Improved endodomains and immune cells comprising the same

Abstract

This disclosure relates to improved endodomains and immune cells comprising the same. Also provided are methods of using immune cells comprising the improved endodomains as well as methods of improving therapeutic cells comprising a CAR.

Description

Attorney Docket No.: CHLU-001 / 001WO IMPROVED ENDODOMAINS AND IMMUNE CELLS COMPRISING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent ApplicationNo. 63 / 506,746filed June 7, 2023, which is incorporated herein by reference in its entirety. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “CHLU- 001_001WO_SeqList.xml”. The XML file is 45,080 bytes, created on June 6, 2024, and is being submitted electronically via USPTO Patent Center. FIELD

[0003] This disclosure relates to improved endodomains and immune cells comprising the same. Also provided are methods of using immune cells comprising the improved endodomains as well as methods of improving therapeutic cells comprising a CAR. BACKGROUND

[0004] Chimeric antigen receptor (CAR) T cells are a cellular therapy used widely in the treatment of hematologic malignancies. This therapy has provided an impressive outcome in hematological malignancies and six CAR-T cell therapies have been approved for clinical use, including 4 CD19-targeted and 2 BCMA-targeted CARs. However, the clinical outcome of CAR-T cell therapy in solid tumors remains limited due to the suppressive tumor microenvironment rendering T cell dysfunction and poor persistence. To overcome this limitation, new strategies for boosting adoptive cell therapy in solid tumors are needed.

[0005] Several cytokines profoundly affect T cell activation, differentiation, and homeostasis, such as interleukin (IL)-7, IL-15, and IL-21 (see Du et al., Front Mol Biosci 8, 67517). For example, IL7 acts as a key mediator of the survival time of tumor-specific T cells in mice (see Pellegrini et al.,Nat Med 15, 528-536).

[0006] Described herein are novel CAR constructs incorporating the intracellular signaling domain of interleukins such as IL-7Ra.Attorney Docket No.: CHLU-001 / 001WO SUMMARY

[0007] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and an intracellular signaling domain of IL-7Ra.

[0008] In some embodiments, the intracellular signaling domain of IL-7Ra comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 6. In some embodiments, the intracellular signaling domain of IL-7Ra comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0009] In some embodiments, the intracellular domain further comprises a CD3ȗ costimulatory domain. In some embodiments, the CD3ȗ costimulatory domain is located C- terminal to the intracellular signaling domain of IL-7Ra. In some embodiments, the one or more costimulatory domains is located N-terminal to the intracellular signaling domain of IL-7Ra. In some embodiments, the one or more costimulatory comprises a 4-1BB costimulatory domain, a CD27 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, or an OX40 costimulatory domain.

[0010] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0011] In some embodiments, the extracellular antigen-binding domain is an scFv. In some embodiments, the extracellular antigen-binding domain binds to a cancer antigen. In some embodiments, the cancer antigen is B7H3, CD19, BCMA, or Folate receptor.

[0012] In another aspect, provided herein is a polynucleotide encoding a CAR described herein.

[0013] In another aspect, provided herein is an expression vector comprising a polynucleotide described herein.

[0014] In another aspect, provided herein is an immune cell comprising a CAR, a polynucleotide or an expression vector described herein. In some embodiments, the cell is a T cell, an NK cell, an NKT cells, a double negative T cell or a gamma / delta T cell. In some emvodiments, the immunce cell is derived from an induced pluripotent stem cell or a hematopietic stem cell. In some embodiments, the cell exhibits increased expansion compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the cell exhibits increased cytokine production compared to a cellAttorney Docket No.: CHLU-001 / 001WO comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the cell exhibits a naïve / stem-like memory T cell phenotype.

[0015] In another aspect, provided herein is a pharmaceutical composition comprising an immune cell described herein and a pharmaceutically acceptable carrier. In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a polynucleotide, an expression vector, an immune cell or a pharmaceutical composition described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is brain cancer, ovarian cancer, breast cancer, cholangiocarcinoma, multiple myeloma or AML. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is a lymphoma.

[0016] In another aspect, provided herein is a method of improving a therapeutic cell comprising a CAR, the method comprising inserting an intracellular signaling domain of IL-7Ra into the CAR. In some embodiments, the intracellular signaling domain of IL-7Ra comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 6. In some embodiments, the intracellular signaling domain of IL-7Ra comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the CAR further comprises a CD3ȗ costimulatory domain is located C-terminal to the intracellular signaling domain of IL-7Ra. In some embodiments, the CAR further comprises one or more costimulatory domains located N- terminal to the intracellular signaling domain of IL-7Ra. In some embodiments, the CAR cell targets B7H3, CD19, BCMA, FOLR1. In some embodiments, the method results in increased expansion compared to the expansion prior to improvement. In some embodiments, the method results in increased cytokine production compared to cytokine production prior to improvement. BRIEF DESCRIPTION OF DRAWINGS

[0017] FIGs.1A-1E. FIG.1A is a schematic representation of CARs with intracellular signaling of IL7 receptor alpha chain of different sizes; long (L, 585 bp), medium (M, 375 bp), and short (S, 171 bp). FIG.1B shows a flow cytometric plot of the expression of the CARs in transduced human T lymphocytes. FIG.1C is a bar graph represents the percentage of transgene expression. FIG.1D shows the expansion capacity of transduced CARs. **** Pௗ<ௗ0.0001 vs. NT; **** Pௗ<ௗ0.0001 vs. B7H3-BB-IL7L; **** Pௗ<ௗ0.0001 vs. B7H3-BB-IL7M. FIG.1E shows the absolute CAR-T cell numbers and percentage of CAR expression. ** Pௗ<ௗ0.01, **** Pௗ<ௗ0.0001 vs. NT control.Attorney Docket No.: CHLU-001 / 001WO

[0018] FIGs.2A-2F. FIG.2A shows CD4+ and CD8+ T cell subsets in transduced human T lymphocytes. FIG.2B shows the frequency of naïve / stem-like memory T cells (TN / TSCM), memory T cells (TCM), effector memory T cells (TEM), and effector memory T cells (TEMRA) in CAR-T cells* Pௗ<ௗ0.05, *** Pௗ<ௗ0.001 vs. B7H3-BB CARs. FIG.2C shows the characterization of the immune phenotype of CD4+ and CD8+ T cell subpopulations. Naïve T cells (TN, CD45RA+CCR7+), central memory T cells (TCM, CD45RA^CCR7+), effector memory T cells (TEM, CD45RA^CCR7^), and effector T cells (TEMRA, CD45RA+CCR7^) in CAR-T cell products on day 11 after transduction. Data are presented as the mean ± S.E.M. (n=3). * Pௗ<ௗ0.05, ** Pௗ<ௗ0.01 vs. B7H3 CAR-T cells (two-way ANOVA). FIG.2Dshows a flow cytometric analysis of intracellular p-STAT5 expression in B7H3-targeting CAR-T cells after stimulation with a NALM-6 for 30 and 60 min. FIG.2E shows a flow cytometric analysis of intracellular pSTAT5 expression in CAR-T cells p-STAT5 after stimulation with U87 cells for 30 or 60 min. FIG.2F shows a flow cytometric analysis of intracellular pSTAT5 expression in CAR-T cells stimulated with LN229 cells for 30 or 60 min. Histograms of pSTAT5 expression in the CD3+ population were generated. The percentage of pSTAT5+ T cells and the relative MFI of pSTAT5 were analyzed and are shown in the bar graphs. The data are presented as the mean ± S.E.M. (n = 4) *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA).

[0019] FIG.3A-3F. FIG.3A shows B7H3 expression in GBM (LN229 and U87) and B- cell acute lymphoblastic leukemia (NALM-6) cell lines screened by flow cytometry using a goat anti-human B7H3 polyclonal antibody as the primary antibody and PE-conjugated anti-goat IgG as the secondary antibody. FIG.3B shows cytotoxicity of CAR-T cells against LN229,U87 glioblastoma and NALM-6 leukemia cells. FIG.3C shows CD25 molecule expressed on CAR-T when co-culturing with LN229, U78 or NALM-6 cells for 72 h. FIG.3D shows CD69 molecule expressed on CAR-T after co-cultured with LN229, U78 or NALM-6 cells for 72 h. FIG.3E shows cytokine production of CAR-T cells stimulated with GBM cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and nonsignificance (ns) vs. B7H3-BB CARs. FIG.3F shows the average MFI of the inhibitory receptors PD1 (upper), TIGIT (middle), and TIM3 (lower) in CAR-T cells cocultured with GBM cells.

[0020] FIGs.4A-4I. FIG.4A is a schematic representation of the experimental procedure for the tumor rechallenge in vitro assay. FIG.4B shows Representative flow cytometric analysis of the rechallenge assay using LN229 cells. FIGs.4C and 4D show the remaining target cells andAttorney Docket No.: CHLU-001 / 001WO effector cells, respectively, from each round of coculture with LN229.. FIG.4E shows a representative flow cytometric analysis of U87 cells in The rechallenge assay. FIGs.4F and 4G show the remaining target and effector CAR-T cells, respectively, from each round of co-culture. FIGs.4H and 4I show the immune phenotype of CAR-T cells after the third re-exposure with U87 cell in tumor rechallenge assay. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. B7H3-BB CARs (one-way ANOVA). FIG.4H flow cytometry analysis of the memory T cell subpopulations identified as follows: naïve T cells (TN, CD45RA+CCR7+), central memory T cells (TCM, CD45RA^CCR7+), effector memory T cells (TEM, CD45RA^CCR7^), and effector T cells (TEMRA, CD45RA+CCR7^) and bar graphs presenting the percent positive cells of phenotype in the CD4+ and CD8+ T cell subpopulations. Data are shown as the mean ± S.E.M. (n=3)*Pௗ<ௗ0.05 TEM of B7H3-L and B7H3-S CAR-T cells vs. B7H3 CAR-T cells (two-way ANOVA). FIG.4I shows the expression of the exhaustion markers including PD-1, TIGIT, TIM-3, LAG-3 on CAR-T cells after the third re-exposure with U87. Data are shown as the mean ± S.E.M. (n=3).*Pௗ<ௗ0.05,**Pௗ<ௗ0.01 (one-way ANOVA).

[0021] FIGs.5A-5C. FIG.5A shows a heatmap illustrating the relative expression of cell proliferation, apoptosis and metabolic process by in vitro co-culturing CAR-T cells against target tumor cells at an E:T ratio of 1:1 in the absence of IL-2 or IL-7, and IL-15 supplementation for 72 h. FIG.5B shows the programmed cell death of CAR-T cells after coculture with GBM cells as a representative flow cytometry plot for one sample and a bar graph of the average results from three donors. * P < 0.05, **** P < 0.0001 vs. live B7-BB CARs; # Pௗ<ௗ0.05, #### Pௗ<ௗ0.0001 vs. apoptotic B7-H3-BB CARs. FIG.5C shows the expression of genes involved in apoptosis, proliferation, and metabolism in CAR-T cells cocultured with LN229 cells.. ++ P < 0.05 vs. prestimulated NT controls, $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001 vs. prestimulated B7H3-BB CARs; ** P < 0.05 vs. poststimulated NT controls; # Pௗ<ௗ0.05 ## Pௗ<ௗ0.01, and #### Pௗ<ௗ0.0001 vs. poststimulated B7H3-BB CARs.

[0022] FIGs.6A-6E. FIG.6A is a schematic representation of the experimental protocol using LN229 cell line-derived xenografts. FIG.6B shows the Tumor volume in LN229 subcutaneously inoculated mice ** P < 0.01; nonsignificance (ns) vs. the NT control group, # P<0.05 vs. the B7H3-BB-treated group. FIG.6C shows the estimated tumor mass in each animal. FIG.6D shows the body weight of mice bearing LN229 tumor cells. FIG.6E shows Kaplan–Attorney Docket No.: CHLU-001 / 001WO Meier survival curves comparing tumor progression over time in the NT-, B7H3-BB- and B7H3- BB-IL7S-treated groups (n = 4 mice per group). *** P < 0.01 vs. B7-H3-BB-treated group.

[0023] FIGs.7A-7H. FIG.7A is a schematic representation of the experimental protocol using U87 cell line-derived xenografts. FIG.7B shows the tumor volume in U87 subcutaneously inoculated mice. ** P < 0.01; *** P < 0.001 vs. the NT control group; # P<0.05 vs. the B7H3- BB-treated group. FIG.7C shows the estimated tumor mass in each animal. FIG.7D shows the body weight of mice bearing U87 tumor cells. FIG.7E shows Kaplan–Meier survival curves comparing tumor progression over time in the NT-, B7H3-BB- and B7H3-BB-IL7S-treated groups (n = 4 mice per group). ** P < 0.01 vs. B7-H3-BB-treated group. FIG.7F shows IL2- independent proliferation of NT, B7H3-BB, and B7H3-BB-IL7S CAR-T cells. The results are presented as the mean ± SEM of three individual donors. FIG.7G shows a representative IHC image of human CD3 staining in tumor tissues from LN229 tumor-bearing mice on day 49. FIG. 7H shows a quantification of CD3-positive cells that infiltrated into tumors. The data are shown as the mean ± S.E.M. (n = 3). *P < 0.05 versus NT; #P < 0.05 versus B7H3- treated group (one- way ANOVA).

[0024] FIGs.8A-8G show the characterization of CD19-targeted CAR-T cells comprising an IL-7S endo-signaling domain. FIG.8A shows a schematic llustration of anti- CD19 CAR-T cells. FIG.8B shows CAR-T cell transduction efficiency in primary CD3+ T cells. FIG.8C and 8D show proliferation expressed as total cell number and fold expansion of CD19- specific CAR-T cells during manufacturing, respectively. FIGs.8E-8G show results of a tumor rechallenge assay. Total effector cells and specific lysis after each round are shown in FIGs.8E and 8F, respectively. Representative flow cytometry data of the tumor rechallenge assay is shown in FIG.8G. Data shown are mean ± S.E.M. from three independent donors (n=3). *p < 0.05, **p < 0.01 by one-way ANOVA.

[0025] FIG.9A-9G show the characterization of BCMA-targeted CAR-T cells comprising an IL-7S endosignaling domain. FIG.9A shows a schematic illustration of anti- BCMA CAR-T cells / FIGs.9B shows CAR-T cell transduction efficiency in primary CD3+ T cells. FIG.9C and 9D show proliferation expressed as total cell number and fold expansion of BCMA-specific CAR-T cells, respectively. FIGs.9E-9G show the results of a t tumor rechallenge assay. Total effector cells and specific lysis after each round are shown in FIGs.9E and 9F, respectively. Representative flow cytometry of tumor rechallenge assay is shown in FIG.Attorney Docket No.: CHLU-001 / 001WO 9G. Data shown are mean ± S.E.M. from three independent donors (n=3). *p < 0.05, **p < 0.01, *** P<0.005 by one-way ANOVA.

[0026] FIGs.10A-10F show the characterization of FOLR1-targeted CAR-T cells comprising an IL-7S endosignaling domain. FIG.10A shows a schematic illustration of anti- FOLR1 CAR-T cells. FIG.10B shows representative flow cytometry plots showing CAR-T cell transduction efficiency and the mean florescent intensity (MFI) of CAR expression on primary CD3+ T cells FIG.10B. FIG.10C shows proliferation expressed as fold expansion and absolute number of FOLR1-specific CAR-T cells. FIGs.10D-10F show results of a ttumor rechallenge assay. FIG.10D shows representative flow cytometry plots. FIGs.10E and 10F show residual target cells and effector cells, respectively. Data shown are mean ± S.E.M. from two independent donors (n=2). *p < 0.05 by one-way ANOVA. DETAILED DESCRIPTION

[0027] It is to be understood that one skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rdedition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18thedition (1990). These texts can, of course, also be referred to in making or using an aspect of the disclosure. Provided herein are chimeric antigen receptors (CARs) comprising an intracellular signaling domain of IL-7R, as well as polynucleotides encoding the CARs, immune cells comprising the polynucleotides or CARs, and methods of using the same. Chimeric Antigen Receptors

[0028] In one aspect, provided herein is a chimeric antigen receptor (CAR) an intracellular signaling domain of IL-7R. The basic structure of CARs is well known in the art, including an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding region. First-generation CARs generally comprise an extracellular antigen-recognizing domain combined with intracellular CD3ȗ domain for signalAttorney Docket No.: CHLU-001 / 001WO transduction. Second-generation CARs usually comprise two intracellular domains: a CD3ȗ costimulatory and an additional costimulatory domain. Third-generation CARs usually comprise three intracellular domains: a CD3ȗ costimulatory and two additional costimulatory domains. See, e.g., Tomasik et al., Front. Immunol., 28 October 2022, Sec. Cancer Immunity and Immunotherapy Volume 13 – 2022.

[0029] In some embodiments of the disclosure, a CAR comprises, in N-terminal to C- terminal order, (i) an extracellular antigen-binding domain, (ii) a transmembrane domain and (iii) an intracellular domain comprising an intracellular signaling domain of IL-7Ra and one or more costimulatory domains.

[0030] CARs of the disclosure may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.

[0031] This disclosure also encompasses CARs comprising one or more conservative amino acid substitutions relative to the sequences disclosed herein. A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue are substitutions that change an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size). For example, lysine, arginine and histidine have similar properties in that they have a basic side-chain, and aspartic acid and glutamic acid have similar properties in that they have an acidic side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have an uncharged polar side-chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have a non-polar side-chain. Also, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have an aromatic side-chain. Thus, it will be obvious to those skilled in the art that, even when substitution of amino acid residues in groups showing similar properties as described above occurs; it will show no significant change in the properties. Extracellular Domains

[0032] The CARs provided herein may comprise an extracellular antigen-binding domain. Any suitable type of antigen-binding domain that can regulate the activity of a receptor in a ligand-dependent manner may be used in the CARs provided herein. Illustrative antigen binding domains include, for example, a single chain variable fragment (scFv), single domainAttorney Docket No.: CHLU-001 / 001WO antibody fragment (sdAb), Vȕ-only domains, and TCR antigen binding domains derived from the TCR Į and ȕ chain variable domains.

[0033] In some embodiments, the extracellular antigen-binding domain is an scFv. An scFv is a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain. The heavy chain variable region and the light chain variable region of an antibody each contain three complementarity-determining regions (CDRs) interposed between flanking stretches known as framework regions. As is well known in the art, the CDRs of an antibody or an scFv generally determine the specificity of an antibody or scFv for its target, while the framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0034] Generally, the light and heavy chain variable regions in an scFv are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain. The scFv generally retains the specificity of the intact antibody from which it is derived.

[0035] Any known scFv may be used in the CARs described herein, for example, the scFv of a known therapeutic antibody.

[0036] In some embodiments, the extracellular antigen-binding domain binds to a cancer antigen. The sequences of scFvs binding to cancer antigens are well-known in the art.

[0037] In some embodiments, the cancer antigen is MUC1, CD19, BCMA, Folate receptor alpha (FOLR1), cMet, GD2, NKG2D, or EpCAM.

[0038] In some embodiments, the cancer antigen is B7H3. In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence DIQMTQSPASLSVSVGETVTITCRTSENIYSNLAWYQQKQGKSPQLLVYVATNLADGVP SRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGASPTFGGGTKLEIKGGGGSGGGGSG GGGSEVKLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQTPEKRLEWVAAINSD GGNTYYPDTVKDRFTISRDSAKHTLYLQMSNLRSEDTALYYCARHEDYRSGFAYWGQ GTLVTVSA (SEQ ID NO: 1). In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellularAttorney Docket No.: CHLU-001 / 001WO antigen-binding domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the extracellular antigen-binding domain consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0039] In some embodiments, the cancer antigen is Folate receptor alpha (FOLR1). In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDT FYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVT VSSGGGGSGGGGSGGGGSDIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYH QKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYT FGGGTKLEIKR (SEQ ID NO: 21). In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the extracellular antigen-binding domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the extracellular antigen-binding domain consists of the amino acid sequence set forth in SEQ ID NO: 21.

[0040] In some embodiments, the cancer antigen is CD19. In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGG GSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS (SEQ ID NO: 22). In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the extracellular antigen-binding domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the extracellular antigen-binding domain consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0041] In some embodiments, the cancer antigen is BCMA. In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence having at least 80%Attorney Docket No.: CHLU-001 / 001WO identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence DAVMTQIPLSLPVSLGDQVSISCRSSQSLENSDGNTYVSWYVQKPGQSPQLLIYRVSIRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGIYFCLQVSHVPFTFGSGTKLEIKRADAAPTVSI FPPSSEQLTSGGASVVCFLNNFYPKDINVSSNRIGGGGSGGGGSGGGGSEVQLQQSGPES VKPGASVKMSCKASGYTFTNYVMHWVKQKPGQGLEWIGYIIPYNDVTKYNEKFKGKT TLTSDKSSSTAYMDLSSLTSEDSAVYSCARWAWDGYFDYWGQGTTLTVSS (SEQ ID NO: 23). In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the extracellular antigen-binding domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the extracellular antigen-binding domain consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0042] “Identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. In some embodiments, the comparison is across the entire length of the molecule. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity, with one of the sequences of the present disclosure. Alignment and percent sequence identity may be determined for the nucleic acid or amino acid sequences provided herein by importing said nucleic acid or amino acid sequences into and using ClustalW (available at https: / / genome.jp / tools-bin / clustalw / ). For example, the ClustalW parameters used for performing the protein sequence alignments found herein were generated using the Gonnet (for protein) weight matrix. In some aspects, the ClustalW parameters used for performing nucleic acid sequence alignments using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix. Other algorithms for aligning amino acid and nucleic acid sequence are known in the art and include, for example, NCBI’s BLAST.Attorney Docket No.: CHLU-001 / 001WO Hinge Domain

[0043] A CAR described herein may further comprise a hinge region connecting the extracellular antigen-binding domain and the transmembrane domain.

[0044] Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo.

[0045] In some embodiments, the hinge domain is a IgG2-CH3-hinge domain. In some embodiments, the hinge domain is a CD8a hinge domain. In some embodiments, the hinge domain is a CD28 hinge domain.

[0046] In some embodiments, the IgG2-CH3 hinge domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence ERKCCVECPPCPGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPE NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO: 2). In some embodiments, the IgG2-CH3 hinge domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgG2-CH3 hinge domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IgG2-CH3 hinge domain consists of the amino acid sequence set forth in SEQ ID NO: 2. Transmembrane Domains

[0047] The CARs of the present disclosure may comprise a transmembrane domain situated between the extracellular domain and the intracellular domain, or between the hinge domain and the intracellular domain. The transmembrane domain may be fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 intracellular domain might also comprise a CD28 transmembrane domain.

[0048] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. Transmembrane regions may comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may be synthetic.Attorney Docket No.: CHLU-001 / 001WO

[0049] In some embodiments, a CAR provided herein comprises a CD28 transmembrane domain. In some embodiments, a CAR provided herein comprises a CD8a transmembrane domain. In some embodiments, a CAR provided herein comprises a TNFRSF19 transmembrane domain.

[0050] In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence TRFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 3). In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the CD28 transmembrane domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 3. Intracellular Domains

[0051] The CARs disclosed herein may comprise an intracellular domain. The intracellular signaling domain of a CAR is generally understood to be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed.

[0052] In some embodiments, the intracellular domain of a CAR described herein comprises an intracellular signaling domain of IL-7Ra and one or more costimulatory domains. The intracellular domain of IL-7Ra may be placed anywhere in the intracellular domain of the CAR. In some embodiments, the intracellular domain of the CAR comprises, in N-terminal to C- terminal direction, a costimulatory domain, an intracellular signaling domain of IL-7Ra and a CD3ȗ domain.

[0053] Without wishing to be bound by theory, it is believed that the intracellular signaling domain of IL-7Ra confers improved properties to a cell comprising the CAR of disclosure, including, for example, improved proliferations, delayed exhaustion, increased anti- tumor activity and / or a stem-like memory T cell (naïve T cell) phenotype. In some embodiments, activation of the IL-7Ra intracellular signaling domain requires binding of the immune cell expressing the CAR to its target (i.e., the antigen to which the extracellular antigen-binding domain binds).Attorney Docket No.: CHLU-001 / 001WO

[0054] In some embodiments, a truncated form of the IL-7Ra intracellular signaling domain may be used in the CARs described herein. Illustrative sequences of the IL-7Ra intracellular signaling domains are set forth in SEQ ID NOs: 4-6:

[0055] In some embodiments, an IL-7Ra intracellular signaling domain comprises the sequence KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFL QDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQE EAYVTMSSFYQNQ (SEQ ID NO: 4). In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein consists essentially of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 7Ra intracellular signaling domain in a CAR disclosed herein consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0056] In some embodiments, an IL-7Ra intracellular signaling domain comprises the sequence KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFL QDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESVAQGQPILTSLGSNQEEAYVTMSS FYQNQ (SEQ ID NO: 5). In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein consists essentially of the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein consists of the amino acid sequence set forth in SEQ ID NO: 5.Attorney Docket No.: CHLU-001 / 001WO

[0057] In some embodiments, an IL-7Ra intracellular signaling domain comprises the sequence KKRIKPIVWPSLPDHKKTLEHLCKKPRKVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (SEQ ID NO: 6). In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the IL- 7Ra intracellular signaling domain in a CAR disclosed herein consists essentially of the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the IL-7Ra intracellular signaling domain in a CAR disclosed herein consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0058] While the entire intracellular signaling domain of a protein can be employed as a costimulatory domain in the CARs described herein, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR of the instant disclosure. The term intracellular domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0059] Examples of costimulatory domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0060] In some embodiments, the one or more costimulatory domain of a CAR described herein comprises a 4-1BB costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, an OX40 costimulatory domain, a CD27 costimulatory domain, a CD137 costimulatory domain, a IL-2Rȕ costimulatory domain, a Fc Receptor gamma (FcRȖ) costimulatory domain, a Fc Receptor beta (FcRȕ) costimulatory domain, a CD2 costimulatory domain, a CD3Ȗ costimulatory domain, a CD3į costimulatory domain, a CD3İ costimulatoryAttorney Docket No.: CHLU-001 / 001WO domain, a CD5 costimulatory domain, a CD7 costimulatory domain, a CD22 costimulatory domain, a CD28H costimulatory domain, a CD30 costimulatory domain, a CD40 costimulatory domain, a CD79a costimulatory domain, a CD79b costimulatory domain, a CD66d costimulatory domain, a DAP-10 costimulatory domain, a LFA1 costimulatory domain, a NKGD2 costimulatory domain, a FN14 costimulatory domain, a HVEM costimulatory domain, a LTBR costimulatory domain, a TNFR1 costimulatory domain, a TNFR2 costimulatory domain, a BAFF-R costimulatory domain, a BCMA costimulatory domain, a TACI costimulatory domain, a TROY costimulatory domain, a RANK costimulatory domain, an EDAR costimulatory domain, a XEDAR costimulatory domain, a GITR costimulatory domain, a LIGHT costimulatory domain, a DR6 costimulatory domain, a CD276 (B7-H3) costimulatory domain, a NGFR costimulatory domain or any combination thereof.

[0061] In some embodiments, the one or more costimulatory domain comprises one or more CD3ȗ costimulatory domains. In some embodiments, the CD3ȗ costimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 7). In some embodiments, the CD3ȗ costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the CD3ȗ costimulatory domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the CD3ȗ costimulatory domain consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0062] In some embodiments, the one or more costimulatory domain comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are signaling motifs found a variety of immune cell signaling proteins. In some embodiments, the intracellular domain contains 1, 2, or 3 ITAMs. ITAMS for use in the CARs of the present disclosure may be derived from, for example, TCRȗ, FcRȖ, FcRȕ, CD3Ȗ, CD3į, CD3İ, CD3ȗ, CD5, CD22, CD79a, CD79b, and CD66d.

[0063] The one or more costimulatory domains within the intracellular domain of the CARs of the instant disclosure may be linked to each other in a random or specified order.Attorney Docket No.: CHLU-001 / 001WO Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker. Illustrative CAR Sequences

[0064] Examples of CAR comprising an IL-7Ra intracellular signaling domain are shown in Table 1. Table 1: Illustrative CAR SequencesAttorney Docket No.: CHLU-001 / 001WOPolynucleotides and Vectors

[0065] In another aspect, the present disclosure is directed to polynucleotide sequences that encode the CARs described herein. In some embodiments, the polynucleotide encodes a fusion protein comprising, in N-terminal to C-terminal direction, the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and the intracellular domain of a CAR described herein.

[0066] In some embodiments, the polynucleotide comprises one or more of: a nucleic acid sequence encoding the extracellular domain of a CAR described herein, a nucleic acid sequence encoding a hinge domain of a CAR described herein, a nucleic acid sequence encoding the transmembrane domain of a CAR described herein, a nucleic acid sequence encoding intracellular signaling domain of IL-7Ra, and a nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein.

[0067] In some embodiments, the polynucleotide comprises, in 5’ to 3’ direction, a nucleic acid sequence encoding the extracellular domain of a CAR described herein, a nucleic acid sequence encoding the transmembrane domain of a CAR described herein, a nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein a nucleic acidAttorney Docket No.: CHLU-001 / 001WO sequence encoding intracellular signaling domain of IL-7Ra, and a nucleic acid sequence encoding a CD3ȗ costimulatory domain.

[0068] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the extracellular domain of a CAR described herein. An illustrative nucleic acid sequence encoding an extracellular antigen-binding domain that binds to B7H3 is set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding the extracellular domain of a CAR described herein is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding the extracellular domain of a CAR described herein comprises the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding the extracellular domain of a CAR described herein consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding the extracellular domain of a CAR described herein consists of the nucleic acid sequence set forth in SEQ ID NO: 8.

[0069] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the hinge domain of a CAR described herein.

[0070] An illustrative nucleic acid sequence encoding a IgG2-CH3 hinge domain is set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding the hinge domain of a CAR described herein is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding the hinge domain of a CAR described herein comprises the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding the hinge domain of a CAR described herein consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding the hinge domain of a CAR described herein consists of the nucleic acid sequence set forth in SEQ ID NO: 9.

[0071] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the transmembrane domain of a CAR described herein. An illustrative nucleic acid sequence encoding a CD28 transmembrane domain is set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of a CARAttorney Docket No.: CHLU-001 / 001WO described herein is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of a CAR described herein comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of a CAR described herein consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of a CAR described herein consists of the nucleic acid sequence set forth in SEQ ID NO: 10.

[0072] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the intracellular domain of a CAR described herein.

[0073] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra. Illustrative nucleic acid sequences encoding an intracellular signaling domain of IL-7Ra are set forth in SEQ ID NOs: 11-13.

[0074] In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra comprises the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist essentially of the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist of the nucleic acid sequence set forth in SEQ ID NO: 11.

[0075] In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra comprises the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist essentially of the nucleic acid sequence set forthAttorney Docket No.: CHLU-001 / 001WO in SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist of the nucleic acid sequence set forth in SEQ ID NO: 12.

[0076] In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra comprises the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist essentially of the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra consist of the nucleic acid sequence set forth in SEQ ID NO: 13.

[0077] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein.

[0078] An illustrative nucleic acid sequence encoding the 4-1BB costimulatory domain is set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein comprises the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding one or more costimulatory domains of a CAR described herein consists of the nucleic acid sequence set forth in SEQ ID NO: 14.

[0079] An illustrative nucleic acid sequence encoding the CD3ȗ costimulatory domain is set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding the CD3ȗ costimulatory domain is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding the CD3ȗ costimulatory domain comprises the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding the CD3ȗ costimulatory domain consistsAttorney Docket No.: CHLU-001 / 001WO essentially of the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding the CD3ȗ costimulatory domain consists of the nucleic acid sequence set forth in SEQ ID NO: 15.

[0080] The DNA encoding a CAR or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein also can be modified. Thus, in one embodiment, the present disclosure includes variants to the nucleic acid sequences that encode encoding a CAR or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the CAR or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR of the present disclosure under at least moderately stringent hybridization conditions.

[0081] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g.20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C – 16.6 (Log10 [Na+]) + 0.41(%(G+C) – 600 / l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1°C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions.

[0082] The polynucleotides encoding CAR described herein or encoding or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein may further comprise a Kozak sequence. A Kozak sequence can function as the start site for protein translation. An illustrativeAttorney Docket No.: CHLU-001 / 001WO Kozak sequence that may be used in the polynucleotides described herein is GCCACC (SEQ ID NO: 16).

[0083] The polynucleotides encoding CAR described herein or encoding or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein may further comprise a Stop codon after the nucleic acid sequence encoding the C-terminal domain of the CAR (e.g., the one or more costimulatory domain). In some embodiments, the Stop coding is TAA.

[0084] Three illustrative polynucleotides encoding CARs disclosed herein are shown in SEQ ID NOs: 17-19 and 27-29 below. Each of SEQ ID NOs: 17-19 comprises, in 5’ to 3’ direction, a nucleic acid sequence encoding an extracellular B7H3 targeting domain, a nucleic acid sequence encoding an IgG2-CH3 hinge domain, a nucleic acid sequence encoding a CD28 transmembrane domain, a nucleic acid encoding a 4-1BB costimulatory domain, a nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra , and a nucleic acid sequence encoding a CD3ȗ costimulatory domain. SEQ ID NO: 27 comprises, in 5’ to 3’ direction, a nucleic acid sequence encoding an extracellular Folate Receptor targeting domain, a nucleic acid sequence encoding an IgG2-CH3 hinge domain, a nucleic acid sequence encoding a CD28 transmembrane domain, a nucleic acid encoding a 4-1BB costimulatory domain, a nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra , and a nucleic acid sequence encoding a CD3ȗ costimulatory domain. SEQ ID NO: 28 comprises, in 5’ to 3’ direction, a nucleic acid sequence encoding an extracellular CD19 targeting domain, a nucleic acid sequence encoding an IgG2-CH3 hinge domain, a nucleic acid sequence encoding a CD28 transmembrane domain, a nucleic acid encoding a 4-1BB costimulatory domain, a nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra , and a nucleic acid sequence encoding a CD3ȗ costimulatory domain. SEQ ID NO: 29 comprises, in 5’ to 3’ direction, a nucleic acid sequence encoding an extracellular BCMA targeting domain, a nucleic acid sequence encoding an IgG2-CH3 hinge domain, a nucleic acid sequence encoding a CD28 transmembrane domain, a nucleic acid encoding a 4-1BB costimulatory domain, a nucleic acid sequence encoding an intracellular signaling domain of IL-7Ra , and a nucleic acid sequence encoding a CD3ȗ costimulatory domain. 1 CTCGAGGCCA CCATGATGGA GTTCGGCCTC TCATGGCTGT TTTTAGTTGC TATCTTAAAG 61 GGGGTCCAGT GTGACATCCA GATGACTCAG TCTCCAGCCT CCCTATCTGT ATCTGTGGGA 121 GAAACTGTCA CCATCACATG TCGAACAAGT GAGAATATTT ACAGTAATTT AGCATGGTATAttorney Docket No.: CHLU-001 / 001WO 181 CAGCAGAAAC AGGGAAAATC TCCTCAGCTC CTGGTCTATG TTGCAACAAA CTTAGCAGAT 241 GGTGTGCCAT CAAGGTTCAG TGGCAGTGGA TCAGGCACAC AGTATTCCCT CAAGATCAAC 301 AGCCTGCAGT CTGAAGATTT TGGGAATTAT TACTGTCAAC ATTTTTGGGG TGCTTCCCCG 361 ACGTTCGGTG GAGGCACCAA GCTGGAAATC AAAGGGGGCG GCGGAAGCGG GGGCGGCGGC 421 AGCGGTGGGG GAGGCTCTGA AGTGAAGTTG GTGGAGTCTG GGGGAGGCTT AGTGAAGCCT 481 GGAGGGTCCC TGAAACTCTC CTGTGCAGCC TCTGGATTCA CCTTCAGTAA TTATGCCATG 541 TCTTGGGTTC GCCAGACTCC AGAAAAGAGG CTGGAGTGGG TCGCAGCCAT TAATAGTGAT 601 GGTGGTAACA CCTACTATCC AGACACTGTG AAGGACCGCT TCACCATCTC CAGAGACAGT 661 GCCAAGCACA CCCTGTACCT GCAAATGAGC AATCTGAGGT CTGAGGACAC AGCCTTATAT 721 TACTGTGCAA GACATGAAGA CTATAGGTCC GGGTTTGCTT ACTGGGGCCA AGGGACTCTG 781 GTCACTGTCT CTGCAGGATC CGAGCGAAAG TGCTGTGTGG AGTGCCCCCC ATGCCCTGGA 841 CAGCCTAGAG AACCACAAGT ATACACTCTG CCACCTAGCC GTGAGGAAAT GACCAAGAAT 901 CAAGTCAGCC TCACCTGCTT AGTGAAGGGA TTCTATCCAT CAGACATTTC CGTCGAGTGG 961 GAATCTAACG GACAGCCGGA GAACAACTAT AAAACTACTC CACCCATGCT CGACAGCGAC 1021 GGTTCCTTCT TCCTCTACAG TAAGCTCACA GTTGATAAGT CAAGGTGGCA GCAAGGAAAT 1081 GTGTTTTCGT GCTCAGTGAT GCACGAGGCG CTGCATAACC ATTACACCCA GAAGAGCCTC 1141 AGCCTCTCCC CTGGAAAGAC GCGTTTTTGG GTACTGGTCG TGGTGGGTGG GGTGCTCGCT 1201 TGCTATTCAT TGTTGGTGAC CGTGGCATTC ATCATCTTCT GGGTAATGCA TAAGAGAGGG 1261 CGGAAGAAAT TACTATACAT ATTCAAGCAA CCCTTCATGCGACTACTCAG 1321 GAGGAGGATG GCTGCTCCTG CCGATTCCCC GAGGAAGAAGCGAGCTGAAG 1381 AAGAGGATCA AGCCTATCGT GTGGCCCTCA CTGCCCGACC ATAAAAAAAC CCTGGAACAC 1441 CTGTGCAAAA AGCCAAGAAA GGTGGCCCAG GGCCAGCCTA TCCTGACCTC CCTCGGCTCA 1501 AACCAGGAAG AAGCATATGT GACCATGTCC AGCTTCTATC AGAACCAACG GGTGAAGTTC 1561 TCTAGAAGTG CCGATGCCCC CGCCTACCAG CAAGGACAGA ACCAGCTGTA CAATGAGTTG 1621 AACTTGGGAC GGAGGGAGGA GTATGACGTC CTGGACAAAC GTCGAGGTCG CGACCCCGAG 1681 ATGGGCGGCA AACCACAACG TCGCAAGAAC CCTCAGGAGG GGCTTTATAA CGAGCTGCAG 1741 AAAGATAAGA TGGCCGAGGC CTACTCTGAA ATCGGAATGA AAGGGGAGCG TAGAAGGGGA 1801 AAAGGTCACG ATGGATTGTA CCAAGGTCTG AGTACGGCGA CCAAGGACAC TTATGACGCA 1861 TTGCATATGC AGGCACTGCC CCCGAGATAA GCATGC (SEQ ID NO: 17)Attorney Docket No.: CHLU-001 / 001WO 1561 GACACCTTTC CACAGCAGTT GGAGGAAAGC GTCTGGGCGG TGATGTGCAG 1621 TCCCCAAATT GCCCAAGTGA AGACGTCGTC AGAGTTTCGG CAGAGACTCT 1681 TCGCTGACCT GTCTGGCTGG GAATGTGAGC CCCCCATTCT GAGTTCATCC 1741 CGCAGTCTTG ACTGTAGGGA GTCCGGGAAG ACGTTTATCA GGACCTGTTA 1801 CTCAGCCTCG GGACAACAAA TTCGACACTC TTTCCCTGCA GTCTGGCATA 1861 CTGACTTTGA ATCCAGTCGC ACAGGGGCAG CAAGCCTGGG AAGTAATCAG 1921 GAGGAGGCCT ACGTGACTAT GAGCTCGTTT AGCGGGTGAA GTTCTCTAGA 1981 AGTGCCGATG CCCCCGCCTA CCAGCAAGGATGTACAATGA GTTGAACTTG 2041 GGACGGAGGG AGGAGTATGA CGTCCTGGAC AAACGTCGAG GTCGCGACCC CGAGATGGGC 2101 GGCAAACCAC AACGTCGCAA GAACCCTCAG GAGGGGCTTT ATAACGAGCT GCAGAAAGAT 2161 AAGATGGCCG AGGCCTACTC TGAAATCGGA ATGAAAGGGG AGCGTAGAAG GGGAAAAGGT 2221 CACGATGGAT TGTACCAAGG TCTGAGTACG GCGACCAAGG ACACTTATGA CGCATTGCAT 2281 ATGCAGGCAC TGCCCCCGAG ATAAGCATGC (SEQ ID NO: 18)1 ATGGAGTTTG GCTTGTCTTG GCTTTTTCTC GTTGCAATTC TGAAAGGCGT TCAGTGCAGC 61 CGAGCCGCCC AGCCCGCAAT GGCTCAAGTT CAGCTACAGC AAAGCGGCGC CGAGCTCGTA 121 AAGCCTGGTG CGTCTGTTAA AATCAGTTGC AAAGCATCCG GCTACAGCTT CACTGGGTAC 181 TTCATGAATT GGGTGAAACA GTCTCACGGC AAAAGTCTCG AATGGATTGG AAGAATCCATAttorney Docket No.: CHLU-001 / 001WO 241 CCCTATGACG GTGATACGTT TTATAACCAG AACTTTAAAG ATAAAGCAAC CCTCACCGTG 301 GACAAGTCTT CCAATACAGC CCACATGGAA CTGCTGTCTC TCACGAGCGA GGACTTCGCC 361 GTCTACTACT GCACCCGCTA CGATGGGTCG CGCGCTATGG ATTACTGGGG ACAGGGCACT 421 ACTGTGACGG TCTCCAGCGG GGGAGGCGGA AGTGGCGGAG GAGGAAGTGG AGGCGGGGGC 481 AGCGATATCG AGCTGACCCA GAGCCCGGCC TCCCTGGCAG TCTCTCTCGG CCAGCGAGCC 541 ATCATCTCAT GTAAGGCTTC GCAATCTGTG TCTTTTGCCG GCACAAGCTT AATGCACTGG 601 TATCACCAGA AGCCGGGGCA GCAACCAAAA CTTTTGATTT ATCGGGCCTC AAACCTGGAA 661 GCAGGAGTCC CTACTAGATT TAGCGGGAGC GGATCGAAGA CAGACTTTAC CCTGAACATT 721 CACCCTGTCG AGGAAGAAGA TGCCGCGACG TACTATTGTC AGCAATCCCG GGAATACCCC 781 TATACATTCG GAGGCGGAAC CAAGTTAGAA ATCAAGCGGG CGGCCGGATC CGAGCGAAAG 841 TGCTGTGTGG AGTGCCCCCC ATGCCCTGGA CAGCCTAGAG AACCACAAGT ATACACTCTG 901 CCACCTAGCC GTGAGGAAAT GACCAAGAAT CAAGTCAGCC TCACCTGCTT AGTGAAGGGA 961 TTCTATCCAT CAGACATTTC CGTCGAGTGG GAATCTAACG GACAGCCGGA GAACAACTAT 1021 AAAACTACTC CACCCATGCT CGACAGCGAC GGTTCCTTCT TCCTCTACAG TAAGCTCACA 1081 GTTGATAAGT CAAGGTGGCA GCAAGGAAAT GTGTTTTCGT GCTCAGTGAT GCACGAGGCG 1141 CTGCATAACC ATTACACCCA GAAGAGCCTC AGCCTCTCCC CTGGAAAGAC GCGTTTTTGG 1201 GTACTGGTCG TGGTGGGTGG GGTGCTCGCT TGCTATTCAT TGTTGGTGAC CGTGGCATTC 1261 ATCATCTTCT GGGTAAGAGA GGGCGGAAGA AATTACTATA CATATTCAAG CAACCCTTCA 1321 TGCGCCCAGT GCAGACTACT CAGGAGGAGG ATGGCTGCTC CTGCCGATTC CCCGAGGAAG 1381 AAGAGGGGGG TTGCGAGCTG CGGGTGAAGT TCTCTAGAAG TGCCGATGCC CCCGCCTACC 1441 AGCAAGGACA GAACCAGCTG TACAATGAGT TGAACTTGGG ACGGAGGGAG GAGTATGACG 1501 TCCTGGACAA ACGTCGAGGT CGCGACCCCG AGATGGGCGG CAAACCACAA CGTCGCAAGA 1561 ACCCTCAGGA GGGGCTTTAT AACGAGCTGC AGAAAGATAA GATGGCCGAG GCCTACTCTG 1621 AAATCGGAAT GAAAGGGGAG CGTAGAAGGG GAAAAGGTCA CGATGGATTG TACCAAGGTC 1681 TGAGTACGGC GACCAAGGAC ACTTATGACG CATTGCATAT GCAGGCACTG CCCCCGAGAT 1741 AA (SEQ ID NO 27) 1 ATGGCTCTGC CTGTTACAGC TCTGCTGCTG CCTCTGGCTC TGCTTCTGCA CGCTGCCAGA 61 CCTGACATCC AGATGACCCA GACCACAAGC AGCCTGTCTG CCAGCCTGGG CGATAGAGTG 121 ACCATCAGCT GTAGAGCCAG CCAGGACATC AGCAAGTACC TGAACTGGTA TCAGCAGAAA 181 CCCGACGGCA CCGTGAAGCT GCTGATCTAC CACACCAGCA GACTGCACAG CGGCGTGCCA 241 AGCAGATTTT CTGGAAGCGG CAGCGGCACC GACTACAGCC TGACCATCTC CAACCTGGAA 301 CAAGAGGATA TCGCTACCTA CTTCTGCCAG CAAGGCAACA CCCTGCCTTA CACCTTTGGC 361 GGAGGCACCA AGCTGGAAAT CACAGGCGGC GGAGGAAGCG GAGGCGGAGG ATCTGGTGGT 421 GGTGGATCTG AAGTGAAACT GCAAGAGTCT GGCCCTGGCC TGGTGGCCCC ATCTCAATCT 481 CTGAGCGTGA CCTGTACCGT CAGCGGAGTG TCCCTGCCTG ATTATGGCGT GTCCTGGATC 541 AGACAGCCTC CTCGGAAAGG ACTGGAATGG CTGGGAGTGA TCTGGGGCAG CGAGACAACC 601 TACTACAACA GCGCCCTGAA GTCCCGGCTG ACCATCATCA AGGACAACTC CAAGAGCCAG 661 GTGTTCCTGA AGATGAACAG CCTCCAGACC GACGACACCG CCATCTACTA TTGCGCCAAG 721 CACTACTACT ACGGCGGCAG CTACGCTATG GACTATTGGG GCCAGGGCAC CAGCGTGACC 781 GTGTCTAGTG GATCCGAGCG AAAGTGCTGT GTGGAGTGCC CCCCATGCCC TGGACAGCCT 841 AGAGAACCAC AAGTATACAC TCTGCCACCT AGCCGTGAGG AAATGACCAA GAATCAAGTC 901 AGCCTCACCT GCTTAGTGAA GGGATTCTAT CCATCAGACA TTTCCGTCGA GTGGGAATCT 961 AACGGACAGC CGGAGAACAA CTATAAAACT ACTCCACCCA TGCTCGACAG CGACGGTTCC 1021 TTCTTCCTCT ACAGTAAGCT CACAGTTGAT AAGTCAAGGT GGCAGCAAGG AAATGTGTTT 1081 TCGTGCTCAG TGATGCACGA GGCGCTGCAT AACCATTACA CCCAGAAGAG CCTCAGCCTC 1141 TCCCCTGGAA AGACGCGTTT TTGGGTACTG GTCGTGGTGG GTGGGGTGCT CGCTTGCTAT 1201 TCATTGTTGG TGACCGTGGC ATTCATCATC TTCTGGGTAA TGCATAAGAG AGGGCGGAAG 1261 AAATTACTAT ACATATTCAA GCAACCCTTC ATGCGCCCAG TGCAGACTAC TCAGGAGGAG 1321 GATGGCTGCT CCTGCCGATT CCCCGAGGAA GAAGAGGGGG GTTGCGAGCT GAAGAAGAGG 1381 ATCAAGCCTA TCGTGTGGCC CTCACTGCCC GACCATAAAA AAACCCTGGA ACACCTGTGC 1441 AAAAAGCCAA GAAAGGTGGC CCAGGGCCAG CCTATCCTGA CCTCCCTCGG CTCAAACCAG 1501 GAAGAAGCAT ATGTGACCAT GTCCAGCTTC TATCAGAACC AACGGGTGAA GTTCTCTAGA 1561 AGTGCCGATG CCCCCGCCTA CCAGCAAGGA CAGAACCAGC TGTACAATGA GTTGAACTTG 1621 GGACGGAGGG AGGAGTATGA CGTCCTGGAC AAACGTCGAG GTCGCGACCC CGAGATGGGC 1681 GGCAAACCAC AACGTCGCAA GAACCCTCAG GAGGGGCTTT ATAACGAGCT GCAGAAAGATAttorney Docket No.: CHLU-001 / 001WO 1741 AAGATGGCCG AGGCCTACTC TGAAATCGGA ATGAAAGGGG AGCGTAGAAG GGGAAAAGGT 1801 CACGATGGAT TGTACCAAGG TCTGAGTACG GCGACCAAGG ACACTTATGA CGCATTGCAT 1861 ATGCAGGCAC TGCCCCCGAG ATAA (SEQ ID NO: 28) 1 ATGGAGTTTG GACTTTCTTG GTTATTCCTG GTGGCTATCC TAAAGGGAGT ACAGTGCGAC 61 GCAGTCATGA CTCAGATCCC ACTCAGCCTC CCAGTGAGCT TGGGAGACCA GGTATCTATA 121 TCCTGCCGGA GCTCCCAGTC ATTGGAAAAT TCTGATGGGA ACACCTATGT TTCATGGTAT 181 GTTCAAAAGC CAGGCCAGTC CCCTCAGTTA TTGATTTATC GCGTGAGTAT TAGATTTTCG 241 GGTGTTCTTG ATCGATTTAG CGGAAGCGGC AGTGGCACCG ACTTTACTCT AAAGATCAGC 301 CGAGTAGAGG CTGAAGATTT GGGCATATAC TTCTGCTTGC AAGTGTCTCA TGTGCCCTTT 361 ACTTTTGGCT CCGGCACCAA ATTAGAGATT AAACGCGCCG ATGCCGCCCC CACCGTTTCC 421 ATCTTCCCGC CCAGCAGCGA GCAGCTCACT AGCGGAGGGG CCAGTGTGGT CTGCTTCCTG 481 AATAATTTCT ACCCGAAAGA TATCAACGTT TCTTCAAATC GCATTGGGGG AGGGGGGAGC 541 GGTGGAGGCG GCTCGGGGGG CGGCGGGTCT GAGGTGCAGC TGCAGCAGAG CGGTCCCGAG 601 TCCGTGAAGC CAGGCGCATC AGTGAAGATG TCCTGTAAAG CGAGTGGCTA CACATTTACA 661 AACTATGTCA TGCACTGGGT CAAGCAGAAG CCCGGTCAGG GGCTAGAGTG GATCGGTTAT 721 ATCATTCCTT ATAATGATGT CACCAAGTAT AACGAGAAGT TTAAAGGTAA GACAACACTC 781 ACAAGTGACA AGAGCTCCTC AACCGCCTAC ATGGACCTGA GCTCTCTGAC CTCAGAGGAT 841 AGTGCGGTCT ATTCATGCGC TCGGTGGGCC TGGGATGGAT ATTTCGACTA CTGGGGTCAA TGACCGTGTC CAGCGGATCC GAGCGAAAGT GCTGTGTGGA GTGCCCCCCA AGCCTAGAGA ACCACAAGTA TACACTCTGC CACCTAGCCG TGAGGAAATG AAGTCAGCCT CACCTGCTTA GTGAAGGGAT TCTATCCATC AGACATTTCC AATCTAACGG ACAGCCGGAG AACAACTATA AAACTACTCC ACCCATGCTC GTTCCTTCTT CCTCTACAGT AAGCTCACAG TTGATAAGTC AAGGTGGCAGTGTTTTCGTG CTCAGTGATG CACGAGGCGC TGCATAACCA TTACACCCAG 1261 AAGAGCCTCA GCCTCTCCCC TGGAAAGACG CGTTTTTGGG TACTGGTCGT GGTGGGTGGG 1321 GTGCTCGCTT GCTATTCATT GTTGGTGACC GTGGCATTCA TCATCTTCTG GGTAATGCAT 1381 AAGAGAGGGC GGAAGAAATT ACTATACATA TTCAAGCAAC CCTTCATGCG CCCAGTGCAG 1441 ACTACTCAGG AGGAGGATGG CTGCTCCTGC CGATTCCCCG AGGAAGAAGA GGGGGGTTGC 1501 GAGCTGAAGA AGAGGATCAA GCCTATCGTG TGGCCCTCAC TGCCCGACCA TAAAAAAACC 1561 CTGGAACACC TGTGCAAAAA GCCAAGAAAG GTGGCCCAGG GCCAGCCTAT CCTGACCTCC 1621 CTCGGCTCAA ACCAGGAAGA AGCATATGTG ACCATGTCCA GCTTCTATCA GAACCAACGG 1681 GTGAAGTTCT CTAGAAGTGC CGATGCCCCC GCCTACCAGC AAGGACAGAA CCAGCTGTAC 1741 AATGAGTTGA ACTTGGGACG GAGGGAGGAG TATGACGTCC TGGACAAACG TCGAGGTCGC 1801 GACCCCGAGA TGGGCGGCAA ACCACAACGT CGCAAGAACC CTCAGGAGGG GCTTTATAAC 1861 GAGCTGCAGA AAGATAAGAT GGCCGAGGCC TACTCTGAAA TCGGAATGAA AGGGGAGCGT 1921 AGAAGGGGAA AAGGTCACGA TGGATTGTAC CAAGGTCTGA GTACGGCGAC CAAGGACACT 1981 TATGACGCAT TGCATATGCA GGCACTGCCC CCGAGATAA

[0085] In another aspect, provided herein is a vector comprising a polynucleotide encoding a CAR described herein. In another aspect, provided herein is a vector encoding or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein.

[0086] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, for example, moloney murine leukemia viruses (MoMuLV). In some embodiments, the vector is a pox vector, a herpes simplex I virus (HSV) vector, an adenovirus vector, or an adeno-associated virus vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is plasmid, a phagemid, a phage derivative, an animalAttorney Docket No.: CHLU-001 / 001WO virus, or a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Further, the expression vector may be provided to cells, such as immune cells, in the form of a viral vector.

[0087] The vectors described herein may comprise a promoter operatively linked to the nucleic acid sequence encoding a CAR disclosed herein or the extracellular domain of a CAR described herein, the transmembrane domain of a CAR described herein, and / or the intracellular domain of a CAR described herein. Illustrative promoters that may be used in the vectors described herein include the immediate early cytomegalovirus (CMV) promoter, the Elongation Growth Factor-1Į (EF-1Į) promoter, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter a U6 promoter, a H1 promoter, a 7SK promoter, a Ef1a promoter.

[0088] The promoter may be a constitutive promoter or an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0089] Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, for example, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and / or self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self- cleaving peptides.

[0090] Vectors of the present disclosure may be used for expression of the CAR in a cell, e.g., an immune cell. Vectors of the present disclosure may also be used for expression of the car in vivo, e.g., in a patient.

[0091] Vectors described herein may further comprise one or more enhancers that regulate the expression of the CAR. Typically, enhancers are located in the region 30-110 base pairs upstream of the transcription start site.Attorney Docket No.: CHLU-001 / 001WO

[0092] In order to assess the expression of a fusion protein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin or ampicillin resistance genes and the like.

[0093] Reporter genes may be used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. Generally, a reporter gene encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity, fluorescence or luminescence. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Examples of reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene Immune Cells

[0094] In another aspect, provided herein are immune cells comprising a polynucleotide, vector, or CAR described herein. In some embodiments, an immune cell comprises a polynucleotide described herein. In some embodiments, an immune cell comprises a vector described herein. In some embodiments, an immune cell comprises a CAR described herein. In some embodiments, an immune cell comprise a polynucleotide described herein expresses the protein encoded by the polynucleotide. In some embodiments, an immune cell comprise a vector described herein expresses the protein encoded by the vector.

[0095] In some embodiments, the immune cell is a T cell, B cell, Natural Killer (NK) cell or a natural killer T (NKT) cell.

[0096] There are several distinct types of T cells which develop upon migration to the thymus, which include, helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells and stem memory T cells. Different types of T cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T cell types will be readily apparent to the ordinarily skilled artisan. In some embodiments, the T cell is an effector T cell or a regulatory T cell. In some embodiments, the T cells is a double negative (CD4- / CD8-) T cells or a gamma / delta T cell. Gamma / delta T cells areAttorney Docket No.: CHLU-001 / 001WO a small subset of CD3-positive T cells which function in the inflammatory response in both the innate and adaptive immune system.

[0097] In some embodiments, the immune cell is autologous to a subject. In some embodiments, the immune cell is allogeneic to a subject.

[0098] In some embodiments, the immune cell is isolated. In some embodiments, the immune cell is ex vivo. In some embodiments, the immune cell is for use as a medicament. In some embodiments, the medicament is for the treatment of cancer in a subject in need thereof.

[0099] Also provided herein is a population of immune cells comprising a polynucleotide, vector or CAR described herein. [000100] For use in adoptive cell therapy, immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the T cells are obtained from a unit of blood collected from a subject by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. The T cells may be isolated from the blood using any suitable method known in the art, including, for example, such as Ficoll™ separation, centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. [000101] Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells can be further isolated by positive or negative selection techniques and optionally enriched by negative selection and / or positive selection using any suitable method known in the art. [000102] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical methods (e.g., calcium phosphate precipitation, particle bombardment, microinjection, or electroporation), by chemical methods (e.g., colloidal dispersion systems, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes), or by biological means (e.g., using a vector such as a viral vector).Attorney Docket No.: CHLU-001 / 001WO [000103] Methods of activating and culturing immune cells comprising a polynucleotide, vector or CAR of the instant disclosure, will be readily apparent to the person of ordinary skill in the art. For example, the T cell Transact (Miltenyi Biotec cat#130-128-758) protocol may be used to activate and expand the T cells described herein. [000104] In some embodiments, T cells of the instant disclosure are expanded and activated in vitro. Generally, T cells may be expanded in vitro by contacting them with T cell stimulatory agents such as anti-CD3 antibodies and / or anti-CD28 antibodies and culturing them for an appropriate amount of time under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air with 5% CO2). [000105] For clinical applications, the isolation, transfection, and expansion of the immune cells may be carried out according to good manufacturing (GMP) standards. [000106] In another aspect, an immune cell comprising a CAR described herein is derived from a stem cell, for example an induced pluripotent stem cell or a hematopietic stem cell. [000107] In another aspect, an immune cell comprising a CAR described herein is an ex vivo cell. [000108] In some aspects, an immune cell comprising a CAR described herein exhibits increased expansion compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the expansion is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, least about 5-fold, at least about 10-fold, at least about 15-fold or at least about 20-fold. Expansion may be measured, for example, as an increase in cell or an increase in confluency over a suitable period (e.g., 24 hours, 48 hours, or 72 hours. [000109] In some aspects, an immune cell comprising a CAR described herein exhibits increased cytokine production compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the cytokine production is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, least about 5-fold, at least about 10-fold, at least about 15-fold or at least about 20-fold. Cytokine production may be measured, for example, by measuring the levels of cytokines in the cell culture medium over a suitableAttorney Docket No.: CHLU-001 / 001WO period (e.g., 24 hours, 48 hours, or 72 hours after the immune cell is exposed to the target antigen. In some embodiments, the cytokine is one or more of TNF-Į, IL-6, IL-10, IL-2, IFN-Ȗ, IL-4, and IL-4. Cytokines may be measured in cell culture medium using any suitable method known in the art including, for example, enzyme-linked immunosorbent assays (ELISAs) or Western Blotting. [000110] In some aspects, an immune cell comprising a CAR described herein exhibits a naïve / stem-like memory T cell phenotype. An immune cell having a naïve / stem-like T cell phenotype may be identified by measuring markers such as CD27 and CD45RA, with naïve cells expressing both molecules, whereas memory and effector cells expressing only CD27 or CD45RA, respectively (see Hamann et al., J Exp Med.1997 Nov 3;186(9):1407-18). In some embodiments, a cell having a naïve / stem-like T cell phenotype is a CD45RO-CD62L+ cell. In some embodiments, the fraction of cells exhibiting a naïve / stem-like memory T cell phenotype in a population of cells comprising a CAR of the present invention is higher than the fraction of cells exhibiting a naïve / stem-like memory T cell phenotype in a population of cells comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the fraction of cells exhibiting a naïve / stem-like memory T cell phenotype is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, least about 5-fold, at least about 10-fold, at least about 15-fold or at least about 20-fold. Markers such as CD27 and CD45RA may be measured by any suitable method known in the art, including, for example, flow cytometry. [000111] In some aspects, an immune cell comprising a CAR described herein exhibits increased anti-tumor activity compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the anti-tumor activity is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, least about 5-fold, at least about 10-fold, at least about 15-fold or at least about 20-fold. Anti-tumor activity may be measured, for example, by measuring the decrease in tumor cells co-cultured with the immune cells comprising a CAR over a suitable period (e.g., 24 hours, 48 hours, or 72 hours. Anti-tumor activity may also be measured in vivo using xenograft models.Attorney Docket No.: CHLU-001 / 001WO [000112] In some aspects, an immune cell comprising a CAR described herein exhibits increased activation compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the immune cell activation is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2- fold, at least about 3-fold, at least about 4-fold, least about 5-fold, at least about 10-fold, at least about 15-fold or at least about 20-fold. Immune cell activation may be measured, for example, by measuring the expression of immune cell activation markers (such as CD25 and / or CD69 for T cells) over a suitable period (e.g., 24 hours, 48 hours, or 72 hours. Immune cell activation markers (such as CD25 and / or CD69 for T cells) may be measured using any suitable method known in the art, including, for example, flow cytometry. [000113] In some aspects, an immune cell comprising a CAR described herein exhibits decreased apoptosis when co-cultured with target cells compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra. In some embodiments, the apoptosis is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. Apoptosis may be measured, for example, by measuring the expression of pro-apoptotic genes (such as bad) or other apoptotic markers such caspase 3 / 7 activity or phosphatidyl serine expression on the outer membrane of the cell as over a suitable period (e.g., 24 hours, 48 hours, or 72 hours. Methods of measuring apoptosis are well known in the art, and commercial kits for such assays are widely available. Pharmaceutical Compositions [000114] In another aspect, provided herein are pharmaceutical compositions comprising the immune cells described herein and optionally a pharmaceutically acceptable carrier. Also provided herein are pharmaceutical compositions comprising a vector described herein and optionally a pharmaceutically acceptable carrier. [000115] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Optional examples of such carriers or diluentsAttorney Docket No.: CHLU-001 / 001WO include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. [000116] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. [000117] In one embodiment, the immune cell or vector is prepared with carriers that will protect the compound against rapid elimination from the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. [000118] In one embodiment, oral or parenteral compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [000119] The formulation can also contain more than one active compound as necessary for the particular indication being treated, optionally those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [000120] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluentAttorney Docket No.: CHLU-001 / 001WO such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [000121] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [000122] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the caseAttorney Docket No.: CHLU-001 / 001WO of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [000123] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [000124] For administration by inhalation, the immune cells or vectors are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. [000125] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the cells or vectors are formulated into ointments, salves, gels, or creams as generally known in the art. [000126] In one embodiment, an immune cell or a vector described herein is prepared with carriers that will protect the immune cells or vector against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,Attorney Docket No.: CHLU-001 / 001WO polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. [000127] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Methods of Treatment [000128] Also provided herein are methods of treating a disease in a subject comprising administering to the subject a vector provided herein, an immune cell provided herein, or a pharmaceutical composition provided herein. Further provided is the use of a vector provided herein, an immune cell provided herein, or a pharmaceutical composition provided herein for the treatment of cancer in a subject. Further provided is the use of a vector provided herein or an immune cell provided herein, for use in the manufacture of a medicament for the treatment of cancer in a subject. [000129] It is to be understood that, unless otherwise stated, any description of a method of treatment or prevention includes use of the CARs, polynucleotides, immune cells and pharmaceutical compositions described herein to provide such treatment or prevention as is described herein. It is to be further understood, unless otherwise stated, any description of a method of treatment or prevention includes use of the peptides to prepare a medicament to treat or prevent such condition. The treatment or prevention includes treatment or prevention of human or non-human animals including rodents and other disease models. [000130] It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the peptides to provide such treatment as is described herein. It is to be further understood, unless otherwise stated, any description of a method of treatment includes use of the peptides to prepare a medicament to treat such condition. The treatment includes treatment of human or non-human animals including rodents and other disease models.Attorney Docket No.: CHLU-001 / 001WO [000131] As used herein, the term “subject” is interchangeable with the term “subject in need thereof” and “patient,” all of which refer to a subject having a disease or having an increased risk of developing the disease. A “subject” includes a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In some embodiments, the mammal is a human. [000132] A subject can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject can have a refractory or resistant a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at start of treatment or may become resistant during treatment. In some embodiments, the subject received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject received at least one prior therapy. [000133] As used herein, the term “treating” or “treat” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder and includes the administration of a vector, an immune cell or a pharmaceutical composition described herein, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model. It is to be appreciated that references to “treating” or “treatment” include the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.Attorney Docket No.: CHLU-001 / 001WO [000134] It is to be understood that a vector, an immune cell or a pharmaceutical composition of the disclosure, can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes. As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder. [000135] In some embodiments, the disease to be treated is cancer. In some embodiments, the disease to be treated is a solid tumor. In some embodiments, the disease to be treated is a hematological malignancy. In some embodiments, the disease to be treated is a lymphoma. In some embodiments, the disease to be treated is multiple myeloma. In some embodiments, the disease to be treated is brain cancer, e.g., glioblastoma. In some embodiments, the disease to be treated is ovarian cancer. In some embodiments, the disease to be treated is breast cancer. In some embodiments, the disease to be treated is cholangiocarcinoma. In some embodiments, the disease to be treated a leukemia, e.g., acute myelogenous leukemia. In some embodiments, the cancer has metastasized. [000136] In some embodiments, a therapeutically effective amount of a vector, an immune cell or a pharmaceutical composition described herein is administered to the subject in need thereof. A therapeutically effective amount of a vector, an immune cell or a pharmaceutical composition described herein relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antigen-binding domain of the CAR encoded by the vector and / or expressed by the immune cell and its target antigen. [000137] As used herein, “treating cancer” includes, but is not limited to, reversing, alleviating or inhibiting the progression of cancer or symptoms associated with cancer or preventing worsening of the severity of cancer or symptoms or conditions associated with cancer. [000138] Efficaciousness of treatment may be determined in association with any known method for diagnosing or treating the particular cancer. Alleviation of one or more symptoms of the cancer may indicate that the vector, immune cell or pharmaceutical composition described herein confers a clinical benefit. An increase in time to recurrence of a tumor may indicate that the vector, immune cell or pharmaceutical composition described herein confers a clinical benefit. A reduction in size of a tumor may indicate that the vector, immune cell or pharmaceutical composition described herein confers a clinical benefit.Attorney Docket No.: CHLU-001 / 001WO [000139] For example, anti-tumor efficacy of a vector, an immune cell or a pharmaceutical composition described herein may be evaluated in humanized mouse models, such as the LN229 or U87 allograft mouse models of glioblastoma. To generate such an allograft model, LN229 or U87 cells may be injected into the right flank region of a mouse, e.g., a NOD-SCID mouse. Once the mice develop tumors of a suitable size (e.g., about 50-70 mm3tumor volume on average), the vector, immune cell or pharmaceutical composition described herein may be administered, for example by daily intraperitoneal injection or intravenous infusion. A suitable control such as an immune cell expressing a CAR that does not comprise an intracellular signaling domain of IL- 7Ra may be included. Tumor volumes may then be measured by caliper and other clinical signs, behavior, and weight loss may be recorded over the course of the study. To quantify treatment efficacy, the relative tumor growth inhibition rate may be calculated as follows: TGI % = (1-T / C) × 100%, where T is tumor volume or tumor weight of treatment group and C is tumor volume or tumor weight of control group. [000140] In one embodiment, the vector, immune cell or pharmaceutical composition described herein are used in combination with other therapies. Accordingly, the application provides a method of treating cancer using a vector, an immune cell or a pharmaceutical composition described herein in combination with at least one additional therapy. The other therapy may be administered prior to, overlapping with, concurrently, and / or after administration of the vector, immune cell or pharmaceutical composition described herein. When administered concurrently, the vector, immune cell or pharmaceutical composition described herein and the other therapeutic may be administered in a single formulation or in separate formulations, and if separately, then optionally, by different modes of administration. The combination of a vector, an immune cell or pharmaceutical composition described herein and one or more other therapies may synergistically act to combat cancer. [000141] For example, the combination therapy can include a vector, an immune cell or a pharmaceutical composition described herein co-administered with one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, anti-neoplastic agents, and / or cytotoxic or cytostatic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.Attorney Docket No.: CHLU-001 / 001WO Methods of improving Therapeutic Cells [000142] In another aspect, provided herein are methods of improving therapeutic cells, such as CAR cells, comprising modifying an existing CAR cell by inserting an intracellular signaling domain of IL-7Ra described herein into the CAR cells. Such methods may result in enhanced proliferation, expansion, cytokine productions and / or potency of the therapeutic cell. [000143] A person of skill in the art will appreciate that the intracellular signaling domain of IL-7Ra may be inserted at any suitable position of the CAR. Preferably, the methods of improving therapeutic cells described herein do not substantially affect the antigen specificity of the receptor comprised by the cells. Thus, in preferred embodiments, a method of improving a therapeutic cell described herein results in an improved cell with substantially the same antigen specificity. “Substantially the same antigen specificity” means that the type of antigen recognized by the receptor is unchanged and that the affinity of a receptor for the antigen is decreased by at most 10% compared to the receptor of the cell prior to improvement. [000144] In some embodiments, the intracellular signaling domain of IL-7Ra is inserted into the intracellular domain of the CAR. In some embodiments, the intracellular signaling domain of IL-7Ra is inserted between an intracellular costimulatory domain and a CD3ȗ costimulatory domain. Thus, in some embodiments, a method of improving therapeutic cells described herein results in a cell comprising a CAR comprising a CD3ȗ costimulatory domain located C-terminal to the intracellular signaling domain of IL-7Ra. In some embodiments, a method of improving therapeutic cells described herein results in a cell comprising a CAR comprising one or more costimulatory domains located N-terminal to the intracellular signaling domain of IL-7Ra. Illustrative co-stimulatory domains include a 4-1BB costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, and an OX40 costimulatory domain. [000145] In some embodiments, a method of improving a therapeutic cell described herein results in increased expansion compared to the expansion prior to improvement. Expansion of a therapeutic cell may be determined using any suitable method known in the art including, for example, cell counts by flow cytometry or microscopy. In some embodiments, a method of improving a therapeutic cell described herein results in an increase in expansion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative toAttorney Docket No.: CHLU-001 / 001WO the expansion of the cell prior to the improvement. In some embodiments, a method of improving a therapeutic cell described herein results in an increase in expansion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the expansion of a cell comprising a CAR without an intracellular signaling domain of IL-7Ra. In some embodiments, a method of improving a therapeutic cell described herein results in increased cytokine production. Cytokine production of a therapeutic cell may be determined using any suitable method known in the art including, for example, measurements of the cytokines in the cell culture medium by ELISA or flow cytometry. In some embodiments, a method of improving a therapeutic cell described herein results in an increase in cytokine production of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the cytokine production by the cell prior to the improvement. In some embodiments, a method of improving a therapeutic cell described herein results in an increase in cytokine production of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the cytokine production by a cell comprising a CAR without an intracellular signaling domain of IL-7Ra. [000146] In some embodiments, a method of improving a therapeutic cell described herein results in increased potency. The potency of a therapeutic cell may be measured using any suitable method known in the art or described herein including, for example, cytotoxicity assays, in vivo efficacy studies, and target cell clearance assays. An example of a method of measuring target cell clearance is flow cytometry. In some embodiments, a method of improving a cell therapy results described herein in an increase in potency of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the potency of the cell prior to the improvement. In some embodiments, a method of improving a cell therapy described herein results in an increase in potency of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the potency of a cell comprising a CAR without an intracellular signaling domain of IL-7Ra.Attorney Docket No.: CHLU-001 / 001WO [000147] In some embodiments, a method of improving a therapeutic cell described herein results in less T cell exhaustion. T cell exhaustion may be assessed using any suitable method known in the art or described herein, including, for example, detection of exhaustion marker expression, functional assays, exhaustion gene expression profiling, metabolic profiling, longitudinal clinical monitoring, or immunohistochemistry. In some embodiments, a method of improving a therapeutic cell described herein results in a decrease in T cell exhaustion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least about 95% relative to the exhaustion of the cell prior to the improvement. In some embodiments, a method of improving a therapeutic cell described herein results in a decrease in T cell exhaustion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2- fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the exhaustion of a cell comprising a CAR without an intracellular signaling domain of IL-7Ra. [000148] In some embodiments, a method of improving a therapeutic cell described herein results in a decrease in tumor size in vivo when the therapeutic cell is administered to a subject. Tumor size in vivo may be determined in any suitable model using, for example bioluminescence imaging or caliper measurement. In some embodiments, a method of improving a therapeutic cell described herein results in a decrease in tumor size of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the decrease in tumor size observed with the cell prior to the improvement. In some embodiments a method of improving a therapeutic cell described herein results in a decrease in tumor size of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the decrease in tumor size observed with a cell comprising a CAR without an intracellular signaling domain of IL-7Ra. [000149] In some embodiments, a method of improving a therapeutic cell described herein results in a prolongation of the duration of response when the therapeutic cell is administered to a subject. The duration of response is generally defined as the interval from response initiation (when the treated subject first shows a complete or partial response to therapy) to the earlier of disease progression or death. Disease relapse is generally measured as the recurrence of tumorAttorney Docket No.: CHLU-001 / 001WO cells after the subject has achieved a complete response. In some embodiments, a method of improving a therapeutic cell described herein results in a prolongation of the duration of response of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the duration of response observed with the cell prior to the improvement. In some embodiments, a method of improving a therapeutic cell described herein results in a prolongation of the duration of response of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the duration of response observed with with a cell comprising a CAR without an intracellular signaling domain of IL-7R. [000150] In some embodiments, the cell therapy being improved by the method described herein is an FDA-approved CAR-T cell therapy. In some embodiments, the cell therapy being improved by the method described herein is Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Breyanzi (lisocabtagene maraleucel), Tecartus (brexucabtagene autoleucel), or Abecma (idecatagene vicleucel). [000151] In some aspects, the cell therapy being improved by the methods described herein is an allogeneic cell therapy. EXAMPLES [000152] The examples described in this section are provided solely for illustration and are not intended to limit the invention. Example 1: Incorporation of the interleukin-7 receptor alpha cytoplasmic domain enhanced B7H3-targeted CAR-T cell therapy in preclinical models of glioblastoma [000153] This example describes the development novel B7H3-targeting CAR constructs incorporating the intracellular^signaling domain of IL-7Ra and the antitumor capacity of these novel CAR-T cells in vitro and in vivo. Materials and Methods Cell lines and cultures [000154] Human glioblastoma cell lines (LN229 and U87) and the human embryonic kidney cell line 293 (293T) used in this study were purchased from ATCC (Manassas, VA,Attorney Docket No.: CHLU-001 / 001WO USA). All of these cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco) supplemented with 1% GlutaMax (Invitrogen, Carlsbad, CA, USA) and 1% penicillin^streptomycin. and incubated at 37°C and 5% CO2. To detach adherence to plastic culture flasks, 0.25% trypsin / 0.02% EDTA solution (Invitrogen, USA) was utilized. All cell cultures were routinely tested for mycoplasma contamination by a MycoAlert Plus Mycoplasma Detection kit (Lonza Bioscience, Basel, Switzerland). CAR construction and transduction of human T cells [000155] PBMCs were isolated from 10 ml peripheral blood samples from healthy donors using a Ficoll histopaque gradient method (Sigma^Aldrich). PBMCs were washed with 1X PBS (Cellgro, MediaTech, VWR) and resuspended in sterile RPMI medium (Cellgro, MediaTech, VWR) supplemented with 10% FBS, 1% penicillin–streptomycin and 1% GlutaMax (Gibco, Life Technologies) to a density of 2 × 106cells / well. T cells were activated in TexMACS medium containing recombinant human IL-2 (50 units / mL) and TransAct CD3 / CD28 reagent for 3 days. The activated T cell was transduced by retroviral plasmids with a determined MOI = 2, and the medium was changed every 3–4 days. Then, the cells were continuously cultured for 11–18 days, and the cells were collected for in vitro and in vivo studies. Cytotoxicity assay [000156] Tumor cells were seeded at a density of 1ௗ×ௗ105cells / well in a 24-well plate (Corning, CA, USA) for 24 hours. Nontransduced T cells (NT) as the control group and B7H3- targeted CAR-T cells (experimental group, named B7H3-BB or B7H3-BB-IL7) were added to a 24-well plate at different E:T ratios (1:1, 0.5:1 and 0.25:1). The plate was then placed for 72 hours at 37°C in a 5% CO2incubator. Flow cytometry was used to detect CD3+ T cells and target cells. Apoptosis analysis [000157] T cells were stained with anti-human CD3 antibody followed by Annexin V antibody and 7-AAD and then analyzed by flow cytometry. For experiments with B7H3-BB-IL7, APOAF-60TST was purchased from Sigma^Aldrich.Attorney Docket No.: CHLU-001 / 001WO T cell activation / exhaustion assays [000158] CAR-T cell activation was evaluated by the expression of CD25 and CD69 on the surface of responding cytolytic effectors as previously described (see Moghimi et al., Nat Commun 12, 511). Briefly, T cells in complete DMEM in the absence of any cytokine addition and anti-human B7H3-BB and B7H3-BB-IL7 CARs were cocultured with B7H3-positive target cells LN229 or U87 at an E:T ratio of 1:1. The tubes were incubated for 72 hours at 37°C in a CO2 incubator. T cells were then collected and analyzed for appearance activation markers on the surface by flow cytometry using anti-human CD3, CD25, and CD69 antibodies (Bioligands). Intracellular STAT5 phosphorylation [000159] Transduced T cells were harvested and resuspended at 4 × 105cells per mL of complete medium without cytokines into a FACS tube and then added at 2 × 105cells / well of target cells. Thirty to 60 minutes later, cells were washed in cold 1X PBS containing 5% FBS (flow buffer) and centrifuged at 250×g for 5 min. Fixative buffer was added to the cells and incubated for 10 minutes at 37qC before ice-cold Perm buffer II was slowly added to the tube and then incubated for 30 minutes at 4°C. Afterward, the tubes were centrifuged, and the supernatant was discarded, followed by another wash step with cold flow buffer. Five microliters of anti-human pSTAT5 and 20 ^L of anti-human CD3 antibodies were then added to the cells. The cells were gently vortexed and then incubated in the dark for 30 minutes at room temperature. Afterward, the cells were washed one more time with cold flow buffer and then immediately analyzed on a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, USA). Data were analyzed using FlowJo software (TreeStar, OR, USA). Tumor rechallenge in vitro [000160] LN229 cells (1 × 105) and T cells (1 × 105) transduced with B7H3-BB or B7H3- BB-IL7S CARs were cocultured in a 24-well plate using fresh culture media without IL-2 or IL- 7 and IL-15. Three days later, the cells were harvested for FACS analysis. CAR-T cells were then replated at a 1:1 E:T ratio with fresh LN229 cells in half-half culture media to start the second, third, and fourth tumor cocultures. After stimulation by the fourth tumor line, T cells were counted, and the coculture was analyzed by FACS analysis.Attorney Docket No.: CHLU-001 / 001WO IL2-independent proliferation assay [000161] B7H3-BB and B7H3-BB-IL7S CAR-T or NT cells (2 × 105) were seeded in a 96- well plate using fresh culture media without IL-2 or IL-7 and IL-15. The viability of trypan blue- stained T cells was analyzed by using Countess cell counting chamber slides and a Countess III automated cell counter (Invitrogen, Carlsbad, CA). Multiplex CBA analysis [000162] Cytokines from the 24-h coculture in which the effector to target cell ratio (E:T) was 1:1 and the culture supernatant were assessed by the BD cytometric bead array (CBA) (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. Flow cytometry and antibodies [000163] A flow cytometric assay was utilized to analyze the expression levels of CD3 and CD276 (B7H3). Briefly, cells were washed once with flow cytometry cell staining buffer (1X PBS containing 2% FBS) to block their Fc receptor. The allophycocyanin (APC) red channel was utilized to distinguish CAR-T cells. Recombinant human B7-H3 protein, Fc Tag (Acro Biosystems), was used to detect the expression of B7H3 CAR on CAR-T cells, which were then stained with allophycocyanin (APC) anti-His Tag (Biolegend, CA, USA). [000164] The antibodies used in flow cytometric immunophenotyping of T cells were as follows: PerCP-Cy5.5 anti-human CD3 (BD Bioscience, USA), PE anti-human CD3 (Biolegend, CA, USA), APC anti-human CD4 (Biolegend, CA, USA), PE anti-human CD8 (BD, NJ, USA), PerCP anti-human CD3 (Biolegend, CA, USA), FITC anti-human CD8 (Biolegend, CA, USA), APC anti-human CD3 (Biolegend, CA, USA), Allexa Fluor 488 anti-Stat5 (pY694, BD Bioscience), APC anti-human CD45RO (Biolegend, CA, US), FITC anti-human CD62L (Biolegend, CA, USA), APC-Cy7 anti-human CD69 (Biolegend, CA, USA), PE anti-human TIGIT (Biolegend, CA, USA), APC anti-human CD366 / TIM3 (Biolegend, CA, USA), and APC anti-human PD1 / CD279 (Biolegend, CA, USA). The stained cells were analyzed using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, USA), and the data were analyzed using FlowJo software v.10 (TreeStar, OR, USA).Attorney Docket No.: CHLU-001 / 001WO RNA isolation and qRT^PCR [000165] Total RNA was extracted with the RNeasy Plus procedure (QIAGEN, Hilden, Germany). RNA (1 μg each sample) was employed in a 20 μL first strand cDNA synthesis using the QuantiTect reverse-transcription system (QIAGEN). qRT^PCR was performed by the CFX96 Real-Time Detection System (Bio-Rad, MA, USA) using Luna Universal qPCR master mix (New England Biolabs). Gene expression profiles were calculated as 2íǻǻCt relative to ȕ- actin. The oligonucleotide primers for qRT^PCR are described inTable 2. Table 2: The oligonucleotide primers for qRT-PCR analysisXenograft mouse models [000166] Six-week-old female NOD / ShiJic-scid Jcl (NOD-SCID) mice (Nomura Siam International Co., Thailand) were engrafted subcutaneously with a 50:50 mixture with 5 × 106tumor cells (B7H3+, LN229, U87) and Matrigel matrix (Corning, NY, USA) into the right flank. Mice were randomly divided into 3 groups, NT, B7H3-BB, and B7H3-BB-IL7S (n = 4 animals per group), when the tumor volumes were between 50 and 70 mm3. CAR-T cells (1 × 107) were administered intravenously with a total volume of 200 μL twice a week. Body weight, tumor size, and the onset of GvHD were monitored weekly. Tumor volume was calculated using the following formula: volume = (length × weight2) / 2. All efforts were made to minimize animal suffering.Attorney Docket No.: CHLU-001 / 001WO Statistical analysis [000167] GraphPad Prism software v.7 (GraphPad, San Diego, CA, USA) was used to analyze and visualize the results. Unpaired two-tailed Student’s t test and one-way or two-way ANOVA followed by Tukey’s multiple comparison test were performed to assess differences between groups or differences between each group and the indicated control. Survival determined from the time of tumor cell injection was analyzed by the Kaplan–Meier method, and differences in survival between groups were compared by the log-rank test. Data are shown as the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 as significant; ns, not significant. Results Next-generation B7H3-specific CARs consist of an intracellular signaling domain of IL-7Ra [000168] To provide three activation signals to CAR-T cells, the cytoplasmic domain of the IL-7 receptor D (IL-7Ra) was incorporated between the intracellular domain of 4-1BB and CD3z. FIGs.1A-1E. show the generation and characterization of B7H3-targeted chimeric antigen receptor T cells consisting of the cytoplasmic domain of IL-7Ra. First, CAR constructs containing three different lengths of the IL-7Ra cytoplasmic domain, designated B7H3-BB- IL7L, B7H3-BB-IL7M, and B7H3-BB-IL7S were designed. FIG.1A shows the modular composition of B7H3 (clone 2H4)-specific and intracellular signaling of IL7 receptor alpha chain with different sizes; long (L, 585 bp), medium (M, 375 bp), and short (S, 171 bp) fused to CD3 zeta-chain signaling receptor. Second-generation B7H3 CAR-T cells containing the intracellular domain of 4-1BB and CD3z (B7H3-BB) were also generated as a control. B7H3-IL7R-L consists of Box 1 motif with long length of intracellular domain with p.I356V and p.K269fs, and Y4493 C-terminal region, while B7H3-IL7R-M includes Box1 motif with medium length of intracellular domain and Y4493 C-terminal region. B7H3-IL7R-S is the shortest length of IL- 7RD cytoplasmic domain that has Box1 motif and Y4493 tyrosine residues. The second generation B7H3 CAR construct containing only intracellular domain of 41BB and CD3z (B7H3) were also generated as a control. Each B7H3 CAR was transduced into activated T cells. [000169] The transduction efficiency of B7-H3 CAR-T cells containing different lengths of the IL-7Ra cytoplasmic domain was comparable to that of conventional B7-H3-BB CAR-TAttorney Docket No.: CHLU-001 / 001WO cells (FIGs.1B and 1C). FIG.1B shows a flow cytometric plot of the expression of B7H3- specific CARs in transduced human T lymphocytes (data form four independent donors). FIG. 1C is a bar graph represents the percentage of transgene expression at days 4 and 11. After T cell cultivation, there were no significant differences in CAR surface expression, cell expansion or absolute CAR-T cell number among B7H3-IL7R-S CAR-T cell, B7H3-IL7R-M CAR-T cell, B7H3-IL7R-L CAR-T cell and conventional B7H3 CAR-T cell (FIG.1D and 1E). FIG.1D shows the expansion capacity of transduced CARs. Activated T cells (2ௗ×ௗ105) from each group were cultured in complete medium at day 0. The number of total T lymphocytes was monitored twice a week using a hemocytometer; data are shown as the mean ± S.E. of 4 independent donors. **** Pௗ<ௗ0.0001 vs. NT; **** Pௗ<ௗ0.0001 vs. B7H3-BB-IL7L; **** Pௗ<ௗ0.0001 vs. B7H3-BB-IL7M. FIG.1E shows the absolute CAR-T cell numbers calculated by total cell number and percentage of CAR expression at days 7 and 11; data are shown as the mean ± S.E. of 4 independent donors. The results were compared with two-way ANOVA. ** Pௗ<ௗ0.01, **** Pௗ<ௗ0.0001 vs. NT control.. Incorporation of the IL7Ra signaling domain in B7H3 CAR-T cell maintains a less- differentiated phenotype and phospho-STAT5 expression in after expansion and antigen stimulation [000170] FIGs.2A-2F show enrichment of the naïve / stem-like memory T cell (TN / TSCM) population and phospho-STAT5 expression in B7H3 CAR-T cells incorporated with IL-7Ra signaling. On day 11 after transduction, the T cell subsets and memory phenotypes of the different constructs using flow cytometry were analyzed, and no difference in the CD4 and CD8 ratios between each construct were observed (FIG.2A showing CD4+ and CD8+ T cell subsets in transduced human T lymphocytes at days 4 and 11; data are shown as the mean ± S.E. of 4 individual donors.). However, the TN / TSCM population was significantly increased in all three constructs that incorporated the IL-7Ra signaling domain compared to conventional B7H3-BB CAR-T cells (FIG.2B, showing the frequency of TN / TSCM, memory T cells (TCM), effector memory T cells (TEM), and effector memory T cells (TEMRA) in CAR-T cells on day 11 after transfection; data shown are representative of 4 independent donors, results were compared with two-way ANOVA. * Pௗ<ௗ0.05, *** Pௗ<ௗ0.001 vs. B7H3-BB CARs). When CD45RA and CCR7 were employed to characterize the memory phenotype, our results were consistent. We observedAttorney Docket No.: CHLU-001 / 001WO a greater proportion of naïve phenotype in CD8+ population of B7H3-IL7R CAR-T cells comparing with conventional B7H3 CAR-T cells (FIG.2C). [000171] In addition, STAT5 phosphorylation was investigated in different B7-H3 CAR-T cells^after stimulation with the GBM cell lines U87 and LN229. Upon stimulation with the B7H3- negative cell line (NALM-6), all CAR-T cells did not activate pSTAT5 in any of the constructs (FIG. 2D), whereas upon. Upon stimulation with B7H3+ target cells, all CARs showed a significant upregulation of pSTAT5 in an antigen-dependent manner (FIGs.2E and 2F). At 30 min after stimulation, no differences in pSTAT5 expression between the second-generation B7H3 CAR-T cells and the B7H3 CAR-T cells with additional IL7RĮ signals was observed. The results are shown as the mean ± SEM of 3^4 individual donors. # Pௗ<ௗ0.05 ## Pௗ<ௗ0.01, ### Pௗ<ௗ0.001 vs. B7-H3-BB CAR-T cells. Upon stimulation with the target antigen, all CAR-T cells demonstrated significantly higher expression of pSTAT5 compared with NT cells. The upregulated pSTAT5 expression subsequently declined after 60 minutes of antigen exposure. Interestingly, the B7H3- BB-IL7S CAR construct significantly slowed the reduction in pSTAT5, suggesting the induction of intracellular in all conditions. Notably, B7H3-IL7R-S CAR-T cells could sustain pSTAT5 expression even after 60 minutes of antigen exposure, implying that the incorporation of IL7R-S in the B7H3 CAR endosignaling domain can induce IL-7R signaling in an antigen-dependent manner (Figure 2E and 2F). In vitro antigenic responses of B7-H3 CAR-T cells incorporated with the IL7Ra cytoplasmic signaling domain. [000172] To investigate the antigenic responses of B7H3-BB-IL7 CAR-T cells in vitro, the cytotoxic function of CAR-T cells against B7H3-positive glioblastoma (GBM) cells (LN229 and U87 cells) was evaluated. FIG.3A shows B7H3 expression in GBM (LN229 and U87) and B- cell acute lymphoblastic leukemia (NALM-6) cell lines screened by flow cytometry using a goat anti-human B7H3 polyclonal antibody as the primary antibody and PE-conjugated anti-goat IgG as the secondary antibody. B7H3-BB and B7H3-BB-IL7 CAR-T cells effectively lysed B7H3- expressing LN229 and U87 cell lines in a dose-dependent manner. FIG.3A-3F show in vitro functional assays of B7-H3 CAR-T cells incorporated with IL7Ra signaling. Notably, B7H3-BB- IL7S demonstrated higher, but not statistically significant, cytotoxicity than B7H3-BB CAR-TAttorney Docket No.: CHLU-001 / 001WO cells at low E:T ratios (FIG.3B, showing cytotoxicity of CAR-T cells against LN229 or U87 glioblastoma cells at different E-to-T cell ratios for 72 h). [000173] Activation markers were also analyzed after encountering the target antigen. As expected, CD25 and CD69 expression was highly upregulated upon B7-H3 encountering target cells in all CAR-T groups. Notably, there was a significant increase in the mean florescence intensity of CD69 expression with the B7H3-BB-IL7S CAR construct (FIGs.3C^3E). FIGs 3C and 3D show CD25 expression and CH69 expression, respectively, on CAR-T cells when cocultured with NALM-6 or GBM cells for 72 h. The results are representative of two individual donors. * P < 0.05 vs. B7H3-BB CAR. Next, cytokine secretion of the CAR-T cells was measured. All CAR-T cells secreted high amounts of various cytokines, including TNF-Į, IL-6, IL-2, IFN-Ȗ, IL-4, and IL-4, after antigen stimulation compared with NT cells. Interestingly, all B7H3-IL7RĮ CAR-T secreted significantly higher levels of IL-4 and IL-10 than conventional B7H3 CAR-T cells, whereas B7H3-IL7R-S and -L significantly secreted lower level of IFN-Ȗ. IL-2 secretion was significant lower in B7H3-IL7R-L and -M compared with 2nd generation B7H3 CAR. There was no statistically significant difference in TNF-Į, and IL-6 concentration (Figure 3F). Furthermore, the expression of immune checkpoint molecules, including PD1, TIGIT and TIM3, were evaluated, after stimulation with target antigens and our present results demonstrated a comparable levels of these molecules across CAR constructs (Figure 3E). Furthermore, the expression of immune checkpoint molecules, including PD1, TIGIT and TIM3, was evaluated after stimulation with target antigens,^and the results demonstrated comparable levels of these molecules across CAR constructs (FIG 3F, showing the average MFI of the inhibitory receptors PD1 (upper), TIGIT (middle), and TIM3 (lower) in CAR-T cells cocultured with GBM cells for 72 h; graphs are presented as the mean ± SEM from three independent donors). B7H3 CAR-T cells composed of short length IL7RĮ (B7H3-IL7R-S) provides superior antitumor activity in a tumor rechallenge assay. [000174] Next, it was investigated whether B7H3-BB-IL7Į CAR-T cells had functional advantages in antitumor activity and T cell survival over conventional B7H3 CAR-T cells using an in vitro tumor-rechallenge assay. FIGs.4A-4D show in vitro antitumor activity and persistence of B7H3-BB-IL-7Ra CAR-T cells in a tumor rechallenge experiment. FIG.4A is aAttorney Docket No.: CHLU-001 / 001WO schematic representation of the experimental procedure for the tumor rechallenge in vitro assay. The experiments were conducted using two different GBM cell lines, LN229 (FIGs.4B-4D) and U87 (FIGs.4E-4G). As shown in FIGs.4B-G, FACS analysis indicated the CAR-mediated proliferation and effector function of T cells after repeated stimulation with GBM cells in the absence of exogenous cytokines. FIGs.4B-4G show cytotoxicity of B7-H3-CAR-T cells against tumor cells (B7H3+; LN229) at a 1:1 effector:target (E:T) ratio for at least 4 rounds. Each round was incubated for 72 h in the absence of IL-2 or IL-7 and IL15. During the initial round of coculture, B7H3 CAR-T cells from all the groups effectively eliminated both GBM cell types. However, B7H3, B7H3- IL7R-M and B7H3-IL7R-L CAR-T cells exhibited a reduction in their cytotoxic function toward both target cells after the fourth exposure. In contrast, B7H3-IL7R-S CAR-T cells maintained their cytotoxic function against GBM cells (FIGs.4C and 4F). Moreover, B7H3-IL7R-S CAR-T cells demonstrated a significantly higher number of effector cells than did conventional B7H3 CAR-T cells after the fourth re-exposure to the GBM cells (FIGs.4D and 4G). Analysis of the phenotypes of CAR-T cells at the end of the third round of tumor rechallenge was also performed. The majority of the remaining CAR-T cells were effector memory cells, and there were no significant differences in the memory phenotype among the B7H3 CAR-T groups. Notably, compared with conventional B7H3 CAR-T cells, B7H3-IL7R-S cells exhibited lower PD1 and LAG-3 upregulation (FIGs.4H and 4I). These findings suggested that the incorporation of a short length of IL7RĮ (IL7R-S) results in better antitumor activity and T cell persistence after repeated antigen exposure than the incorporation of other constructs. B7H3-IL7R-S CAR-T cells exhibited distinct transcriptional expression profiles [000175] Incorporation of the cytoplasmic domain of IL7RĮ using the IL7R-S construct in B7H3 CAR-T cells exhibited higher TN / TSCM memory phenotype with better antitumor activity and effector T cell proliferation than conventional B7H3 CAR-T cell. To understand the molecular mechanism behind the enhanced function of B7H3-IL7R-S CAR-T cells, RNA-sequencing (RNA- seq) was performed to identify transcriptional expression profiles after antigen stimulation of B7H3-specific CAR-T cells incorporating cytoplasmic domain of IL7RĮ that consists of the Box1 motif and Y4493 tyrosine residues (FIG. 5A). RNA-seq analysis indicated that differentially expressed genes (DEGs) associated with cell proliferation were increased, while DEGs linked to apoptosis were decreased. Similarly, alterations in metabolic process were estimated in B7H3Attorney Docket No.: CHLU-001 / 001WO CAR-T cells incorporating IL7R-S. This finding suggests that anti-apoptosis may as a significant factor to improve function of B7H3-IL7R-S CAR-T cells. [000176] B7H3-IL7R-S CAR-T cells decreased apoptosis induction with dysregulated PIM1-related metabolism. FIGs.5B-5C show that incorporation of IL-7RĮ signaling reduced apoptosis and modulated metabolic gene expression profiles in B7H3 CAR-T cells. As shown in FIGs.5B and 5C, B7H3- ILR7-S CAR-T cells exhibited lower apoptosis than conventional second-generation B7H3 CAR constructs when cocultured with both GBM cell lines (B7H3 - ILR7-S CAR vs. B7H3- CAR: P < 0.0001 for coculturing LN229 cells; B7H3- IL7R-S CAR vs. B7H3- CAR: P < 0.0001 for coculturing U87 cells). FIG.5B is a flow cytometric plot showing the programmed cell death of CAR-T cells after coculture with GBM cells for 72 h. Apoptotic cells were gated from CD3+ cells, and the percentages of live and apoptotic cells are shown. FIG.5C is a bar graph showing the mean ± SEM of 2^4 individual donors. * P < 0.05, **** P < 0.0001 vs. live B7- CARs; # Pௗ<ௗ0.05, #### Pௗ<ௗ0.0001 vs. apoptotic B7-H3 CARs. [000177] qRT^PCR analysis revealed a reduction in the expression of proapoptotic genes, such as BAD, in IL7RĮ-containing CAR-T cells (B7H3 -IL7R-S CAR vs. B7H3 CAR: P < 0.01) but no alteration in antiapoptotic gene expression. Surprisingly, B7H3- -IL7R-S CAR-T cells also increased PIM1-mediated survival and metabolism after stimulation. [000178] Before stimulation with tumor antigens, decreased GLUT1 was observed in B7H3 CAR-T cells compared to NT cells; however, increased GLUT1 mRNA was detected in B7H3 - IL7R-S CAR cells (B7H3 -IL7R-S CAR vs. B7H3 CAR: P < 0.001). [000179] A heatmap analysis revealed greater expression of transcriptional regulators associated with T cell proliferation, such as FYN, ZAP70, PTPRC, IL10, TWSG1, and CD46, in B7H3-IL7R-S cells than in B7H3 CAR-T cells. Conversely, genes related to apoptotic processes and programmed cell death, including ST3GAL, BAX, BBC3, and CD274, were downregulated in B7H3-IL7R-S CAR-T cells compared to conventional B7H3 CAR-T cells. There was also significant upregulation of antiapoptotic genes such as BCL3 and ADAM8 in B7H3-IL7RS CAR- T cells (FIG.5A). Furthermore, we investigated whether the incorporation of a shorter IL7RĮ signaling domain reduced activation-induced cell death in B7H3 CAR-T cells. As shown in FIG. 5B, B7H3-IL7R-S cells exhibited lower apoptosis levels than did conventional second- generation B7H3 CAR-T cells when cocultured with LN229 and U87 cells, supporting the downregulation of genes associated with apoptosis observed via GO analysis.Attorney Docket No.: CHLU-001 / 001WO [000180] There was no notable change in the expression of hexokinase 2 (HK2). TGF-ȕ expression was lower in the B7-H3 CAR-T groups than in the NT group after tumor stimulation (FIG.5C). FIG.5C shows results of a quantitative RT^PCR analysis shows the expression of genes involved in apoptosis, proliferation, and metabolism in CAR-T cells cocultured with LN229 cells for 72 h. The ȕ^actin housekeeping gene was used for relative quantification of gene expression. The results are representative of three independent donors. ++ P < 0.05 vs. prestimulated NT controls, $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001 vs. prestimulated B7H3-BB CARs; ** P < 0.05 vs. poststimulated NT controls; # Pௗ<ௗ0.05 ## Pௗ<ௗ0.01, and #### Pௗ<ௗ0.0001 vs. poststimulated B7H3- CARs. [000181] These findings indicate that IL-7R signaling promotes T cell survival, leading to superior antitumor activity and persistence of B7H4- -IL7R-S CAR-T cells in vitro. B7H3- -IL7R-S CAR-T cells eliminate tumor progression and prolong survival in GBM Xenograft Models in vivo [000182] As B7H3- -IL7R-S exhibited superior antitumor activity and a favorable profile compared with other intrinsic IL-7RĮ constructs, the antitumor responses of B7H3 -IL7R-S in comparison with conventional B7H3 was tested in xenograft models of glioblastoma. FIGs.6A- 6E show antitumor responses of B7-H3 CAR-T cells in a xenograft mouse model of GBM. A schematic representation of the experimental protocol using LN229 cell line-derived xenografts tumor xenografts is illustrated in FIG.6A. Mice bearing LN229 glioblastoma (5 × 106) were treated with B7H3, B7H3-IL7R-S CAR-T or NT cells (1 × 107) after 7 days of tumor engraftment. [000183] Analysis of tumor volume revealed that B7H3 CAR-T cells only slowed tumor progression (FIGs.6B and 6C) without a significantly enhanced survival rate in LN229 tumor- bearing mice. FIG.6B shows the Tumor volume in LN229 subcutaneously inoculated mice recorded every week using a caliper, tumor volume was calculated by the formula (lengthௗ×ௗwidth2) / 2. Data are represented as the mean ± S.E. of 4 individual mice; results were compared with one-way ANOVA. ** P < 0.01; non significance (ns) vs. the NT control group, # P<0.05 vs. the B7H3 -treated group. FIG.6C shows the estimated tumor mass in each animal. In contrast, B7H3-IL7R-S CAR-T cells showed significant tumor eradication and prolonged survival compared to B7H3 CAR-T cells (Kaplan^Meier, log-rank tests and Cox regressionAttorney Docket No.: CHLU-001 / 001WO analyses were used; P < 0.001) (FIG.6E, showing Kaplan–Meier survival curves comparing tumor progression over time in the NT-, B7H3 - and B7H3 -IL7R-S-treated groups (n = 4 mice per group). *** P < 0.01 vs. B7-H3 -treated group). No difference in the weight of mice was observed among the three groups (FIG.6D, showing the body weight of mice bearing LN229 tumor cells was monitored pre- and postinfusion of CAR-T cells). [000184] Furthermore, the antitumor efficacy of anti-B7H3 -IL7R-S CAR-T cells was also assessed in mice bearing highly invasive U87 tumor xenografts. FIG.7A is a schematic representation of the experimental protocol using U87 cell line-derived xenografts. Mice bearing U87 glioblastoma (5 × 106) were treated with B7H3, B7H3-IL7R-S CAR-T or NT cells (1 × 107) after 7 days of tumor engraftment. The administration of B7H3- -IL7R-S CAR-T cells resulted in potent antitumor activity with a growth inhibition rate of 84.4% (FIGs.7B and 7C). FIG.7B shows the tumor volume in U87 subcutaneously inoculated mice recorded every week using a caliper, tumor volume was calculated by the formula (lengthௗ×ௗwidth2) / 2. Data are represented as the mean ± S.E. of 4 individual animals. The results were compared with one-way ANOVA. ** P < 0.01; *** P < 0.001 vs. the NT control group; # P<0.05 vs. the B7H3- treated group. FIG.7C shows the estimated tumor mass in each animal. [000185] In addition, as indicated in FIG.7E, a trend of longer disease-free survival was observed in mice treated with B7H3- IL7R-S CAR-T cells (Kaplan^Meier, log-rank tests and Cox regression analyses were used; P < 0.01) (FIG.7E, showing Kaplan–Meier survival curves comparing tumor progression over time in the NT-, B7H3 - and B7H3- IL7R-S-treated groups (n = 4 mice per group). ** P < 0.01 vs. B7-H3 -treated group). And no difference in the weight of mice was observed among the three experimental groups (FIG.7D, showing shows the body weight of mice bearing U87 tumor cells was monitored pre- and postinfusion of CAR-T cells). These results demonstrated the enhanced antitumor activity of B7H3-BB-IL7S CAR-T cells in two preclinical models of highly invasive GBM. FIG.7F shows IL2-independent proliferation of NT, B7H3, and B7H3- IL7R-S CAR-T cells in the absence of IL-2 or IL-7 and IL15. Total T cells were recorded every other day using a Countess III automated cell counter. The results are presented as the mean ± SEM of three individual donors.Attorney Docket No.: CHLU-001 / 001WO B7-H3 CAR-T cells were not stimulated by c-expressing IL7 in a largely IL2- independent manner [000186] Unlike constitutively expressed IL-7 signaling, the B7H3- -IL7R-S CAR construct was designed to require target engagement for the activation of IL-7R signaling. To determine whether this insertion of the IL-7RĮ cytoplasmic domain resulted in the self- proliferation of CAR-T cells, non-transfected (NT), B7H3, and B7H3 -IL7R-S CAR-T cells were cultured in the absence of IL-2 supplementation and monitored the cell count over time. [000187] As shown in FIG.7F, B7H3- -IL7R-S CAR-T cells showed a higher proliferation rate than B7H3- CAR-T cells and NT cells. However, the total cell number decreased over time, and on day 70 of analysis, no viable cells were detected. This finding suggests that incorporation of the IL-7Ra signaling domain increased CAR-T cell persistence without transforming into IL- 2-independent T cell growth. [000188] In addition, immunohistochemistry (IHC) of tumor biopsies obtained from mice bearing LN229 cells demonstrated greater CD3+ T cell infiltration in the B7H3-IL7R-S group than in the B7H3 and NT groups (FIGs.7H and 7G). These results demonstrated the enhanced antitumor activity of B7H3-IL7R-S CAR-T cells in xenograft mouse models. Example 2: CD19 CAR-T cells With IL-7RS endo-signaling domain exhibits better persistence and antitumor activity in lymphoma [000189] Schematic illustration of anti-CD19 CAR-T cells constructed with a retroviral vector encoding a signal peptide, the CD19-specific scFv, the hinge / spacer, and CD28 transmembrane domain with either 41BB-only (CD19-BB) or 41BB-IL7S signaling domain (CD19-BB-IL7S), and the CD3ȗ cytoplasmic domain are shown in FIG.8A. (B) The plots showing CAR-T cell [000190] Transduction efficiency of CAR-T expression on primary CD3+ T cells on day 4 and 11 is shown in FIG.8B. Proliferation expressed as total cell number and fold expansion of CD19-specific CAR-T cells during manufacturing are shown in FIGs.8C and D, respectively. [000191] A tumor rechallenge assay was performed without exogenous cytokines and initial culture was performed using a 1:1 E:T ratio of CD-19-BB, CD19-BBIL7S CAR-T or NT controls cocultured with Raji cell line. After 48 h, flow cytometry analysis was acquired for a sample collection and Raji cells were re-introduced into each treatment well. This process was repeated for eight rounds. Total effector cells and specific lysis after each round are shown inAttorney Docket No.: CHLU-001 / 001WO FIGs.8E and 8F, respectively. Representative flow cytometry data of the tumor rechallenge assay is shown in FIG.8G. Data shown are mean ± S.E.M. from three independent donors (n=3). *p < 0.05, **p < 0.01 by one-way ANOVA. Example 3: BCMA CAR-T cell With IL-7RS endo-signaling domainexhibits better persistence and antitumor activity in Multiple Myeloma. [000192] A schematic illustration of anti-BCMA CAR-T cells constructed with a retroviral vector encoding a signal peptide, the CD19-specific scFv, the hinge / spacer, and CD28 transmembrane domain with either 41BB-only (BCMA-BB) or 41BB-IL7S signaling domain (BCMA-BB-IL7S), and the CD3ȗ cytoplasmic domain is shown in FIG.9A. CAR-T cell transduction efficiency of CAR expression on primary CD3+ T cells on day 4 and 11 is shown in FIG.9B. [000193] Proliferation expressed as total cell number and fold expansion of BCMA-specific CAR-T cells during manufacturing are shown in FIGs.9C and 9D, respectively. [000194] A tumor rechallenge assay was performed without exogenous cytokines and initial culture was performed using a 1:1 E:T ratio of BCMA-BB, BCMA-BBIL7S CAR-T or NT controls cocultured with RPMI8226 cell line. After 48 h, flow cytometry analysis was acquired for a sample collection and Raji cells were re-introduced into each treatment well. This was repeated for five rounds. Total effector cells and specific lysis after each round are sonw in FIGs.9E and 9F. Representative flow cytometry of tumor rechallenge assay is shown in FIG. 9G. Data shown are mean ± S.E.M. from three independent donors (n=3). *p < 0.05, **p < 0.01, *** P<0.005 by one-way ANOVA. Example 4: FOLR1 CAR-T cell With IL-7RS endo-signaling domain exhibits better persistence and antitumor activity in ovarian cancer. [000195] A schematic illustration of anti-FOLR1 CAR-T cells constructed with a retroviral vector encoding a signal peptide, the FOLR1-specific scFv, the hinge / spacer, and CD28 transmembrane domain with either 41BB-only (FOLR1-BB) or 41BB-IL7S signaling domain (FOLR1-BB-IL7S), and the CD3ȗ cytoplasmic domain followed by a truncated CD19 (dCD19) to facilitate expression of transduced CAR-T is shown in FIG.10A. [000196] Representative flow cytometry plots showing CAR-T cell transduction efficiency and the mean florescent intensity (MFI) of CAR-T expression on primary CD3+ T cells on day 4 and 11 are shonw in FIG.10B.Attorney Docket No.: CHLU-001 / 001WO [000197] Proliferation expressed as fold expansion and absolute number of FOLR1-specific CAR-T cells during manufacturing are shown in FIG.10C. [000198] A tumor rechallenge assay was performed without exogenous cytokines and initial culture was performed using a 4:1 E:T ratio of FOLR1-BB, FOLR1-BB-IL7S CAR-T or NT controls cocultured with SKOV3 cell line. After 72 h, flow cytometry analysis was acquired for a sample collection and SKOV3 cells were re-introduced into each treatment well. This was repeated for four rounds. Representative flow cytometry plots are shown in FIG.10D. Residual target cells and effector cells counted by flow cytometer with a fixed acquisition volume are shown in FIGs 10E and 10F, respectivleyt.. Data shown are mean ± S.E.M. from two independent donors (n=2). *p < 0.05 by one-way ANOVA.

Claims

Attorney Docket No.: CHLU-001 / 001WO CLAIMS 1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and an intracellular signaling domain of IL-7Ra.

2. The CAR of claim 1, wherein the intracellular signaling domain of IL-7Ra comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:

6.

3. The CAR of claim 1, wherein the intracellular signaling domain of IL-7Ra comprises the amino acid sequence set forth in SEQ ID NO:

6.

4. The CAR of any one of claims 1-3, wherein the intracellular domain further comprises a CD3ȗ costimulatory domain.

5. The CAR of claim 4, wherein the CD3ȗ costimulatory domain is located C-terminal to the intracellular signaling domain of IL-7Ra.

6. The CAR of any one of claims 1-5, wherein the one or more costimulatory domains is located N-terminal to the intracellular signaling domain of IL-7Ra.

7. The CAR of any one of claims 1-6, wherein the one or more costimulatory comprises a 4- 1BB costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, or an OX40 costimulatory domain.

8. The CAR of any one of claims 1-7, wherein the transmembrane domain comprises a CD28 transmembrane domain.

9. The CAR of any one of claims 1-8, wherein the extracellular antigen-binding domain is an scFv.

10. The CAR of any one of claims 1-9, wherein the extracellular antigen-binding domain binds to a cancer antigen.Attorney Docket No.: CHLU-001 / 001WO 11. The CAR of claim 10, wherein the cancer antigen is B7H3, CD19, BCMA, or Folate receptor alpha (FOLR1).

12. A polynucleotide encoding the CAR of any one of claims 1-11.

13. An expression vector comprising the polynucleotide of claim 12.

14. An immune cell comprising the CAR of any one of claims 1-11, the polynucleotide of claims 8 or the expression vector of claim 9.

15. The immune cell of claim 14, wherein the cell is a T cell, an NK cell, an NKT cells, a double negative T cell or a gamma / delta T cell.

16. The immune cell of claim 14 or 15, wherein the immunce cell is derived from an induced pluripotent stem cell or a hematopietic stem cell.

17. The immune cell of any one of claims 14-16, wherein the cell exhibits increased expansion compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra.

18. The immune cell of any one of claims 14-17, wherein the cell exhibits increased cytokine production compared to a cell comprising a CAR that does not contain an intracellular signaling domain of IL-7Ra.

19. The immune cell of any one of claims 14-18, wherein the cell exhibits a naïve / stem-like memory T cell phenotype.

20. A pharmaceutical composition comprising the immune cell of any one of claims 14-19 and a pharmaceutically acceptable carrier.

21. A method of treating cancer in a subject in need thereof, comprising administering to the subject the polynucleotide of claim 12, the expression vector of claim 13 or the immune cell of any one of claims 14-19 or the pharmaceutical composition of claim 20.

22. The method of claim 21 where the cancer is a solid tumor.Attorney Docket No.: CHLU-001 / 001WO 23. The method of claim 21, wherein the cancer is brain cancer, ovarian cancer, breast cancer, cholangiocarcinoma, multiple myeloma or AML.

24. The method of claim 21, wherein the cancer is glioblastoma.

25. The method of claim 21, wherein the cancer is a lymphoma.

26. A method of improving a therapeutic cell comprising a CAR, the method comprising inserting an intracellular signaling domain of IL-7Ra into the CAR.

27. The method of claim 26, wherein the intracellular signaling domain of IL-7Ra comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:

6.

28. The method of claim 26, wherein the intracellular signaling domain of IL-7Ra comprises the amino acid sequence set forth in SEQ ID NO:

6.

29. The method of any one of claims 26-28, wherein the CAR further comprises a CD3ȗ costimulatory domain is located C-terminal to the intracellular signaling domain of IL-7Ra.

30. The method of any one of claims 26-29, wherein the CAR further comprises one or more costimulatory domains located N-terminal to the intracellular signaling domain of IL-7Ra.

31. The method of any one of claims 26-30, wherein the CAR cell targets B7H3, CD19, BCMA, FOLR1.

32. The method of any one of claims 26-31, wherein the method results in increased expansion compared to the expansion prior to improvement.

33. The method of any one of claims 26-32, wherein the method results in increased cytokine production compared to cytokine production prior to improvement.

34. The method of any one of claims 26-33, wherein the therapeutic cell is allogeneic to the subject.

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