IL-13 receptor α2-targeted T cell immunotherapy with zeta-chain-associated protein kinase 70 (ZAP-70)-mediated targeting

Abstract

To provide nucleic acids and cells having the same for improved further T cell immunotherapies.SOLUTION: The invention provides a nucleic acid encoding a zetakine including a specific sequence. Based thereon, the invention includes cells having a zetakine which specifically binds to the IL-13 receptor α2 (IL13Ra2) with 50-times higher affinity than wild-type IL-13, and cell-based immunotherapy targeting cancer cells, such as cells of solid tumors, using compositions comprising such cells. In some embodiments, the receptors include spacer regions, such as particular spacer regions designed to provide certain advantages.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 643,139, entitled "IL-13 Receptor α2-Targeted T Cell Immunotherapy with Zeta-Cytokine Directed Specificity", filed on Mar. 14, 2018, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application is filed with an electronic sequence listing. The sequence listing is provided as a file named SCRI179WOSEQLIST, created on Mar. 12, 2019, and is approximately 35 kb in size. The information described in this electronic sequence listing is hereby incorporated by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein include cell-based immunotherapies that target cancer cells (such as solid tumor cells) using cells having a zeta-cytokine, a receptor having specificity conferred by a cell membrane-bound IL-13 mutein, such as a cell having a zeta-cytokine that specifically binds to IL-13 receptor α2 (IL13Ra2) with an affinity 50-fold that of wild-type IL-13, and compositions comprising such cells. In some embodiments, the receptor includes a spacer region, such as a spacer region designed to provide certain desired properties to the receptor and selected to have a particular predetermined length.

Background Art

[0004] Despite the fact that our understanding of brain cancer has been deepening, the mortality rate has remained at a certain level over the past decade, and innovative new treatments are urgently needed. Based on remarkable clinical data showing complete remission in patients with B-cell malignancies after administration of CD19-targeted CAR-expressing T cells, T-cell immunotherapy is now regarded as promising in cancer treatment. However, further improved T-cell immunotherapy is still required. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] Some embodiments of the methods and compositions provided herein are nucleic acids encoding zetakine, a receptor directed by a cell membrane-bound IL-13 mutein, wherein the receptor zetakine comprises an extracellular domain comprising a mutein of IL-13 and a spacer; a transmembrane domain; and an intracellular signaling region and wherein the spacer is located between the mutein and the transmembrane domain characterized in that it comprises a nucleic acid.

[0006] In some embodiments, the mutein of IL-13 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, the mutein of IL-13 comprises the amino acid sequence of SEQ ID NO: 16.

[0007] In some embodiments, the spacer is a peptide spacer.

[0008] In some embodiments, the peptide spacer is 110 amino acids or less in length and 1 amino acid or more or 2 amino acids or more in length, for example, 15 amino acids or less in length and 1 amino acid or more or 2 amino acids or more in length.

[0009] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (short spacer), an IgG4 hinge-CH3 spacer (medium-length spacer), or an IgG4 hinge-CH2-CH3 spacer (long spacer).

[0010] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (medium-length spacer).

[0011] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2-CH3 spacer (long spacer).

[0012] In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm).

[0015] In some embodiments, the intracellular signaling domain comprises a combination of a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and combinations thereof, and all or a part of the CD3ζ domain. In some embodiments, the intracellular signaling region comprises a signal transduction functional part of the CD3ζ domain and a costimulatory functional part of the 4-1BB domain.

[0016] Some embodiments further comprise a sequence encoding a marker. In some embodiments, the marker comprises a truncated form of a cell surface receptor terminus, and the marker may be EGFRt.

[0017] Some embodiments further comprise a transgene encoding a dihydrofolate reductase configured to be selectable by methotrexate. In some embodiments, the transgene encoding the dihydrofolate reductase is a double mutant of dihydrofolate reductase (DHFRdm). In some embodiments, the double mutant of dihydrofolate reductase comprises an L22F amino acid mutation and an F31S amino acid mutation.

[0018] Some embodiments further comprise a sequence encoding a ribosome skip sequence.

[0019] In some embodiments, the ribosome skip sequence comprises P2A or T2A.

[0020] Some of the embodiments of the methods and compositions provided herein comprise an expression vector comprising a nucleic acid according to any of the embodiments provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector or an adenoviral vector.

[0021] Some embodiments of the methods and compositions provided herein include chimeric receptor polypeptides encoded by nucleic acids according to any of the embodiments provided herein.

[0022] Some embodiments of the methods and compositions provided herein include host cells comprising nucleic acids according to any of the embodiments provided herein.

[0023] In some embodiments, the host cell is a T cell or a progenitor T cell.

[0024] In some embodiments, the host cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocyte is a central memory T cell, and the central memory T cell is CD45RO+, CD62L+, and CD8+.

[0025] In some embodiments, the host cell is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocyte is a naive CD4+ T cell, and the naive CD4+ T cell is CD45RA+, CD62L+, and CD4+, and CD45RO-.

[0026] In some embodiments, the host cell is a progenitor T cell.

[0027] In some embodiments, the host cell is a hematopoietic stem cell.

[0028] Some embodiments of the methods and compositions provided herein include compositions comprising a host cell according to any of the embodiments provided herein and a pharmaceutically acceptable additive.

[0029] Some embodiments of the methods and compositions provided herein include a method of making a host cell (such as, for example, a host cell according to any of the embodiments described herein), comprising: introducing a nucleic acid according to any of the embodiments described herein into a lymphocyte; culturing said lymphocytes in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody and at least one homeostatic cytokine; and selecting said lymphocytes with a selection agent configured to selectively enrich for cells transduced with said nucleic acid or a vector comprising said nucleic acid; The present invention includes a method comprising the steps of:

[0030] In some embodiments, the selection agent comprises methotrexate.

[0031] In some embodiments, the lymphocytes have a phenotype of CD45RA-, CD45RO+ and CD62L+. In some embodiments, the lymphocytes are CD8+ or CD4+.

[0032] In some embodiments, the cytokine is IL-15, IL-7 and / or IL-21.

[0033] Some embodiments further comprise introducing into the host cell a second polynucleotide encoding a marker protein, hi some embodiments, the marker protein is EGFRt.

[0034] Some embodiments of the methods and compositions provided herein include host cells according to any of the embodiments described herein for use in pharmaceuticals or for use in the treatment or suppression of cancers or solid tumors that express interleukin-13 receptor alpha 2 (IL-13Ra2). In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is an IL13Rα-positive malignant tumor. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM).

[0035] Some embodiments of the methods and compositions provided herein are methods of treating, suppressing or alleviating cancer in a subject, the method comprising administering to a subject in need of treatment, suppression or alleviation of cancer a host cell according to any of the embodiments provided herein. In some embodiments, the cancer is an IL13Rα-positive malignant tumor. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is a glioma or glioblastoma. In some embodiments, the cancer is a glioma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0036] Some embodiments further include the step of administering an adjuvant therapy selected from chemotherapy and radiotherapy. In some embodiments, the chemotherapeutic agent is electrochemotherapy, an alkylating agent, an antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, and thioguanine), an antitumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, a DNA intercalator, or a checkpoint inhibitor (checkpoint kinases CHK1, CHK2).

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0051] Some embodiments of the methods and compositions provided herein relate to an IL13 zeta cytokine that is specifically bindable to the interleukin 13 receptor alpha 2 subunit (IL-13Rα2) or a chimeric antigen receptor (CAR) configured to specifically bind to IL-13Rα2. Some embodiments include nucleic acids encoding such CARs, host cells comprising the CAR, and methods of treatment utilizing host cells having the CAR, the methods of treatment including, for example, providing the host cells as a medicament to a subject in need thereof, thereby treating or suppressing, for example, a disease or condition mediated through the interleukin 13 receptor alpha 2 subunit (IL-13Rα2) (such as cancer, including IL-13Ra2-positive malignancies including gliomas and glioblastomas).

[0052] IL-13Rα2 has been previously reported to be overexpressed in metastatic or late-stage Basal-like breast cancer (BLBC) (Papageorgis et al. Breast Cancer Research, 2015; 17 (1); this reference is hereby incorporated by reference in its entirety). Also, based on publicly available data, a correlation has been found between the prediction of progression-free survival and high expression of IL-13Rα2. Also, in a subtype of BLBC that tends to rapidly spread to the lungs, high expression of IL-13Rα2 has been observed. Furthermore, IL-13Rα2 has been found to stimulate the proliferation of human glioma cells and also to stimulate metastasis via the Src / PI3K / Akt / mTOR signaling pathway (Tu et al. Tumour Biol. 2016 Nov; 37(11):14701-14709; this reference is hereby incorporated by reference in its entirety). For example, therapies targeting IL13Ra2, such as therapies using chimeric receptors, have been previously reported (see, for example, Brown et al Clin Cancer Res 2015; Brown et al N Engl J Med 2016; Brown et al Mol Ther 2017; WO2014072888(A1); in these references, anti-IL-13 receptor α2 (IL-13-Ra2) antibodies and antibody-drug conjugates for cancer treatment have been reported; these references are hereby incorporated by reference in their entirety). Such therapies include those using a binding domain or antigen recognition domain (such as zetakine, etc.) based on an IL13 variant (such as E13Y), or therapies containing these domains. Interleukin 13 receptor alpha chain variant 2 (IL-13Rα2) is desirable as a target for adoptive T cell therapy. More specifically, the expression of IL-13Rα2 is used as a prognostic marker for patient survival in glioblastoma multiforme (GBM) and is involved in promoting tumor progression.

[0053] IL-13Ra2 is expressed in more than 80% of high-grade gliomas such as glioblastoma multiforme, but in these tumors, there are no target epitopes and no overall expression of a specific target is observed, indicating that targeted therapy is avoided. Therefore, in some embodiments of the present invention, to overcome such constraints, gliomas are treated or suppressed by a therapy using multiple targets. It is considered that a combinatorial method combining the IL-13 zeta cytokine with another targeted therapy such as a chimeric antigen receptor can overcome the avoidance of therapy in tumors without target epitopes. This new therapy provided herein has been tested in in vitro and orthotopic xenograft tumor models. In some embodiments, a therapy such as immunotherapy directed by the IL-13 zeta cytokine is useful by itself for glioblastoma and other IL-13Ra2-positive malignant tumors or for specific subjects having these diseases, or as part of a cure for these diseases or subjects.

[0054] Some of the embodiments provided herein include chimeric receptors such as zeta cytokines. In some embodiments, the zeta cytokine includes a ligand-binding domain, a spacer region, a transmembrane domain, and an intracellular signaling region (usually including a first signaling domain and a co-stimulatory signaling domain). Some of the embodiments provided herein include second-generation IL-13 zeta cytokines, for example, second-generation IL-13 zeta cytokines that include a selected or designed specific spacer region. In some aspects, the zeta cytokines provided herein include zeta cytokines with improved therapeutic efficacy or anti-tumor action or anti-tumor response in vivo, or zeta cytokines configured to improve therapeutic efficacy or anti-tumor action or anti-tumor response in vivo. In some respects, some of the embodiments provided herein are based on the observation described herein that a specific zeta cytokine in response to a target antigen was able to induce the production of more cytokines compared to a similar or identical reference chimeric receptor except for the selected spacer region. Also, in some respects, some of the embodiments provided herein are based on the observation described herein that a specific zeta cytokine was able to suppress tumor growth over a long period of time in an orthotopic xenograft tumor model and significantly increase the median survival rate.

[0055] Definitions of Terms As used herein, the terms "nucleic acid" or "nucleic acid molecule" have their ordinary and customary meaning in light of this specification, and include, for example, polynucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease action or exonuclease action, but are not limited thereto. Nucleic acid molecules may be composed of natural nucleotide monomers (such as DNA, RNA, etc.), monomers composed of analogs of natural nucleotides (such as enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety, or the pyrimidine base moiety or purine base moiety. Modifications of the sugar moiety include, for example, substitution of one or more hydroxyl groups by halogen, alkyl group, amine or azide group, and the sugar moiety may be etherified or esterified. Further, the entire sugar moiety may be substituted with a structurally similar or electronically similar structure, such as, for example, an azasugar or a carbocyclic sugar analog. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or bonds similar thereto. Bonds similar to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphorosenoate bonds, phosphorodiselenoate bonds, phosphoroanilothioate bonds, phosphororanilidate bonds, phosphoramidate bonds, and the like. "Nucleic acid molecule" also includes so-called "peptide nucleic acids", which contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. In some embodiments, a nucleic acid sequence encoding a protein is provided. In some embodiments, the nucleic acid is RNA or DNA.

[0056] As used herein, "vector", "expression vector" or "construct" has its ordinary and common meaning in light of this specification. For example, it refers to a nucleic acid used to introduce a heterologous nucleic acid into a cell, which contains various regulatory factors and thus can include, but is not limited to, a nucleic acid capable of expressing a heterologous nucleic acid in a cell. Examples of vectors include, but are not limited to, plasmids, minicircles, yeast, or viral genomes. In some embodiments, the vector is a plasmid, minicircle or viral genome. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a foamy virus vector, an adenovirus vector, a retrovirus vector, or a lentivirus vector.

[0057] In some embodiments, vectors and sequences modified or optimized by codon optimization etc. are provided. As codon optimization, there is a design process of changing specific codons to codons known to be able to maximize the protein expression efficiency in a desired cell (preferably a human cell). In some embodiments, codon optimization is described, and by optimizing codons using algorithms known to those skilled in the art, synthetic gene transcripts optimized to increase protein yield can be produced. Programs including algorithms for codon optimization are known to those skilled in the art. Such programs include, for example, OptimumGene TMExamples include algorithms and the GeneGPS (registered trademark) algorithm. Furthermore, synthetic sequences with optimized codons are commercially available, for example, from Integrated DNA Technologies and other DNA sequencing services. In some embodiments, nucleic acid sequences of genes encoding full-length gene transcripts with codons optimized for expression in humans are described. In some embodiments, the gene is optimized to have codons selected such that protein expression in human cells is increased to the maximum extent possible, and the codons so selected can increase the concentration of the protein or CAR in T cells.

[0058] Optimizing codons can also reduce the formation of the secondary structure of the polynucleotide. In some embodiments, optimizing codons can also lower the overall GC / AT ratio. If codon optimization is carried out too strictly, an undesirable secondary structure may be formed, or an undesirable GC content may result in the formation of a secondary structure. Such secondary structures affect transcription efficiency. By using a program such as GeneOptimizer after optimizing codon usage frequency, the formation of secondary structures can be avoided or the GC content can be optimized. Such additional programs can be used to further optimize or troubleshoot after the initial codon optimization, thereby limiting the formation of secondary structures that may occur after the first optimization. Other programs for optimization are known to those skilled in the art. In some embodiments, the nucleic acid comprises a sequence that has been codon-optimized for expression in humans and / or for removal of secondary structures and / or for lowering the overall GC / AT ratio. In some embodiments, the sequence is optimized to avoid the formation of secondary structures. In some embodiments, the sequence is optimized such that the overall GC / AT ratio is decreased.

[0059] Some embodiments include a polypeptide sequence or a conservatively variant sequence thereof (e.g., a sequence in which the polypeptide sequence has been conservatively substituted). In some embodiments, "conservative amino acid substitution" refers to an amino acid substitution in which functionally equivalent amino acids are substituted. Conservative amino acid changes result in synonymous changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids having equivalent polarity have equivalent function and result in synonymous changes in the amino acid sequence of the resulting peptide. Substituting a particular nonpolar residue with a smaller nonpolar residue may also be considered a "conservative substitution", even if these residues belong to separate groups (e.g., substitution of phenylalanine with smaller isoleucine). Families of amino acid residues having similar side chains are defined in the art. Some of the families included in conservative amino acid substitutions are shown in Table 1. [Table 1]

[0060] As used herein, the term "chimeric antigen receptor" has its ordinary and customary meaning in light of this specification, and includes, for example, a synthetically designed receptor in which a ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder is linked via a spacer domain to one or more intracellular signaling domains (such as a co-stimulatory domain) of a T cell or other receptor, but is not limited thereto. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs). By using a vector such as a retroviral vector or a lentiviral vector to transfer the coding sequence of the chimeric receptor into T cells, the specificity of a monoclonal antibody or its binding fragment can be transplanted into T cells. A CAR is a genetically engineered T cell receptor designed to direct T cells to target cells expressing a specific cell surface antigen. By a method called adoptive cell transfer, first T cells are obtained from a subject, and then the T cells are genetically engineered to be able to express a receptor capable of exerting specificity against an antigen. Then, these T cells are reintroduced into the patient. The reintroduced T cells can recognize and target the antigen. Such a CAR is a genetically engineered receptor capable of transplanting any specificity into cells expressing an immune receptor. Some researchers also understand that a chimeric antigen receptor, i.e., a "CAR", includes an antibody or antibody fragment, a spacer, a signaling domain, and a transmembrane region. Since the various components or domains (such as an epitope-binding region (such as an antibody fragment, scFv, or a portion thereof), a spacer, a transmembrane domain, or a signaling domain, etc.) included in the CARs described herein are genetically engineered and thus surprising effects are obtained, in the overall disclosure of this specification, the components of the CARs can often be distinguished as separate entities. Since the various components included in the CARs have various variations, for example, such variations enhance the binding affinity for a specific epitope or antigen.

[0061] By transferring the CAR coding sequence into T cells using a vector, the specificity of a monoclonal antibody or its binding fragment or scFv can be transplanted into T cells. When using CARs to treat a subject in need of treatment, a technique called adoptive cell transfer is used. In this technique, T cells are collected from the subject to be treated, and the resulting T cells are genetically engineered to express a CAR specific for an antigen. The T cells that have become capable of recognizing and targeting the antigen due to the expression of the CAR are reintroduced into the patient.

[0062] As used herein, the term "ligand" has its ordinary and customary meaning in light of this specification, and includes, but is not limited to, substances capable of forming a complex with a biomolecule. Examples of ligands include, but are not limited to, substrates, proteins, small molecules, inhibitors, activators, nucleic acids, and neurotransmitters. Ligands bind by intermolecular forces, such as ionic bonds, hydrogen bonds, or van der Waals interactions. When a ligand binds to a receptor protein, its three-dimensional structure changes, thereby enabling the function of the ligand to be exerted. The binding strength of a ligand, also called binding affinity, is determined by direct interaction and dissociation. A ligand may be bound by a "ligand-binding domain." A "ligand-binding domain" may refer to, for example, a conserved sequence found in a structure capable of binding to a specific ligand or a specific epitope on a protein. A ligand-binding domain or ligand-binding moiety may include an antibody or its binding fragment or scFv, a ligand of a receptor or its variant, a peptide, and / or a polypeptide affinity molecule or polypeptide binding partner. A ligand-binding domain may be a specific protein domain or an epitope on a protein specific for one or more ligands, but is not limited thereto.

[0063] As used herein, the term "single-chain variable fragment" or "scFv" has its ordinary and customary meaning in light of this specification, and includes, for example, but is not limited to, a fusion protein comprising the variable heavy chain (VH) and variable light chain (VL) of an immunoglobulin linked by a short linker peptide. Without limiting the present invention in any way, this linker may contain glycine to obtain flexibility and may contain hydrophilic amino acids (such as serine or threonine) to obtain solubility. The linker can link the N-terminus of VH to the C-terminus of VL, or can also link the C-terminus of VH to the N-terminus of VL. In some embodiments, the ligand-binding domain present on the CAR is a single-chain variable fragment (scFV). In some embodiments, the scFv domain present on the CAR is specific for IL-13 receptor α2 (IL13Rα2) present on tumor cells.

[0064] In some embodiments, the extracellular domain comprises at least one peptide spacer. In some embodiments, the peptide spacer is 15 amino acids or shorter in length and 1 amino acid or longer or 2 amino acids or longer in length. In some embodiments, the spacer is a polypeptide chain. In some embodiments, the length of the polypeptide chain is, for example, 3 amino acids in length, 5 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, 28 amino acids in length, 29 amino acids in length, 30 amino acids in length, 31 amino acids in length, 32 amino acids in length, 33 amino acids in length, 34 amino acids in length, 35 amino acids in length, 36 amino acids in length, 37 amino acids in length, 38 amino acids in length, 39 amino acids in length, 40 amino acids in length, 41 amino acids in length, 42 amino acids in length, 43 amino acids in length, 44 amino acids in length, 45 amino acids in length, 46 amino acids in length, 47 amino acids in length, 48 amino acids in length, 49 amino acids in length, 50 amino acids in length, 51 amino acids in length, 52 amino acids in length, 53 amino acids in length, 54 amino acids in length, 55 amino acids in length, 56 amino acids in length, 57 amino acids in length, 58 amino acids in length, 59 amino acids in length, 60 amino acids in length, 61 amino acids in length, 62 amino acids in length, 63 amino acids in length, 64 amino acids in length, 65 amino acids in length, 66 amino acids in length, 67 amino acids in length, 68 amino acids in length, 69 amino acids in length, 70 amino acids in length, 71 amino acids in length, 72 amino acids in length, 73 amino acids in length, 74 amino acids in length, 75 amino acids in length, 76 amino acids in length, 77 amino acids in length, 78 amino acids in length, 79 amino acids in length, 80 amino acids in length, 81 amino acids in length, 82 amino acids in length, 83 amino acids in length, 84 amino acids in length, 85 amino acids in length, 86 amino acids in length, 87 amino acids in length, 88 amino acids in length, 89 amino acids in length, 90 amino acids in length, 91 amino acids in length, 92 amino acids in length, 93 amino acids in length, 94 amino acids in length, 95 amino acids in length, 96 amino acids in length, 97 amino acids in length, 98 amino acids in length, 99 amino acids in length, 100 amino acids in length, 101 amino acids in length, 102 amino acids in length, 103 amino acids in length, 104 amino acids in length, 105 amino acids in length, 106 amino acids in length, 107 amino acids in length, 108 amino acids in length, 109 amino acids in length,Length of 110 amino acids, length of 111 amino acids, length of 112 amino acids, length of 113 amino acids, length of 114 amino acids, length of 115 amino acids, length of 116 amino acids, length of 117 amino acids, length of 118 amino acids, length of 119 amino acids, length of 120 amino acids, length of 121 amino acids, length of 122 amino acids, length of 123 amino acids, length of 124 amino acids, length of 125 amino acids, length of 126 amino acids, length of 127 amino acids, length of 128 amino acids, length of 129 amino acids, length of 130 amino acids, length of 131 amino acids, length of 132 amino acids, length of 133 amino acids, length of 134 amino acids, length of 135 amino acids, length of 136 amino acids, length of 137 amino acids, length of 138 amino acids, length of 139 amino acids, length of 140 amino acids, length of 141 amino acids, length of 142 amino acids, length of 143 amino acids, length of 144 amino acids, length of 145 amino acids, length of 146 amino acids, length of 147 amino acids, length of 148 amino acids, length of 149 amino acids, length of 150 amino acids, length of 151 amino acids, length of 152 amino acids, length of 153 amino acids, length of 154 amino acids, length of 155 amino acids, length of 156 amino acids, length of 157 amino acids, length of 158 amino acids, length of 159 amino acids, length of 160 amino acids, length of 161 amino acids, length of 162 amino acids, length of 163 amino acids, length of 164 amino acids, length of 165 amino acids, length of 166 amino acids, length of 167 amino acids, length of 168 amino acids, length of 169 amino acids, length of 170 amino acids, length of 171 amino acids, length of 172 amino acids, length of 173 amino acids, length of 174 amino acids, length of 175 amino acids, length of 176 amino acids, length of 177 amino acids, length of 178 amino acids, length of 179 amino acids, length of 180 amino acids, length of 181 amino acids, length of 182 amino acids, length of 183 amino acids, length of 184 amino acids, length of 185 amino acids, length of 186 amino acids, length of 187 amino acids, length of 188 amino acids, length of 189 amino acids, length of 190 amino acids, length of 191 amino acids, length of 192 amino acids, length of 193 amino acids, length of 194 amino acids, length of 195 amino acids, length of 196 amino acids, length of 197 amino acids, length of 198 amino acids, length of 199 amino acids, length of 200 amino acids, length of 201 amino acids, length of 202 amino acids, length of 203 amino acids, length of 204 amino acids, length of 205 amino acids, length of 206 amino acids, length of 207 amino acids, length of 208 amino acids, length of 209 amino acids, length of 210 amino acids, length of 211 amino acids, length of 212 amino acids, length of 213 amino acids, length of 214 amino acids, length of 215 amino acids, length of 216 amino acids, length of 217 amino acids, length of 218 amino acids, length of 219 amino acids, length of 220 amino acids,It may be a length of 221 amino acids, 222 amino acids, 223 amino acids, 224 amino acids, 225 amino acids, 226 amino acids, 227 amino acids, 228 amino acids, 229 amino acids, 230 amino acids, 231 amino acids, 232 amino acids, 233 amino acids, 234 amino acids, 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids or 240 amino acids, or a length within the range defined by any two of these lengths. In the chimeric receptor, the spacer may constitute a polypeptide chain of a desired length, for example, by including any of 20 amino acids in any order. The 20 amino acids include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the spacer is located between the scFV or ligand-binding domain and the transmembrane region in the chimeric receptor. By customizing, selecting, constructing or optimizing the spacer to have a desired length, the binding affinity of the scFv domain or ligand-binding domain for the target cell may be improved, thereby enhancing cytotoxicity. In some embodiments, the linker or spacer located between the scFv domain or ligand-binding domain and the transmembrane domain may be 25 to 55 amino acids in length (for example, at least 25 amino acids in length, at least 26 amino acids in length, at least 27 amino acids in length, at least 28 amino acids in length, at least 29 amino acids in length, at least 30 amino acids in length, at least 31 amino acids in length, at least 32 amino acids in length, at least 33 amino acids in length, at least 34 amino acids in length, at least 35 amino acids in length, at least 36 amino acids in length, at least 37 amino acids in length, at least 38 amino acids in length, at least 39 amino acids in length, at least 40 amino acids in length, at least 41 amino acids in length, at least 42 amino acids in length, at least 43 amino acids in length, at least 44 amino acids in length, at least 45 amino acids in length, at least 46 amino acids in length, at least 47 amino acids in length,at least 48 amino acids in length, at least 49 amino acids in length, at least 50 amino acids in length, at least 51 amino acids in length, at least 52 amino acids in length, at least 53 amino acids in length, at least 54 amino acids in length or at least 55 amino acids in length, or an amino acid length within a range defined by any two of these lengths). Examples of spacers include a spacer consisting of only the IgG4 hinge, a spacer consisting of the IgG4 hinge linked to the CH2 domain and the CH3 domain, or a spacer consisting of the IgG4 hinge linked to the CH3 domain. Also, examples of spacers include those described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013), International Patent Application WO2014031687, U.S. Patent No. 8,822,647, U.S. Published Application No. 2014 / 0271635 (these documents are hereby expressly incorporated by reference in their entirety).

[0065] In some embodiments, as the signaling domain of the CAR, such as the first signaling domain or the costimulatory signaling domain, the intracellular domain or cytoplasmic domain of a protein or receptor protein that interacts with intracellular components can be mentioned. These intracellular domains can relay signals or be involved in signal relay. In some aspects, such interactions occur through information transmission by the intracellular domain via specific protein-protein interactions or protein-ligand interactions with effector molecules or effector proteins, and then a chain reaction of signal transduction occurs, enabling the signal to be sent to its destination. In some embodiments, the costimulatory domain is mentioned as the signaling domain. In some aspects, the costimulatory domain includes a signaling portion that provides a signal to T cells. This signaling portion provides a signal to enhance responses such as T cell effector responses, such as immune responses, activation, proliferation, differentiation, cytokine secretion, cytolytic activity, perforin activity, or granzyme activity, in addition to the first signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex. In some embodiments, as the intracellular signaling domain and / or costimulatory domain, the whole or part of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3, or the whole or part of a ligand that specifically binds to CD83 can be mentioned, but is not limited thereto.

[0066] In some embodiments, the compositions, cells, and vectors of the present invention include a marker sequence or a nucleic acid encoding the marker sequence, and examples of the marker sequence include a protein that serves as a label for cells. In some of the cell embodiments of the present invention, the cells of the present invention co-express a marker protein of a specific chimeric protein expressed on the cell. In some of the cell embodiments provided herein, the chimeric receptor is co-expressed with a specific marker protein. In some of the cell embodiments provided herein, the cell includes a nucleic acid encoding a chimeric receptor. Examples of the marker include a selectable marker sequence, such as a gene introduced into a vector or a cell to confer a property that enables artificial selection. The selectable marker sequence or the marker sequence may be a screenable marker, and by using such a marker sequence, a researcher can distinguish desired cells from undesired cells or enrich specific types of cells. In some embodiments, a vector encoding a chimeric receptor including a marker sequence encoding a cell surface selectable marker is provided. In the embodiments described herein, a CAR including a selectable marker sequence that may be selectable in experiments such as flow cytometry is provided. In some embodiments, the marker is a Her2tG protein or an EGFRt protein.

[0067] As used herein, "methotrexate (MTX)" has its ordinary and customary meaning in light of this specification, and includes, for example, but is not limited to, antimetabolites and folic acid antimetabolites. Methotrexate (MTX) acts by suppressing folic acid metabolism. In some embodiments, a method of generating genetically engineered multiplex T cells for adoptive T cell immunotherapy is provided. In the broadest sense, the method of the invention may include the step of providing a gene delivery polynucleotide according to any of the embodiments described herein, and the step of selecting cells containing the gene delivery polynucleotide by adding a selection reagent. In some of the embodiments described herein, the selection reagent includes a selection agent. In some embodiments, the selection reagent is MTX.

[0068] As used herein, the term "dihydrofolate reductase" or "DHFR" has its ordinary and customary meaning in light of this specification, and includes, for example, but is not limited to, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid using NADPH as an electron donor, and this tetrahydrofolic acid can be converted into a cofactor derived from tetrahydrofolic acid used in C1 transfer reactions. In some of the embodiments of this specification, a gene delivery polynucleotide is provided. In some embodiments, the gene delivery polynucleotide includes at least one selectable marker cassette encoding a double mutant of dihydrofolate reductase (DHFRdm).

[0069] As used herein, the "ribosome skip sequence" refers to a sequence that has the function of causing the ribosome during translation to "skip" the ribosome skip sequence, thereby preventing the formation of peptide bonds and initiating translation from the region immediately following the ribosome skip sequence. For example, since some viruses have ribosome skip sequences, multiple proteins can be continuously translated from a single nucleic acid, and the translated proteins can be obtained as individual proteins without being linked by peptide bonds. As used herein, the ribosome skip sequence is used as a "linker" sequence. In some of the nucleic acid embodiments provided herein, the nucleic acid of the present invention contains a ribosome skip sequence between the sequence encoding the chimeric receptor and the sequence encoding the marker protein, so that the chimeric receptor and the marker protein are co-expressed without being linked by peptide bonds. In some embodiments, the ribosome skip sequence is a P2A sequence, a T2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the ribosome skip sequence is a T2A sequence.

[0070] As used herein, "zetakine" may refer to a specific type of CAR that includes a cytokine that can specifically bind to a cytokine receptor in its ligand-binding domain. In some embodiments, such cytokines include human cytokines, such as IL-13. In some embodiments, this IL-13 contains mutations in its sequence and exhibits high affinity for the IL-13 receptor α2.

[0071] As used herein, "mutant" has its ordinary and general meaning in light of this specification, and examples include, but are not limited to, proteins obtained by mutation. "Mutant of IL-13" may be a mutant of IL-13 that may have at least one or at least two amino acid substitutions.

[0072] As used herein, the term "interleukin-13 receptor alpha 2 subunit (IL-13Rα2)", also known as CD213A2 (cluster of differentiation 213A2), has its ordinary and customary meaning in the context of this specification. For example, in humans, it includes, but is not limited to, the cell membrane-bound protein encoded by the IL-13RA2 gene. IL-13Rα2 is closely related to IL-13Rα1, which is one of the subunits of the interleukin-13 receptor complex. IL-13Rα2 usually binds to IL-13 with high affinity, but has no functional cytoplasmic domain and is not considered to function as a signal transduction mediator. On the other hand, although IL-13Rα2 cannot directly bind to IL-4, it can control the actions of IL-13 and IL-4. Furthermore, it has been reported that IL-13Rα2 plays a certain role in the intracellular trafficking of IL-13.

[0073] As used herein, the term "T cell", i.e., "T lymphocyte", may be a T cell obtained from any mammalian species, such as a T cell obtained from a monkey, dog, human, etc., but is preferably a T cell obtained from a primate. In some embodiments, the T cell is, for example, of the same species as the recipient subject to whom the T cell is to be administered, such as in the form of a therapeutic composition, or the recipient subject to whom the T cell has been administered (the same species but derived from a different donor). In some embodiments, the T cell is autologous (the donor and recipient are the same). In some embodiments, the T cell is syngeneic (the donor and recipient are different but are monozygotic twins).

[0074] As used herein, the term "cytotoxic T lymphocyte (CTL)" has its ordinary and customary meaning in the context of this specification. For example, it includes, but is not limited to, T lymphocytes that express CD8 on their surface (e.g., CD8 + T cells). In some embodiments, such cells are "memory" T cells that have experienced an antigen (T MIt is preferably a (cell). In some embodiments, the cell is a cytotoxic T lymphocyte. As used herein, a "central memory" T cell (or "T CM ") is a cytotoxic T lymphocyte (CTL) that has experienced an antigen and expresses CD62L, CCR-7, and / or CD45RO on its surface compared to naive cells, but does not express CD45RA or has reduced expression of CD45RA. In some embodiments, the cell is a central memory T cell (T CM ). In some embodiments, central memory cells may have positive expression of CD62L, CCR7, CD28, CD127, CD45RO, or CD95, or a combination thereof, and reduced expression of CD54RA compared to naive cells. As used herein, an "effector memory" T cell (or "T EM ") is a T cell that has experienced an antigen and does not express CD62L or has reduced expression of CD62L on its surface compared to central memory cells, and does not express CD45RA or has reduced expression of CD45RA compared to naive cells. In some embodiments, the cell is an effector memory T cell. In some embodiments, effector memory cells have negative expression of CD62L and / or CCR7 compared to naive cells or central memory cells, and the expression of CD28 and / or CD45RA may be positive or negative.

[0075] Mature T cells express CD4 as a surface protein and are called CD4+ T cells. CD4+ T cells are generally regarded as being destined to function as helper T cells in the immune system. For example, when an antigen-presenting cell expresses an antigen on class II MHC, CD4+ cells assist the antigen-presenting cell via a combination of cell-cell interactions (such as CD40 and CD40L) and cytokines. However, there are rare exceptions, for example, regulatory T cells, natural killer cells or a subgroup of cytotoxic T cells also express CD4. None of these CD4+ expressing T cell populations are considered helper T cells.

[0076] As used herein, the term "central memory" T cells (or "T CM cells") has its ordinary and customary meaning in the context of this specification and includes, for example, antigen-experienced cytotoxic T lymphocytes (CTLs) that express CD62L, CCR-7, CD45RO, or any combination thereof on their surface and do not express CD45RA or have reduced expression of CD45RA, but is not limited thereto. In some embodiments, central memory cells have positive expression of CD62L, CCR7, CD28, CD127, CD45RO, or CD95, or any combination thereof, and reduced expression of CD54RA, compared to naive cells.

[0077] As used herein, the term "effector memory" T cells (or "T EMThe term ")", as used herein, has its ordinary and common meaning in light of this specification, and includes, for example, T cells that have experienced an antigen and do not express CD62L or have reduced expression of CD62L on their surface compared to central memory cells, and do not express CD45RA or have reduced expression of CD45RA compared to naive cells, but is not limited thereto. In some embodiments, effector memory cells have negative expression of CD62L or CCR7 or both, and positive or negative expression of CD28 or CD45RA or both, compared to naive cells or central memory cells.

[0078] As used herein, the term "naive" T cell has its ordinary and common meaning in light of this specification, and includes, for example, T lymphocytes that have not experienced an antigen and express CD62L or CD45RA or both, and do not express CD45RO, compared to central memory cells or effector memory cells, but is not limited thereto. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, and examples of phenotypic markers of naive T cells include CD62L, CCR7, CD28, CD127, or CD45RA, or any combination thereof.

[0079] As used herein, the term "effector" T cell or "T" E " has its ordinary and common meaning in light of this specification, and includes, for example, cytotoxic T lymphocytes that have experienced an antigen and do not express CD62L, CCR7, or CD28, or have reduced expression of CD62L, CCR7, or CD28, or cytotoxic T lymphocytes that are positive for granzyme B or perforin or a combination thereof, but is not limited thereto.

[0080] As used herein, the term "precursor T cell" has its ordinary and customary meaning in light of this specification, and includes, for example, lymphoid progenitor cells that can migrate to the thymus and become precursor T cells and that do not express a T cell receptor, but is not limited thereto. All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (lymphoid progenitor cells) derived from hematopoietic stem cells colonize the thymus, proliferate by cell division, and create a large population of immature thymocytes. The earliest thymocytes do not express either CD4 or CD8, and are thus classified as double-negative (CD4 - CD8 - ) cells. As they develop, they become double-positive thymocytes (CD4 + CD8 + ) and ultimately mature into single-positive (CD4 + CD8 - or CD4 - CD8 + ) thymocytes, and are then released from the thymus into peripheral tissues.

[0081] As used herein, the term "pharmaceutical additive" or "pharmaceutical carrier" has its ordinary and customary meaning in light of this specification, and includes, for example, a pharmaceutically active agent for the purpose of treatment or therapy or a carrier or inert medium used as a solvent for formulating and / or administering T cells, but is not limited thereto. Carriers include polymeric micelles, liposomes, lipoprotein-based carriers, nanoparticle carriers, dendrimers or other carriers for T cells known to those of skill in the art. Ideal carriers or additives include those that are non-toxic and have biocompatibility, non-immunogenicity and biodegradability, are not recognized by the host's defense mechanisms, or have any combination of such properties.

[0082] As used herein, "subject" or "patient" refers to any organism that may use or be administered the embodiments described herein for purposes such as experiments, diagnosis, prevention, and / or treatment. Examples of subjects or patients include animals. In some embodiments, the subject is a mouse, rat, rabbit, non-human primate, or human. In some embodiments, the subject is a cow, sheep, pig, horse, dog, cat, primate, or human.

[0083] Interleukin 13 receptor α2 (IL-13Rα2) has been found to be highly expressed in various cancer cells such as pancreatic cancer, breast cancer, ovarian cancer, and malignant glioma (e.g., glioblastoma). In addition, IL-13Rα2 may be overexpressed in most human patients with high-grade astrocytoma (see PLoS One. 2013 Oct 16; 8(10):e77719, which is hereby incorporated by reference in its entirety). Furthermore, suppressing the expression level of IL13RA2 in various models of cancer cells may significantly delay tumor growth (Breast Cancer Research, 2015; 17 (1), which is hereby incorporated by reference in its entirety). Most normal tissues do not express IL-13RA2, and in such cases, it is assumed that even if IL-13RA2 is expressed, the expression level is low. In the case of glioblastoma multiforme (GBM), the high expression of IL13Rα2 may be a prognostic marker indicating tumor progression and low patient survival rate.

[0084] Some of the embodiments provided herein include chimeric receptors, such as chimeric receptors having specificity for solid tumors (e.g., zeta cytokines). In some embodiments, the extracellular domain includes a binding domain that includes a variant of IL13 (e.g., IL13 E13Y), and this binding domain is linked to the transmembrane domain of the chimeric receptor via a spacer region. In some embodiments, this spacer region typically extends from the binding domain to the transmembrane domain. In some embodiments, the spacer includes a polypeptide having a length greater than 12 amino acids, 15 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, or 100 amino acids, or greater than about 12 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, or about 100 amino acids, or at least 12 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or at least 100 amino acids, or at least about 12 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, or at least about 100 amino acids, including, for example, a polypeptide having a length greater than 50 or 100 amino acids and less than 150 amino acids, less than 200 amino acids, or less than 220 amino acids, including, for example, a polypeptide having a length of 50-220 amino acids, 50-150 amino acids, or a polypeptide having a length of 110 amino acids or about 110 amino acids.In some embodiments, the transmembrane domain is or comprises the CD28 transmembrane domain (CD28tm), followed by a costimulatory domain after this transmembrane domain, and this costimulatory domain is, for example, a costimulatory domain derived from the intracellular segment of human 4-1BB (CD137) and a first signaling domain such as the signaling domain of CD3ζ.

[0085] In some embodiments, the nucleic acid encoding the chimeric receptor further comprises a sequence encoding a marker, and the sequence encoding this marker may be operably linked to the same promoter as the promoter to which the nucleic acid encoding the chimeric receptor is linked. In some aspects, the marker is a truncated form of a cell surface receptor, for example, a truncated form of the epidermal growth factor receptor (EGFRt), CD19 (CD19t) or HER2 (Her2t) or other receptors. In some aspects, the nucleic acid encoding the marker is separated from the nucleic acid encoding the chimeric receptor by a skip sequence such as a P2A ribosome skip sequence, a T2A ribosome skip sequence or an IRES. In some aspects, the nucleic acid further comprises a transgene encoding a double mutant of dihydrofolate reductase (DHFRdm), and the transgene encoding this DHFRdm may be added, for example, to facilitate the selection of cells (such as therapeutic T cell products) expressing the construct product by methotrexate.

[0086] Specific Nucleic Acids Some of the embodiments of the methods and compositions provided herein include a nucleic acid encoding a zeta cytokine, which is a receptor directed by a cell membrane-bound IL-13 mutein. In some embodiments, the nucleic acid is a) a first polynucleotide encoding an extracellular domain; b) a second polynucleotide encoding a mutein of IL-13; c) a third polynucleotide encoding a transmembrane domain; and d) A fourth polynucleotide encoding an intracellular signaling domain is included. In some embodiments, the nucleic acid further comprises a polynucleotide encoding a marker polypeptide (such as EGFRt, etc.). In some embodiments, the nucleic acid further comprises a polynucleotide encoding a selectable marker (such as DHFRdm, etc.). In some embodiments, the nucleic acid comprises a ribosome skip sequence.

[0087] In some embodiments, the mutein of IL13 comprises an amino acid sequence having an identity (%) with the amino acid sequence of SEQ ID NO: 16, consists of such an amino acid sequence, or consists essentially of such an amino acid sequence. In some of such embodiments, the sequence identity with SEQ ID NO: 16 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%, or a percentage between any two of these percentages. In some embodiments, the mutein of IL13 comprises the amino acid sequence of SEQ ID NO: 16, consists of this amino acid sequence, or consists essentially of this amino acid sequence.

[0088] In some embodiments, the extracellular domain comprises at least one peptide spacer. In some embodiments, the peptide spacer is 15 amino acids or less in length and 1 amino acid or more or 2 amino acids or more in length. In some embodiments, the spacer is a polypeptide chain. In some embodiments, the spacer comprises an IgG4 hinge spacer or a portion thereof. In some embodiments, the spacer comprises a hinge region of a human antibody or a portion thereof. In some embodiments of the method, the spacer comprises a hinge region of IgG4 or a portion thereof. In some embodiments, the IgG4 hinge region is a modified IgG4 hinge or a portion thereof. As used herein, a "modified IgG4 hinge" may refer to a hinge region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with the amino acid sequence of the hinge region shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or a hinge region having sequence identity within a range defined by any two of these percentages. In some embodiments, the spacer is a short (S) spacer, a medium-length (M) spacer or a long (L) spacer. The short (S) spacer comprises the sequence shown in SEQ ID NO: 9. The medium-length (M) spacer comprises the sequence shown in SEQ ID NO: 10. The long (L) spacer comprises the sequence shown in SEQ ID NO: 11.

[0089] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (short (S) spacer), an IgG4 hinge-CH3 spacer (medium-length (M) spacer), or an IgG4 hinge-CH2-CH3 spacer. In some embodiments, the spacer comprises an IgG4-CH3 spacer (medium-length (M) spacer).

[0090] In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having a sequence identity (%) with the amino acid sequence of SEQ ID NO: 10. In some of such embodiments, the sequence identity with SEQ ID NO: 10 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%, or a percentage between any two of these percentages. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10.

[0091] In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having a sequence identity (%) with the amino acid sequence of SEQ ID NO: 11. In some of such embodiments, the sequence identity with SEQ ID NO: 11 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%, or a percentage between any two of these percentages. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11.

[0092] In some embodiments, the spacer has at least 10 to 229 amino acids, at least 10 to 200 amino acids, at least 10 to 175 amino acids, at least 10 to 150 amino acids, at least 10 to 125 amino acids, at least 10 to 100 amino acids, at least 10 to 75 amino acids, at least 10 to 50 amino acids, at least 10 to 40 amino acids, at least 10 to 30 amino acids, at least 10 to 20 amino acids, or at least 10 to 15 amino acids, or a number of amino acids represented by any integer between the two ends of these ranges. In some embodiments, the spacer region has 1 to 12 amino acids, 1 to 119 amino acids, or 1 to 229 amino acids. In some embodiments, the spacer is more than 1 amino acid in length and less than 250 amino acids in length, more than 1 amino acid in length and less than 200 amino acids in length, more than 1 amino acid in length and less than 150 amino acids in length, more than 1 amino acid in length and less than 100 amino acids in length, more than 1 amino acid in length and less than 75 amino acids in length, more than 1 amino acid in length and less than 50 amino acids in length, more than 1 amino acid in length and less than 25 amino acids in length, more than 1 amino acid in length and less than 20 amino acids in length, more than 1 amino acid in length and less than 15 amino acids in length, more than 1 amino acid in length and less than 12 amino acids in length, or more than 1 amino acid in length and less than 10 amino acids in length. In some embodiments, the spacer is 10 to 250 amino acids in length, 10 to 150 amino acids in length, 10 to 100 amino acids in length, 10 to 50 amino acids in length, 10 to 25 amino acids in length, 10 to 15 amino acids in length, 15 to 250 amino acids in length, 15 to 150 amino acids in length, 15 to 100 amino acids in length, 15 to 50 amino acids in length, 15 to 25 amino acids in length, 25 to 250 amino acids in length, 25 to 100 amino acids in length, 25 to 50 amino acids in length, 50 to 250 amino acids in length, 50 to 150 amino acids in length, 50 to 100 amino acids in length, 100 to 250 amino acids in length, 100 to 150 amino acids in length, or 150 to 250 amino acids in length. Representative spacers include a spacer consisting of only the IgG4 hinge, a spacer consisting of an IgG4 hinge linked to the CH2 and CH3 domains, or a spacer consisting of an IgG4 hinge linked to the CH3 domain.Also, representative spacers include, but are not limited to, the spacers described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013), International Patent Application WO2014031687, U.S. Patent No. 8,822,647, and U.S. Published Application No. 2014 / 0271635 (these documents are hereby expressly incorporated by reference in their entirety).

[0093] In some embodiments, the "transmembrane domain" is a hydrophobic protein region that penetrates the bilayer of the cell membrane and serves to anchor proteins embedded in the biological membrane. The topology of the transmembrane domain may be, but is not limited to, a transmembrane α-helix. In some embodiments, the transmembrane domain includes the CD28 transmembrane sequence or a fragment thereof, and the CD28 transmembrane sequence or a fragment thereof has a length, for example, of 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, or 28 amino acids, or a length within the range defined by any two of these lengths. In some embodiments, the CD28 transmembrane sequence or a fragment thereof is 28 amino acids in length.

[0094] In some embodiments, the intracellular signaling domain includes a combination of a co-stimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and combinations thereof, and the whole or a part of the CD3ζ domain. In some embodiments, the intracellular signaling domain includes the signal-transducing functional part of the CD3ζ domain and the co-stimulatory functional part of the 4-1BB domain.

[0095] In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some aspects, the marker is an end-truncated form of a cell surface receptor, for example, an end-truncated form of epidermal growth factor receptor (EGFRt), CD19 (CD19t), or HER2 (Her2t), or other receptors. In some embodiments, the marker sequence is an end-truncated form of a cell surface receptor and may be EGFRt.

[0096] In some embodiments, the nucleic acid further comprises a transgene encoding dihydrofolate reductase configured to be selectable by methotrexate. In some embodiments, the transgene encoding dihydrofolate reductase is a double mutant of dihydrofolate reductase (DHFRdm). In some embodiments, the double mutant of dihydrofolate reductase comprises an L22F amino acid mutation and an F31S amino acid mutation.

[0097] In some embodiments, the nucleic acid further comprises a sequence encoding a ribosome skip sequence. In some embodiments, the ribosome skip sequence comprises P2A or T2A.

[0098] In some embodiments, the nucleic acid is modified such that the GC / AT ratio of the entire nucleic acid is reduced. In some embodiments, the nucleic acid is codon-optimized for expression in humans.

[0099] Some of the embodiments of the methods and compositions provided herein include expression vectors comprising nucleic acids according to any of the embodiments of the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector or an adenoviral vector. In some embodiments, the receptor zeta cytokine comprises an extracellular domain comprising an IL-13 mutein and a spacer; a transmembrane domain; and an intracellular signaling region, and the spacer is located between the mutein and the transmembrane domain.

[0100] Specific Chimeric Receptors Some embodiments of the methods and compositions provided herein include a chimeric receptor polypeptide encoded by a nucleic acid according to any embodiment of the invention or by a vector according to any embodiment of the invention. Some embodiments of the methods and compositions provided herein include a zeta cytokine, which is a receptor directed by a cell membrane-bound IL13 mutein and encoded by a nucleic acid according to any embodiment of the invention or by a vector according to any embodiment of the invention.

[0101] Specific Host Cells Some embodiments of the methods and compositions provided herein include host cells comprising a nucleic acid according to any embodiment of the invention or an expression vector according to any embodiment of the invention. In some embodiments, the host cell comprises a genetically engineered cell. In some embodiments, the host cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocyte is a central memory T cell, and the central memory T cell is CD45RO+, CD62L+, and CD8+. In some embodiments, the host cell is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocyte is a naive CD4+ T cell, and the naive CD4+ T cell is CD45RA+, CD62L+, and CD4+ and CD45RO-. In some embodiments, the host cell is a progenitor T cell. In some embodiments, the host cell is a hematopoietic stem cell.

[0102] Some embodiments of the methods and compositions provided herein provide host cells according to any of the embodiments of the invention or compositions according to any of the embodiments of the invention for use in the treatment or suppression of cancer or solid tumors that express IL-13 receptor α2. The composition includes a host cell according to any of the embodiments of the invention and a pharmaceutical additive. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13Rα-positive malignant tumor. In some embodiments, the cancer is a brain cancer or a brain tumor. Thus, some embodiments relate to host cells according to any of the embodiments described herein for use in a pharmaceutical or for the treatment or suppression of cancers such as IL13Rα-positive malignant tumors, glioblastoma multiforme (GBM), gliomas, and brain cancers including, but not limited to, glioblastomas.

[0103] Specific Compositions Some embodiments of the methods and compositions provided herein include compositions comprising a host cell according to any of the embodiments of the invention and a pharmaceutically acceptable additive.

[0104] Specific Methods for Producing Host Cells Some embodiments of the methods and compositions provided herein include methods of making a host cell according to any of the embodiments of the invention. Some such embodiments include a) introducing a nucleic acid according to any of the embodiments of the invention or an expression vector according to any of the embodiments of the invention into lymphocytes; b) culturing the lymphocytes in the presence of an anti-CD3 antibody or an anti-CD28 antibody and at least one constant cytokine; and c) selecting the lymphocytes with a selection reagent configured to selectively enrich the cells transduced with the nucleic acid or the vector including. In some embodiments, the selection reagent is methotrexate. In some embodiments, the lymphocytes have a CD45RA−, CD45RO+ or CD62L+ phenotype, or any combination thereof. In some embodiments, the lymphocytes are CD8+ or CD4+. In some embodiments, the cytokine is IL-15, IL-7 or IL-21, or any combination thereof. In some embodiments, the method further comprises introducing into the host cell a second nucleic acid encoding a marker protein. In some embodiments, the marker protein is EGFRt. In some embodiments, the expression vector comprises a nucleic acid according to any of the embodiments of the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector or an adenoviral vector.

[0105] In some embodiments, the host cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocyte is a central memory T cell, and the central memory T cell is CD45RO+, CD62L+ or CD8+, or any combination thereof. In some embodiments, the host cell is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocyte is a naive CD4+ T cell, and the naive CD4+ T cell is CD45RA+, CD62L+ or CD4+, or any combination thereof, and is CD45RO−. In some embodiments, the host cell is a progenitor T cell. In some embodiments, the host cell is a hematopoietic stem cell.

[0106] Specific Therapeutic Methods Some of the embodiments of the methods and compositions provided herein include the use of the host cells of the invention in a method of treatment. Some of such embodiments include the use of the cells in the treatment, inhibition or alleviation of cancer or solid tumors that express IL-13 receptor α2 (IL-13Ra2). In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL-13Rα-positive malignancy. In some embodiments, the cancer is a brain cancer or a brain tumor.

[0107] Some embodiments of the methods and compositions provided herein are methods of performing adoptive cell therapy in a subject having cancer or a tumor, the method comprising administering to the subject a host cell according to any of the embodiments of the present invention or a composition according to an embodiment of the present invention. The composition comprises a host cell according to any of the embodiments of the present invention and a pharmaceutically acceptable additive. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13Rα-positive malignant tumor. In some embodiments, the cancer is a brain cancer. In some embodiments, the subject is a subject selected for administration of combination therapy. In some embodiments, the combination therapy comprises administration of a chemotherapeutic agent. In some embodiments, the combination therapy comprises administration of radiation therapy. In some embodiments, the chemotherapeutic agent comprises electrochemotherapy, an alkylating agent, an antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin or thioguanine), an antitumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, a DNA intercalator or a checkpoint inhibitor (checkpoint kinase CHK1 or CHK2). In some embodiments, the cancer is glioma.

Example

[0108] Example 1 - Construction of IL-13 (E13Y) Zeta-Cytokine CAR Various IL-13(E13Y) zeta cytokine CARs with different spacer regions were constructed. As shown in Figure 1, the nucleic acid encoding the IL-13(E13Y) zeta cytokine CAR comprised a leader sequence (EF1p); a polynucleotide encoding a mutein of IL-13 (E13Y); a polynucleotide encoding any one of three spacers; a polynucleotide encoding a CD28tm transmembrane sequence; and a polynucleotide encoding a signaling domain containing a 4-1BB domain as the first signaling domain and a CD3ζ domain as the co-stimulatory domain. The three spacers were a short "S" spacer containing a modified IgG4 hinge; a medium-length "M" spacer containing a modified IgG4 hinge and the CH3 region of immunoglobulin (IgG4 hinge-CH3); and a long "L" spacer containing a modified IgG4 hinge, the CH2 domain of immunoglobulin, and the CH3 domain of immunoglobulin (IgG4 hinge-CH2-CH3). The L spacer contained two mutations (L235D, N297Q) in the CH2 domain, which could reduce the possibility of undesirable effects caused by the interaction between the CAR and the Fc receptor (FcR). Furthermore, the CAR contained a self-cleaving ribosome skip sequence 2A peptide (T2A). In some constructs, the lentivector further contained a transgene encoding a double mutant of dihydrofolate reductase (DHFRdm) configured to be selectable by methotrexate. The mutein of IL-13 is shown in SEQ ID NO: 16. An example of the sequence of the mutein of IL-13 is described in the literature by Kahlon et al. (Kahlon KS et al., Cancer Res. 2004; this literature is incorporated herein by reference in its entirety). As described herein, the E13Y mutation improved the selective binding to IL-13Rα2.

[0109] Example 2 - In Vitro Comparison of Various CARs with Different Spacers According to the method shown in FIG. 2A, various T cells with different CARs were prepared. First, CD8+ T cells were selected from peripheral blood mononuclear cells (PBMCs) using microbeads. After stimulating the selected cells with CD3 / CD28 microbeads, the construct was transduced using lentivirus. In some repeated experiments (*), a construct further containing T2A-DHFRdm inserted in-frame downstream of EGFRt was transduced into the CD8 T cells to co-express T2A-DHFRdm, enabling the selection of MTX-resistant T cells that co-express the IL13 zeta cytokine CAR in a functionally significant amount. Next, the cells were stained by flow cytometry to identify the cells expressing the zeta cytokine CAR, and the cells were further selected using the EGFRt marker. Representative flow cytometry data shown in FIG. 2B indicated that CD8+ T cells were efficiently selected, and then, by utilizing the expression of the surrogate marker (EGFRt), almost pure IL13 zeta cytokine CAR-expressing CD8+ T cells could be enriched or selected (*).

[0110] Furthermore, the specific lysis of IL13Ra2-expressing target cells by CD8+ T cells containing the zeta cytokine CAR with an S spacer, an M spacer, or an L spacer, and the cytokine expression by the CD8+ T cells in the presence of target cells were evaluated. First, the expression of IL13Ra2 in U87 cells, U251T cells, and DAOY cells as target cells was analyzed (FIG. 3A). In the 4-hour 51 Cr cytotoxicity assay, the labeled U87 tumor cell line, U251T tumor cell line, and DAOY tumor cell line were co-cultured with Mock T cells or various second-generation IL13 zeta cytokine CAR T cells with different spacers (FIG. 3B). Specific lysis was induced in the IL13 zeta cytokine CAR T cells with an M spacer and the IL13 zeta cytokine CAR T cells with an L spacer, but the IL13 zeta cytokine CAR T cells with a short spacer could not efficiently target IL13Ra2. The data showed the mean ± SD of three different donors.

[0111] In the cytokine release assay, T cells containing the zeta cytokine CAR were cultured with target cells for 24 hours. The cell-free supernatant was collected, and the secretion of TNFα, IFNγ, and IL-2 was measured. CD8+ T cells containing the zeta cytokine CAR with an M spacer and CD8+ T cells containing the zeta cytokine CAR with an L spacer had high cytokine production, but cytokine production was hardly observed in CD8+ T cells containing the zeta cytokine CAR with an S spacer (Figure 3C).

[0112] Example 3 - Antitumor Activity of IL13 Zeta-Cytokine CAR In Vivo The in vivo antitumor activity of the IL13 zeta cytokine CAR containing an M spacer or an L spacer was tested. On day 0, U87 glioblastoma cells labeled with ffLuc (0.2×10 6 cells) were intracranially injected into the forebrain of NSG mice. On day 7, CD8+ T cells transduced with the second-generation IL13 zeta cytokine CAR containing a medium-length spacer or a long spacer were administered to mice (n = 5 per group) at various doses (2×10 6 cells or 1×10 6 cells), or Mock T cells were administered to the mice.

[0113] As an indicator of tumor burden, the total flux (photons / second) from U87 cells was measured. As shown in Figure 4A, the tumor burden decreased over time in mice treated with cells containing the IL13 zeta cytokine CAR with an L spacer compared to Mock-treated mice and mice treated with the IL13 zeta cytokine CAR with an L spacer. The Kaplan-Meier survival curves showed an improvement in survival rate in mice administered cells containing the IL13 zeta cytokine CAR with an L spacer at various doses (Figure 4B).

[0114] Example 4 - Analysis of IL13 Zeta-Cytokine CAR In Vitro Cells containing an IL13 zeta cytokine CAR with an M spacer or an L spacer were generated by a method substantially similar to the method shown in FIG. 5A. Briefly, CD4+ T cells and CD8+ T cells were isolated and co-cultured. After 2 days, the T cells were transduced with an IL-13 zeta cytokine with an M spacer or an IL-13 zeta cytokine with an L spacer. After the end of stimulation (S1D13), the cells were analyzed for the CD4:CD8 ratio, marker expression, and direct expression of the zeta cytokine by staining with an anti-IL-13 antibody (FIG. 5B).

[0115] The cells were tested in an in vitro functional analysis (FIG. 6). After co-culturing T cells directed by the IL-13 zeta cytokine with IL-13Ra2-expressing target cells, a chromium release assay was performed to evaluate the degree of specific lysis. In this assay, T cells on day 15 of stimulation (S1D15) were co-cultured with various target cell lines, and the ratio of effector cells to target cells was varied. K562-OKT3 cells were used as a positive control to activate T cells by induction of the TCR complex, and the other cell lines expressed IL-13Ra2 to varying degrees (see the upper table). The lower table shows the ratio of CD4 to CD8 in the T cells and the positive rate of the surrogate marker. The assay was performed by normalizing the total number of surrogate marker-positive cells. As a result of the test, it was shown that lysis against IL-13Ra2-positive target cells was induced by both the zeta cytokine with an M spacer and the zeta cytokine with an L spacer.

[0116] Example 5 - Antitumor Activity of IL13 Zeta-Cytokine CAR In Vivo As shown in Fig. 7, IL-13 zeta cytokine with M spacer and IL-13 zeta cytokine with L spacer were observed to suppress tumor growth in a glioblastoma (GBM) model. The cells prepared in Example 4 and the cells on the 15th day after stimulation (S1D15) were tested in an orthotopic xenograft tumor model. This model was prepared by intracranial injection of GFP:ffluc-expressing U87 glioblastoma cells into NOD-Scid IL2yR-null mice. Seven days later, T cells directed by IL-13 zeta cytokine were injected ipsilateral to the GFP:ffluc-expressing U87 tumor. Evaluation of the flux generated from the ffluc-expressing U87 tumor showed that the tumor burden in vivo was reduced by IL-13 zeta cytokine-expressing T cells with M spacer or L spacer, and thus these IL-13 zeta cytokine-expressing T cells were observed to suppress tumor growth (Fig. 7). Statistical analysis of the change in flux 26 days after tumor inoculation showed that IL-13 zeta cytokine-expressing T cells with M spacer or L spacer significantly suppressed the occurrence of flux compared to Mock T cells (without IL-13 zeta cytokine).

[0117] Example 6 - Analysis of IL13 Zeta-Cytokine CAR In Vitro T cells directed by IL-13 zeta cytokine were analyzed by a chromium release assay. In this assay, rapidly cultured T cells on day 14 (S1R1D14) were co-cultured with various target cell lines, and at this time, the ratio of effector cells to target cells was varied. K562-OKT3 cells were used as a positive control to activate T cells by induction of the TCR complex, and the other cell lines expressed IL-13Ra2 to varying degrees (see the upper table). To demonstrate specific lysis of IL-13Ra2, mock T cells that had not been transduced for zeta cytokine expression were incorporated into this test as a negative control. The lower table shows the ratio of CD4 to CD8 in T cells and the positive rate of surrogate markers. The assay was performed by normalizing the total number of marker-positive cells. As a result of the test, it was shown that lysis of IL-13Ra2-positive target cells was induced by either the zeta cytokine with the M spacer or the zeta cytokine with the L spacer (Figure 8).

[0118] In another test, T cells expressing IL-13 zeta cytokine were co-cultured with various target cell lines for 24 hours. Here, the ratio of effector cells to target cells was set to 2:1. From the test data, it was shown that the zeta cytokines tested were able to specifically bind to IL-13Ra2 and induce cytokine release (Figure 9).

[0119] A list of specific amino acid sequences and nucleotide sequences in the embodiments provided herein is shown in Table 2. [Table 2] TIFF0007696958000003.tif233168TIFF0007696958000004.tif233168TIFF0007696958000005.tif233168TIFF0007696958000006.tif233168TIFF0007696958000007.tif234168TIFF0007696958000008.tif229169TIFF0007696958000009.tif234168TIFF0007696958000010.tif235168TIFF0007696958000011.tif233168TIFF0007696958000012.tif234169TIFF0007696958000013.tif233169TIFF0007696958000014.tif235169TIFF0007696958000015.tif235168TIFF0007696958000016.tif235169TIFF0007696958000017.tif64169

[0120] As used herein, the term "comprising" is synonymous with the terms "including", "containing", or "characterized by", and has an open-ended and inclusive meaning and does not exclude additional elements or steps not described herein.

[0121] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention can be modified, and the manufacturing methods and apparatuses can also be modified. Such modifications can be readily understood by those skilled in the art in view of the practice of the invention or the disclosure disclosed herein. Accordingly, the present invention is not limited to the specific embodiments disclosed herein, but encompasses all possible modifications and other aspects within the true scope and spirit of the present invention.

[0122] All references, including but not limited to published patent applications, unpublished patent applications, patents, and academic literature, cited herein are hereby incorporated by reference in their entirety and form part of this specification. If a document, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the description of this specification shall be adopted and / or given precedence over such conflicting matter.

[0123] The present invention includes the following inventions. [1] A nucleic acid encoding a zetakine, which is a receptor imparted with directivity by a cell membrane-bound IL-13 mutein, wherein the receptor zetakine comprises an extracellular domain containing a mutein of IL-13 and a spacer; a transmembrane domain; and an intracellular signaling region, and the spacer is located between the mutein and the transmembrane domain. [2] The nucleic acid according to [1], wherein the mutein of IL-13 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. [3] The nucleic acid according to [1] or [2], wherein the mutein of IL-13 comprises the amino acid sequence of SEQ ID NO: 16. [4] The nucleic acid according to any one of [1] to [3], wherein the spacer is a peptide spacer. [5] The nucleic acid according to any one of [1] to [4], wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (short spacer), an IgG4 hinge-CH3 spacer (medium-length spacer), or an IgG4 hinge-CH2-CH3 spacer (long spacer). [6] The nucleic acid according to any one of [1] to [5], wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (medium-length spacer). [7] The nucleic acid according to any one of [1] to [6], wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2-CH3 spacer (long spacer). [8] The nucleic acid according to any one of [1] to [7], wherein the spacer comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10, consists of this sequence, or consists essentially of this sequence. [9] The nucleic acid according to any one of [1] to [8], wherein the spacer comprises the amino acid sequence of SEQ ID NO: 10, consists of this sequence, or consists essentially of this sequence.

[10] The nucleic acid according to any one of [1] to [9], wherein the spacer comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, consists of this sequence, or consists essentially of this sequence.

[11] The nucleic acid according to any one of [1] to

[10] , wherein the spacer comprises the amino acid sequence of SEQ ID NO: 11, consists of this sequence, or consists essentially of this sequence.

[12] The nucleic acid according to any one of [1] to

[11] , wherein the transmembrane domain comprises the CD28 transmembrane domain (CD28tm).

[13] The nucleic acid according to any one of [1] to

[12] , wherein the intracellular signaling domain comprises a combination of a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3 and combinations thereof, and the whole or a part of the CD3ζ domain.

[14] The nucleic acid according to any one of [1] to

[13] , wherein the intracellular signaling region comprises the signal transduction functional part of the CD3ζ domain and the costimulatory functional part of the 4-1BB domain.

[15] The nucleic acid according to any one of [1] to

[14] , further comprising a sequence encoding a marker.

[16] The nucleic acid according to

[15] , wherein the marker comprises a truncated form of the cell surface receptor terminus, and the marker may be EGFRt.

[17] The nucleic acid according to any one of [1] to

[16] , further comprising a transgene encoding a dihydrofolate reductase configured to be selectable by methotrexate.

[18] The nucleic acid according to

[17] , wherein the transgene encoding the dihydrofolate reductase is a double mutant of dihydrofolate reductase (DHFRdm).

[19] The nucleic acid according to

[17] or

[18] , wherein the double mutant of the dihydrofolate reductase comprises an L22F amino acid mutation and an F31S amino acid mutation.

[20] The nucleic acid according to any one of [1] to

[19] , further comprising a sequence encoding a ribosome skipping sequence.

[21] The nucleic acid according to

[20] , wherein the ribosome skipping sequence comprises P2A or T2A.

[22] An expression vector comprising the nucleic acid according to any one of [1] to

[21] .

[23] The expression vector according to

[22] , which is a viral vector.

[24] The expression vector according to

[22] or

[23] , which is a lentiviral vector or an adenoviral vector.

[25] A chimeric receptor polypeptide encoded by the nucleic acid according to any one of [1] to

[21] .

[26] A host cell comprising the nucleic acid according to any one of [1] to

[21] .

[27] The host cell according to

[26] , which is a T cell or a progenitor T cell.

[28] The host cell according to

[26] or

[27] , which is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells.

[29] The host cell according to

[28] , wherein the CD8+ cytotoxic T lymphocyte is a central memory T cell, and the central memory T cell is CD45RO+, CD62L+ and CD8+.

[30] The host cell according to

[26] or

[27] , which is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

[31] The host cell according to

[30] , wherein the CD4+ helper lymphocytes are naive CD4+ T cells, and the naive CD4+ T cells are CD45RA+, CD62L+ and CD4+, and CD45RO-.

[32] The host cell according to any one of

[26] to

[31] , which is a progenitor T cell.

[33] The host cell according to any one of

[26] to

[32] , which is a hematopoietic stem cell.

[34] A composition comprising the host cell according to any one of

[26] to

[32] and a pharmaceutically acceptable additive.

[35] A method for producing a host cell according to any one of

[26] to

[32] , comprising the steps of introducing the nucleic acid according to any one of [1] to

[21] into lymphocytes; culturing the lymphocytes in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody and at least one constant cytokine; and selecting the lymphocytes with a selection reagent configured to selectively enrich the cells transfected with the nucleic acid or a vector containing the nucleic acid.

[36] A method for producing a host cell, comprising the steps of introducing the nucleic acid according to any one of [1] to

[21] into lymphocytes; culturing the lymphocytes in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody and at least one constant cytokine; and selecting the lymphocytes with a selection reagent configured to selectively enrich the cells transfected with the nucleic acid or a vector containing the nucleic acid.

[37] The method according to

[35] or

[36] , wherein the selection reagent is methotrexate.

[38] The method according to any one of

[35] to

[37] , wherein the lymphocytes have a phenotype of CD45RA-, CD45RO+ and CD62L+.

[39] The method according to any one of

[35] to

[38] , wherein the lymphocytes are CD8+ or CD4+.

[40] The method according to any one of

[35] to

[39] , wherein the cytokine is IL-15, IL-7 and / or IL-21. The method according to any one of

[35] to

[40] , further comprising the step of introducing a second nucleic acid encoding a marker protein into the host cell. The method according to

[41] , wherein the marker protein is EGFRt. The host cell according to any one of

[26] to

[32] , for use in a medicament or for use in the treatment or suppression of cancer or solid tumors that express IL-13 receptor α2 (IL-13Ra2). The use according to

[43] , wherein the cancer is a brain cancer. The use according to

[43] or

[44] , wherein the cancer is an IL-13Rα-positive malignant tumor. The use according to any one of

[43] to

[45] , wherein the cancer is glioblastoma. The use according to any one of

[43] to

[46] , wherein the cancer is glioblastoma multiforme (GBM). A method for treating, suppressing or alleviating cancer in a subject, comprising the step of administering to a subject in need of treatment, suppression or alleviation of cancer a host cell according to any one of

[26] to

[32] . The method according to

[48] , wherein the cancer is an IL13Rα-positive malignant tumor. The method according to

[48] or

[49] , wherein the cancer is a brain cancer. The method according to any one of

[48] to

[50] , wherein the cancer is glioma or glioblastoma. The method according to any one of

[48] to

[51] , wherein the cancer is glioma. The method according to any one of

[48] to

[51] , wherein the cancer is glioblastoma multiforme (GBM). The method according to any one of

[48] to

[53] , further comprising the step of administering an additional therapy selected from chemotherapy and radiotherapy.

[55] The method according to

[54] , wherein the chemotherapeutic agent comprises electrochemotherapy, alkylating agent, antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda (registered trademark)), cladribine, clofarabine, cytarabine (Ara-C (registered trademark)), floxuridine, fludarabine, gemcitabine (Gemzar (registered trademark)), hydroxyurea, methotrexate, pemetrexed (Alimta (registered trademark)), pentostatin, and thioguanine), antitumor antibiotic, topoisomerase inhibitor, mitotic inhibitor, corticosteroid, DNA intercalator, or checkpoint inhibitor (checkpoint kinases CHK1, CHK2).

[56] The method according to any one of

[48] to

[55] , wherein the subject is a mammal.

[57] The method according to any one of

[48] to

[56] , wherein the subject is a human.

Claims

1. A nucleic acid encoding a zeta cytokine, which is a receptor imparted with directivity by an IL-13 mutein, wherein the receptor zeta cytokine comprises an extracellular domain containing a mutein of IL-13; a spacer; a transmembrane domain; and an intracellular signaling region and the spacer consists of an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 10 and having a length within the range of 114 to 124 amino acids in length, the spacer links the mutein to the transmembrane domain, and the transmembrane domain links the spacer to the intracellular signaling region, a nucleic acid.

2. The nucleic acid according to claim 1, wherein the mutein comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

17.

3. The nucleic acid according to claim 1 or 2, wherein the mutein comprises the amino acid sequence shown in SEQ ID NO:

17.

4. The nucleic acid according to any one of claims 1 to 3, wherein the transmembrane domain comprises the CD28 transmembrane domain.

5. The nucleic acid according to any one of claims 1 to 4, wherein the intracellular signaling region comprises a combination of a co-stimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3 and combinations thereof and the whole or a part of the CD3ζ domain.

6. The nucleic acid according to any one of claims 1 to 5, wherein the intracellular signaling region comprises a part of the CD3ζ domain and the co-stimulatory part of the 4-1BB domain.

7. The nucleic acid according to any one of claims 1 to 6, further comprising an array encoding a marker protein.

8. The nucleic acid according to claim 7, wherein the marker protein comprises a truncated epidermal growth factor receptor or dihydrofolate reductase (DHFR).

9. The nucleic acid according to claim 8, wherein the DHFR is a double mutant of DHFR (DHFRdm).

10. The nucleic acid according to claim 9, wherein the DHFRdm comprises an L22F amino acid mutation and an F31S amino acid mutation.

11. The nucleic acid according to any one of claims 1 to 10, further comprising a ribosome skip sequence.

12. The nucleic acid according to claim 11, wherein the ribosome skip sequence comprises P2A or T2A.

13. An expression vector comprising the nucleic acid according to any one of claims 1 to 12.

14. The expression vector according to claim 13, which is a viral vector.

15. The expression vector according to claim 13 or 14, which is a lentiviral vector or an adenoviral vector.

16. Zeta cytokine, which is a receptor conferred with directivity by an IL-13 mutein encoded by the nucleic acid according to any one of claims 1 to 12.

17. A cell comprising the nucleic acid according to any one of claims 1 to 12.

18. The cell according to claim 17, which is a lymphocyte.

19. The cell according to claim 18, wherein the lymphocyte has a phenotype of CD45RA−, CD45RO+ and CD62L+.

20. The cell according to any one of claims 17 to 19, which is a naive CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell or a CD45RO+CD62L+CD8+ T cell.

21. The cell according to any one of claims 17 to 19, which is a naive CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell or a CD45RA+CD62L+CD4+CD45RO− T cell.

22. The cell according to claim 17, which is a progenitor T cell.

23. The cell according to claim 22, wherein the progenitor T cell is a hematopoietic stem cell.

24. A composition comprising the cell according to any one of claims 17 to 23 and a pharmaceutically acceptable carrier.

25. A method for producing a genetically engineered cell comprising the nucleic acid according to any one of claims 1 to 12, comprising: introducing the nucleic acid according to any one of claims 1 to 12 into a cell to produce a genetically engineered cell; culturing the genetically engineered cell in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody and at least one constant region cytokine; and concentrating the cultured genetically engineered cell The method comprising.

26. The method according to claim 25, wherein the nucleic acid further comprises a sequence encoding a marker protein, and the concentrating step comprises culturing the genetically engineered cell after culture with a reagent that selects for the expression of the marker protein.

27. The method according to claim 25 or 26, wherein the cell is a lymphocyte.

28. The method according to claim 27, wherein the lymphocytes have a phenotype of CD45RA−, CD45RO+ and CD62L+. **Claim 29** The method according to any one of claims 25 to 28, wherein the cells are naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells or CD45RO+CD62L+CD8+ T cells. **Claim 30** The method according to any one of claims 25 to 28, wherein the cells are naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, or CD45RA+CD62L+CD4+CD45RO− T cells. **Claim 31** The method according to claim 25 or 26, wherein the cells are progenitor T cells. **Claim 32** The method according to claim 31, wherein the progenitor T cells are hematopoietic stem cells. **Claim 33** The method according to any one of claims 25 to 32, wherein the at least one homeostatic cytokine is IL-15, IL-7, IL-21 or a combination thereof. **Claim 34** The method according to any one of claims 26 to 33, wherein the marker protein is a truncated epidermal growth factor receptor. **Claim 35** Use of the cells according to any one of claims 17 to 23 in the manufacture of a medicament for the treatment or suppression of a cancer expressing IL-13 receptor α2 (IL-13Ra2). **Claim 36** The use according to claim 35, wherein the cancer includes a brain cancer. **Claim 37** The use according to claim 36, wherein the brain cancer is glioma, glioblastoma or glioblastoma multiforme. **Claim 38** The use according to any one of claims 35 to 37, wherein the cancer is an IL-13Rα-positive malignant tumor.

39. The composition according to claim 24, which is for the treatment of cancer.

40. The composition according to claim 39, wherein the cancer is an IL-13Rα-positive malignant tumor.

41. The composition according to claim 39 or 40, wherein the cancer includes brain cancer.

42. The composition according to claim 41, wherein the brain cancer is glioma, glioblastoma or glioblastoma multiforme.

43. The composition according to any one of claims 39 to 42, which is for administration to a subject undergoing a therapy selected from chemotherapy and radiotherapy.

44. The composition according to claim 43, wherein the chemotherapy includes electrochemotherapy, alkylating agent, antitumor antibiotic, topoisomerase inhibitor, mitotic inhibitor, corticosteroid, DNA intercalator, checkpoint inhibitor or a combination thereof.

45. The composition according to claim 43, wherein the chemotherapy includes 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine or a combination thereof.

46. The composition according to any one of claims 43 to 45, which is for administration to a mammal.

47. The composition according to any one of claims 43 to 46, which is for administration to a human.

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