GCTAGC);Sitio de restricción de Rsrll de 3' (CGGACCG) GCTAGCCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTG AGTTACCACACCCAGCATTCCTCCTGATCCCAGAGGTGCAGCTGGTGG AGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCT TGTGCCGCCTCCGGCTTCACCTTCAGTAGGAATGGCATGTCTTGGGTG AGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGGCCACCGTTAGTAG TGGTGGTAGTTACATCTACTATGCAGACAGTGTGAAGGGGCGGTTCAC CATCTCCAGGGACAACGCCAAGAACTCCCTGTACCTCCAGATGAACTC CCTGAGGGCCGAGGATACCGCCGTGTACTACTGTGCCAGACAAGGGA CTACGGCACTAGCTACGAGGTTCTTCGATGTCTGGGGCCAGGGCACC CTGGTGACCGTGTCCTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTC CGGTGGTGGTGGTTCTGACATCCAGATGACCCAGTCCCCCTCTTCTCT GTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTAAGGCCAGTCA GGATGTAGGTACTGCTGTAGCCTGGTATCAGCAGAAGCCTGGCAAGGC TCCCAAGCTGCTGATCTACTCGGCATCCTACCGGTCCACTGGCGTGCC TTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCAT CTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCCAGCACCA TTATAGTGCTCCGTGGACGTTTGGCGGCGGAACAAAGGTGGAGATCAA GGAATCTAAGTACGGACQG; 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Sequence SEQ ID NO:63 ScFv VLVH of IL13Ra2 Ab02 GCTAGCCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTG AGTTACCACACCCAGCATTCCTCCTGATCCCAGATATTCAGATGACCCA GAGCCCGAGCAGCCTGAGCGCGAGCGTGGGGCGATCGCGTGACCATTA CCTGCACCGCGAGCCTGAGCGTGAGCAGCACCTATCTGCATTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCACCAGCA ACCTGGCGAGCGGCGTGCCGAGCCGCTTAGGCGACCGACCGCCGACCGA IAGCAGCCIGCAGCCAGGAAGAI III GC GACCTATTATTGCCATCAGTATCATCGCAGCCCGCTGACCTTTTGGCGG CGGCACCAAAGTGGAAATTAAAGGTGGTGGTGGTTCTGGCGGCGGCG GCTCCGGTGGTGGTGGTTCTGAAGTGCAGCTGGTGGAAGGCGGGGGG GGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCCGGCGA GCGGCTTTACCTTTACCAAATATGGCGTGCATTGGGTGCGCCAGGCGC CGGGCAAAGGCCTGGAATGGGTGGCGGTGAAATGGGCGGGCGGCAG CACCGATTATAACAGCGCGGGATCGACCGCCGATTGATT TAACGCGAAAAACAGCCTGTATCTGCAGATGAACAGCCCTGCGCGCGGA AGATACCGCGGTGTATTATTGCGCGCGCGATCGATCCGATGCGATGGA TTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGAATCTAAGTA CGGACCG SEQ ID NO:64 scFv VHV2L de Ab32L de: AÓ02-VL; AbO2-VH;ATG start codon; 5' Nhel restriction site (GCTAGC); 3' Rsrll restriction site (CGGACCG) GCTAGCCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTG AGTTACCACACCCAGCATTCCTCCTGATCCCAGAAGTGCAGCTGGTGG CTGCGCGGCGAGCGGCTTTACCTTTACCAAATATGGCGTGCATTGGGT GCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGGTGAAATGG GCGGGCGGCAGCACCGATTATAACGCCGCTGATGAGCCGCTTTAC CATTAGCCGCGATAACGCGAAAAACAGCTGAGCGCTGACT CCTGCGCGCCGGAAGATACCGCGGTGTATTATTGCGCGCGCGATCATC GCGATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGC AGCGGTGGTGGTGGTTCTGGCGGCGGCTCCGGTGGTGGTGGGTT GGCGATCGCGTGACCATTACCTGCACCGCGAGCCTGAGCGTGAGCAG CACCTATCTGCATTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACT; 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Característica(s) Secuencia GCTGATTTATAGCACCAGCAACCTGGCGAGCGGCGTGCCGAGCCGCT TTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCC TGCAGCCGGAAGAI II IGCGACCTATTTATTGCCATCAGTATCATCGCAG CCCGCTGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAAGAATCTAA GTACGGACCG SEQ ID NO:65 AbO1-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVRQAPGKGLEWV ATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QGTTALATRFFDVWGQGTLVTVSS SEQ ID NO:66 Ab01 HCDR1 SRNGMS SEQ ID NO:67 Ab01 HCDR2 TVSSGGSYIYYADSVKG SEQ ID NO:68 Ab01 HCDR3 QGTTALATRFFDV SEQ ID NO:69 AbO1-VL DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYS ASYRSTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSAPWTFGG GTKVEIK SEQ ID NO:70 Ab01 LCDR1 KASQDVGTAVA SEQ ID NO:71 Ab01 LCDRLSCAASGFTFTKYGVHWVRQAPGKGLEWV Ab02 HCDR3 DHRDAMDY SEQ ID NO:77AbO2-VL DIQMTQSPSSLSASVGDRVTITCTASLSVSSTYLHWYQQKPGKAPKLUYS TSNLASGVPSRFSGSGSGTDFTLTISSL 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Sequence QPEDFATYYCHQYHRSPLTFGGGTKVEIK SEQ ID NO:78 Ab02 LCDR1 TASLSVSSTYLH SEQ ID NO:79 Ab02 LCDR2 STSNLAS SEQ ID NQ:80 Ab02 LCDR3 SE HQYHR:DDHm ID NO:78 MVGSLNCIVAVSQNMGIGKNGDFPWPPLRNESRYFQRMTTTSSVEGKQN LVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLT EQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFEFFFPEF IDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND SEQ ID NO:82 DHFRdm ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTCCCAGAACATGGGC ATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATC CAGATATTCAGACCACGACCAGGACTGACTGACTGACTGATCTAATC AATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGA ATCGACCTTTAAAGGGTAGAATTAATTTAGTTCAGCAGAGAACTCAA GGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGCC TAAAACTTACTGAACAACCAGAATTAGCAACATAGTAGATTAGTC GGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCAG GCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGA CACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAAT ACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAGAGCAGATCAT AATTTGAAGTATGAGAAGAATGATTAA SEQ ID NO:83 GMCSFssLíder ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAG CATTCCTCCTGATCCCA SEQ ID NO:84 AbO1-VL GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGC GACAGAGTGACCATCACCTGTAAGGCCAGTCAGGATGTAGGTACTGCT GTAGCCTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATC TACTCGGCATCCTACCGGTCCACTGGCGTGCCTTCCAGATTCTCCGGC TCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCT GAGGATTTCGCCACCTACTACTGCCAGCACCATTATAGTGCTCCGTGG 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Sequence ACGTTTGGCGGCGGAACAAAGGTGGAGATCAAG SEQ ID NO:85 AbO1-VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCG ATGGCATGTCTTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGG GTGGCCACCGTTAGTAGTGGTGGTAGTTACATCTACTATGCAGACAGT GTGAAGGGGCGGTTCACCATCCAGGGACAACGCCAAGAACTCCCT CTGTGCCAGACAAGGGACTACGGCACTAGCTACGAGGTTCTTCGATGT CTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCT SEQ ID NO:86 AbO2-VL GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGAGCGTGGGG CGATCGCGTGACCATTAGCCGAGCGAGCGAGCGA CCTATCTGCATTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGC TGATTTATAGCACCAGCAACCTGGCGAGCGGCGTGCCGAGCCGCTTTA GCGGCAGCGGCAGCGGCACCGAI II IACCCTGACCATTAGCAGCCTG CAGCCGGAAGATTTTGCGACCTTATTTGCCATCAGCAGTCATTC CGCTGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAA SEQ ID NO:87 Ab02-VH GAAGTGCAGCTGGTGGAAAGCGGCGGCCTGGTGCAGCCGGGCG GCAGCCTGCGCCTGAGCTGCGGCGAGCGGCTTTACCTTTACCAAAT ATGGCGTGCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGGTGAAATGGGCGGGCGGCAGCACCGATTATAACAGCGCGCT GATGAGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACAGCCTGTA TCTGCAGATGAACAGCCCTGCGCGCGGAAGATACCGGTGTTATTG CGCGCGCGATCGATCGATGCGATGGGATTTAGGGCCGATTGGCCGATT SEQ ID NO:88 Ligator of (Gly- Gly-Gly-Ser)x3 GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCT SEQ ID NO:89 VLVH of scFv of Ab01 GACATCCAGATGACCCA GACAGAGTGACCATCACCTGTAAGGCCAGTCAGGATGTAGGTACTGCT GTAGCCTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATC 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Característica(s) Secuencia TACTCGGCATCCTACCGGTCCACTGGCGTGCCTTCCAGATTCTCCGGC TCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCT GAGGATTTCGCCACCTACTACTGCCAGCACCATTATAGTGCTCCGTGG ACGTTTGGCGGCGGAACAAAGGTGGAGATCAAGGGTGGTGGTGGTTC TGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTGGTG GAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTC TTGTGCCGCCTCCGGCTTCACCTTCAGTAGGAATGGCATGTCTTGGGT GAGGCAGGCCCCTGGCAAGGGCCTGGAGGTGGGTGGCCACCGTTAGTA GTGGTGGTAGTTACATCTACTATGCAGACAGTGTGAAGGGGCGGTTCA CCATCTCCAGGGACAACGCCAAGAACTCCCTGTACCTCCAGATGAACT CCCTGAGGGCCGAGGATACCGCCGTGTACTACTGTGCCAGACAAGGG ACTACGGCACTAGCTACGAGGTTCTTCGATGTCTGGGGCCAGGGCACC CTGGTGACCGTGTCCTCT SEQ ID NQ:90 VHVL de scFv de Ab01 GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCG GCTCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCAGTAGGA ATGGCATGTCTTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGG GTGGCCACCGTTAGTAGTGGTGGTGTAGTTACATCTACTATGCAGACAGT GTGAAGGGGCGGTTCACCATCTCCAGGGACAACGCCAAGAACTCCCT GTACCTCCAGATGAACTCCCTGAGGGCCGAGGATACCGCCGTGTACTACTGTGCCAGACAAGGGACTACGGCACTAGCTACGAGGTTCTTCGATGT CTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCTGGTGGTGGTGGTT CTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCCAGATGACC CAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATC ACCTGTAAGGCCAGTCAGGATGTAGGTACTGCTGTAGCCTGGTATCAG CAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTCGGCATCCTAC CGGTCCACTGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACC GATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCT ACTACTGCCAGCACCATTATAGTGCTCCGTGGACGTTTGGCGGCGGAA CAAAGGTGGAGATCAAG SEQ ID NO:91 VLVH de scFv de GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGG 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Sequence Ab02 CGATCGCGTGACCATTACCTGCACCGCGAGCCTGAGCGTGAGCAGCA CCTATCTGCATTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGC TGATTTATAGCACCAGCAACCTGGCGAGCGGCGTGCCGAGCCGCTTTA GCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTG CAGCCGGAAGAI II IGCGACCTATTATTGCCATCAGTATCATCGCAGCC CGCTGACCTTTTGGCGGCGGCACCAAAGTGGAAATTAAAGGTGGTGGT GGTTCTGGCGGCGGCCGTCCGGGTTTGGGTGGGCT GGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGC CTGAGCTGCGCGGCGAGCGGCTTTACCTTTACCAAATGGCGTGCAT TGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGGTGA TTTACCATTAGCCGCGATAACGCGAAAAACAGCCTGTATCTGCAGATGA ACAGCCTGCGCGCGGAAGATACCGCGGTGTTATTGCGCGCGCGAT CATCGCGATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGT GAGCAGC SEQ ID NO:92 VHVL of scFv of Ab02 GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCG GCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTACCAAAT ATGGCGTGCATTGGGTGCGCCAGGCGCGGGCAAAGGCCTGGAATGG GTGGCGGTGAAATGGGCGCCAGGCCGATTGATGAGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACAGCCTGTA TCTGCAGATGAACAGCCCTGCGCGCGGAAGATACCGCGGTGTTATTG CGCGCGCGATCCGCGATGCGATGGATTATTGGGGCCAGGGCACCC TGGTGACCGTGAGCAGCGGTGGTGGTGGGGGGCGTCGTC CGGTGGTGGTGGTTCTGATATTCAGATGACCCAGAGCCCGAGCAGCCT GAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCACCGCGAGCC TGAGCGTGAGCAGCACCTATCTGCATTGGTATCAGCAGAAACCGGCA AAGCGCCGAAACTGCTGATTTAGCAGCCAGCCGAGCGAGCGCC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGAI II IACCCT GACCATTAGCAGCCTGCAGCCGGAAGAI II IGCGACCTATTATTGCCAT CAGTATCATCGCAGCCCGCTGACCTTTTGGCGGCGGCACCAAAGTGGA AATTAAA SEQ ID NO: Feature(s) Sequence SEQ ID NO:93 Spacer S GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT SEQ ID NO:94 CD28tm ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGGCCTGCTACAG CCTGCTGGTCACCATGGGTTGTTGTT NO:95 41-BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTGA GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTG SEQ ID NO:96 CD3( CGGGAAGTTCAAGCCAGCCAGCCGACCGCCGAGCGCCGATT CCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTA CGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGC AAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCA AGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTC CACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGC CCCCAAGG SEQ ID NO:97 T2A CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGAGGAGGAGGGCC IDGCC NO:98 EGFRt CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAGACTCACTCT CCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGT GGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAA AGGAAATCACAGGGI III IGCTGATTCAGGCTTGGCCTGAAAACAGGA CGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCA AGCAACATGGTCAGTTTTCTCTTGCAGTCGATCATGCATCCATCC CTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATT TCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACT GTTTGGGACCTCCGGTCAGAAAACCAAAATTAAGCAACAGAGGTGA AAACAGCTGCAAGGCCACAGGCCAGGCCGCTGCCGTTGTT CGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCCTTGCCGG AATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGA 7CRRQn / n7n7 / 3 / YIAI SEQ ID NO: Feature(s) Secuencia GGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCA CCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGG GACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACT GCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTG GTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCC AAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAAC GAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCC TCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG SEQ ID NO:99 DHFRdm ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGC ATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATC CAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAG AATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGA ATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAA GGAACCTCCACAAGGAGCTCAI II ICTTTCCAGAAGTCTAGATGATGCC TTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCT GGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAG GCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGA CACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAAT ACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA SEQ ID NQ:100 Spacer M GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTGGCCAGCCTAGA GAACCCCAGGTGTACACCCTGCCTCCCAGGAAGAGATGACCAA GAACCAGGTGTCCCTGACCTGCCCTGCCCAGCCTTGACCTT TATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAGAACAACTACA AGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTCCTGTACT CCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTC AGCTGCAGCGTGATGCACGAGGAGCCCAACCACCTACCACCA GTCCCTGAGCCTGAGCCTGGGCAAG SEQ ID NO:101 Spacer L ATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCGA CGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAGCCCAAGGACCCCC zcRRQn / nznz / a / YiAi SEQ ID NO: Feature(s) Sequence TGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGT GGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCCCAGA GCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCC AGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCTCGA GCCCCAGGTGTACACCCTGCCTGACCGAGCCGAGACCAAGA CCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGA CCACCCCTCCCGTGCTGGACAGCGACGGCAGCTCTTCCTGTACAGCC GGCTGACCGTGGACAAGCCAGCCGGTGAAGGGACCGTCGCTA TGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAG CCTGAGCCTGTCCCTGGGCAAG [000142] With respect to the use of plural and / or singular terms herein, those skilled in the art may translate from plural to singular and / or from singular to plural as appropriate for the context and / or application. The various singular / plural permutations can be set out expressly in the present for the sake of clarity.[000143] It shall be understood by those skilled in the art that, in general, the terms used herein, and especially in the appended claims (for example, the bodies of the appended claims), are generally proposed as “open” terms (for example, the term “including” should be construed as “including but not limited to,” the term “having” should be construed as “having at least,” the term “includes” should be construed as “includes but not limited to,” etc.). It shall be understood by those skilled in the art that if a specific number of an introduced claim citation is proposed, this intention shall be explicitly stated in the claim, and in the absence of such a statement, this intention is not present. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases “at least one” and “one or more” to introduce claim citations.However, the use of these phrases should not be considered to imply that the introduction of a claim quotation by the indefinite articles “a” or “an” limits any particular claim containing this introduced claim quotation to modalities containing only one of these quotations, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “one” or “an” (for example, “one” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same applies to the use of definite articles used to introduce claim quotations.Furthermore, even if a specific number of an introduced claim citation is explicitly cited, those skilled in the art will recognize that this citation should ordinarily be interpreted as meaning at least the cited number (e.g., the simple citation of “two citations,” without any other modifiers, means at least two citations, or two or more citations). Moreover, in those cases where a convention analogous to “at least one of A, B, and C, etc.” is used, this construction is generally intended to mean that a person skilled in the art would understand the convention (e.g., a system having at least one of A, B, and C) would include, but not be limited to, systems having A alone, B alone, C alone, A and B jointly, A and C jointly, B and C jointly, and / or A, B, and C jointly, etc.).In general, this construction is intended to mean that a person skilled in the art would understand the convention (for example, a system having at least one of A, B, or C would include, but not be limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will also be understood by those skilled in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or figures, should be understood to include the possibilities of one of the terms, any one of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B". [000144] Furthermore, where features or aspects of the description are described in terms of Markush groups, those skilled in the technique will recognize that the description is also therefore described in terms of any individual member or subgroup of members of the Markush group. [000145] Any of the characteristics of a modality from the first to the eighth aspect is applicable to all aspects and modalities identified herein. Furthermore, any of the characteristics of a modality from the first to the eighth aspect is independently combinable, partially or totally, with other modalities described herein in any way; for example, one, two, or three or more modalities may be combined totally or partially. In addition, any of the characteristics of a modality from the first to the eighth aspect may be made optional for other aspects or modalities.

Claims

1. A nucleic acid encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: a ligand-binding domain that binds to or interacts with an IL-13 alpha 2 receptor (IL13Ra2); a polypeptide spacer between the ligand-binding domain and a transmembrane domain; the transmembrane domain; and an intracellular signaling region.

2. The nucleic acid of claim 1, wherein the ligand-binding domain comprises: a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NQ:20, or conservative variations thereof; a heavy chain complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO:21, or conservative variations thereof; and / or a heavy chain complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:22, or conservative variations thereof.

3. The nucleic acid of claim 1 or 2, wherein the ligand-binding domain comprises: a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 23, or conservative variations thereof; a light chain complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 24, or conservative variations thereof; and / or a light chain complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 25, or conservative variations thereof.

4. The nucleic acid of any of claims 1-3, wherein the ligand-binding domain comprises a variable heavy chain (VH) domain comprising a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO:

18.

5. The nucleic acid of any of claims 1-4, wherein the ligand-binding domain comprises a variable light chain (VL) domain comprising a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO:

19.

6. The nucleic acid of any of claims 1-5, wherein the ligand-binding domain comprises: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:20; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:21; and / or a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

22.

7. The nucleic acid of any of claims 1-6, wherein the ligand-binding domain comprises: zcRRQn / nznz / a / YiAi a light chain CDR1 having the amino acid sequence of SEQ ID NO:23; a light chain CDR2 having the amino acid sequence of SEQ ID NO:24; and / or a light chain CDR3 having the amino acid sequence of SEQ ID NO:

25.

8. The nucleic acid of any of claims 1-7, wherein the ligand-binding domain comprises a VH domain comprising a polypeptide having the amino acid sequence of SEQ ID NO:

18.

9. The nucleic acid of any of claims 1-8, wherein the ligand-binding domain comprises a VL domain having the amino acid sequence of SEQ ID NO:

19.

10. The nucleic acid of any of claims 1-9, wherein the ligand-binding domain is an antibody fragment, such as an antigen-binding fragment.

11. The nucleic acid of any of claims 1-10, wherein the ligand-binding domain is a single-strand variable fragment (scFv).

12. The nucleic acid of any of claims 1-11, wherein the scFv comprises a VL-VH orientation.

13. The nucleic acid of any of claims 1-12, wherein the single-strand variable fragment (scFv) comprises a sequence having at least 95% identity with the nucleotide sequence of SEQ ID NO:

61.

14. The nucleic acid of any of claims 1-13, wherein the single-strand variable fragment (scFv) comprises the nucleotide sequence of SEQ ID NO:

61.

15. The nucleic acid of any of claims 1-14, wherein the separator comprises an amino acid sequence of X1PPX2P.

16. The nucleic acid of any of claims 1-15, wherein the spacer region comprises a portion of a hinge region of a human antibody or a modified variant thereof.

17. The nucleic acid of any of claims 1-16, wherein the spacer is 15 amino acids or less but not less than 1 or 2 amino acids.

18. The nucleic acid of any of claims 1-17, wherein the spacer comprises, consists of, or essentially consists of a sequence having at least 95% identity with the amino acid sequence of SEQ ID NQ:

09.

19. The nucleic acid of any of claims 1-18, wherein the spacer comprises, consists of, or essentially consists of an amino acid sequence of SEQ ID NQ:09, or conservative substitutions thereof.

20. The nucleic acid of any of claims 1-19, wherein the spacer comprises, consists of, or essentially consists of, an lgG4 (S) hinge spacer.

21. The nucleic acid of any of claims 1-20, wherein the spacer comprises, consists of, or essentially consists of, an lgG4-CH3 (M) hinge spacer.

22. The nucleic acid of any of claims 1-21, wherein the spacer comprises, consists of, or essentially consists of, an lgG4-CH2 (L234D, N297A)-CHE (L) hinge spacer.

23. The nucleic acid of any of claims 1-22, wherein the intracellular signaling region comprises primary and costimulatory signaling domains, optionally comprising all or a portion of a CD3-zeta in combination with a costimulatory domain selected from the group consisting of signaling domains of CD27, CD28, 4-1 BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, and B7-H3 and combinations thereof.

24. The nucleic acid of claim 23, wherein the intracellular signaling region comprises a CD3-zeta signaling portion and a 4-1 BB signaling portion.

25. The nucleic acid of any of claims 1-24, wherein the transmembrane domain comprises a CD28 transmembrane domain (CD28tm).

26. The nucleic acid of any of claims 1-25, further comprising a nucleic acid encoding a marker sequence.

27. The nucleic acid of claim 26, wherein the marker sequence is a truncated receptor and optionally is an EGFRt, a HER2t, or a CD19t.

28. The nucleic acid of any of claims 1-27, further comprising a dihydrofolate reductase transgene configured for methotrexate selection.

29. The nucleic acid of claim 28, wherein the dihydrofolate reductase transgene is a dihydrofolate reductase double mutator (DHFRdm).

30. The nucleic acid of claim 29, wherein the dihydrofolate reductase double mutant comprises amino acid mutations of L22F and F31S.

31. An expression vector comprising the nucleic acid of any of claims 1-30.

32. The expression vector of claim 31, wherein the vector is a viral vector.

33. The expression vector of claim 30 or 31, wherein the vector is a lentiviral or adenoviral vector.

34. A chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid of any of claims 1-30.

35. A host cell comprising the nucleic acid of any of claims 1-30.

36. The host cell of claim 35, wherein the host cell is a CD8+ cytotoxic T lymphocyte cell selected from the group consisting of untreated CD8+ T cells, CD8+ central memory T cells, CD8+ memory effector T cells, and CD8+ bulk T cells.

37. The host cell of claim 36, wherein the CD8+ cytotoxic T lymphocyte cell is a central memory T cell, and wherein the central memory T cell is positive for CD45RO+, CD62L+, and CD8+.

38. The host cell of claim 35, wherein the host cell is a CD4+ helper T lymphocyte cell selected from the group consisting of untreated CD4+ T cells, CD4+ central memory T cells, CD4+ memory effector T cells, and CD4+ bulk T cells.

39. The host cell of claim 38, wherein the CD4+ helper lymphocyte cell is a previously untreated CD4+ T cell, and wherein the previously untreated CD4+ T cell is positive for CD45RA+, CD62L+ and CD4+ and negative for CD45RO.

40. The host cell of any of claims 35-39, wherein the host cell is a precursor T cell.

41. The host cell of any of claims 35-40, wherein the host cell is a hematopoietic stem cell.

42. A pharmaceutical composition comprising the host cell of any of claims 35-41, and a pharmaceutically acceptable excipient.

43. A method for preparing the host cell of any of claims 35-41, comprising: introducing the nucleic acid of any of claims 1-30 into a cell; and culturing the cell in the presence of anti-CD3 antibody and / or anti-CD28 antibody, and at least one homeostatic cytokine under conditions sufficient for the cells to expand; and selecting the cell with a selection reagent; wherein the selection reagent is configured to selectively enrich cells transduced with the nucleic acid or vector.

44. A method for preparing a host cell, comprising: introducing the nucleic acid of any of claims 1-30 into a cell; and culturing the cell in the presence of anti-CD3 antibody and / or anti-CD28 antibody, and at least one homeostatic cytokine under conditions sufficient for the cells to expand; and selecting the cell with a selection reagent; wherein the selection reagent is configured to selectively enrich cells transduced with the nucleic acid or vector.

45. The method of claim 43 or 44, wherein the cell is a lymphocyte.

46. ​​The method of claim 45, wherein the lymphocyte has a CD45RA-, CD45RO+ and CD62L+ phenotype.

47. The method of claim 45 or 46, wherein the lymphocyte is CD8+ or CD4+.

48. The method of any of claims 43 to 47, wherein the selection reagent is methotrexate.

49. The method of any of claims 43-48, wherein the cytokine is IL-15, II-7 and / or 11-21.

50. The method of any of claims 43-49, further comprising introducing a second nucleic acid into the host cell, wherein the second nucleic acid encodes for a marker protein.

51. The method of claim 50, wherein the marker protein is EGFRt.

52. The host cell of any of claims 35-41 for use in a medicament or for use in the treatment or inhibition of a cancer or solid tumor expressing an IL-13a2 receptor.

53. The use of claim 52, wherein the cancer comprises brain cancer.

54. The use of claim 52 or 53, wherein the cancer is a glioblastoma or glioma tumor.

55. The use of claim 54, wherein the cancer is glioblastoma multiforme (GBM).

56. The use of claim 54, wherein the cancer is a glioma.

57. A method for treating, inhibiting, or improving cancer in a subject, comprising: administering the host cell of any of claims 35-41 to the subject in need thereof.

58. The method of claim 57, wherein cancer is a malignancy positive for IL13Ra.

59. The method of claim 57 or 58, wherein the cancer is brain cancer.

60. The method of any of claims 57-59, wherein the cancer is a glioblastoma or glioma tumor.

61. The method of any of claims 57-60, wherein the cancer is a glioma.

62. The method of any of claims 57-60, wherein the cancer is glioblastoma multiforme (GBM).

63. The method of any of claims 57 to 62, further comprising administering an additional therapy selected from chemotherapy and radiation therapy.

64. The method of claim 63, wherein the chemotherapeutic drug comprises electrochemotherapy, alkylating agent, antimetabolite (for example, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (XelodaMR), Cladribine, Clofarabine, Cytarabine (Ara-CMR), Floxuridine, Fludarabine, Gemcitabine (GemzarMR), Hydroxyurea, Methotrexate, Pemetrexed (AlimtaMR), Pentostatin, and Thioguanine), antitumor antibiotic, topoisomerase inhibitor, mitotic inhibitor, corticosteroid, DNA intercalation agent, or checkpoint inhibitor (CHK1, CHK2 checkpoint kinase).

65. The method of any of claims 57-64, wherein the subject is a mammal.

66. The method of any of claims 57-65, wherein the subject is human.