NISHIMURA CHRISTOPHER (US)
CLAIMS What is claimed: 1. 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 costimulatory domain. 2. The CAR of claim 1, wherein the TMIGD2 costimulatory domain comprises the intracellular region of TMIGD2. 3. The CAR of claim 2, wherein the TMIGD2 costimulatory domain comprises a sequence selected from the group consisting of residues 172−282 of SEQ ID NO:3, 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 costimulatory domain comprises an amino acid sequence with at least 75% identity to a sequence selected from the group consisting of residues 172−282 of SEQ ID NO:3, 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 a tumor-associated antigen. 6. The CAR of claim 5, wherein the tumor-associated antigen is 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; LTβR; 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; α-fetoprotein (AFP); Frizzled; OX40; PRAME; and SSX- 2 antigen. 7. The CAR of any one of claims 1-6, wherein the antigen binding domain comprises an 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 (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. 11. The CAR of 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 set forth in SEQ ID NO:2. 14. The CAR of claim 13, wherein the hinge region comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth 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 set forth in SEQ ID NO:1. 17. The CAR of claim 15, wherein the transmembrane region comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth in SEQ ID NO:1. 18. The CAR of any one of claims 1-17, wherein the effector domain is a CD3ζ effector domain. 19. The CAR of claim 18, wherein the effector domain comprises the amino acid sequence set forth in SEQ ID NO:6. 20. The CAR of claim 18, wherein the effector domain comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth in SEQ ID NO:5. 21. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172−282 of SEQ ID NO:3, 172−278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:1. 22. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172−282 of SEQ ID NO:3, 172−278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:6. 23. The CAR of any one of claims 1-20, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172−282 of SEQ ID NO:3, 172−278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:1; and (c) the sequence set forth in SEQ ID NO:6. 24. The CAR of claim 22 or 23, wherein the CAR further comprises the sequence set forth in SEQ ID NO:2. 25. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR of any one of claims 1-24. 26. A vector comprising a nucleic acid sequence encoding the CAR of 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 of claim 27, wherein the expression control sequence is a promoter. 29. The vector of any one of claims 26-28, further comprising a nucleic acid sequence encoding a self-cleaving peptide. 30. The vector of claim 29, wherein the self-cleaving peptide is a 2A self- cleaving peptide. 31. The vector of 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 3’ of the nucleic acid sequence encoding the CAR. 35. The vector of 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 5’ 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 of 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 of 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 of claim 40, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, an NK cell, a macrophage, other immune cell, or any combination thereof. 42. The cell of claim 40, wherein the T cell is a naïve T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, an NK cell, a macrophage, other immune cell, 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 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 comprising administering to the subject an effective amount of the cell of any one of claims 37-44. 46. The method of claim 45, wherein the disease or condition is a malignancy. 47. The method of claim 46, wherein the malignancy is a 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, renal cancer, brain cancer, stomach cancer, small intestine cancer, bone cancer, cervix cancer, endometrium cancer, eye cancer, gallbladder cancer, thyroid cancer, thymus 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 hematologic malignancy. 51. A method of eliciting an immune response against a tumor-associated antigen that specifically binds the CAR of any one of claims 1-24 in a subject, comprising administering to the subject an effective amount of the cell of any one of claims 37-44. 52. A composition comprising a CAR of any one of claims 1-24 and a pharmaceutically acceptable excipient, carrier, or diluent. 53. A composition comprising a cell of any one of claims 37-44 and a pharmaceutically acceptable excipient, carrier, or diluent. |
[0174] In any of the embodiments described herein, nucleic acid molecules encoding CARs may be codon-optimized for a particular cell using known techniques (Scholten et al., 2006). Codon optimization can be performed using, e.g., the GenScript® OptimumGene TM tool. Codon-optimized sequences include sequences that are partially or fully codon-optimized. [0175] A nucleic acid molecule encoding a CAR of this disclosure can be inserted into an expression vector, such as a viral vector, for transduction into a cell, such as a T cell. In some embodiments, an expression construct of the present disclosure comprises a nucleic acid molecule encoding a CAR provided herein and, optionally, further encoding a self-cleaving peptide and/or EGFRt marker operably linked to an expression control sequence such as a promoter. [0176] In certain embodiments, nucleic acid molecules of the present disclosure may be operatively linked to certain elements of the vector. For example, polynucleotide sequences that are needed to affect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may 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 possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. [0177] In certain embodiments, the expression construct is comprised in a vector which may integrate into a cell’s genome or promote integration of the nucleic acid molecule insert upon introduction into the cell and thereby replicate along with the cell’s genome, such as a viral vector. Viral vectors include retrovirus, adenovirus, parvovirus, coronavirus, negative strand RNA viruses, positive strand RNA viruses, and double- stranded DNA viruses. (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). [0178] Construction of an expression vector that is used for genetically engineering and producing a CAR of interest can be accomplished by using any suitable molecular biology engineering techniques known in the art. To obtain efficient transcription and translation, a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence, such as a leader sequence and particularly a promoter operably linked to the nucleotide sequence encoding the immunogen. Methods for making CARs of the present disclosure are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No.7,446,191; U.S. Patent Publ. No.2010/065818; U.S. Patent No.8,822,647; PCT Publ. No. WO 2014/031687; U.S. Patent No.7,514,537; Brentjens et al., 2007; and Walseng et al., 2017; the techniques of which are herein incorporated by reference. [0179] In certain embodiments, nucleic acid molecules of the present disclosure are used to transfect/transduce a cell, such as a T cell, an NK cell, a macrophage or another immune cell, for use in adoptive transfer therapy. Cells may be induced to incorporate the vector or other material by use of a viral vector, transformation 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 the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. In certain embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a naïve T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof. Methods for transfecting/transducing T cells with polynucleotides have been previously described (U.S. Patent Application Pub. No. US 2004/0087025) as have adoptive transfer procedures using T cells of 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 Publ. No.2011/0243972; U.S. Patent Publ. No.2011/0189141), such that adaptation of these methodologies to the presently disclosed CARs of the present disclosure is within the scope of the present disclosure. [0180] Functional characterization of CARs described herein may be performed according to any art-accepted methodologies for assaying T cell and/or NK cell activity, including determination of T cell and/or NK cell binding, activation or induction and also including determination of T cell and/or NK cell responses that are antigen-specific. Examples include determination of 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 measures of T cell and/or NK cell functions. Procedures for performing these and similar assays are described herein and/or may be found, for example, in 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. Kits [0181] In some embodiments, kits are 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 self-cleaving peptide, and/or (c) one or more reagents for transducing the vector or nucleic acid molecule into a cell. In certain embodiments, the kits further comprise instructions for use. Methods of Use [0182] The present disclosure also provides methods for treating a disease or condition, wherein the methods comprise administering to a subject in need thereof an effective amount of a composition, cell, or unit dose of the present disclosure, wherein the disease or condition expresses or is otherwise associated with the antigen that is specifically bound by a CAR provided herein. In certain embodiments, the disease or condition is a hyperproliferative or proliferative disease, such as a cancer, an autoimmune disease, or an infectious disease (e.g., viral, bacterial, fungal, or parasitic). [0183] In certain embodiments, a subject to be treated using the methods provided herein is a human. In other embodiments, the subject is a non-human animal, for example in a veterinary or medical research setting. In those embodiments where the subject is a human, the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. Cells according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art. [0184] A composition, cell, or unit dose of the present disclosure may be administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter the target antigen or cells. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the undesired type or level or activity of the tagged cells, the particular form of the active ingredient; and the method of administration. [0185] In some embodiments, the disease or condition is a malignancy. In some embodiments, the malignancy is cancer. In general, cancers treatable by presently disclosed methods and compositions include carcinomas, sarcomas, gliomas, lymphomas, leukemias, myelomas, cancers of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer, gastric cancer, non- small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's 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, linitis plastic, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optice nerve glioma, a mixed glioma, Hodgkin’s lymphoma, a B-cell lymphoma, non-Hodgkin’s lymphoma (NHL), Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma, Waldenström's macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, Sezary syndrome, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi’s sarcoma; liposarcoma; pleomorphic sarcoma; or synovial sarcoma; lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma); an ovarian carcinoma (e.g., Surface epithelial-stromal tumor (Adenocarcinoma) or ovarian epithelial carcinoma (which includes serous tumor, endometrioid tumor and mucinous cystadenocarcinoma), Epidermoid (Squamous cell carcinoma), Embryonal carcinoma and choriocarcinoma ( germ cell tumors)); a kidney carcinoma (e.g., Renal adenocarcinoma, hypernephroma, Transitional cell carcinoma (renal pelvis), Squamous cell carcinoma, Bellini duct carcinoma, Clear cell adenocarcinoma, Transitional cell carcinoma, Carcinoid tumor of the renal pelvis); an adrenal carcinoma (e.g., Adrenocortical carcinoma), a carcinoma of the testis (e.g., Germ cell carcinoma (Seminoma, Choriocarcinoma, Embryonal carciroma, Teratocarcinoma), Serous carcinoma); Gastric carcinoma (e.g., Adenocarcinoma); an intestinal carcinoma (e.g., Adenocarcinoma of the duodenum); a colorectal carcinoma; or a skin carcinoma (e.g., Basal cell carcinoma, Squamous cell carcinoma); ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate. [0186] 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, renal cancer, brain cancer, stomach cancer, thyroid cancer, anus cancer, bone cancer, cervix cancer, endometrium cancer, esophagus cancer, eye cancer, gallbladder cancer, thymus, sarcoma and osteosarcoma. [0187] In some embodiments, the cancer comprises a hematologic malignancy. [0188] In some embodiments, the disease or condition is a “hyperproliferative disorder” and “proliferative disorder.” In some embodiments, the hyperproliferative disorders and proliferative disorders is one or more of tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre malignant cells. In some embodiments, the cancer comprises a solid tumor. [0189] In certain embodiments, the methods provided herein comprise administering a cell expressing a CAR of the present disclosure, a composition comprising the cell, or a unit dose thereof. The amount of cells in a composition is at least one cell (for example, one CAR-modified CD8+ T cell subpopulation; one CAR- modified CD4+ T cell subpopulation; one CAR-modified NK cell subpopulation) or is more typically greater than 10 2 cells, for example, up to 10 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells, or 10 10 cells or more, such as about 10 11 cells/m 2 . In certain embodiments, the cells are administered in a range from about 10 5 to about 10 11 cells/m 2 , preferably in a 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 upon the ultimate use for which the composition is intended as well as the type of cells included therein. For example, cells modified to contain a CAR specific for one or more antigens will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For uses provided herein, cells are generally in a volume of a liter or less, 500 mls or less, 250 mls or less, or 100 mls or less. In embodiments, the density of the desired cells is typically greater than 10 4 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater. The cells may be administered as a single infusion or in multiple infusions over a range of time. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cells. In any of the presently disclosed embodiments, the cell is an allogeneic cell, a syngeneic cell, or an autologous cell. [0190] Also contemplated are pharmaceutical compositions that comprise cells expressing the CARs as disclosed herein and a pharmaceutically acceptable carrier, diluent, and/or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In embodiments, compositions comprising cells as disclosed herein further comprise a suitable infusion media. [0191] Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the undesired type or level or activity of the tagged cells, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art. [0192] Certain methods of treatment or prevention contemplated herein include administering a cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the chromosome of the cell. For example, such a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen- presenting cells, NK cells) in order to administer a desired, CAR-expressing T-cell composition to a subject as an 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, an NK cell, or any combination thereof. In certain embodiments, the immune system cell is a naïve T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, an NK cell, or any combination thereof. In particular embodiments, the cell is a CD4+ T cell. In particular embodiments, the cell is a CD8+ T cell. In particular embodiments, the cell is an NK cell. [0193] As used herein, administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be affected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., CAR-expressing recombinant (i.e., engineered) cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof). [0194] In certain embodiments, a plurality of doses of a cell as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks. [0195] In still further embodiments, the subject being treated is further receiving immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof. In yet further embodiments, the subject being treated has received a non-myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two to at least three months after the non-myeloablative hematopoietic cell transplant. [0196] An effective amount of a pharmaceutical composition (e.g., cell, CAR, unit dose, or composition) refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. [0197] Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy. For example, in certain embodiments, a combination therapy comprises administering a CAR (or an engineered cell expressing the same) with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, a combination therapy comprises administering CAR of the present disclosure (or an engineered cell expressing the same) with an agonist of a stimulatory immune checkpoint agent. In further embodiments, a combination therapy comprises administering a CAR of the present disclosure (or an engineered cell expressing the same) with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof. [0198] Cytokines are used to manipulate host immune response towards anticancer activity (see, e.g., Floros & Tarhini, 2015). Cytokines useful for promoting immune anticancer or antitumor response 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, singly or in any combination with the binding proteins or cells expressing the same of this disclosure. [0199] Various embodiments of the technology are described above. It will be appreciated that details set forth above are provided to describe the embodiments in a manner sufficient to enable a person skilled in the relevant art to make and use the disclosed embodiments. Several of the details and advantages, however, may not be necessary to practice some embodiments. Additionally, some well-known structures or functions may not be shown or described in detail, so as to avoid unnecessarily obscuring the relevant description of the various embodiments. Although some embodiments may be within the scope of the technology, they may not be described in detail with respect to the Figures. Furthermore, features, structures, or characteristics of various embodiments may be combined in any suitable manner. Moreover, one skilled in the art will recognize that there are a number of other technologies that could be used to perform functions similar to those described above. While processes or blocks are presented in a given order, alternative embodiments may perform routines having stages, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and/or modified. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel or may be performed at different times. The headings provided herein are for convenience only and do not interpret the scope or meaning of the described technology. [0200] Various embodiments of the invention are set forth herein below in paragraphs 201 to 253: [0201] A chimeric antigen receptor (CAR) comprising an extracellular region comprising an antigen binding domain, a transmembrane region, and an intracellular region comprising an effector domain and a TMIGD2 costimulatory domain. [0202] The CAR of paragraph 201, wherein the TMIGD2 costimulatory domain comprises the intracellular region of TMIGD2. [0203] The CAR of paragraph 202, wherein the TMIGD2 costimulatory domain comprises a sequence selected from the group consisting of residues 172 ^282 of SEQ ID NO:3, 172 ^278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5. [0204] The CAR of paragraph 203, wherein the TMIGD2 costimulatory domain comprises an amino acid sequence with at least 75% identity to a sequence selected from the group consisting of residues 172 ^282 of SEQ ID NO:3, 172 ^278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5. [0205] The CAR of any one of paragraphs 201-204, wherein the antigen binding domain specifically binds a tumor-associated antigen. [0206] The CAR of paragraph 205, wherein the tumor-associated antigen is 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; LTβR; 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; α-fetoprotein (AFP); Frizzled; OX40; PRAME; and SSX-2 antigen. [0207] The CAR of any one of paragraphs 201-206, wherein the antigen binding domain comprises an scFv. [0208] The CAR of any one of paragraphs 201-207, wherein the antigen binding domain comprises a linker. [0209] The CAR of paragraph 208, wherein the linker is a glycine-serine linker. [0210] 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. [0211] The CAR of any one of paragraphs 201-210, wherein the extracellular region further comprises an N-terminal leader sequence. [0212] The CAR of any one of paragraphs 201-211, wherein the extracellular region further comprises a hinge region. [0213] The CAR of paragraph 212, wherein the hinge region comprises the amino acid sequence set forth in SEQ ID NO:2. [0214] The CAR of paragraph 213, wherein the hinge region comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth in SEQ ID NO:2. [0215] The CAR of any one of paragraphs 201-214, wherein the transmembrane region comprises a CD8α transmembrane region. [0216] The CAR of paragraph 215, wherein the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:1. [0217] The CAR of paragraph 215, wherein the transmembrane region comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth in SEQ ID NO:1. [0218] The CAR of any one of paragraphs 201-217, wherein the effector domain is a CD3ζ effector domain. [0219] The CAR of paragraph 218, wherein the effector domain comprises the amino acid sequence set forth in SEQ ID NO:6. [0220] The CAR of paragraph 218, wherein the effector domain comprises an amino acid sequence with at least 75% identity to the amino acid sequence set forth in SEQ ID NO:5. [0221] The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172 ^282 of SEQ ID NO:3, 172 ^278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:1. [0222] The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172 ^282 of SEQ ID NO:3, 172 ^278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:6. [0223] The CAR of any one of paragraphs 201-220, wherein the CAR comprises (a) a sequence selected from the group consisting of residues 172 ^282 of SEQ ID NO:3, 172 ^278 of SEQ ID NO:4, and residues 52-162 of SEQ ID NO:5; and (b) the sequence set forth in SEQ ID NO:1; and (c) the sequence set forth in SEQ ID NO:6. [0224] The CAR of paragraph 222 or 223, wherein the CAR further comprises the sequence set forth in SEQ ID NO:2. [0225] An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR of any one of paragraphs 201-223. [0226] A vector comprising a nucleic acid sequence encoding the CAR of any one of paragraphs 201-223. [0227] The vector of paragraph 226, wherein the nucleic acid sequence encoding the CAR is operably linked to an expression control sequence. [0228] The vector of paragraph 227, wherein the expression control sequence is a promoter. [0229] The vector of any one of paragraphs 226-228, further comprising a nucleic acid sequence encoding a self-cleaving peptide. [0230] The vector of paragraph 229, wherein the self-cleaving peptide is a 2A self- cleaving peptide. [0231] The vector of paragraph 230, wherein the 2A self-cleaving peptide is a P2A peptide. [0232] The vector of any one of paragraphs 226-231, further comprising a nucleic acid sequence encoding a transduction marker polypeptide. [0233] 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. [0234] The vector of any one of paragraphs 230-233, wherein the nucleic acid sequence encoding the self-cleaving peptide is 3’ of the nucleic acid sequence encoding the CAR. [0235] 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 5’ of the nucleic acid sequence encoding the marker polypeptide. [0236] The vector of any one of paragraphs 226-235, wherein the vector is a viral vector. [0237] An isolated cell expressing the CAR of any one of paragraphs 201-225. [0238] The cell of paragraph 237, wherein the cell comprises the nucleic acid molecule of paragraph 218. [0239] The cell of paragraphs 237 or 238, wherein the cell comprises a vector of any one of paragraphs 225-234. [0240] The cell of any one of paragraphs 237-239, wherein the cell is a T cell, a natural killer (NK) cell, a macrophage, or other immune cell. [0241] 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, an NK cell, a macrophage, other immune cell, or any combination thereof. [0242] The cell of paragraph 240, wherein the T cell is a naïve T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, an NK cell, a macrophage, other immune cell, or any combination thereof. [0243] The cell of any one of paragraphs 237-242, wherein the cell further expresses a transduction marker on its surface. [0244] 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. [0245] A method of treating a disease or condition in a subject in need thereof comprising administering to the subject an effective amount of the cell of any one of paragraphs 237-244. [0246] The method of paragraph 245, wherein the disease or condition is a malignancy. [0247] The method of paragraph 246, wherein the malignancy is a cancer. [0248] The method of paragraph 247, 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, renal cancer, brain cancer, stomach cancer, small intestine cancer, bone cancer, cervix cancer, endometrium cancer, eye cancer, gallbladder cancer, thyroid cancer, thymus cancer, sarcoma, and osteosarcoma. [0249] The method of paragraphs 247 or 248, wherein the cancer comprises a solid tumor. [0250] The method of any one of paragraphs 247-249, wherein the cancer comprises a hematologic malignancy. [0251] A method of eliciting an immune response against a tumor-associated antigen that specifically binds the CAR of any one of paragraphs 201-225 in a subject, comprising administering to the subject an effective amount of the cell of any one of paragraphs 237-244. [0252] A composition comprising a CAR of any one of paragraphs 201-225 and a pharmaceutically acceptable excipient, carrier, or diluent. [0253] A composition comprising a cell of any one of paragraphs 237-244 and a pharmaceutically acceptable excipient, carrier, or diluent. [0254] 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. [0255] These and other changes can be made in light of the above Detailed Description. While the above description details certain embodiments and describes the best mode contemplated, no matter how detailed, various changes can be made. Implementation details may vary considerably, while still being encompassed by the technology disclosed herein. As noted above, particular terminology used when describing certain features or aspects of the technology should not be taken to imply that the terminology is being redefined herein to be restricted to any specific characteristics, features, or aspects of the technology with which that terminology is associated. [0256] The foregoing is merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein. [0257] The following example is illustrative of several embodiments of the present technology: EXAMPLES Example 1: CAR-T Cell Therapy Using CAR-T Cells comprising TMIGD2 as a Costimulatory Molecule [0258] The following example demonstrates the generation of a new CAR-T vector containing the cytoplasmic tail of TMIGD2 (transmembrane and immunoglobulin domain containing 2), wherein the CAR-T vector is capable of transducing normal T cells and can kill human tumor cells. Generation of a CAR-T Vector with TMIGD2 [0259] A CAR-T vector using TMIGD2 as a costimulatory molecule has been developed (Figure 1). The new vector contains an 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. TGMID2 CAR-T Mediated Killing of Human Tumor Cells [0260] As a proof of principle, VH and VL of a mAb against human CD19 were cloned into the TMIGD2 vector shown in Figure 1 and experiments for CAR-T mediated killing of human tumor cells were performed. 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 non-transduced T cells, and with CAR-T transduced T cells. The results showed that CAR-T cells, but not non- transduced T cells, killed almost all of the Raji tumor cells (Figure 2), demonstrating that the CAR-T cells including a TMIGD2 costimulatory domain are able to efficiently kill tumor cells. In Vivo Therapeutic Efficacy of CD19-TMIGD2 CAR-T Cells [0261] This example demonstrates the in vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells in a Raji lymphoma model. NSG TM mice were intravenously (I.V.) injected with 0.25 x 10^6 Raji tumor cells on day 0, and with 10^7 non-transduced T cells or CD19- TMIGD2 CAR-T cells on day 3 and 10, respectively. The mice were monitored for survival. As demonstrated in Figure 3, the survival of the mice received CD19-TMIGD2 CAR-T cells was significantly improved compared to the non-transduced control mice. Example 2: CAR-T Cell Therapy Using anti B7-H3 CAR-T Cells comprising TMIGD2 as a Costimulatory Molecule [0262] The following example demonstrates the generation of anti-B7-H3 CAR-T cells, which incorporate a vector containing the cytoplasmic tail of TMIGD2 (transmembrane and immunoglobulin domain containing 2), wherein the CAR-T vector transduces normal T cells and kills human tumor cells expressing B7-H3 in vitro and in vivo. Experimental Methods Cell lines [0263] NIH 3T3 cells (mouse fibroblast) were obtained from the American Type Culture Collection (ATCC); NSO cells (mouse multiple myeloma) from Department of Cell Biology, Albert Einstein College of Medicine, HEK293T cells (human epithelial kidney) from Department of Cell Biology, Albert Einstein College of Medicine, U118 cells (human glioblastoma) from the ATCC, HCC827 cell (human lung adenocarcinoma) from the ATCC, THP-1 cells (human acute monocytic leukemia) from the ATCC, Raji cells (B cell Burkitts lymphoma) from the ATCC, Jurkat (NFAT) cells (T cell leukemia) from BPS Bioscience, AsPC-1 cells (pancreatic adenocarcinoma) from the ATCC, PANC-1 cells (pancreatic ductal epitheloid carcinoma) from the ATCC, and Phoenix-AMPHO cells (human epithelial kidney) from the ATCC. Cell lines were grown in DMEM or RPMI 1640 media supplemented with 10% FBS, 1% penicillin, and 1% streptomycin and cultured in a humidified incubator at 37°C and 5% CO2. Transfection of tumor cell lines [0264] Phoenix-AMPHO cells were co-transfected with a MSCV-YFP plasmid containing the protein-of-interest (mouse B7-H3, human B7-H3, cynomolgus B7-H3, or Luciferase-tdTomato (-Luc)) and the pCMV-VSV-G plasmid. 48 and 72 hour viral supernatant was collected and used to transfect tumor cell lines using polybrene. Transfected cells were sorted at least twice to ensure pure populations using a BD FACS Aria II cell sorter. Generation of anti-B7-H3 monoclonal antibodies [0265] C57BL/6 mice were immunized using a recombinant protein comprised of the IgV domain of human B7-H3 fused to a human Fc fragment. After immunization, hybridomas were generated by fusing NSO myeloma cells and mouse splenocytes using standard methods. Antibody-producing hybridomas were screened by flow cytometry to ensure specific binding to mouse and human B7-H3. Afterwards, hybridomas were thrice subcloned by single cell dilution and then expanded in the cell compartment of bioreactor flasks in DMEM high glucose media (supplemented with 10% ultra-low IgG FBS, 10% NCTC-109, 1% penicillin, 1% streptomycin, and 1% non-essential amino acids. The media compartment of the bioreactor flasks contained DMEM high glucose media supplemented with 1% penicillin and 1% streptomycin. Antibody supernatant from the cell compartment was collected and stored at 4°C until purification using Protein G resin -packed columns. Purified B7-H3 mAbs were analyzed using SDS-PAGE prior to affinity determination, isotype determination, and VH and VL sequencing. B7-H3 mAb affinity determination [0266] Anti-B7-H3 mAb affinity 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. Afterwards, the protein-loaded biosensors were placed into solutions containing serial dilutions of anti-B7-H3 mAbs. Kon, Koff, and KD were determined by analysis using a 1:1 binding model. Generation of CAR constructs [0267] Anti-B7-H3 single chain variable fragments (scFvs) were generated by cloning the native mAb signal peptide sequence to the VH and VL regions of anti-B7-H3 mAbs connected by a G4S linker. The scFv was connected to a human CD8ɑ hinge and transmembrane domain, the intracellular region of various costimulatory proteins (CD28, 4-1BB, TMIGD2, CD28-4-1BB, or TMIGD2-4-1BB), followed by the intracellular domain of human CD3ζ. A self-cleaving P2A peptide sequence was then inserted followed by the 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 a self-cleaving T2A peptide sequence, followed by a firefly luciferase gene sequence. The entire CAR sequence was cloned into the pLVX-Zsgreen lentiviral expression plasmid under the control of an EF1ɑ promoter. A commercially available CD19-CD28-4-1BB CAR was also modified to include the P2A-EGFRt and T2A-Luciferase sequences. Production and determination of viral titer from CAR lentivirus [0268] HEK293T cells were co-transfected with the psPAX packaging plasmid, pMD2.G envelope plasmid, and CAR plasmid. 48- and 72-hour viral supernatant was collected, concentrated 100X, and subsequently pooled to ensure equivalent titer. Virus titer was determined by transducing activated human T cells with the CAR lentivirus as described below. Viral titer was calculated based on EGFR+ T cells. Isolation of human T cells [0269] Leukopaks from healthy human donors were obtained 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 media and stored in liquid nitrogen or were used immediately. Generation of CAR-T cells [0270] Fresh or thawed T cells were activated for 24 hours on an OKT3 (1 ug/mL) and CD28 (1 ug/mL) antibody-coated 24-well plate in CTS OpTmizer media supplemented with OpTmizer T-cell expansion supplement, 10% FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin, IL-7, and IL-15. Activated T cells were then transduced on non-tissue culture treated plates coated with RetroNectin reagent (19 ug/mL) and CAR lentivirus (MOI of approximately 10). CAR-T cells were then expanded for at least 7 days before use in experiments. Prior to all experiments, CAR-T cell transduction efficiency was normalized to 50% CAR+ cells (for in vitro cytotoxicity screening assay) or to the lowest efficiency donor by adding of non-transduced T cells. If necessary, CAR-T cells were purified using anti-phycoerythrin (PE) microbeads following anti-EGFR-PE staining. In vitro coculture killing assays [0271] 0.1x10 6 CAR-T cells and either HCC827-Luc (0.01x10 6 cells), U118-Luc, (0.01x10 6 cells), and THP-1 (0.01x10 6 cells) were plated in T cell media without the addition of cytokines. 3-5 days later, the tumor cells were enumerated with flow cytometry. For the Incucyte time-lapse cytotoxicity assays, 5x10 3 HCC827-Luc or U118- Luc cells were plated one day prior to imaging. Tumor-coated plates were then placed in the Incucyte machine and imaged every 4 hours at 4 locations per well for a total of 112 hours.16 hours after the initial imaging, 0.1x10 6 CAR-T cells were gently added to the wells. Imaging data was analyzed using the Incucyte software. Flow Cytometry [0272] 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 include CD45, CD3, CD4, CD8, CD45RA, CCR7, EGFR, G4S linker, PD-1, TIM-3, LAG-3, B7-H3, CD69, and CD33. Viability was determined using 7-amino-actinomycin D (7-AAD), zombie NIR, ghost violet 510, and 4’,6-diamidino-2-phenylindole (DAPI). In some experiments, cells were fixed using a 2% paraformaldehyde (PFA) solution prior to analysis. All samples were acquired using a BD LSRII or Cytek Aurora flow cytometer. 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. Flow cytometry cytokine analysis [0273] 0.4x10 6 CAR-T cells and 0.1x10 6 HCC827 tumor cells were cocultured for 24 hours in 24-well plates. Supernatants from the cocultures were collected and stored at -80°C until analysis. The concentration 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 was determined using the flow cytometry-based LEGENDplex human Th cytokine panel kit on non-diluted or 1:5 diluted supernatant samples. Data was analyzed using the LEGENDplex online data analysis software suite. RNA isolation [0274] For in vitro RNA isolation, CAR-T cells were gently removed from coculture wells and plated into fresh wells for 30 minutes to allow excess tumor cells to attach to the plate. CAR-T cells were gently removed and live cells enriched using Lymphoprep density gradient centrifugation. T cells were further purified isolated using a CD3+ selection beads. RNA was extracted and stored at -80°C. RNA sequencing (40 million paired-end reads) after library preparation (using the NEBNext Ultra II kit) was performed by Admera Health. For in vivo RNA isolation, lung-infiltrating T cells were isolated from mouse lungs. Briefly, 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 media. Next, the single cell suspension was layered on a discontinuous density gradient Percoll solutions (40% and 80%), and the immune cells at the 40% 80% interface were collected. A second density gradient centrifugation using Lymphoprep was performed to further enrich the live cell population. Mouse cells were then depleted two times. Afterwards, T cells from the same donor transduced with the same CAR were pooled, and RNA was isolated as described above. RNA sequencing (40 million paired- end reads) after library preparation (using the SMARTseq V4 with NExtera XT kit) was performed by Admera Health B7-H3 CAR vs CD19 CAR RNA sequencing analysis [0275] Reads from RNA sequencing were aligned using STAR alignment software (version 2.6.1b) to a reference human genome (hg38; downloaded in 11/2019 from the UCSC genome browser). Gene-mapped fragments were counted using HTseq software (version 0.6.1). Genes with an average expression count of 1 or more in either group were considered expressed. Differentially expressed genes (DEGs) were determined by an adjusted p value of <0.05 and log2 fold change > 0.5. Principle 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 ranked gene lists determined by multiplying the - log10 p-value and the sign of the log2(fold change). B7-H3 CAR in vitro and in vivo RNA sequencing analysis [0276] Paired-end reads from RNA sequencing were aligned using the STAR aligner (version 2.7.9a) to the human reference genome (hg38). The alignment data were then used by the RSEM software (version 1.3.3) to quantify expression levels of individual genes (both coding and non-coding) in the GENCODE annotation (version 41), yielding estimated read counts and transcripts per million (TPMs). Genes with a TPM < 1 in all samples were excluded from further analysis. Differentially expressed genes (DEGs) were determined by an adjusted p value of <0.05 and log2 fold change > 0.5. Principal component analysis (PCA) and differential expression analysis were then performed using the DESeq2 software (version 1.38.3). Gene Set Enrichment Analysis (GSEA) was performed using the Hallmark, KEGG, and Reactome gene sets after ranking genes by multiplying the -log10(p-value) and the sign of log2(fold change) for each gene. Gene sets with a false discovery rate (FDR) < 0.05 were considered enriched. Over representation analysis was also performed on DEGs or significantly differentially expressed genes (adjusted p-value < 0.05) using the cluster. Profiler (version 4.6.2) enrichGO function. Pathways with an adjusted p value < 0.05 were considered enriched in either the up- or down-regulated genes. Gene sets describing T cell dysfunction and metabolism were obtained from the literature, and the inhibitory protein list was manually curated. Seahorse metabolic assay [0277] 0.1x10 6 CAR-T cells were cocultured alone or with 5x10 3 HCC827 tumor cells for one or seven days. CAR-T cells were collected from the wells and transferred to a new well for 30 minutes to allow for attachment of any tumor cells. CAR-T cells were re-collected and analyzed using the Seahorse T cell metabolic profiling kit on Seahorse PDL-coated plates. After transfer to PDL-coated plates, CAR-T cells were rested 5 minutes before centrifugation to allow for even cell distribution on the bottom of the well. Seahorse plates were run on a Seahorse XF96 analyzer at baseline and after injections of Oligomycin A, Bam15, and Rotenone/Antimycin A. Data was analyzed using Agilent Seahorse Analytics online software. %ATP from mitochondria was obtained by the following formula: 100% - %ATP from glycolysis. Jurkat (NFAT) T cell activation experiment [0278] Jurkat (NFAT) cells were transduced with CAR constructs as described above. Triplicate wells in a 96-well plate were coated with either OKT3 (1 μg/mL) or 0.2x10 5 tumor cells (U118, HCC827, or AsPC-1) overnight.0.2x10 5 Raji tumor cells were added to additional triplicate wells. 0.1x10 6 non-transduced or CAR-transduced Jurkat (NFAT) cells were added to each well and incubated for 6 hours. The Bio-Glo Luciferase assay system was used to detect the Jurkat (NFAT) luciferase. Luciferase signal was acquired on a plate reader. Chronic antigen exposure [0279] 0.2x10 6 CAR-T cells were plated in 96-well plates in triplicate wells using T cell media without cytokines. 0.1x10 5 HCC827 tumor cells were added to each well. Every 3-4 days, CAR-T cells were gently removed from the well and transferred to a new well. A small cell aliquot was then analyzed using flow cytometry. Unanalyzed CAR-T cells were centrifuged, and additional supernatant was removed such that one half of the original volume remained in the well.0.1x10 5 HCC827 parental cells in fresh T cell media were then added at an equivalent volume. This was repeated for 17 days. Mice [0280] For all in vivo experiments, 8-12 week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used. NSG mice were purchased from The Jackson Laboratory and bred at the Albert Einstein College of Medicine. Mice were housed in a specific-pathogen free animal facility under a 12-hour light/dark cycle with food and water freely available. All experimental procedures were approved by the Albert Einstein Institutional Animal Care and Use Committee. Bioluminescent imaging [0281] Bioluminescent imaging was performed using the IVIS Spectrum in vivo imaging system and analyzed using Living Image software (version 3.0). Images were acquired 10-15 minutes after intraperitoneal (I.P.) injection of D-Luciferin (150 μg/g of mouse weight). In vivo lung cancer model [0282] 0.5x10 6 HCC827-Luc cells were injected I.V. in the lateral tail vein of NSG mice in 100μL of sterile PBS. Three days later, tumor engraftment was confirmed via IVIS imaging and experimental groups were normalized to ensure equivalent baseline tumor burden. One injection of 10x10 6 CAR-T cells was then given I.V. into the lateral tail vein, followed by an additional injection one week later. Tumor burden was tracked via IVIS imaging over the course of 100 days. Survival was also tracked during this time. In vivo glioblastoma model [0283] U118-Luc tumor cells were resuspended at 5x10 3 cells/μL in sterile PBS. A small hole was drilled in the skull using an 18-gauge needle 1 mm lateral and 1mm anterior to bregma using the freehand method in NSG mice anesthetized with continuous isoflurane (2%). 2 μL of tumor cells were injected into the hole perpendicular to the benchtop surface using a blunt end Hamilton syringe fitted with a sterile pre-cut pipette tip which exposed 1mm of the syringe needle over the course of one minute. Meloxicam (5 mg/kg) was administered pre-emptively and for two days after tumor implantation.7 days later, tumor engraftment was confirmed using IVIS imaging. Mice were then allocated into experimental groups normalized for baseline tumor burden. Afterwards, 1x10 6 CAR-T cells were injected intratumorally (I.T.) using the same surgical preparation described above in a 2μL volume of PBS. Tumor burden and survival was tracked via IVIS imaging for 100 days. In vivo pancreatic cancer model [0284] PANC-1-Luc tumor cells were resuspended at 1x10 4 cells/50 μL of sterile PBS. The pancreas was expressed though a 1 cm incision made in the skin and musculature of NSG mice. 50 μL of tumor cells were injected into the head of the pancreas using a 27 gauge needle. Meloxicam was administered pre-emptively and for three days after tumor implantation. Seven days later, tumor engraftment was confirmed using IVIS imaging. Mice were then allocated into experimental groups normalized for baseline tumor burden. 10x10 6 CAR-T cells were then injected I.V. into the lateral tail vein, followed by another injection 7 days later. Tumor burden and survival was tracked via IVIS imaging for 100 days. In vivo CAR-T cell persistence model [0285] 0.5x10 6 parental HCC827 tumor cells were I.V. injected into the lateral tail vein. Three days later, a single dose of 10x10 6 CAR-Luc T cells was injected I.V. into the lateral tail vein. CAR-Luciferase T cell signal was tracked using IVIS imaging over the course of 46 days. Immune cell isolation from mouse blood and organs [0286] To obtain immune cells from mouse blood, blood was taken from the lateral tail vein in heparinized capillary tubes. Red blood cells (RBCs) were then lyzed. Remaining cells were then washed twice with PBS before flow cytometry analysis. Mice were euthanized and organs of interest were directly removed (spleens) or perfused with PBS then removed (lungs). Spleens were then transferred to C tubes and mechanically dissociated. The cell suspension was strained through a 40 μM filter. To obtain immune cells from the lungs, single cell suspensions were made using enzymatic digestion, and immune cells isolated using discontinuous Percoll gradients. RBC lysis buffer was performed to remove RBCs. Statistics [0287] Statistical analysis was performed using GraphPad prism software (version 9.4.1). As described in each figure legend, testing between groups was performed using an unpaired two-tailed Student’s t-test, one-way ANVOA, Kruskal-Wallis test, two-way ANOVA, or Mantel-Cox log-rank test. The data are shown as individual values, or as individual values and the mean ± SEM. A P value of less than 0.05 was considered statistically significant. Generation and validation of anti-B7-H3 monoclonal antibodies [0288] Six anti-B7-H3 mAb clones denoted as 1G5, 15F9, 23B2, 8B12, 12B4, and 24D12 that bound to both mouse and human B7-H3 at affinities ranging from 0.32 nM to 11.08 nM using biolayer interferometry (BLI) were obtained. All of the anti-B7-H3 mAbs bound to mouse and human B7-H3 were validated that each stably expressed on 3T3 cells by flow cytometry. Additionally, each anti-B7-H3 mAbs was tested to confirm that each could bind to non-human primate cynomolgus B7-H3. All anti-B7-H3 mAbs were able to detect cynomolgus B7-H3 stably expressed on 3T3 cells by flow cytometry. The anti-B7-H3 mAbs were able to recognize human, mouse, and cynomolgus B7-H3. In vitro screening of anti-B7-H3 CAR-T cells [0289] Using a single chain variable fragment (scFv) derived from anti-B7-H3 mAb clone 8B12, anti-B7-H3 CARs were constructed by sequentially linking the scFv to a human CD8ɑ hinge and transmembrane (H/TM) domain, the intracellular domain of various costimulatory proteins, and the intracellular domain of CD3ζ. After the CD3ζ sequence, a self-cleaving P2A peptide and a truncated human epidermal growth factor receptor (hEGFRt) were included (Figure 4A). The hEGFRt protein was modified by removing two of four extracellular domains and all intracellular domains to prevent antigen binding and signaling. As it can still be recognized by anti-EGFR antibodies, it functioned as marker of transduction efficiency. The following costimulatory domains were utilized in the CAR constructs: CD28 (B7-H3.28.ζ CAR), 4-1BB (B7-H3.BB.ζ CAR) , TMIGD2 (B7-H3.TMI.ζ CAR), CD28-4-1BB (B7-H3.28.BB.ζ CAR), or TMIGD2-4-1BB (B7-H3.TMI.BB.ζ CAR). An anti-CD19 CAR was similarly generated using an anti-CD19 scFv and with the CD28-4-1BB costimulatory domains to serve as an irrelevant target control (CD19.28.BB.ζ) (Figure 4A). On primary human T cells, all CAR constructs were efficiently expressed with transduction efficiencies regularly greater than 85% CAR+ as determined by expression of the hEGFRt protein, and there was no difference in memory phenotype or expansion between the CAR-T cell constructs (Figure 4H-K). [0290] The in vitro anti-tumor response of the B7-H3 CAR-T cells was tested against multiple tumor types which were B7-H3+ by flow cytometry. The CAR-T cell transduction efficiency was lowered to 50% using non-transduced T cells to better reflect transduction efficiencies seen in other clinical trials. U118 glioblastoma (GBM) and HCC827 lung cancer cell lines were stably transfected with a plasmid expressing Luciferase and tdTomato (-Luc) proteins to allow for tumor cell discrimination. B7-H3.TMI.ζ and B7- H3.28.BB.ζ CAR-T cells showed tumor cell killing against 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 our CARs were effective against non- solid tumors as well, we also examined if our CAR-T cells could 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). Although donor-to-donor variability was high, it was consistently found that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed anti-tumor responses across all cell lines tested. [0291] A time-lapse imaging cytotoxicity assay was performed by repeatedly imaging U118-Luc or HCC827-Luc tumor cells alone or cocultured with B7-H3.TMI.ζ, B7- H3.28.BB.ζ, or CD19.28.BB.ζ CAR-T cells to determine if there were differences in the kinetics of tumor cell killing between these two CARs. Live tumor cell growth was tracked based on tdTomato signal and morphological exclusion of dead cells. Against U118-Luc tumor cells, control CD19.28.BB.ζ CAR-T cells showed similar tumor growth to tumor cells alone, whereas both the B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells cleared the tumor cells (Figure 4E). This finding was recapitulated in HCC827-Luc cocultures (Figure 4F). [0292] The cytokine release profile of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells was evaluated using a multiplexed 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 coculture with HCC827 tumor cells, both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR- T cells showed significantly higher cytokine release across all cytokines tested compared to control CD19.28.BB.ζ CAR-T cells (Figure 4G). B7-H3.TMI.ζ CAR-T cells released significantly less cytokines than B7-H3.28.BB.ζ CAR-T cells across apart from IL-6. [0293] These results demonstrated that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed equivalent cytotoxic responses and killing kinetics in vitro. While the cytokine secretion profile of these CAR-T cells was similar, B7-H3.TMI.ζ CAR-T cells mostly secreted lower amounts of these cytokines compared to B7-H3.28.BB.ζ CAR-T cells. B7-H3.TMI.ζ CAR-T cells show anti-tumor responses in vivo [0294] The in vivo anti-tumor responses of the lead B7-H3 CARs, B7-H3.TMI.ζ and B7-H3.28.BB.ζ, alongside control CD19.28.BB.ζ, were assessed in three solid tumor models. All tumor cell lines (HCC827, U118, and PANC-1) were stably transfected with a plasmid containing Luciferase and tdTomato (-Luc) to allow for in vivo tracking by bioluminescent imaging. The CARs 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 (I.V.) into NSG mice followed by injection of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, or control CD19.28.BB.ζ CAR-T cells I.V. three and ten days later (Figure 5A). B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells reduced tumor burden and showed a concomitant increase of overall survival compared to CD19.28.BB.ζ CAR-T (Figures 5B-5D). There was no significant difference in tumor cell signal or survival between the two B7-H3 CAR-T cells (Figure 5B-5D). [0295] The CAR-T cell therapy was explored in an orthoptic GBM model. U118-Luc cells were intracranially injected into the right cerebral hemisphere of NSG mice followed by an injection of CAR-T cells intratumorally (I.T.) seven days later (Figure 5E), as it has been shown that intratumorally injected CAR-T cells confer superior anti-tumor responses than I.V. injected CAR-T cells at equivalents low doses. Both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed anti-tumor responses against U118-Luc cells compared to CD19.28.BB.ζ CAR-T cells (Figure 5F and 5G). There was no difference in tumor burden between the B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells (Figure 5F and 5G). While both B7-H3 CAR-T cells improved overall survival compared to CD19 CAR-T cells, B7-H3.TMI.ζ CAR-T cells had superior survival outcomes compared to B7- H3.28.BB.ζ CAR-T cells (Figure 5H). No significant tumor burden prior to death in B7- H3.28.BB.ζ CAR-T cell-treated mice was observed, suggesting that the tumor was not the cause of death in this cohort of mice. [0296] The CAR-T cell therapy was explored in an orthotopic model of pancreatic cancer. NSG mice with were orthotopically injected with PANC-1-Luc tumor cells into the pancreas, followed by I.V. injections of CAR-T cells seven and fourteen days later (Figure 5I). Both B7-H3 CAR-T cells reduced tumor burden and improved overall survival compared to control CD19.28.BB.ζ CAR-T cells (Figures 5J to 5L). B7-H3.TMI.ζ CAR-T cells had less tumor burden compared to B7-H3.28.BB.ζ CAR-T cells, although this only reached statistical significance on day 21 (Figure 5K). Similarly, there was a trend towards improved survival in B7-H3.TMI.ζ CAR-T cells compared to B7-H3.28.BB.ζ CAR- T cells, with four of seven B7-H3.TMI.ζ CAR-T cell-treated alive 100 days after tumor cell injection compared to one out of seven B7-H3.28.BB.ζ CAR-T cell-treated mice. [0297] Together, these experiments demonstrate that B7-H3.TMI.ζ and B7- H3.28.BB.ζ CAR-T cells show anti-tumor responses against multiple tumor models in vivo. Further, B7-H3.TMI.ζ CAR-T cells demonstrate equivalent or superior outcomes in a tumor-dependent manner. B7-H3.TMI.ζ CAR-T cells persist in vivo [0298] As the B7-H3 CAR-T cells were determined to be cytolytic in multiple tumor models in vivo, the expansion and persistence of B7-H3 CAR-T cells in vivo was examined. The original CAR constructs were modified to include a self-cleaving T2A peptide followed by a luciferase (CAR-Luc) to allow for in vivo CAR-T cell tracking using bioluminescent imaging (Figure 6A). NSG mice were injected with parental HCC827 cells that do not express a luciferase, followed by a single, sub-therapeutic injection of B7- H3.TMI.ζ-Luc, B7-H3.28.BB.ζ-Luc, or CD19.28.BB.ζ-Luc CAR-T cells I.V. (Figure 6B). On day 7, CAR-Luc signal was detectable in all constructs and there was no difference between any group; beginning on day 21, both B7-H3 CAR-Luc T cells showed significantly higher signal 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 signal than CD19.28.BB.ζ-Luc CAR-T cells, suggesting that B7-H3.TMI.ζ-Luc CAR-T cells can persist longer that B7-H3.28.BB.ζ-Luc CAR-T cells in vivo, although the latter did approach statistical significance (Figure 6C to 6E). [0299] 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 that was higher than CD19.28.BB.ζ-Luc CAR-T cells (Figure 6F). Examining 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 at equivalent numbers between the B7-H3 CAR-Luc T cell constructs (Figure 6G to 6I). Taken together, these data show that both B7-H3 CAR Luc-T cells can expand and persist in vivo in an antigen-dependent manner. Further, B7-H3.TMI.ζ-Luc CAR-T cells show modestly improved persistence compared to B7-H3.28.BB.ζ-Luc CAR-T at late timepoints, likely due to CAR persistence in locations other than the lungs, spleen, and blood. B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells show transcriptomic differences in vitro [0300] RNA sequencing comparing B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells to CD19.28.BB.ζ CAR-T cells after coculture with HCC827 tumor cells for 24 hours in vitro was performed. Compared to CD19.28.BB.ζ CAR-T cells, both B7-H3.TMI.ζ and B7- H3.28.BB.ζ CAR-T cells showed broad transcriptomic differences (Figure 7A). 307 shared differentially expressed genes (DEGs) were found, as well as 198 and 153 unique DEGs between the B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells, respectively (Figure 7B). Gene set enrichment analysis (GSEA) revealed many enriched pathways in both B7-H3 CAR-T cells compared to CD19 CAR-T cells, with a high degree of similarity in terms of the pathways expressed, but variability in the order of their enrichment (Figure 7C). Interestingly, among the top enriched pathways, the “Oxidative phosphorylation” pathway in B7-H3.TMI.ζ CAR-T cells was found to be comparable to the “Glycolysis” pathway in the B7-H3.28.BB.ζ CAR-T cells (Figure 7C), suggesting metabolic differences between these two CAR constructs. [0301] The Seahorse T cell metabolic profiling assay was utilized to measure changes in the %ATP generated from glycolysis and mitochondria after coculture and at baseline (cultured in the absence of tumor cells) to validate the mechanistic findings. In this assay, the %ATP generated from glycolysis and the %ATP generated from mitochondria sum to 100%, thus these values are dependent on one another. Upon acute 24-hour stimulation, B7-H3.TMI.ζ CAR-T cells had a lower %ATP generated from glycolysis (Figure 7D, left graph) and a concomitant higher %ATP generated from mitochondria (Figure 7D, right graph) at baseline and after coculture compared to B7- H3.28.BB.ζ CAR-T cells. After chronic stimulation with repeated tumor cell additions, B7- H3.TMI.ζ CAR-T cells did not show differences in the %ATP generated from glycolysis or mitochondria at baseline (Figure 7E, left graph), but did show a lower %ATP generated from glycolysis and concomitant higher %ATP generated from mitochondria after coculture (Figure 7E, right graph). It was observed that B7-H3.TMI.ζ CAR-T cells maintained their metabolic signature while B7-H3.28.BB.ζ CAR-T cells modestly increased their glycolytic energy expenditure with an accompanying decrease in mitochondrial energy expenditure (Figures 7D and 7E). These functional metabolic assays recapitulated the RNA sequencing experimental findings. [0302] To directly compare B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells, an additional RNA sequencing experiment was performed from RNA isolated after 72 hours of coculture with HCC827 tumor cells. Broad transcriptomic differences between these two B7-H3 CARs were present, with a total of 1328 DEGs identified (Figure 7F). GSEA analysis revealed four metabolic were negatively enriched in B7-H3-TMIGD2 CAR-T cells, including “Oxidative phosphorylation”, “Fatty acid metabolism”, and “Adipogenesis”; “Glycolysis” approached significance (FDR = 0.05) (Figure 7G). These results suggest that B7-H3.28.BB.ζ CAR-T cells were overall more metabolically active than B7-H3.TMI.ζ CAR-T cells after 72 hours of coculture. To determine which pathway(s) contributed the largest role, an overrepresentation analysis (ORA) was performed by examining what pathways were overrepresented among the most highly expressed DEGs (p adj < 0.05; fold change > 1.5). ORA analysis revealed “Glycolytic process” as the only overrepresented metabolic pathway in B7-H3.28.BB.ζ CAR-T cells, with other pathways broadly describing hypoxia or nucleotide processes. (Figure 7H). B7-H3.TMI.ζ CAR-T cells broadly showed changes pathways associated with transcription, mitosis, and ubiquitination (Figure 7H). [0303] Using a previously published gene set describing key enzymes, regulatory proteins, accessory proteins, and other related genes in glycolysis and oxidative phosphorylation, eight upregulated DEGs were found associated with the classical glycolysis enzymes or regulation of glycolysis (HK1, PGAM1, TPI1, ALDOC, ALDOA, PFKFB3, and PFKFB4), compared to only two DEGs associated with a subunit or other function in oxidative phosphorylation (NFDFB1 and AK2) in B7-H3.28.BB.ζ CAR-T cells compared to B7-H3.TMI.ζ CAR-T cells (Figure 7I). Together, these data suggest that B7- H3.28.BB.ζ CAR-T cells utilize the glycolytic pathway more so than other pathways for their metabolic needs. B7-H3.TMI.ζ CAR-T cells show distinct transcriptional programs in vivo [0304] The tumor microenvironment was examined to determine the impact transcriptional programs would have in the B7-H3 CAR-T cells. RNA sequencing was performed from lung-infiltrating T cells collected from the lungs of lung-tumor bearing mice 7 days after B7-H3 CAR-T cell injection. Broad transcriptomic differences were found and 945 DEGs between our B7-H3 CAR-T cells (Figure 8A). Examining the top enriched pathways by GSEA analysis revealed B7-H3.TMI.ζ CAR T cells were positively enriched for pathways broadly describing RNA- and DNA-associated processes (Figure 8B). ORA analysis revealed that B7-H3.28.BB.ζ CAR T cells showed overrepresented pathways broadly encompassing cytokines and chemokine pathways among others, and while B7-H3.TMI.ζ CAR T cells did not show statistically significant overrepresented pathways (p adj < 0.05) due to few genes meeting the log2 fold change cutoff, overrepresented pathways approaching significance (p < 0.1) described lysosomal and vacuole pathways (Figure 8C). Using the same metabolism gene set as before, metabolism-associated gene signatures were analyzed in this data set. Six DEGs were found associated with glycolysis enzymes and regulatory proteins upregulated in B7- H3.28.BB.ζ CAR T cells (PFKM, GAPDH, PFKFB3, PFKFB2, HIF1A, and PFKFB4) and no genes associated with oxidative phosphorylation (Figure 8D). [0305] Using a recently described T cell dysfunction gene signature, 12 DEGs were found upregulated in the B7-H3.28.BB.ζ CAR T cells (IL2RA, PLS3, DUSP4, GZMB, PHLDA1, CSF1, TNFRSF18, NDFIP2, AHI1, CDK6, LAYN, AND HAVCR2) compared to only one in B7-H3.TMI.ζ CAR T cells (KLRC1) (Figure 8E). Examining a hand-curated gene list of inhibitory proteins, it was found that B7-H3.TMI.ζ CAR T cells showed six downregulated DEGs (BTLA, HAVCR2, PDCD1, CTLA4, PDCDLG2, and CD274), compared to B7-H3.28.BB.ζ CAR T cells (Figure 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 showing a less glycolytic, less dysfunctional, and less inhibited phenotype compared to the latter. [0306] Common significant genes (p adj < 0.05) were analyzed between the in vivo and in vitro RNA sequencing experiments that directly compared the two B7-H3 CAR-T cells. 67 shared genes were found in B7-H3.28.BB.ζ CAR and 85 genes in the B7- H3.TMI.ζ CAR (Figure 8G). ORA analysis of these common genes revealed numerous overrepresented pathways in both CAR constructs (Figure 8H). In the B7-H3.28.BB.ζ CAR, pathways broadly related to hypoxia and nucleotide metabolism were present among others; notably, “glycolytic process” also appeared in this list. In the B7-H3.TMI.ζ CAR, pathways broadly related to endosomes, lysosomes, autophagy, and others were present. Taken tougher, these results show that, when examining common DEGs across experiments, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells show distinct pathway signatures. B7-H3.TMI.ζ CAR-T cells show distinct phenotypic changes after chronic antigen exposure [0307] Persistent exposure to antigens can lead to dysfunctional phenotypes and suboptimal effector responses in CAR-T cells. In this setting, different CAR constructions can significantly influence the expression of a variety cell surface markers. An in vitro model of chronic antigen exposure (CAE) was adapted wherein CAR T cells were continuously cultured with sufficient HCC827 tumor cells so that the tumor cells were always present in the coculture to examine if the TMIGD2 and CD28-4-1BB costimulatory domains would also differentially alter cell surface protein expression. The phenotype of CD3+, CD4+, and CD8+ CAR+ B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were compared at each analysis timepoint over the course of 17 days of coculture with HCC827 tumor cells. Describing first the results from the total CD3+ CAR+ population, global changes between the two B7-H3 CAR constructs were observed via t-distributed stochastic neighbor embedding (t-SNE) analysis generated from CAR+ cells at all timepoints (Figure 9A). CAR expression decreased in both B7-H3 CAR-T cells, with B7- H3.TMI.ζ CAR-T cells showing fewer CAR+ cells on day 17 (Figure 9B). This finding demonstrates CAR downregulation post-tumor encounter. Looking at PD-1+ TIM-3+ LAG-3+ exhausted CAR-T cells, it was found that B7-H3.TMI.ζ CAR-T cells acquired fewer exhausted cells by days 13 and 17 than B7-H3.28.BB.ζ CAR-T cells (Figure 9C). As differences in the expression of PD-1, TIM-3, LAG-3, or a combination could underlie this finding, each protein was subsequently individually examined. No differences in PD- 1 or TIM-3 expression were detected (Figure 9C). By contrast, it was found that LAG-3 expression in B7-H3.TMI.ζ CAR T cells was significantly lower on days 13 and 17 compared to B7-H3.28.BB.ζ CAR T cells (Figure 9C). [0308] When examining memory phenotypes, a significantly enriched population of central memory cells (CD45RA- CCR7+) cells in B7-H3.TMI.ζ CAR-T cells was found beginning on day 10 onwards, while on Day 6 the opposite was true (Figure 9C). A higher percentage of naïve T cells (CD45R+ CCR7+) in B7-H3.28.BB.ζ were observed on day 17, and no differences in effector memory (CD45RA- CCR7-) or terminally differentiated EMRA cells (CD45RA+ CCR7-) between the groups (Figure 9C). It was also found that B7-H3.TMI.ζ CAR-T cells exhibited higher CD69+ expression on days 3 and 6, equivalent expression on day 10, and higher expression on days 13 and 17 compared to B7- H3.28.BB.ζ CAR-T cells (Figure 9C). [0309] It was found that while both B7-H3 CAR-T cells showed a trend of decreasing CD4+ T cells and increasing CD8+ T cells, B7-H3.TMI.ζ CAR-T cells showed significantly lower CD4+ and significantly higher CD8+ T cells compared to B7-H3.28.BB.ζ CAR-T cells beginning on day 13 (Figure 9C). The DEGs from the in vivo RNA sequencing data (Figure 8) were reexamined to validate the finding, given that this experimental design more closely mimics CAE than the other experiments. The CD4 gene was found to be a significant downregulated DEG (p adj < 0.05; log2 fold change > ±0.5) and the CD8A gene was significantly upregulated (p adj < 0.05; log2 fold change = 0.4206) in B7- H3.TMI.ζ CAR T cells; the CD8B gene showed a non-significant (p adj = 0.161; log2 fold change = 0.31) upregulation in B7-H3.TMI.ζ CAR-T cells as well. To confirm this finding at the protein level, lung-infiltrating and splenic T cells were analyzed from lung-tumor bearing mice 46 days post tumor injection. One donor was the same as in Figure 6, while another was a separate donor. B7-H3.TMI.ζ CAR-T cells showed a higher percentage of CD8+ T cells and lower percent of CD4+ T cells compared to B7-H3.28.BB.ζ CAR-T cells in lung-infiltrating T cells, whereas splenic T cells showed no difference in either population. [0310] The trends described above were largely replicated in CD4+ CAR+ and CD8+ CAR+ populations with only minor changes in temporal dynamics (Figure 9E-9F) except for notable exceptions described below. B7-H3.TMI.ζ CD4+ CAR+ T cells expressed more TIM-3 day 10, and lower TIM-3 on days 13 and 17 compared to B7- H3.28.BB.ζ CD4+ CAR+ CAR-T cells (Figure 9E). They also showed equivalent percent of CD69+ cells on day 17 due to a decrease in CD69 expression. B7-H3.TMI.ζ CD8+ CAR+ T cells showed a lower percentage of TEMRA cells on Days 10 and 13 compared to B7-H3.28.BB.ζ CAR-T cells (Figure 9F). [0311] It has been reported that the antigen affinity and T cell receptor (TCR) signaling strength can alter T cell memory formation in CD8+ T cells, and that TCR signal strength can affect the expression of PD-1 and LAG-3. As the B7-H3 CARs utilize the same scFv and thus share the same antigen affinity, the TMIGD2 and CD28-4-1BB costimulatory domains were examined to determine if they altered T cell activation strength as a mechanism underlying the phenotypic differences described above. A T cell activation reporter cell line, Jurkat (NFAT) cells—which express firefly luciferase via NFAT response elements— was transduced with the B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and CD19.28.BB.ζ CARs. Non-transduced Jurkat (NFAT) cells or CAR-transduced Jurkat (NFAT) cells were cultured either alone, with plate-bound activating anti-CD3 OKT3 antibody, with CD19+ B7-H3- cells (Raji), or with CD19- B7-H3+ cell lines (HCC827, AsPC-1, and U118) (Figure 9D). All Jurkat (NFAT) cells signaled in response to OKT3 stimulation and did not signal 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 broadly similar degrees, but not to Raji cells. Notably, the level was nearly identical for the HCC827 cell line used for CAE stimulation and all RNA sequencing experiments. CD19.28.BB.ζ-transduced Jurkat (NFAT) cells signaled in response to Raji cells but not HCC827, AsPC-1, and U118 tumor cell lines. Taken together, these results show that B7-H3.TMI.ζ CAR-T cells acquire differences in memory, exhaustion, activation, and CD4/CD8 phenotype upon CAE, and that this effect is not mediated by differences in signal strength. Discussion [0312] B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were the top performing CARs using in vitro killing assays. B7-H3.TMI.ζ CAR-T cells were also efficacious in multiple solid tumor models, showing equivalent or superior outcomes to B7-H3.28.BB.ζ CAR-T cells. Additionally, B7-H3.TMI.ζ CAR-T cells showed unique transcriptomic, metabolomic, and phenotypic profiles, indicating that TMIGD2 costimulation offers distinct benefits from CD28-41BB costimulation. [0313] Using in vitro killing assays with lowered 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 costimulation may be superior to current FDA-approved CD28 and 4-1BB costimulatory domains. Comparing the two lead constructs, the B7-H3.TMI.ζ CAR-T cells released a lower concentration of cytokines. Given that cytokine release syndrome (CRS) may be mediated in part by cytokines released from CAR-T cells or cells activated by CAR-T cells (e.g. macrophages and monocytes), TMIGD2-based CARs may represent a safer costimulatory domain as well. [0314] Unexpectedly, combining TMIGD2 and 4-1BB signaling using a third- generation CAR did not show cytotoxicity against two solid tumor cells lines (HCC827 and U118). Jurkat (NFAT) cells transduced with our B7-H3.TMI.BB.ζ CAR showed reduced activation compared to B7-H3.28.BB.ζ CAR-transduced cells. [0315] In the orthotopic GBM model, but not other tumor models, it was noted an unexpected toxicity in mice treated with B7-H3.28.BB.ζ CAR-T cells but not B7-H3.TMI.ζ CAR-T cells, suggesting that the latter is safer in this context. As these mice died without any significant tumor burden, it is likely that the CAR-T cells rather than the tumor cells were the underlying cause. As B7-H3.28.BB.ζ CAR-T cells exhibit significantly higher levels of cytokine release than B7-H3.TMI.ζ CAR-T cells, localized cytokine release syndrome may be a factor. A similar effect has been reported in the clinic, described as a “local” or “compartmental” cytokine release syndrome. This toxicity could also be magnified by the I.T. injection of CAR-T cells in this model compared to I.V. administration. [0316] CAR costimulatory domains have significant impacts on CAR-T cell metabolism. CAR-T cells with a CD28 costimulatory domain utilize glycolytic metabolism, whereas a 4-1BB costimulatory domain utilizes oxidative metabolism. Further, analysis of a CD19 CAR-T cell product from one clinical trial has demonstrated that non- responders and partial responders show enrichment of glycolysis gene signatures. In the constructs, B7-H3.28.BB.ζ CAR-T cells showed a metabolic profile more reliant on glycolysis than B7-H3.TMI.ζ CAR-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. [0317] Compared to B7-H3.28.BB.ζ CAR-T cells, B7-H3.TMI.ζ CAR-T cells reduced dysfunction- and exhaustion-associated phenotypes which are involved in CAR-T cell anti-tumor responses, and can be modified by the choice of costimulatory domain. Therefore, TMIGD2 costimulation is a new method to prevent T cell dysfunction and exhaustion. [0318] It was found that upon CAE, but not during the initial generation, B7-H3.TMI.ζ CAR-T cells showed a time-dependent enrichment of central memory cells. A higher percentage of this population is associated with better outcomes in CAR-T cell therapy. In addition, CAR-T cells generated from bulk CD8+ T cells compared to central memory- enriched populations show an increased risk for CRS. The TMIGD2 costimulatory domain can increase central memory cells and may be beneficial for improving therapeutic efficacy and safety. 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