Chimeric antigen receptors comprising TMIGD2 co-stimulatory domains and related methods of use

Abstract

The present technology provides a chimeric antigen receptor (CAR) comprising a TMIGD2 co-stimulatory domain. In certain embodiments, these CARs comprise an intracellular region, a transmembrane region, and an extracellular region, the intracellular region comprising a TMIGD2 costimulatory domain and an effector domain, the extracellular region comprising an antigen binding domain and optionally a hinge region and / or a leader sequence. In other embodiments, these CARs comprise an intracellular region, a transmembrane region, and an extracellular region, the intracellular region comprising a TMIGD2 costimulatory domain and an effector domain, the extracellular region comprising an antigen binding domain that specifically binds to the tumor associated antigen B7-H3, and optionally a hinge region and / or a leader sequence. Also provided are nucleic acids, vectors, and cells associated with these CARs, as well as methods of using the CARs, nucleic acids, vectors, and cells to treat conditions associated with expression of particular antigens.

Description

[0001] Cross-references to related applications and priority claims

[0002] This application claims priority to U.S. Provisional Application No. 63 / 368,549, filed on July 15, 2022, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federal Funding

[0004] This invention was made with government support under CA175495-08 and CA175495-06 awarded by the National Institutes of Health (NIH) / National Cancer Institute (NCI). The government has certain rights in this invention.

[0005] Incorporation by Reference into the Sequence Listing

[0006] This application contains a sequence listing in accordance with ST.26, which is filed in XML format via the Patent Center and is hereby incorporated by reference in its entirety. The .xml copy was created on July 14, 2023, named 1298078025WO00.xml, and is 25KB in size. Background Art

[0007] Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells or NK cells or macrophages or other immune cells is a potent antigen-specific therapy for the treatment of diseases such as human malignancies. CAR-expressing T cells or NK cells or macrophages or other immune cells target tumor-associated antigens expressed on the surface of malignant cells. Since CAR-T or NK cells or macrophages or other immune cell-based therapeutic strategies target antigens already expressed on the cell surface, problems associated with tumor escape mechanisms involving the major histocompatibility complex can be overcome.

[0008] Advances in elucidating the mechanisms and molecules that regulate immune responses have provided many therapeutic targets for treating cancer. For example, co-stimulatory and co-inhibitory molecules play a central role in regulating T cell immune responses. However, despite impressive patient responses to antibody agents targeting these co-stimulatory and co-inhibitory molecules, including, for example, anti-PD-1 / PD-L1, checkpoint inhibition therapy still fails in many patients.

[0009] There remains a need for new compositions and methods that can improve the effectiveness of these agents, particularly those involving CAR-modified T cells. Summary of the invention

[0010] In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular region comprising an antigen binding domain; (b) a transmembrane region; and (c) an intracellular region comprising an effector domain and a TMIGD2 co-stimulatory domain.

[0011] In some embodiments, the TMIGD2 co-stimulatory domain comprises an intracellular region of TMIGD2. In some embodiments, the TMIGD2 co-stimulatory domain comprises a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5. In some embodiments, the TMIGD2 co-stimulatory domain comprises an amino acid sequence having at least 75% identity to a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5.

[0012] In some embodiments, the antigen binding domain specifically binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; LewisA; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptide bound to HLA; hTERT peptide bound to HLA; tyrosinase peptide bound to HLA; WT-1 peptide bound to HLA; LT βR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME; and SSX-2 antigens.

[0013] In some embodiments, the antigen binding domain includes scFv. In some embodiments, the antigen binding domain includes a joint. In some embodiments, the joint is a glycine-serine joint. In some embodiments, the glycine-serine joint includes (GlyxSery)z, wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0, and z is an integer from 1 to 10.

[0014] In some embodiments, the extracellular region further comprises an N-terminal leader sequence. In some embodiments, the extracellular region further comprises a hinge region. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 2.

[0015] In some embodiments, the transmembrane region comprises a CD8α transmembrane region.In some embodiments, the transmembrane region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:1.

[0016] In some embodiments, the effector domain is a CD3 zeta effector domain. In some embodiments, the effector domain comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the effector domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 5.

[0017] In some embodiments, the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 1. In another embodiment, the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 6. In yet another embodiment, the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 1; and (c) the sequence shown in SEQ ID NO: 6. In some embodiments, the CAR further comprises the amino acid sequence shown in SEQ ID NO: 2.

[0018] In some aspects, the present disclosure provides an isolated polynucleotide encoding any of the CARs described herein.

[0019] In some aspects, the disclosure provides an expression vector comprising any of the isolated polynucleotides described herein operably linked to an expression control sequence. In some embodiments, the expression control sequence is a promoter. In some embodiments, the expression control sequence is a promoter.

[0020] In some embodiments, the expression vector further comprises a polynucleotide encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is a P2A peptide. In some embodiments, the polynucleotide encoding the self-cleaving peptide is on the 3' side of the polynucleotide encoding the CAR. In some embodiments, the polynucleotide encoding the self-cleaving peptide is on the 5' side of the polynucleotide encoding the marker polypeptide.

[0021] In some embodiments, the expression vector further comprises an isolated polynucleotide encoding a transduction marker polypeptide. In some embodiments, the transduction marker polypeptide is a truncated form of epidermal growth factor receptor (EGFRt) or a portion or variant thereof or GFP or a portion or variant thereof.

[0022] In some embodiments, the vector is a viral vector.

[0023] In some aspects, the present disclosure provides a host cell expressing any of the CARs of the present disclosure and / or comprising any of the isolated polynucleotides of the present disclosure and / or comprising an expression vector of the present disclosure.

[0024] In some embodiments, the host cell is a T cell, a natural killer (NK) cell, a macrophage, or other immune cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, a CD4 - CD8 - Double negative T cells, NK cells, macrophages, other immune cells, or any combination thereof. In some embodiments, the T cells are naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, NK cells, macrophages, other immune cells, or any combination thereof.

[0025] In some embodiments, the host cell also expresses a transduction marker at its cell surface. In some embodiments, the transduction marker is a truncated form of the epidermal growth factor receptor (EGFRt) or a portion or variant thereof or GFP or a portion or variant thereof.

[0026] In some aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a host cell of the present disclosure.

[0027] In some embodiments, the disease or condition is a malignant tumor. In some embodiments, the malignant tumor is cancer. In some embodiments, the cancer is selected from prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, stomach cancer, thyroid cancer, anal cancer, small intestine cancer, bone cancer, cervical cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, thymic cancer, sarcoma and osteosarcoma. In some embodiments, cancer includes solid tumors. In some embodiments, cancer includes hematological malignancies.

[0028] In some aspects, the present disclosure provides a method of eliciting an immune response against a tumor-associated antigen that specifically binds to a CAR of the present disclosure, the method comprising administering an effective amount of any of the host cells of the present disclosure to a subject in need thereof.

[0029] In some aspects, the present disclosure provides a composition comprising a CAR of the present disclosure and a pharmaceutically acceptable excipient, carrier or diluent.

[0030] In some aspects, the present disclosure provides a composition comprising the cells of the present disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0031] These and other embodiments of the present disclosure are disclosed in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of a CAR vector according to an embodiment of the present disclosure. The vector encodes a CAR comprising an IgG1 Vk leader sequence, VL and VH domains with an intermediate linker sequence, a CD8a hinge and transmembrane region, a TMIGD2 intracellular tail co-stimulatory domain, and a CD3ζ effector domain. The vector also comprises a self-cleaving peptide P2A, a GMCSFR leader, and a polypeptide marker EGFRt.

[0033] Figure 2 Representative graphs are depicted showing that T cells expressing a CAR according to one embodiment of the present disclosure kill Raji human tumor cells.

[0034] Figure 3 The in vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells in the Raji lymphoma model is shown. Statistics were calculated using the log-rank (Mantel-Cox) test. N=3, p=0.0295.

[0035] Figure 4A Schematic diagram showing the design of example B7-H3 and CD19 CAR constructs.

[0036] Figure 4B Representative graphs are depicted showing single killing co-culture assays of B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cells and U118-Luc tumor cells. CAR transduction efficiency was adjusted to 50%.

[0037] Figure 4C Representative graphs are depicted showing single killing co-culture assays of B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cells and HCC827-Luc tumor cells. CAR transduction efficiency was adjusted to 50%.

[0038] Figure 4D Representative graphs are depicted showing single killing co-culture assays of B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cells and THP-1 tumor cells. CAR transduction efficiency was adjusted to 50%.

[0039] Figure 4E Representative graphs are depicted showing tumor growth curves from time lapse co-culture assays of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells and U118-Luc tumor cells, where tumor burden was quantified based on changes in signal area (tdTomato) over time.

[0040] Figure 4F Representative graphs are depicted showing tumor growth curves from time-lapse co-culture assays of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells and HCC827-Luc tumor cells, where tumor burden was quantified based on changes in signal area (tdTomato) over time.

[0041] Figure 4G Representative graphs are depicted showing cytokine release by B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T after 24 h of co-culture with HCC827 tumor cells.

[0042] Figure 4H Gating strategies for detecting B7-H3 and CD19 CAR-T cells and memory subsets are shown.

[0043] Fig. 4I Representative flow cytometry graphs and bar graphs showing B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cell transduction efficiency are depicted.

[0044] Figure 4J Representative graphs are depicted showing memory populations of B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cell constructs.

[0045] Figure 4K Representative graphs are depicted showing summary data on the expansion of B7-H3.28.ζ, B7-H3.BB.ζ, B7-H3.TMI.ζ, B7-H3.28.BB.ζ, B7-H3.TMI.BB.ζ, CD19.28.BB.ζ CAR-T cell constructs in the absence of tumor antigen.

[0046] Figure 5A Schematic representation of the representative experimental design timeline for in vivo HCC827-Luc cell killing assay using TMIGD2 CAR-T cells.

[0047] Figure 5B Shown in accordance with Figure 5A The experimental design is partially schematically shown in Figure 4. Representative bioluminescence images of mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted HCC827-Luc cells.

[0048] Figure 5C A representative diagram is shown, which shows that Figure 5A The experimental design is partially schematically shown in Figure 5. Quantification of tumor signals from individual mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted HCC827-Luc cells and comparison of summary data of experimental groups.

[0049] Figure 5D A representative graph is shown showing the Figure 5C Kaplan-Meier survival curves of the B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and CD19.28.BB.ζ CAR-T cell experimental groups.

[0050] Figure 5E Schematic representation of the timeline for a representative experimental design for an in vivo U118-Luc cell killing assay using TMIGD2 CAR-T cells.

[0051] Fig. 5F Shown in accordance with Figure 5E The experimental design is partially schematically shown in Figure 4. Representative bioluminescent images of mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted U118-Luc cells.

[0052] Figure 5G A representative diagram is shown, which shows that Figure 5E The experimental design is partially schematically shown in Figure 5. Quantification of tumor signals from individual mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted U118-Luc cells and comparison of summary data of experimental groups.

[0053] Figure 5H A representative graph is shown showing the Fig. 5F Kaplan-Meier survival curves of the B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and CD19.28.BB.ζ CAR-T cell experimental groups.

[0054] Fig.5I Schematic representation of the timeline for a representative experimental design for an in vivo PANC-1-Luc cell killing assay using TMIGD2 CAR-T cells.

[0055] Figure 5J Shown in accordance with Fig.5I The experimental design is partially schematically shown in Figure 4. Representative bioluminescent images of mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted PANC-1-Luc cells.

[0056] Figure 5K A representative diagram is shown, which shows that Fig.5I The experimental design is partially schematically shown in Figure 5. Quantification of tumor signals from individual mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted PANC-1-Luc cells and comparison of summary data of experimental groups.

[0057] Figure 5L A representative graph is shown showing the Fig.5I Kaplan-Meier survival curves of the B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and CD19.28.BB.ζ CAR-T cell experimental groups.

[0058] Fig. 6A Schematic diagram showing the design of example B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-Luc constructs.

[0059] Figure 6BSchematic representation of the representative experimental design timeline for in vivo HCC827-Luc cell killing assay using TMIGD2 CAR-T cells.

[0060] Figure 6C Shown in accordance with Figure 6B The experimental design is partially schematically shown in Figure 4. Representative bioluminescent images of mice at representative survival time points after administration of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells targeting transplanted UCC827-Luc cells.

[0061] Fig.6D A representative diagram is shown, which shows the Figure 6B The experimental design is partially illustrated in FIG. 5 . Line graphs of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T luciferase signals from individual mice.

[0062] Fig. 6E A representative diagram is shown, which shows the Figure 6B The experimental design is partially schematically shown in Figure 4. Bar graph comparing B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T luciferase signals.

[0063] Fig. 6F Representative graphs are depicted showing the peak bioluminescent signals of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells from each mouse during the experiment.

[0064] Figure 6G Representative graphs are depicted showing the quantification of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells in lung tissues in the experiments.

[0065] Figure 6H Representative graphs are depicted showing the quantification of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells in spleen tissues in the experiments.

[0066] Fig.6I Representative graphs are depicted showing the quantification of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, CD19.28.BB.ζ CAR-T cells in the blood in the experiment.

[0067] Fig. 7A A heat map is shown, which plots the Figure 4AFigure 3. All differentially expressed genes (DEGs) of B7-H3.TMI.ζCAR-T cells and B7-H3.28.BB.ζCAR-T cells compared with CD19.28.BB.ζCAR-T cells shown in (p adj < 0.05; fold change > 0.5).

[0068] Figure 7B A representative graph showing a Venn diagram containing unique and shared DEGs between B7-H3.TMI.ζCAR-T cells and B7-H3.28.BB.ζCAR-T cells is depicted.

[0069] Figure 7C Representative graphs are depicted showing gene set enrichment analysis (GSEA) pathway analyses obtained from Reactome and Hallmark datasets comparing B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells to CD19.28.BB.ζ CAR-T cells.

[0070] Fig.7D Representative graphs are depicted showing a comparison of the percentage of ATP generated from glycolysis and mitochondria in B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells cultured in the absence of tumor cells (baseline) or with HCC827 tumor cells (co-culture) for 24 hours.

[0071] Fig. 7E Representative graphs are depicted showing a comparison of the percentage of ATP generated from glycolysis and mitochondria in B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells cultured in the absence of tumor cells (baseline) or with HCC827 tumor cells (co-culture) for 7 days with repeated addition of tumor cells every two days.

[0072] Figure 7F A heat map is shown, which depicts the comparison of DEGs from B7-H3.TMI.ζCAR-T cells and B7-H3.28.BB.ζCAR-T cells.

[0073] Figure 7G Representative graphs showing GSEA pathway analysis of RNA sequencing data obtained from Reactome and Hallmark datasets for B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells are depicted.

[0074] Figure 7HRepresentative graphs are depicted showing dot plots of overrepresentation analysis of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells containing highly expressed DEGs (fold change >±1.5).

[0075] Fig.7I Shown is a heat map of DEGs from the curated gene set depicting glycolysis and oxidative phosphorylation proteins of B7-H3.TMI.ζ compared to B7-H3.28.BB.ζ CAR-T cells.

[0076] Fig. 8A Shown is a heat map depicting the comparison of DEGs in vivo lung-infiltrating T cells from mice injected with B7-H3.TMI.ζCAR-T cells and in vivo lung-infiltrating T cells from mice injected with B7-H3.28.BB.ζCAR-T cells.

[0077] Figure 8B Representative graphs showing GSEA pathway analysis of DEGs are depicted.

[0078] Figure 8C Representative graphs showing over-representation analysis of highly expressed DEGs (fold change > ± 1.5) by B7-H3.TMI.ζ compared to B7-H3.28.BB.ζ CAR-T cells are depicted.

[0079] Fig.8D Shown is a heat map plotting the average gene expression of DEGs found in the metabolic gene signature of B7-H3.TMI.ζ compared to B7-H3.28.BB.ζ CAR-T cells.

[0080] Fig. 8E Shown is a heat map plotting the average gene expression of DEGs found in the T cell dysfunction gene signature for B7-H3.TMI.ζ compared to B7-H3.28.BB.ζ CAR-T cells.

[0081] Fig.8F Shown is a heat map plotting the average gene expression of DEGs found in the inhibitory protein gene signature of B7-H3.TMI.ζ compared to B7-H3.28.BB.ζ CAR-T cells.

[0082] Figure 8G A representative graph is depicted showing a Venn diagram containing overlapping significant genes (p adj < 0.05) in B7-H3.TMI.ζ or B7-H3.28.BB.ζ CAR-T cells from in vivo and in vitro RNA sequencing experiments directly comparing the two constructs.

[0083] Figure 8H A representative diagram is shown, which shows Figure 8G ORA analysis of shared genes between B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells.

[0084] Fig.9A Shown are tSNE plots from the CAR+ population comparing phenotypic analysis of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells after CAE with HCC827 tumor cells.

[0085] Fig. 9B CAR transduction efficiency during chronic antigen exposure (CAE) is shown, which involves phenotypic analysis comparing B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells after CAE with HCC827 tumor cells.

[0086] Fig. 9C Representative graphs showing cell surface marker usage by phenotypically exhausted, memory, activated, and CD4 / CD8 CAR-T cell populations during CAE are depicted.

[0087] Fig.9D Depicted are representative graphs showing luciferase signals from untransduced and B7-H3.TMI.ζ, B7-H3.28.BB.ζ, or CD19-28.BB.ζCAR-transduced Jurkat (NFAT-responsive luciferase reporter) cells after culture under the indicated conditions.

[0088] Fig.9E Representative graphs showing phenotypic analysis of CAR+CD4+ T cells during CAE are depicted.

[0089] Fig.9F Representative graphs showing phenotypic analysis of CAR+CD8+ T cells during CAE are depicted. DETAILED DESCRIPTION

[0090] The present disclosure provides CARs comprising a TMIGD2 co-stimulatory domain, as well as nucleic acid molecules and vectors encoding these CARs, cells expressing these CARs, and methods of using the CARs, nucleic acid molecules, vectors and cells.

[0091] HHLA2 (HERV-H LTR associated 2) is a functional member of the B7 family, and TMIGD2 (NCBI accession number NP_653216.2) has been previously identified as a co-stimulatory receptor for HHLA2 (Zhao 2013; Janakiram 2015a; Janakiram 2015b). TMIGD2 has at least three isoforms: isoform 1 (SEQ ID NO: 3, NCBI NP_653216.2), isoform 2 (SEQ ID NO: 4, NCBI NP_001162597.1) and isoform 3 (SEQ ID NO: 5, NCBI NP_001295161.1). Exemplary DNA sequences encoding isoforms 1-3 are shown in SEQ ID NO: 9 (NCBI NM_144615.), SEQ ID NO: 10 (NCBI NM_001169126.1), and SEQ ID NO: 11 (NCBI NM_001308232), respectively.

[0092] Although most CARs incorporate CD28 or 41-BB as co-stimulatory molecules, the present disclosure relates to CARs that use TMIGD2 as a co-stimulatory molecule. TMIGD2 is expressed on T cells. After antigen stimulation, almost all T cells lose TMIGD2 expression, and this loss is associated with increased levels of PD-1, exhaustion, and senescence (Zhu 2013; Crespo 2017; Janakiram 2017). The use of TMIGD2 to provide co-stimulation in CAR-T cell therapy provides a unique opportunity to maintain TMIGD2 co-stimulatory signaling and preserve functionally active CAR-T cells.

[0093] Exemplary CARs disclosed herein include (a) an extracellular region comprising a binding domain (e.g., scFv); (b) a transmembrane region; and (c) an intracellular region comprising an effector domain and a TMIGD2 costimulatory domain. The TMIGD2 costimulatory domain may include all or part of the intracellular region of any TMIGD2 isoform (including any one of isoforms 1-3), or a variant thereof.

[0094] CAR of the present disclosure can be used for cellular immunotherapy (e.g., T cells and / or natural killer (NK) cells) to treat diseases associated with the expression of one or more antigens, such as cancer. In some embodiments, when administered to a subject with malignant cells expressing one or more cancer-associated antigens, CAR of the present disclosure reduces and / or suppresses the growth, area, volume and / or spread of malignant cells, eliminates (e.g., kills) malignant cells, and / or increases the survival of the subject to a greater extent and / or for a longer period of time compared to cells that do not contain CAR of the present disclosure.

[0095] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed herein should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments are included herein.

[0096] References throughout this specification to "one example," "an example," "an embodiment," "an embodiment," "an aspect," or "aspect" mean that a particular feature, structure, or characteristic described in conjunction with the example is included in at least one example of the present disclosure. Thus, the expressions "in one example," "in an example," "one embodiment," "an embodiment," "an aspect," or "aspect" appearing in various places throughout this specification do not necessarily all refer to the same example, embodiment, and / or aspect.

[0097] The headings provided herein are for convenience only and are not intended to limit or interpret the scope or meaning of the disclosure.

[0098] definition

[0099] Unless otherwise stated, any concentration range, percentage range, ratio range or integer range mentioned herein should be understood to include the value of any integer within the recorded range, and include its fraction (such as one tenth and one hundredth of an integer) when appropriate. In addition, unless otherwise stated, any numerical range recorded herein should be understood to include any integer within the recorded range. As used herein, unless otherwise stated, the term "about" refers to ± 20% of the indicated range, value or structure. It should be understood that, as used herein, the term "a / an" refers to "one or more" in the listed districts. Words using the singular or plural number also include the plural or singular number, respectively. The word "or" used in a list referring to two or more items covers all the following explanations of the word: any item in the list, all items in the list, and any combination of items in the list. Furthermore, the expression "at least one of A, B, and C, etc." is intended to be meaningful so that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, and C" will include but is not limited to systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally such construction is intended to be meaningful so that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, or C" will include but is not limited to systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). As used herein, the terms "comprising," "having," and "including" are used synonymously, and these terms and variations thereof are intended to be interpreted as non-limiting.

[0100] "Nucleic acid molecule" or "polynucleotide" refers to a polymeric compound comprising covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), including cDNA, genomic DNA, and synthetic DNA, any of which may be single-stranded or double-stranded. Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences encoding the same amino acid sequence.

[0101] "Percentage identity" or "percentage sequence identity" relative to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after the sequences are aligned and a gap is introduced (if necessary) to achieve maximum percentage sequence identity and any conservative substitution is not considered as part of the sequence identity. The comparison for the purpose of determining percentage amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software or other software suitable for nucleic acid sequences. Appropriate parameters for aligning sequences can be determined, including algorithms required for achieving maximum alignment over the entire length of the compared sequences. However, for purposes of this article, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office (Washington, D.C. 20559) together with user documentation, and is registered in the U.S. Copyright Office with registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) and can also be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change.

[0102] In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A with, and or to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with, and or to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0103] "Conservative substitutions" refer to amino acid substitutions that do not significantly affect or change the binding properties of a particular protein. Typically, conservative substitutions are substitutions in which the substituted amino acid residue is replaced with an amino acid residue with a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, for substitution purposes, an aliphatic grouping may include Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. More information can be found in Creighton (1984) Proteins , WH Freeman and Company. In certain embodiments, the variant proteins, peptides, polypeptides and amino acid sequences of the present disclosure may contain one or more conservative substitutions relative to a reference amino acid sequence.

[0104] The term "isolated" refers to material that is removed from its original environment (e.g., from its natural environment if it is naturally occurring). Such a nucleic acid could be part of a vector and / or such a nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide.

[0105] As used herein, "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that contains only a domain, motif, part or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, part or fragment of the parent or reference compound. In certain embodiments, the functional portion refers to the "signaling portion" of an effector molecule, effector domain, costimulatory molecule or costimulatory domain.

[0106] As used herein, the term "expression" refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule (such as a gene). The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The expressed nucleic acid molecule is usually operably linked to an expression control sequence (e.g., a promoter).

[0107] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one nucleic acid molecule is affected by the other nucleic acid molecule.

[0108] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence that can affect the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter for realizing transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The carrier can be a plasmid, a phage particle, a virus, or just a potential genome insert. Once transformed into a suitable host, the carrier can replicate and play a role independently of the host genome, or can be integrated into the genome itself in some cases. Herein, "plasmid", "expression plasmid", "virus" and "vector" are usually used interchangeably.

[0109] In the context of inserting a nucleic acid molecule into a cell, the term "introducing" refers to "transfection" or "transformation" or "transduction", and includes descriptions of incorporating nucleic acid molecules into eukaryotic cells, where the nucleic acid molecules can be incorporated into the genome of the cell and converted into autonomous replicons. As used herein, the terms "engineered", "recombinant" or "non-natural" refer to organisms, microorganisms, cells, nucleic acid molecules or vectors that include at least one genetic change or have been modified by the introduction of exogenous nucleic acid molecules, wherein such changes or modifications are introduced by genetic engineering. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, CARs or enzymes, or other functional disruptions of other nucleic acid molecules, additions, deletions, substitutions or cellular genetic material.

[0110] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. The construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome." vector " is a nucleic acid molecule capable of transporting another nucleic acid molecule. The vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector or a linear or circular DNA or RNA molecule, which may include a chromosome, a non-chromosome, a semisynthetic or a synthetic nucleic acid molecule. Exemplary vectors include vectors capable of autonomous replication (episomal vectors), vectors capable of delivering polynucleotides to a cell genome (e.g., viral vectors) or vectors capable of expressing nucleic acid molecules connected thereto (expression vectors).

[0111] As used herein, "enrichment" or "depletion" with respect to the amount of a cell type in a mixture refers to an increase in the amount of the "enriched" type, a decrease in the amount of the "depleted" cell, or both, in a cell mixture resulting from one or more enrichment or depletion processes or steps. In certain embodiments, the amount of a certain cell type in a mixture will be enriched, while the amount of a different cell type will be depleted, such as enrichment for CD4 + CD8 + cells, or enriched CD8 + CD4 + cells, or a combination thereof.

[0112] "Chimeric antigen receptor" (CAR) refers to the CAR of the present disclosure, which is engineered to contain two or more naturally occurring (or engineered) amino acid sequences, which are connected together in a manner that is not naturally present or not naturally present in a specific cell, and when present on the surface of the cell, the CAR can act as a receptor. The CAR of the present disclosure includes an extracellular portion, which includes an antigen binding domain, such as an antigen binding domain obtained or derived from an immunoglobulin, such as an scFv derived from an antibody connected to a transmembrane region and one or more intracellular signaling domains (optionally containing a costimulatory domain) (see, e.g., Sadelain et al., 2013; see also Harris & Kranz, 2016; Stone et al., 2014).

[0113] The term "variable region" or "variable domain" refers to the antibody heavy or light chain involved in antigen binding. H ) and light chain (V L ) each generally comprises four commonly conserved framework regions (FRs) and three CDRs. The framework regions separate the CDRs and the CDRs are located between the framework regions.

[0114] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", and are known in the art to refer to amino acid sequences within the variable region of an antibody, which are generally conferred antigen specificity and / or binding affinity and are separated from each other in the primary structure by framework sequences. In some cases, framework amino acids may also contribute to binding. Typically, there are three CDRs in each variable region. Variable domain sequences can be aligned based on numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow annotation of equivalent residue positions and comparison of different molecules using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15: 298-300).

[0115] As used herein, "antigen" refers to an immunogenic molecule that stimulates an immune response. This immune response may involve antibody production, activation of specific immune competent cells, or both. Antigens can be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is apparent that antigens can be synthesized, recombinantly produced, or derived from biological samples. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express the antigen.

[0116] As used herein, a "binding domain" (also referred to as a "binding region") refers to a molecule or portion thereof, such as an scFv, that has the ability to specifically and non-covalently associate, combine or combine with a target. Binding domains include any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner of a biomolecule, molecular complex or other target of interest. Exemplary binding domains include single-chain immunoglobulin variable regions, receptor extracellular domains, ligands or synthetic polypeptides selected for their specific ability to bind to a biomolecule, molecular complex or other target of interest.

[0117] As used herein, "effector domain" is an intracellular portion or domain of a CAR or receptor that can directly or indirectly promote a biological or physiological response in a cell when receiving an appropriate signal. In certain embodiments, the effector domain is from a protein or a portion thereof or a protein complex that receives a signal and triggers a signal from the effector domain when it binds to a target or homologous molecule or when a protein or a portion thereof or a protein complex binds directly to a target or homologous molecule.

[0118] As used herein, a "transmembrane region" is a portion of a transmembrane protein that can be inserted into or span the cell membrane.

[0119] "Treatment" or "treatment" or "improvement" refers to the medical management of a disease, disorder or condition of a subject. Typically, an appropriate dose or treatment regimen is administered in an amount sufficient to cause a therapeutic or preventive benefit, the dose or treatment regimen comprising cells expressing the CAR of the present disclosure and optionally an adjuvant. Therapeutic or preventive benefits include improving clinical outcomes; alleviating or relieving symptoms associated with the disease; reducing the occurrence of symptoms; improving quality of life; extending the disease-free state; reducing the extent of the disease; stabilizing the disease state; delaying disease progression; alleviating; surviving; prolonging survival; or any combination thereof.

[0120] As used herein, "hyperproliferative disorder" and "proliferative disorder" refer to excessive growth or proliferation compared to normal or non-diseased cells. Exemplary hyperproliferative disorders and proliferative disorders include tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells.

[0121] In addition, "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymphoid or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignancies, angiogenesis associated with malignancy, or other forms of cancer.

[0122] A "therapeutically effective amount" or "effective amount" of a cell expressing a CAR of the present disclosure refers to an amount of CAR-expressing cells sufficient to produce a therapeutic effect, including improving clinical outcomes in a statistically significant manner; alleviating or relieving symptoms associated with the disease; reducing the occurrence of symptoms; improving quality of life; prolonging the disease-free state; reducing the extent of the disease; stabilizing the disease state; delaying disease progression; alleviating; surviving; or prolonging survival. When referring to a single active ingredient or a cell expressing a single active ingredient administered alone, a therapeutically effective amount refers to the effect of the ingredient or the cell expressing the ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amount of an active ingredient or a combination of an auxiliary active ingredient and a cell expressing an active ingredient that produces a therapeutic effect, whether administered continuously or simultaneously.

[0123] The term "pharmaceutically acceptable" with respect to a carrier, excipient or diluent means that the carrier, excipient or diluent is suitable for administration to humans or other non-human mammalian subjects and is generally considered safe or does not cause serious adverse events.

[0124] As used herein, the term "adoptive immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease-antigen-specific immune cells, such as T cells. Adoptive cell immunotherapy can be autologous (immune cells come from the recipient), allogeneic (immune cells come from a donor of the same species), or syngeneic (immune cells come from a donor that is genetically identical to the recipient).

[0125] "T cells" or "T lymphocytes" are cells of the immune system that mature in the thymus and produce T cell receptors (TCRs), including αβT cells and γδT cells. T cells can be naive (not exposed to antigen; unlike T cells, they are not exposed to antigen). CM Compared with normal T cells, the expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA increased, and the expression of CD45RO decreased), memory T cells (T M T cells (antigen experienced and long lived) and effector cells (antigen experienced, cytotoxic). M They can be further divided into central memory T cells (T CM Compared with naive T cells, the expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95 was increased, and the expression of CD54RA was decreased) and effector memory T cells (T EM , with naive T cells or T CM Compared with the control group, the expression of CD62L, CCR7, CD28, and CD45RA was decreased, and the expression of CD127 was increased).

[0126] As used herein, "natural killer cells" or "NK cells" refer to cells that are activated in response to interferon or macrophage-derived cytokines (including viral infection), and the acquired immune response generates antigen-specific cytotoxic T cells that can clear the infection and express CD56.

[0127] In addition, it should be understood that the present disclosure discloses a single construct or a group of constructs derived from various combinations of structures and subunits described herein, and the disclosure level is the same as if each construct or group of constructs were individually set forth. Therefore, the selection of a specific structure or a specific subunit is within the scope of the present disclosure.

[0128] The terminology used in the specification is intended to be interpreted in its broadest reasonable manner even when used in conjunction with a detailed description of certain embodiments.

[0129] Chimeric Antigen Receptor (CAR)

[0130] Provided herein are CARs comprising TMIGD2 costimulatory domains and compositions comprising these CARs. In certain embodiments, provided herein are CARs comprising (a) an extracellular region comprising a binding domain specifically bound to a tumor-associated antigen expressed on a cell (such as a malignant cell), (b) a transmembrane region, and (c) an intracellular region comprising an effector domain and a TMIGD2 costimulatory domain. Also provided herein are cells expressing these CARs and compositions thereof, and unit doses of these cells and compositions. In some embodiments, these unit doses include (i) a composition comprising at least about 50% modified CD4+T cells, and (ii) a composition comprising at least about 50% modified CD8+T cells, and a ratio of about 1: 1, wherein the unit dose contains naive T cells of a reduced amount or is substantially free of naive T cells.

[0131] TMIGD2 co-stimulatory domain

[0132] The costimulatory domain refers to a portion of the intracellular domain that includes a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for lymphocytes to respond efficiently to antigens.

[0133] CAR provided herein contains TMIGD2 costimulatory domain, which includes all or part of the intracellular region of TMIGD2, or a variant thereof. In certain embodiments, the TMIGD2 intracellular region is derived from TMIGD2 isoform 1 (SEQ ID NO: 3), isoform 2 (SEQ ID NO: 4) or isoform 3 (SEQ ID NO: 5). In certain embodiments in which the TMIGD2 costimulatory domain includes a portion of the intracellular region of TMIGD2, the portion is sufficient to transduce signals generally associated with TMIGD2 binding to HHLA2.

[0134] In certain embodiments, the TMIGD2 intracellular region is derived from isoform 1 or 3 and comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3 or residues 52-162 of SEQ ID NO:5, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3 or residues 52-162 of SEQ ID NO:5, or a portion thereof.

[0135] In certain embodiments, the TMIGD2 intracellular region is derived from isoform 2 and comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-278 of SEQ ID NO:4, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 172-278 of SEQ ID NO:4, or a portion thereof.

[0136] In certain embodiments where the TMIGD2 costimulatory domain comprises an amino acid sequence having less than 100% identity to the TMIGD2 intracellular region or a portion thereof (e.g., the TMIGD2 intracellular region of isoforms 1-3), all substitutions resulting in sequence differences are conservative substitutions. In other embodiments, the TMIGD2 costimulatory domain may comprise one or more non-conservative substitutions relative to the TMIGD2 intracellular region. In certain embodiments, the TMIGD2 costimulatory domain comprises one, two, three, four or five or more conservative substitutions relative to the TMIGD2 intracellular region.

[0137] Intracellular effector domain

[0138] In certain embodiments, the CAR provided herein comprises an intracellular effector domain. In some embodiments, the intracellular effector domain is a CD3 zeta effector domain or a functional portion or variant thereof.

[0139] In some embodiments, the CD3 zeta effector domain comprises, consists of, or consists essentially of an amino acid sequence of SEQ ID NO:6, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.

[0140] In certain embodiments where CAR provided herein comprises an intracellular effector domain, the effector domain is directly adjacent to the TMIGD2 costimulatory domain. In other embodiments, the effector domain and the TMIGD2 costimulatory domain are separated by one or more amino acids.

[0141] Transmembrane region

[0142] In certain embodiments, the CAR provided herein comprises a transmembrane region connecting the extracellular region and the intracellular region.

[0143] In certain embodiments, the transmembrane region is derived from CD8α. In certain of these embodiments, the transmembrane region comprises, consists of, or consists essentially of an amino acid sequence of SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1.

[0144] Extracellular region

[0145] In certain embodiments, the CARs provided herein comprise an extracellular region that includes a target-specific binding element (also referred to as an antigen binding domain).

[0146] In certain embodiments, the antigen binding domain in the extracellular region is derived from an antibody and comprises an antigen binding portion thereof. For example, in certain embodiments, the antigen binding domain comprises an antibody V H All or part of the chain (e.g., V H variable domain or one or more CDRs thereof), antibody V L All or part of the chain (e.g., V L In some of these embodiments, the antigen binding domain is an scFv. In some of these embodiments, the scFv linker domain comprises a V L and V H Peptide linkers between components. For example, scFv can be designed so that V L The C-terminus of the domain is connected to the V H The N-terminal end of the domain ((N)V L (C)-Connector-(N)V H (C)), or make V H The C-terminus of the domain is connected to the V L N-terminal (N)V H (C)-Connector-(N)V L (C). Exemplary linkers include those having a glycine-serine amino acid chain comprising Gly x Ser y , wherein x and y are each independently an integer from 0 to 10, provided that x and y are not simultaneously 0 (e.g., (Gly 4 Ser) 2 ;(Gly 3 Ser) 2 ;Gly 2 Ser; or a combination thereof, such as (Gly 3Ser) 2 Gly 2 Ser).

[0147] Anti-B7-H3 CAR-T binding domain sequences useful in the present technology are described, for example, in PCT / US2020 / 66002 entitled “Chimeric Antigen Receptors Targeting B7-H3 (CD276) and Associated Methods” and PCT / US2019 / 61887 entitled “Monoclonal antibodies against IgV domain of B7-H3 and uses thereof”, the techniques and sequences of which are incorporated herein by reference in their entirety.

[0148] In some embodiments, the binding domain specifically binds to one or more tumor-associated antigens selected from the group consisting of: HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptide bound to HLA; hTERT peptide bound to HLA; tyrosinase peptide bound to HLA; WT-1 peptide bound to HLA; LTβ R; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME; and / or SSX-2 antigens.

[0149] The sources of binding domains are known in the art, including known antibodies, methods for generating antibodies, and binding domains described herein. In some embodiments, the tumor-associated antigen is CD19. In particular embodiments, the binding domain is derived from an anti-CD19 antibody, such as, for example, FMC-63 antibody, MOR208, blinatumomab, MEDI-551, Meck patent anti-CD19 antibody, Xmab5871, or MDX-1342.

[0150] In some embodiments, the antigen-specific receptor binding domain is derived from FMC-63 antibody, MOR208, blinatumomab, MEDI-551, Merck's proprietary anti-CD19 antibody, Xmab5871 or MDX-1342, having a VH or (i.e., and / or) VL having at least about 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more amino acid sequence identity with the antibody variable region or its scFv from FMC-63 antibody, MOR208, blinatumomab, MEDI-551, Merck's proprietary anti-CD19 antibody, Xmab5871 or MDX-1342, or having a CDR or functional CDR variant according to any of these antibodies.

[0151] In some embodiments, the antigen binding domain is directly adjacent to the transmembrane domain. In other embodiments, there are one or more intermediate residues connecting the antigen binding domain and the transmembrane domain, such as a hinge region. In certain embodiments in which CAR comprises a hinge region, the hinge region is derived from CD8α, and in some of these embodiments, the hinge region comprises, consists of, or is substantially composed of: SEQ ID NO: 2 or a portion thereof of an amino acid sequence, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2.

[0152] In some embodiments, the extracellular region further comprises a leader sequence, which includes but is not limited to a leader peptide. For example, the leader peptide can be an IgG1 V K The leader domain or a portion or variant thereof, which is in contact with the V H Domain or V L The N-terminal end of the domain binds.

[0153] Specific implementation plan

[0154] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain and (b) an intracellular CD3 zeta effector domain or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD3 zeta effector domain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:6, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4. NO:6 or a portion thereof having a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some of these embodiments, the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain are directly adjacent to each other. In other embodiments, there are one or more amino acids separating the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 of SEQ ID NO:3 or a portion thereof and the amino acid sequence shown in SEQ ID NO:6. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 52-162 of SEQ ID NO:5 or a portion thereof and the amino acid sequence shown in SEQ ID NO:6. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-278 of SEQ ID NO:4 or a portion thereof and the amino acid sequence shown in SEQ ID NO:6.

[0155] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain and (b) a CD8α transmembrane region or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD8α transmembrane region comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4 NO: The sequence shown in 1 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In certain embodiments, the CD8α transmembrane region is directly adjacent to the TMIGD2 co-stimulatory domain, and in other embodiments, there are one or more amino acids separating the CD8α transmembrane region from the TMIGD2 co-stimulatory domain. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 or a portion thereof of SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 52-162 or a portion thereof of SEQ ID NO:5 and the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-278 or a portion thereof of SEQ ID NO:4 and the amino acid sequence shown in SEQ ID NO:1.

[0156] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, and (c) a CD8α transmembrane region or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD3 zeta effector domain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:6, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4. NO:6 or a portion thereof having a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and (c) the CD8α transmembrane region comprises, consists of or consists essentially of the following: the amino acid sequence shown in SEQ ID NO:1 or a portion thereof, or a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or a portion thereof. In certain embodiments, the TMIGD2 costimulatory domain is located between the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the CD3ζ effector domain is located between the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the CD8α transmembrane region is directly adjacent to the CD3ζ effector domain or the TMIGD2 costimulatory domain, and in other embodiments, there are one or more amino acids separating the CD8α transmembrane region from the CD3ζ effector domain or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, and in other embodiments, there are one or more amino acids separating the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 of SEQ ID NO:3 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence shown in SEQ ID NO:1.In certain embodiments, the CAR provided herein comprises the amino acid sequence set forth in residues 52-162 of SEQ ID NO: 5 or a portion thereof, the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the CAR provided herein comprises the amino acid sequence set forth in residues 172-278 of SEQ ID NO: 4 or a portion thereof, the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence set forth in SEQ ID NO: 1.

[0157] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, (c) a CD8α transmembrane region or a portion or variant thereof, and (d) a CD8α hinge region or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD3 zeta effector domain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:6, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4. NO:6 or a portion thereof having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (c) the CD8α transmembrane region comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and (d) the CD8α hinge region comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. NO:2 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In certain embodiments, the TMIGD2 costimulatory domain is located between the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the CD3ζ effector domain is located between the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the CD8α transmembrane region is directly adjacent to the CD3ζ effector domain or the TMIGD2 costimulatory domain, and in other embodiments, there are one or more amino acids separating the CD8α transmembrane region and the CD3ζ effector domain or the TMIGD2 costimulatory domain.Similarly, in certain embodiments, the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more amino acids separating the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain. In certain embodiments, the CD8α hinge region is directly adjacent to the CD8α transmembrane region. In other embodiments, there are one or more amino acids separating the hinge region and the transmembrane region. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 of SEQ ID NO:3 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:2. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 52-162 of SEQ ID NO:5 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:2. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-278 of SEQ ID NO:4 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:2.

[0158] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain, (b) a CD8α transmembrane region or a portion or variant thereof, and (c) a CD8α hinge region. In certain of these embodiments, the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; (b) the CD8α transmembrane region comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; NO:1 or a portion thereof having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and (c) the CD8α hinge region comprises, consists of or consists essentially of the following: the amino acid sequence shown in SEQ ID NO:2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 or a portion thereof. In certain embodiments, the CD8α transmembrane region is directly adjacent to the TMIGD2 costimulatory domain, while in other embodiments, there are one or more amino acids separating the CD8α transmembrane region from the TMIGD2 costimulatory domain. In certain embodiments, the CD8α hinge region is directly adjacent to the CD8α transmembrane region. In other embodiments, there are one or more amino acids separating the hinge region from the transmembrane region. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 of SEQ ID NO:3 or a portion thereof, the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:2. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 52-162 of SEQ ID NO:5 or a portion thereof, the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:2.In certain embodiments, the CAR provided herein comprises the amino acid sequence set forth in residues 172-278 of SEQ ID NO:4, or a portion thereof, the amino acid sequence set forth in SEQ ID NO:1, and the amino acid sequence set forth in SEQ ID NO:2.

[0159] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, (c) a CD8α transmembrane region or a portion or variant thereof, and (d) an extracellular region comprising an antigen binding domain. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD3 zeta effector domain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:6, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4. NO:6 or a portion thereof having a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (c) the CD8α transmembrane region comprises, consists of or consists essentially of the following: the amino acid sequence shown in SEQ ID NO:1 or a portion thereof, or a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or a portion thereof; and (d) the antigen binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the TMIGD2 costimulatory domain is located between the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the CD3ζ effector domain is located between the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the CD8α transmembrane region is directly adjacent to the CD3ζ effector domain or the TMIGD2 costimulatory domain, while in other embodiments, there are one or more amino acids separating the CD8α transmembrane region from the CD3ζ effector domain or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more amino acids separating the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain.Likewise, in certain embodiments, the antigen binding domain is directly adjacent to the CD8α transmembrane region, while in other embodiments, there are one or more amino acids separating the antigen binding domain from the CD8α transmembrane region. In certain of these embodiments, the amino acids separating the antigen binding domain from the CD8α transmembrane region comprise a hinge region, and in certain of these embodiments, the hinge region is a CD8α hinge region comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0160] In certain embodiments, the CAR provided herein comprises (a) a TMIGD2 co-stimulatory domain, (b) a CD8α transmembrane region or a portion or variant thereof, and (c) an extracellular region comprising an antigen binding domain. In certain of these embodiments, (a) the TMIGD2 costimulatory domain comprises, consists of, or consists essentially of the amino acid sequence set forth in residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4, or a portion thereof; and (b) the CD8α transmembrane region comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or residues 172-278 of SEQ ID NO:4. NO: 1 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and (c) the antigen binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the CD8α transmembrane region is directly adjacent to the TMIGD2 costimulatory domain, while in other embodiments, there are one or more amino acids separating the CD8α transmembrane region from the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the antigen binding domain is directly adjacent to the CD8α transmembrane region, while in other embodiments, there are one or more amino acids separating the antigen binding domain from the CD8α transmembrane region. In some of these embodiments, the amino acids separating the antigen binding domain from the CD8α transmembrane region comprise a hinge region, and in some of these embodiments, the hinge region is a CD8α hinge region comprising, consisting of, or consisting essentially of the amino acid sequence shown in SEQ ID NO:2 or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 or a portion thereof.

[0161] In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-282 of SEQ ID NO:3 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence shown in SEQ ID NO:2, and an antigen binding domain. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 52-162 of SEQ ID NO:5 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence shown in SEQ ID NO:2, and an antigen binding domain. In certain embodiments, the CAR provided herein comprises the amino acid sequence shown in residues 172-278 of SEQ ID NO:4 or a portion thereof, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence shown in SEQ ID NO:2, and an antigen binding domain.

[0162] Table 1. Exemplary CAR components

[0163]

[0164]

[0165] *The intracellular regions of TMIGD2 isoforms 1, 2, and 3 are underlined.

[0166] Nucleic acid molecules, vectors and cells

[0167] In certain embodiments, provided herein are nucleic acid molecules encoding any one or more of the CARs provided herein, and compositions and vectors comprising these nucleic acid molecules. Also provided herein are cells comprising these nucleic acid molecules or vectors and compositions thereof, and unit doses of these cells and compositions. In some embodiments, these unit doses include (i) a composition comprising at least about 50% modified CD4+T cells, and (ii) a composition comprising at least about 50% modified CD8+T cells in combination, with a ratio of about 1: 1, wherein the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells.

[0168] TMIGD2 co-stimulatory domain

[0169] In certain embodiments, the nucleic acid molecules provided herein encode a CAR comprising a TMIGD2 co-stimulatory domain derived from TMIGD2 isoform 1 (SEQ ID NO: 9), isoform 2 (SEQ ID NO: 10), or isoform 3 (SEQ ID NO: 11).

[0170] In certain embodiments, the nucleic acid molecules provided herein encode residues 172-282 of SEQ ID NO:3, or a portion thereof, residues 52-162 of SEQ ID NO:5, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 172-282 of SEQ ID NO:3 or residues 52-162 of SEQ ID NO:5, or a portion thereof. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9 or nucleotides 154-489 of SEQ ID NO:11, or a portion thereof.

[0171] In certain embodiments, the nucleic acid molecules provided herein encode residues 172-278 of SEQ ID NO: 4, or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of nucleotides 514-837 of SEQ ID NO: 10, or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0172] Intracellular effector domain

[0173] In certain embodiments, the nucleic acid molecules provided herein encode CARs comprising an intracellular effector domain. In some embodiments, the intracellular effector domain is a CD3 zeta effector domain or a functional portion or variant thereof.

[0174] In certain embodiments, the nucleic acid molecules provided herein encode the amino acid sequence of SEQ ID NO:6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6 or a portion thereof. In certain of these embodiments, the nucleic acid molecules comprise, consist of or consist essentially of the nucleotide sequence shown in SEQ ID NO:12 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO:12 or a portion thereof.

[0175] Transmembrane region

[0176] In certain embodiments, the nucleic acid molecules provided herein encode CARs comprising a transmembrane domain. In some embodiments, the transmembrane domain is a CD8 alpha transmembrane domain or a variant thereof.

[0177] In certain embodiments, the nucleic acid molecules provided herein encode the amino acid sequence of SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In certain of these embodiments, the nucleic acid molecule comprises, consists of or consists essentially of the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0178] Extracellular region

[0179] In certain embodiments, the nucleic acid molecules provided herein include a nucleic acid sequence encoding an extracellular region. In some of these embodiments, the extracellular region includes an antigen binding domain, and in some of these embodiments, the antigen binding domain specifically binds to a tumor-associated antigen selected from the following: HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptide bound to HLA; hTERT peptide bound to HLA; tyrosinase peptide bound to HLA; WT-1 peptide bound to HLA; LTβ R; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME; and / or SSX-2 antigens.

[0180] In certain embodiments, the extracellular region also includes a hinge region connecting the antigen binding domain to the transmembrane domain. In some of these embodiments, the hinge region is a CD8α hinge region. In certain embodiments, the nucleic acid molecules provided herein encode SEQ ID NO:2 or a portion thereof of the CD8α hinge region or with SEQ ID NO:2 or a portion thereof having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity. In some of these embodiments, the nucleic acid molecule comprises the following, is composed of or is substantially composed of: SEQ ID NO:8 or a portion thereof shown in the nucleotide sequence, or with SEQ ID NO:8 having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity.

[0181] Markers and self-cleaving peptides

[0182] In certain embodiments, the nucleic acid molecules provided herein include nucleotide sequences encoding markers such as GFP and / or EGFRt. In some of these embodiments, the nucleic acid molecule encodes an EGFRt encoding marker of SEQ ID NO: 15 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15 or a portion thereof. In some of these embodiments, the nucleic acid molecule includes, consists of or is substantially composed of the nucleotide sequence shown in SEQ ID NO: 18 or a portion thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 18.

[0183] In certain embodiments, the nucleic acid molecules provided herein comprise a nucleotide sequence encoding a self-cleaving peptide such as a 2A peptide. Exemplary 2A peptides are P2A and F2A.

[0184] In some of these embodiments, the nucleic acid molecule encodes a P2A self-cleaving peptide of SEQ ID NO: 13 or a portion thereof or SEQ ID NO: 14 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 or a portion thereof or SEQ ID NO: 14 or a portion thereof. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of a nucleotide sequence as set forth in SEQ ID NO: 16 or a portion thereof or SEQ ID NO: 17 or a portion thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 16 or SEQ ID NO: 17.

[0185] In certain embodiments, the nucleic acid molecules provided herein comprising nucleotide sequences encoding markers such as GFP and / or EGFRt are linked to signal leader sequences. An exemplary sequence is the GMCSFRα chain signal sequence, which directs surface expression, linked to EGFRt.

[0186] Specific implementation plan

[0187] In certain embodiments, the nucleic acid molecules provided herein encode (a) TMIGD2 costimulatory domain and (b) intracellular CD3ζ effector domain or a portion or variant thereof. In certain of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO:10 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO:10 or a portion thereof; and (b) SEQ ID NO:12 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO:10 or a portion thereof; NO: 12 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain and the nucleic acid sequence encoding the CD3 zeta effector domain are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD3 zeta effector domain. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0188] In certain embodiments, the nucleic acid molecules provided herein encode (a) TMIGD2 costimulatory domain and (b) CD8α transmembrane region or a portion thereof, or a variant or a portion thereof or a variant thereof. In certain of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO:10 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO:10 or a portion thereof; and (b) SEQ ID NO:7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-837 of SEQ ID NO:10 or a portion thereof; The nucleotide sequence shown in ID NO:7 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain and the nucleic acid sequence encoding the CD8α transmembrane region are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD8α transmembrane region. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, SEQ ID NO: 16 and SEQ ID NO: 18 both or a portion thereof, or SEQ ID NO: 17 and SEQ ID NO: 18 both or a portion thereof.

[0189] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, and (c) a CD8α transmembrane region or a portion or variant thereof, or a portion or variant thereof. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a portion thereof; (b) SEQ ID NO:12, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, NO:12 or a portion thereof, the nucleotide sequence shown has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and (c) SEQ ID NO:7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:7 or a portion thereof. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the nucleic acid sequence encoding the CD3ζ effector domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the nucleic acid sequence encoding the CD8α transmembrane region is directly adjacent to the nucleic acid sequence encoding the CD3ζ effector domain or the TMIGD2 costimulatory domain, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain.In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or portions thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or portions thereof.

[0190] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, (c) a CD8α transmembrane region or a portion or variant thereof or a portion or variant thereof, and (d) a CD8α hinge region or a portion or variant thereof. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a portion thereof; (b) SEQ ID NO:12, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, NO:12 or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in NO:12 or a portion thereof; (c) SEQ ID NO:7 or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:7 or a portion thereof; and (d) SEQ ID NO:8 or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:8 or a portion thereof. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the nucleic acid sequence encoding the CD3ζ effector domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the nucleic acid sequence encoding the CD8α transmembrane region is directly adjacent to the nucleic acid sequence encoding the CD3ζ effector domain or the TMIGD2 costimulatory domain, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the CD8α transmembrane region and the CD3ζ effector domain or the TMIGD2 costimulatory domain.Similarly, in certain embodiments, the nucleic acid sequences encoding the TMIGD2 co-stimulatory domain and the CD3 ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the TMIGD2 co-stimulatory domain and the intracellular CD3 ζ effector domain. In certain embodiments, the nucleic acid sequence encoding the CD8 α hinge region is directly adjacent to the nucleic acid sequence encoding the CD8 α transmembrane region, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the hinge region and the transmembrane region. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, SEQ ID NO: 16 and SEQ ID NO: 18 both or a portion thereof, or SEQ ID NO: 17 and SEQ ID NO: 18 both or a portion thereof.

[0191] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) a CD8α transmembrane region or a portion or variant thereof or a portion or variant thereof, and (c) a CD8α hinge region or a portion or variant thereof. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a portion thereof; (b) SEQ ID NO:7, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, NO:7 or a portion thereof, the nucleotide sequence shown has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and (c) SEQ ID NO:8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:8 or a portion thereof. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the nucleic acid sequence encoding the CD3ζ effector domain is located between the nucleic acid sequence encoding the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the nucleic acid sequence encoding the CD8α transmembrane region is directly adjacent to the nucleic acid sequence encoding the CD3ζ effector domain or the TMIGD2 costimulatory domain, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain. In certain embodiments, the nucleic acid sequence encoding the CD8α hinge region is directly adjacent to the nucleic acid sequence encoding the CD8α transmembrane region, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the hinge region and the transmembrane region.In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or portions thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or portions thereof.

[0192] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) an intracellular CD3ζ effector domain or a portion or variant thereof, (c) a CD8α transmembrane region or a portion or variant thereof or a portion or variant thereof, and (d) an extracellular region comprising an antigen binding domain. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a portion thereof; (b) SEQ ID NO:12, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, NO:12 or a portion thereof, the nucleotide sequence shown has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (c) SEQ ID NO:7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:7 or a portion thereof; and (d) the antigen binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain is located between the nucleic acid sequences encoding the CD8α transmembrane region and the CD3ζ effector domain. In other embodiments, the nucleic acid sequence encoding the CD3ζ effector domain is located between the nucleic acid sequences encoding the CD8α transmembrane region and the TMIGD2 costimulatory domain. In certain embodiments, the nucleic acid sequence encoding the CD8α transmembrane region is directly adjacent to the nucleic acid sequence encoding the CD3ζ effector domain or the TMIGD2 costimulatory domain, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the CD8α transmembrane region and the CD3ζ effector domain or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD3ζ effector domain are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 costimulatory domain and the intracellular CD3ζ effector domain.Likewise, in certain embodiments, the antigen binding domain is directly adjacent to the CD8α transmembrane region, while in other embodiments, there are one or more nucleotides separating the antigen binding domain from the CD8α transmembrane region. In certain of these embodiments, the nucleotides separating the antigen binding domain from the CD8α transmembrane region comprise a hinge region, and in certain of these embodiments, the hinge region is a CD8α hinge region comprising, consisting of, or consisting essentially of the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or portions thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or portions thereof.

[0193] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) a CD8α transmembrane region or a portion or variant thereof or a portion or variant thereof, and (c) an extracellular region comprising an antigen binding domain. In some of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, nucleotides 154-489 of SEQ ID NO:11, or nucleotides 514-837 of SEQ ID NO:10, or a portion thereof; (b) SEQ ID NO:7, or a portion thereof, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 514-849 of SEQ ID NO:9, or a portion thereof, The nucleotide sequence shown in NO:7 or a portion thereof has a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and (c) the antigen binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 costimulatory domain and the nucleic acid sequence encoding the CD8α transmembrane region are directly adjacent to each other, while in other embodiments, there are one or more nucleotides separating the nucleic acid sequences encoding the TMIGD2 costimulatory domain and the CD8α transmembrane region. In certain embodiments, the antigen binding domain is directly adjacent to the CD8α transmembrane region, while in other embodiments, there are one or more nucleotides separating the antigen binding domain from the CD8α transmembrane region. In some of these embodiments, the nucleotides separating the antigen binding domain from the CD8α transmembrane region comprise a hinge region, and in some of these embodiments, the hinge region is a CD8α hinge region comprising, consisting of, or consisting essentially of the nucleotide sequence shown in SEQ ID NO:8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO:8 or a portion thereof.In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or portions thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or portions thereof.

[0194] In certain embodiments, the nucleic acid molecules provided herein comprise the nucleotide sequence shown in nucleotides 514-849 of SEQ ID NO:9 or a portion thereof, the nucleotide sequence shown in SEQ ID NO:12, the nucleotide sequence shown in SEQ ID NO:7, the nucleotide sequence shown in SEQ ID NO:8, and a nucleic acid sequence encoding an antigen binding domain. In certain embodiments, the nucleic acid molecules provided herein comprise the nucleotide sequence shown in nucleotides 514-837 of SEQ ID NO:10 or a portion thereof, the nucleotide sequence shown in SEQ ID NO:12, the nucleotide sequence shown in SEQ ID NO:7, the nucleotide sequence shown in SEQ ID NO:8, and a nucleic acid sequence encoding an antigen binding domain. In certain embodiments, the nucleic acid molecules provided herein comprise the nucleotide sequence shown in nucleotides 154-489 of SEQ ID NO:11 or a portion thereof, the nucleotide sequence shown in SEQ ID NO:12, the nucleotide sequence shown in SEQ ID NO:7, the nucleotide sequence shown in SEQ ID NO:8, and a nucleic acid sequence encoding an antigen binding domain.

[0195] Table 2. Exemplary CAR components

[0196]

[0197]

[0198]

[0199] *Nucleic acids encoding the intracellular regions of TMIGD2 isoforms 1, 2 and 3 are underlined.

[0200] Table 3. Exemplary lytic peptides and encoded markers

[0201]

[0202]

[0203]

[0204] In any of the embodiments described herein, the nucleic acid molecule encoding the CAR can be codon optimized for a specific cell using known techniques (Scholten et al., 2006). For example, OptimumGene TM Tools for codon optimization. Codon-optimized sequences include partially or fully codon-optimized sequences.

[0205] The nucleic acid molecules encoding the CAR of the present disclosure can be inserted into expression vectors such as viral vectors to be transduced into cells such as T cells. In some embodiments, the expression constructs of the present disclosure include nucleic acid molecules encoding the CAR provided herein and optionally also encoding a self-cleaving peptide and / or EGFRt marker operably connected to an expression control sequence such as a promoter.

[0206] In certain embodiments, the nucleic acid molecules of the present disclosure can be operably linked to certain elements of the vector. For example, a polynucleotide sequence can be operably linked that is required to affect the expression and processing of the coding sequence to which it is linked. Expression control sequences can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and sequences that may enhance protein secretion. If the expression control sequence is adjacent to the target gene and the expression control sequence controls the target gene in trans or at a distance, they can be operably linked.

[0207] In certain embodiments, the expression construct is contained in a vector that can be integrated into the genome of the cell or promote the integration of the nucleic acid molecule insert after introduction into the cell and thereby replicate with the genome of the cell, such as a viral vector. Viral vectors include retroviruses, adenoviruses, parvoviruses, coronaviruses, negative-strand RNA viruses, positive-strand RNA viruses, and double-stranded DNA viruses. (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, 3rd edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0208] The construction of expression vectors for genetic engineering and production of target CARs can be achieved by using any suitable molecular biological engineering techniques known in the art. In order to obtain efficient transcription and translation, the polynucleotides in each recombinant expression construct include at least one appropriate expression control sequence, such as a leader sequence, particularly a promoter operably linked to a nucleotide sequence encoding an immunogen. The method for manufacturing the CAR disclosed herein is described in, for example, U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014 / 031687; U.S. Patent No. 7,514,537; Brentjens et al., 2007; and Walseng et al., 2017; its technology is incorporated herein by reference.

[0209] In certain embodiments, nucleic acid molecules of the present disclosure are used for transfection / transduction cells, such as T cells, NK cells, macrophages or another immune cell, for use in adoptive transfer therapy. Cells can be induced to incorporate carriers or other materials using viral vectors, conversion via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection or other methods. (Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989)). T cells and / or NK cells can be collected using known techniques, and various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding with antibodies, flow cytometry or immunomagnetic selection. In certain embodiments, T cells are CD4+T cells, CD8+T cells, CD4-CD8- double negative T cells, naive T cells, central memory T cells, effector memory T cells, stem cell memory T cells or any combination thereof. Methods for transfecting / transducing T cells with polynucleotides have been previously described (U.S. Patent Application Publication No. US2004 / 0087025) and adoptive transfer procedures using T cells with desired target specificity (Schmitt et al., 2009; Dossett et al., 2009; Till et al., 2008; Wang et al., 2007; Kuball et al., 2007; Leen et al., 2007; U.S. Patent Publication No. 2011 / 0243972; U.S. Patent Publication No. 2011 / 0189141), and therefore it is within the scope of the present disclosure to apply these methods to the CARs currently disclosed in the present disclosure.

[0210] T cell and / or NK cell activity can be determined according to any method accepted in the art to perform functional characterization of CAR described herein, including determining T cell and / or NK cell binding, activation or induction, and also including determining antigen-specific T cell and / or NK cell responses.Examples include determining intracellular calcium, T cell proliferation, T cell and / or NK cell cytokine release, antigen-specific T cell and / or NK cell stimulation, MHC-restricted T cell and / or NK cell stimulation, cytotoxic activity, changes in T cell and / or NK cell phenotypic marker expression, phosphorylation of certain T cell and / or NK cell proteins, and other measurements of T cell and / or NK cell function.Procedures for carrying out these and similar determinations are described herein and / or can be found in, for example, Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and references cited therein.

[0211] Reagent test kit

[0212] In some embodiments, a kit is provided, comprising (a) a CAR vector as disclosed herein, (b) a CAR nucleic acid molecule polynucleotide as disclosed herein optionally encoding a marker peptide and / or a self-cleaving peptide, and / or (c) one or more reagents for transducing a vector or nucleic acid molecule into a cell. In certain embodiments, the kit also includes instructions for use.

[0213] How to use

[0214] The disclosure also provides a method for treating a disease or condition, wherein the method includes applying an effective amount of the present disclosure composition, cell or unit dose to a subject in need, wherein the disease or condition expresses an antigen specifically bound by CAR provided herein or otherwise is associated with an antigen specifically bound by CAR provided herein. In certain embodiments, the disease or condition is hyperproliferative or proliferative disease, such as cancer, autoimmune disease or infectious disease (e.g., virus, bacteria, fungus or parasite).

[0215] In certain embodiments, the subject treated using the methods provided herein is a human. In other embodiments, the subject is a non-human animal, such as in a veterinary or medical research setting. In those embodiments in which the subject is a human, the subject can be male or female and can be of any suitable age, including infants, teenagers, youth, adults, and elderly subjects. Cells according to the present disclosure can be administered in a manner suitable for the disease, condition, or illness to be treated as determined by a person skilled in the art of medicine.

[0216] The compositions, cells or unit doses of the present disclosure can be administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into the lymph nodes or into the cerebrospinal fluid to encounter the target antigen or cell. The appropriate dose, suitable duration and frequency of administration of the composition will be determined by factors such as the patient's condition; body size; type and severity of the disease, condition or disorder; undesirable type or level or activity of the marker cells, the specific form of the active ingredient; and the method of administration.

[0217] In some embodiments, the disease or condition is a malignant tumor. In some embodiments, the malignant tumor is cancer. In general, cancers that can be treated by the currently disclosed methods and compositions include carcinomas, sarcomas, gliomas, lymphomas, leukemias, myeloma, head and neck cancer, melanomas, pancreatic cancer, bile duct cancer, hepatocellular carcinoma, breast cancer, gastric cancer, non-small cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small cell lung cancer, colorectal cancer, glioblastoma, Askin tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft tissue sarcoma, angiosarcoma, phyllodes cystosarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid tumors, desmofibroma, Hyperplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, angiosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, cystic gastrocnemius, vipoma, bile duct carcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, ependymoma, astrocytoma, oligodendroglioma Glioma, brain stem glioma, optic nerve glioma, mixed glioma, Hodgkin lymphoma, B cell lymphoma, non-Hodgkin lymphoma (NHL), Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma, Waldenstrom's macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, mediastinal (thymic) large B cell lymphoma sarcoma; angiosarcoma (fibroblastic sarcoma); dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing's sarcoma; gastrointestinal stromal tumor; leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi's sarcoma; liposarcoma; pleomorphic sarcoma; or synovial sarcoma; lung cancer (e.g., adenocarcinoma, squamous cell carcinoma (epidermoid carcinoma); squamous cell carcinoma; adenocarcinoma; adenosquamous carcinoma; anaplastic carcinoma; large cell carcinoma; small cell carcinoma;Breast cancer (e.g., ductal carcinoma in situ (non-invasive), lobular carcinoma in situ (non-invasive), invasive ductal carcinoma, invasive lobular carcinoma, non-invasive carcinoma); liver cancer (e.g., hepatocellular carcinoma, cholangiocarcinomas or biliary duct cancer); large cell undifferentiated carcinoma, bronchoalveolar carcinoma); ovarian cancer (e.g., surface epithelial-stromal tumors (adenocarcinomas) or ovarian epithelial carcinomas (which include serous tumors, endometrioid tumors, and mucinous cystadenocarcinomas), epidermoid (squamous cell carcinoma), embryonal carcinoma, and choriocarcinoma (germ cell tumor)); kidney cancer (e.g., renal adenocarcinoma, adrenal tumor, transitional cell carcinoma (renal pelvis), squamous cell carcinoma, Bellini duct carcinoma, clear cell adenocarcinoma, transitional cell carcinoma, renal pelvic carcinoid tumor ); adrenal cancer (e.g., adrenocortical carcinoma), testicular cancer (e.g., germ cell cancer (seminoma, choriocarcinoma, embryonal carcinoma, teratoma), serous carcinoma); gastric cancer (e.g., adenocarcinoma); intestinal cancer (e.g., duodenal adenocarcinoma); colorectal cancer; or skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma); ovarian cancer, ovarian epithelial carcinoma, cervical adenocarcinoma or small cell carcinoma, pancreatic cancer, colorectal cancer (e.g., adenocarcinoma or squamous cell carcinoma), lung cancer, breast ductal carcinoma, or prostate adenocarcinoma. ;

[0218] In some embodiments, the cancer is one or more of: prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, stomach cancer, thyroid cancer, anal cancer, bone cancer, cervical cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, thymic cancer, sarcoma, and osteosarcoma.

[0219] In some embodiments, the cancer comprises a hematological malignancy.

[0220] In some embodiments, the disease or condition is a "hyperproliferative disorder" and a "proliferative disorder". In some embodiments, the hyperproliferative disorder and proliferative disorder are one or more of a tumor, a cancer, a neoplastic tissue, a carcinoma, a sarcoma, a malignant cell, a premalignant cell. In some embodiments, the cancer comprises a solid tumor.

[0221] In certain embodiments, the methods provided herein include administering a cell expressing a CAR disclosed herein, a composition comprising the cell, or a unit dose thereof. The amount of cells in the composition is at least one cell (e.g., a CAR-modified CD8+T cell subset; a CAR-modified CD4+T cell subset; a CAR-modified NK cell subset) or more typically greater than 10 2 cells, e.g., up to 10 6 , up to 10 7 , up to 10 8cells, up to 10 9 cells, or 10 10 cells or more, such as about 10 11 Cells / m 2 In certain embodiments, the cells administered are about 10 5 to about 10 11 Cells / m 2 in the range of about 10 5 or about 10 6 to about 10 9 or about 10 10 Cells / m 2 The number of cells will depend on the intended end use of the composition and the type of cells contained therein. For example, cells modified to contain CARs specific for one or more antigens will constitute a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more such cells. For the purposes provided herein, the volume of the cells is typically 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the density of the desired cells is typically greater than 10 4 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 cells / ml or greater. The cells can be administered as a single infusion or as multiple infusions over a certain time frame. Clinically relevant numbers of immune cells can be distributed to cumulatively equal to or greater than 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 In any of the presently disclosed embodiments, the cells are allogeneic cells, syngeneic cells, or autologous cells.

[0222] Also contemplated are pharmaceutical compositions comprising cells expressing CAR as disclosed herein and pharmaceutically acceptable carriers, diluents and / or excipients. Suitable excipients include water, saline, dextrose, glycerol, etc. and combinations thereof. In embodiments, the composition comprising cells as disclosed herein further comprises a suitable infusion medium.

[0223] Pharmaceutical compositions can be applied in a manner suitable for the disease or condition to be treated (or prevented) as determined by medical field technicians. The appropriate dose and suitable duration and frequency of application of the composition will be determined by factors such as the patient's health, the patient's body shape (i.e., weight, mass or body surface area), the type and severity of the patient's condition, the bad type or level or activity of the marker cells, the specific form of the active ingredient and the method of administration. In general, appropriate dosage and treatment regimen provide one or more compositions in an amount sufficient to provide treatment and / or prevention benefits (such as described herein, including improved clinical results, such as more frequent complete or partial remission or longer disease-free period and / or total survival period or the reduction of symptom severity). For preventive use, the dose should be sufficient to prevent, delay the onset of the disease related to the disease or condition or reduce its severity. The preventive benefits of the immunogenic composition administered according to the method described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained thereby by appropriate statistics, biology and clinical methods and techniques, all of which can be easily practiced by those skilled in the art.

[0224] Certain treatments or prevention methods covered herein include administering cells (which may be autologous, allogeneic or isogenic) comprising desired polynucleotides as described herein, which are stably integrated into the chromosomes of cells. For example, autologous, allogeneic or isogenic immune system cells (e.g., T cells, antigen presenting cells, NK cells) can be used to generate such cell compositions in vitro, so as to administer the desired, CAR-expressing T cell compositions to the subject as adoptive immunotherapy. In certain embodiments, the cell is a hematopoietic progenitor cell or a human immune cell. In certain embodiments, the immune system cell is a CD4+T cell, a CD8+T cell, a CD4-CD8-double negative T cell, a NK cell, or any combination thereof. In certain embodiments, the immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, a NK cell, or any combination thereof. In a particular embodiment, the cell is a CD4+T cell. In a particular embodiment, the cell is a CD8+T cell. In a particular embodiment, the cell is a NK cell.

[0225] As used herein, the administration of a composition refers to delivering it to a subject, regardless of the route or mode of delivery. Administration can be performed continuously or intermittently, or parenterally. Administration can be used to treat subjects who have been confirmed to have an identified condition, disease, or morbidity, or to treat subjects who are susceptible to or at risk of such a condition, disease, or morbidity. Co-administration with adjuvant therapy may include simultaneous and / or sequential delivery of multiple agents (e.g., CAR expressing recombinant (i.e., engineered) cells with one or more cytokines; immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low doses of mycophenolic acid prodrugs, or any combination thereof) in any order and any dosing schedule.

[0226] In certain embodiments, multiple doses of cells as described herein are administered to a subject, which may be administered at intervals of about two weeks to about four weeks between administrations.

[0227] In yet further embodiments, the subject receiving treatment is also receiving immunosuppressive therapy, such as a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low dose of mycophenolic acid prodrug, or any combination thereof. In yet further embodiments, the subject receiving treatment has received a non-myeloablative or myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two months to at least three months after the non-myeloablative hematopoietic cell transplant.

[0228] An effective amount of a pharmaceutical composition (e.g., a cell, a CAR, a unit dose, or a composition) refers to an amount sufficient to achieve a desired clinical outcome or beneficial treatment at a desired dose and for a desired period of time, as described herein. An effective amount can be delivered in one or more administrations.

[0229] According to the method disclosed herein, it may also include administering one or more additional agents to treat a disease or condition with a combination therapy. For example, in certain embodiments, the combination therapy includes administering CAR (or an engineered cell expressing it) and (contemporaneously, simultaneously or sequentially) an immune checkpoint inhibitor. In some embodiments, the combination therapy includes administering an agonist of a CAR (or an engineered cell expressing it) and a stimulatory immune checkpoint agent of the present disclosure. In a further embodiment, the combination therapy includes administering a CAR (or an engineered cell expressing it) of the present disclosure and a second therapy, such as a chemotherapeutic agent, radiotherapy, surgery, an antibody, or any combination thereof.

[0230] Cytokines are used to manipulate host immune responses to exert anti-cancer activity (see, e.g., Floros and Tarhini, 2015). Cytokines that can be used to promote immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-24, IFN-γ, TNF-ɑ, and GM-CSF, alone or in any combination with the binding proteins of the present disclosure or cells expressing the same.

[0231] Various embodiments of the present technology are described above. It should be understood that the details set forth above are provided to describe the embodiments in a manner sufficient to enable a person skilled in the relevant art to manufacture and use the disclosed embodiments. However, some details and advantages may not be necessary for practicing some embodiments. In addition, some well-known structures or functions may not be shown or described in detail to avoid unnecessarily obscuring the relevant descriptions of various embodiments. Although some embodiments may be within the scope of the present technology, they may not be described in detail in conjunction with the drawings. In addition, the features, structures or characteristics of various embodiments may be combined in any suitable manner. Moreover, those skilled in the art will recognize that there are many other technologies that can be used to perform functions similar to those described above. Although the process or block is presented in a given order, the alternative embodiment can perform routines with stages or adopt systems with blocks in different orders, and can delete, move, add, subdivide, combine and / or modify some processes or blocks. Each of these processes or blocks can be implemented in various different ways. In addition, although the process or block is sometimes shown as being executed in series, these processes or blocks can be executed in parallel instead or can be executed at different times. The titles provided herein are only for convenience and do not explain the scope or meaning of the described technology.

[0232] Various embodiments of the present invention are set forth in paragraphs 201 to 253 below:

[0233] 201. A chimeric antigen receptor (CAR), comprising an extracellular region containing an antigen binding domain, a transmembrane region, and an intracellular region comprising an effector domain and a TMIGD2 co-stimulatory domain.

[0234] 202. The CAR of paragraph 201, wherein the TMIGD2 co-stimulatory domain comprises the intracellular region of TMIGD2.

[0235] 203. The CAR of paragraph 202, wherein the TMIGD2 co-stimulatory domain comprises a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5.

[0236] 204. The CAR of paragraph 203, wherein the TMIGD2 co-stimulatory domain comprises an amino acid sequence that is at least 75% identical to a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5.

[0237] 205. The CAR of any of paragraphs 201-204, wherein the antigen binding domain specifically binds to a tumor-associated antigen.

[0238] 206. The CAR of paragraph 205, wherein the tumor-associated antigen is selected from HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptide bound to HLA; hTERT peptide bound to HLA; tyrosinase peptide bound to HLA; WT-1 peptide bound to HLA; LT βR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME; and SSX-2 antigens.

[0239] 207. The CAR of any of paragraphs 201-206, wherein the antigen binding domain comprises a scFv.

[0240] 208. The CAR of any one of paragraphs 201-207, wherein the antigen binding domain comprises a linker.

[0241] 209. The CAR of paragraph 208, wherein the linker is a glycine-serine linker.

[0242] 210. The CAR of paragraph 209, wherein the glycine-serine linker comprises (GlyxSery)z, wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0, and z is an integer from 1 to 10.

[0243] 211. The CAR of any one of paragraphs 201-210, wherein the extracellular region further comprises an N-terminal leader sequence.

[0244] 212. The CAR of any one of paragraphs 201-211, wherein the extracellular region further comprises a hinge region.

[0245] 213. The CAR of paragraph 212, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:2.

[0246] 214. The CAR of paragraph 213, wherein the hinge region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:2.

[0247] 215. The CAR of any one of paragraphs 201-214, wherein the transmembrane region comprises a CD8α transmembrane region.

[0248] 216. The CAR of paragraph 215, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:1.

[0249] 217. The CAR of paragraph 215, wherein the transmembrane region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:1.

[0250] 218. The CAR of any one of paragraphs 201-217, wherein the effector domain is a CD3ζ effector domain.

[0251] 219. The CAR of paragraph 218, wherein the effector domain comprises the amino acid sequence shown in SEQ ID NO:6.

[0252] 220. The CAR of paragraph 218, wherein the effector domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:5.

[0253] 221. The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence shown in SEQ ID NO:1.

[0254] 222. The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 6.

[0255] 223. The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence shown in SEQ ID NO:1; and (c) the sequence shown in SEQ ID NO:6.

[0256] 224. The CAR of paragraph 222 or 223, wherein the CAR further comprises the sequence shown in SEQ ID NO:2.

[0257] 225. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR of any one of paragraphs 201-223.

[0258] 226. A vector comprising a nucleic acid sequence encoding the CAR of any one of paragraphs 201-223.

[0259] 227. The vector of paragraph 226, wherein the nucleic acid sequence encoding CAR is operably linked to an expression control sequence.

[0260] 228. The vector of paragraph 227, wherein the expression control sequence is a promoter.

[0261] 229. The vector of any of paragraphs 226-228, further comprising a nucleic acid sequence encoding a self-cleaving peptide.

[0262] 230. The vector of paragraph 229, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0263] 231. The vector of paragraph 230, wherein the 2A self-cleaving peptide is a P2A peptide.

[0264] 232. The vector of any of paragraphs 226-231, further comprising a nucleic acid sequence encoding a transduction marker polypeptide.

[0265] 233. The vector of paragraph 232, wherein the transduction marker polypeptide is a truncated form of epidermal growth factor receptor (EGFRt) or GFP, or a portion or variant thereof.

[0266] 234. The vector of any one of paragraphs 230-233, wherein the nucleic acid sequence encoding the self-cleaving peptide is on the 3′ side of the nucleic acid sequence encoding the CAR.

[0267] 235. The vector of paragraph 233 or 234, wherein the vector comprises a nucleic acid sequence encoding a self-cleaving peptide, and wherein the nucleic acid sequence encoding the self-cleaving peptide is on the 5' side of the nucleic acid sequence encoding the marker polypeptide.

[0268] 236. The vector of any of paragraphs 226-235, wherein the vector is a viral vector.

[0269] 237. An isolated cell expressing the CAR of any of paragraphs 201-225.

[0270] 238. The cell of paragraph 237, wherein the cell comprises the nucleic acid molecule of paragraph 218.

[0271] 239. The cell of paragraph 237 or 238, wherein the cell comprises the vector of any one of paragraphs 225-234.

[0272] 240. The cell of any of paragraphs 237-239, wherein the cell is a T cell, a natural killer (NK) cell, a macrophage or other immune cell.

[0273] 241. The cell of paragraph 240, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a NK cell, a macrophage, other immune cell, or any combination thereof.

[0274] 242. The cell of paragraph 240, wherein the T cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, a NK cell, a macrophage, other immune cell, or any combination thereof.

[0275] 243. The cell of any of paragraphs 237-242, wherein the cell further expresses a transduction marker on its surface.

[0276] 244. The cell of paragraph 243, wherein the transduction marker is a truncated form of epidermal growth factor receptor (EGFRt) or GFP, or a portion or variant thereof.

[0277] 245. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the cells of any of paragraphs 237-244.

[0278] 246. The method of paragraph 245, wherein the disease or condition is a malignancy.

[0279] 247. The method of paragraph 246, wherein the malignancy is cancer.

[0280] 248. The method of paragraph 247, wherein the cancer is selected from prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, stomach cancer, small intestine cancer, bone cancer, cervical cancer, endometrial cancer, eye cancer, gallbladder cancer, thyroid cancer, thymic cancer, sarcoma and osteosarcoma.

[0281] 249. The method of paragraph 247 or 248, wherein the cancer comprises a solid tumor.

[0282] 250. The method of any of paragraphs 247-249, wherein the cancer comprises a hematological malignancy.

[0283] 251. A method of eliciting an immune response in a subject against a tumor-associated antigen that specifically binds to the CAR of any of paragraphs 201-225, comprising administering to the subject an effective amount of the cells of any of paragraphs 237-244.

[0284] 252. A composition comprising the CAR of any one of paragraphs 201-225 and a pharmaceutically acceptable excipient, carrier or diluent.

[0285] 253. A composition comprising the cell of any of paragraphs 237-244 and a pharmaceutically acceptable excipient, carrier or diluent.

[0286] Any patents, applications, and other references cited herein are incorporated herein by reference.Aspects of the described technology can be modified, if necessary, to employ the systems, functions, and concepts of the various references described above to provide yet further embodiments.

[0287] These and other changes can be made in light of the above detailed description. Although the above description details certain embodiments and describes the best mode contemplated, various changes can be made no matter how detailed. Implementation details may vary greatly and still be covered by the technology disclosed herein. As described above, the use of a particular term in describing certain features or aspects of the technology should not be taken as implying that the term is redefined herein to be limited to any particular characteristic, feature, or aspect of the technology associated with the term.

[0288] The foregoing is intended only to illustrate various embodiments of the present invention. The specific modifications discussed above should not be construed as limiting the scope of the present invention. It is apparent to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present invention, and it should be understood that such equivalent embodiments will be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

[0289] The following examples illustrate several implementations of the present technology:

[0290] Example

[0291] Example 1: CAR-T cell therapy using CAR-T cells containing TMIGD2 as a co-stimulatory molecule

[0292] The following examples demonstrate the generation of a novel CAR-T vector containing a cytoplasmic tail of TMIGD2 (transmembrane and immunoglobulin domain containing 2), wherein the CAR-T vector is capable of transducing normal T cells and killing human tumor cells.

[0293] Generation of CAR-T vectors with TMIGD2

[0294] CAR-T vectors using TMIGD2 as a co-stimulatory molecule have been developed ( Figure 1 ). The novel vector contains antibody signal leader, VH, VL, human CD8a hinge and transmembrane region, human TMIGD2 intracellular tail, human CD3ζ, P2A, human GMCSFR signal leader and human EGFRt.

[0295] TGMID2 CAR-T-mediated killing of human tumor cells

[0296] As a proof of principle, the VH and VL of the anti-human CD19 mAb were cloned into Figure 1 The TMIGD2 vector shown in the figure was used to perform a CAR-T-mediated human tumor cell killing experiment. T cells from normal PBMCs were transfected with the TMIGD2 vector to generate CAR-T cells. Human Raji tumor cells expressing the CD19 antigen were then incubated alone, with untransduced T cells, and with CAR-T-transduced T cells. The results showed that CAR-T cells (but not untransduced T cells) killed almost all Raji tumor cells ( Figure 2 ), indicating that CAR-T cells containing the TMIGD2 co-stimulatory domain can efficiently kill tumor cells.

[0297] In vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells

[0298] This example demonstrates the in vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells in the Raji lymphoma model. TM Mice were injected intravenously (IV) with 0.25 x 10^6 Raji tumor cells and 10^7 untransduced T cells or CD19-TMIGD2 CAR-T cells on days 3 and 10, respectively. The survival of the mice was monitored. Figure 3 As shown in , the survival rate of mice receiving CD19-TMIGD2 CAR-T cells was significantly improved compared with non-transduced control mice.

[0299] Example 2: CAR-T cell therapy using anti-B7-H3 CAR-T cells containing TMIGD2 as a co-stimulatory molecule Law

[0300] The following examples demonstrate the generation of anti-B7-H3 CAR-T cells incorporating a vector containing a cytoplasmic tail of TMIGD2 (containing transmembrane and immunoglobulin domain 2), wherein the CAR-T vector transduces normal T cells and kills human tumor cells expressing B7-H3 in vitro and in vivo.

[0301] Experimental methods

[0302] Cell lines

[0303] NIH 3T3 cells (mouse fibroblasts) were obtained from the American Type Culture Collection (ATCC); NSO cells (mouse multiple myeloma) were obtained from the Department of Cell Biology, Albert Einstein College of Medicine, HEK293T cells (human renal epithelial) were obtained from the Department of Cell Biology, Albert Einstein College of Medicine, U118 cells (human glioblastoma) were obtained from ATCC, HCC827 cells (human lung adenocarcinoma) were obtained from ATCC, THP-1 cells (human acute monocytic leukemia) were obtained from ATCC, Raji cells (B-cell Burkitt's lymphoma) were obtained from ATCC, Jurkat (NFAT) cells (T-cell leukemia) were obtained from BPS Bioscience, AsPC-1 cells (pancreatic adenocarcinoma) were obtained from ATCC, PANC-1 cells (pancreatic ductal epithelioid carcinoma) were obtained from ATCC, and Phoenix-AMPHO cells (human renal epithelial) were obtained from ATCC. Cell lines were grown in DMEM or RPMI 1640 medium supplemented with 10% FBS, 1% penicillin and 1% streptomycin and cultured in a humidified incubator at 37°C and 5% CO2.

[0304] Transfection of tumor cell lines

[0305] Phoenix-AMPHO cells were co-transfected with MSCV-YFP plasmid and pCMV-VSV-G plasmid containing the protein of interest (mouse B7-H3, human B7-H3, cynomolgus monkey B7-H3 or luciferase-tdTomato (-Luc)). 48 and 72 hour viral supernatants were collected and used to transfect tumor cell lines using polybrene. Transfected cells were sorted at least twice using a BD FACS Aria II cell sorter to ensure pure populations.

[0306] Generation of anti-B7-H3 monoclonal antibodies

[0307] C57BL / 6 mice were immunized with a recombinant protein consisting of a fusion of the IgV domain of human B7-H3 with a human Fc fragment. After immunization, hybridomas were generated by fusing NSO myeloma cells and mouse spleen cells using standard methods. Hybridomas producing antibodies were screened by flow cytometry to ensure specific binding to mouse and human B7-H3. Thereafter, the hybridomas were subcloned three times by single cell dilution and then amplified in DMEM high glucose medium (supplemented with 10% ultra-low IgG FBS, 10% NCTC-109, 1% penicillin, 1% streptomycin, and 1% non-essential amino acids) in the cell compartment of a bioreactor flask. The culture medium compartment of the bioreactor flask contained DMEM high glucose medium supplemented with 1% penicillin and 1% streptomycin. Antibody supernatants from the cell compartment were collected and stored at 4°C until purified using a Protein G resin packed column. Purified B7-H3 mAbs were analyzed using SDS-PAGE prior to affinity determination, isotype determination, and VH and VL sequencing.

[0308] B7-H3 mAb affinity assay

[0309] The affinity of anti-B7-H3 mAbs to mouse and human B7-H3 was determined by biolayer interferometry. Recombinant mouse and human B7-H3-Fc proteins were loaded onto mouse or human capture biosensors, respectively. Thereafter, the protein-loaded biosensors were placed in solutions containing serial dilutions of anti-B7-H3 mAbs. Kon, Koff, and KD were determined analytically using a 1:1 binding model.

[0310] Generation of CAR constructs

[0311] The anti-B7-H3 single-chain variable fragment (scFv) was generated by cloning the native mAb signal peptide sequence into the VH and VL regions of the anti-B7-H3 mAb connected by a G4S linker. The scFv was linked to the hinge and transmembrane domains of human CD8ɑ, the intracellular regions of multiple co-stimulatory proteins (CD28, 4-1BB, TMIGD2, CD28-4-1BB, or TMIGD2-4-1BB), and then the intracellular domain of human CD3ζ. The self-cleaving P2A peptide sequence was then inserted, followed by a signal peptide from the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR)-ɑ chain and a truncated human epidermal growth factor receptor (hEGFRt) protein for CAR detection. For in vivo CAR-T cell persistence experiments, the hEGFRt protein was followed by the self-cleaving T2A peptide sequence, followed by the firefly luciferase gene sequence. The entire CAR sequence was cloned into the pLVX-Zsgreen lentiviral expression plasmid under the control of the EF1ɑ promoter. The commercially available CD19-CD28-4-1BB CAR was also modified to include the P2A-EGFRt and T2A-luciferase sequences.

[0312] Production of CAR lentivirus and determination of viral titer

[0313] HEK293T cells were co-transfected with psPAX packaging plasmid, pMD2.G envelope plasmid and CAR plasmid. Viral supernatants at 48 and 72 hours were collected, concentrated 100 times, and then pooled to ensure equivalent titers. Viral titers were determined by transducing activated human T cells with CAR lentivirus as described below. Viral titers were calculated based on EGFR+T cells.

[0314] Isolation of human T cells

[0315] Leukopaks were obtained from healthy human donors and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Lymphoprep. T cells were purified by negative enrichment. T cells were either frozen in freezing medium and stored in liquid nitrogen or used immediately.

[0316] Generation of CAR-T cells

[0317] Fresh or thawed T cells were activated for 24 hours in CTS OpTmizer medium on 24-well plates coated with OKT3 (1ug / mL) and CD28 (1ug / mL) antibodies, which were supplemented with OpTmizer T cell expansion supplements, 10% FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin, IL-7 and IL-15. The activated T cells were then transduced onto non-tissue culture treated plates coated with RetroNectin reagent (19ug / mL) and CAR lentivirus (MOI of approximately 10). The CAR-T cells were then expanded for at least 7 days before being used in the experiment. Before all experiments, the CAR-T cell transduction efficiency was standardized based on 50% CAR+ cells (for in vitro cytotoxicity screening assays) or the lowest efficiency donor by adding untransduced T cells. If necessary, CAR-T cells were purified using anti-phycoerythrin (PE) microbeads after anti-EGFR-PE staining.

[0318] In vitro co-culture killing assay

[0319] 0.1x10 6 CAR-T cells and HCC827-Luc (0.01x10 6 cells), U118-Luc (0.01x10 6 cells) or THP-1 (0.01x10 6 5x10 cells) were seeded in T cell culture medium without added cytokines. After 3-5 days, tumor cells were counted by flow cytometry. For the Incucyte time-lapse cytotoxicity assay, 5x10 3 The tumor-coated plates were then placed in the Incucyte machine and imaged every 4 hours at 4 locations in each well for a total of 112 hours. 16 hours after initial imaging, 0.1x10 6 CAR-T cells. Imaging data were analyzed using Incucyte software.

[0320] Flow cytometry

[0321] Cells were stained using antibodies conjugated to the following fluorophores: fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), APC-Fire 750, PE-cyanine (Cy) 7, peridinin-chlorophyll-protein (PerCP)-Cy5.5, brilliant violet (BV)-421, BV-711, alexa fluor (AF)-532, AF-488, brilliant ultra violet (BUV)-496, BUV-395, BUV-496, and BUV-737. Expression of tdTomato and YFP was also used to distinguish cell populations. Antibody targets included CD45, CD3, CD4, CD8, CD45RA, CCR7, EGFR, G4S linker, PD-1, TIM-3, LAG-3, B7-H3, CD69, and CD33. 7-aminoactinomycin D (7-AAD), zombie NIR, ghost violet 510, and 4',6-diamidino-2-phenylindole (DAPI) were used to determine viability. In some experiments, cells were fixed with 2% paraformaldehyde (PFA) solution before analysis. All samples were acquired using BD LSRII or Cytek Aurora flow cytometers. Data analysis was performed on FlowJo (or SpectroFlo). t-distributed stochastic neighbor embedding (t-SNE) plots were generated using the t-SNE plugin on FlowJo.

[0322] Flow cytometry cytokine analysis

[0323] 0.4x10 6 CAR-T cells and 0.1x10 6 HCC827 tumor cells were co-cultured in 24-well plates for 24 h. Supernatants from co-cultures were collected and stored at -80°C until analysis. The concentrations of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF-ɑ were determined using the LEGENDplex Human Th Cytokine Panel Kit for flow cytometry-based supernatant samples at undiluted or 1:5 dilution. Data were analyzed using the LEGENDplex online data analysis software suite.

[0324] RNA Isolation

[0325] For in vitro RNA separation, CAR-T cells were gently removed from the co-culture wells and placed in fresh wells for 30 minutes to allow excess tumor cells to attach to the plate. CAR-T cells were gently removed and live cells were enriched using Lymphoprep density gradient centrifugation. CD3+ selection beads were used to further purify and separate T cells. RNA was extracted and stored at -80 ° C. RNA sequencing (40 million double-end reads) after library preparation (using NEBNext Ultra II kit) was performed by Admera Health. For in vivo RNA separation, lung-infiltrating T cells were isolated from mouse lungs. In brief, single cell suspensions were prepared from lungs by enzymatic digestion (collagenase IV (200 IU.mL), dispase (0.5 IU / mL) and DNase I (100 U / mL) in RPMI 1640 medium). Next, the single cell suspension was layered on a discontinuous density gradient Percoll solution (40% and 80%), and immune cells at the 40% 80% interface were collected. Lymphoprep was used for a second density gradient centrifugation to further enrich the live cell population. The mouse cells were then depleted twice. Thereafter, T cells from the same donor transduced with the same CAR were pooled and RNA was isolated as described above. RNA sequencing (40 million double-end reads) after library preparation (using SMARTseq V4 and NExtera XT kits) was performed by Admera Health.

[0326] B7-H3 CAR and CD19 CAR RNA sequencing analysis

[0327] Reads from RNA sequencing were aligned to the reference human genome (hg38; downloaded from the UCSC genome browser in November 2019) using STAR alignment software (version 2.6.1b). Gene mapping fragments were counted using HTseq software (version 0.6.1). Genes with an average expression count of 1 or more in any group were considered expressed. Differentially expressed genes (DEGs) were determined by an adjusted p-value of <0.05 and a log2 fold change of >0.5. Principal component analysis and differential expression analysis were then performed using DESeq2 software (version 3.10). Gene set enrichment analysis (GSEA) was performed using the Hallmark, Kegg, and Reactome databases, using a ranked gene list determined by multiplying the -log10 p-value with the sign of the log2 (fold change).

[0328] In vitro and in vivo RNA sequencing analysis of B7-H3 CAR

[0329] The double-end reads from RNA sequencing were aligned with human reference genome (hg38) using STAR aligner (version 2.7.9a).Then, the alignment data was used by RSEM software (version 1.3.3) to quantify the expression level of individual genes (coding and non-coding) in GENCODE annotation (version 41), so as to obtain estimated read counts and transcripts per million (TPM).Genes with TPM<1 in all samples were excluded from further analysis.Differentially expressed genes (DEGs) were determined by adjusted p-values ​​of <0.05 and log2 fold changes of >0.5.Then principal component analysis (PCA) and differential expression analysis were performed using DESeq2 software (version 1.38.3).After gene sorting by multiplying the sign of -log10 (p-value) with log2 (fold change) for each gene, gene set enrichment analysis (GSEA) was performed using Hallmark, KEGG and Reactome gene sets. Gene sets with a false discovery rate (FDR) < 0.05 were considered enriched. Overrepresentation analysis was also performed using cluster.Profiler (version 4.6.2) enrichGO function for DEGs or significantly differentially expressed genes (adjusted p-value < 0.05). Pathways with adjusted p-value < 0.05 were considered enriched in up-regulated or down-regulated genes. Gene sets describing T cell dysfunction and metabolism were obtained from the literature, and a list of inhibitory proteins was manually curated.

[0330] Seahorse Metabolic Assay

[0331] 0.1x10 6 CAR-T cells were cultured alone or with 5x10 3 HCC827 tumor cells were co-cultured for one or seven days. CAR-T cells were collected from the wells and transferred to new wells for 30 minutes to allow attachment of any tumor cells. CAR-T cells were re-collected and analyzed on Seahorse PDL-coated plates using the Seahorse T Cell Metabolic Analysis Kit. After transfer to the PDL-coated plate, the CAR-T cells were allowed to rest for 5 minutes and then centrifuged to allow the cells to be evenly distributed on the bottom of the well. The Seahorse plate was run on a Seahorse XF96 analyzer at baseline and after injection of oligomycin A, Bam15, and rotenone / antimycin A. Data were analyzed using Agilent Seahorse Analytics online software. % ATP from mitochondria was obtained by the following formula: 100% - % ATP from glycolysis.

[0332] Jurkat (NFAT) T cell activation assay

[0333] Jurkat (NFAT) cells were transduced with CAR constructs as described above. OKT3 (1 μg / mL) or 0.2x10 5 Tumor cells (U118, HCC827 or AsPC-1) were coated overnight in triplicate wells of a 96-well plate. 0.2 x 10 5 Raji tumor cells. Add 0.1x10 6 Untransduced or CAR-transduced Jurkat (NFAT) cells were incubated for 6 hours. Jurkat (NFAT) luciferase was detected using the Bio-Glo luciferase assay system. Luciferase signals were acquired on a plate reader.

[0334] Chronic antigen exposure

[0335] Use T cell culture medium without cytokines to transfer 0.2x10 6 CAR-T cells were seeded in triplicate wells of a 96-well plate. 0.1x10 5 HCC827 tumor cells. Every 3-4 days, the CAR-T cells were gently removed from the well and transferred to a new well. A small aliquot of the cells was then analyzed using flow cytometry. The unanalyzed CAR-T cells were centrifuged and the extra supernatant was removed so that half of the original volume remained in the well. An equal volume of 0.1x10 5 This operation was repeated for 17 days.

[0336] Mouse

[0337] For all in vivo experiments, 8-12 week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were used. NSG mice were purchased from Jackson Laboratory and maintained at the Albert Einstein College of Medicine. Mice were housed in a specific pathogen-free animal facility with free access to food and water under a 12 h light / dark cycle. All experimental procedures were approved by the Albert Einstein Institutional Animal Care and Use Committee.

[0338] Bioluminescence imaging

[0339] Bioluminescence imaging was performed using an IVIS Spectrum in vivo imaging system and analyzed using Living Image software (version 3.0). Images were acquired 10-15 minutes after intraperitoneal (IP) injection of D-luciferin (150 μg / g mouse body weight).

[0340] In vivo lung cancer model

[0341] 0.5x10 6 HCC827-Luc cells were injected IV into the lateral tail vein of NSG mice in 100 μL sterile PBS. Three days later, tumor engraftment was confirmed via IVIS imaging and the experimental groups were normalized to ensure equivalent baseline tumor burden. Then, 10x10 6 The researchers injected 10 CAR-T cells into the ovary and then injected them again one week later. Tumor burden was followed via IVIS imaging over 100 days. Survival was also followed during this period.

[0342] In vivo glioblastoma model

[0343] U118-Luc tumor cells were cultured at 5×10 3 The cells were resuspended in sterile PBS at a concentration of 10 cells / μL. Using an 18-gauge needle, a small hole was drilled manually 1 mm lateral and 1 mm anterior to the bregma in the skull of NSG mice anesthetized with continuous isoflurane (2%). 2 μL of tumor cells were injected into the hole perpendicular to the workbench surface using a blunt-end Hamilton syringe equipped with a sterile pre-cut pipette tip, with 1 mm of the syringe needle exposed from the pipette tip within one minute. Meloxicam (5 mg / kg) was administered preemptively and two days after tumor implantation. After 7 days, tumor engraftment was confirmed using IVIS imaging. The mice were then assigned to experimental groups that were standardized for baseline tumor burden. Thereafter, 1x10 6 CAR-T cells. Tumor burden and survival were tracked via IVIS imaging over 100 days.

[0344] In vivo pancreatic cancer model

[0345] PANC-1-Luc tumor cells were cultured at 1x10 4 The pancreas was expressed by making a 1 cm incision in the skin and muscle tissue of NSG mice. 50 μL of tumor cells were injected into the pancreatic head using a 27-gauge needle. Meloxicam was administered preemptively and three days after tumor implantation. Seven days later, tumor engraftment was confirmed using IVIS imaging. Mice were then assigned to experimental groups that were normalized for baseline tumor burden. 10x10 6 CAR-T cells were injected once more 7 days later. Tumor burden and survival were tracked via IVIS imaging over 100 days.

[0346] In vivo CAR-T cell persistence model

[0347] 0.5x106 Three days later, a single dose of 10x10 6 CAR-Luciferase T cells were imaged over 46 days.

[0348] Isolation of immune cells from mouse blood and organs

[0349] To obtain immune cells from mouse blood, blood was taken from the lateral tail vein and placed in a heparinized capillary. Red blood cells (RBC) were then lysed. The remaining cells were then washed twice with PBS before flow cytometry analysis. The mice were euthanized and the organ of interest was removed directly (spleen) or perfused with PBS and then removed (lungs). The spleen was then transferred to a C tube and mechanically separated. The cell suspension was filtered through a 40 μM filter. To obtain immune cells from the lungs, a single cell suspension was prepared using enzymatic digestion and immune cells were separated using a discontinuous Percoll gradient. RBCs were removed using RBC lysis buffer.

[0350] statistics

[0351] Statistical analysis was performed using GraphPad prism software (version 9.4.1). As described in each figure legend, unpaired two-tailed Student's t test, one-way ANVOA, Kruskal-Wallis test, two-way ANOVA or Mantel-Cox log-rank test were used to test between groups. Data are shown in the form of a single value or a single value and mean ± SEM. P values ​​less than 0.05 were considered to be statistically significant.

[0352] Generation and validation of anti-B7-H3 monoclonal antibodies

[0353] Six anti-B7-H3 mAb clones were obtained, denoted as 1G5, 15F9, 23B2, 8B12, 12B4 and 24D12, which were measured using biolayer interferometry (BLI) to bind to mouse and human B7-H3 with an affinity ranging from 0.32nM to 11.08nM. All anti-B7-H3 mAbs that bind to mouse and human B7-H3 were stably expressed on 3T3 cells by flow cytometry. In addition, each anti-B7-H3 mAb was tested to confirm that each can bind to non-human primate cynomolgus monkey B7-H3. By flow cytometry, all anti-B7-H3 mAbs were able to detect cynomolgus monkey B7-H3 stably expressed on 3T3 cells. Anti-B7-H3 mAbs can recognize human, mouse and cynomolgus monkey B7-H3.

[0354] In vitro screening of anti-B7-H3 CAR-T cells

[0355] The anti-B7-H3 CAR was constructed using a single-chain variable fragment (scFv) derived from the anti-B7-H3 mAb clone 8B12 by sequentially linking the scFv to the hinge and transmembrane (H / TM) domains of human CD8α, the intracellular domains of multiple co-stimulatory proteins, and the intracellular domain of CD3ζ. Following the CD3ζ sequence, a self-cleaving P2A peptide and a truncated human epidermal growth factor receptor (hEGFRt) ( Figure 4A ). The hEGFRt protein was modified by removing two of the four extracellular domains and all intracellular domains to prevent antigen binding and signaling. Since it can still be recognized by anti-EGFR antibodies, it serves as a marker for transduction efficiency. The following co-stimulatory domains were used in the CAR constructs: CD28 (B7-H3.28.ζCAR), 4-1BB (B7-H3.BB.ζCAR), TMIGD2 (B7-H3.TMI.ζCAR), CD28-4-1BB (B7-H3.28.BB.ζCAR) or TMIGD2-4-1BB (B7-H3.TMI.BB.ζCAR). Similarly, anti-CD19 CAR was generated using anti-CD19 scFv, and the CD28-4-1BB co-stimulatory domain was used as an irrelevant target control (CD19.28.BB.ζ)( Figure 4A On primary human T cells, all CAR constructs were efficiently expressed, as determined by expression of hEGFRt protein, with transduction efficiencies generally above 85% CAR+, and no differences in memory phenotype or expansion between CAR-T cell constructs ( Figure 4H -K).

[0356] The in vitro anti-tumor response of B7-H3 CAR-T cells against a variety of B7-H3+ tumor types was tested by flow cytometry. The CAR-T cell transduction efficiency was reduced to 50% using untransduced T cells to better reflect the transduction efficiency seen in other clinical trials. U118 glioblastoma (GBM) and HCC827 lung cancer cell lines were stably transfected with plasmids expressing luciferase and tdTomato (-Luc) protein to allow tumor cell differentiation. B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells exhibited tumor cell killing of U118-Luc cells ( Figure 4B ). B7-H3.28.ζ, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed tumor cell killing against HCC827-Luc cells ( Figure 4C). To confirm that our CAR is also effective against non-solid tumors, we also examined whether our CAR-T cells can target the THP-1 acute monocytic leukemia (AML) cell line. B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and B7-H3.TMI.BB.ζ CAR-T cells showed significant tumor cell killing against THP-1 cells ( Figure 4D Despite the wide variation between donors, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were consistently found to exhibit anti-tumor responses in all cell lines tested.

[0357] Time-lapse imaging cytotoxicity assays were performed to determine whether there were differences in tumor cell killing kinetics between the two CARs by repeatedly imaging U118-Luc or HCC827-Luc tumor cells alone or co-cultured with B7-H3.TMI.ζ, B7-H3.28.BB.ζ, or CD19.28.BB.ζ CAR-T cells. The growth of live tumor cells was tracked based on tdTomato signaling and morphological exclusion of dead cells. For U118-Luc tumor cells, control CD19.28.BB.ζ CAR-T cells exhibited similar tumor growth as tumor cells alone, while both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells eliminated tumor cells ( Figure 4E This finding was replicated in HCC827-Luc co-cultures ( Figure 4F ).

[0358] The cytokine release properties of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T were evaluated using a multiplex flow cytometry bead assay measuring the following cytokines: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF-ɑ. After co-culture with HCC827 tumor cells, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed significantly higher cytokine release in all cytokines tested compared to control CD19.28.BB.ζ CAR-T cells (Figure 5—figure supplement 1A). Figure 4G ). Except for IL-6, B7-H3.TMI.ζCAR-T cells released significantly fewer cytokines than B7-H3.28.BB.ζCAR-T cells.

[0359] These results indicate that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells exhibit equivalent cytotoxic responses and killing kinetics in vitro. Although the cytokine secretion properties of these CAR-T cells are similar, B7-H3.TMI.ζ CAR-T cells mostly secrete lower amounts of these cytokines compared to B7-H3.28.BB.ζ CAR-T cells.

[0360] B7-H3.TMI.ζCAR-T cells exhibit anti-tumor responses in vivo

[0361] The in vivo anti-tumor response of the lead B7-H3 CAR, B7-H3.TMI.ζ and B7-H3.28.BB.ζ and the control CD19.28.BB.ζ were evaluated in three solid tumor models. All tumor cell lines (HCC827, U118 and PANC-1) were stably transfected with plasmids containing luciferase and tdTomato (-Luc) to allow in vivo tracking by bioluminescent imaging. CAR B7-H3.TMI.ζ and B7-H3.28.BB.ζ were tested in a metastatic lung cancer model. HCC827-Luc tumor cells were injected intravenously (IV) into NSG mice, and then B7-H3.TMI.ζ, B7-H3.28.BB.ζ or control CD19.28.BB.ζ CAR-T cells were injected IV three days and ten days later ( Figure 5A ). B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells reduced tumor burden and showed increased overall survival compared with CD19.28.BB.ζ CAR-T ( Figures 5B-5D There were no significant differences in tumor cell signaling or survival between the two B7-H3CAR-T cells ( Figures 5B-5D ).

[0362] CAR-T cell therapy was explored in an orthoptic GBM model. U118-Luc cells were injected intracranially into the right cerebral hemisphere of NSG mice, followed by intratumoral (IT) injection of CAR-T cells seven days later. Figure 5E ), because intratumorally injected CAR-T cells have been shown to have superior anti-tumor responses than IV-injected CAR-T cells at equivalent low doses. Compared with CD19.28.BB.ζCAR-T cells, both B7-H3.TMI.ζ and B7-H3.28.BB.ζCAR-T cells showed anti-tumor responses against U118-Luc cells ( Fig. 5F and 5G There was no difference in tumor burden between B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells ( Fig. 5F and 5G Although both B7-H3 CAR-T cells improved overall survival compared with CD19 CAR-T cells, the survival results of B7-H3.TMI.ζCAR-T cells were superior to those of B7-H3.28.BB.ζCAR-T cells ( Figure 5H In mice treated with B7-H3.28.BB.ζCAR-T cells, no significant tumor burden was observed before death, indicating that the tumor was not the cause of death in this group of mice.

[0363] CAR-T cell therapy was explored in an orthotopic model of pancreatic cancer. PANC-1-Luc tumor cells were injected orthotopically into the pancreas of NSG mice, followed by IV injection of CAR-T cells seven and fourteen days later. Fig.5I Both B7-H3 CAR-T cells reduced tumor burden and improved overall survival compared with control CD19.28.BB.ζCAR-T cells ( Figures 5J to 5L ). B7-H3.TMI.ζCAR-T cells had a smaller tumor burden compared to B7-H3.28.BB.ζCAR-T cells, but this only reached statistical significance on day 21 ( Figure 5K Similarly, there was a trend toward improved survival in B7-H3.TMI.ζCAR-T cells compared with B7-H3.28.BB.ζCAR-T cells, with four of seven mice treated with B7-H3.TMI.ζCAR-T cells still alive 100 days after tumor cell injection, compared with only one of seven mice treated with B7-H3.28.BB.ζCAR-T cells.

[0364] In summary, these experiments showed that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells exhibited antitumor responses against multiple tumor models in vivo. In addition, B7-H3.TMI.ζ CAR-T cells showed equivalent or superior results in a tumor-dependent manner.

[0365] B7-H3.TMI.ζCAR-T cells persist in vivo

[0366] Since B7-H3 CAR-T cells were determined to be cytolytic in vivo in multiple tumor models, the expansion and persistence of B7-H3 CAR-T cells in vivo were examined. The original CAR construct was modified to include a self-cleaving T2A peptide followed by luciferase (CAR-Luc) to allow in vivo CAR-T cell tracking using bioluminescent imaging ( Fig. 6A). NSG mice were injected with parental HCC827 cells that do not express luciferase, followed by a single subtherapeutic IV injection of B7-H3.TMI.ζ-Luc, B7-H3.28.BB.ζ-Luc, or CD19.28.BB.ζ-Luc CAR-T cells ( Figure 6B ). At day 7, CAR-Luc signals were detectable in all constructs and did not differ between any groups; starting from day 21, both B7-H3CAR-Luc T cells showed significantly higher signals than CD19 CAR-Luc T cells; however, by day 46, B7-H3.TMI.ζ-Luc but not B7-H3.28.BB.ζ-Luc CAR-T cells showed significantly higher CAR-Luc signals than CD19.28.BB.ζ-Luc CAR-T cells, suggesting that B7-H3.TMI.ζ-Luc CAR-T cells can persist longer in vivo than B7-H3.28.BB.ζ-Luc CAR-T cells, although the latter did approach statistical significance ( Figures 6C to 6E ).

[0367] Examination of the peak CAR-Luc signal from each mouse throughout the experiment revealed that B7-H3.TMI.ζ-Luc and B7-H3.28.BBζ-Luc CAR-T cells showed equivalent expansion above that of CD19.28.BB.ζ-Luc CAR-T cells ( Fig. 6F Examination of the number of T cells in the lungs, spleen, and blood of CAR-Luc treated mice at the end of the experiment revealed that B7-H3-CAR-Luc T cells were present in greater numbers than CD19.28.BB.ζ-Luc CAR-T cells, and that the numbers were comparable between the B7-H3 CAR-Luc T cell constructs ( Figures 6G to 6I ). Taken together, these data suggest that both B7-H3 CAR Luc-T cells can expand and persist in vivo in an antigen-dependent manner. In addition, B7-H3.TMI.ζ-Luc CAR-T cells showed moderately improved persistence at later time points compared to B7-H3.28.BB.ζ-Luc CAR-T, which may be due to the persistence of CAR in locations other than the lungs, spleen, and blood.

[0368] B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells exhibit transcriptome differences in vitro

[0369] RNA sequencing was performed to compare B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells with CD19.28.BB.ζ CAR-T cells after 24 h of in vitro co-culture with HCC827 tumor cells. Both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed extensive transcriptome differences compared with CD19.28.BB.ζ CAR-T cells ( Fig. 7A ). 307 shared differentially expressed genes (DEGs) and 198 and 153 unique DEGs were found between B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells ( Figure 7B ). Gene set enrichment analysis (GSEA) revealed many enriched pathways between the two B7-H3 CAR-T cells and CD19 CAR-T cells, with high similarity in terms of the pathways expressed, but differences in their enrichment order ( Figure 7C Interestingly, among the most enriched pathways, the “oxidative phosphorylation” pathway in B7-H3.TMI.ζCAR-T cells was found to be comparable to the “glycolysis” pathway in B7-H3.28.BB.ζCAR-T cells ( Figure 7C ), which suggests metabolic differences between these two CAR constructs.

[0370] The Seahorse T cell metabolic profiling assay was used to measure changes in % ATP generated from glycolysis and mitochondria after co-culture and at baseline (cultured in the absence of tumor cells) to validate the mechanistic findings. In this assay, the sum of % ATP generated from glycolysis and % ATP generated from mitochondria is 100%, so these values ​​are interdependent. Upon acute 24-hour stimulation, B7-H3.TMI.ζCAR-T cells had lower % ATP generated from glycolysis at baseline and after co-culture compared to B7-H3.28.BB.ζCAR-T cells. Fig.7D , left panel) and a concomitant higher %ATP generated from mitochondria ( Fig.7D , right). After chronic stimulation with repeated addition of tumor cells, B7-H3.TMI.ζCAR-T cells showed no difference in % ATP generated from glycolysis or mitochondria at baseline ( Fig. 7E , left), but did show a lower %ATP generated from glycolysis and a concomitant higher %ATP generated from mitochondria after co-culture ( Fig. 7E , right). It was observed that B7-H3.TMI.ζCAR-T cells maintained their metabolic characteristics, while B7-H3.28.BB.ζCAR-T cells moderately increased their glycolytic energy consumption, accompanied by a decrease in mitochondrial energy consumption ( Fig.7D and 7E). These functional metabolic assays reproduced the results of RNA sequencing experiments.

[0371] To directly compare B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells, additional RNA sequencing experiments were performed on RNA isolated after 72 h of co-culture with HCC827 tumor cells. Extensive transcriptome differences were found between these two B7-H3 CARs, with a total of 1328 DEGs identified ( Figure 7F ). GSEA analysis revealed that four metabolic pathways were negatively enriched in B7-H3-TMIGD2 CAR-T cells, including "oxidative phosphorylation", "fatty acid metabolism" and "lipogenesis"; "glycolysis" was close to significance (FDR = 0.05) ( Figure 7G ). These results indicate that after 72 h of co-culture, the metabolic activity of B7-H3.28.BB.ζCAR-T cells was generally higher than that of B7-H3.TMI.ζCAR-T cells. To determine which pathway(s) contributed the most, overrepresentation analysis (ORA) was performed by examining which pathways were overrepresented in the most highly expressed DEGs (p adj < 0.05; fold change > 1.5). ORA analysis revealed that "glycolysis process" was the only overrepresented metabolic pathway in B7-H3.28.BB.ζCAR-T cells, while other pathways broadly described hypoxia or nucleotide processes. ( Figure 7H B7-H3.TMI.ζCAR-T cells showed extensive changes in pathways related to transcription, mitosis, and ubiquitination ( Figure 7H ).

[0372] Using previously published gene sets describing key enzymes, regulatory proteins, accessory proteins, and other related genes in glycolysis and oxidative phosphorylation, eight upregulated DEGs were found to be related to the regulation of classical glycolytic enzymes or glycolysis (HK1, PGAM1, TPI1, ALDOC, ALDOA, PFKFB3, and PFKFB4), and only two DEGs were related to subunits or other functions in oxidative phosphorylation (NFDFB1 and AK2) in B7-H3.28.BB.ζCAR-T cells compared with B7-H3.TMI.ζCAR-T cells ( Fig.7I Together, these data suggest that B7-H3.28.BB.ζCAR-T cells utilize the glycolytic pathway more than other pathways to meet their metabolic needs.

[0373] B7-H3.TMI.ζCAR-T cells exhibit distinct transcriptional programs in vivo

[0374] The tumor microenvironment was examined to determine the impact of transcriptional programs in B7-H3 CAR-T cells. RNA sequencing was performed on lung-infiltrating T cells collected from the lungs of lung tumor-bearing mice 7 days after B7-H3 CAR-T cell injection. We found extensive transcriptome differences between our B7-H3 CAR-T cells and 945 DEGs ( Fig. 8A ). Inspection of the most enriched pathways by GSEA analysis revealed that B7-H3.TMI.ζCAR T cells were positively enriched in pathways that broadly describe RNA- and DNA-related processes ( Figure 8B ORA analysis revealed that B7-H3.28.BB.ζCAR T cells showed over-represented pathways, which widely covered cytokine and chemokine pathways, while B7-H3.TMI.ζCAR T cells did not show statistically significant over-represented pathways (p adj < 0.05) due to few genes reaching the log2 fold change cutoff value. The over-represented pathways close to significance (p < 0.1) described lysosomal and vacuolar pathways ( Figure 8C ). Using the same metabolic gene set as before, we analyzed the metabolic-related gene signatures in this dataset. We found that six DEGs were associated with glycolytic enzymes and regulatory proteins upregulated in B7-H3.28.BB.ζCAR T cells (PFKM, GAPDH, PFKFB3, PFKFB2, HIF1A, and PFKFB4), while no genes related to oxidative phosphorylation ( Fig.8D ).

[0375] Using a recently described gene signature for T cell dysfunction, we found that 12 DEGs were upregulated in B7-H3.28.BB.ζCAR T cells (IL2RA, PLS3, DUSP4, GZMB, PHLDA1, CSF1, TNFRSF18, NDFIP2, AHI1, CDK6, LAYN, and HAVCR2), compared to only one DEG upregulated in B7-H3.TMI.ζCAR T cells (KLRC1) ( Fig. 8E ). Inspection of the manually curated list of inhibitory protein genes revealed that B7-H3.28.BB.ζCAR T cells showed six downregulated DEGs (BTLA, HAVCR2, PDCD1, CTLA4, PDCDLG2, and CD274) compared with B7-H3.28.BB.ζCAR T cells ( Fig.8F Together, these data suggest that B7-H3.TMI.ζ and B7-H3.28.BB.ζ function differently within the in vivo tumor microenvironment, with the former displaying a less glycolytic, less dysfunctional, and less suppressive phenotype than the latter.

[0376] The common significant genes (p adj < 0.05) were analyzed between in vivo and in vitro RNA sequencing experiments directly comparing the two B7-H3 CAR-T cells. 67 shared genes were found in B7-H3.28.BB.ζCAR and 85 shared genes were found in B7-H3.TMI.ζCAR ( Figure 8G ORA analysis of these common genes revealed a large number of over-represented pathways in both CAR constructs ( Figure 8H ). In B7-H3.28.BB.ζCAR, there are pathways broadly related to hypoxia and nucleotide metabolism, etc.; it is worth noting that "glycolysis process" also appears in this list. In B7-H3.TMI.ζCAR, there are pathways broadly related to endosomes, lysosomes, autophagy, etc. Taken together, these results indicate that B7-H3.TMI.ζ and B7-H3.28.BB.ζCAR-T cells display different pathway characteristics when examining the common DEGs in the experiment.

[0377] B7-H3.TMI.ζCAR-T cells show obvious phenotypic changes after chronic antigen exposure

[0378] Continuous exposure to antigens can lead to dysfunctional phenotypes and suboptimal effector responses in CAR-T cells. In this context, different CAR constructs can significantly affect the expression of multiple cell surface markers. An in vitro model of chronic antigen exposure (CAE) was adapted, in which CAR T cells were continuously cultured with sufficient HCC827 tumor cells so that tumor cells were always present in the co-culture to examine whether TMIGD2 and CD28-4-1BB co-stimulatory domains would also differentially alter cell surface protein expression. During 17 days of co-culture with HCC827 tumor cells, the phenotypes of CD3+, CD4+, and CD8+CAR+B7-H3.TMI.ζ and B7-H3.28.BB.ζCAR-T cells were compared at each analyzed time point. First, the results of the total CD3+CAR+ population are described, and global changes between the two B7-H3 CAR constructs were observed via t-distributed random neighbor embedding (t-SNE) analysis generated from CAR+ cells at all time points ( Fig.9A CAR expression was reduced in both B7-H3 CAR-T cells, and B7-H3.TMI.ζCAR-T cells showed fewer CAR+ cells on day 17 ( Fig. 9B ). This finding suggests that CAR is downregulated after encountering the tumor. Observing PD-1+TIM-3+LAG-3+ depleted CAR-T cells, it was found that B7-H3.TMI.ζCAR-T cells acquired fewer depleted cells than B7-H3.28.BB.ζCAR-T cells by day 13 and day 17 ( Fig. 9CAs differences in expression of PD-1, TIM-3, LAG-3, or a combination could underlie this finding, each protein was subsequently examined individually. No differences in PD-1 or TIM-3 expression were detected ( Fig. 9C In contrast, LAG-3 expression in B7-H3.TMI.ζCAR T cells was found to be significantly lower compared with B7-H3.28.BB.ζCAR T cells at days 13 and 17 ( Fig. 9C ).

[0379] When examining the memory phenotype, a significant enrichment of the central memory cell (CD45RA-CCR7+) population was found in B7-H3.TMI.ζCAR-T cells starting from day 10, whereas the opposite was true at day 6 ( Fig. 9C On day 17, a higher proportion of naive T cells (CD45R+CCR7+) was observed in B7-H3.28.BB.ζ, whereas no differences were found between the groups in effector memory (CD45RA-CCR7-) or terminally differentiated EMRA cells (CD45RA+CCR7-). Fig. 9C ). It was also found that B7-H3.TMI.ζCAR-T cells showed higher CD69+ expression on days 3 and 6, equivalent expression on day 10, and higher expression on days 13 and 17 compared with B7-H3.28.BB.ζCAR-T cells ( Fig. 9C ).

[0380] The results showed that although both B7-H3 CAR-T cells showed a trend of decreased CD4+T cells and increased CD8+T cells, from day 13 onwards, B7-H3.TMI.ζCAR-T cells showed significantly lower CD4+T cells and significantly higher CD8+T cells compared with B7-H3.28.BB.ζCAR-T cells ( Fig. 9C). Given that this experimental design more closely mimics CAE than other experiments, DEGs from in vivo RNA sequencing data (Figure 8) were re-examined to verify this finding. It was found that the CD4 gene was a significantly downregulated DEG in B7-H3.TMI.ζCAR T cells (p adj <0.05; log2 fold change> ± 0.5), while the CD8A gene was significantly upregulated (p adj <0.05; log2 fold change = 0.4206); the CD8B gene also showed insignificant (p adj = 0.161; log2 fold change = 0.31) upregulation in B7-H3.TMI.ζCAR-T cells. To confirm this finding at the protein level, lung-infiltrating T cells and spleen T cells in lung-bearing mice were analyzed 46 days after tumor injection. One donor is the same as in Figure 6, and the other is a separate donor. Compared with B7-H3.28.BB.ζCAR-T cells, B7-H3.TMI.ζCAR-T cells showed a higher percentage of CD8+ T cells and a lower percentage of CD4+ T cells in lung-infiltrating T cells, while splenic T cells showed no differences in either population.

[0381] The above trends were largely replicated in the CD4+CAR+ and CD8+CAR+ populations, with only minor changes in the temporal dynamics ( Figures 9E-9F ), with notable exceptions described below. Compared with B7-H3.28.BB.ζCD4+CAR+CAR-T cells, B7-H3.TMI.ζCD4+CAR+T cells expressed more TIM-3 on day 10 and less TIM-3 on days 13 and 17 ( Fig.9E ). They also showed equivalent percentages of CD69+ cells on day 17 due to reduced CD69 expression. B7-H3.28.BB.ζCAR-T cells showed lower percentages of TEMRA cells on days 10 and 13 ( Fig.9F ).

[0382] It has been reported that antigen affinity and T cell receptor (TCR) signaling intensity can change T cell memory formation in CD8+T cells, and TCR signal intensity can affect the expression of PD-1 and LAG-3. Since B7-H3 CAR utilizes the same scFv and therefore shares the same antigen affinity, TMIGD2 and CD28-4-1BB costimulatory domains were examined to determine whether they change T cell activation intensity as a potential mechanism for the above phenotypic differences. Jurkat (NFAT) cells, a T cell activation reporter cell line, were transduced with B7-H3.TMI.ζ, B7-H3.28.BB.ζ and CD19.28.BB.ζ CARs, which express firefly luciferase via NFAT response elements. Untransduced Jurkat (NFAT) cells or CAR-transduced Jurkat (NFAT) cells were cultured alone or with plate-bound activating anti-CD3 OKT3 antibody, or with CD19+B7-H3- cells (Raji), or with CD19-B7-H3+ cell lines (HCC827, AsPC-1, and U118) ( Fig.9D ). All Jurkat (NFAT) cells signaled in response to OKT3 stimulation, but not when cultured alone. B7-H3.TMI.ζ- and B7-H3.28.BB.ζ-transduced Jurkat (NFAT) cells signaled in response to HCC827, AsPC-1, and U118 tumor cell lines to approximately the same extent, but not in response to Raji cells. It should be noted that the levels of the HCC827 cell line used for CAE stimulation and all RNA sequencing experiments were almost identical. CD19.28.BB.ζ-transduced Jurkat (NFAT) cells signaled in response to Raji cells, but not in response to HCC827, AsPC-1, and U118 tumor cell lines. Taken together, these results show that B7-H3.TMI.ζCAR-T cells acquired differences in memory, exhaustion, activation, and CD4 / CD8 phenotypes after CAE, and that this effect was not mediated by differences in signal intensity.

[0383] discuss

[0384] Using in vitro killing assays, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were the best performing CARs. B7-H3.TMI.ζ CAR-T cells were also effective in multiple solid tumor models, showing equivalent or superior results to B7-H3.28.BB.ζ CAR-T cells. In addition, B7-H3.TMI.ζ CAR-T cells showed unique transcriptomic, metabolomic and phenotypic characteristics, indicating that TMIGD2 co-stimulation provides significant benefits over CD28-41BB co-stimulation.

[0385] Using an in vitro killing assay with reduced transduction efficiency, it was found that only B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells could kill three different tumor cell lines. TMIGD2 co-stimulation may be superior to the currently FDA-approved CD28 and 4-1BB co-stimulatory domains. Comparing these two main constructs, B7-H3.TMI.ζ CAR-T cells release lower concentrations of cytokines. Given that cytokine release syndrome (CRS) may be mediated in part by cytokines released from CAR-T cells or CAR-T cell-activated cells (e.g., macrophages and monocytes), TMIGD2-based CARs may also be safer co-stimulatory domains.

[0386] Unexpectedly, the use of third-generation CARs in combination with TMIGD2 and 4-1BB signaling showed no cytotoxicity against two solid tumor cell lines (HCC827 and U118). Jurkat (NFAT) cells transduced with our B7-H3.TMI.BB.ζCAR showed reduced activation compared to B7-H3.28.BB.ζCAR-transduced cells.

[0387] In an orthotopic GBM model, but not in other tumor models, unexpected toxicity was noted in mice treated with B7-H3.28.BB.ζCAR-T cells, but not in mice treated with B7-H3.TMI.ζCAR-T cells, suggesting that the latter is safer in this setting. Since these mice died without any significant tumor burden, it is possible that CAR-T cells, rather than tumor cells, were the underlying cause. Since B7-H3.28.BB.ζCAR-T cells exhibited significantly higher levels of cytokine release than B7-H3.TMI.ζCAR-T cells, localized cytokine release syndrome may be a factor. Similar effects have been reported clinically and have been described as “localized” or “compartmental” cytokine release syndrome. This toxicity may also be amplified in this model by IT injection of CAR-T cells compared to IV administration.

[0388] CAR co-stimulatory domains have a significant impact on CAR-T cell metabolism. CAR-T cells with CD28 co-stimulatory domains utilize glycolytic metabolism, while 4-1BB co-stimulatory domains utilize oxidative metabolism. In addition, analysis of CD19 CAR-T cell products from a clinical trial has shown that non-responders and partial responders exhibit enrichment of glycolytic gene signatures. Among constructs, B7-H3.28.BB.ζCAR-T cells exhibited a metabolic signature that was more dependent on glycolysis than B7-H3.TMI.ζCAR-T cells based on RNA sequencing pathway analysis, overrepresentation analysis, acute and chronic stimulation Seahorse metabolic assays, and analysis of common DEGs between RNA sequencing experiments.

[0389] Compared with B7-H3.28.BB.ζCAR-T cells, B7-H3.TMI.ζCAR-T cells reduced the dysfunction and exhaustion-related phenotypes involved in CAR-T cell antitumor responses and could be modified by the choice of costimulatory domains. Therefore, TMIGD2 costimulation is a new approach to prevent T cell dysfunction and exhaustion.

[0390] It was found that B7-H3.TMI.ζCAR-T cells showed time-dependent enrichment of central memory cells after CAE, but during initial generation. The higher the percentage of this population, the better the outcome of CAR-T cell therapy. In addition, CAR-T cells generated from a large number of CD8+T cells showed an increased risk of CRS compared with central memory-enriched populations. The TMIGD2 co-stimulatory domain can increase central memory cells and may be beneficial for improving therapeutic efficacy and safety.

[0391] Unexpected enrichment of CD8+ T cells was found in B7-H3.28.BB.ζCAR-T cells after CAE. This finding is consistent with the phenotypic analysis of lung-infiltrating T cells in vivo and reanalysis of in vivo RNA sequencing gene expression data. CD8+CAR-T cells constitute a larger population than CD4+CAR-T cells after infusion of large amounts of CAR-T cell products and after infusion of CAR-T cell products with a 1:1 ratio of CD8:CD4 T cells. TMIGD2 co-stimulation improves the proliferation of CD8+CAR-T cells compared with CD28-4-1BB co-stimulation, and / or TMIGD2 co-stimulation improves the survival of CD8+CAR-T cells.

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Claims

1. A chimeric antigen receptor (CAR), comprising: (a) an extracellular region comprising an antigen binding domain; (b) a transmembrane region; and (c) The intracellular region contains the effector domain and the TMIGD2 co-stimulatory domain.

2. The CAR of claim 1, wherein the TMIGD2 co-stimulatory domain comprises the intracellular region of TMIGD2.

3. The CAR of claim 2, wherein the TMIGD2 co-stimulatory domain comprises a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:

5.

4. The CAR of claim 3, wherein the TMIGD2 co-stimulatory domain comprises an amino acid sequence having at least 75% identity with a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:

5.

5. The CAR of any one of claims 1-4, wherein the antigen binding domain specifically binds to a tumor-associated antigen.

6. The CAR according to claim 5, wherein the tumor-associated antigen is selected from HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptide bound to HLA; hTERT peptide bound to HLA; tyrosinase peptide bound to HLA; WT-1 peptide bound to HLA; LT βR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME; and SSX-2 antigens.

7. The CAR of any one of claims 1-6, wherein the antigen binding domain comprises a scFv.

8. The CAR of any one of claims 1-7, wherein the antigen binding domain comprises a linker.

9. The CAR of claim 8, wherein the linker is a glycine-serine linker.

10. The CAR of claim 9, wherein the glycine-serine linker comprises (Gly x Ser y ) z , wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0, and z is an integer from 1 to 10.

11. The CAR according to any one of claims 1-10, wherein the extracellular region further comprises an N-terminal leader sequence.

12. The CAR of any one of claims 1-11, wherein the extracellular region further comprises a hinge region.

13. The CAR of claim 12, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

2.

14. The CAR of claim 13, wherein the hinge region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:

2.

15. The CAR of any one of claims 1-14, wherein the transmembrane region comprises a CD8α transmembrane region.

16. The CAR of claim 15, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

1.

17. The CAR of claim 15, wherein the transmembrane region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:

1.

18. The CAR of any one of claims 1-17, wherein the effector domain is a CD3 zeta effector domain.

19. The CAR of claim 18, wherein the effector domain comprises the amino acid sequence shown in SEQ ID NO:

6.

20. The CAR of claim 18, wherein the effector domain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:

5.

21. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO:

1.

22. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO: 3, residues 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO:

6.

23. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from residues 172-282 of SEQ ID NO:3, residues 172-278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence shown in SEQ ID NO:1; and (c) the sequence shown in SEQ ID NO:

6.

24. The CAR according to claim 22 or 23, wherein the CAR further comprises the sequence shown in SEQ ID NO:

2.

25. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR according to any one of claims 1-24.

26. A vector comprising a nucleic acid sequence encoding the CAR according to any one of claims 1-24.

27. The vector of claim 26, wherein the nucleic acid sequence encoding the CAR is operably linked to an expression control sequence.

28. The vector according to claim 27, wherein the expression control sequence is a promoter.

29. The vector according to any one of claims 26 to 28, further comprising a nucleic acid sequence encoding a self-cleaving peptide.

30. The vector according to claim 29, wherein the self-cleaving peptide is a 2A self-cleaving peptide. The vector according to claim 30 , wherein the 2A self-cleaving peptide is a P2A peptide.

32. The vector of any one of claims 26-31, further comprising a nucleic acid sequence encoding a transduction marker polypeptide.

33. The vector of claim 32, wherein the transduction marker polypeptide is a truncated form of epidermal growth factor receptor (EGFRt) or GFP, or a portion or variant thereof.

34. The vector of any one of claims 29-31, wherein the nucleic acid sequence encoding the self-cleaving peptide is on the 3' side of the nucleic acid sequence encoding the CAR.

35. The vector according to claim 32 or 33, wherein the vector comprises a nucleic acid sequence encoding a self-cleaving peptide, and wherein the nucleic acid sequence encoding the self-cleaving peptide is on the 5' side of the nucleic acid sequence encoding the marker polypeptide.

36. The vector of any one of claims 26-35, wherein the vector is a viral vector.

37. An isolated cell expressing the CAR according to any one of claims 1-24.

38. The cell of claim 37, wherein the cell comprises the nucleic acid molecule of claim 25.

39. The cell of claim 37 or 38, wherein the cell comprises a vector according to any one of claims 26-36.

40. The cell of any one of claims 37-39, wherein the cell is a T cell, a natural killer (NK) cell, a macrophage or other immune cell.

41. The cell according to claim 40, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4 - CD8 - Double negative T cells, NK cells, macrophages, other immune cells, or any combination thereof.

42. The cell of claim 40, wherein the T cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, a NK cell, a macrophage, other immune cells, or any combination thereof.

43. The cell of any one of claims 37-42, wherein the cell further expresses a transduction marker on its surface.

44. The cell of claim 43, wherein the transduction marker is a truncated form of the epidermal growth factor receptor (EGFRt) or GFP, or a portion or variant thereof.

45. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell according to any one of claims 37-44.

46. ​​The method of claim 45, wherein the disease or condition is malignancy.

47. The method of claim 46, wherein the malignancy is cancer.

48. The method of claim 47, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, stomach cancer, small intestine cancer, bone cancer, cervical cancer, endometrial cancer, eye cancer, gallbladder cancer, thyroid cancer, thymic cancer, sarcoma, and osteosarcoma.

49. The method of claim 47 or 48, wherein the cancer comprises a solid tumor.

50. The method of any one of claims 47 to 49, wherein the cancer comprises a hematological malignancy.

51. A method of eliciting an immune response in a subject against a tumor-associated antigen that specifically binds to a CAR according to any one of claims 1-24, the method comprising administering to the subject an effective amount of a cell according to any one of claims 37-44.

52. A composition comprising the CAR according to any one of claims 1-24 and a pharmaceutically acceptable excipient, carrier or diluent.

53. A composition comprising a cell according to any one of claims 37-44 and a pharmaceutically acceptable excipient, carrier or diluent.

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