Anti-il13rα2 antibody compositions and uses thereof

Abstract

The present technology relates generally to compositions that specifically recognize and bind to IL13Rα2 polypeptides. The compositions of the present technology are useful in methods for diagnosing or treating IL13Rα2-associated pathologies (e.g., cancer).

Description

ANTI-IL13Ra2 ANTIBODY COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Appl. No. 63 / 587,145, filed October 1, 2023, which is incorporated herein by reference in its entirety for any and all purposes.TECHNICAL FIELD

[0002] The present disclosure provides compositions that specifically bind to IL13Ra2, including antibodies such as human, humanized, or chimeric antibodies, antibody fragments, and fusion proteins thereof. The compositions of the present technology bind to and neutralize the activity of IL13Ra2 polypeptides and are useful for diagnosing or treating IL13Ra2-associated pathologies (e.g., cancer).BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] High expression of IL13Ra2 was firstly observed in glioblastoma (GBM), and has emerged as a promising tumor-associated antigen and biomarker in multiple tumors. Ovarian cancer expresses as much as 83% of the tumors IL13Ra2, clear cell carcinomas (75%). Its expression has been observed in some cell lines of pancreatic cancer and melanoma and highly aggressive variants of breast cancer with the capacity to metastasize to lung and brain. IL13Ra2 overexpression is enriched in basal respect to luminal primary breast tumors, as well as in a subset of metastatic basal breast cancer cells. In colorectal cancer, high IL13Ra2 expression was found in about 66% of the cases. High IL13Ra2 expression is associated to poor outcomes in renal clear cell carcinomas. It is also overexpressed in cutaneous T-cell lymphoma cells. Thus, IL13Ra2 expression in cancer appears to be quite transversal, being present in tumors from different lineages (epithelial, mesenchymal, hematopoietic). IL13Ra2 expression has been associated to major invasion and more metastatic capacity of cancer cells with lower survival.

[0005] Accordingly, there is an urgent need for therapeutic agents that target and eliminate IL13Ra2 expressing cancers.SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: (a) the VH comprises a VH- CDR1 sequence comprising SEQ ID NO: 2, a VH-CDR2 sequence comprising SEQ ID NO: 3, and a VH-CDR3 sequence comprising SEQ ID NO: 4; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 6, a VL-CDR2 sequence comprising SEQ ID NO: 7, and a VL-CDR3 sequence comprising SEQ ID NO: 8; (b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 10, a VH-CDR2 sequence comprising SEQ ID NO: 11, and a VH-CDR3 sequence comprising SEQ ID NO: 12; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 14, a VL-CDR2 sequence comprising SEQ ID NO: 15, and a VL-CDR3 sequence comprising SEQ ID NO: 16; (c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 18, a VH-CDR2 sequence comprising SEQ ID NO: 19, and a VH-CDR3 sequence comprising SEQ ID NO: 20; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 22, a VL-CDR2 sequence comprising SEQ ID NO: 23, and a VL-CDR3 sequence comprising SEQ ID NO: 24; (d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 26, a VH-CDR2 sequence comprising SEQ ID NO: 27, and a VH-CDR3 sequence comprising SEQ ID NO: 28; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 30, a VL-CDR2 sequence comprising SEQ ID NO: 31, and a VL-CDR3 sequence comprising SEQ ID NO: 32; (e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 34, a VH-CDR2 sequence comprising SEQ ID NO: 35, and a VH-CDR3 sequence comprising SEQ ID NO: 36; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL-CDR3 sequence comprising SEQ ID NO: 40; or (f) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 42, a VH-CDR2 sequence comprising SEQ ID NO: 43, and a VH-CDR3 sequence comprising SEQ ID NO: 44; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 46, a VL-CDR2 sequence comprising SEQ ID NO: 47, and a VL-CDR3 sequence comprising SEQ ID NO: 48, wherein the antibody or antigen binding fragment specifically binds to an IL13Ra2 polypeptide.

[0007] In some embodiments of the antibody or antigen binding fragment described herein, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41 and / or the VL comprises the amino acid sequence of any one of SEQ ID NOs: 5,13, 21, 29, 37 or 45. In other embodiments of the antibody or antigen binding fragment disclosed herein, the VH comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41 and / or the VL comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45. The antibody or antigen binding fragment may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or multi-specific antibody. Additionally or alternatively, in some embodiments, the multi-specific antibody or antigen binding fragment binds to a B-cell, a T-cell, a NK cell, a myeloid cell, a plasma cell, a mastcell or a DOTA hapten. In certain embodiments, the multi-specific antibody or antigen binding fragment binds to CD3, CD4, CD8, CD20, CD 19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46 and KIR.

[0008] Additionally or alternatively, in some embodiments, the antibody or antigen binding fragment further comprises a Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE. In certain embodiments, the antibody comprises an IgGl constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A and L235A. In other embodiments, the antibody comprises an IgG4 constant region comprising a S228P mutation. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv.

[0009] Additionally or alternatively, in some embodiments of the antibody or antigen binding fragment disclosed herein, the IL13Ra2 polypeptide comprises amino acid residues 27-343 of SEQ ID NO: 49. In some embodiments, the antibody lacks a-l,6-fucose modifications.

[0010] In one aspect, the present disclosure provides a recombinant nucleic acid molecule encoding any and all embodiments of the antibody or antigen binding fragment described herein. The recombinant nucleic acid molecule may comprise DNA or mRNA. Also disclosed herein are vectors comprising any and all embodiments of the recombinant nucleic acid molecules of the present technology. In certain embodiments, the vector is a viral vector, a retroviral vector, or a plasmid. In another aspect, the present disclosure provides a host cell comprising the recombinant nucleic acid molecule of the present technology, or the vector of the present technology.

[0011] In another aspect, the present disclosure provides a composition comprising (a) any and all embodiments of the antibodies or antigen binding fragments described herein, the recombinant nucleic acid molecules described herein, or the vectors described herein, and (b) a pharmaceutically-acceptable carrier, wherein the antibody or antigen binding fragment is optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof.

[0012] In yet another aspect, the present disclosure provides a kit comprising any and all embodiments of the antibody or antigen binding fragment disclosed herein and instructions for use. The antibody or antigen binding fragment may be coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the antibody or antigen binding fragment of the present technology.

[0013] In one aspect, the present disclosure provides an engineered immune cell comprising: a receptor comprising any and all embodiments of the antibody or antigen binding fragment of the present technology, and / or a nucleic acid molecule encoding the receptor. The receptor may be a T cell receptor or a chimeric antigen receptor. In some embodiments, the receptor is expressed on the surface of the engineered immune cell. Additionally or alternatively, in some embodiments, the antigen binding fragment of the receptor is an scFv, a Fab, or a F(ab)2.

[0014] Additionally or alternatively, in some embodiments, the nucleic acid molecule encoding the receptor is operably linked to a promoter. The promoter may be a constitutive promoter or a conditional promoter. In certain embodiments, the conditional promoter is inducible by binding of the receptor to an IL13Ra2 antigen. Additionally or alternatively, in some embodiments, the receptor is linked to a reporter or a selection marker such as GFP or LNGFR or an anti-DOTA scFv. In a further embodiment, the receptor is linked to the reporter or selection marker via a self-cleaving linker (e.g., P2A linker).

[0015] In some embodiments of the engineered immune cells of the present technology, the chimeric antigen receptor comprises (i) an extracellular antigen binding domain; (ii) atransmembrane domain; and (iii) an intracellular domain. Additionally or alternatively, in certain embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv), such as a human scFv. In any of the preceding embodiments of the engineered immune cell disclosed herein, the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain. Additionally or alternatively, in some embodiments of the engineered immune cell disclosed herein, the intracellular domain comprises one or more costimulatory domains. Examples of costimulatory domains include, but are not limited to, a CD28 costimulatory domain, a 4- IBB costimulatory domain, an 0X40 costimulatory domain, an ICOS costimulatory domain, a DAP- 10 costimulatory domain, a PD-1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, a CD3^-chain, or any combination thereof.

[0016] In any and all embodiments of the engineered immune cell disclosed herein, the engineered immune cell is a lymphocyte, such as a tumor infiltrating lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a B cell, or a natural killer (NK) cell. Additionally or alternatively, in some embodiments, the engineered immune cell is derived from an autologous donor or an allogenic donor.

[0017] In one aspect, the present disclosure provides a method for treating an IL13Ra2- associated pathology in a subject in need thereof comprising administering an effective amount of any and all embodiments of the antibody or antigen binding fragment described herein, any and all embodiments of the recombinant nucleic acid molecule described herein, any and all embodiments of the vector described herein or any and all embodiments of the composition described herein.

[0018] In one aspect, the present disclosure provides a method for treating an IL13Ra2- associated pathology in a subject in need thereof comprising administering an effective amount of any and all embodiments of the engineered immune cells disclosed herein. Additionally or alternatively, in some embodiments, the method further comprises administering a cytokine to the subject. The cytokine may be administered prior to, during, or subsequent to administration of the engineered immune cells. Examples of cytokines include, but are not limited to interferon a, interferon P, interferon y, complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19,CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL1O, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

[0019] Examples of IL13Ra2-associated pathologies that can be treated by the antibodies of the present technology include, but are not limited to: helminthic and parasitic infections (such as schistosomiasis), chronic hepatitis, allergic / inflammatory diseases (such as lung fibrosis, asthma, inflammatory bowel disease), and cancer (e.g., glioblastoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, melanoma, clear cell carcinomas, pancreatic cancer, T-cell lymphoma cells, and metastases thereof).

[0020] In any of the foregoing embodiments of the methods described herein, the engineered immune cells, the antibody or antigen binding fragment, the recombinant nucleic acid molecule, the vector or the composition is administered pleurally, parenterally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.

[0021] Additionally or alternatively, in certain embodiments, the methods of the present technology further comprise administering an additional cancer therapy. In some embodiments, the additional cancer therapy is selected from among chemotherapy, radiation therapy, immune checkpoint blockade therapy, anti -cancer nucleic acids or proteins, anticancer viruses or microorganisms, and any combinations thereof.

[0022] In one aspect, the present disclosure provides a method for preparing immune cells for cancer therapy, comprising isolating immune cells from a donor subject, transducing the immune cells with any and all embodiments of the recombinant nucleic acid molecules described herein or any and all embodiments of the vectors of the present technology; and administering the transduced immune cells and an anti-cancer monoclonal antibody to a recipient subject. The donor subject and the recipient subject may be the same or different. Additionally or alternatively, in some embodiments, the immune cells isolated from the donor subject comprise one or more lymphocytes, such as tumor infiltrating lymphocytes, T cells, CD4+ T cells, CD8+ T cells, B cells, or natural killer (NK) cells.

[0023] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of any and all embodiments of the antibody or antigen binding fragment disclosed herein, wherein the antibody or antigen binding fragment is configured to localize to a cancer cell expressing IL13Ra2 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting radioactive levels emitted by the antibody or antigen binding fragment that are higher than a reference value. In some embodiments, the subject is diagnosed with or is suspected of having cancer. Radioactive levels emitted by the antibody or antigen binding fragment may be detected using positron emission tomography or single photon emission computed tomography.

[0024] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody or antigen binding fragment of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger-emitter, or any combination thereof. Examples of beta particle-emitting isotopes include86Y,90Y,89Sr,165Dy,186Re,188Re,177Lu, and67Cu. In some embodiments of the method, nonspecific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via N297A mutation in Fc region, which results in aglycosylation).BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1. Heavy chain variable domains (VH) and light chain variable domains (VL) amino acid sequences of the 9C02 antibody (SEQ ID NOs: 1 and 5, respectively), 3F10 antibody (SEQ ID NOs: 9 and 13, respectively), 3C05 antibody (SEQ ID NOs: 17 and 21, respectively), 11E02 (SEQ ID NOs: 25 and 29, respectively), 1 A10 antibody (SEQ ID NOs: 33 and 37, respectively), and 9H03 antibody (SEQ ID NOs: 41 and 45, respectively). The VH and VL CDR1-3 sequences (IMGT definition) for the six anti-IL13Ra2 antibodies of the present technology are indicated in bold font.

[0026] FIG. 2 shows in vivo PET imaging data results for the anti-IL13Ra2 antibodies of the present technology.

[0027] FIG. 3 shows immunoreactivity assay results with radiolabeled anti-IL13Ra2 antibodies of the present technology with both human IL13Ral and IL13Ra2 antigens.

[0028] FIG. 4 shows saturation binding assay results with89Zr-labelled anti-IL13Ra2 antibodies of the present technology.

[0029] FIG. 5 shows cell uptake results with89Zr-labelled anti-IL13Ra2 antibodies of the present technology.

[0030] FIG. 6 shows a representative flow cytometry plot using Biotin-Proteus for anti- DOTA huC825 antibody following transduction of T cells with KLG3-CAR transgenecarrying retroviral vector.

[0031] FIG. 7 shows specific lysis of tumor cells at different effector to target ratios at 24 h post co-culture as measured by total bioluminescence of the tumor cells.

[0032] FIG. 8 shows antigen-specific production of IFN-y by KLG3-based CAR and huC825-19BBz T cells.

[0033] FIG. 9 shows antigen-specific production of IL-2 by KLG3-based CAR and huC825-19BBz T cells.

[0034] FIG. 10A shows a schematic map of the SFG_KLG3_BBZ_ P2A_C825_Fcmut plasmid vector. FIG. 10B shows the nucleic acid sequences of the components of the FG_KLG3_BBZ_ P2A_C825_Fcmut plasmid vector (represented as SEQ ID NOs: 85-89).DETAILED DESCRIPTION

[0035] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0036] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (\ 999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,'Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg el al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0037] Glioblastoma (GBM) is the most aggressive and most common malignant primary brain tumor. Interleukin 13 receptor alpha 2 (IL13Ra2) is a cell surface receptor that is frequently expressed in GBM but has negligible expression in healthy tissue. Its expression is also corelated with poor prognosis. So far, no human antibody specifically targeting IL13Ra2 is clinically available. As demonstrated in the Examples herein, the anti-IL13Ra2 targeting antibodies are useful in methods for detecting and treating IL13Ra2-associated pathologies (e.g., cancer).Definitions

[0038] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0039] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0040] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0041] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor) modified lymphocytes and CAR (z.e. chimeric antigen receptor) modified lymphocytes. In another embodiment, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. In another embodiment, TILs, T- cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one embodiment, the adoptive cell therapeutic composition comprises T cells.

[0042] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refers to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming apolypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0043] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0044] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M-1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997.

[0045] More particularly, antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constantregion and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat el al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0046] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds IL13Ra2 protein will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). “Immunoglobulin-related compositions” as used herein, refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multi-specific antibodies, bispecific antibodies, etc., as well as antibody fragments. An antibody or antigen binding fragment thereof specifically binds to an antigen.

[0047] As used herein, the term “antibody-related polypeptide” means antigen-binding antibody fragments, including single-chain antibodies, that can comprise the variable region(s) alone, or in combination, with all or part of the following polypeptide elements:hinge region, CHi, CH2, and CH3 domains of an antibody molecule. Also included in the technology are any combinations of variable region(s) and hinge region, CHi, CH2, and CH3 domains. Antibody-related molecules useful in the present methods, e.g., but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHi domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward el al.. Nature 341 : 544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). As such “antibody fragments” or “antigen binding fragments” can comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments or antigen binding fragments include Fab, Fab1, F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0048] As used herein, the terms “single-chain antibodies” or “single-chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, VL and VH. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimer, trimer or other polymers. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv(scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0049] Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0050] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or syntheticcompound. In some embodiments, the target antigen may be a polypeptide (e.g., an IL13Ra2 polypeptide). An antigen may also be administered to an animal to generate an immune response in the animal.

[0051] The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment useful in the present technology include scFv, (SCFV)2, SCFVFC, Fab, Fab' and F(ab')2, but are not limited thereto.

[0052] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration.

[0053] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0054] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.

[0055] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1 x 106cells, at least about 1 x 107cells, at least about 1 x 108cells, at least about 1 x 109cells, at least about 1 x 1010cells, at least about 1 x 1011cells, at least about I x lO12cells, or more cells expressing similar or different phenotypes.

[0056] As used herein, the term “CDR-grafted antibody” means an antibody in which at least one CDR of an “acceptor” antibody is replaced by a CDR “graft” from a “donor” antibody possessing a desirable antigen specificity.

[0057] As used herein, the term “chimeric antibody” means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et al., PCT / US86 / 02269; Akira et aL, European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et aL, European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et aL, Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et aL, J. Immunol 139: 3521-3526, 1987; Sun et aL, Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et aL, Cancer Res 47: 999-1005, 1987; Wood et aL, Nature 314: 446-449, 1885; and Shaw et al., J Natl. Cancer Inst. 80: 1553-1559, 1988.

[0058] As used herein, the term “consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.

[0059] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et aL, J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison.Table A. CDR Definitions'Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept, of Health andHuman Services, “Sequences of proteins of immunological interest” (1991).2Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997).3Residue numbering follows the nomenclature of MacCallum el al.. J. Mol. Biol. 262:732- 745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008).4Residue numbering follows the nomenclature of Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Pliickthun, J. Mol. Biol., 309:657-670 (2001).

[0060] 5Residue numbering follows the nomenclature of Honegger and Pliickthun, J. Mol. Biol., 309:657-670 (2001).

[0061] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0062] The term “chimeric antigen receptor (CAR)”, as used herein, refers to an artificially constructed hybrid single-chain protein or single-chain polypeptide containing an extracellular target-binding (e.g., antigen -binding) domain, linked directly or indirectly to a transmembrane domain (“TM domain”, e.g., the transmembrane domain of a costimulatory molecule), which is in turn linked directly or indirectly to an intracellular signaling domain (ISD) comprising a primary immune cell signaling domain (e.g., one involved in T cell or NK cell activation). The extracellular target-binding domain can be a single-chain variable fragment derived from an antibody (scFv). In addition to scFvs, other single chain antigen binding domains can be used in CAR, e.g., tandem scFvs, single-domain antibody fragments (VHHS or sdAbs), single domain bispecific antibodies (BsAbs), intrabodies, nanobodies, immunokines in a single chain format, and Fab, Fab’, or (Fab’)2 in single chain formats. The extracellular target-binding domain can be joined to the TM domain via a flexible hinge / spacer region. The intracellular signaling domain (ISD) comprises a primary signaling sequence, or primary immune cell signaling sequence, which can be from an antigen-dependent, TCR-associated T cell activation molecule, e.g., a portion of the intracellular domain of CD3i (TCRQ, FcRy, FcRp, CD3y, CD35, CD3s, CD5, CD79a, CD79b, or CD66d. The ISD can further comprise a costimulatory signaling sequence; e.g., a portion of the intracellular domain of an antigen-independent, costimulatory molecule such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, DaplO, or the like. Characteristics of CARs include their ability to redirect immune cell (e.g, T cell or NK cell) specificity and reactivity toward a selected target in either MHC-restricted (in cases of TCR-mimic antibodies) or non-MHC-restricted (in cases of antibodies against cell surface proteins) manners, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives immune cells (e.g, T cells or NK cells) expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0063] There are currently three generations of CARs. The “first generation” CARs are typically single-chain polypeptides composed of an scFv as the antigen-binding domain fused to a transmembrane domain fused to the cytoplasmic / intracellular domain, which comprises a primary immune cell signaling sequence such as the intracellular domain from the CD3(^ chain, which is the primary transmitter of signals from endogenous TCRs. The “first generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. The “second generation” CARs add intracellular domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, 0X40) to the primary immune cell signaling sequence of the CAR to provide additional signals to the T cell. Thus, the “second generation” CARs comprise fragments that provide costimulation (e.g., CD28 or 4-IBB) and activation (e.g., CD3Q. Preclinical studies have indicated that the “second generation” CARs can improve the antitumor activity of T cells.

[0064] The “third generation” CARs comprise those that provide multiple costimulation (e.g., CD28 and 4-IBB) and activation (e.g., CD3Q. Examples of CAR T therapies are described, see, e.g, US Patent No. 10,221,245 describing CAR CTL019 which has an anti- CD33 extracellular target-binding domain, a transmembrane domain from CD8, a costimulatory domain from 4-IBB, and a primary signaling domain from CD3(^, as well as US Patent No. 9,855,298 which describes a CAR having an anti-CD33 extracellular targetbinding domain, a costimulatory domain from CD28, and a primary signaling domain from CD3

[0065] As used herein, the term, “co-stimulatory signaling domain,” or “co-stimulatory domain”, refers to the portion of the CAR comprising the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4- IBB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and co-stimulatory signaling domains can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0066] As used herein, the term “chimeric co-stimulatory receptor” or “CCR” refers to a chimeric receptor that binds to an antigen and provides co-stimulatory signals, but does not provide a T-cell activation signal.

[0067] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0068] As used herein, the term “effector cell” means an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid orlymphoid origin, e.g, lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and carry out specific immune functions. An effector cell can induce antibody-dependent cell-mediated cytotoxicity (ADCC), e.g, a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcaR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens.

[0069] As used herein, the term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, an “epitope” of the IL13Ra2 protein is a region of the protein to which the anti- IL13Ra2 antibodies of the present technology specifically bind. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for anti-IL13Ra2 antibodies which bind to an epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if an anti-IL13Ra2 antibody binds the same site or epitope as an anti-IL13Ra2 antibody of the present technology. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of IL13Ra2 protein can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

[0070] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of themRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell.

[0071] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0072] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or sample obtained from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0073] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using softwareprograms known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0074] As used herein, a "host cell" is a cell that is used in to receive, maintain, reproduce and amplify a vector. A host cell also can be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.

[0075] As used herein, “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, nomore than 3. The humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0076] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35B (Hl), 50-65 (H2) and 95-102 (H3) in the VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (Hl), 52A-55 (H2) and 96-101 (H3) in the VH(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0077] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0078] As used herein, the term “intact antibody” or “intact immunoglobulin” means an antibody that has at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FRi, CDRi, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0079] As used herein the term “immune cell” refers to any cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified.

[0080] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0081] As used herein, the term “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors orother chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0082] As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and / or interaction.

[0083] The term “linker” refers to synthetic sequences (e.g., amino acid sequences) that connect or link two sequences, e.g., that link two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of amino acid sequences.

[0084] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white lymphocyte populations including tissue specific and specialized varieties. It encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-l cells, B-2 cells, and anergic AN1 / T3 cell populations. As used herein “tumor-infiltrating lymphocytes” or TILs refer to white blood cells that have left the bloodstream and migrated into a tumor.

[0085] As used herein, the term “T-cell” includes naive T cells, CD4+ T cells, CD8+ T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, chimeric B cells, and antigen-specific T cells.

[0086] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonalantibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (See, e.g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0087] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0088] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungalcompounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0089] As used herein, the term “polyclonal antibody” means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0090] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0091] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0092] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, hasbeen modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0093] As used herein, “regulatory region” of a nucleic acid molecule means a cisacting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0094] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0095] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0096] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0097] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0098] As used herein, “specifically binds” refers to a polypeptide or fragment thereof (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 104M, I O5M, 106M, 107M, 10"8M, 109M, 10I OM, 10 " M, or 10l 2M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide (e.g., an IL13Ra2 polypeptide), or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0099] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0100] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0101] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv)inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0102] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0103] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.

[0104] As used herein, a vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0105] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0106] Amino acid sequence modification(s) of the anti-IL13Ra2 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an anti- IL13Ra2 antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to obtain the antibody of interest, as long as the obtained antibody possesses the desired properties. The modification also includes the change of the pattern of glycosylation of the protein. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. “Conservative substitutions” are shown in the Table below.

[0107] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can beperformed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays may be selected for further development.Immunoglobulin-related Compositions of the Present Technology

[0108] The present technology describes methods and compositions for the generation and use of anti-IL13Ra2 immunoglobulin-related compositions (e.g., anti-IL13Ra2 antibodies or antigen binding fragments thereof). Anti-IL13Ra2 immunoglobulin-related compositions within the scope of the present technology include, e.g., but are not limited to, monoclonal, chimeric, humanized, bispecific, multispecific antibodies and diabodies that specifically bind the target polypeptide, a homolog, derivative or a fragment thereof. The present disclosure also provides antigen binding fragments of any of the anti-IL13Ra2 antibodies disclosed herein, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab’, scFv, and Fv. The VH and VL CDR sequences of the IL13Ra2 antibodies of the present disclosure based on the IMGT annotation system are summarized below:

[0109] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: (a) the VH comprises a VH- CDR1 sequence comprising SEQ ID NO: 2, a VH-CDR2 sequence comprising SEQ ID NO: 3, and a VH-CDR3 sequence comprising SEQ ID NO: 4; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 6, a VL-CDR2 sequence comprising SEQ ID NO: 7, and a VL-CDR3 sequence comprising SEQ ID NO: 8; (b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 10, a VH-CDR2 sequence comprising SEQ ID NO: 11, and a VH-CDR3 sequence comprising SEQ ID NO: 12; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 14, a VL-CDR2 sequence comprising SEQ ID NO: 15, and a VL-CDR3 sequence comprising SEQ ID NO: 16; (c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 18, a VH-CDR2 sequence comprising SEQ ID NO: 19, and a VH-CDR3 sequence comprising SEQ ID NO: 20; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 22, a VL-CDR2 sequence comprising SEQ ID NO: 23, and a VL-CDR3 sequence comprising SEQ ID NO: 24; (d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 26, a VH-CDR2 sequence comprising SEQ ID NO: 27, and a VH-CDR3 sequence comprising SEQ ID NO: 28; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 30, a VL-CDR2 sequence comprising SEQ ID NO: 31, and a VL-CDR3 sequence comprising SEQ ID NO: 32; (e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 34, a VH-CDR2 sequence comprising SEQ ID NO: 35, and a VH-CDR3 sequence comprising SEQ ID NO: 36; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL-CDR3 sequence comprising SEQ ID NO: 40; or (f) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 42, a VH-CDR2 sequence comprising SEQ ID NO: 43, and a VH-CDR3 sequence comprising SEQ ID NO: 44; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 46, a VL-CDR2 sequence comprising SEQ ID NO: 47, and a VL-CDR3 sequence comprising SEQ ID NO: 48, wherein the antibody or antigen binding fragment specifically binds to an IL13Ra2 polypeptide.

[0110] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: (a) the VH comprises the aminoacid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41; and / or (b) the VL comprises the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.

[0111] In any of the above embodiments of the immunoglobulin-related compositions, the heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL) sequences form an antigen binding site that binds to the extracellular domain of an IL13Ra2 polypeptide. In some embodiments, the VH and VL sequences are components of the same polypeptide chain. In other embodiments, the VH and VL sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0112] In some embodiments, the immunoglobulin-related compositions of the present technology bind to the extracellular domain of an IL13Ra2 polypeptide. In certain embodiments, the epitope is a conformational epitope or non-conformational epitope. In some embodiments, the IL13Ra2 polypeptide has the amino acid sequence of SEQ ID NO: 49.

[0113] UniProt Ref: Q14627 Homo sapiens IL-13 receptor subunit alpha-2 (SEQ ID NO: 49):

[0114] MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYL QWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTL LPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIG VLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGS SENKPIRS S YFTFQLQNIVKPLPP VYLTFTRES SCEIKLKWSIPLGPIPARCFDYEIEIRE DDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEG EDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEFFCDT

[0115] Additionally or alternatively, in some embodiments, the antibody or antigen binding fragment binds to the extracellular domain of an IL13Ra2 polypeptide. In certain embodiments, the IL13Ra2 polypeptide comprises amino acid residues 27-343 of SEQ ID NO: 49.

[0116] In any of the above embodiments, the antibody further comprises a Fc domain of any isotype, e.g., but are not limited to, IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAi and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include:

[0117] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 50)

[0118] APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKA QASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP SHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVE EGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAP VKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQP GSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0119] Human IgGl constant region, Uniprot: P01857 (SEQ ID NO: 51)

[0120] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 52)

[0122] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 53)

[0124] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0125] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 54)

[0126] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDIS STRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVI AELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQ VQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQ DTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESH PNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLL PPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPG RYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0127] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 55)

[0128] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQ S SGL YSLS S VVT VP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCP SCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0129] Human IgAl constant region, Uniprot: P01876 (SEQ ID NO: 56)

[0130] ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTA RNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTP PTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSS GKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLT WASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAG KPTHVNVSVVMAEVDGTCY

[0131] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 57)

[0132] ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTA RNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPP PCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPER DLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0133] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 58)

[0134] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C

[0135] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NOS: 50-57. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NO: 58.

[0136] In some embodiments, the immunoglobulin-related compositions of the present technology bind specifically to at least one IL13Ra2 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology bind at least one IL13Ra2 polypeptide with a dissociation constant (KD) of about 103M, 104M, I 05M, 106M, 107M, 108M, 109M, 1010M, 101 1M, or 1012M. In certain embodiments, the immunoglobulin-related compositions are monoclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multi-specific antibodies. In some embodiments, the antibodies comprise a human antibody framework region.

[0137] In certain embodiments, the immunoglobulin-related composition includes one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a “conservative substitution” as defined herein.

[0138] Additionally or alternatively, in some embodiments, the multi-specific antibodies of the present disclosure bind to CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransf erase, p 15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gplOO), GnT-V intron V sequence (N- acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), P-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD 138, EGFR, EGP-1, EGP-2, VEGF, P1GF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, Ll-CAM, Lewis Y (Ley) antigen, E-cadherin, V- cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD 137, CD99, GloboH, CD24, STEAP1, B7H3, Poly sialic Acid, 0X40, 0X40- ligand, peptide MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1- 6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel 17, LMP2, or WT1), or a small molecule DOTA hapten.

[0139] In certain embodiments, the immunoglobulin-related compositions contain an IgGl constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A and L235A. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions contain an IgG4 constant region comprising a S228P mutation.

[0140] In some aspects, the anti-IL13Ra2 immunoglobulin-related compositions described herein contain structural modifications to facilitate rapid binding and cell uptake and / or slow release. In some aspects, the anti-IL13Ra2 immunoglobulin-related composition of the present technology (e.g., an antibody) may contain a deletion in the CH2 constant heavy chain region to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a Fab fragment is used to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a F(ab)'2 fragment is used to facilitate rapid binding and cell uptake and / or slow release.

[0141] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0142] In another aspect, the present technology provides a host cell or vector expressing any nucleic acid sequence encoding any and all embodiments of the immunoglobulin-related compositions described herein.

[0143] The immunoglobulin-related compositions of the present technology (e.g., an anti- IL13Ra2 antibody) can be monospecific, bispecific, trispecific or of greater multispecificity. Multi-specific antibodies can be specific for different epitopes of one or more IL13Ra2 polypeptides or can be specific for both the IL13Ra2 polypeptide(s) as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g, WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0144] The immunoglobulin-related compositions of the present technology can further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

[0145] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen binding fragment may be optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof. For a chemical bond or physical bond, a functional group on the immunoglobulin-related composition typically associates with a functional group on theagent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

[0146] The functional groups on the agent and immunoglobulin-related composition can associate directly. For example, a functional group (e.g., a sulfhydryl group) on an agent can associate with a functional group (e.g., sulfhydryl group) on an immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate through a cross-linking agent (z.e., linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents associated with a conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with an agent depends on the number of functional groups present on the agent.

[0147] In yet another embodiment, the conjugate comprises one immunoglobulin- related composition associated to one agent. In one embodiment, a conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to an immunoglobulin-related composition by any method known to those in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

[0148] The agent may also be chemically bonded to the immunoglobulin-related composition by means of cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can, for example, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. web-site can provide assistance. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V., Amsterdam, The Netherlands.

[0149] Alternatively, the functional group on the agent and immunoglobulin-related composition can be the same. Homobifunctional cross-linkers are typically used to cross-link identical functional groups. Examples of homobifunctional cross-linkers include EGS (z.e., ethylene glycol bisfsuccinimidylsuccinate]), DSS (z.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP i.e., 3,3'- dithiobisfsulfosuccinimidylpropionate])), DPDPB (z.e., 1 ,4-di-[3'-(2'-pyridyldithio)- propionamido]butane), and BMH (z.e., bis-maleimidohexane). Such homobifunctional cross-linkers are also available from Pierce Biotechnology, Inc.

[0150] In other instances, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The web-site of Pierce Biotechnology, Inc. described above can also provide assistance to one skilled in the art in choosing suitable cross-linkers which can be cleaved by, for example, enzymes in the cell. Thus the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (z.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), Sulfo-LC- SPDP (z.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (z.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (z.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (z.e., N- succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (z.e., 3-[(2- aminoethyl)dithio]propionic acid HC1).

[0151] In another embodiment, a conjugate comprises at least one agent physically bonded with at least one immunoglobulin-related composition. Any method known to those in the art can be employed to physically bond the agents with the immunoglobulin-related compositions. For example, the immunoglobulin-related compositions and agents can be mixed together by any method known to those in the art. The order of mixing is not important. For instance, agents can be physically mixed with immunoglobulin-related compositions by any method known to those in the art. For example, the immunoglobulin- related compositions and agents can be placed in a container and agitated, by for example, shaking the container, to mix the immunoglobulin-related compositions and agents.

[0152] The immunoglobulin-related compositions can be modified by any method known to those in the art. For instance, the immunoglobulin-related composition may be modified by means of cross-linking agents or functional groups, as described above.A. Methods of Preparing Anti-ILl 3Ra2 Antibodies of the Present Technology

[0153] General Overview. Initially, a target polypeptide is chosen to which an antibody of the present technology can be raised. For example, an antibody may be raised against the full-length IL13Ra2 protein, or to a portion of the extracellular domain of the IL13Ra2 protein. Techniques for generating antibodies directed to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xeno or human mice, hybridomas, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from IL13Ra2 protein containing the extracellular domain which is capable of eliciting an immune response. In certain embodiments, the IL13Ra2 polypeptide comprises amino acid residues 27-343 of SEQ ID NO: 49.

[0154] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, which are directed to IL13Ra2 protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0155] Anti-ILl 3Ra2 antibodies that can be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains and / or antibody fragments. Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments have been described. See U.S. Pat. No. 5,648,237.

[0156] Generally, an antibody is obtained from an originating species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain or both, of an originating species antibody having specificity for a target polypeptide antigen is obtained. An originating species is any species which was useful to generate the antibody of the present technology or library of antibodies, e.g., rat, mouse, rabbit, chicken, monkey, human, and the like.

[0157] Phage or phagemid display technologies are useful techniques to derive the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology, can be carried out in E. coli.

[0158] Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present technology These include, but are not limited to, nucleic acid sequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. It is appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates sequence homology variations of up to 25% as calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.) so long as such a variant forms an operative antibody which recognizes IL13Ra2 proteins. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc. Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences or to create variations in coding regions and / or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like.

[0159] Preparation of Polyclonal Antisera and Immunogens. Methods of generating antibodies or antibody fragments of the present technology typically include immunizing asubject (generally a non-human subject such as a mouse or rabbit) with a purified IL13Ra2 protein or fragment thereof or with a cell expressing the IL13Ra2 protein or fragment thereof. An appropriate immunogenic preparation can contain, e.g., a recombinantly- expressed IL13Ra2 protein or a chemically-synthesized IL13Ra2 peptide. The extracellular domain of the IL13Ra2 protein, or a portion or fragment thereof, can be used as an immunogen to generate an anti-IL13Ra2 antibody that binds to the IL13Ra2 protein, or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation. In certain embodiments, the extracellular domain comprises amino acid residues 27-343 of SEQ ID NO: 49. The full-length IL13Ra2 protein or fragments thereof, are useful as fragments as immunogens. In some embodiments, an IL13Ra2 fragment comprises the extracellular domain of the IL13Ra2 protein, or a portion or fragment thereof (e.g., an IL13Ra2 polypeptide comprising amino acid residues 27-343 of SEQ ID NO: 49), such that an antibody raised against the peptide forms a specific immune complex with the IL13Ra2 protein. In some embodiments, the antigenic IL13Ra2 peptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid residues. Longer antigenic peptides are sometimes desirable over shorter antigenic peptides, depending on use and according to methods well known to those skilled in the art. Multimers of a given epitope are sometimes more effective than a monomer.

[0160] If needed, the immunogenicity of the IL13Ra2 protein (or fragment thereof) can be increased by fusion or conjugation to a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. One can also combine the IL13Ra2 protein with a conventional adjuvant such as Freund’s complete or incomplete adjuvant to increase the subject’s immune reaction to the polypeptide. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette- Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0161] In describing the present technology, immune responses may be described as either “primary” or “secondary” immune responses. A primary immune response, which isalso described as a “protective” immune response, refers to an immune response produced in an individual as a result of some initial exposure (e.g, the initial “immunization”) to a particular antigen, e.g., IL13Ra2 protein. In some embodiments, the immunization can occur as a result of vaccinating the individual with a vaccine containing the antigen. For example, the vaccine can be an IL13Ra2 vaccine comprising one or more IL13Ra2 protein- derived antigens. A primary immune response can become weakened or attenuated over time and can even disappear or at least become so attenuated that it cannot be detected. Accordingly, the present technology also relates to a “secondary” immune response, which is also described here as a “memory immune response.” The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already been produced.

[0162] Thus, a secondary immune response can be elicited, e.g., to enhance an existing immune response that has become weakened or attenuated, or to recreate a previous immune response that has either disappeared or can no longer be detected. The secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells that were generated at the first presentation of the antigen. Delayed type hypersensitivity (DTH) reactions are a type of cellular secondary or memory immune response that are mediated by CD4+T cells. A first exposure to an antigen primes the immune system and additional exposure(s) results in a DTH.

[0163] Following appropriate immunization, the anti-IL13Ra2 antibody can be prepared from the subject’s serum. If desired, the antibody molecules directed against the IL13Ra2 protein can be isolated from the mammal (e.g, from the blood) and further purified by well- known techniques, such as polypeptide A chromatography to obtain the IgG fraction.

[0164] Monoclonal Antibody. In one embodiment of the present technology, the antibody is an anti-IL13Ra2 monoclonal antibody. For example, in some embodiments, the anti-IL13Ra2 monoclonal antibody may be a human or a mouse anti-IL13Ra2 monoclonal antibody. For preparation of monoclonal antibodies directed towards the IL13Ra2 protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique See, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridomatechnique See, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77- 96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (See, e.g., Cote, et al. , 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids that encode regions of antibodies can be isolated. PCR utilizing primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then DNAs encoding antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences also can be fused to DNAs encoding other proteins - e.g., a bacteriophage coat, or a bacterial cell surface protein - for expression and display of the fusion polypeptides on phage or bacteria. Amplified sequences can then be expressed and further selected or isolated based, e.g., on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the IL13Ra2 protein. Alternatively, hybridomas expressing anti-IL13Ra2 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject’s spleen using routine methods. See, e.g., Milstein et al., (Galfire and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods will produce monoclonal antibodies of varying specificity (i.e., for different epitopes) and affinity. A selected monoclonal antibody with the desired properties, e.g., IL13Ra2 binding, can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of IL13Ra2 protein. Also, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of the IL13Ra2 protein. Other manipulations include substituting or deleting particular amino acyl residues that contribute to instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve affinity of the antibody of the IL13Ra2 protein.

[0165] Hybridoma Technique. In some embodiments, the antibody of the present technology is an anti-IL13Ra2 monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow el al.. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al. , Monoclonal Antibodies And T- Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0166] Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, anti-IL13Ra2 antibodies, can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for an IL13Ra2 polypeptide, e.g., a polypeptide or derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 99I ^wdon et aL, Advances in Immunology M'. 191-280, 1994; PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al. , WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC);WO 91 / 17271 (Affymax); and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0167] Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and / or screening.

[0168] Expression of Recombinant Anti-ILl 3Ra2 Antibodies. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding an anti- IL13Ra2 antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti-IL13Ra2 antibody chains, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti-IL13Ra2 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-IL13Ra2 antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence)by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos. 6,291,160 and 6,680,192.

[0169] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression of a construct in that subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-IL13Ra2 antibody, and the collection and purification of the anti-IL13Ra2 antibody, e.g., cross-reacting anti- IL13Ra2 antibodies. See generally, U.S. 2002 / 0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, c.g, ampicillin-resistance or hygromycin-resi stance, to permit detection of those cells transformed with the desired DNA sequences. Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576,195.

[0170] The recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein with IL13Ra2 binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operably-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcript! on / translati on system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-IL13Ra2 antibody), include, e.g., but are not limited to, promoters of 3 -phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-IL13Ra2 antibody of the present technology is operably-linked to an araB promoter and expressible in a host cell. See U.S. Pat. 5,028,530. The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., anti-IL13Ra2 antibody, etc.).

[0171] Another aspect of the present technology pertains to anti-IL13Ra2 antibodyexpressing host cells, which contain a nucleic acid encoding one or more anti-IL13Ra2 antibodies. The recombinant expression vectors of the present technology can be designed for expression of an anti-IL13Ra2 antibody in prokaryotic or eukaryotic cells. For example, an anti-IL13Ra2 antibody can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides having a predetermined property, e.g., anti-IL13Ra2 antibody, via expression of stochastically generated polynucleotide sequences has been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

[0172] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S- transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0173] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et aL, (1988) Gene 69: 301-315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion has been described by Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g, an anti-IL13Ra2 antibody, in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (See, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the present technology can be carried out by standard DNA synthesis techniques.

[0174] In another embodiment, the anti-IL13Ra2 antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 3Q 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, an anti-IL13Ra2 antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, e.g., anti-IL13Ra2 antibody, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, etal., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0175] In yet another embodiment, a nucleic acid encoding an anti-IL13Ra2 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187- 195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are useful for expression of the anti-IL13Ra2 antibody of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0176] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissuespecific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1 : 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0177] Another aspect of the present methods pertains to host cells into which a recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0178] A host cell can be any prokaryotic or eukaryotic cell. For example, an anti- IL13Ra2 antibody can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al. , J Immunol . 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0179] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0180] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti-IL13Ra2 antibody or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0181] A host cell that includes an anti-IL13Ra2 antibody of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) recombinant anti-IL13Ra2 antibody. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the anti-IL13Ra2 antibody has been introduced) in a suitable medium such that the anti-IL13Ra2 antibody is produced. In another embodiment, the method further comprises the step of isolating the anti-IL13Ra2 antibody from the medium or the host cell. Once expressed, collections of the anti-IL13Ra2 antibody, e.g., the anti-IL13Ra2 antibodies or the anti-IL13Ra2 antibody- related polypeptides are purified from culture media and host cells. The anti-IL13Ra2 antibody can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like. In one embodiment,the anti-IL13Ra2 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually, anti-IL13Ra2 antibody chains are expressed with signal sequences and are thus released to the culture media. However, if the anti-IL13Ra2 antibody chains are not naturally secreted by host cells, the anti-IL13Ra2 antibody chains can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel electrophoresis and the like (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).

[0182] Polynucleotides encoding anti-IL13Ra2 antibodies, e.g., the anti-IL13Ra2 antibody coding sequences, can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and / or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or P-lactoglobulin. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0183] Single-Chain Antibodies. In one embodiment, the anti-IL13Ra2 antibody of the present technology is a single-chain anti-IL13Ra2 antibody. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to an IL13Ra2 protein (See, e.g., U.S. Pat. No. 4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al. , Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0184] Chimeric and Humanized Antibodies. In one embodiment, the anti-IL13Ra2 antibody of the present technology is a chimeric anti-IL13Ra2 antibody. In one embodiment, the anti-IL13Ra2 antibody of the present technology is a humanized anti- IL13Ra2 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptorantibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.

[0185] Recombinant anti-IL13Ra2 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the anti-IL13Ra2 antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-IL13Ra2 antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT / US86 / 02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, etal., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999- 1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321 : 552-525; Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141 : 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. No. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7 : 805-814, 1994; Roguska et al., PNAS 91 : 969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, a cDNA encoding a murine anti- IL13Ra2 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al.,WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) roc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Pat. No. 6,180,370; U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0186] In one embodiment, the present technology provides the construction of humanized anti-IL13Ra2 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized anti-IL13Ra2 antibodies, heavy and light chain immunoglobulins.

[0187] CDR Antibodies . In some embodiments, the anti-IL13Ra2 antibody of the present technology is an anti-IL13Ra2 CDR antibody. Generally the donor and acceptor antibodies used to generate the anti-IL13Ra2 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single VH or VL of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VH and VL. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one needs to replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to IL13Ra2 protein. Methods for generating CDR- grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and Winter U.S. 5,225,539; and EP 0682040. Methods useful to prepare VH and VL polypeptides are taught by Winter et al., U.S. Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694.

[0188] After selecting suitable framework region candidates from the same family and / or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (z.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and / or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site-directed mutagenesis.

[0189] Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0190] This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-IL13Ra2 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR See, e.g., US 5,585,089, especially columns 12-16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified anti-IL13Ra2 CDR-grafted antibody significantly compared to the same antibody with a fully human FR.

[0191] Multi-specific Antibodies (e.g., Bispecific Antibodies (BsAbs)). A bispecific antibody is an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. BsAbs can be made, for example, by combining heavy chains and / or light chains that recognize different epitopes of the same or different antigen. In some embodiments, by molecular function, a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH / VL pair), and binds a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds.

[0192] Multi-specific antibodies, such as bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) have at least one arm that specifically binds to, for example, IL13Ra2 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B-cell, a T-cell, a NK cell, a myeloid cell, a plasma cell, or a mast-cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD 19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46 and KIR. In certain embodiments, the BsAbs are capable of binding to tumor cells that express IL13Ra2 antigen on the cell surface. In some embodiments, the BsAbs have been engineered to facilitate killing of tumor cells by directing (or recruiting) cytotoxic T cells to a tumor site. Other exemplary BsAbs include those with a first antigen binding site specific for IL13Ra2 and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA-chelates described herein, Biotin, fluorescein, or those disclosed in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937- 5946).

[0193] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that either utilize the full immunoglobulin framework (e.g., IgG), single chain variable fragment (scFv), or combinations thereof. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, a scFv with a single binding site for one antigen and a Fabfragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs comprise two single chain variable fragments (scFvs) in tandem have been designed such that an scFv that binds a tumor antigen (e.g., IL13Ra2) is linked with an scFv that engages T cells (e.g., by binding CD3). In this way, T cells are recruited to a tumor site such that they can mediate cytotoxic killing of the tumor cells. See e.g., Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321 :974- 977 (2008)). In some embodiments, BsAbs of the present technology comprise two single chain variable fragments (scFvs) in tandem have been designed such that an scFv that binds a tumor antigen (e.g., IL13Ra2) is linked with an scFv that engages a small molecule DOTA hapten.

[0194] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10): 1221-1225 (1997). Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et al., Nature Biotech.15: 159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0195] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some certain embodiments, a BsAb according to the present technology comprises an immunoglobulin, which immunoglobulin comprises a heavy chain and a light chain, and an scFv. In some certain embodiments, the scFv is linked to the C-terminal end of the heavy chain of any IL13Ra2 immunoglobulin disclosed herein. In some certain embodiments, scFvs are linked to the C-terminal end of the light chain of any IL13Ra2 immunoglobulin disclosed herein. In various embodiments, scFvs are linked to heavy or light chains via a linker sequence. Appropriate linker sequencesnecessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the VL and Vkappa domains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain. The resulting scFv-CHl construct is excised and ligated into a vector containing a DNA sequence encoding the VH region of an anti-IL13Ra2 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell for the expression of the bispecific fusion protein.

[0196] In some embodiments, a linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., first and / or second antigen binding sites). In some embodiments, a linker is employed in a BsAb described herein based on specific properties imparted to the BsAb such as, for example, an increase in stability. In some embodiments, a BsAb of the present technology comprises a G4S linker. In some certain embodiments, a BsAb of the present technology comprises a (G4S)nlinker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more.

[0197] Fc Modifications. In some embodiments, the anti-IL13Ra2 antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an FcyR), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann etal., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make a direct contact with an Fc receptor such as an FcyR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop.

[0198] In some embodiments, an anti-IL13Ra2 antibody of the present technology has an altered affinity for activating and / or inhibitory receptors, having a variant Fc region with one or more amino acid modifications, wherein said one or more amino acid modification isa N297 substitution with alanine, a L234 substitution with alanine, a L235 substitution with alanine, or a K322 substitution with alanine.

[0199] Glycosylation Modifications . In some embodiments, anti-IL13Ra2 antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnTl -deficient CHO cells.

[0200] In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g., IL13Ra2), without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, "glycosylation sites" include any specific amino acid sequence in an antibody to which an oligosaccharide (z.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.

[0201] Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, an Fc-gly coform (hIL13Ra2-IgGln) that lacks certain oligosaccharides including fucose and terminal N- acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function.

[0202] In some embodiments, the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site.Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some certain embodiments, the presenttechnology includes deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine.

[0203] Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, BiotechnoL Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et a / . , 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publications WO 00 / 61739A1 ; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0204] Fusion Proteins. In one embodiment, the anti-IL13Ra2 antibody of the present technology is a fusion protein. The anti-IL13Ra2 antibodies of the present technology, when fused to a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences. Moreover, fusion proteins of the present technology can also be engineered to improve characteristics of the anti-IL13Ra2 antibodies. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the anti-IL13Ra2 antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to an anti-IL13Ra2 antibody to facilitate purification. Such regions can be removed prior to final preparation of the anti-IL13Ra2 antibody. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routine techniques in the art. The anti-IL13Ra2 antibody of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In select embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et aL, Cell 37: 767, 1984.

[0205] Thus, any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein show an increased half-life in vivo.

[0206] Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis el al., J. Biochem. 270: 3958- 3964, 1995.

[0207] Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232 262. Alternatively, deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired. For example, the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett el al.. J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0208] Labeled Anti-ILl 3Ra2 antibodies. In one embodiment, the anti-IL13Ra2 antibody of the present technology is coupled with a label moiety, i.e., detectable group. The particular label or detectable group conjugated to the anti-IL13Ra2 antibody is not a critical aspect of the technology, so long as it does not significantly interfere with thespecific binding of the anti-IL13Ra2 antibody of the present technology to the IL13Ra2 protein. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well -developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g, Dynabeads™), fluorescent dyes (e.g, fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H,14C,35S,125I,121I,131I,112In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging),18F,nC,150,89Zr (for Positron emission tomography), "mTC,n iIn (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6thEd., Molecular Probes, Inc., Eugene OR.).

[0209] The label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0210] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti -ligands can be used. Where a ligand has a natural anti -ligand, e.g., biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-IL13Ra2 antibody.

[0211] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moi eties, include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties, include, but are not limited to, e.g., luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signalproducing systems which can be used, see U.S. Pat. No. 4,391,904.

[0212] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0213] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies, e.g., the anti-IL13Ra2 antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.B. Identifying and Characterizing the Anti-ILl 3Ra2 Antibodies of the Present Technology

[0214] Methods for identifying and / or screening the anti-ILl 3Ra2 antibodies of the present technology. Methods useful to identify and screen antibodies against IL13Ra2 polypeptides for those that possess the desired specificity to IL13Ra2 protein (e.g., those that bind to the extracellular domain of IL13Ra2 protein) include any immunologically- mediated techniques known within the art. Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art.For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A et cd., Immunity, 2: 373-80, 1995); (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989);(4) mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian etal., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0215] Similarly, products of an immune response in either a model organism (e.g., mouse) or a human subject can also be detected by various methods that are well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be measured using3H-thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells together with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and(5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of the PBMCs expressing these markers.

[0216] In one embodiment, anti-IL13Ra2 antibodies of the present technology are selected using display of IL13Ra2 peptides on the surface of replicable genetic packages. See, e.g., U.S. Pat. Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for producing / selecting a filamentous bacteriophage particle containing a phagemid genome encoding for a binding molecule with a desired specificity has been described. See, e.g., EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0217] In some embodiments, anti-IL13Ra2 antibodies of the present technology are selected using display of IL13Ra2 peptides on the surface of a yeast host cell. Methods useful for the isolation of scFv polypeptides by yeast surface display have been described by Kieke etal., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0218] In some embodiments, anti-IL13Ra2 antibodies of the present technology are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91 : 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0219] In certain embodiments, anti-IL13Ra2 antibodies of the present technology are selected using tRNA display of IL13Ra2 peptides. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. BioL, 9: 741- 46, 2002.

[0220] In one embodiment, anti-IL13Ra2 antibodies of the present technology are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0221] In some embodiments, anti-IL13Ra2 antibodies of the present technology are expressed in the periplasm of gram negative bacteria and mixed with labeled IL13Ra2 protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for IL13Ra2 protein, the concentration of the labeled IL13Ra2 protein bound to the anti- IL13Ra2 antibodies is increased and allows the cells to be isolated from the rest of the library as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Pat. Publication No. 2004 / 0058403.

[0222] After selection of the desired anti-IL13Ra2 antibodies, it is contemplated that said antibodies can be produced in large volume by any technique known to those skilled in the art, e.g., prokaryotic or eukaryotic cell expression and the like. The anti-IL13Ra2 antibodies which are, e.g., but not limited to, anti-IL13Ra2 hybrid antibodies or fragments can be produced by using conventional techniques to construct an expression vector that encodes an antibody heavy chain in which the CDRs and, if necessary, a minimal portion ofthe variable region framework, that are required to retain original species antibody binding specificity (as engineered according to the techniques described herein) are derived from the originating species antibody and the remainder of the antibody is derived from a target species immunoglobulin which can be manipulated as described herein, thereby producing a vector for the expression of a hybrid antibody heavy chain.

[0223] Measurement of IL 13Ra2 Binding. In some embodiments, an IL13Ra2 binding assay refers to an assay format wherein IL13Ra2 protein and an anti-IL13Ra2 antibody are mixed under conditions suitable for binding between the IL13Ra2 protein and the anti- IL13Ra2 antibody and assessing the amount of binding between the IL13Ra2 protein and the anti-IL13Ra2 antibody. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of the IL13Ra2 protein, the amount of the binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, radioimmunoassays, scintillation proximity assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like.Biophysical assays for the direct measurement of IL13Ra2 protein binding to anti-IL13Ra2 antibody are, e.g., nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips) and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the specific binding of a candidate anti-IL13Ra2 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-IL13Ra2 antibody, the candidate anti- IL13Ra2 antibody is useful as an anti-IL13Ra2 antibody of the present technology.C. Diagnostic Uses o f the Anti-ILl 3Ra2 Antibodies o f the Present Technology

[0224] General. The anti-IL13Ra2 antibodies of the present technology are useful in methods known in the art relating to the localization and / or quantitation of IL13Ra2 protein (e.g., for use in measuring levels of the IL13Ra2 protein within appropriate physiological samples, for use in imaging the polypeptide, and the like). Antibodies of the present technology are useful to isolate an IL13Ra2 protein by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-IL13Ra2 antibody of the present technology can facilitate the purification of natural immunoreactive IL13Ra2 proteins from biological samples, e.g., mammalian sera or cells as well as recombinantly-producedimmunoreactive IL13Ra2 proteins expressed in a host system. Moreover, anti-IL13Ra2 antibodies can be used to detect an immunoreactive IL13Ra2 protein (e.g., in plasma, a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the immunoreactive polypeptide. The anti-IL13Ra2 antibodies of the present technology can be used diagnostically to monitor immunoreactive IL13Ra2 protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As noted above, the detection can be facilitated by coupling (z.e., physically linking) the anti-IL13Ra2 antibodies of the present technology to a detectable substance.

[0225] Detection ofIL13Ra2 protein. An exemplary method for detecting the presence or absence of an immunoreactive IL13Ra2 protein in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti- IL13Ra2 antibody of the present technology capable of detecting an immunoreactive IL13Ra2 protein such that the presence of an immunoreactive IL13Ra2 protein is detected in the biological sample. Detection may be accomplished by means of a detectable label attached to the antibody.

[0226] The term “labeled” with regard to the anti-IL13Ra2 antibody is intended to encompass direct labeling of the antibody by coupling (z.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.

[0227] In some embodiments, the anti-IL13Ra2 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, anti-IL13Ra2 antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other label.

[0228] Examples of suitable chromogenic labels include diaminobenzidine and 4- hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, A-5-steroid isomerase, yeast-alcoholdehydrogenase, a-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.

[0229] Examples of suitable radioisotopic labels include3H,i nIn,125I,1311,32P,35S,14C,51Cr,57TO,58CO,59Fe,75Se,152Eu,90Y,67Cu,217Ci,211At,212Pb,47Sc,109Pd, etc.mIn is an exemplary isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the125I or131I-labeled IL 13Ra2 -binding antibodies by the liver. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example,n iIn coupled to monoclonal antibodies with 1-(P- isothiocyanatobenzyl)-DPTA exhibits little uptake in non-tumorous tissues, particularly the liver, and enhances specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861- 870 (1987)). Examples of suitable non-radioactive isotopic labels include157Gd,55Mn,162Dy,52Tr, and56Fe.

[0230] Examples of suitable fluorescent labels include an152Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, a Green Fluorescent Protein (GFP) label, an o-phthaldehyde label, and a fluorescamine label. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0231] Examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label. Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron.

[0232] The detection method of the present technology can be used to detect an immunoreactive IL13Ra2 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of an immunoreactive IL13Ra2 protein include enzyme linked immunosorbent assays (ELISAs), FACS, immunohistochemical staining, Western blots, immunoprecipitations, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detection of an immunoreactive IL13Ra2 protein include introducing into asubject a labeled anti-IL13Ra2 antibody. For example, the anti-IL13Ra2 antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains IL13Ra2 protein molecules from the test subject.

[0233] Immunoassay and Imaging. An anti-IL13Ra2 antibody of the present technology can be used to assay immunoreactive IL13Ra2 protein levels in a biological sample (e.g., human plasma) using antibody -based techniques. For example, protein expression in tissues can be studied with classical immunohistological or immunohistochemical methods. Jalkanen, M. etal., J Cell. Biol. 101 : 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine (125I,121I,131I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium ("mTc), and fluorescent labels, such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

[0234] In addition to assaying immunoreactive IL13Ra2 protein levels in a biological sample, anti-IL13Ra2 antibodies of the present technology may be used for in vivo imaging of IL13Ra2. Antibodies useful for this method include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the anti-IL13Ra2 antibodies by labeling of nutrients for the relevant scFv clone.

[0235] An anti-IL13Ra2 antibody which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (e.g.,131I,112In, "mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc. The labeled anti-IL13Ra2 antibody will thenaccumulate at the location of cells which contain the specific target polypeptide. For example, labeled anti-IL13Ra2 antibodies of the present technology will accumulate within the subject in cells and tissues in which the IL13Ra2 protein has localized.

[0236] Thus, the present technology provides a diagnostic method of a medical condition, which involves: (a) assaying the expression of immunoreactive IL13Ra2 protein by measuring binding of an anti- IL13Ra2 antibody of the present technology in cells or body fluid of an individual; (b) comparing the amount of immunoreactive IL13Ra2 protein present in the sample with a standard reference, wherein an increase or decrease in immunoreactive IL13Ra2 protein levels compared to the standard is indicative of a medical condition.

[0237] Affinity Purification. The anti-IL13Ra2 antibodies of the present technology may be used to purify immunoreactive IL13Ra2 protein from a sample. In some embodiments, the antibodies are immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzyrn. 34 Academic Press, N.Y. (1974)).

[0238] The simplest method to bind the antigen to the antibody-support matrix is to collect the beads in a column and pass the antigen solution down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using low flow rates. The immobilized antibody captures the antigen as it flows past. Alternatively, an antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction has been completed, the slurry is passed into a column for collection of the beads. The beads are washed using a suitable washing buffer and then the pure or substantially pure antigen is eluted.

[0239] An antibody or polypeptide of interest can be conjugated to a solid support, such as a bead. In addition, a first solid support such as a bead can also be conjugated, if desired, to a second solid support, which can be a second bead or other support, by any suitablemeans, including those disclosed herein for conjugation of a polypeptide to a support. Accordingly, any of the conjugation methods and means disclosed herein with reference to conjugation of a polypeptide to a solid support can also be applied for conjugation of a first support to a second support, where the first and second solid support can be the same or different.

[0240] Appropriate linkers, which can be cross-linking agents, for use for conjugating a polypeptide to a solid support include a variety of agents that can react with a functional group present on a surface of the support, or with the polypeptide, or both. Reagents useful as cross-linking agents include homo-bi-functional and, in particular, hetero-bi-functional reagents. Useful bi-functional cross-linking agents include, but are not limited to, A-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected to provide a selectively cleavable bond between a polypeptide and the solid support. For example, a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means for cleaving a polypeptide from a solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and US. Pat. No. 5,643,722). Other cross-linking reagents are well-known in the art. (See, e.g., Wong (1991), supra, and Hermanson (1996), supra).

[0241] An antibody or polypeptide can be immobilized on a solid support, such as a bead, through a covalent amide bond formed between a carboxyl group functionalized bead and the amino terminus of the polypeptide or, conversely, through a covalent amide bond formed between an amino group functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bi-functional trityl linker can be attached to the support, e.g., to the 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino group or a carboxyl group on the resin via an amino resin. Using a bi-functional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoroacetic acid to ensure that the polypeptide is cleaved and can be removed. In such a case, the polypeptide can be deposited as a beadless patch at the bottom of a well of a solid support or on the flat surface of a solid support. After addition of a matrix solution, the polypeptide can be desorbed into a MS.

[0242] Hydrophobic trityl linkers can also be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution, e.g., a matrix solution containing 3 -HP A, to cleave an amino linked trityl group from the polypeptide. Acid lability can also be changed.For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to the appropriate / ^-substituted, or more acid-labile tritylamine derivatives, of the polypeptide, z.e., trityl ether and tritylamine bonds can be made to the polypeptide. Accordingly, a polypeptide can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, under typical MS conditions, where a matrix, such as 3- HPA acts as an acid.

[0243] Orthogonally cleavable linkers can also be useful for binding a first solid support, e.g., a bead to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from a second solid support, without cleaving the polypeptide from the support; the polypeptide then can be cleaved from the bead at a later time. For example, a disulfide linker, which can be cleaved using a reducing agent, such as DTT, can be employed to bind a bead to a second solid support, and an acid cleavable bi-functional trityl group could be used to immobilize a polypeptide to the support. As desired, the linkage of the polypeptide to the solid support can be cleaved first, e.g., leaving the linkage between the first and second support intact. Trityl linkers can provide a covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

[0244] For example, a bead can be bound to a second support through a linking group which can be selected to have a length and a chemical nature such that high density binding of the beads to the solid support, or high density binding of the polypeptides to the beads, is promoted. Such a linking group can have, e.g., “tree-like” structure, thereby providing a multiplicity of functional groups per attachment site on a solid support. Examples of such linking group; include polylysine, polyglutamic acid, penta-erythrole and / / v.s-hydroxy- aminomethane.

[0245] Noncovalent Binding Association. An antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support, through a noncovalent interaction. For example, a magnetic bead made of a ferromagnetic material, which is capable of being magnetized, can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the solid support can be provided with an ionic or hydrophobic moiety,which can allow the interaction of an ionic or hydrophobic moiety, respectively, with a polypeptide, e.g., a polypeptide containing an attached trityl group or with a second solid support having hydrophobic character.

[0246] A solid support can also be provided with a member of a specific binding pair and, therefore, can be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, a bead coated with avidin or with streptavidin can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or derivative of biotin, such as iminobiotin.

[0247] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, e.g., can be incorporated into either a polypeptide or a solid support and, conversely, avidin or other biotin binding moiety would be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzyme, and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen to which it interacts specifically, and other such pairs knows to those skilled in the art.

[0248] The anti-IL13Ra2 antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides methods using the antibodies in the diagnosis of IL13Ra2 activity in a subject. Anti-IL13Ra2 antibodies of the present technology may be selected such that they have any level of epitope binding specificity and very high binding affinity to an IL13Ra2 protein. In general, the higher the binding affinity of an antibody the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing target polypeptide. Accordingly, anti-IL13Ra2 antibodies of the present technology useful in diagnostic assays usually have binding affinities of about 108M’1, 109M’1, 1010M’1, 1011M'1or 1012M'1. Further, it is desirable that anti-IL13Ra2 antibodies used as diagnostic reagents have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 h, at least five (5) h, or at least one (1) hour.

[0249] Anti-IL13Ra2 antibodies can be used to detect an immunoreactive IL13Ra2 protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow &-n-Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject is at an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of IL13Ra2 protein in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsied body tissue.

[0250] Immunometric or sandwich assays are one format for the diagnostic methods of the present technology. See U.S. Pat. No. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, e.g., an anti-IL13Ra2 antibody or a population of anti- IL13Ra2 antibodies immobilized to a solid phase, and another anti-IL13Ra2 antibody or a population of anti-IL13Ra2 antibodies in solution. Typically, the solution anti-IL13Ra2 antibody or population of anti-IL13Ra2 antibodies is labeled. If an antibody population is used, the population can contain antibodies binding to different epitope specificities within the target polypeptide. Accordingly, the same population can be used for both solid phase and solution antibody. If anti-IL13Ra2 monoclonal antibodies are used, first and second IL13Ra2 monoclonal antibodies having different binding specificities are used for the solid and solution phase. Solid phase (also referred to as “capture”) and solution (also referred to as “detection”) antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the IL13Ra2 protein with the anti-IL13Ra2 antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the anti- IL13Ra2 antibody being used as a reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, typically by detecting a label linked to the solid phase through binding of labeled solution antibody. Usually for a given pair of antibodies orpopulations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of the immunoreactive IL13Ra2 protein in samples being tested are then read by interpolation from the calibration curve (z.e., standard curve). Analyte can be measured either from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of the IL13Ra2 protein in a sample.

[0251] Suitable supports for use in the above methods include, e.g., nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway N.J.), and the like. Immobilization can be by absorption or by covalent attachment. Optionally, anti-IL13Ra2 antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.

[0252] In some embodiments, the present disclosure provides an anti-IL13Ra2 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule which is administered conjugated to an antibody moiety, z.e., antibody or antibody fragment, or subfragment, and is useful in diagnosing or detecting a disease by locating the cells containing the antigen.

[0253] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incorporated in its entirety by reference. In some embodiments, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to whichare attached a multiplicity of chelating groups for binding the ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose. Chelates may be coupled to the antibodies of the present technology using standard chemistries. The chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radio-imaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the IL13Ra2 antibodies of the present technology.

[0254] Macrocyclic chelates such as NOTA (l,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of use with a variety of metals and radiometals, such as radionuclides of gallium, yttrium and copper, respectively. Such metal -chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Examples of other DOTA chelates include (i) DOTA-Phe- Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D- Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)- NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D- Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D- Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe- D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D- Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)- D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; and (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0255] Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as223Ra for RAIT are also contemplated.

[0256] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of an antibody (or antigen binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a cancer cell expressing IL13Ra2 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting radioactive levels emitted by the antibody that are higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value may be calculated by measuring the radioactive levels present in non-tumor (normal) tissues, and computing the average radioactive levels present in non-tumor (normal) tissues ± standard deviation. In some embodiments, the ratio of radioactive levels between a tumor and normal tissue is about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1.

[0257] In some embodiments, the subject is diagnosed with or is suspected of having an IL13Ra2-associated cancer. Radioactive levels emitted by the antibody may be detected using positron emission tomography or single photon emission computed tomography.

[0258] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger-emitter, or any combination thereof. Examples of beta particle-emitting isotopes include86Y,90Y,89Sr,165Dy,186Re,188Re,177Lu, and67Cu. Examples of alpha particle-emitting isotopes include213Bi,211At,225Ac,152Dy,212Bi,223Ra,219Rn,215Po,211Bi,221Fr,217At, and255Fm. Examples of Auger-emitters includei nIn,67Ga,51Cr,58Co, "mTc,103mRh,195mPt,119Sb,161HO,189mOs,192Ir,2O1T1, and203Pb. In some embodiments of the method, nonspecific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via N297A mutation in Fc region, which results in aglycosylation). The therapeutic effectiveness of such an immunoconjugate may be determined by computing the area underthe curve (AUC) tumor: AUC normal tissue ratio. In some embodiments, the immunoconjugate has a AUC tumor: AUC normal tissue ratio of about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1.Vectors for Adoptive Cell Therapy

[0259] Many expression vectors are available and known to those of skill in the art and can be used for expression of anti-IL13Ra2 polypeptides provided herein. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0260] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g. a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0261] Expression of the antibodies or antigen-binding fragments thereof can be controlled by any promoter / enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bemoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797(1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Set. USA I '. 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the P- lactamase promoter (Jay et al., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 50:21-25(1983)); see also "UsefulProteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella etal., Nature 505:209-213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 1 15-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 55:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409(1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 55:647-658 (1984); Adams et al., Nature 515:533- 538 (1985); Alexander et al., Mol. Cell Biol. 7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes andDevel. 1 :268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5: 1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes andDevel. 7: 161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 15:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 254: 1372- 1378 (1986)).

[0262] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the antibody, or portion thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the antibody chain and signals required for efficient polyadenylation of the transcript, ribosome bindingsites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0263] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.

[0264] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.

[0265] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 poly A signal.

[0266] Genetic modification of engineered immune cells (e.g., T cells, NK cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding a receptor (e.g., TCR, CAR)comprising the anti-IL13Ra2 antibodies or antigen binding fragments thereof can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0267] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g, using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g, using a natural or chemically modified RNA) can be used.

[0268] For initial genetic modification of the cells to provide a receptor (e.g., TCR, CAR) comprising the anti-IL13Ra2 immunoglobulin-related composition expressing cells, a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two costimulatory ligands, retroviral gene transfer (transduction) likewise proves effective.Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller, et al. Mol. Cell. Biol. 5:431-437 (1985)); PA317 (Miller, et al. Mol. Cell. Biol. 6:2895-2902 (1986)); and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non -amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0269] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. Blood 80: 1418-1422(1992), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. Exp. Hemat. 22:223-230 (1994); and Hughes, et al. J. Clin. Invest. 89: 1817 (1992).

[0270] Transducing viral vectors can be used to express a co-stimulatory ligand and / or secretes a cytokine (e.g., 4-1 BBL and / or IL- 12) in an engineered immune cell. Preferably, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., Journal of Virology 71 :6641-6649, 1997;Naldini et al., Science 272:263 267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614, (1988); Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61(1990); Sharp, The Lancet 337 : 1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S- 83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No. 5,399,346).

[0271] In certain non-limiting embodiments, the vector expressing a presently disclosed anti-IL13Ra2 immunoglobulin-related composition is a retroviral vector, e.g., an oncoretroviral vector.

[0272] Non-viral approaches can also be employed for the expression of a protein in cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat'L Acad. Sci. U.S.A. 84:7413, (1987); Ono et al., Neuroscience Letters 17:259 (1990); Brigham et al., Am. J. Med. Sci. 298:278, (1989); Staubinger et al., Methods in Enzymology 101 :512 (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621 (1988); Wu et al., Journal of Biological Chemistry 264: 16985 (1989)), or by microinjection under surgical conditions (Wolff et al., Science 247: 1465 (1990)). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases ortargeted nucleases (e.g., Zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression may be obtained by RNA electroporation.

[0273] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g., the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0274] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.Polypeptides and Analogs and Polynucleotides

[0275] Also included in the presently disclosed subject matter are anti-IL13Ra2 antibodies or antigen binding fragments thereof, CD3(^, CD8, CD28, etc. polypeptides or fragments thereof, and polynucleotides encoding thereof that are modified in ways that enhance their anti -tumor activity when expressed in an engineered immune cell. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence. Such alterations may comprise certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further comprises analogs of any naturally-occurring polypeptide of the presently disclosed subject matter. Analogs can differ from a naturally- occurring polypeptide of the presently disclosed subject matter by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the presently disclosed subject matter can generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%), about 98%, about 99% or more identity or homology with all or part of a naturally-occurring amino, acid sequence of the presently disclosed subject matter. The length of sequence comparison is atleast about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence. Modifications comprise in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the presently disclosed subject matter by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L- amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta (P) or gamma (y) amino acids.

[0276] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains of the presently disclosed subject matter. A fragment can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, a fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, a fragment is at least about 60 to about 80, about 100, about 200, about 300 or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of ordinary skill in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0277] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein of the present technology. Such analogs are administered according to methods of the presently disclosed subject matter. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the antineoplasticactivity of the original polypeptide when expressed in an engineered immune cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. The protein analogs can be relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0278] In accordance with the presently disclosed subject matter, the polynucleotides encoding anti-IL13Ra2 antibodies or antigen binding fragments thereof, CD3 , CD8, CD28 etc. can be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that are involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis- elements in transcription and translation. Any suitable codon optimization methods or technologies that are known to ones skilled in the art can be used to modify the polynucleotides of the presently disclosed subject matter, including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron.Engineered Immune Cells of the Present Technology

[0279] The presently disclosed subject matter provides engineered immune cells expressing a receptor or other cell-surface ligand comprising an extracellular antigenbinding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain comprises any and all embodiments of the anti- IL13Ra2 antigen binding fragments described herein. In certain embodiments immune cells can be transduced with a presently disclosed anti-IL13Ra2 polypeptide construct such that the cells express the anti-IL13Ra2 polypeptide. In some embodiments, the receptor, e.g. a T cell receptor (TCR), is non-native receptor (e.g., not endogenous to the immune cells). In some embodiments, the non-native receptor is a truncated receptor, a genetically modified receptor, an antibody, or other ligand capable of interacting with a target cell. In some embodiments, the receptor is a chimeric antigen receptor (CAR), for example, a T cell CAR that binds to an IL13Ra2 antigen.

[0280] Presently disclosed engineered immune cells can further include at least one recombinant or exogenous co-stimulatory ligand. For example, presently disclosed engineered immune cells can be further transduced with at least one co-stimulatory ligand,such that the engineered immune cells co-expresses or is induced to co-express the IL 13Ra2 -targeted CAR and the at least one co-stimulatory ligand. The interaction between the IL 13Ra2 -targeted CAR and at least one co-stimulatory ligand provides a non-antigen - specific signal important for full activation of an immune cell (e.g., T cell). Co-stimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta O-TP), CD257 / B cellactivating factor (B AFF) / Bly s / THANK / Tall- 1, glucocorticoid-induced TNF Receptor ligand (GITRL), and T F-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins — they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, PD-L1 / (B7-H1) that ligands for PD-1. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the engineered immune cell comprises one recombinant co-stimulatory ligand that is 4-1BBL. In certain embodiments, the engineered immune cell comprises two recombinant co-stimulatory ligands that are 4-1BBL and CD80. CARs comprising at least one co-stimulatory ligand are described in U.S. Patent No. 8,389,282, which is incorporated by reference in its entirety.

[0281] Furthermore, a presently disclosed engineered immune cells can further comprise at least one exogenous cytokine. For example, a presently disclosed engineered immune cell can be further transduced with at least one cytokine, such that the engineered immune cells secrete the at least one cytokine as well as expresses the IL 13Ra2 -targetedCAR. In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL- 3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21. In certain embodiments, the cytokine is IL-12.

[0282] The presently disclosed subject matter also provides methods of using such cells for the treatment of a tumor. The engineered immune cells of the presently disclosed subject matter can be cells of the lymphoid lineage or myeloid lineage. Non-limiting examples of immune cells of the myeloid lineage include neutrophils, monocytes, macrophages, eosinophils, erythrocytes, megakaryocytes, and platelets. The lymphoid lineage, comprising B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell -like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. In certain embodiments, the T cells express Foxp3 to achieve and maintain a T regulatory phenotype.

[0283] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0284] The engineered immune cells of the presently disclosed subject matter may be white blood cells (e.g., T cells, B cells, neutrophils, NK cells, etc.).

[0285] The engineered immune cells of the presently disclosed subject matter express an extracellular antigen-binding domain (e.g., a human scFv, a Fab that is optionally crosslinked, or a F(ab)2) that specifically binds to an IL13Ra2 antigen, for the treatment of an IL13Ra2-associated pathology such as cancer, e.g., for treatment of a solid or liquidtumor. Such engineered immune cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment of IL13Ra2-associated pathologies. In some embodiments, the immune cell is a lymphocyte, such as a T cell, a B cell or a natural killer (NK) cell. In certain embodiments, the engineered immune cell is a T cell. The T cell can be a CD4+T cell or a CD8+T cell. In certain embodiments, the T cell is a CD4+T cell. In certain embodiments, the T cell is a CD8+T cell.

[0286] The engineered immune cells can be generated from peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., et al., Nat Rev Cancer 3 :35-45 (2003) (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R.A. et al., Science 314: 126-129 (2006) (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising the a and P heterodimer), in Panelli et al. J Immunol 164:495-504 (2000); Panelli et al. J Immunol 164:4382-4392 (2000) (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and in Dupont et al. Cancer Res 65:5417-5427 (2005); Papanicolaou et al. Blood 102:2498-2505 (2003) (disclosing selectively in v / Yro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). The engineered immune cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0287] In certain embodiments, presently disclosed engineered immune cells (e.g., T cells) expresses from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a presently disclosed IL 13Ra2 -targeted CAR.

[0288] For example, the higher the CAR expression level in an engineered immune cell, the greater cytotoxicity and cytokine production the engineered immune cell exhibits. Additionally, or alternatively, the cytotoxicity and cytokine production of a presently disclosed engineered immune cell (e.g., T cell) are proportional to the expression level of IL13Ra2 antigen in a target tissue or a target cell. For example, the higher the expression level of human IL13Ra2 antigen in the target, the greater cytotoxicity and cytokine production the engineered immune cell exhibits.

[0289] The unpurified source of immune cells may be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-immune cell initially. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and / or stages of differentiation for both positive and negative selections.

[0290] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. Preferably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.

[0291] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0292] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels. The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Preferably, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.

[0293] In some embodiments, the engineered immune cells comprise one or more additional modifications. For example, in some embodiments, the engineered immune cells comprise and express (is transduced to express) an antigen recognizing receptor that binds to a second antigen that is different than an IL13Ra2 antigen. The inclusion of an antigen recognizing receptor in addition to a presently disclosed CAR on the engineered immune cell can increase the avidity of the CAR or the engineered immune cell comprising the same on a targeted cell, especially, the CAR is one that has a low binding affinity to an IL13Ra2 antigen, e.g., a Kd of about 2 x 10'8M or more, about 5 x 10'8M or more, about 8 x 10'8Mor more, about 9 x 10'8M or more, about 1 x 10'7M or more, about 2 x 10'7M or more, or about 5 x 10'7M or more.

[0294] In certain embodiments, the antigen recognizing receptor is a chimeric costimulatory receptor (CCR). CCR is described in Krause, et al., J. Exp. Med. 188(4):619- 626(1998), and US20020018783, the contents of which are incorporated by reference in their entireties. CCRs mimic co-stimulatory signals, but unlike, CARs, do not provide a T- cell activation signal, e.g., CCRs lack a CD3(^ polypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen- presenting cell. A combinatorial antigen recognition, i.e., use of a CCR in combination with a CAR, can augment T-cell reactivity against the dual-antigen expressing T cells, thereby improving selective tumor targeting. Kloss et al., describe a strategy that integrates combinatorial antigen recognition, split signaling, and, critically, balanced strength of T-cell activation and costimulation to generate T cells that eliminate target cells that express a combination of antigens while sparing cells that express each antigen individually (Kloss et al., Nature Biotechnology 3 l(l):71-75 (2013)). With this approach, T-cell activation requires CAR-mediated recognition of one antigen, whereas costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach diminishes the efficiency of T-cell activation to a level where it is ineffective without rescue provided by simultaneous CCR recognition of the second antigen. In certain embodiments, the CCR comprises an extracellular antigenbinding domain that binds to an antigen different than an IL13Ra2 antigen, a transmembrane domain, and a co-stimulatory signaling region that comprises at least one co-stimulatory molecule, including, but not limited to, CD28, 4- IBB, 0X40, ICOS, PD-1, CTLA-4, LAG-3, 2B4, and BTLA. In certain embodiments, the co-stimulatory signaling region of the CCR comprises one co-stimulatory signaling molecule. In certain embodiments, the one co-stimulatory signaling molecule is CD28. In certain embodiments, the one co-stimulatory signaling molecule is 4-1BB. In certain embodiments, the co- stimulatory signaling region of the CCR comprises two co-stimulatory signaling molecules. In certain embodiments, the two co-stimulatory signaling molecules are CD28 and 4-1BB. A second antigen is selected so that expression of both the IL13Ra2 antigen and the second antigen is restricted to the targeted cells (e.g., cancerous tissue or cancerous cells). Similar to a CAR, the extracellular antigen-binding domain can be a scFv, a Fab, a F(ab)2; or afusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the CCR comprises a scFv that binds to CD 138, transmembrane domain comprising a CD28 polypeptide, and a co-stimulatory signaling region comprising two co-stimulatory signaling molecules that are CD28 and 4-1BB.

[0295] In certain embodiments, the antigen recognizing receptor is a truncated CAR. A “truncated CAR” is different from a CAR by lacking an intracellular signaling domain. For example, a truncated CAR comprises an extracellular antigen-binding domain and a transmembrane domain, and lacks an intracellular signaling domain. In accordance with the presently disclosed subject matter, the truncated CAR has a high binding affinity to the second antigen expressed on the targeted cells. The truncated CAR functions as an adhesion molecule that enhances the avidity of a presently disclosed CAR, especially, one that has a low binding affinity to an IL13Ra2 antigen, thereby improving the efficacy of the presently disclosed CAR or engineered immune cell (e.g., T cell) comprising thereof. In certain embodiments, the truncated CAR comprises an extracellular antigen-binding domain that binds to CD 138, a transmembrane domain comprising a CD8 polypeptide. A presently disclosed T cell comprises or is transduced to express a presently disclosed CAR targeting an IL13Ra2 antigen and a truncated CAR targeting CD 138. In certain embodiments, the targeted cells are solid tumor cells. In some embodiments, the engineered immune cells are further modified to suppress expression of one or more genes. In some embodiments, the engineered immune cells are further modified via genome editing. Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U.S. Patent Nos. 7,888,121 ;7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861 ; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231 ; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960, the disclosures of which are incorporated by reference in their entireties. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or theinsertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR / Cas system with an engineered crRNA / tracr RNA ('single guide RNA') to guide specific cleavage. In some embodiments, the engineered immune cells are modified to disrupt or reduce expression of an endogenous T-cell receptor gene (see, e.g. WO 2014153470, which is incorporated by reference in its entirety). In some embodiments, the engineered immune cells are modified to result in disruption or inhibition of PD1, PDL-1 or CTLA-4 (see, e.g. U.S. Patent Publication 20140120622), or other immunosuppressive factors known in the art (Wu et al. (2015) Oncoimmunology 4(7): el016700, Mahoney et al. (2015) Nature Reviews Drug Discovery 14, 561-584).Chimeric Antigen Receptors

[0296] The present disclosure provides a chimeric antigen receptor (CAR) that specifically binds to IL13Ra2. The present disclosure relates to engineered immune cells, CAR-encoding nucleic acids, and methods of making such CARs, engineered immune cells, and nucleic acids comprising such CARs. The present disclosure provides methods of treatment for the use of these CARs and engineered immune cells for the treatment of conditions associated with malignant cells expressing IL13R2a2 (e.g., cancer).

[0297] In some embodiments, the engineered immune cells provided herein express at least one chimeric antigen receptor (CAR). CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. For example, CARs can be used to graft the specificity of a monoclonal antibody onto an immune cell, such as a T cell. In some embodiments, transfer of the coding sequence of the CAR is facilitated by a nucleic acid vector, such as a retroviral vector.

[0298] There are currently three generations of CARs. In some embodiments, the engineered immune cells provided herein express a “first generation” CAR. “First generation” CARs are typically composed of an extracellular antigen binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain fused to cytoplasmic / intracellular domain of the T cell receptor (TCR) chain. “First generation” CARs typically have the intracellular domain from the CD3(^ chain, which is the primary transmitter of signals from endogenous TCRs. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through theirCD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0299] In some embodiments, the engineered immune cells provided herein express a “second generation” CAR. “Second generation” CARs add intracellular domains from various co-stimulatory molecules (e.g., CD28, 4- IBB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. “Second generation” CARs comprise those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (e.g., CD3Q. Preclinical studies have indicated that “Second Generation” CARs can improve the antitumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL).

[0300] In some embodiments, the engineered immune cells provided herein express a “third generation” CAR. “Third generation” CARs comprise those that provide multiple costimulation (e.g., CD28 and 4-1BB) and activation (e.g., CD3Q.

[0301] In accordance with the presently disclosed subject matter, the CARs of the engineered immune cells provided herein comprise an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain.Extracellular Antigen-Binding Domain of a CAR

[0302] The presently disclosed subject matter provides engineered immune cells expressing a CAR comprising any and all embodiments of the anti-IL13Ra2 antibodies or antigen binding fragments described herein.

[0303] In one aspect, the present disclosure provides an engineered immune cell comprising: a CAR including an anti-IL13Ra2 antibody or antigen binding fragment comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 2, a VH-CDR2 sequence comprising SEQ ID NO: 3, and a VH- CDR3 sequence comprising SEQ ID NO: 4; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 6, a VL-CDR2 sequence comprising SEQ ID NO: 7, and a VL- CDR3 sequence comprising SEQ ID NO: 8; (b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 10, a VH-CDR2 sequence comprising SEQ ID NO: 11, and a VH- CDR3 sequence comprising SEQ ID NO: 12; and the VL comprises a VL-CDR1 sequencecomprising SEQ ID NO: 14, a VL-CDR2 sequence comprising SEQ ID NO: 15, and a VL- CDR3 sequence comprising SEQ ID NO: 16; (c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 18, a VH-CDR2 sequence comprising SEQ ID NO: 19, and a VH- CDR3 sequence comprising SEQ ID NO: 20; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 22, a VL-CDR2 sequence comprising SEQ ID NO: 23, and a VL- CDR3 sequence comprising SEQ ID NO: 24; (d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 26, a VH-CDR2 sequence comprising SEQ ID NO: 27, and a VH- CDR3 sequence comprising SEQ ID NO: 28; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 30, a VL-CDR2 sequence comprising SEQ ID NO: 31, and a VL- CDR3 sequence comprising SEQ ID NO: 32; (e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 34, a VH-CDR2 sequence comprising SEQ ID NO: 35, and a VH- CDR3 sequence comprising SEQ ID NO: 36; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL- CDR3 sequence comprising SEQ ID NO: 40; or (f) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 42, a VH-CDR2 sequence comprising SEQ ID NO: 43, and a VH- CDR3 sequence comprising SEQ ID NO: 44; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 46, a VL-CDR2 sequence comprising SEQ ID NO: 47, and a VL- CDR3 sequence comprising SEQ ID NO: 48, and / or a nucleic acid encoding the anti- IL13Ra2 antibody or antigen binding fragment.

[0304] In certain embodiments, the extracellular antigen-binding domain of a CAR specifically binds an IL13Ra2 antigen. In certain embodiments, the extracellular antigenbinding domain is derived from a monoclonal antibody (mAb) that binds to an IL13Ra2 antigen. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a Fab, which is optionally crosslinked. In some embodiments, the extracellular binding domain comprises a F(ab)2. In some embodiments, any of the foregoing molecules are comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a human scFv that binds specifically to an IL13Ra2 antigen. In certain embodiments, the scFv is identified by screening scFv phage library with an IL13Ra2-Fc fusion protein.

[0305] In certain embodiments, the extracellular antigen-binding domain of a presently disclosed CAR has a high binding specificity and high binding affinity to an IL13Ra2antigen. For example, in some embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, in a human scFv or an analog thereof) binds to an IL13Ra2 antigen with a dissociation constant (Ka) of about 1 x 10'5M or less. In certain embodiments, the Ka is about 5 x 10'6M or less, about 1 x 10'6M or less, about 5 x 10'7M or less, about 1 x 10'7M or less, about 5 x 10'8M or less, about 1 x 10'8M or less, about 5 x 10'9or less, about 4 x 10'9or less, about 3 x 10'9or less, about 2 x 10'9or less, or about 1 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 3 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 3 x 10'9to about 2 x 10'7.

[0306] Binding of the extracellular antigen-binding domain (embodiment, for example, in a human scFv or an analog thereof) of a presently disclosed IL 13 Ra2 -targeted CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or a scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the IL 13Ra2 -targeted CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the human scFv of a presently disclosed IL 13 Ra2 -targeted CAR is labeled with GFP.

[0307] In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to an IL13Ra2 antigen that is expressed by a tumor cell. In some embodiments, the extracellular antigen -binding domain of the expressed CAR binds to an IL13Ra2 antigen that is expressed on the surface of a tumor cell. In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to an IL13Ra2 antigen that is expressed on the surface of a tumor cell in combination with an MHC protein. Insome embodiments, the MHC protein is a MHC class I protein. In some embodiments, the MHC Class I protein is an HLA-A, HLA-B, or HLA-C molecule. In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to an IL13Ra2 antigen that is expressed on the surface of a tumor cell not in combination with an MHC protein.

[0308] In certain embodiments, the extracellular antigen-binding domain (e.g., human scFv) comprises a heavy chain variable region and a light chain variable region, optionally linked with a linker sequence, for example a (G4S)nlinker peptide (n= 1-15) (SEQ ID NO: 59), between the heavy chain variable region and the light chain variable region. In certain embodiments, the extracellular antigen-binding domain is a human scFv-Fc fusion protein or full length human IgG with VH and VL regions.

[0309] In certain non-limiting embodiments, an extracellular antigen-binding domain of the presently disclosed CAR can comprise a linker connecting the heavy chain variable region and light chain variable region of the extracellular antigen-binding domain. As used herein, the term “linker” refers to a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together (e.g., to couple VH and VL domains). In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO: 59.

[0310] In addition, the extracellular antigen-binding domain can comprise a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum. Signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway.

[0311] In certain embodiments, the signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 60 as provided below:

[0312] MALPVTALLLPLALLLHAARP (SEQ ID NO: 60).

[0313] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 60 is set forth in SEQ ID NO: 61, which is provided below:

[0314] atggccctgccagtaacggctctgctgctgccacttgctctgctcctccatgcagccaggcct (SEQ ID NO: 61).

[0315] In certain embodiments, the signal peptide comprises a CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 62 as provided below:

[0316] MALPVTALLLPLALLLHA (SEQ ID NO: 62).

[0317] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 62 is set forth in SEQ ID NO: 63, which is provided below:

[0318] ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTG CATGCA (SEQ ID NO: 63).Transmembrane Domain of a CAR

[0319] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (e.g., a transmembrane peptide not based on a protein associated with the immune response), or a combination thereof.

[0320] In certain embodiments, the transmembrane domain of a presently disclosed CAR comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: PI0747 or NP006130 (SEQ ID NO: 64), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 64 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively, or additionally, in nonlimiting various embodiments, the CD28 polypeptide has an amino acid sequence of aminoacids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 64. In certain embodiments, the CAR of the presently disclosed comprises a transmembrane domain comprising a CD28 polypeptide, and an intracellular domain comprising a co-stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain and the intracellular domain has an amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 64.

[0321] SEQ ID NO: 64 is provided below:

[0322] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNALSCKYSYNLFSREFR ASLHKGLDSAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYQTDI YFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 64)

[0323] In accordance with the presently disclosed subject matter, a “CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide comprised in the transmembrane domain and the intracellular domain (e.g., the co-stimulatory signaling region) of the presently disclosed CAR (amino acids 114 to 220 of SEQ ID NO: 64) comprises nucleic acids having the sequence set forth in SEQ ID NO: 65 as provided below.

[0324] attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacac ctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagt aacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccc cgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO: 65)

[0325] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. The CD8 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%) homologous to SEQ ID NO: 66 (homology herein may be determined using standard software such as BLAST or FASTA) as provided below, or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 66 which is at least 20, or at least 30,or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively, or additionally, in various embodiments, the CD8 polypeptide has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 66.

[0326] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSN PTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDF RRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRP WKSGDKPSLSARYV (SEQ ID NO: 66)

[0327] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 67 as provided below:

[0328] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCN (SEQ ID NO: 67)

[0329] In accordance with the presently disclosed subject matter, a “CD8 nucleic acid molecule” refers to a polynucleotide encoding a CD8 polypeptide. In certain embodiments, the CD8 nucleic acid molecule encoding the CD8 polypeptide comprised in the transmembrane domain of the presently disclosed CAR (SEQ ID NO: 67) comprises nucleic acids having the sequence set forth in SEQ ID NO: 68 as provided below.

[0330] CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGAT CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGG GCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGC CCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTG CAAC (SEQ ID NO: 68)

[0331] In certain non-limiting embodiments, a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition while preserving the activating activity of the CAR. In certain non-limiting embodiments, the spacer region can be the hinge region from IgGl, the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide (e.g., SEQ ID NO: 64), a portion of a CD8 polypeptide (e.g., SEQ IDNO: 66), a variation of any of the foregoing which is at least about 80%, at least about 85%>, at least about 90%, or at least about 95% homologous thereto, or a synthetic spacer sequence. In certain non-limiting embodiments, the spacer region may have a length between about 1-50 (e.g., 5-25, 10-30, or 30-50) amino acids.Intracellular Domain of a CAR

[0332] In certain non-limiting embodiments, an intracellular domain of the CAR can comprise a CD3(^ polypeptide, which can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T cell). CD3(^ comprises 3 ITAMs, and transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T cell) after antigen is bound. The CD3(^ polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: NP 932170 (SEQ ID NO: 69), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3(^ polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 70 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively, or additionally, in various embodiments, the CD3(^ polypeptide has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 70. In certain embodiments, the CD3(^ polypeptide has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 70.

[0333] SEQ ID NO: 70 is provided below:

[0334] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (SEQ ID NO: 70)

[0335] In certain embodiments, the CD3(^ polypeptide has the amino acid sequence set forth in SEQ ID NO: 71, which is provided below:

[0336] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQ ALPPR (SEQ ID NO: 71)

[0337] In certain embodiments, the CD3(^ polypeptide has the amino acid sequence set forth in SEQ ID NO: 72, which is provided below:

[0338] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (SEQ ID NO: 72)

[0339] In accordance with the presently disclosed subject matter, a “CD3(^ nucleic acid molecule” refers to a polynucleotide encoding a CD3(^ polypeptide. In certain embodiments, the CD3(^ nucleic acid molecule encoding the CD3^ polypeptide (SEQ ID NO: 71) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 73 as provided below.

[0340] agagtgaagttcagcaggagcgcagagccccccgcgtaccagcagggccagaaccagctctataacgagctca atctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgc cggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggcc ctgccccctcgcg (SEQ ID NO: 73)

[0341] In certain embodiments, the CD3(^ nucleic acid molecule encoding the CD3(^ polypeptide (SEQ ID NO: 72) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 74 as provided below.

[0342] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 74)

[0343] In certain non-limiting embodiments, an intracellular domain of the CAR further comprises at least one signaling region. The at least one signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof.

[0344] In certain embodiments, the signaling region is a co-stimulatory signaling region.

[0345] In certain embodiments, the co-stimulatory signaling region comprises at least one co-stimulatory molecule, which can provide optimal lymphocyte activation. As used herein, “co-stimulatory molecules” refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen. The at least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co- stimulatory response, i.e., an intracellular response that effects the stimulation provided when an antigen binds to its CAR molecule. Co-stimulatory ligands, include, but are not limited to CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD- LI. As one example, a 4-1BB ligand (i.e., 4-1BBL) may bind to 4-1BB (also known as “CD 137”) for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR+T cell. CARs comprising an intracellular domain that comprises a co-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 are disclosed in U.S. 7,446,190, which is herein incorporated by reference in its entirety. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises two co-stimulatory molecules: CD28 and 4- IBB or CD28 and 0X40.

[0346] 4- IBB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. The 4- IBB polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: P41273 or NP 001552 (SEQ ID NO: 75) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0347] SEQ ID NO: 75 is provided below:

[0348] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPP NSF S S AGGQRTCDICRQCKGVFRTRKEC S STSNAECDCTPGFHCLGAGC SMCEQDC KQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLGTKERDWCGPSPAD LSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 75).

[0349] In certain embodiments, the 4- IBB co-stimulatory domain has the amino acid sequence set forth in SEQ ID NO: 76, which is provided below:

[0350] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 76)

[0351] In accordance with the presently disclosed subject matter, a “4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4- IBB polypeptide. In certain embodiments, the 4- IBB nucleic acid molecule encoding the 4- IBB polypeptide (SEQ ID NO: 76) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 77 as provided below.

[0352] AAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTAT GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG AAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 77).

[0353] An 0X40 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: P43489 or NP 003318 (SEQ ID NO: 78), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0354] SEQ ID NO: 78 is provided below:

[0355] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRP GNGMVSRC SRSQNT VCRPCGPGFYNDWS SKPCKPCTWCNLRSGSERKQLCTATQD TVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPAS NSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA AILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTL AKI (SEQ ID NO: 78).

[0356] In accordance with the presently disclosed subject matter, an “0X40 nucleic acid molecule” refers to a polynucleotide encoding an 0X40 polypeptide.

[0357] An ICOS polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: NP_036224 (SEQ ID NO: 79) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0358] SEQ ID NO: 79 is provided below:

[0359] MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQ FKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANY YFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVWCILGCILICWLT KKKYSSSVHDPNGEYMFMRATAKKSRLTDVTL (SEQ ID NO: 79)

[0360] In accordance with the presently disclosed subject matter, an “ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide.

[0361] CTLA-4 is an inhibitory receptor expressed by activated T cells, which when engaged by its corresponding ligands (CD80 and CD86; B7-1 and B7-2, respectively), mediates activated T cell inhibition or anergy. In both preclinical and clinical studies, CTLA-4 blockade by systemic antibody infusion, enhanced the endogenous anti-tumor response albeit, in the clinical setting, with significant unforeseen toxicities.

[0362] CTLA-4 contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternate splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. The intracellular domain is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif able to bind PI3K, PP2A and SHP-2 and one proline-rich motif able to bind SH3 containing proteins. One role of CTLA-4 in inhibiting T cell responses seem to be directly via SHP-2 and PP2A dephosphorylation of TCR-proximal signaling proteins such as CD3 and LAT. CTLA-4 can also affect signaling indirectly via competing with CD28 for CD80 / 86 binding. CTLA-4 has also been shown to bind and / or interact with PI3K, CD80, AP2M1, and PPP2R5A.

[0363] In accordance with the presently disclosed subject matter, a CTLA-4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: P16410.3 (SEQ ID NO: 80) (homology herein may be determined using standard software such as BLAST or FASTA) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0364] SEQ ID NO: 80 is provided below:

[0365] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLAS SRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG TSSGNQLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLL WILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPI N (SEQ ID NO: 80).

[0366] In accordance with the presently disclosed subject matter, a “CTLA-4 nucleic acid molecule” refers to a polynucleotide encoding a CTLA-4 polypeptide.

[0367] PD-1 is a negative immune regulator of activated T cells upon engagement with its corresponding ligands PD-L1 and PD-L2 expressed on endogenous macrophages and dendritic cells. PD-1 is a type I membrane protein of 268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein's structure comprises an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine- based switch motif, that PD-1 negatively regulates TCR signals. SHP- 1 and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism tumor cells may evade the host immune system. In pre-clinical and clinical trials, PD-1 blockade by antagonistic antibodies induced anti -tumor responses mediated through the host endogenous immune system. In accordance with the presently disclosed subject matter, a PD-1 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to NCBI Reference No: NP_005009.2 (SEQ ID NO: 81) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0368] SEQ ID NO: 81 is provided below:

[0369] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPR PAGQFQTLVVGWGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVP VFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSA QPLRPEDGHCSWPL (SEQ ID NO: 81).

[0370] In accordance with the presently disclosed subject matter, a “PD-1 nucleic acid molecule” refers to a polynucleotide encoding a PD-1 polypeptide.

[0371] Lymphocyte-activation protein 3 (LAG-3) is a negative immune regulator of immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains. The LAG3 gene contains 8 exons. The sequence data, exon / intron organization, and chromosomal localization all indicate a close relationship of LAG3 to CD4. LAG3 has also been designated CD223 (cluster of differentiation 223).

[0372] In accordance with the presently disclosed subject matter, a LAG-3 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: P18627.5 (SEQ ID NO: 82) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0373] SEQ ID NO: 82 is provided below:

[0374] MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPWWAQEGAPAQLPCSPTIP LQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSV GPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALS CRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRE SPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQ AGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSS LDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGR APGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQS KIEELEQEPEPEPEPEPEPEPEPEPEQL (SEQ ID NO: 82).

[0375] In accordance with the presently disclosed subject matter, a “LAG-3 nucleic acid molecule” refers to a polynucleotide encoding a LAG-3 polypeptide. Natural Killer Cell Receptor 2B4 (2B4) mediates non-MHC restricted cell killing on NK cells and subsets of T cells. To date, the function of 2B4 is still under investigation, with the 2B4-S isoform believed to be an activating receptor, and the 2B4-L isoform believed to be a negative immune regulator of immune cells. 2B4 becomes engaged upon binding its high-affinity ligand, CD48. 2B4 contains a tyrosine-based switch motif, a molecular switch that allows the protein to associate with various phosphatases. 2B4 has also been designated CD244 (cluster of differentiation 244).

[0376] In accordance with the presently disclosed subject matter, a 2B4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: Q9BZW8.2 (SEQ ID NO: 83) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0377] SEQ ID NO: 83 is provided below:

[0378] MLGQWTLILLLLLKVYQGKGCQGSADHWSISGVPLQLQPNSIQTKVDSI AWKKLLPSQNGFHHILKWENGSLPSNTSNDRFSFIVKNLSLLIKAAQQQDSGLYCLE VTSISGKVQTATFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGN VSYAWYRGSKLIQTAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQ NAHQEFRFWPFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDL KTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSP SFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 83). In accordance with the presently disclosed subject matter, a “2B4 nucleic acid molecule” refers to a polynucleotide encoding a 2B4 polypeptide.

[0379] B- and T-lymphocyte attenuator (BTLA) expression is induced during activation of T cells, and BTLA remains expressed on Thl cells but not Th2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homolog, B7H4. However, unlike PD-1 and CTLA-4, BTLA displays T-Cell inhibition via interaction with tumor necrosis family receptors (TNF- R), not just the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses.BTLA activation has been shown to inhibit the function of human CD8+cancer-specific T cells. BTLA has also been designated as CD272 (cluster of differentiation 272).

[0380] In accordance with the presently disclosed subject matter, a BTLA polypeptide can have an amino acid sequence that is at least about 85%>, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss- Prot Ref. No.: Q7Z6A9.3 (SEQ ID NO: 84) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0381] SEQ ID NO: 84 is provided below:

[0382] MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILA GDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLP NDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGG LPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLL SETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKE APTEYASICVRS (SEQ ID NO: 84).

[0383] In accordance with the presently disclosed subject matter, a “BTLA nucleic acid molecule” refers to a polynucleotide encoding a BTLA polypeptide.Therapeutic Methods of the Present Technology

[0384] In one aspect, the immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) of the present technology are useful for the treatment of IL13Ra2-associated pathologies, including but not limited to, helminthic and parasitic infections (such as schistosomiasis), chronic hepatitis, allergic / inflammatory diseases (such as lung fibrosis, asthma, inflammatory bowel disease), glioblastoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, melanoma, clear cell carcinomas, pancreatic cancer, T-cell lymphoma cells, and metastases thereof. Such treatment can be used in patients identified as having pathologically high levels of the IL13Ra2 (e.g., those diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating an IL13Ra2-associated pathology in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen binding fragment thereof) of the present technology.

[0385] In one aspect, the present disclosure provides a method for treating an IL13Ra2- associated pathology in a subject in need thereof comprising administering an effective amount of any and all embodiments of the antibody or antigen binding fragment disclosed herein.

[0386] In one aspect, the present disclosure provides a method for treating an IL13Ra2- associated pathology in a subject in need thereof comprising administering an effective amount of any and all embodiments of the recombinant nucleic acid molecules or vector described herein.

[0387] In one aspect, the present disclosure provides a method for treating an IL13Ra2- associated pathology in a subject in need thereof comprising administering an effective amount of any and all embodiments of the engineered immune cells described herein. Additionally or alternatively, in some embodiments, the method further comprises administering a cytokine to the subject. The cytokine may be administered prior to, during, or subsequent to administration of the engineered immune cells. Examples of cytokines include, but are not limited to interferon a, interferon P, interferon y, complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

[0388] In one aspect, the present disclosure provides a method for preparing immune cells for therapy, comprising isolating immune cells from a donor subject, transducing the immune cells with any and all embodiments of the recombinant nucleic acid molecules described herein or any and all embodiments of the vectors of the present technology; and administering the transduced immune cells to a recipient subject. The donor subject and the recipient subject may be the same or different. Additionally or alternatively, in some embodiments, the immune cells isolated from the donor subject comprise one or more lymphocytes, such as tumor infiltrating lymphocytes, T cells, CD4+ T cells, CD8+ T cells, B cells, or natural killer (NK) cells.

[0389] Examples of IL13Ra2-associated pathologies that can be treated by the antibodies of the present technology include, but are not limited to: helminthic and parasitic infections (such as schistosomiasis), chronic hepatitis, allergic / inflammatory diseases (such as lung fibrosis, asthma, inflammatory bowel disease), glioblastoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, melanoma, clear cell carcinomas, pancreatic cancer, T-cell lymphoma cells, and metastases thereof.

[0390] Combination therapy. The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of IL13Ra2- associated cancers. For example, the antibodies of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent.

[0391] In certain embodiments, the methods of the present technology further comprise administering an additional cancer therapy. In some embodiments, the additional cancer therapy is selected from among chemotherapy, anti -cancer monoclonal antibody therapy, radiation therapy, immune checkpoint blockade therapy, anti-cancer nucleic acids or proteins, anti-cancer viruses or microorganisms, and any combinations thereof.

[0392] In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10- ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, proteinbound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracy clines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof. Examples of other anti-cancer therapeutic agents include, but are not limited to alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGFZEGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapy agents and targeted biological therapy agents (e.g.,therapeutic peptides described in US 6306832, WO 2012007137, WO 2005000889, WO 2010096603 etc.).

[0393] In any and all embodiments of the methods disclosed herein, the immune checkpoint inhibitor may include one or more of an anti-PD-1 antibody, an anti-PD- L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-lBB antibody, an anti-CD73 antibody, an anti-GITR antibody, an anti-LAG-3 antibody, an anti -0X40 antibody, an anti-TIGIT antibody, an anti-B7-H3 antibody, an anti- B7-H4 antibody, or an anti-BTLA antibody. In some embodiments, the immune checkpoint inhibitor may include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, ticlimumab, JTX-4014, Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308),Tislelizumab (BGB-A317), Toripalimab (JS 001), Dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, AMP-514, KN035, CK-301, AUNP12, CA-170, or BMS-986189.

[0394] The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of helminthic and parasitic infections (such as schistosomiasis, opisthorchiasis etc.), chronic hepatitis, or allergic / inflammatory diseases (such as lung fibrosis, asthma, inflammatory bowel disease). For example, the antibodies of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent-selected from the group consisting of anthelmintics, antimalarials (e.g., chloroquine, hydroxychloroquine),, antiinflammatory agents, antiviral medications (e.g., entecavir, tenofovir, lamivudine, adefovir, telbivudine), interferon alfa, glucocorticoids (e.g., hydrocortisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, budesonide, beclomethasone, fluticasone, flunisolide, ciclesonide, fenoterol, pirbuterol, salbutamol, terbutaline, formoterol, salmeterol, clenbuterol, prokaterol, bambuterol etc.), aminosalicylates (e.g., sulfasalazine, mesalazine, sulfapyridine, 5-aminosalicylic acid), immunosuppressants / cytostatics (e.g., cyclosporin A, tacrolimus, azathioprine, 6- mercaptopurine, methotrexate, cyclophosphamide, minocycline, leflunomide, pimecrolimus, 6-thioguanine, hydroxyurea, fumaric acid esters, mycophenolic acid etc.), antibacterial chemotherapeutics (e.g., metronidazole, ornidazole, ofloxacin, ciprofloxacin), antidiarrheal drugs (diphenoxylate, loperamide), spasmoanalgesics, NSAIDs (e.g., indomethacin, diclofenac, ibuprofen, piroxicam, tenoxicam, meloxicam, nimesulide, celecoxib, parecoxib,lumiracoxib), antirheumatic drugs (e.g., auranofin, aurothiomalate, d-penicillamine), cyclooxygenase (COX) inhibitors, cromones (e.g., cromoglycate, nedocromil), and cytokine and histamine release inhibitors (tranilast), antileukotrienes (e.g., montelukast), phosphodiesterase-4 inhibitors (roflumilast), P2-mimetics, theophylline- methylxanthines (e.g., theophylline, aminophylline, etophylline), tiotropium bromide, ichtamol, dithranol, salicylic acid, urea, corticosteroids (e.g., mometasone, budesonide), fluorouracil, selective vitamin A derivatives (e.g., retinoids such as acitretin, adapalene, tazarotene), and vitamin D3 analogues (e.g., calcitriol, calcipotriol, tacalcitol, oxacal ci tri ol, paricalcitol).

[0395] Examples of additional therapeutic agents that treat helminthic and parasitic infections include, but are not limited to, praziquantel (PZQ), oxamniquine (OXA), ivermectin, artesunate (AS), artemether (ART), mefloquine (MFQ), Artemisinin, P- aminopropionitrile-monofumarate salt, P-aminopropionitrile, atorvastatin (AV), medroxyprogesterone acetate, edelfosine (EDLF), albendazole (ABZ), arabino-galactan- ABZ complex, mefloquine, tribendimidine (TBD), ibuprofen, naproxen, miconazole (MCZ), clotrimazole (CTZ), M. armillaris, Resveratrol, Sylimarin, Limonin, a-Lipoic acid (ALA), B. trimera, 4-Hydroxyquinolin-2(lH)-one (BDHQ), A. sativum, A. sativum + A. cepa, N. sativa, N-acetyl-cysteine, curcumin, melatonin, aqueous extract of Thunbergia laurifolia, xanthumol, nitazoxanide, oxantel pamoate, and benzimidazoles (e.g., mebendazole, triclabendazole, flubendazole, fenbendazole, oxfendazole, thiabendazole, oxibendazole).

[0396] In any of the foregoing embodiments of the methods described herein, the engineered immune cells, the antibody or antigen binding fragment, the recombinant nucleic acid molecule, or the vector is administered pleurally, parenterally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.

[0397] PRIT. In one aspect, the present disclosure provides a method for detecting tumors in a subject in need thereof comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multi-specific antibody or antigen binding fragment of the present technology that binds to the radiolabeled DOTA hapten and an IL13Ra2 antigen, wherein the complex is configured to localize to a tumor expressing the IL13Ra2 antigen recognized by the multi-specific antibody or antigen binding fragment of the complex; and (b) detecting the presence of tumors in the subject bydetecting radioactive levels emitted by the complex that are higher than a reference value. In some embodiments, the subject is human.

[0398] In one aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multi-specific antibody or antigen binding fragment of the present technology that binds to the radiolabeled DOTA hapten and an IL13Ra2 antigen, wherein the complex is configured to localize to a tumor expressing the IL13Ra2 antigen recognized by the multi-specific antibody or antigen binding fragment of the complex; (b) detecting radioactive levels emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the radioactive levels emitted by the complex are higher than a reference value. In some embodiments, the subject is human.

[0399] Also disclosed herein is a method for selecting a subject for pretargeted radioimmunotherapy comprising (a) administering to the subject an effective amount of the multi-specific antibody or antigen binding fragment of the present technology that binds to a radiolabeled DOTA hapten and an IL13Ra2 antigen, wherein the multi-specific antibody is configured to localize to a tumor expressing the IL13Ra2 recognized by the multi-specific antibody or antigen binding fragment; (b) administering an effective amount of a radiolabeled-DOTA hapten to the subject, wherein the radiolabeled-DOTA hapten is configured to bind to the multi-specific antibody or antigen binding fragment; (c) detecting radioactive levels emitted by the multi-specific antibody; and (d) selecting the subject for pretargeted radioimmunotherapy when the radioactive levels emitted by the multi-specific antibody are higher than a reference value.

[0400] Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr- Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D- Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D- Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D- Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D- Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D- Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D- Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu- D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys- NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D- Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; (xix) Ac-D-Lys(DOTA)-D-Tyr- D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2and (xx) DOTA. The radiolabel may be an alpha particle-emitting isotope, a beta particle-emitting isotope, or an Auger-emitter. Examples of radiolabels include213Bi,211At,225Ac,152Dy,212Bi,223Ra,219Rn,215Po,211Bi,221Fr,217At,255Fm,86Y,90Y,89Sr,165Dy,186Re,188Re,177Lu,67Cu,n iIn,67Ga,51Cr,58Co, "mTc,103mRh,195mPt,119Sb,161HO,189mOs,192Ir,2O1T1,203Pb,68Ga,227Th, or64Cu.

[0401] In some embodiments of the methods disclosed herein, the radioactive levels emitted by the complex are detected using positron emission tomography or single photon emission computed tomography.

[0402] Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with, or is suspected of having glioblastoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, melanoma, clear cell carcinomas, pancreatic cancer, T-cell lymphoma cells, and metastases thereof.

[0403] Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In certain embodiments, the complex is administered into the cerebral spinal fluid or blood of the subject.

[0404] In some embodiments of the methods disclosed herein, the radioactive levels emitted by the complex are detected between 2 to 120 hours after the complex is administered. In certain embodiments of the methods disclosed herein, the radioactive levels emitted by the complex are expressed as the percentage injected dose per gram tissue (%ID / g). The reference value may be calculated by measuring the radioactive levels present in non-tumor (normal) tissues, and computing the average radioactive levels present in non-tumor (normal) tissues ± standard deviation. In some embodiments, the reference value is the standard uptake value (SUV). See Thie JA, JNucl Med. 45(9): 1431-4 (2004). In some embodiments, the ratio of radioactive levels between a tumor and normal tissue isabout 2:l, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0405] In another aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with cancer comprising (a) administering to the subject an effective amount of the multi-specific antibody or antigen binding fragment of the present technology that binds to a radiolabeled DOTA hapten and an IL13Ra2 antigen, wherein the multi-specific antibody is configured to localize to a tumor expressing the IL13Ra2 recognized by the multi-specific antibody or antigen binding fragment; and (b) administering an effective amount of a radiolabeled-DOTA hapten to the subject, wherein the radiolabeled-DOTA hapten is configured to bind to the multi-specific antibody or antigen binding fragment. In some embodiments, the subject is human.

[0406] The anti-DOTA x IL13Ra2 multi-specific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) sufficient for it to saturate tumor cells. In some embodiments, unbound anti-DOTA x IL13Ra2 multi-specific antibody is removed from the blood stream after administration of the anti-DOTA x IL13Ra2 multi-specific antibody. In some embodiments, the radiolabeled-DOTA hapten is administered after a time period that may be sufficient to permit clearance of unbound anti- DOTA x IL13Ra2 multi-specific antibody.

[0407] The radiolabeled-DOTA hapten may be administered at any time between 1 minute to 4 or more days following administration of the anti-DOTA x IL13Ra2 multispecific antibody. For example, in some embodiments, the radiolabeled-DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 hours, 72 hours, 96 hours, or any range therein, following administration of the anti-DOTA x IL13Ra2 multi-specific antibody. Alternatively, the radiolabeled-DOTA hapten may be administered at any time after 4 or more days following administration of the anti-DOTA x IL13Ra2 multi-specific antibody.

[0408] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a clearing agent to the subject prior to administration of the radiolab el ed-DO TA hapten. A clearing agent can be any molecule (dextran or dendrimer or polymer) that can be conjugated with C825-hapten. In some embodiments, the clearing agent is no more than 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD, or 5kD. In some embodiments, the clearing agent is a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD dextran-DOTA-Bn (Y), 500 kD dextran-DOTA-Bn (Lu), or 500 kD dextran-DOTA-Bn (In) etc.).

[0409] In some embodiments, the clearing agent and the radiolab el ed-D OTA hapten are administered without further administration of the anti-DOTA x IL13Ra2 multi-specific antibody or antigen binding fragment of the present technology. For example, in some embodiments, an anti-DOTA x IL13Ra2 multi-specific antibody or antigen binding fragment of the present technology is administered according to a regimen that includes at least one cycle of: (i) administration of the anti-DOTA x IL13Ra2 multi-specific antibody or antigen binding fragment of the present technology (optionally so that relevant tumor cells are saturated); (ii) administration of a radiolabeled-DOTA hapten and, optionally a clearing agent; (iii) optional additional administration of the radiolabeled-DOTA hapten and / or the clearing agent, without additional administration of the anti-DOTA x IL13Ra2 multi-specific antibody. In some embodiments, the method may comprise multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0410] Additionally or alternatively, in some embodiments of the method, the anti- DOTA x IL13Ra2 multi-specific antibody and / or the radiolabeled-DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, intratumorally, orally or intranasally.

[0411] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with cancer comprising administering to the subject an effective amount of a complex comprising a radiolabe...

Claims

WHAT IS CLAIMED IS1. An antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein:(a) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 2, a VH- CDR2 sequence comprising SEQ ID NO: 3, and a VH-CDR3 sequence comprising SEQ ID NO: 4; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 6, a VL-CDR2 sequence comprising SEQ ID NO: 7, and a VL-CDR3 sequence comprising SEQ ID NO: 8;(b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 10, a VH-CDR2 sequence comprising SEQ ID NO: 11, and a VH-CDR3 sequence comprising SEQ ID NO: 12; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 14, a VL-CDR2 sequence comprising SEQ ID NO: 15, and a VL-CDR3 sequence comprising SEQ ID NO: 16;(c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 18, a VH-CDR2 sequence comprising SEQ ID NO: 19, and a VH-CDR3 sequence comprising SEQ ID NO: 20; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 22, a VL-CDR2 sequence comprising SEQ ID NO: 23, and a VL-CDR3 sequence comprising SEQ ID NO: 24;(d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 26, a VH-CDR2 sequence comprising SEQ ID NO: 27, and a VH-CDR3 sequence comprising SEQ ID NO: 28; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 30, a VL-CDR2 sequence comprising SEQ ID NO: 31, and a VL-CDR3 sequence comprising SEQ ID NO: 32;(e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 34, a VH-CDR2 sequence comprising SEQ ID NO: 35, and a VH-CDR3 sequence comprising SEQ ID NO: 36; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL-CDR3 sequence comprising SEQ ID NO: 40; or(f) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 42, a VH-CDR2 sequence comprising SEQ ID NO: 43, and a VH-CDR3 sequence comprising SEQ ID NO: 44; and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 46, a VL-CDR2 sequence comprising SEQ ID NO: 47, and a VL-CDR3 sequence comprising SEQ ID NO: 48, wherein the antibody or antigen binding fragment specifically binds to an IL13Ra2 polypeptide.

2. The antibody or antigen binding fragment of claim 1, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41 and / or the VL comprises the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.

3. The antibody or antigen binding fragment of claim 1, wherein the VH comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 9, 17, 25, 33 or 41 and / or the VL comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.

4. The antibody or antigen binding fragment of any one of claims 1-3, further comprising a Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE.

5. The antibody of claim 4, comprising an IgGl constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A and L235A.

6. The antibody of claim 4, comprising an IgG4 constant region comprising a S228P mutation.

7. The antigen binding fragment of any one of claims 1-3, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv.

8. The antibody or antigen binding fragment of any one of claims 1-7, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or multi-specific antibody.

9. The multi-specific antibody of claim 8, wherein the multi-specific antibody or antigen binding fragment binds to a B-cell, a T-cell, a NK cell, a myeloid cell, a plasma cell, a mast-cell or a DOTA hapten.

10. The multi-specific antibody of claim 9, wherein the multi-specific antibody or antigen binding fragment binds to CD3, CD4, CD8, CD20, CD 19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46 and KIR.

11. The antibody or antigen binding fragment of any one of claims 1-10, wherein the IL13Ra2 polypeptide comprises amino acid residues 27-343 of SEQ ID NO: 49.

12. The antibody of any one of claims 1-6 or 8-11, wherein the antibody lacks a- 1,6-fucose modifications.

13. A recombinant nucleic acid molecule encoding the antibody or antigen binding fragment of any one of claims 1-12.

14. The recombinant nucleic acid molecule of claim 13, wherein the recombinant nucleic acid molecule comprises DNA or mRNA.

15. A vector comprising the recombinant nucleic acid molecule of claim 13 or 14, optionally wherein the vector is a viral vector, a retroviral vector, or a plasmid.

16. A host cell comprising the recombinant nucleic acid molecule of claim 13 or 14, or the vector of claim 15.

17. A composition comprising (a) the antibody or antigen binding fragment of any one of claims 1-12, the recombinant nucleic acid molecule of claim 13 or 14, or the vector of claim 15, and (b) a pharmaceutically-acceptable carrier, wherein the antibody or antigen binding fragment is optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof.

18. A kit comprising the antibody or antigen binding fragment of any one of claims 1-12 and instructions for use.

19. The kit of claim 18, wherein the antibody or antigen binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

20. The kit of claim 18 or 19, further comprising a secondary antibody that specifically binds to the antibody or antigen binding fragment of any one of claims 1-12.

21. An engineered immune cell comprising: a receptor comprising the antibody or antigen binding fragment of any one of claims 1-12, and / or a nucleic acid molecule encoding the receptor.

22. The engineered immune cell of claim 21, wherein the receptor is a T cell receptor (TCR).

23. The engineered immune cell of claim 21, wherein the receptor is a chimeric antigen receptor (CAR).

24. The engineered immune cell of any one of claims 21-23, wherein the receptor is expressed on the surface of the engineered immune cell.

25. The engineered immune cell of any one of claims 21-24, wherein the nucleic acid molecule encoding the receptor is operably linked to a promoter.

26. The engineered immune cell of claim 25, wherein the promoter is a constitutive promoter or a conditional promoter.

27. The engineered immune cell of claim 26, wherein the conditional promoter is inducible by binding of the receptor to an IL13Ra2 antigen.

28. The engineered immune cell of any one of claims 21-27, wherein the antigen binding fragment of the receptor is an scFv, a Fab, or a F(ab)2.

29. The engineered immune cell of any one of claims 21-28, wherein the receptor is linked to a reporter or a selection marker.

30. The engineered immune cell of claim 29, wherein the reporter or selection marker is GFP or LNGFR or an anti-DOTA scFv.

31. The engineered immune cell of any one of claims 29-30, wherein the receptor is linked to the reporter or selection marker via a self-cleaving linker.

32. The engineered immune cell of any one of claims 23-31, wherein the chimeric antigen receptor comprises (i) an extracellular antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain.

33. The engineered immune cell of claim 32, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) or a human scFv.

34. The engineered immune cell of any one of claims 32-33, wherein the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain.

35. The engineered immune cell of any one of claims 32-34, wherein the intracellular domain comprises one or more costimulatory domains.

36. The engineered immune cell of claim 35, wherein the one or more costimulatory domains are selected from among a CD28 costimulatory domain, a 4- IBB costimulatory domain, an 0X40 costimulatory domain, an ICOS costimulatory domain, a DAP- 10 costimulatory domain, a PD-1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, a CD3^-chain, or any combination thereof.

37. The engineered immune cell of any of claims 21-36, wherein the engineered immune cell is a lymphocyte.

38. The engineered immune cell of claim 37, wherein the lymphocyte is a tumor infiltrating lymphocyte, a T cell, a B cell, or a natural killer (NK) cell.

39. The engineered immune cell of claim 38, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

40. The engineered immune cell of any of claims 21-39, wherein the engineered immune cell is derived from an autologous donor or an allogenic donor.

41. A method for treating an IL13Ra2-associated pathology in a subject in need thereof comprising administering an effective amount of the antibody or antigen binding fragment of any one of claims 1-12, the recombinant nucleic acid molecule of claim 13 or 14, the vector of claim 15 or the composition of claim 17.

42. A method for treating an IL13Ra2-associated pathology in a subject in need thereof comprising administering an effective amount of the engineered immune cells of any of claims 21-40.

43. The method of claim 42, further comprising administering a cytokine to the subject.

44. The method of claim 43, wherein the cytokine is administered prior to, during, or subsequent to administration of the engineered immune cells.

45. The method of claim 43 or 44, wherein the cytokine is selected from a group consisting of interferon a, interferon P, interferon y, complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

46. The method of any one of claims 41-45, wherein the IL13Ra2-associated pathology is cancer, a helminthic or parasitic infection, chronic hepatitis, or an allergic / inflammatory disease.

47. The method of claim 46, wherein the cancer is selected from among glioblastoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, melanoma, clear cell carcinomas, pancreatic cancer, T-cell lymphoma cells, and metastases thereof.

48. The method of any one of claims 41-47, wherein the engineered immune cells, the antibody or antigen binding fragment, the recombinant nucleic acid molecule, the vector or the composition is administered pleurally, parenterally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.

49. The method of any one of claims 41-48, further comprising administering an additional cancer therapy.

50. The method of claim 49, wherein the additional cancer therapy is selected from among chemotherapy, radiation therapy, immune checkpoint blockade therapy, anticancer nucleic acids or proteins, anti -cancer viruses or microorganisms, and any combinations thereof.

51. A method for preparing immune cells for cancer therapy, comprising isolating immune cells from a donor subject, transducing the immune cells with the recombinant nucleic acid molecule of any of claims 13-14 or the vector of claim 15; and administering the transduced immune cells to a recipient subject.

52. The method of claim 51, wherein the donor subject and the recipient subject are the same.

53. The method of claim 51, wherein the donor subject and the recipient subject are different.

54. The method of any one of claims 51-53, wherein the immune cells isolated from the donor subject comprise one or more lymphocytes.

55. The method of claim 54, wherein the one or more lymphocytes is a tumor infiltrating lymphocyte, a T cell, a B cell, or a natural killer (NK) cell.

56. The method of claim 54 or 55, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

57. A method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of the antibody or antigen binding fragment of any one of claims 1-12, wherein the antibody or antigen binding fragment is configured to localize to a cancer cell expressing IL13Ra2 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting radioactive levels emitted by the antibody or antigen binding fragment that are higher than a reference value.

58. The method of claim 57, wherein the subject is diagnosed with or is suspected of having cancer.

59. The method of claim 57 or 58, wherein the radioactive levels emitted by the antibody or antigen binding fragment are detected using positron emission tomography or single photon emission computed tomography.

60. The method of any one of claims 57-59, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen binding fragment of any one of claims 1-12 conjugated to a radionuclide.

61. The method of claim 60, wherein the radionuclide is an alpha particleemitting isotope, a beta particle-emitting isotope, an Auger-emitter, or any combination thereof.

62. The method of claim 61, wherein the beta particle-emitting isotope is selected from the group consisting of86Y,90Y,89Sr,165Dy,186Re,188Re,177Lu, and67Cu.

Images