CAR T cell therapy and IFNγ

Abstract

Provided herein, inter alia, are compositions comprising chimeric antigen receptor (CAR) modified immune cells, methods of formulation, and methods useful for the treatment of cancer and leukemia.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 168,210, filed March 30, 2021, the contents of which are incorporated herein by reference in their entirety. The present disclosure relates to chimeric antigen receptor (CAR) recombinant immune cells, methods for their formulation, and methods of use.

[0002] SEQUENCE LISTING: This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on June 10, 2022, is named 40056-0070WO1_SL.txt and is 84,079 bytes in size. [Background technology]

[0003] Glioblastoma (GBM) is one of the most lethal cancers with extremely limited treatment options (Non-Patent Documents 1-3). Despite aggressive standard treatments, tumor recurrence is almost inevitable and nearly fatal, and most patients do not survive more than two years after diagnosis. Advances in immunotherapy have led to ongoing attempts to develop therapeutic strategies to induce antitumor immune responses in GBM, including adoptive transfer of chimeric antigen receptor (CAR) T cells. Clinical studies evaluating CAR T cells in GBM have demonstrated early evidence of safety and bioactivity in selected patients, but responses have nevertheless been limited. The challenges for efficient CAR T cell therapy for solid tumors such as GBM are multifactorial. Tumor heterogeneity and cellular plasticity lead to the proliferation of antigen-losing tumor variants and treatment failure. The tumor microenvironment in GBM tumors is bone marrow-rich and has a low T cell population, presenting specific challenges for CAR T cells.

[0004] IL13Rα2-CAR T therapy has shown some promise in the treatment of GBM, despite the heterogeneous expression of IL13Rα2 by tumor cells (Non-Patent Document 4). Responses were associated with increased CNS inflammatory cytokines and infiltration of endogenous immune cells (Non-Patent Document 4). Consistent with this observation, a recent longitudinal analysis of immune monitoring after HER2-CAR T cell therapy showed evidence of endogenous immune reactivity that may contribute to the patient's favorable response (Non-Patent Document 5). Proinflammatory cytokines secreted by CAR T cells, such as IFNγ, may play an important role in the activation and programming of immune infiltrates in the GBM TME. IFNγ can activate macrophages (Non-Patent Document 6) and microglia (Non-Patent Document 7), recruit and activate cytotoxic T cells, polarize CD4+ T cells into Th1 effector cells, and impair the development and function of tumor-promoting Tregs (Non-Patent Documents 8-10). IFN can further act as a major signal (Non-Patent Document 12) to promote the activation and priming of tumor-reactive T cells (Non-Patent Document 11). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] McGranahan T, Therkelsen KE, Ahmad S, Nagpal S.Current State of Immunotherapy for Treatment of Glioblastoma.Current treatment options in oncology 2019,20(3):24. [Non-Patent Document 2] Siegel RL, Miller KD, Jemal A. Cancer statistics,2018.CA:a cancer journal for Protestant 2018,68(1):7-30. [Non-Patent Document 3] Alexander BM,Cloughesy TF.Adult Glioblastoma.Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2017,35(21):2402-2409. [Non-Patent Document 4] Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al.Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy.

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[0006] Disclosed herein are immune system cells, e.g., T cells or NK cells, that express both a CAR that targets a tumor antigen and human IFNγ encoded by a nucleic acid molecule ("recombinant human IFNγ"), e.g., immune cells with a nucleic acid molecule encoding a CAR and human IFNγ. Without being bound by any theory, the co-expression appears to enhance one or more of immune cell activation, immune cell proliferation, and tumor cell killing by endogenous cells that recognize the tumor cells. The CAR may include a targeting domain that is an scFv that targets a tumor antigen (e.g., an scFv that targets CD19) or a ligand that binds to a receptor on a tumor cell (e.g., IL-13 or a variant thereof). Thus, the cell can carry a nucleic acid molecule that encodes a CAR and human IFNγ. The expression of the CAR and human IFNγ can be under the control of the same expression control sequence or different expression control sequences. The cell can carry a nucleic acid molecule that encodes a single amino acid sequence that includes a CAR and human interferon gamma. For example, the amino acid sequence of the CAR may be followed by a ribosomal skip sequence, followed by an amino acid sequence comprising human IFNγ. The amino acid sequence may include at least one signal sequence for secretion of the protein (e.g., a signal sequence for secretion of the CAR and a signal sequence for expression of human IFNγ). In an embodiment, the nucleic acid of the present disclosure may be a non-endogenous nucleic acid.

[0007] Immune cells expressing CAR and interferon can target and kill cancer cells expressing the CAR target. In addition, they can activate the killing of cancer cells that do not express the CAR target, for example, by activating natural and adaptive immune subsets in the tumor microenvironment. Thus, they are useful for treating tumors that contain both cancer cells expressing the CAR target and cancer cells that do not express the CAR target or have very low expression of the CAR target. The human IFNγ may comprise the following amino acid sequence:

[0008] [ka] The human IFNγ amino acid sequence may be preceded by a signal sequence that directs secretion of human interferon gamma from a eukaryotic cell (e.g., a human cell). Thus, the human interferon gamma precursor may be used (signal sequence underlined).

[0009] [ka] CARs can target tumor antigens, non-limiting examples of which include: Suitable IL-13 CARs include variants of human IL-13 that include the following amino acid sequence:

[0010] [ka] Sequence of wild type human IL13 (signal sequence underlined):

[0011] [ka] The IL-13 CAR can be, for example, 30a spacer (e.g., comprising an amino acid sequence as set forth in any of SEQ ID NOs: 2-12); a transmembrane domain (e.g., comprising an amino acid sequence as set forth in any of SEQ ID NOs: 13-20); a costimulatory domain (e.g., comprising an amino acid sequence as set forth in any of SEQ ID NOs: 22-25); and, in some cases, a linker having 3-15 amino acid residues (e.g., GGG); and a mutant IL13 comprising a CD3 zeta cytoplasmic domain (a mutant thereof comprising an amino acid sequence as set forth in any of SEQ ID NOs: 21 or 50-56). Useful CARs can comprise an amino acid sequence as set forth in any of SEQ ID NOs: 70-76.

[0012] [ka] SEQ ID NO:76 Provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is represented by SEQ ID NO: 30 The present invention relates to a nucleic acid molecule comprising a targeting domain comprising an amino acid sequence represented by SEQ ID NO: 1, a spacer, a transmembrane domain, a costimulatory domain, and a CD3ζ signaling domain. In one embodiment, the transmembrane domain is selected from a CD4 transmembrane domain or a variant thereof having one to five amino acid modifications, a CD8 transmembrane domain or a variant thereof having one to five amino acid modifications, a CD28 transmembrane domain or a variant thereof having one to five amino acid modifications; and the IL13 receptor targeting domain is selected from a CD4 transmembrane domain or a variant thereof having one to five amino acid modifications, a CD8 transmembrane domain or a variant thereof having one to five amino acid modifications; 30(in some cases, Y at position 13 is not substituted); the costimulatory domain is selected from a 41 BB costimulatory domain or a variant thereof having one to five amino acid modifications; a CD28 costimulatory domain or a variant thereof having one to five amino acid modifications; a CD28gg costimulatory domain or a variant thereof having one to five amino acid modifications, wherein the costimulatory domain is a 41 BB costimulatory domain; The BB costimulatory domain comprises an amino acid sequence represented by SEQ ID NO: 24 or a variant thereof having one to five amino acids modified; the CD3ζ signaling domain comprises an amino acid sequence represented by SEQ ID NO: 21 or a variant thereof having an amino acid sequence represented by any of SEQ ID NOs: 50 to 56; a linker having 3 to 15 amino acid residues is located between the costimulatory domain and the CD3ζ signaling domain or a variant thereof; the CAR comprises an amino acid sequence represented by SEQ ID NOs: 70 to 76 or a variant thereof having one to five amino acids modified; the CAR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 70 to 76; the CAR comprises an amino acid sequence having not more than 5, 4, 3, 2, or 1 single amino acid substitution and / or deletion compared to any of SEQ ID NOs: 70 to 76. Also disclosed is an expression vector comprising any of the above nucleic acid molecules. Also disclosed is a viral vector comprising any of the above nucleic acid molecules.

[0013] CARs may comprise scFvs targeting any cancer cell antigen, such as CD19, MUC16, MUC1, tMUC1, CAIX, CEA, CD20, CD22, CD30, HER-2, MAGEA3, p53, PSCA BCMA, CD123, CD44V6, integrin B7, ICAM-1, CD70, CEA, GD2, PSMA, B7H3, CD33, Flt3, CLL1, folate receptor, EGFR, CD7, EGFRvIII, glypican 3, CD5, ROR1, CS1, AFP, CD133, and TAG-72. CARs may comprise ligands, such as IL-13 or a variant thereof, chlorotoxin or a variant thereof, and the like. Thus, CARs useful for co-expression include those described in International Publication No. WO2016 / 044811, International Publication No. WO2017 / 079694, International Publication No. WO2017 / 066481, and International Publication No. WO2017 / 062628.

[0014] Also disclosed are populations of human T cells, NK cells, bone marrow cells, gamma delta T cells, or iPSC-derived effector cells containing any of the above nucleic acid molecules.Also disclosed are populations of human T cells containing any of the above expression vectors or viral vectors.In an embodiment, the population of human T cells comprises central memory T cells, naive memory T cells, panT cells, or PBMCs substantially depleted of CD25+ cells and CD14+ cells.

[0015] Also disclosed herein is a method of treating a patient suffering from cancer (e.g., brain cancer (glioblastoma), pancreatic cancer, melanoma, neuroblastoma, liver cancer, sarcoma, colorectal cancer, gastric cancer, ovarian cancer, fallopian tube cancer, thyroid cancer, bladder cancer, cervical cancer, digestive system cancer, head and neck cancer, osteosarcoma, renal cell carcinoma, prostate cancer, breast cancer, or lung cancer) comprising administering a population of autologous or allogeneic human T cells harboring a nucleic acid disclosed herein. In some embodiments, the cells are administered locally or systemically, and in single or repeated doses. Also disclosed herein is a method of preparing a CAR T cell, comprising providing a population of autologous or allogeneic human T cells and transducing the T cells with a vector comprising a nucleic acid molecule disclosed herein.

[0016] Also disclosed are T cells carrying a vector or nucleic acid expressing a CAR and IFNγ. In an embodiment, at least 20%, 30%, or 40% of the transduced human T cells are central memory T cells and at least 30% of the transduced human T cells are CD4+ and CD62L+ or CD8+ and CD62L+. In an embodiment, the population of human T cells is selected from the group consisting of SEQ ID NO: 30or 70-76, or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted); the population of T cells may include one or more of effector T cells, effector memory cells, central memory T cells, stem central memory cells, and naive T cells; the population of human T cells includes central memory T cells (TCM cells), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are TCM cells, or the population of T cells includes a combination of central memory T cells, naive T cells, and stem central memory cells (TCM / SCM / N cells), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are TCM / SCM / N cells. In certain embodiments, the population of T cells includes effector T cells and effector memory cells. In some embodiments, the population of T cells includes both CD4+ and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+, at least 3% of the CD3+ T cells are CD8+, and at least 70, 80, or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the CD3+ cells are CD4+, and at least 4%, 5%, 8%, 10%, 20% of the CD3+ cells are CD8+ cells). In some embodiments, the population of human T cells is autologous to the patient. In some embodiments, the population of human T cells is allogeneic to the patient. In some embodiments, the T cells expressing CAR and IFNγ are interchangeable with the descriptions IL13Rα2-IFNγCAR T cells, IL13Rα2-CAR / IFNγT cells, and IL13 CAR T-IFNγ cells, among others.

[0017] In certain embodiments, the spacer domain is selected from the group consisting of an IgG4(EQ) spacer domain, an IgG4(HL-CH3) spacer domain, and an IgG4(CH3) spacer domain; the spacer domain comprises SEQ ID NO: 10; the spacer domain comprises SEQ ID NO: 9; the spacer domain comprises SEQ ID NO: 12; the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD8 transmembrane domain, and a CD28 transmembrane domain; and the costimulatory domain is selected from a CD28 costimulatory domain, a CD28gg costimulatory domain, and a 41-BB costimulatory domain.

[0018] Further provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor being represented by SEQ ID NO: 30 or 109, 110, 111, 112, 113 consecutive amino acids of SEQ ID NO: 30 Also disclosed are nucleic acid molecules comprising a targeting domain comprising an amino acid sequence comprising a mutant IL13 domain comprising one, two, three, four or five single amino acid changes throughout the entire sequence; a spacer domain; a transmembrane domain; a costimulatory domain and a CD3 zeta signaling domain. In one embodiment, the spacer domain comprises an amino acid sequence represented by any of SEQ ID NOs: 2-12, and the costimulatory domain comprises an amino acid sequence represented by any of SEQ ID NOs: 22-25 and is a CD3 zeta domain or a mutant thereof. In one embodiment, the CAR comprises a CD28 costimulatory domain and a mutant CD3 zeta domain. Also disclosed herein are vectors or expression vectors comprising the nucleic acid molecules; populations of human T cells or NK cells carrying the nucleic acid molecules disclosed herein. In one embodiment, the population of human T cells comprises central memory T cells, naive memory T cells, panT cells, or PBMCs substantially depleted of CD25+ and CD14+ cells.

[0019] Also disclosed herein are methods of treating a patient suffering from glioblastoma, pancreatic ductal adenocarcinoma, melanoma, ovarian cancer, renal cell carcinoma, breast cancer, or lung cancer, comprising administering a population of autologous or allogeneic cells carrying a nucleic acid molecule disclosed herein, in some embodiments, the cells are administered locally, systemically, or intracerebroventricularly, and in single or repeated doses. Also disclosed herein is a method of preparing CAR T cells, comprising providing a population of autologous or allogeneic human T cells or NK cells, and transducing the cells with a vector comprising a nucleic acid molecule disclosed herein. Also disclosed herein are polypeptides encoded by the nucleic acids disclosed herein. In certain embodiments, the NK cells are derived from umbilical cord blood, peripheral blood, or stem cells.

[0020] The CAR or polypeptide may be expressed with additional sequences useful for monitoring expression, such as a T2A or P2A skip sequence and a truncated EGFR or truncated CD19 or LNGFR (which may consist of or include the amino acid sequence set forth in SEQ ID NO: 31). A non-endogenous or exogenous nucleic acid molecule (or polypeptide) is a nucleic acid molecule (or polypeptide) that is not endogenously present in a cell. The term includes recombinant nucleic acid molecules (or polypeptides) that are expressed in a cell. An exogenous nucleic acid is a nucleic acid that is not present in a native wild-type cell; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence or by position / location. An exogenous nucleic acid molecule can be introduced into a cell or a precursor of the cell by genetic recombination. An exogenous nucleic acid molecule encoding a polypeptide can be linked to expression control sequences and can include a sequence encoding a signal sequence, either or both of which may be heterologous to the sequence encoding the signal sequence.

[0021] Spacer Region The CAR or polypeptide disclosed herein may include a spacer located between the targeting domain (i.e., IL13 or a variant thereof) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least a portion of a human Fc region, such as a hinge portion or a CH3 domain of a human Fc region or a variant thereof. Table 1 below provides various spacers that can be used in the CARs disclosed herein. Table 1: Examples of spacers

[0022] [Table 1] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that resides between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (also referred to as CH3 or ΔCH2) or both the CH3 and CH2 domains. The immunoglobulin-derived sequence can include one or more amino acid modifications, e.g., substitutions of 1, 2, 3, 4 or 5 amino acids, e.g., substitutions that reduce off-target binding. The spacer region may also include an IgG4 hinge region with the sequence ESKYGPPCPSCP (SEQ ID NO: 4) or ESKYGPPCPPCP (SEQ ID NO: 3). The spacer region may also include the hinge sequence ESKYGPPCPPCP (SEQ ID NO: 3), followed by the linker sequence GGSSGGGGSG (SEQ ID NO: 2), followed by the IgG4 CH3 sequence

[0023] [ka] (SEQ ID NO: 12). Thus, the entire spacer region can include the sequence:

[0024] [ka] (Sequence number 9).

[0025] Transmembrane domain A variety of transmembrane domains can be used in the CAR. In some cases, the transmembrane domain is the CD28 transmembrane domain, which is represented by FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 14). In some cases, the CD28 transmembrane domain has 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes compared to SEQ ID NO: 14. Table 2 includes examples of suitable transmembrane domains. When a spacer region is present, the transmembrane domain (TM) is located at the carboxy terminus of the spacer region. Table 2: Examples of transmembrane domains

[0026] [Table 2] Costimulatory domain The costimulatory domain may be any domain suitable for use with the CD3ζ signaling domain. In some cases, the costimulatory domain is a CD28 costimulatory domain that comprises a sequence at least 90%, at least 95%, at least 98% identical or completely identical to RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 22). In some cases, the 4-1BB costimulatory domain has 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes compared to SEQ ID NO: 22. The costimulatory domain(s) is located between the transmembrane domain and the CD3ζ signaling domain. Table 3 provides examples of suitable costimulatory domains along with the sequence of the CD3ζ signaling domain. In some cases, the costimulatory domain is represented by RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 22). Table 3: Examples of CD3 ζ domains and costimulatory domains

[0027] [Table 3] In one embodiment, the costimulatory domain is selected from the group consisting of a costimulatory domain shown in Table 3 or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified, a CD28 costimulatory domain or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified, a 4-1BB costimulatory domain or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified, and an OX40 costimulatory domain or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified. In one embodiment, there are two costimulatory domains, for example, a CD28 costimulatory domain or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified (e.g., substituted) and a 4-1BB costimulatory domain or a variant thereof in which 1 to 5 (e.g., 1 or 2) amino acids are modified (e.g., substituted). In one embodiment, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions. The costimulatory domain is amino-terminal to the CD3ζ signaling domain, and a short linker of 2-10, e.g., 3 amino acids (e.g., GGG) can be placed between the costimulatory domain and the CD3ζ signaling domain.

[0028] CD3ζ Signaling Domain The CD3 ζ signaling domain can be any domain suitable for use in combination with the CD3 ζ signaling domain. In some cases, the CD3 ζ signaling domain is

[0029] [ka] and / or a sequence that is at least 90%, at least 95%, at least 98% identical or completely identical to In some cases, the CD3ζ signaling domain has 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes compared to SEQ ID NO: 21. In some cases, the CD3ζ signaling domain comprises any of SEQ ID NOs: 50-56. These mutant CD3ζ signaling domains have a Y to F mutation in one or more ITAM domains. In some cases, mutants with inactivating mutations in ITAMs 2 and 3 are preferably used.

[0030] Human IFN-γ An IFNγ domain is a domain that includes at least a functional portion of mature human IFNγ (eg, amino acids 24-161 of human IFNγ; GenBank NP_000610) or a functional portion of mature human IFNγ. Mature human IFNγ contains the following sequence:

[0031] [ka] Immature human IFNγ (including the signal sequence) has the following sequence:

[0032] [ka] In one embodiment, the human IFNγ has the following sequence:

[0033] [ka] Includes: In some cases, the IFNγ domain is SEQ ID NO:1 or SEQ ID NO: 29 or sequence number 32 For example, SEQ ID NO: 1 or SEQ ID NO: 2 have 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 32 In one embodiment, the IFNγ domain provided herein has one, two or all three amino acid changes of K74A, E75Y and N83R. 29 or sequence number 32In one embodiment, the IFNγ comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 32 At least one amino acid is substituted with an amino acid residue selected from positions corresponding to Q1, D2, P3, K6, Q64, Q67, K68, E71, T72, K74, E75, D76, N78, V79, K80, N83, S84, K86, R89, D90, and any combination thereof. In one embodiment, the IFNγ domain provided herein is SEQ ID NO:1 or SEQ ID NO: 32 and further comprising at least one sequence identical to SEQ ID NO: 1 or SEQ ID NO: 32 and any combination thereof. In some embodiments, mutants of IFNγ can also be used. Several IFNγ mutants are known in the art and may be useful (Mendoza JL, et.al., (2019) Nature 567:56; International Publication WO2020 / 028275).

[0034] Cleaved EGFR or Cleaved CD19 or LNGFR In an embodiment, the CAR or peptide disclosed herein comprises a ribosomal skipping sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27) and the following sequence:

[0035] [ka] The truncated EGFR includes a sequence that is at least 90%, at least 95%, at least 98% identical or completely identical to a truncated EGFR represented by the formula: In some cases, the truncated EGFR has 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes compared to SEQ ID NO:28.

[0036] In an embodiment, the CAR or peptide disclosed herein comprises a ribosomal skipping sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27) and the following sequence:

[0037] [ka] In some cases, the truncated CD19t has 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes compared to SEQ ID NO:26.

[0038] In an embodiment, the CAR or peptide disclosed herein comprises a ribosomal skipping sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27) and the following sequence:

[0039] [ka] The nucleic acid sequence includes a sequence that is at least 90%, at least 95%, at least 98% identical or completely identical to a tEGFR represented by the formula:

[0040] In one embodiment, the CAR or peptide disclosed herein comprises a ribosomal skip sequence and the following sequence:

[0041] [ka] In some cases, the truncated LNGFR comprises a sequence at least 90%, at least 95%, at least 98% identical or completely identical to a truncated LNGFR represented by SEQ ID NO: 33 In comparison to, 1, 2, 3, 4 or 5 amino acid (preferably conservative) changes have been made.

[0042] Other ribosomal skipping sequences useful in the CARs or peptides disclosed herein include T2A, which has a sequence at least 95% identical to the sequence represented by EGRGSLLTCGDVEENPGP (SEQ ID NO: 46), or P2A, which has a sequence at least 95% identical to the sequence represented by GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 47). In some cases, the ribosomal skipping sequence has 1, 2, 3, 4, or 5 amino acids (preferably conservative) changed compared to SEQ ID NO: 46 or 47.

[0043] Amino acid modification refers to the substitution, insertion, and / or deletion of amino acids in a protein or peptide sequence. An "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. Substitutions can be made in a non-conservative manner (i.e., by changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to another group) or in a conservative manner (i.e., by changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to the same group) to change the amino acids in the resulting protein. Such conservative changes generally result in less change in the structure and function of the resulting protein. The following are examples of different classifications of amino acids: 1) amino acids with non-polar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (pH 6.0). Another grouping may be amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0044] In some cases, CARs can be produced using vectors in which the CAR open reading frame is followed by a truncated EGFR (EGFRt) lacking a ribosomal skip sequence and a cytoplasmic signaling tail, or a truncated CD19R or LNGFR. This arrangement provides an inert, non-immunogenic surface marker that allows accurate measurement of genetically modified cells in co-expression of EGFRt, positive selection of genetically modified cells, and efficient cell tracking of therapeutic NK cells in vivo after adoptive transfer. Efficient control of proliferation to avoid cytokine storm and off-target toxicity is critical for the success of NK cell immunotherapy. EGFRt, CD19t, or LNGFR incorporated into CAR lentiviral or retroviral vectors can act as suicide genes to eliminate CAR+ cells in the event of treatment-related toxicity.

[0045] In some cases, the nucleic acid molecules disclosed herein include a promoter that controls both the expression of CAR and human interferon gamma. In other cases, the nucleic acid molecules disclosed herein include a first promoter that controls the expression of CAR and a second promoter that controls the expression of human interferon gamma. In some cases, the first and second promoters are the same, and in some cases, they are different. In some embodiments, the first promoter is a strong constitutive or inducible promoter. In some embodiments, the second promoter is a weaker promoter than the first promoter or is an inducible promoter. Useful promoters are well known in the art. For example, a synthetic NFAT promoter can be used in the nucleic acid encoding the CAR construct. Useful promoters may include one or more of CMV, EF1, SV40, PKG1, PKG100, Ubc, tetracycline, doxycycline, NFAT, and any other constitutive or inducible promoter. In some embodiments, an NFAT recognition element can be used (TGGAGGAAAAACTGTTTCATACAGAAGGCG; SEQ ID NO: 34In one embodiment, a useful promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 repeats of an NFAT recognition element. In one embodiment, a useful promoter comprises the sequence represented by SEQ ID NO: 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 and 48 Contains one or more of the following:

[0046] [ka]

[0047] [ka] The CAR or polypeptide disclosed herein may be produced by any means known in the art, but is preferably produced using recombinant DNA technology. Nucleic acids encoding several regions of the chimeric receptor may be advantageously prepared and assembled into complete coding sequences by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding regions are preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably immune cells (e.g., T cells), most preferably autologous T cells. The CAR or polypeptide may be transiently expressed in a cell population by an mRNA encoding the CAR or polypeptide. The mRNA can be introduced into immune cells by electroporation (Wiesinger et al. 2019 Cancers (Basel) 11:1198). In one embodiment, disclosed herein is a method of increasing survival of a subject with cancer comprising administering a composition comprising a CAR immune cell disclosed herein. Disclosed herein, in one embodiment, is a method of treating cancer in a patient comprising administering a composition comprising the CAR immune cells disclosed herein. In one embodiment, disclosed herein is a method of reducing or ameliorating symptoms associated with cancer in a patient comprising administering a composition comprising a CAR immune cell disclosed herein. In some embodiments, compositions comprising the CAR NK cells or CAR T cells disclosed herein are administered locally or systemically. In some embodiments, compositions comprising the CAR immune cells disclosed herein are administered in a single dose or multiple doses. In some embodiments, compositions comprising the CAR immune cells disclosed herein are administered to patients with cancer, pathogen infection, autoimmune disorders, or allogeneic transplants. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety for any and all purposes. Other features and advantages of the described compositions and methods will be apparent from the following detailed description and drawings, and from the claims. The present invention will now be described with reference to the drawings. [Brief description of the drawings]

[0048] [Figure 1]Figures 1A-1D: mIL13BBζ generation and phenotypic characterization of mIL13BBζ CAR T cells. 1A, Schematic showing mouse IL13Rα2-CAR T (mIL13BBζ CAR T) construct. 1B and 1C, Flow cytometry and graphs showing phenotypic changes of mouse CAR T cells from day 0 to day 4. 1D, In vitro killing of mIL13BBζ CAR T cells against IL13Rα2+ and IL13Rα2-K-Luc cells. [Diagram 2] Figures 2A-2I: Murine IL13BBζ CAR T cells have potent antitumor activity. 2A, Flow cytometry (left panel) and bar graph summarizing CAR+ T cell ratios demonstrating transduction efficiency (right). 2B and 2C, Immunofluorescence and flow cytometry staining confirmed the transduction of mIL13Rα2 in KR158 (K-Luc) glioma cells. 2D, In vitro killing of mIL13BBζ CAR T cells against K-Luc cells (E:T, 1:3). 2E, Luminex detected IFNγ and TNFα levels. 2F, Schematic showing the in vivo experimental design. 2G, Hematoxylin and eosin (H&E) images showed infiltrating K-Luc in untreated and CAR T-treated brains. 2H, Survival curves of mice bearing K-Luc-mIL13Rα2+ tumors in untreated and CAR T-treated groups. 2I, Bioluminescence images (BLI; top) and flux values ​​(bottom) show tumor growth in untreated and CAR T-treated groups. Data from at least three independent experiments are shown as mean ± sem (2D and 2E) and analyzed by unpaired two-tailed Student's t-test. Differences between survival curves were analyzed by log-rank (Mantel-Cox) test (2H). *p<0.05, **p<0.01, and ***p<0.001 for the indicated comparisons. [Diagram 3]Figure 3A-3F: mIL13BBζ CAR T cells had potent antitumor activity and induced endogenous memory immune responses against GL261-Luc tumors. 3A, Hematoxylin and eosin (H&E) images showed the morphology of GL261-Luc tumors. 3B, Immunofluorescence and flow cytometry staining confirmed the transduction of IL13Rα2 in GL261-Luc glioma cells. 3C, In vitro killing of mIL13BBζ CAR T cells against IL13Rα2+GL261-Luc glioma cells (E:T, 1:3). 3D, Luminex ELISA detected IFNγ and TNFα levels. 3E, Survival curves of mice bearing IL13Rα2+GL261-Luc glioma tumors in untreated and CAR T-treated groups. 3F, Survival of mice cured by CAR T therapy and rechallenged with IL13Rα2-negative GL261-Luc. Data from at least two independent experiments are presented as mean ± sem (3C) and analyzed by unpaired two-tailed Student's t-test. For comparison, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Differences between survival curves (3E) were analyzed by log-rank (Mantel-Cox) tested by log-rank (Mantel-Cox) test. [Figure 4] Figures 4A-4E: mIL13BBζ CAR T cells have superior antitumor activity in immunocompetent hosts. 4A, Schematic diagram showing in vivo experimental design with CAR administration on day 4 or day 7. 4B, Bioluminescence images (BLI; top) and flux values ​​(bottom) showing tumor growth in untreated and CAR T-treated groups in a 4-day-old tumor model. 4C, Survival curves of mice bearing 4-day-old K-Luc IL13Rα2+ tumors in untreated and CAR T-treated groups. Bioluminescence images (BLI; top) and flux values ​​(bottom) showing tumor growth in untreated and CAR T-treated groups in a 7-day-old tumor model. Survival curves of mice bearing 7-day-old K-Luc IL13Rα2+ tumors in untreated and CAR T-treated groups. Differences between survival curves (4C, 4E) were analyzed by log-rank (Mantel-Cox) test. [Diagram 5]Figure 5a-5f: CAR T cells induce endogenous memory immune responses and the generation of antigen-specific T cells. a, Survival of mice cured by CAR T therapy and rechallenged with IL13Rα2-negative K-Luc tumors. b, Bioluminescence (BLI) images (top) and flux values ​​(bottom) show tumor growth in survivors of naive control and CAR T therapy groups. c, Overview of experimental design. d, In vitro killing and e, proliferation of endogenous T cells isolated from untreated or CAR T-treated mice against K-Luc-mIL13Rα2+ cells (E:T, 10:1). f, Evaluation of the in vivo killing capacity of CAR T cells and endogenous T cells isolated from untreated or CAR T-treated cells in mice bearing tumors (K-Luc). Data include at least two independent experiments. Each symbol represents one individual. Data are presented as mean ± sem (d and e) and analyzed by unpaired two-tailed Student's t-test. Differences between survival curves were analyzed by the log-rank (Mantel-Cox) test (a). *p<0.05, **p<0.01, and ***p<0.001 for the indicated comparisons. [Figure 6] Figure 6a-6b: mIL13BBζ CAR T cells have potent antitumor activity but fail to induce endogenous memory immune responses in a small tumor model. a, Survival curves of mice bearing 4-day-old K-Luc IL13Rα2+ tumors in untreated and CAR T-treated groups. b, Survival of mice cured by CAR T therapy and rechallenged with IL13Rα2-negative K-Luc tumors. Differences between survival curves (a) were analyzed by log-rank (Mantel-Cox) test. [Figure 7]Figure 7a-7e: Comparison of survival in mice bearing mixed antigen tumors. a, Schematic of the in vivo experimental design on days 4 and 7. b, Flow cytometry showing different levels of IL13Rα2. c, Survival curves of K-Luc IL13Rα2+ tumor-bearing mice on day 4 in untreated and CAR T-treated groups. d, Survival curves of K-Luc IL13Rα2+ tumor-bearing mice on day 7 in untreated and CAR T-treated groups. e: Quantification of CD11b and CD3 cells after flow cytometry sorting of untreated K-Luc tumor-bearing mice. Each symbol represents one individual. Data are shown as mean ± sem. (e) Analyzed by two-tailed Student's t-test. For comparison, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Differences between survival curves (c, d) were analyzed by log-rank (Mantel-Cox) test. [Figure 8] Figure 8: Flow cytometric sorting of endogenous and CAR T cells. Representation of flow cytometric sorting of endogenous (CD3+CD19-) and CAR T (CD3+CD19+) populations. [Figure 9]Figure 9a-9g: CAR T cells activate endogenous T cells in the glioma tumor microenvironment. a, Nanostring analysis shows global changes in gene expression of intratumoral T cells (CD3+) isolated from untreated or CAR T-treated mice 3 days after treatment. b, UMAP plot shows changes in the lymphoid compartment in the glioma TME after CAR T therapy. c, Feature plot demonstrates enriched pathways within CD8 and CD4 T cell subclusters after treatment. d, Heat map of enrichment scores (GSEA analysis) shows enriched pathways in T cell subclusters. e, Experimental design demonstrating adoptive transfer of CD45.1+ mock or CAR T cells (top) and flow cytometry analysis shows the frequency of endogenous (CD3+CD45.2+) or adoptively transferred T cells (CD3+CD45.1+) in the glioma TME (bottom). f, Bar graphs compare numbers and phenotypic characterization (CD69, Ki67, and GZMB) of adoptively transferred mock (CD3+CD45.1+) or CAR T cells (CD3+CD45.1+CD19+). g, Bar graphs compare endogenous T cell (CD3+CD45.2+) numbers and phenotypic characterization (CD69, Ki67 GZMB, and IFNγ) in untreated, mock, or CAR T treated mice (n=5 / group). Data include at least two independent experiments. Data are presented as mean±sem (F and G) and analyzed by unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, and ***p<0.001 for the indicated comparisons. [Figure 10] Figure 10a-10d: Single cell RNA sequencing of intratumoral immune cells. a, UMAP plot merging unprocessed and CAR T-treated data of intratumoral immune cells only. b, Feature plot of immune subset-specific marker gene expression. c, Change in frequency of lymphoid subcluster (top) and violin plot (bottom) shows lymphoid-specific markers. d, Bar graph shows change in frequency of myeloid subcluster (top) and violin plot (bottom) shows myeloid-specific markers. [Figure 11]Figures 11a-11b: Single-cell RNA sequencing identifies the phenotype of intratumoral T cells. UMAP plots demonstrate enhancement of genes associated with memory stem-like T cells after CAR T therapy. a, UMAP plots show reduced expression of genes associated with regulatory T cells after CAR T therapy. [Figure 12] Figure 12: Expression of genes associated with T cell activation in intratumoral T cells. qPCR analysis shows genes associated with T cell activation. Data are shown as mean ± sem and analyzed by unpaired two-tailed Student's t-test. For comparison, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 13] Figure 13a-13f: CAR T cells activate resident myeloid populations in the glioma tumor microenvironment. a, UMAP shows changes in intratumoral myeloid cells from CAR T-treated or untreated mice. b, Enrichment plot of IFNγ signaling pathways in intratumoral macrophage and microglial cells in CAR T-treated compared to untreated, identified by GSEA computational methods. c, GSEA analysis reveals upregulation of population-specific activation pathways in myeloid subclusters (MP: macrophages; MG: microglia; DC: dendritic cells; Neu: neutrophils). d, Nanostring analysis shows global changes in gene expression in myeloid cells (CD11b+) isolated from CAR T-treated vs. untreated mice. e, UMAP shows relative expression levels of antigen-presenting gene signatures at the single-cell level within the myeloid compartment. f, Histogram (left) and bar graph (right) show intratumoral CD11b+CD45.2+ cells expressing MHCII, MHCI, CD86, and IFNγ. Data are shown as mean ± sem. Analyzed by unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for the indicated comparisons. [Figure 14]Figures 14a-14j: Deficiency of IFNγ in CAR T cells impairs antitumor activity and host immune cell activation. a, Schematic of experimental design. b, Comparison of percentage CAR positivity, viability, proliferation, and CAR 4:CAR 8 ratio in CAR Twt and CAR TIFNγ- / -. c, In vitro killing of CAR Twt and CAR TIFNγ- / - versus K-Luc-IL13Rα2+ cells (E:T, 1:1). d, Flow cytometry shows intracellular cytokine levels (TNFα, GZMB, and IFNγ) in wt and IFNγ- / - CAR T cells. e, Bioluminescence (BLI) images (top) and flux values ​​(bottom) show tumor growth in untreated, CAR Twt, or CAR TIFNγ- / -. f, Survival curves of mice bearing K-Luc-IL13Rα2+ tumors in untreated, CAR Twt, and CAR TIFNγ- / - groups. g, Heatmap shows normalized expression of genes associated with immune activation and suppression in tumors. h, Bar graph (left) and flow cytometry plot (left) comparing CAR T cell (CD3+CD19+) numbers and activation phenotype (CD69). i, Bar graph (left) and flow cytometry plot (right) comparing endogenous T cell (CD3+CD19) numbers and activation phenotype (CD69). j, Histogram (left) and bar graph (right) showing phenotype in bone marrow (CD11b+) compartment. Data include at least two independent experiments. Each symbol represents one individual. Data are presented as mean ± sem (h, i, and j) and analyzed by unpaired two-tailed Student's t test. Differences between survival curves (f) were analyzed by log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, and ***p<0.001 for the indicated comparisons. [Figure 15]Defective IFNγ production by CAR T cells impairs antitumor activity and host immune cell activation. A, Schematic of experimental design. B, Comparison of CAR positivity, survival, expansion rate and CD4:CD8 ratio in CAR Twt and CAR TIFNγ− / −. C, In vitro killing of CAR Twt and CAR TIFNγ− / − against K-Luc-mIL13Rα2+ cells (E:T, 1:1). D, Representative flow cytometry plots show intracellular cytokine levels (TNFα, GZMB and IFNγ) in wt and IFNγ− / − CAR T cells after exposure to K-Luc-mIL13Rα2+ tumors. E, Representative bioluminescence (BLI) images (top) and flux values ​​(bottom) show tumor growth in untreated, CAR Twt or CAR TIFNγ− / −. Individual mice are represented by dotted lines and median flux is indicated by bold line. F, Survival curves of mice bearing K-Luc-mIL13Rα2+ tumors in untreated, CAR Twt-, and CAR TIFNγ- / -treated groups. G, Heatmap shows normalized expression of genes associated with immune activation and suppression in the TME. H, Bar graph (left) and representative flow cytometry plot (right) comparing CAR T cell (CD3+CD19+) numbers and activation phenotype (CD69). I. Bar graph (left) and representative flow cytometry plot (right) comparing endogenous T cell (CD3+CD19-) numbers and activation phenotype (CD69). J, Representative histogram (left) and bar graph (right) showing phenotype in the myeloid (CD11b+) compartment. Data are representative of at least two independent experiments. Each symbol represents one individual (H, I, and J). Data are presented as mean ± sem and analyzed by unpaired two-tailed Student's t-test. Differences between survival curves (F) were analyzed by log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, analyzed by rank (Mantel-Cox) test. [Figure 16]Figures 16a-16d: CAR T cells promote monocyte differentiation and generation of M1-type macrophages. a, Scheme of experimental design. b, Flow cytometry (left) and bar graphs (right) show phenotypic changes in monocytes after incubation with different conditioned media. c, Microscopy images show morphological changes in monocytes after incubation with different conditioned media. d, qPCR analysis of genes related to M1 macrophage phenotype. Data are shown as mean ± sem. Analyzed by unpaired two-tailed Student's t-test. For comparison, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 17] Figures 17a-17k: CAR T cells can activate immune cells of GBM patients. a, Scheme of experimental design. b, Flow cytometry, c, Microscopic images and d, Bar graph summary of phenotypic changes of patient macrophages after incubation in conditioned medium. e, Flow cytometry and f, Summary of phenotypic changes in patient T cells after incubation in conditioned medium. g, Schematic of study design for patients undergoing CAR T therapy. h, Flow cytometry shows intracellular IFNγ levels in patient T cells obtained before CAR T therapy (Pre-CAR) and during response to CAR T therapy after 4 h stimulation after co-culture with irradiated autologous tumor (Post-CAR). i, T cell counts after incubation with autologous irradiated (Irr.) patient tumor. j, In vitro killing by T cells against autologous (UPN109) or non-specific tumor line (K562) at E:T ratio of 10:1. k, Flow cytometry demonstrates IL13Rα2 expression in patient autologous (UPN109) tumors. Each symbol represents one replicate. Data are shown as mean ± sem. (d, f, i and j) Analyzed by unpaired two-tailed Student's t-test. For comparison, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 18] Figure 18: List of primers used in the studies disclosed herein. SEQ ID NOs: 77-106, 89-90, 107-108, respectively, are disclosed in order of appearance. [Figure 19]FIG. 19: List of antibodies used in the studies disclosed herein. [Figure 20] Figures 20A-20D: Molecular design and generation of different IL13Rα2-IFNγ CAR T cell constructs and aspects of preparation of CAR T cells co-expressing interferon gamma. 20A. Construct design of IL13 CAR T co-expressing IFNγ compared to standard IL13 CAR T. 20B. Schematic of transduction and IL13 CAR T cell production. T cells are isolated and activated in the presence of CD3 / CD28 antibodies (1:1) followed by IL13 CAR transduction. 2C. Flow cytometry showed the percentage of CAR positive cells using IL13 as a marker of CAR expression. 20D. IFNγ levels in different IL13 CAR T cells during production (left panel) and ELISA confirmed IFNγ expression and secretion in CAR T cells, confirming transduction and expression of IFNγ within the construct. [Figure 21] Figures 21A-21E: Results of functional and phenotypic assessment of murine IL13 CAR T cells and IL13Rα2-IFNγ CAR T cells. Murine IL13 CAR T cells or IL13 CAR T cells co-expressing IFNγ were co-cultured with murine glioma tumors at a 1 effector:3 target ratio. 21A. Microscopic images demonstrate the killing capacity of the different IL13 CAR T constructs (untransduced mock T cells, IL13 CAR T only, and IL13 CAR IFNγ T cells). 21B. T cell counts after 24 hours of co-culture. 21C. Tumor cell counts after 24 hours of co-culture. 21D. Bar graph shows T cell activation ratio measured by CD69 expression after 24 hours of co-culture. 21E. Flow cytometry analysis demonstrated comparable exhausted (PD-1+Tim3+) and differentiated (CD62L+CD45RA+) phenotypes in murine IL13Rα2-IFNγ CAR T cells and IL13Rα2-IFNγ CAR T cells. [Figure 22]Figures 22A-22C: Functional and phenotypic evaluation of human IL13 CAR cells and IL13Rα2-IFNγCAR T cells. Human IL13 CAR T cells or IL13 CAR T-IFNγ cells were co-cultured with patient-derived glioma tumors (1 effector:25 targets). 22A. T cell numbers after 24 hours of co-culture. 22B. Tumor cell numbers measured after 24 hours of co-culture. 22C. Flow cytometry analysis showed comparable exhausted (PD-1+Tim3+) and differentiated (CD62L+CD45RA+) phenotypes in human IL13Rα2-IFNγCAR T cells and IL13Rα2-IFNγCAR T cells. [Figure 23] IL13Rα2-IFNγ CAR T cells T cells induced an abscopal effect in a multilesion metastatic melanoma model. Tumors were injected into both flanks. Once the tumors reached a given size, IL13Rα2 CAR T cells or IL13Rα2-IFNγ CAR T cells were administered locally into one tumor. Both tumors were measured for antitumor activity. Line graphs showed the change in tumor volume in tumors on the opposite side of treatment. [Figure 24] Amino acid sequences represented by various human IL13 CARs with various domains indicated (SEQ ID NOs: 70-72). [Diagram 25] Amino acid sequences represented by various human IL13 CARs with the various domains indicated (SEQ ID NOs: 73-75). [Figure 26] Figures 26A-26B: IL13Rα2-IFNγ CAR T cells can reprogram macrophages. 26A. Schematic of transduction and CAR T cell production. 26B. Bar graph demonstrated reprogramming of macrophages by IL13Rα2-IFNγ CAR T cells. Bar graph showed qPCR analysis of genes associated with pro-inflammatory and metabolically active macrophages when incubated with supernatant collected during CAR product manufacturing or exposed to exogenous IFNγ. Each data point represents one replicate. [Figure 27]Figures 27A-27C: Generation of inducible IL13Rα2-IFNγ CAR T cells with a synthetic NFAT promoter. 27A. Schematic showing the molecular design of inducible IFNγ expression upon CAR T activation. 27B. Illustration showing the experimental design. Briefly, cells were transduced with NFAT-eGFP-CAR T cell construct and co-cultured with IL13Rα2+ and IL13Rα2- tumors. Upon stimulation and activation by antigen-positive tumors, GFP is expressed and detectable. 27C. Flow cytometry demonstrated expression of GFP in cells transduced and activated by antigen-positive tumors. [Figure 28] Figures 28A-28C: IL13Rα2-IFNγ CAR T cells were more effective in targeting medium / low IL13Rα2 antigen expressing tumors in vivo compared to standard IL13Rα2 CAR T cells. 28A. Schematic showing experimental design. 28B. Bar graphs showed tumor progression in mice bearing high IL13Rα2 antigen expressing tumors after treatment with IL13Rα2 CAR T cells or IL13Rα2-IFNγ CAR T cells. 28C. Bar graphs showed tumor progression in mice bearing medium-IL13Rα2 antigen expressing tumors after treatment with IL13Rα2 CAR T cells or IL13Rα2-IFNγ CAR T cells. [Figure 29] Figures 29A-29D: IL13Rα2-IFNγ CAR T cells and IL13Rα2-IFNγlow CAR variant expressed different levels of IFNγ. 29A. Schematic showing construct design with IFNγ under the control of promoters of different strengths. 29B. Schematic showing experimental design showing transduction and supernatant collection. 29C. Bar graph showing IFNγ levels during ex vivo expansion. 29D. Bar graph showing number of surviving tumors after 5 days of co-culture of CAR T cells with tumors (effector:target ratio of 1:50). [Diagram 30]Figures 30A-30C: IL13Rα2-CAR T cells and IL13Rα2-CAR-NFAT / IFNγT cells show comparable killing capabilities. 30A. Scheme of construct design shows IFNγ under the control of the NFAT promoter. 30B. Schematic of experimental design demonstrating co-culture of different CARs in the presence of antigen-positive tumors. 30C. Graph shows cytotoxic function of IL13Rα2-CAR T compared to IL13Rα2-CAR-NFAT / IFNγT cells. [Diagram 31] Figures 31A-31B: IL13Rα2-IFNγ CAR T cells synergize with myeloid cells to enhance antitumor function. 32A. For the experiment, T cells transduced with IL13Rα2-IFNγ CAR T cells or IL13Rα2 CAR T cells were co-cultured with macrophages and antigen-positive tumor cells. 32B. Bar graphs show the number of tumors in ternary co-cultures with IL13Rα2-IFNγ CAR T cells or IL13Rα2 CAR T cells with or without macrophages or tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0050] Working Example Materials and Methods Mice and cell lines C57BL / 6 / J, CD45.1 (B6.SJL-PtprCaPeppcb / BoyJ), Thy1.1 (B6.PL-Thy1a / CyJ), IFNγR- / - (B6.129S7-Ifngr1tm1Agt / J), and IFNγ- / - (B6.129S7-Ifngtm1Ts / J) mice were purchased from The Jackson Laboratory. NOD / Scid IL2RγCnull (NSG) mice were bred at City of Hope. All mouse experiments were approved by the City of Hope Institutional Animal Care and Use Committee (IACUC). Luciferase-expressing mouse GL261 (GL261-Luc) and KR158B (K-Luc) glioma cells were transduced with lentivirus to generate mouse IL13Rα2 (mIL13Rα2)-expressing sublines (GL261-Luc-mIL13Rα2 and K-Luc-mIL13Rα2). These tumor lines were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 25 mM HEPES (Irvine Scientific, Santana, CA), and 2 mM L-glutamine (Lonza). Cell surface expression of mIL13Rα2 was confirmed by flow cytometry and immunofluorescence imaging. Patient-derived glioma cells (PBT030-2-ffLuc) were isolated from a GBM patient resection per a protocol approved by the COH IRB and maintained as described above. All tumor lines were authenticated for desired antigen / marker expression by flow cytometry, and cells were tested for mycoplasma and maintained in culture for no more than 1-3 months.

[0051] CAR T cell production Human CAR T cells: Naïve and memory T cells were isolated from healthy donors at City of Hope by protocols approved by the COH IRB (12, 32). The construction of IL13Rα2-targeted CAR and CAR transduction have been described in references 12, 33, 34. Briefly, primary T cells were stimulated with Dynabeads Human T expander CD3 / CD28 (Invitrogen) (T cells:beads = 1:2) for 24 h and transduced with CAR lentivirus (multiplicity of infection [MOI] = 0.5). Seven days after CAR transduction, CD3 / CD28 beads were removed, cells were resuspended, and expanded for an additional 10–15 days in X-VIVO 15 medium (Lonza) containing 10% FCS, 50 U / ml recombinant human IL-2, and 0.5 ng / ml recombinant human IL-15, followed by ex vivo expansion.

[0052] Mouse CAR T cells A mouse IL13BBζ chimeric antigen receptor was constructed in MSCV retroviral backbone (Addgene) containing extracellular mouse IL13 and mouse CD8 hinge, mouse CD4 transmembrane domain, and intracellular mouse 4-1BB costimulatory and mouse CD3ζ signals. After T2A ribosomal skip, a truncated mouse CD19 was inserted as a transduction marker. The resulting plasmid was transfected into Plate cells (kindly provided by Dr. Zuoming Sun Laboratory) using Fugene (Promega). After 48 hours, the supernatant was harvested and filtered using a 0.2 μm filter. The retroviral supernatant was aliquoted and frozen until the time of transduction. Mouse T cells were isolated from the spleens of naive C57BL / 6J mice or the appropriate strains (CD45.1, Thy1.1, or IFNγ- / -) using the EasySep Mouse T cell Isolation Kit (STEMCELL Technologies) and stimulated with Dynabead Mouse T-Activator CD3 / CD28 beads (Gibco) at a 1:1 ratio. T cells were transduced with retrovirus-containing supernatants (as above) on RetroNectin-coated plates (Takara Bio USA). Cells were then grown for 4 days in RPMI-1640 (Lonza) supplemented with 10% FBS (Hyclone Laboratories), 55 mM 2-mercaptoethanol (Gibco), 50 U / mL recombinant human-IL-2 (Novartis), and 10 ng / mL recombinant mouse IL-7 (Peprotech). Prior to in vitro and in vivo experiments, T cells were de-beaded and CAR expression was determined by flow cytometry.

[0053] qRT-PCR analysis RNA was isolated from demyelinated brain tissue (either bulk tissue or flow-sorted cells) using the RNeasy Mini Kit (Qiagen). cDNA was reverse transcribed using SuperScript VILO Mastermix (Life Technologies) according to the manufacturer's instructions. qPCR reactions were performed as previously described (35). Primers used are listed in Figure 18.

[0054] In vivo studies All mouse experiments were performed using protocols approved by the City of Hope IACUC. Orthotopic GBM models were generated as previously described (36). 1 × 10 5Orthotopic tumor models were established by implanting tumor cells intracranially (ic). Engraftment was verified by bioluminescence imaging 1 day before CAR T cell injection. Mice were randomized into groups based on bioluminescence signal. Four or seven days after tumor injection, mice were implanted with 1×10 6 Patients were treated intracranially with mIL13BBζ-CAR T cells. Tumor burden was monitored with SPECTRAL LagoX (Spectral Instruments Imaging) and analyzed using Aura software (v2.3.1, Spectral Instruments Imaging). Survival curves were generated with GraphPad Prism Software (v8). For the rechallenge experiment, tumor clearance was monitored by tumor rechallenge with K-Luc or 5 × 10 4 The results were verified by bioluminescence imaging before tumor rechallenge with GL261-Luc cells. For subcutaneous studies, 1 x 10 in PBS 6 K-Luc-mIL13Rα2 was injected into the right and left flanks of 8-10 week old C57BL / 6J donor mice. Tumors were allowed to establish for 8 days, after which 1 × 10 6 CAR T cells were injected directly into the tumor. After 3 days, tumor masses were harvested, manually dissociated, and sorted by flow cytometry into CD3+CD19- (endogenous T cells) or CD3+CD19+ (CAR T cells) using a BD AriaSORP (BD Biosciences). Purified T cell populations were used as effector cells in in vitro co-cultures at a 10:1 (effector:target) ratio as described below, or re-injected back into 8-day-old subcutaneous K-Luc-mIL13Rα2 tumors, whose tumor volumes were measured over time using calipers. Mice were also monitored by the Center for Comparative Medicine at City of Hope for survival and any symptoms related to tumor progression, with euthanasia applied according to American Veterinary Medical Association Guidelines.

[0055] In vitro cytotoxicity For assessment of CAR T cell proliferation and cytotoxic activity, K-Luc-mIL13Rα2 or GL261-Luc-mIL13Rα2 tumor cells were co-cultured with mouse CAR T cells at a CAR+tumor ratio of 1:3 for 48 hours. For co-culture with in vivo primed effector T cells, T cells were plated at an effector:tumor ratio of 10:1 for 72 hours. Cells were stained with anti-CD3, CD8, and CD19. Absolute numbers of viable tumor and CAR T cells were assessed by flow cytometry. For degranulation assays, CAR T cells and tumor cells were co-cultured at an effector:tumor ratio of 1:1 in the presence of GolgiStop protein trafficking inhibitor (BD Biosciences) for 5 hours. Cell mixtures were stained with anti-CD3, CD8, and CD19, followed by intracellular staining with anti-IFNγ (BD Biosciences), GZMB, and TNFα (eBiosciences) antibodies and analyzed by flow cytometry. All samples were acquired on a MACSQuant Analyzer (Miltenyi Biotec) and analyzed with FlowJo software (v10.7) and GraphPad Prism (v8).

[0056] Patient sample analysis Conditioned medium was generated by seeding patient-derived glioma cells with human CAR T cells or combinations at a 1:1 ratio for 24 hours. Supernatants were collected and centrifuged to remove cellular debris. Peripheral blood from GBM patients (obtained from scheduled blood draws in clinical protocols approved by City of Hope) was lysed with PharmLyse buffer (BD Biosciences). CD3 and CD14 cells were isolated using a selection kit (STEMCELL Technologies). CD14 and CD3 positive cells were incubated with conditioned medium in the presence or absence of IFNγR neutralizing antibody (R&D Systems). For macrophage differentiation, CD14 cells were incubated in the presence of M-CSF (BioLegend) for 7 days and then exposed to conditioned medium in the presence or absence of IFNγR neutralizing antibody (R&D Systems). After 48 hours, cells were visualized using a Keyence microscope and phenotyped by flow cytometry. Evaluation of intrinsic responses in unique responders to CAR T therapy (ref) was performed as previously reported (37). Briefly, T cells were isolated from whole blood before and during treatment. Every 2 days, T cells were incubated with irradiated (40 Gy) autologous tumor cells in the presence of IL2 (50 U / ml). After 14 days, T cells were purified and counted. T cells were cultured with fresh autologous tumor or irrelevant tumor lines at a 10:1 (effector:target) ratio, and tumor counts were measured after 3 days. IFNγ production was measured by stimulating T cells with a cell stimulation cocktail for an additional 4 hours, followed by flow cytometry for intracellular IFNγ.

[0057] Flow cytometry assay Live tumor cells grown in vitro were stained with unconjugated goat anti-mouse IL13Rα2 (R&D Systems) followed by secondary donkey anti-goat NL637 (R&D Systems). Live mouse CAR T cells were stained with CD8 (BioLegend), CD3, CD4, CD62L (eBiosciences) or CD45RA (BD Biosciences). CD19 (BD Biosciences) was used as a surrogate to detect CAR. Brains from euthanized mice were removed at the indicated time points and cut along the coronal and sagittal planes using a rodent brain matrix to obtain 4x4mm sections centered on the injection site. These sections were hand minced and then passed through a 40μm filter. Myelin was removed using Myelin Removal Beads II and LS magnetic columns (Miltenyi Biotec) according to the manufacturer's instructions, and cells were then counted. Cells were stained and analyzed using flow cytometry. For flow sorting, cells were stained with the indicated antibodies (Figure 19) and sorted using BD AriaSORP (BD Biosciences). For gene expression analysis of the TME, RNA from the remaining cells was lysed.

[0058] Immunofluorescence and immunohistochemistry For immunofluorescence, K-Luc and GL261-Luc parental or mIL13Rα2-transduced cells were cultured on coverslips and stained with unconjugated goat anti-mouse IL13Rα2 (R&D Systems) followed by secondary donkey anti-goat NL637 (R&D Systems) and actin. Slides were imaged using a confocal microscope (Zeiss confocal microscope) as described previously ( 38 ). For immunohistochemistry, mice were euthanized 3 days after CAR T injection and perfused with PBS followed by 4% PFA. Whole brains were dissected and incubated in 4% PFA for 3 days followed by 70% ethanol for 3 days before embedding in paraffin. 10 μM cross sections were cut and stained with H&E, CD3 (ab16669, Abcam) or F4 / 80 (ab6640, Abcam). Slides were digitized at 40x magnification using a NanoZoomer 2.0-HT digital slide scanner (Hamamatsu).

[0059] Bioplex Cytokine Analysis To assess the CAR T cell cytokine profile, mIL13BBζ CAR+ T cells and tumor cells (GL261-Luc-mIL13Rα2 or K-Luc-mIL13Rα2) were incubated at a 1:1 ratio for 1 day without exogenous cytokines. Cell-free supernatants were collected and assayed using ProcartaPlex Mouse Th1 / Th2 Cytokine Panel 11 plex (ThermoFisher Scientific) according to the manufacturer's instructions and acquired with a Bio-Plex 3D Suspension Array System (Bio-Rad Laboratories).

[0060] NanoString Gene Expression Analysis RNA was purified from flow-sorted CD3+ or CD11b+ sorted cells using the SohERSPlus micro kit according to the manufacturer's instructions (Qiagen, Germantown, MD, USA). RNA samples were then quantified and qualified using a Nanodrop 1000 spectrophotometer (ThermoFisher, Waltham, MA, USA) and Bioanalyser Tape station (Agilent, Santa Clara, CA, USA) assay. Subsequent Nanostring analysis was performed at concentrations of 35 ng / well and 25 ng / well for CD3+ and CD11b+ cells, respectively. Samples were analyzed based on the nCounter® Mouse PanCancer Immune Profiling Gene Expression Panel (NanoString Technologies, Seattle, WA, USA): hybridization reactions were performed at 65°C for 18 hours. A fully automated nCounter FLEX analysis system was used, consisting of an automated nCounter® Prep Station and an nCounter® Digital Analyzer optical scanner (NanoString Technologies, Seattle, WA, USA). Normalization was performed by using the geometric mean of the positive control counts and normalization genes present in the CodeSet Content: samples with normalization factors outside the 0.3-3.0 range were excluded, as were samples with reference factors outside the 0.10-10.0 range. Gene expression analysis was performed using nSolver v3.0 and Advanced analysis module software (NanoString Technologies, Seattle, WA, USA).

[0061] Single-cell RNA sequencing Seven days after K-Luc-mIL13Rα2 engraftment, CAR T cells were either injected or not injected into the tumor as described above. Brains from CAR T-treated or untreated mice (n=3 per group) were harvested and pooled 3 days after CAR T cell injection, manually minced, and myelin-removed before flow sorting on a BD AriaSORP (BD Biosciences) for live (DAPI-) CD45-PE+ (BD Biosciences) cells. Single cell suspensions were processed using the Chromium Single Cell 3'v3 Reagent Kit (10xGenomics) and loaded onto a Chromium Single Cell Chip (10xGenomics) according to the manufacturer's instructions. Raw sequence data from each of the two experiments were aligned to the mouse genome (mm10), respectively, and expression data were generated at single-cell resolution according to 10xGenomics using the cellranger count command. Genomics (https: / / support.10xgenomics.com / single-cell-gene expression / software / pipelines / latest / using / count). The R package Seurat 39 was used to perform gene and cell filtering, normalization, principal component analysis, variable gene discovery, clustering analysis, and Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction. Briefly, matrices containing per-gene expression data were imported to create Seurat objects separately for CAR T-untreated and CAR T-treated samples. Cells with <200 detectable genes and a ratio of mitochondrial genes >10% were further removed. Data were then merged and log-normalized for subsequent analysis. Principal component analysis (PCA) was performed for dimensionality reduction, and the first 20 principal components were used for clustering analysis at a resolution of 0.6. Clusters were visualized with UMAP embedding. Using the Immunologic Genome Project (ImmGen), 40, 41 the expression levels of the following markers were further plotted using VlnPlot to facilitate cell type identification:They were Itagm, Cd3e, Cd19, Cd79a, Nkh7, Cd68 and Cd8a. Upon identification of the lymphoid and myeloid parent clusters, for each of them, we followed the above strategy of sub-clustering to generate daughter clusters. In collaboration with ImmGen, the key markers for distinguishing the myeloid daughter clusters were Itgam, Cd68, S100a9, Itgax, Tmem119 and P2ry12, while lymphoid Cd3e, Cd4, Cd8a, Cd79a and Ncr1. To further visualize the average expression of the modules of genes D74, H2-Aa, H2-Ab1, H2-Eb1 and MARCKS across populations in the myeloid daughter clusters, the AddModuleScore function was used to generate features that could be rendered using FeaturePlot.

[0062] Gene set enrichment analysis Differentially expressed (DE) genes between untreated and CAR T-treated in each myeloid and lymphoid parent and daughter cluster were detected with the function FindAllMarkers. Gene ontology (GO), Kyoto encyclopedia of genes and genomes pathways, and Immunologic signatures collection (ImmuneSigDB) (42) analyses were performed with the complete list of DE genes for each cluster using the GSEA function implemented in the clusterProfiler package (43) and then plotted using ggplot2 (H. Wickham. ggplot2: elegant graphics for data analysis. Springer-Verlag New York, 2016).

[0063] statistical analysis Statistical significance was determined using Student's t-test (two groups) or one-way ANOVA analysis with Bonferroni (three or more groups). Survival was plotted using Kaplan-Meier survival curves and statistical significance was determined by the log-rank (Mantel-Cox) test. All analyses were performed using GraphPad Prism software (v5). *, P<0.05; **, P<0.01; ***, P<0.001. EXAMPLES

[0064] Murine IL13Rα2-CAR T cells mediate potent antitumor activity in an immunocompetent model of GBM We established an immune-competent mouse model of the clinical IL13Rα2-CAR T cell platform described above. We constructed a mouse counterpart to the human IL13Rα2-targeted CAR, composed of an IL-13(E12Y) tumor-targeting domain, mouse CD8 hinge (mCD8h), mouse CD8 transmembrane domain (mCD8tm), mouse 4-1BB costimulatory domain (m4-1BB) and mouse CD3 zeta (mCD3ζ) (12). A T2A skip sequence separates the CAR from a truncated mouse CD19 (mCD19t) used for cell tracking (Figure 1A). The engineering process resulted in a transduction efficiency of 70-85%, as assessed by the frequency of CD19t+ cells (Figure 2A). Phenotypic analysis showed that, similar to human CAR T cells (12), murine IL13Rα2-targeted CAR T cells (mIL13BBζ CAR T cells) contained comparable numbers of CD4+ and CD8+ T cell subsets with mixed early memory (CD62L+) and effector (CD62L-) T cell populations at day 4 of ex vivo expansion (Figure 1B and Figure 1C). We designed a therapeutic setting based on two syngeneic immunocompetent mouse glioma models, K-Luc and GL261-Luc. K-Luc, a firefly luciferase modified subline of KR15813, was used because it recapitulates the highly invasive characteristics of GBM (Figure 2B). This tumor line was derived from a spontaneous glioma arising from an Nf1, Trp53 mutant mouse and is poorly immunogenic, as shown by its non-responsiveness to anti-PD-1 checkpoint therapy (14). As a second model, we also used GL261 engineered to express ffLuc (GL261-luc), a non-invasive "bulky" glioma (Figure 3A) that was generated by chemical induction, contains multiple mutations, and, in contrast to K-Luc, is responsive to anti-PD-1 immunotherapy (15, 16). Both tumor lines were engineered to express mouse IL13Rα2 (mIL13Rα2) (Figures 2C and 3B).mIL13BBζ CAR T cells specifically killed mIL13Rα2-recombinant K-Luc and GL261-Luc cells (Figures 2D and 3C), which were associated with the production of the inflammatory cytokines IFNγ and TNFα (Figures 2E and 3D), and did not respond to the IL13Rα2-negative parental tumor line in vitro (Figure 1D). Next, we evaluated CAR T cell antitumor activity against orthotopically implanted glioma tumors in C57BL / 6 immunocompetent mice. In IL13Rα2+K-Luc and GL261-Luc tumor models, a single intratumoral administration of mIL13BBζ CAR T cells 7 days after tumor injection mediated potent in vivo antitumor activity and conferred a significant survival benefit (Figures 2F-2I and 3E). We compared anti-K-Luc activity in C57BL / 6 mice with tumors implanted in an immunodeficient NOD-scid IL2Rgnull (NSG) model that lacks adaptive immune subsets. In a smaller 4-day-old tumor model, antitumor activity in C57BL / 6 and NSG was comparable, and CAR T cell functionality was comparable in both mouse strains (Figures 4A-4C). Surprisingly, however, in larger tumor models (day 7) where the immune microenvironment was more established, the in vivo response to T cell therapy was superior in immunocompetent C57BL / 6 compared to immunodeficient NSG mice (p<0.001) (Figures 4D-4E). This observation indicates that tumor-infiltrating immune cells may enhance the antitumor effect of CAR T cell therapy in GBM. EXAMPLES

[0065] CAR T therapy can promote immunological memory and the generation of tumor-specific T cells To evaluate whether CAR T cells have the ability to induce endogenous antitumor immunity, mice cured after CAR T cell therapy were challenged with IL13Rα2-negative parental tumors. Indeed, when the engrafted tumors (engrafted for 7 days prior to CAR T therapy) were larger, cured mice in the immunocompetent C57BL / 6 model successfully rejected tumor rechallenge with IL13Rα2-negative K-Luc (Figure 5A and Figure 5B) and GL261-Luc (Figure 3F) parental tumor cells, demonstrating that CAR T cells can promote immune memory in two independent tumor models that differ in their responsiveness to anti-PD 1 immunotherapy (14, 16). The ability of CAR T cells to induce endogenous immunity against IL13Rα2-negative tumor cells still required a more established TME, as cured mice in the small tumor model (day 4) were unable to mount an antitumor response after rechallenge with parental tumors (Figure 6). This observation suggests that tumor exposure is not sufficient to induce endogenous antitumor immunity, but rather the establishment of immune memory requires CAR T cell therapy and host immune infiltrates. In a similar series of experiments, we compared the antitumor activity of mIL13BBζ CAR T cells against IL13Rα2+K-Luc (100% IL13Rα2+ cells) versus a mixture of IL13Rα2+K-Luc and IL13Rα2-parental K-Luc tumor cells (1:1 ratio). Supporting the notion that CAR T cells can promote endogenous immune responses against antigen-negative tumor cells, mIL13BBζ CAR T cells mediated comparable survival benefits against tumors with allogeneic and heterogeneous IL13Rα2 antigen expression (p<0.001; Figures 7A and 7B). Again, CAR T cell therapy was less effective against mixed antigen tumors (1:1 ratio) in a small tumor model (day 4) (p<0.001; Figures 7C and 7D), and responses against IL13Rα2-negative tumors required the establishment of a tumor microenvironment. Thus, these observations suggest that altering the TME of GBM with CAR T cell therapy enhances antitumor responses and promotes endogenous antitumor memory responses. To more directly assess whether CAR T therapy can enhance the generation of tumor-specific T cells, we isolated intratumoral endogenous (CD3+CD19-) and CAR T cells (CD3+CD19+) from untreated and CAR T cell-treated mice in an IL13Rα2+K-Luc glioma model (day 3 post-treatment) (Figure 8) and either co-cultured with tumor cells ex vivo or adoptively transferred into tumor-bearing mice (Figure 5C). In vitro, endogenous T cells isolated from CAR T cell-treated tumors, but not control untreated groups, demonstrated enhanced killing of IL13Rα2+K-Luc cells and T cell proliferation in co-culture assays (10:1, effector:target ratio; 72 hours) (Figures 5D and 5E). To assess in vivo function, endogenous T cells from untreated and CAR T cell-treated mice were isolated and adoptively transferred into IL13Rα2+K-Luc tumor-bearing mice. Measurement of tumor progression demonstrated that mice injected with endogenous T cells isolated from CAR T-treated mice had significantly reduced tumor growth compared to the control group (Figure 5F). Thus, these results demonstrated that CAR T cells have the ability to promote antigen spreading and the generation of tumor-specific T cell responses. EXAMPLES

[0066] CAR T cells activate innate and adaptive immune subsets in the tumor microenvironment To elucidate immune-related changes in the TME that occur concomitantly with the establishment of endogenous antitumor immunity following CAR T cell therapy, we investigated both lymphoid and myeloid compartments by gene and protein expression profiling. First, for the lymphoid compartment, we performed nanostring analysis of purified intratumoral CD3+ cells and demonstrated global changes at the transcriptome level in CAR T-treated mice compared to untreated (Figure 9A). To increase resolution and more precisely define subpopulations, we performed scRNAseq on CD45 cells isolated from untreated or CAR T-treated mice (Figures 10A and 10B). We then computationally separated lymphoid and myeloid populations and reanalyzed the scRNAseq data at a higher granularity. This approach yielded nine distinct lymphoid subpopulations broadly defined by the distribution of classical marker genes, including three distinct subsets of CD8+ T cells (CD8_L2, CD8_L3, and CD8_L4), two subsets of CD4+ T cells (CD4_L1, CD4_L6), one subset of NK cells, two subsets of B cells, and one subset resembling γδ T cells (Figure 9B and Figure 10C). Interestingly, the frequency of CD8_L2 did not change, but the frequency of CD8_L3 and CD8_L4 subclusters increased after CAR T treatment (Figure 10C). Focusing on the T cell subclusters, CD8_L3 was mainly observed after treatment and was characterized by the expression of Cxcr3 associated with T cell trafficking (Fig. 11a), as well as Itgae (CD103), Cd74 (Hladr) and Ifitm1 (IFN-inducible transmembrane protein 1) (Fig. 9C), corresponding to an activated resident memory CD8 T cell phenotype. CD8_L4 expanded after treatment and was identified as a highly activated effector T cell with higher expression of Ki67, Cd74 and Gzma genes (Fig. 9C). Within the CD4 subset, the frequency of the CD4_L1 cluster did not change after treatment, and the expression of Il 7 r, Tcf7 and Itga 4 genes associated with effector memory CD4 T cells in the CAR T treatment group was moderately increased (Figs. 10C and 11A).Intratumoral regulatory T cells (Tregs), defined by the subcluster CD4_L6 based on the expression of CD4, Foxp3, GITR (Tnfrsf18) and Ctla4, were reduced after CAR T therapy (Figure 11B). Overall, gene set enrichment analysis (GSEA) revealed that most T cell subclusters were enriched for gene signatures associated with activation and Th1 responses (Figure 9D). Thus, the above studies demonstrated that CAR T cells can dramatically alter the lymphoid compartment within tumors, resulting in clusters of activated, memory or effector T cell populations.

[0067] To further characterize T cell populations following CAR T cell therapy at the cellular level and distinguish between changes in endogenous versus adoptively transferred T cells, isogenically mismatched CD45.1 CAR T cells were used to treat IL13Rα2+K-Luc tumors implanted in CD45.2 mice (Figure 9e). Following intracranial delivery, CAR T cells (CD3+CD45.1+CD19+), unlike mock-transduced T cells (CD3+CD45.1+CD19-), showed significantly increased T cell numbers and expression of markers of activation (CD69+), cytotoxic function (GZMB+), and proliferation (Ki67+) (Figure 9f), indicating that the observed effector activity was CAR-dependent. Interestingly, only after CAR T therapy, there was a significant increase in the number of endogenous T cells (CD3+CD45.2+) with an activated (CD69+), proliferative (Ki67+), and cytotoxic phenotype (GZMB+), which was not detected in untreated or sham-treated controls (Figure 9G). This was consistent with qPCR analysis demonstrating upregulation of GZMA, GZMB, and PRF1 genes in intratumoral CD3+ T cells after CAR T therapy (Figure 12). The above results demonstrated that CAR T cells enhance endogenous T cell activation and proliferation, consistent with our previous findings that endogenous intratumoral T cells isolated after CAR T cell therapy have antitumor activity (Figures 5D-5F). We next evaluated changes in innate myeloid cells such as microglia / macrophages, since they are the predominant immune population in glioma tumors and have crucial functions in glioma pathogenesis (17). Analysis of intratumoral myeloid populations at the single cell level identified 17 distinct myeloid subpopulations that were significantly remodeled following CAR T therapy (Figure 13A). We observed interesting complexity and dynamics of intratumoral monocyte / macrophage / microglia / DC compartments in glioma TME. Some macrophage / monocyte subpopulations decreased in frequency, while others increased, reshaping the TME. We identified seven major monocyte / macrophage (Itgam, Cd68), four microglia (Tmem119 and P2ry12), four DC and two clusters of neutrophils (S100A9) subpopulations. Gene set enrichment analysis (GSEA) clearly enriched genes associated with IFNγ-stimulated macrophages and microglia in the CAR-treated group (Figure 13B). Further evaluation of the major myeloid populations (macrophages, microglia, and neutrophils) identified higher expression of genes associated with mature and IFNγ-activated macrophages as well as stimulated neutrophils (Figure 13C), further confirming that resident innate immune cells are exposed to IFNγ-mediated activation.

[0068] Nanostring analysis of intratumoral microglia / macrophage cells (CD11b+) from the TME 3 days after CAR T therapy showed enrichment of genes mediating antigen processing and presentation (e.g., Cd74, H2-Ab1, H2-Aa, H2-Eb1) (Figure 13D). Further analysis using scRNAseq revealed that a majority of macrophage / microglia subclusters may be involved in antigen processing and presentation (Figure 13E). Evaluating CAR T cell-induced changes in resident microglia / macrophage populations by flow cytometry, we found an increase in the frequency and number of activated brain-resident macrophages / microglia cells (CD86+, MHCIhigh, MHCIIhigh) in CAR T-treated mice (Figure 13F). Evaluation of the myeloid compartment also revealed a significant increase in the frequency of the inflammatory cytokine IFNγ+ (Figure 13F). Collectively, these data indicate that CAR T therapy alters the GBM immune landscape and activates host innate and adaptive immune cells. These results also further elucidate the key function of IFNγ in inducing local immune cell activation. EXAMPLES

[0069] Deficiency of IFNγ in CAR T cells impairs in vivo antitumor activity and host immune cell activation Given that myeloid cells constitute the largest population in the glioma TME and our scRNAseq analysis identified gene signatures associated with IFNγ stimulation within macrophage and microglia subclusters (Figure 13B), we reasoned that IFNγ produced by stimulated CAR T cells may be involved in regulating the activation of resident macrophage / microglia cells and the subsequent priming and induction of adaptive immune responses. IFNγ is one of the key effector cytokines abundantly produced by CAR T cells upon activation and is a prototypic macrophage activator (18). To investigate whether IFNγ secreted by CAR T cells is responsible for the observed changes in the phenotype and function of resident macrophages / microglial cells, CAR T cells were cultured in WT (CAR T wt ) or IFNγ- / - (CAR T IFNγ- / - ) mice (Figure 14A) and characterized accordingly. wt and CAR T IFNγ- / - ) showed comparable therapeutic outcomes (Figure 14B). Next, we performed in vitro killing assays to validate the functionality of CAR T cells from IFNγ- / - mice compared to CAR T cells from WT mice, which demonstrated comparable killing efficacy at a 1:1 effector-to-target ratio (Figure 14C). Evaluation of CAR T multifunctionality showed that CAR T wt and CAR T IFNγ- / - It was demonstrated that TNFα and GZMB were produced equally in CAR T cells. IFNγ- / - The defect in IFNγ production in CAR T cells was predicted (Figure 14D). IFNγ- / - Mice receiving CAR T wtCAR T cells had lower overall survival compared to mice treated with IFNγ-deficiency, indicating reduced anti-cancer activity and reduced survival due to IFNγ-deficiency in CAR T cells (Figure 14E and Figure 14F). Analysis of the entire TME showed that mice receiving CAR T cells had increased expression of genes involved in activation and inflammatory cytokines, and conversely, decreased expression of genes involved in suppressive phenotype and function of intratumoral immune infiltrates (Figure 14G), indicating that defective IFNγ secretion by CAR T cells alters the glioma TME. IFNγ is a pleiotropic cytokine that induces activation of CD8 T cells (9), promotes polarization of Th1 CD4 cells (19), and reprograms or activates macrophage / microglial cells (6, 7). Therefore, we next evaluated whether the lack of IFNγ secreted by CAR T cells affects host immune cells. Flow cytometric analysis of the TME 3 days after CAR T cell therapy revealed a significant decline in the number of endogenous and CAR T cells, which correlated with a decrease in activated (CD69+) T cells (Figures 14H and 14I). IFNγ- / - Compared with CAR T cells, wt The frequency of MHCI+ / MHCII+ and CD86+ macrophage / microglial cell activation was significantly increased in tumor-bearing mice receiving CAR T (Figure 14J). Thus, IFNγ production as a result of CAR T antitumor activity activates and reactivates T cells, enhancing the activation and antigen-presenting capacity of macrophages / microglial cells. EXAMPLES

[0070] Defective IFNγ signaling in the host reduces CAR T antitumor activity in vivo Previous studies have reported IFNγ signaling as a signature of response to immunotherapy, such as anti-PD-1 therapy (20). To investigate whether host IFNγ signaling functions in CAR T-mediated immune responses, we performed a CAR T wt The cells were adoptively transferred into K-Luc-bearing WT or IFNγR− / − mice (Figure 15A). wtIFNγR− / − mice receiving CAR T cells had poorer survival, suggesting that defective IFNγ signaling in host immune cells reduced the anti-tumor activity of CAR T cells and overall survival (Figure 15B and Figure 15C). Gene expression analysis of the TME revealed that defective IFNγ signaling in the host during CAR therapy reduced expression of genes involved in activation and proinflammatory responses (CD40, NOS2, TNFα, GZMA, GZMB, PRF1, and IFNγ) (Figure 15D). To further explore CAR T cell-mediated changes in the immune landscape, we used isogenically mismatched (Thy1.1+) CAR T cells to treat inflammatory conditions. wt Cells were adoptively transferred into K-Luc-bearing WT or IFNγR− / − mice. Flow cytometry analysis of the TME demonstrated significantly increased activation of macrophage / microglial cells (CD11b+CD86+MHCI+MHCII+) in WT mice compared with IFNγR− / − mice as early as 3 days after CAR T cell therapy (Figure 15E). Compared with WT mice, the number of endogenous T cells (Thy1.2+CD3+) activated with the capacity to proliferate and produce effector cytokines was significantly lower in IFNγR− / − mice (Figure 15F). Interestingly, compared to WT mice, adoptively transferred CAR T cells (Thy1.1+CD3+) displayed significantly higher numbers, proliferative (Ki67+) and cytotoxic phenotypes (IFNγ+ and GZMB+) compared to IFNγR− / − mice (FIG. 15G), suggesting a potential IFNγ positive feedback from host immune cells promoting CAR T cell activity in the TME. Thus, the above results establish that an interaction exists between host and adoptively transferred immune cells and that host IFNγ signaling in the glioma TME is necessary to mount a strong immune response during CAR T therapy. To investigate whether IFNγ secreted by CAR T cells was responsible for the observed changes in resident macrophage / microglial cell phenotype and function, wild-type (CAR T wt ) or IFNγ- / - (CAR T IFNγ- / -) mice (Figure 15A) and characterized accordingly. wt and CAR T IFNγ- / - ) showed comparable therapeutic products with some differences in the ratio of CAR 4:CAR 8 T cells (P<0.05; Figure 15B). Next, we verified the functionality of CAR T cells from IFNγ- / - mice by performing in vitro killing assays compared to CAR T cells from WT mice, which demonstrated comparable killing efficacy at an E:T ratio of 1:1 (Figure 15C). Evaluation of CAR T cell polyfunctionality was performed using CAR T wt and CAR T IFNγ- / - We demonstrated equivalent production of TNFα and GZMB in both CAR T cells. IFNγ- / - Defective IFNγ production in CAR T cells was predicted (Figure 15D). IFNγ- / - Mice receiving CAR T wt CAR T cells showed poor overall survival compared to mice treated with IFNγ-positive mice, indicating that IFNγ deficiency in CAR T cells compromised their anticancer activity in vivo (Figures 15E and 15F). Gene expression analysis of tumors and associated TME 3 days after CAR T cell therapy demonstrated that CAR T wt We found enhanced expression in genes involved in activation and pro-inflammatory cytokines in mice receiving CAR T cells, and on the other hand, reduced expression of genes involved in the suppressive phenotype and function of intratumoral immune infiltrates (Figure 15G), indicating that defective IFNγ secretion by CAR T cells alters the glioma TME. IFNγ is a pleiotropic cytokine that induces activation of CD8 T cells, promotes polarization of Th1 CD4 cells, and reprograms or activates macrophage / microglial cells. Therefore, we next evaluated whether defective IFNγ secreted by CAR T cells affects host immune cells. Flow cytometry analysis of the TME 3 days after CAR T cell therapy revealed a significant decrease in T cell numbers, both in endogenous and CAR T cells, which correlated with a decrease in activated (CD69+) T cells (Figures 15H and 15I). Furthermore, CAR T IFNγ- / -Compared with CAR T cells, wt We observed a significant increase in the frequency of MHCI+ / MHCII+ and CD86+ macrophage / microglial cell activation in mice receiving CAR T cells (Figure 15J). Importantly, the lack of IFNγ secretion by CAR T cells led to the CAR T wt Compared with CAR T cells, IFNγ- / - Mice receiving the cells resulted in higher M2-type tumor macrophages. Thus, IFNγ production as a result of CAR T cell antitumor activity led to the activation and reactivation of T cells and reprogramming of macrophage / microglial cells, enhancing their activation and antigen-presenting capacity. EXAMPLES

[0071] Human CAR T cell therapy modulates patient-host immune cells We next investigated the impact of CAR T cell antitumor activity on human endogenous immune cells in GBM patients. To clinically evaluate whether CAR T cells can promote the activation of GBM patient monocytes or macrophages, we developed an in vitro assay to phenotypically characterize patient myeloid populations in the presence of CAR T cell antitumor activity. Supernatants from co-cultures of human CAR T cells against patient-derived glioma tumors were collected and then incubated with glioma patient-derived monocytes (Figure 16A), ex vivo differentiated patient macrophages, or total CD3+ patient T cells (Figure 17A). Phenotypic and morphological assessment revealed that conditioned medium (CM) from CAR T-tumor co-cultures promoted differentiation and activation of patient-derived monocytes or macrophages, as evidenced by increased expression of activation markers (CD14+CD86+ / CD80+ and CD14+HLADr+), which correlated with enhanced expression of genes associated with classical M1 macrophages (Figures 16B-16D and 17B-17D). Accordingly, exposure to CM from CAR T-tumor co-cultures induced activation of T cells isolated from GBM patient blood, as evidenced by enhanced expression of CD69 (Figures 17E and 17F). Importantly, blocking IFNγ signaling in macrophages and T cells reduced activation (Figures 17B-17F), highlighting the impact of IFNγ in CAR T-mediated activation of host immune cells in GBM patients. Taken together, these findings suggest that IFNγ secreted by CAR T cell antitumor function promotes the polarization of monocytes into activated macrophages, further inducing the activation of differentiated macrophages, and promoting T cell activation.

[0072] Having demonstrated our immune-competent mouse model of GBM, we finally aimed to evaluate whether CAR T cells have the ability to induce the generation of tumor-specific T cells in a clinical setting. To precisely investigate this phenomenon, we evaluated samples from case reports that showed complete responses and were uniquely responsive to CAR T therapy (4). T cells were isolated from blood before CAR T cell treatment (Pre-CAR T) and during response to CAR T therapy (Post-CAR T) (Figure 17G). The isolated T cells were stimulated and expanded in the presence of irradiated autologous tumor cells. Flow cytometric evaluation of the T cell population clearly demonstrated enhanced tumor reactivity as indicated by enhanced intracellular IFNγ and proliferation of isolated T cells during response to pre-initiated CAR T cell therapy (Figure 17H ​​and Figure 17I). Importantly, in patient-derived co-cultures of ex vivo expanded T cells with autologous patient glioma tumors or unrelated tumor cell lines, T cells isolated in response to CAR T therapy demonstrated tumor-specific killing of autologous versus unrelated tumor cells (Figure 17J). These results took into account that the tumor cells were IL13Rα2 negative (Figure 17K). These findings suggest that effective CAR T therapy may stimulate host immune cells to promote the generation of tumor-specific T cell responses, further confirming our preclinical studies that host immune cells play a critical role in the success of CAR T therapy. EXAMPLES

[0073] Co-expression of IL-13 CAR T and interferon gamma We investigated the impact of co-expression of interferon gamma by creating an expression cassette in which the IL-13 CAR of Example 1 (Figure 1A) is linked to immature mouse interferon gamma via a T2A skip sequence (Figure 20A). We designed and constructed IL13Rα2-CAR / IFNg. Incorporation of IFNg is demonstrated for mouse and human platforms. Equivalent transduction and expansion of CAR T cells is feasible in the CAR cassette (Figure 20A). The vectors were sequenced and verified. Mouse T cells were isolated and transduced with either IL-13 CAR and truncated CD19 (lacking the signaling domain) or IL-13 CAR and vector expressing mouse interferon gamma (Figure 20B). Culture supernatants were collected and flow cytometry was used to evaluate the presence of IL-13 as a marker of CAR expression. Both constructs expressed the IL-13 CAR, with transduction efficiency >50% by FACS (Figure 20C). To further confirm that the IL13Rα2-CAR / IFNg vector confers the ability of T cells to express and secrete IFNg, supernatants were harvested from ex vivo expanded expanded IL13Rα2-CAR / IFNg and IL13Rα2-CAR T cells and IFNg levels were measured by ELISA (Figure 1D). Measurement of interferon gamma production by ELISA showed that only the IL-13 CAR T-interferon gamma construct expressed interferon gamma (Figure 20D).

[0074] Next, we evaluated the phenotype of CAR T cells. Our study demonstrated no phenotypic differences between IL13Rα2-CAR / IFNg and IL13Rα2-CAR in mouse (FIG. 21E) and human (FIG. 22C) T cells. Briefly, mouse IL-13 CAR T cells and mouse IL-13 CAR-Interferon gamma T cells were co-cultured with mouse glioma tumor cells at an effector:target ratio of 1:3 for 24 hours. As shown in FIG. 21A-21C, both IL-13 CAR-Interferon gamma T cells showed superior proliferation and tumor cell killing. T cell activation was assessed by measuring CD69 expression. As seen in FIG. 21D, T cell activation was similar for both IL-13 CAR T cells and IL-13 CAR T-Interferon.

[0075] CAR T cells expressing human IL-13 CAR (human IL-13 with E13Y mutation, human CD8 hinge, human CD8 TM, human 4-1BB costimulatory domain and human CD3 zeta) were generated with or without co-expression of human interferon gamma. Human IL-13 CAR T cells and human IL-13 CAR-interferon gamma T cells were co-cultured with patient-derived glioma tumor cells at an effector:target ratio of 1:25 for 24 hours. T cells and tumor cells were evaluated. As seen in Figure 22A, IL-13 CAR-interferon gamma T cells showed superior proliferation and nearly similar T cell killing. Importantly, when co-cultured with antigen-positive tumors, there is enhanced proliferation of IL13Rα2-CAR / IFNg T cells compared to IL13Rα2-CAR T cells in mice (Figure 21C) and humans (Figure 22B). In both mice and humans, a slightly enhanced killing activity was observed in IL13Rα2-CAR / IFNgT cells compared with IL13Rα2-IFNgCAR cells.

[0076] Importantly, to confirm that the secreted IFNg from IL13Rα2-CAR / IFNg T cells is functional and has immune stimulatory components, supernatants from ex vivo expanded T cells engineered to constitutively express IFNg were added to macrophage cultures (Figure 26A). Macrophages exposed to IL13Rα2-CAR / IFNg culture conditions exhibited a more pro-inflammatory and metabolically active phenotype compared to supernatants collected from IL13Rα2-CAR T cells (Figure 26B). Together, these results demonstrate that IL13Rα2-CAR / IFNg is functional to express and secrete IFNγ and activate endogenous innate immune populations such as macrophages.

[0077] We performed a pilot study to evaluate the antitumor activity of IL13Rα2-CAR / IFNg and IL13Rα2-CAR in mice with high (FIG. 28B; left) or medium / low (FIG. 28C; right) tumors in our synthetic model. IL13Rα2-CAR in tumors with IL13Rα2 antigen was compared. The function of the two CARs was comparable in high antigen-expressing tumors, but the antitumor activity in mice with medium antigen-expressing tumors treated with IL13Rα2-CAR / IFNg was significantly altered compared to IL13Rα2-CAR.

[0078] As part of the global impact of IFNg, we also tested whether IL13Rα2-IFNgCAR shows superior antitumor activity against metastatic disease or tumors at distant sites. Therefore, mouse IL-13 CAR T cells and mouse IL-13 CAR-interferon gamma T cells were evaluated in a mouse model of metastatic melanoma (Figure 23). Briefly, tumor cells were injected into both flanks of mice. When the tumor reached a certain size, mouse IL-13 CAR T cells and mouse IL-13 CAR-interferon gamma T cells were injected locally into one tumor. Tumor size was measured bilaterally. The graph in Figure 23 shows the tumor volume of the non-treated side. As can be seen, IL-13 CAR T-interferon gamma T cells showed a greater abscopal effect than IL-13 CAR T cells. Interestingly, our studies in melanoma-bearing mice found that IL13Rα2-CAR / IFNg T cells have superior ability to target distant tumors. These findings strongly highlight IL13Rα2-CAR / IFNg as an important therapeutic approach for cancers with multifocal lesions such as GBM.

[0079] The experiments were designed to test the in vivo functional activity of IL13Rα2-IFNgCAR in a syngeneic immunocompetent glioma model and NSG mice implanted with IL13Rα2+ primary brain tumor lines (PBT). We demonstrated that IL13Rα2-CAR / IFNgT cells have superior antitumor activity in mice with medium / low antigen tumors and eradicate tumors at distant sites in a metastatic melanoma model. To evaluate the importance of IFNg as an immune stimulant and the therapeutic importance of IL13Rα2-CAR / IFNgT cells, we developed a ternary co-culture system with CAR T cells, macrophages, and tumor cells (Figures 31A-31B). The results suggest that in the presence of other immune cells such as macrophages, IL13Rα2-CAR / IFNγT cells exhibit significantly superior antitumor function and stimulate endogenous immunity. EXAMPLES

[0080] Optimization of co-expression of IL-13 CAR T and interferon gamma We also designed and constructed different IL13Rα2-CAR / IFNg variants to prioritize both efficacy and safety by optimizing and regulating IFNγ expression. We successfully designed and sequence checked the constructs shown in Figure 29A. T cells were transduced with the CAR / IFNg variants and the supernatants were collected to confirm IFNg production and co-cultured with IL13Rα2+ tumors to confirm functionality (Figure 29B). The CAR / IFNg variants were evaluated for different levels of IFNγ expression and secretion. Compared to the EF1 promoter, which is a strong promoter, the pkg100 promoter is a weaker promoter. CAR T cells with IFNγ under the pkg100 promoter showed reduced IFNγ levels (Figure 29C). IL13Rα2-CAR / IFNγ low T cells address safety concerns related to overproduction of IFNγ. Next, to confirm cytotoxicity function, we used IL13Rα2-CAR / IFNγ low T cells were co-cultured with IL13Rα2+ tumors at an effector-to-target ratio of 1:50 for 5 days. The number of viable tumors was assessed to determine whether IL13Rα2-CAR / IFNγ low The T cells were shown to have cytotoxic function comparable to standard IL13Rα2-CAR T cells (Figure 29D).

[0081] We designed an inducible construct system that uses a synthetic NFAT promoter to control IFNγ expression. This construct was designed to control the expression of a gene of interest to ensure that IFNg is expressed only when CAR T cells are activated. As a proof of concept, we placed GFP under the control of the NFAT promoter. Our studies demonstrated that upon CAR activation in the presence of IL13Rα2 antigen-positive tumors, the NFAT promoter is functional and can induce GFP expression (Figures 27A-27C). We then replaced the GFP gene with IFNγ (Figure 30A). T cells transduced with the construct were co-cultured with IL13Rα2+ tumors at an effector-to-target ratio of 1:50 for 5 days (Figure 30B). Our results confirm that the NFAT CAR is functional and exhibits a killing capacity comparable to standard IL13Rα2-CAR T cells (Figure 30C). These results indicate that the incorporation of a synthetic NFAT promoter into the CAR construct does not impair CAR T cell function. References

[0082] [Table 4]

[0083] [Table 5]

[0084] [Table 6]

[0085] [Table 7]

[0086] [Table 8] Other embodiments Although the invention has been described in conjunction with the detailed description thereof, it is to be understood that the above description is intended to illustrate, but not to limit, the scope of the invention.

Claims

1. 1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises: a targeting domain comprising a ligand selected from IL-13 or a variant thereof and chlorotoxin or a variant thereof, a spacer, a transmembrane domain, a costimulatory domain, and a CD3ζ signaling domain, and a nucleotide sequence encoding a polypeptide comprising human interferon gamma or a variant thereof comprising a native human interferon gamma signal sequence for secretion of human interferon gamma or a signal sequence for secretion of human interferon gamma that is different from said native human interferon gamma signal sequence.

2. The nucleic acid molecule of claim 1, comprising a promoter that controls expression of both CAR and human interferon gamma.

3. 2. The nucleic acid molecule of claim 1, wherein a first promoter controls expression of the CAR and a second promoter controls expression of human interferon gamma or a variant thereof.

4. The nucleic acid molecule of claim 3 , wherein the first promoter is a constitutive promoter and the second promoter is a constitutive promoter or an inducible promoter.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein a nucleotide sequence encoding a 2A skip sequence is located between a nucleotide sequence encoding a CAR and a nucleotide sequence encoding human interferon gamma or a variant thereof, Optionally, the 2A skip sequence is selected from the group consisting of T2A, P2A, E2A and F2A.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein the human interferon gamma or a variant thereof comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:

29.

7. The nucleic acid molecule according to any one of claims 1 to 6, wherein the CAR comprises an amino acid sequence represented by SEQ ID NO:

30.

8. 8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a NKG2D transmembrane domain. Optionally, the transmembrane domain is a CD28 transmembrane domain or a CD8 transmembrane domain.

9. 9. The nucleic acid molecule of any one of claims 1 to 8, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, or a 2B4 costimulatory domain. In some cases, the costimulatory domain comprises an amino acid sequence represented by any of SEQ ID NOs: 22-25 and 66.

10. A nucleic acid molecule according to any one of claims 1 to 9, wherein the CD3ζ signaling domain comprises an amino acid sequence represented by SEQ ID NO:21 or a variant thereof comprising an amino acid sequence represented by any one of SEQ ID NOs:50 to 56.

11. The nucleic acid molecule according to any one of claims 1 to 10, wherein a linker having 3 to 15 amino acid residues is located between the costimulatory domain and the CD3ζ signaling domain or a variant thereof.

12. The nucleic acid molecule according to any one of claims 1 to 11, wherein the spacer comprises any one of the amino acid sequences represented by SEQ ID NOs: 2 to 12 and 44.

13. The nucleic acid molecule according to any one of claims 1 to 12, wherein the CAR comprises an amino acid sequence represented by any one of SEQ ID NOs: 70 to 76, or a variant thereof in which 1 to 5 amino acids have been modified.

14. A population of human T cells comprising a nucleic acid molecule according to any one of claims 1 to 13.

15. 15. The population of human T cells of claim 14 for use in a method for treating cancer in a patient, said method comprising administering the population of human T cells of claim 14, wherein said population of human T cells is autologous or allogeneic, and wherein the cancer is targeted by a targeting domain of a CAR.

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