Antigen-specific T cell receptors and chimeric antigen receptors and methods of use in immune signaling modulation for cancer immunotherapy

Abstract

The present invention relates to T cell receptors (TCRs) for the cancer / testis antigens NY-ESO-1 and CT83, which are presented by multiple HLA molecules. Preferred TCRs of the present invention, derived from human T cells, exhibit high affinity and antigen specificity in vitro and in vivo. The present invention also relates to modulation of TCR-T CAR-T cell signaling and functional persistence in cancer immunotherapy. In one aspect, the present disclosure relates to methods for identifying and functionally validating TCRs from cancer antigen-specific T cells.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for the identification and functional validation of T cell receptors (TCRs) from tumor antigen-specific T cells, modulation of TCR-T cells and chimeric antigen receptor (CAR)-T cells to increase and prolong T cell persistence and reduce T cell exhaustion by direct manipulation of TCR or CAR signaling domains and knockdown / knockout of negative signaling molecules, and methods of using TCRs, TCR-T, and CAR-T cells in the treatment of cancer. The present invention also relates to identified polypeptides comprising one or more alpha and beta chains of T cell receptors ("TCRs") specific for cancer antigens, such as NY-ESO-1 ("ESO-1 TCR"), CT83 ("CT83-TCR"), and viral antigens, such as human cytomegalovirus (HCMV) PP65 and (HCMV) IE1 TCR, nucleic acids and recombinant vectors encoding the polypeptides, and cells comprising the nucleic acids or recombinant vectors. [Background technology]

[0002] Background of the Invention The host immune system includes innate and adaptive immune systems that recognize and eliminate exogenous or endogenous antigens derived from pathogens or abnormal tissues, including cancer cells. Schreiber, RD, Old, LJ, & Smyth, MJ, Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science 331, 1565-1570, doi:10.1126 / science.1203486 (2011); Vesely, MD, Kershaw, MH, Schreiber, RD, & Smyth, MJ, Natural innate and adaptive immunity to cancer. Annual review of immunology 29, 235-271, doi:10.1146 / annurev-immunol-031210-101324 (2011). Various types of immune cells contribute to the recognition, suppression, and rejection of cancer cells. Although tumor-reactive T lymphocytes (T cells) have been demonstrated to play a direct role in tumor rejection, clinical responses to early cancer immunotherapy have been limited due to several factors, particularly immunosuppression. More recently, the identification of immune checkpoints has led to the development of targeted immunotherapies. Depletion or inhibition of immunosuppressive checkpoints, such as programmed cell death-1 protein (PD1) and its ligand PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and other checkpoint inhibitors, has greatly enhanced antitumor immunity and demonstrated impressive and durable clinical responses in patients with many types of cancer.Callahan, MK, et al., Anti-CTLA-4 antibody therapy: immune monitoring during clinical development of a novel immunotherapy. Seminars in oncology 37,473-484, doi:10.1053 / j.seminoncol.2010.09.001 (2010), Chambers, CA, Kuhns, MS, Egen, JG & Allison, JP, CTLA-4-mediated inhibition in regulation. of T cell responses:mechanisms and manipulation in tumor immunotherapy.Annual review of immunology 19,565-594,doi:10.1146 / annurev.immunol.19.1.565(2001), Zhu,Y.,Yao,S.&Chen,L.Cell surface signaling molecules in the control of immune responses:a tide model,Immunity 34, 466-478, doi:10.1016 / j.immuni.2011.04.008 (2011); Wang, H.Y. & Wang, R.F. Regulatory T cells and cancer. Current opinion in immunology 19, 217-223, doi:10.1016 / j.coi.2007.02.004 (2007); Joyce, J.A. & Fearon, D.T.T. cell exclusion, immune privilege, and the tumor microenvironment. Science 348, 74-80, doi:10.1126 / science.aaa6204 (2015). Benefiting from breakthroughs in cancer immunotherapy, T cell-based immunotherapy has been successfully applied in recent years to treat human cancers, including or excluding melanoma, renal cell carcinoma, and lymphoma, with varying degrees of tumor regression.

[0003] CD8+ and CD4+ T cells are key components of T cell-based antitumor immunity. CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), specifically recognize epitope complexes bound to class I molecules of the major histocompatibility complex (MHC-human leukocyte antigen, or HLA in humans) via their T cell receptors (TCRs) and can kill cells when the complexes are presented on the cell surface. CD4+ T cells, primarily referred to as T helper cells (Th cells), are another type of T cell that plays an important role in the immune system. CD4+ T cells specifically recognize epitope complexes bound to class II molecules of the MHC via their TCRs, releasing cytokines that can regulate the immune system. CD4+ T cells are also essential for the activation of other immune cells, which may or may not include CD8+ T cells, B lymphocytes, and macrophages.

[0004] To initiate tumor-specific T cell responses, tumor antigens are processed and degraded into peptides containing 9–13 amino acids (called epitopes) in tumor cells or other antigen-processing cells (APCs) via the proteasome pathway (for major histocompatibility complex (MHC) class I molecule binding) or the endosomal / lysosomal pathway (for MHC class II molecule binding). The end products of such antigen processing bind to specific types of MHC class I or II molecules on APCs, are transported to the cell surface, and can activate CD8+ or CD4+ T cells when the epitope-HLA complex specifically binds to the TCR on the T cell surface. The cytotoxic activity of CD8+ T cells can directly kill tumor cells. However, other studies have demonstrated that CD4+ T cells also play a role in antitumor immunity. Wang, RF & Rosenberg, SA Human tumor antigens for cancer vaccine development. Immunological reviews 170, 85-100 (1999). Wang, RF The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends in immunology 22, 269-276 (2001). Furthermore, a subset of CD4+ T cells (CD4 CTLs) has cytotoxic activity and is directly involved in tumor cell killing in an HLA class II-restricted manner. Takeuchi,A.&Saito,T.CD4 CTL,a Cytotoxic Subset of CD4(+)T Cells,Their Differentiation and Function.Frontiers in immunology 8,194,doi:10.3389 / fimmu.2017.00194(2017),Wang,RF&Wang,HYImmune targets and neoantigens for cancer immunotherapy and precision medicine.Cell research 27,11-37,doi:10.1038 / cr.2016.155(2017). Chimeric antigen receptor (CAR)-engineered T cells have provided durable clinical benefits against hematologic cancers, including leukemia and lymphoma. CD19-CAR-T products have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of lymphoma and leukemia. June, C.H. & Sadelain, M. Chimeric Antigen Receptor Therapy. N Engl J Med 379, 64–73, doi:10.1056 / NEJMra1706169 (2018). However, CAR-T cell therapy has not performed well in treating solid tumors. Furthermore, approximately 30–50% of CD19-CAR-T-treated cancer patients who achieve remission experience disease relapse within 12 months of treatment. Shah,NN&Fry,TJMechanisms of resistance to CAR T cell therapy.Nat Rev Clin Oncol 16,372-385,doi:10.1038 / s41571-019-0184-6(2019);Park,JH et al., Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia.N Engl J Med 378,449-459,doi:10.1056 / NEJMoa1709919(2018);Maude, SL et al., Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.N Engl J Med 378, 439-448, doi:10.1056 / NEJMoa1709866(2018). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Schreiber , RD , Old , LJ .& Smyth , MJ , Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion .

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[0006] overview In one aspect, the present disclosure relates to methods for identifying and functionally validating TCRs from cancer antigen-specific T cells. For example, in some aspects, the present disclosure relates to methods for identifying and functionally validating TCRs from NY-ESO-1-specific, CT83-specific, human cytomegalovirus (HCMV)-pp65-specific, and / or HCMV-IE-1-specific T cells.

[0007] In some aspects, the disclosure features a method of detecting and cloning TCRs from cancer antigen-specific T cells in a human subject (by way of non-limiting example, cancer antigen-specific T cells can include or exclude any of NY-ESO-1-specific, CT83-specific, HCMV-pp65-specific, and / or HCMV-IE-1-specific T cells), the method comprising: a) in vitro stimulating naive T cells with a cancer antigen, e.g., a class I or class II HLA-restricted epitope complex (by way of non-limiting example, a class II HLA-DP4-restricted NY-ESO-1 epitope complex, a class I HLA-A2-restricted CT83 epitope complex, a class I HLA-A2-restricted HCMV-pp65 epitope complex, and / or a class I HLA-A2-restricted HCMV-IE-1 epitope complex). b) detecting in vitro and in vivo T cell populations specific for cancer antigen epitopes (non-limiting examples include or exclude any of NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1 epitopes); c) selecting cancer antigen epitope-specific CD4+ or CD8+ T cell populations (non-limiting examples include or exclude any of NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1 epitope-specific CD4+ or CD8+ T cell populations); and d) isolating single T cells from the selected T cells. e) obtaining T cell V(D)J sequences from the T cells by single-cell next-generation sequencing; f) synthesizing primers for TCR cloning based on the sequences; g) amplifying alpha and beta chain TCR variable regions from pooled T cells stimulated with cancer antigen(s) (non-limiting examples include or exclude any of the NY-ESO-1 epitope, CT83 epitope, HCMV-pp65 epitope, and / or HCMV-IE-1 epitope); and h) assembling the amplified alpha and beta chain TCR variable regions into a vector to form a complete TCR construct.In some aspects, the method further comprises j) transducing the cloned TCR into naive CD4+ or CD8+ T cells, i) measuring the transduced T cell activity, and k) screening the transduced T cells in vitro and in vivo for binding to, recognition of, and / or activation by multiple targets (e.g., one or more peptides, one or more cells or cell lines, transfected cell lines, and / or one or more tumor cell lines). In one aspect, the TCR from a cancer antigen-specific T cell identified by the method of the preceding aspect or other aspects and embodiments described herein can bind and / or recognize any cancer antigen described herein, including any cancer antigen or fragment or epitope thereof as described in any aspect or embodiment described herein, including any cancer antigen described in the discussion of "cancer antigen" and "tumor antigen."

[0008] In one aspect, also disclosed herein is a method of identifying epitope-specific T cells and TCRs of any preceding aspect or of any aspect or embodiment disclosed herein, wherein the method comprises administering to the subject a cell line comprising one or more cells or cell lines (e.g., which may include or exclude HEK293 cells, HEK293T cells, Cos-7 cells, 586-mel cells, 624-mel cells, MDA-MB-231 cells, MDA-MB-436 cells, E0771 cells, HTB-21 cells). In one aspect, the one or more tumor cell lines are selected from the group consisting of B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, breast cancer, renal cancer, genitourinary cancer, lung cancer, and / or urinary tract cancer. The cell line may include or exclude any cell line selected from the group consisting of human esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma, and may optionally be selected from HEK293 cells, HEK293T cells, Cos-7 cells, 586-mel cells, 624-mel cells, MDA-MB-231 cells, MDA-MB-436 cells, E0771 cells, and HTB-21 cells.

[0009] Also disclosed herein in one aspect is a method of identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein, wherein one or more cells or cell lines are engineered to express an MHC class I or class II molecule.

[0010] Also disclosed herein are methods of identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein, wherein the measured T cell activity (which may include or exclude release of cytokines, including, for example, but not limited to, IFN-α, TGF-β, lymphotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25) is measured by any immunodetection method disclosed herein. In some embodiments, T cell activity may be measured by, for example, but not limited to, ELISA, chemiluminescence, ELISPOT, intracellular cytokine staining, or chromium release, or any other immunodetection method disclosed herein.

[0011] In an aspect, also disclosed herein is a method of identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein, wherein the cancer is selected from the group consisting of B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, pre-cancerous carcinoma, ovarian cancer, pancreatic cancer ... pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, pan The cancer is selected from the group consisting of prostate cancer, skin cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, breast cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma, and can include or exclude any of the group consisting of these.

[0012] In one aspect, also disclosed herein are cancer epitopes, which can include or exclude epitopes from any cancer or tumor antigen recognized or bound by cancer antigen-specific T cells and TCRs identified by the method of detecting or identifying cancer antigen / epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein.

[0013] In one aspect, the cancer epitopes can include or exclude epitopes of NY-ESO-1 identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the above aspects or any aspect or embodiment disclosed herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence SLLMWITQCFLPVF (SEQ ID NO: 1) disclosed herein, and variants thereof.

[0014] Also disclosed herein, in one aspect, are cancer epitopes that can include or exclude epitopes of CT83 identified by the methods for detecting or identifying epitope-specific T cells and TCRs of any of the above aspects or any aspect or embodiment disclosed herein. In some aspects, the epitope can consist essentially of the identified epitope. The basic and essential characteristic of an epitope is that it is a portion of a target protein that can be recognized or bound by a TCR in the context of either class I or class II MHC. For example, disclosed herein are polypeptides comprising the amino acid sequence KLVELEHTL (SEQ ID NO: 2) disclosed herein, and variants thereof.

[0015] In one aspect, the cancer epitope may include or exclude an epitope of HCMV-pp65 identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the above aspects or any aspect or embodiment disclosed herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence NLVPMVATV (SEQ ID NO: 26) and variants thereof disclosed herein.

[0016] In one aspect, the cancer epitopes can include or exclude epitopes of HCMV-IE-1 identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the above aspects or any aspect or embodiment disclosed herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence VLEETSVML (SEQ ID NO: 31) and variants thereof disclosed herein.

[0017] In one aspect, the present disclosure relates to a composition comprising one or more alpha and / or beta chains of a T cell receptor ("TCR") specific for a cancer antigen, identified or detected by the method of identifying or detecting epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein. In some aspects, the alpha and / or beta chains of the TCR recognize and / or bind to a cancer antigen, such as, but not limited to, NY-ESO-1 ("ESO-1 TCR") and / or CT83 ("CT83-TCR"). In some aspects, the alpha and / or beta chains of the TCR recognize and / or bind to a cancer antigen of viral origin, such as a viral antigen expressed on a human or mammalian cancer cell. In some aspects, the alpha and / or beta chains of the TCR recognize and / or bind to a cancer antigen, such as, but not limited to, a protein derived from human cytomegalovirus (HCMV). In some embodiments, the alpha and / or beta chains of the TCR recognize and / or bind to cancer antigens that are HCMV pp65 and HCMV IE-1 proteins, or fragments or epitopes thereof. In some embodiments, the alpha and / or beta chains of the TCR recognize and / or bind to HCMV pp65 (amino acids 495-503) and / or HCMV IE-1 (amino acids 316-324). In some embodiments, the alpha and / or beta chains of the TCR recognize and / or bind to cancer antigens, which can include or exclude any of the cancer antigens disclosed above.

[0018] In some aspects, the present disclosure relates to compositions comprising, for example, one or more alpha chains or regions and / or one or more beta chains or regions of a T cell receptor specific for a cancer antigen. In some aspects, the present disclosure relates to compositions comprising, for example, one or more alpha chains / regions or beta chains / regions of a T cell receptor specific for a cancer antigen, which can include or exclude any of NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), and / or HCMV-IE-1 (IEI-TCR), or any combination thereof. In some embodiments, the composition comprises, for example, at least one polypeptide comprising an alpha chain or region of a T cell receptor specific for NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), pp65 (pp65-TCR), or IE-1 (IE-1-TCR), and at least one polypeptide comprising a beta chain of a T cell receptor specific for NY-ESO-1 (ESO-1 TCR), or CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), or HCMV-IE-1 (IE-1-TCR). In some embodiments, the compositions comprise, for example, one polypeptide comprising an alpha chain or region of a T cell receptor specific for NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), pp65 (pp65-TCR), or IE-1 (IE-1-TCR), respectively, and one polypeptide comprising a beta chain or region of a T cell receptor specific for NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), or HCMV-IE-1 (IE-1-TCR).

[0019] In one aspect, also disclosed herein is the alpha variable region of a cancer antigen-specific TCR detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein. In one aspect, the alpha variable region can include or exclude the alpha variable region of the DP4-ESO-1 TCR, the alpha variable region of the A2-CT83 TCR, the alpha variable region of the A2-pp65 TCR, and / or the alpha region of the A2-IE-1-TCR, for use with any variable region sequence or epitope-specific sequence detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein and / or identified herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY (SEQ ID NO: 3) (the alpha variable region of the DP4-ESO-1 TCR). As another example, disclosed herein is a polypeptide comprising the amino acid sequence MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 5) (the alpha variable region of the A2-CT83 TCR). As another example, disclosed herein is a polypeptide comprising the amino acid sequence MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPN (SEQ ID NO: 29) (the alpha variable region of the A2-pp65 TCR).As another example, disclosed herein is a polypeptide comprising the amino acid sequence MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGHIYGGSQGNLIFGKGTKLSVKPN (SEQ ID NO: 32) (alpha variable region of A2-IE-1-TCR). Also disclosed herein are fragments or variants of any polypeptide or polypeptide fragment of any of the foregoing aspects or any embodiment disclosed herein that bind to antigen with the same specificity as the reference (full-length and unmodified) receptor. In some aspects, variants comprise conservative amino acid substitutions as further disclosed herein. Substitutions into any alpha variable region disclosed herein can include or exclude substitutions in one or more of the six CDRs of the TCR.

[0020] In one aspect, also disclosed herein is a beta variable region of a cancer antigen-specific TCR for use with any variable region sequence or epitope-specific sequence identified by the method of identifying epitope-specific T cells and TCRs of any preceding aspect and / or identified herein. In one aspect, the beta variable region can include or exclude the beta variable region of the DP4-ESO-1 TCR, the beta variable region of the A2-CT83 TCR, the beta variable region of the A2-pp65 TCR, and / or the beta region of the A2-IE-1-TCR for use with any variable region sequence or epitope-specific sequence detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein. In one aspect, also disclosed herein is a beta variable region of the cancer-specific TCR DP4-ESO-1 TCR for use with any variable region sequence or epitope-specific sequence identified by the method of identifying epitope-specific T cells and TCRs of any of the preceding aspects and / or identified herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE (SEQ ID NO: 4). In one aspect, also disclosed herein is a beta variable region of the A2-CT83 TCR for use with any variable region sequence or epitope-specific sequence detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the preceding aspects and / or identified herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE (SEQ ID NO: 6).

[0013] In one aspect, also disclosed herein is a beta variable region of the A2-pp65 TCR for use with any variable region sequence or epitope-specific sequence detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the preceding aspects and / or identified herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPITGTGDYGYTFGSGTRLTVVE (SEQ ID NO: 30). In one aspect, also disclosed herein is a beta variable region of the A2-IE-1 TCR for use with any variable region sequence or epitope-specific sequence detected or identified by the method of detecting or identifying epitope-specific T cells and TCRs of any of the preceding aspects and / or identified herein. For example, disclosed herein is a polypeptide comprising the amino acid sequence MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHHQGPLETQYFGPGTRLLVLE (SEQ ID NO: 33). Variants of any polypeptide or polypeptide fragment in any of the foregoing aspects or any embodiment disclosed herein are also disclosed herein. In some aspects, variants comprise conservative amino acid substitutions as further disclosed herein. Substitutions into any of the beta variable regions disclosed herein can include or exclude substitutions in one or more of the six CDRs of the TCR.

[0021] Also disclosed herein are variants of any polypeptide or polypeptide fragment disclosed in any of the above aspects or in any embodiment disclosed herein, comprising conservative amino acid substitutions, which may include or exclude substitutions in one or more of the six CDRs of the TCR.

[0022] Also disclosed herein are variants of any polypeptide or polypeptide fragment disclosed herein, comprising conservative amino acid substitutions, which may include or exclude substitutions in one or more of the six CDRs of the TCR.

[0023] In one aspect, the present disclosure includes chimeric TCRs comprising a TCR variable region fused to a modified human constant region or to a non-human constant region, which may be modified or unmodified. In some aspects, the chimeric TCR comprises a cancer antigen-specific TCR variable region fused to a non-human, e.g., murine, TCR constant region. In some aspects, the TCR variable region comprises alpha and beta chain variable regions fused to alpha and beta TCR constant regions, respectively, and may include any of the alpha and / or beta chains of any other aspect of the present disclosure. In some aspects, modifying the TCR variable region or fusing it to a non-human constant region reduces mispairing between the chimeric TCR and an endogenous TCR. In some aspects, the chimeric TCR comprises a variable region, which may include or exclude any one of the CT83 TCR variable region, NY-ESO-1 TCR variable region, pp65 TCR variable region, and / or IE-1 TCR variable region, fused to a non-human TCR constant region, e.g., a murine TCR constant region. Among other embodiments, chimeric TCRs reduce mispairing between the chimeric TCR and the endogenous TCR of transduced T cells. For example, in some embodiments, a chimeric CT83 TCR reduces mispairing between the chimeric CT83 TCR (MC) and the endogenous TCR (HC). In other embodiments, for example, a chimeric NY-ESO-1 TCR reduces mispairing between the chimeric NY-ESO-1 (MC) and the endogenous TCR (HC). In other embodiments, for example, a chimeric pp65 TCR reduces mispairing between the chimeric pp65 (MC) and the endogenous TCR (HC). In other embodiments, for example, a chimeric IE-1 TCR reduces mispairing between the chimeric IE-1 (MC) and the endogenous TCR (HC).

[0024] In one aspect, the present disclosure includes chimeric TCRs comprising a TCR variable region fused to a modified human constant region or a non-human constant region, which may be unmodified or modified. In some aspects, the chimeric TCR comprises a cancer antigen-specific TCR variable region fused to a non-human, e.g., murine, TCR constant region. In some aspects, the TCR variable region comprises alpha and beta chain variable regions fused to alpha and beta TCR constant regions, respectively, and can include any of the alpha and / or beta chains of any other aspect of the present disclosure. In some aspects, the chimeric TCR comprises a TCR variable region that includes or excludes any of the CT83 TCR variable region, the NY-ESO-1 TCR variable region, the pp65 TCR variable region, or the IE-1 TCR fused to a non-human, e.g., murine, TCR constant region. Among other aspects, the chimeric TCR reduces mispairing between the chimeric TCR and the endogenous TCR of the transduced T cell. For example, in some embodiments, a chimeric CT83 TCR reduces mispairing between a chimeric CT83 TCR (MC) and an endogenous TCR (HC). In other embodiments, for example, a chimeric NY-ESO-1 TCR reduces mispairing or reduces mispairing between a chimeric NY-ESO-1 (MC) and an endogenous TCR (HC). In other embodiments, for example, a chimeric pp65 TCR reduces mispairing or reduces mispairing between a chimeric pp65 (MC) and an endogenous TCR (HC). In other embodiments, for example, a chimeric IE-1 TCR reduces mispairing or reduces mispairing between a chimeric IE-1 (MC) and an endogenous TCR (HC).

[0025] Also featured are nucleic acids encoding any of the above epitopes, receptor chains and / or polypeptides, or any other polypeptides disclosed in any aspect or embodiment herein, recombinant nucleic acids comprising the above nucleic acids, vectors or constructs comprising the above recombinant nucleic acids, and cells transduced with one or more of the above nucleic acids or vectors.

[0026] In one aspect, nucleic acids encoding polypeptides comprising an epitope of any of the above aspects are also disclosed herein.

[0027] Also disclosed herein, in one aspect, is a nucleic acid encoding a polypeptide TCR alpha and / or beta variable region or chain of any of the preceding aspects or any embodiment disclosed herein. In one aspect, the nucleic acid of the disclosure encodes any one of SEQ ID NOs: 3-6, 10-11, 12-15, 20-25, 27-30, 32, or 33. In one aspect, the nucleic acid has the sequence of any one of SEQ ID NOs: 41-50, which encode the TCR variable regions described below. [Table A] In one embodiment, the nucleic acid sequence may have one or more codon substitutions that do not change the sequence of the encoded polypeptide.

[0028] In some aspects, the disclosure also features nucleic acids encoding any of the TCR regions or chains of the disclosure. In some aspects, the nucleic acids can further comprise signaling components. In some aspects, the signaling components can include or exclude ZAP327 (SEQ ID NO: 17) or ZAP300 (SEQ ID NO: 16), or another signaling component derived from the ZAP70 kinase domain. In some aspects, the signaling components confer increased persistence and / or anti-tumor activity to cells engineered with these nucleic acids.

[0029] Also disclosed herein are compositions of one or more therapeutically effective amounts of the TCR alpha or beta variable regions of any of the preceding aspects or any aspect or embodiment disclosed herein. In some aspects, the compositions may also include any signaling component of any of the preceding aspects, including or excluding another signaling component derived from, for example, ZAP327 (SEQ ID NO: 17) or ZAP300 (SEQ ID NO: 16), or the ZAP70 kinase domain.

[0030] Also disclosed herein are compositions comprising a therapeutically effective amount of one or more TCR T cells, wherein the TCR T cells have been engineered to express any of the nucleic acids of any of the foregoing aspects or any aspect or embodiment disclosed herein. In some aspects, the TCR T cells can be engineered to express, for example, a receptor (which can include or exclude a T cell receptor) that recognizes one or more cancer antigens or neoantigens of any of the foregoing aspects or any aspect or embodiment disclosed herein. In some aspects, the TCR T cells can express one or more TCR alpha and / or one or more beta variable regions of any of the foregoing aspects of the disclosure or any aspect or embodiment disclosed herein. In some aspects, these TCR T cells can also include or exclude another signaling component derived from ZAP327 (SEQ ID NO: 17) or ZAP300 (SEQ ID NO: 16), or the ZAP70 kinase domain. In some aspects, engineered TCR T cells that include and / or express signaling components exhibit surprisingly high persistence and / or anti-tumor activity.

[0031] In one embodiment, the engineered or transduced T cells do not express endogenous TCR(α / β). For example, in some embodiments, the endogenous TCR(α / β) is knocked out using CRISPR technology, e.g., CRISPR / Cas9 technology (Legut, M., Dolton, G., Mian, A.A., Ottmann, O.G. & Sewell, A.K. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood 131, 311-322 (2018)), or CRISPR / Cas12a technology, prior to transduction with a cancer antigen-specific TCR construct.

[0032] In one aspect, nucleic acids encoding siRNAs, e.g., shRNAs, for knocking down genes to enhance the anti-tumor activity of TCR-transduced T cells in vivo are also disclosed herein. The nucleic acid sequence of the shRNA stem targets negative signaling molecules of the immune system, such as checkpoint proteins and / or immunosuppressive proteins. In some aspects, shRNA targets can include, but are not limited to, programmed cell death protein (PD1) (SEQ ID NO: 7), von Hippel-Lindau tumor suppressor (VHL) (SEQ ID NO: 8), and / or protein phosphatase 2 regulatory subunit B delta (PPP2R2D) (SEQ ID NO: 9). In some aspects, PPP2R2D mRNA sequences that can be targeted can include or exclude part or all of PPP2R2D transcript variant 1 (SEQ ID NO: 18); or PPP2R2D transcript variant 3 (SEQ ID NO: 19). In some embodiments, nucleic acids encoding antisense RNA or DNA can also be used to knock down genes or reduce expression of genes encoding negative signaling molecules. In some aspects, any of the nucleic acids of any of the foregoing aspects or embodiments described herein may also include any of these nucleic acids encoding siRNA / shRNA.

[0033] In one aspect, there is also disclosed herein a method of stimulating an immunological response against, or treating, inhibiting and / or preventing cancer, the method comprising administering to a subject a therapeutically effective amount of a composition comprising an epitope or TCR alpha or beta variable region of any preceding aspect or any aspect or embodiment disclosed herein, and / or identifying by a method of detecting or identifying epitope-specific T cells and TCRs of any preceding aspect or any aspect or embodiment disclosed herein.

[0034] In one aspect, the present disclosure also features T cells expressing a chimeric antigen receptor and a CAR. In some aspects, the CAR construct comprises an antigen recognition moiety (e.g., a single-chain variable fragment (ScFv)), a transmembrane domain, and an intracellular T cell activation moiety (consisting of a signaling domain, e.g., a CD28 or 4-1BB costimulatory signaling domain fused to a ZAP300 (SEQ ID NO: 16) or ZAP327 (SEQ ID NO: 17) signaling domain or other signaling domain from ZAP70. In some aspects, the CAR can include or exclude an antigen recognition moiety, e.g., an ScFv, that specifically binds, for example, CD19, BCMA, B7-H3, mesothelin, or HER-2.

[0035] In another aspect, one or more TCR alpha and / or beta variable regions of any of the above aspects or any aspect or embodiment disclosed herein may also further comprise a ZAP300 or ZAP327 moiety or other signaling moiety derived from the ZAP70 kinase domain.

[0036] In one aspect, the present disclosure also relates to a method of using any TCR-T cell or CAR-T cell of any of the above aspects or any aspect or embodiment disclosed herein in the treatment of cancer.

[0037] T cell recognition of target antigens is HLA-restricted, whereas chimeric antigen receptor (CAR)-T cell recognition of targets is not HLA-dependent. As disclosed herein, modulation of TCR-T cell and CAR-T signaling and function in vivo is crucial for prolonging T cell persistence (and reducing T cell exhaustion) by direct modulation of the TCR or CAR signaling domains or knockdown / knockout of negative signaling molecules, which can include or exclude PD-1, VHL, PPP2R2D, and epigenetic factors, which can include or exclude JMJD3 and LSD1.

[0038] In one aspect, the present disclosure also features methods and strategies for extending the persistence of TCR-T and CAR-T cells by direct manipulation of the TCR or CAR signaling domain or by knockdown / knockout of negative signaling molecules. In some aspects, the present disclosure also features methods for enhancing the persistence of CAR-T and TCR cells by expression of chemokine receptors and shRNA knockouts in the TCR or CAR construct. In some aspects, negative signaling molecules are, for example, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3). In certain embodiments, negative signaling molecules are epigenetic factors that may include or exclude, for example, PD-1, VHL, PPP2R2D, and JMJD3 and LSD1. In some embodiments, treatment with any of the engineered TCR T cells of the present disclosure, wherein the T cells contain and / or express signaling components and / or have negative signaling molecule knockdown, surprisingly reduces relapse / cancer recurrence after initial treatment and initial reduction in cancer / tumor burden.

[0039] In one aspect, the present disclosure also features a method for enhancing T cell trafficking to tumor cells in vivo by forced expression of a chemokine receptor. In some aspects, the expression of the chemokine receptor is forced by fusing any of the CAR or TCR constructs of any of the foregoing aspects or embodiments described herein with the chemokine receptor. In some aspects, the chemokine receptor is CCR5, CXCR3, and / or CCR2. In some aspects, the chemokine receptor is CCR5. In some aspects, expression of chemokine receptors and shRNA knockouts can be used in any of the TCR or CAR constructs of any of the foregoing aspects or embodiments described herein. In certain embodiments, for example, the following are provided: (Item 1) A composition comprising one or more polypeptides comprising an alpha variable region or one or more beta variable regions of a T cell receptor (TCR) specific for NY-ESO-1 (NY-ESO-1 TCR), CT83 (CT83-TCR), HCMV pp65 (HCMV pp65 TCR), or HCMV IE-1 (HCMV IE-1 TCR), or any combination thereof. (Item 2) (a) at least one polypeptide comprising an alpha chain or region of a T cell receptor specific for NY-ESO-1 (NY-ESO-1 TCR) and at least one polypeptide comprising a beta chain of a T cell receptor specific for NY-ESO-1 (NY-ESO-1 TCR); (b) at least one polypeptide comprising an alpha chain or region of a T cell receptor specific for CT83 (CT83-TCR) and at least one polypeptide comprising a beta chain of a T cell receptor specific for CT83 (CT83-TCR); (c) at least one polypeptide comprising an alpha chain or region of a T cell receptor specific for HCMV pp65 (HCMV pp65 TCR) and at least one polypeptide comprising a beta chain of a T cell receptor specific for pp65 (pp65 TCR); and (d) at least one polypeptide comprising an alpha chain or region of a T cell receptor specific for HCMV IE-1 (HCMV IE-1-TCR) and at least one polypeptide comprising a beta chain of a T cell receptor specific for HCMV IE-1 (HCMV IE-1-TCR). and at least one polypeptide comprising the beta chain of TCR (IE-1-TCR). (Item 3) The composition comprises the amino acid sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQGSTLGRLYFGRGTQLTVWPD (SEQ ID NO: 27) or MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTG a polypeptide comprising TSLTVIPN (SEQ ID NO:29), a fragment or variant thereof which binds to an antigen with the same specificity as a reference (full length and unmodified) receptor, or a polypeptide comprising an amino acid sequence with 85%, 90%, or 95% homology to a polypeptide having the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29, or having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29, or having a sequence with one or two conservative amino acid substitutions to an amino acid sequence with 95% homology to the sequence of SEQ ID NO:27 or SEQ ID NO:29; and comprising the alpha variable region of an HLA-A2-restricted HCMV pp65 TCR comprising Optionally, the composition further comprises the amino acid sequence MLSPDLPDSAWNTRLLCRVMLCLLGAGSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSGYHSELNMSSLELGDSALYFCASSLENNQPQHFGDGTRLSILE (SEQ ID NO: 28) or MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPIT a polypeptide comprising the beta variable region of an HLA-A2 restricted HCMV pp65 TCR, comprising a polypeptide comprising GTGDYGYTFGSGTRLTVVE (SEQ ID NO: 30), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full length and unmodified) receptor, a sequence having 85%, 90% or 95% homology to a polypeptide comprising the sequence of SEQ ID NO: 28 or SEQ ID NO: 30, or having one or two conservative amino acid substitutions to that of SEQ ID NO: 28 or SEQ ID NO: 30, or an amino acid sequence having 95% homology to the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30, 2. The composition of item 1, wherein the TCR optionally further comprises SEQ ID NO: 27 and SEQ ID NO: 28, or further optionally comprises SEQ ID NO: 29 and SEQ ID NO: 30. (Item 4) The composition comprises the alpha and beta variable regions of a TCR specific for NY-ESO-1 or CT83, and when the TCR is specific for NY-ESO-1, the alpha variable region optionally comprises the amino acid sequence METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY (SEQ ID NO: 3). TCR polypeptides, fragments or variants thereof that bind to antigen with the same specificity as a reference (full-length and unmodified) receptor, polypeptides comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO:3, polypeptides having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:3, or polypeptides having one or two conservative amino acid substitutions to an amino acid sequence having 95% homology to the sequence of SEQ ID NO:3, and DP4-ESO-1 TCR the beta variable region optionally comprises a polypeptide comprising the amino acid sequence MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE (SEQ ID NO: 4), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 4, a polypeptide with one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 4, or a polypeptide with one or two conservative amino acid substitutions to an amino acid sequence with 95% homology to the sequence of SEQ ID NO: 4, and and wherein the alpha variable region optionally comprises an A2-CT83 TCR, a polypeptide comprising the amino acid sequence MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 5), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 5, a polypeptide with one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 5, or a polypeptide with one or two conservative amino acid substitutions to an amino acid sequence with 95% homology to the sequence of SEQ ID NO: 5, 2. The composition of claim 1, wherein the beta variable region of the TCR optionally comprises an A2-CT83 TCR polypeptide comprising the amino acid sequence MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE (SEQ ID NO: 6), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full-length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 6, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 6, or a polypeptide having one or two conservative amino acid substitutions to an amino acid sequence with 95% homology to the sequence of SEQ ID NO: 6. (Item 5) The composition comprises an alpha variable region of an HLA-A2-restricted HCMV IE-1 TCR, wherein the alpha variable region of an HLA-A2-restricted HCMV IE-1 TCR is a polypeptide comprising the amino acid sequence MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGHIYGGSQGNLIFGKGTKLSVKPN (SEQ ID NO: 32), a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 32, a polypeptide having one or two conservative amino acid substitutions relative to SEQ ID NO: 32, or a polypeptide having one or two conservative amino acid substitutions relative to an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 32, and optionally the composition is an HLA-A2-restricted HCMV IE-1 TCR. 2. The composition of item 1, further comprising a beta variable region of a TCR, wherein the beta variable region of the HLA-A2-restricted IE-1 TCR comprises a polypeptide comprising the amino acid sequence MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHHQGPLETQYFGPGTRLLVLE (SEQ ID NO: 33), a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% identity to the amino acid sequence of sequence of SEQ ID NO: 336, a polypeptide having one or two conservative amino acid substitutions relative to SEQ ID NO: 33, or a polypeptide having one or two conservative amino acid substitutions relative to that of an amino acid sequence having 95% identity to the amino acid sequence of sequence of SEQ ID NO: 33. (Item 6) A. A chimeric TCR polypeptide comprising a cancer antigen-specific TCR variable region fused to a constant region selected from a modified human TCR alpha or beta constant region and a non-human TCR alpha or beta constant region, optionally a murine TCR alpha or beta constant region, wherein the alpha and beta variable regions of the TCR variable region fused to the modified or non-human alpha or beta constant chain region are: a. Any combination of alpha and beta TCR variable regions according to either item 3 or 5; or b. DP4-ESO-1 comprising the alpha and beta variable regions of a TCR specific for a cancer antigen selected from NY-ESO-1 or CT83, and if the TCR is specific for NY-ESO-1, the alpha variable region optionally comprising the amino acid sequence METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY (SEQ ID NO: 3). TCR polypeptides, fragments or variants thereof that bind to antigen with the same specificity as a reference (full-length and unmodified) receptor, polypeptides comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO:3, polypeptides having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:3, or polypeptides having one or two conservative amino acid substitutions to an amino acid sequence having 95% homology to the sequence of SEQ ID NO:3, and DP4-ESO-1 TCR optionally, the beta variable region of comprises a polypeptide comprising the amino acid sequence MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE (SEQ ID NO: 4), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 4, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 4, or a polypeptide having one or two conservative amino acid substitutions to an amino acid sequence having 95% homology to the sequence of SEQ ID NO: 4, and c. If the TCR is specific for CT83, the alpha variable region optionally comprises an A2-CT83 TCR, a polypeptide comprising the amino acid sequence MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 5), a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full-length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 5, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 5, or a polypeptide having one or two conservative amino acid substitutions to an amino acid sequence having 95% homology to the sequence of SEQ ID NO: 5, A2-CT83, wherein the beta variable region of the TCR optionally comprises the amino acid sequence MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE (SEQ ID NO: 6). Chimeric TCR polypeptides, including a TCR polypeptide, a fragment or variant thereof that binds to an antigen with the same specificity as a reference (full-length and unmodified) receptor, a polypeptide comprising an amino acid sequence having 85%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 6, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 6, or a polypeptide having one or two conservative amino acid substitutions to an amino acid sequence having 95% homology to the sequence of SEQ ID NO: 6. (Item 7) The alpha chain constant region is selected from a modified human TCR alpha constant chain region (TRAC) comprising the sequence IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS) (SEQ ID NO: 10), and a mouse alpha chain constant region (trac) comprising the sequence IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 13). and, The beta chain constant region is a modified human TCR beta constant (co) having the sequence: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG) (SEQ ID NO: 12). A modified human TCR beta constant region type 1 (T constant region type 2), with the sequence: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF: (SEQ ID NO: 11) RBC1), mouse beta chain constant region type 1 (trbc1) having the sequence DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 14), and The chimeric TCR receptor of item 6, wherein the chimeric TCR receptor is selected from a mouse beta chain constant region type 2 (trbc2) having PPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 15). (Item 8) 8. The chimeric TCR receptor of item 7, wherein the TCR is specific for a cancer antigen selected from NY-ESO-1, CT83 ("CT83-TCR"), HCMV PP65, and HCMV IE1. (Item 9) The chimeric TCR receptor is specific for CT83 and comprises a chimeric alpha chain comprising an HLA-A2-restricted CT83 TCR alpha chain variable region fused to a mouse alpha constant domain, the chimeric TCR receptor comprising a polypeptide selected from a polypeptide having SEQ ID NO:20, a polypeptide comprising an amino acid sequence having 95% sequence identity to the amino acid sequence of SEQ ID NO:20, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:20, and a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence having 95% sequence identity to the sequence of SEQ ID NO:20; and the chimeric TCR receptor specific for CT83 comprises an HLA-A2-restricted CT83 TCR alpha chain variable region fused to a mouse beta constant domain selected from a polypeptide having SEQ ID NO:21, a polypeptide having 95% sequence identity to SEQ ID NO:21, a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:21, and a polypeptide having one or two conservative amino acid substitutions to the amino acid sequence having 95% sequence identity to the sequence of SEQ ID NO:21. 8. The chimeric TCR receptor of item 7, further comprising a chimeric beta chain comprising a TCR beta chain variable domain. (Item 10) the chimeric TCR receptor is specific for NY-ESO-1; HLA-A2 restricted NY-ESO-1 TCR(S2) alpha chain variable domain fused to a murine alpha constant domain having SEQ ID NO: 22; a polypeptide having 95% sequence identity to SEQ ID NO: 22; A polypeptide having one or two conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 22; a polypeptide having one or two conservative amino acid substitutions relative to an amino acid sequence having 95% sequence identity with the sequence of SEQ ID NO: 22; HLA-A2 restricted NY-ESO-1 TCR(S5) alpha chain variable domain fused to a murine alpha constant domain having SEQ ID NO: 24; a polypeptide having 95% sequence identity to SEQ ID NO: 24; a polypeptide having one or two conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:24, and a polypeptide having one or two conservative amino acid substitutions relative to an amino acid sequence having 95% sequence identity with the sequence of SEQ ID NO:24; and The chimeric TCR receptor specific for NY-ESO-1 is HLA-A2 restricted NY-ESO-1 TCR(S2)(G50A, A51E) beta chain variable domain fused to murine beta constant domain 2 comprising SEQ ID NO: 23; a polypeptide having 95% sequence identity to SEQ ID NO: 23; A polypeptide having one or two conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 23; A polypeptide having one or two conservative amino acid substitutions relative to an amino acid sequence having 95% sequence identity with the sequence of SEQ ID NO: 23; HLA-A2 restricted NY-ESO-1 TCR(S5)(G50A, A51E, A97L) beta chain variable domain fused to murine beta constant domain 2 with SEQ ID NO: 25 a polypeptide having 95% sequence identity to SEQ ID NO: 25; A polypeptide having one or two conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 25, and 8. The chimeric TCR receptor of item 7, further comprising a chimeric beta chain selected from polypeptides having one or two conservative amino acid substitutions relative to an amino acid sequence having 95% sequence identity to the sequence of SEQ ID NO: 25. (Item 11) 11. The chimeric TCR receptor of any one of items 6 to 9 or 10, wherein the TCR is fused to a signaling moiety optionally selected from ZAP300 (SEQ ID NO: 16) or ZAP327 (SEQ ID NO: 17). (Item 12) 11. A nucleic acid, vector, or cell comprising the nucleic acid or vector encoding any of the sequences of the polypeptides in the composition or chimeric TCR of any one of items 6, 9, or 10, and optionally further encoding a signaling component, wherein the signaling component is optionally selected from ZAP300 (SEQ ID NO: 16) or ZAP327 (SEQ ID NO: 17). (Item 13) 11. A composition comprising a therapeutically effective amount of one or more TCR T cells, wherein the TCR T cells are engineered to express any of the TCR receptors of items 6, 9 or 10. (Item 14) i6 An isolated nucleic acid encoding an shRNA for knocking down a gene for enhancing the anti-tumor activity of TCR-transduced T cells in vivo, wherein the nucleic acid sequence of the shRNA targets a negative signaling molecule of the immune system, optionally selected from a checkpoint protein and / or an immunosuppressive protein. (Item 15) 15. The isolated nucleic acid of item 14, wherein the shRNA target is selected from programmed cell death protein (PD1), (SEQ ID NO: 7), von Hippel-Lindau tumor suppressor (VHL) (SEQ ID NO: 8), and / or protein phosphatase 2 regulatory subunit B delta (PPP2R2D) (SEQ ID NO: 9). (Item 16) 11. A method of stimulating an immunological response against cancer or treating, inhibiting and / or preventing cancer, the method comprising administering to a subject a composition comprising a therapeutically effective amount of a composition comprising a TCR alpha or beta variable region polypeptide of any of paragraphs 6, 9 or 10. (Item 17) 1. A composition comprising a chimeric antigen receptor or T cells expressing a CAR, wherein the CAR comprises an antigen recognition portion, a transmembrane domain, and an intracellular T cell activation portion, wherein the intracellular T cell activation portion is optionally selected from a CD28 or 4-1BB costimulatory signaling domain fused to a signaling domain, and further wherein the signaling domain is optionally selected from ZAP300 (SEQ ID NO: 16) or ZAP327 (SEQ ID NO: 17), and wherein the antigen recognition portion is optionally a single chain variable fragment (ScFv). (Item 18) 18. A method of treating cancer in a subject having or suspected of having cancer by administering to the subject a composition comprising the TCR-T T cells or CAR-T T cells of item 13 or 17. (Item 19) A method for prolonging T cell persistence or reducing T cell exhaustion in a subject by modulating TCR-T cell signaling and function by administering to the subject a composition comprising the TCR-T cells or CAR-T cells of item 13 or 17. (Item 20) 20. The method of paragraph 19, wherein the TCR or CAR signaling domain is regulated or knocked down by a negative signaling molecule selected from PD-1, VHL, PPP2R2D, and epigenetic factors, which can include or exclude JMJD3 and LSD1. (Item 21) 21. The method of paragraph 20, wherein the TCR or CAR signaling domain is regulated or knocked down by a negative signaling molecule selected from PD-1, VHL, PPP2R2D, and epigenetic factors, which can include or exclude JMJD3 and LSD1. (Item 22) 1. A method for extending the persistence of TCR-T and CAR-T cells by direct manipulation of the TCR or CAR signaling domain or by knockdown / knockout of negative signaling molecules, wherein the negative signaling molecules are selected from indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death-ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3). (Item 23) 23. The method of item 22, wherein the negative signaling molecules are epigenetic factors that may include or exclude PD-1, VHL, PPP2R2D, and JMJD3 and LSD1. (Item 24) 24. The method of claim 23, further comprising forcing expression of a chemokine receptor, thereby enhancing T cell trafficking to tumor cells, wherein forcing expression of a chemokine receptor comprises fusing the CAR or TCR with a chemokine receptor, optionally selected from CCR5, CCR2, and CXCR3. [Brief explanation of the drawings]

[0040] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods.

[0041] [Figure 1] Figure 1 shows the generation and characterization of single T cell clones from an HLA-DP4-presenting NY-ESO-1-reactive T cell line. T cell clones were isolated from the HLA-DP4-presenting NY-ESO-1-reactive cell line. After expansion of each T cell clone, antigen recognition was screened using a peptide containing amino acids 157 to 170 of NY-ESO-1 (SEQ ID NO: 1) presented by HLA-DP4-positive APCs.

[0042] [Figure 2] FIG. 2 shows a map of the construction of the DP4-ESO-1 TCR from one T cell clone into the pMSGV vector.

[0043] [Figure 3A-3B] Figures 3A and 3B show the transduction of DP4-ESO-1 TCR in naive CD4+ T cells. Figure 3A shows the DP4-ESO-1 TCR transduction efficiency in naive CD4+ T cells measured by TCR-specific antibody staining and flow cytometry. Naive CD4+ T cells were transduced with retroviral supernatants produced from different DP4-ESO-1 TCR PG-13 clones. After transduction, DP4-ESO-1 TCR expression was tested, and the average transduction efficiency was 60-70%. Figure 3B shows functional testing of DP4-ESO-1 TCR-transduced CD4+ T cells.

[0044] [Figures 4A-4C]Figures 4A, 4B, and 4C show functional characterization of DP4-ESO-1 TCR-T cells. Figure 4A shows T cell recognition of peptides. DP4-ESO-1 TCR-T cells recognized the NY-ESO-1 157-170 peptide presented by HLA-DP4+ cells. Figure 4B shows T cell recognition of naturally processed NY-ESO-1. DP4-ESO-1 TCR-T cells recognized 293T cells transfected with full-length NY-ESO-1, HLA-DPAl, and HLA-DP4. Figure 4C shows that the DP4-ESO-1 TCR functioned only in CD4+ T cells. Compared to CD8+ T cells transduced with the DP4-ESO-1 TCR, only CD4+ T cells recognized NY-ESO-1 157-170, indicating that TCR function was restricted to CD4+ T cells.

[0045] [Figure 5A] Figures 5A, 5B, and 5C show the improved in vivo antitumor function of DP4-ESO-1 TCR in combination with A2-ESO-1 TCR against MDA-MB-231 / DP4 / ESO. Figure 5A shows the migration of A2-ESO-1 TCR (luciferase-labeled) transduced CD8+ T cells injected in vivo, tracked by luciferase imaging. Figure 5B shows the monitoring of in vivo growth of MDA-MB-231 / DP4 / ESO treated with different groups of T cells. Figure 5C shows a comparison of tumor size treated with different groups of T cells when the mice were sacrificed. [Figure 5B-5C]Figures 5A, 5B, and 5C show the improved in vivo antitumor function of DP4-ESO-1 TCR in combination with A2-ESO-1 TCR against MDA-MB-231 / DP4 / ESO. Figure 5A shows the migration of A2-ESO-1 TCR (luciferase-labeled) transduced CD8+ T cells injected in vivo, tracked by luciferase imaging. Figure 5B shows the monitoring of in vivo growth of MDA-MB-231 / DP4 / ESO treated with different groups of T cells. Figure 5C shows a comparison of tumor size treated with different groups of T cells when the mice were sacrificed.

[0046] [Figures 6A-6B] Figures 6A, 6B, 6C and 6D show the generation and characterization of HLA-A2 restricted CT83-specific T cells. Figure 6A shows that in vitro peptide-stimulated T cells were generated and tested for their ability to recognize 293T / CT83 PEP90-98 (a peptide containing amino acids 90-98 of CT83 (SEQ ID NO: 2)) compared with 293T / control peptide. Figure 6B shows that A2-CT83-specific T cells recognized 293T cells transfected with Ii-CT83, CT83-GFP plasmid DNA, or pulsed with CT83 PEP9-98 (a positive control), but not control 293T cells. Figure 6C shows that A2-CT83-specific T cells were tested for their ability to recognize the human breast cancer cell line MDA-MB-231 (expressing HLA-A2 and CT83), but not MDA-MB-436 (HL-A2-CT83). Figure 6D shows that recognition of MDA-MB-231 cells can be blocked by anti-MHC I antibodies, but not anti-MHC II antibodies. [Figure 6C-6D]Figures 6A, 6B, 6C and 6D show the generation and characterization of HLA-A2 restricted CT83-specific T cells. Figure 6A shows that in vitro peptide-stimulated T cells were generated and tested for their ability to recognize 293T / CT83 PEP90-98 (a peptide containing amino acids 90-98 of CT83 (SEQ ID NO: 2)) compared with 293T / control peptide. Figure 6B shows that A2-CT83-specific T cells recognized 293T cells transfected with Ii-CT83, CT83-GFP plasmid DNA, or pulsed with CT83 PEP9-98 (a positive control), but not control 293T cells. Figure 6C shows that A2-CT83-specific T cells were tested for their ability to recognize the human breast cancer cell line MDA-MB-231 (expressing HLA-A2 and CT83), but not MDA-MB-436 (HL-A2-CT83). Figure 6D shows that recognition of MDA-MB-231 cells can be blocked by anti-MHC I antibodies, but not anti-MHC II antibodies.

[0047] [Figures 7A-7B] Figures 7A and 7B show that vaccination with CT83-PEP90-98 inhibits breast cancer cells. Figure 7A shows a schematic diagram of the experimental design and schedule. Figure 7B shows that tumor-bearing mice were treated by intravenous vaccination with TAT-CT83 PEP90-98-CMI nanoparticles but not with TAT-CT83 PEP66-74-CMI. (CMI stands for CpG, MPLA, and poly(I:C).) **P value < 0.01.

[0048] [Figure 8A-8B]Figures 8A, 8B, 8C, 8D, and 8E show the construction and characterization of HLA-A2-restricted CT83-specific TCRs. Figure 8A shows a flowchart of TCR sequencing, cloning, and construction from a FACS-purified A2-CT83-specific T cell population using 10x single-cell barcoding technology and next-generation sequencing. Figure 8B shows functional analysis of A2-CT83 TCR-transduced T cells and vector-transduced PBMCs against 293T, 293T / CT83-GFP, Cos-7 and Cos-7 / Ii-CT83, and Cos-7-A2 / Ii-CT83 cells. Figure 8C shows that A2-CT83 TCR-T cells specifically recognized 293T cells pulsed with CT83 PEP90-98 compared to 293T cells pulsed with other CT83 peptides. Figure 8D shows that A2-CT83 TCR-T cells specifically recognized MDA-MB-231 cells (expressing CT83 and HLA-A2) but did not recognize MCF7 (A2+CT83-), HTB-2 (A2+CT83-), or MDA-MB-436 (A2-CT83+) cells that expressed only one of the polypeptides. Figure 8E shows that A2-CT83 TCR-T cells were able to recognize CT83+ and HLA-A2+ lung cancer cells (HOP92 / A2, NCI-H358 / A, and NCI-H838 / A2) but were unable to recognize CT83+HLA-A2- tumor cells (HOP92, NCI-H358, and NCI-838). These results demonstrate that the A2-CT83 TCR can specifically recognize the naturally processed CT83 epitope presented by HLA-A2 molecules. [Figure 8C-8D]Figures 8A, 8B, 8C, 8D, and 8E show the construction and characterization of HLA-A2-restricted CT83-specific TCRs. Figure 8A shows a flowchart of TCR sequencing, cloning, and construction from a FACS-purified A2-CT83-specific T cell population using 10x single-cell barcoding technology and next-generation sequencing. Figure 8B shows functional analysis of A2-CT83 TCR-transduced T cells and vector-transduced PBMCs against 293T, 293T / CT83-GFP, Cos-7 and Cos-7 / Ii-CT83, and Cos-7-A2 / Ii-CT83 cells. Figure 8C shows that A2-CT83 TCR-T cells specifically recognized 293T cells pulsed with CT83 PEP90-98 compared to 293T cells pulsed with other CT83 peptides. Figure 8D shows that A2-CT83 TCR-T cells specifically recognized MDA-MB-231 cells (expressing CT83 and HLA-A2) but did not recognize MCF7 (A2+CT83-), HTB-2 (A2+CT83-), or MDA-MB-436 (A2-CT83+) cells that expressed only one of the polypeptides. Figure 8E shows that A2-CT83 TCR-T cells were able to recognize CT83+ and HLA-A2+ lung cancer cells (HOP92 / A2, NCI-H358 / A, and NCI-H838 / A2) but were unable to recognize CT83+HLA-A2- tumor cells (HOP92, NCI-H358, and NCI-838). These results demonstrate that the A2-CT83 TCR can specifically recognize the naturally processed CT83 epitope presented by HLA-A2 molecules. [Figure 8E]Figures 8A, 8B, 8C, 8D, and 8E show the construction and characterization of HLA-A2-restricted CT83-specific TCRs. Figure 8A shows a flowchart of TCR sequencing, cloning, and construction from a FACS-purified A2-CT83-specific T cell population using 10x single-cell barcoding technology and next-generation sequencing. Figure 8B shows functional analysis of A2-CT83 TCR-transduced T cells and vector-transduced PBMCs against 293T, 293T / CT83-GFP, Cos-7 and Cos-7 / Ii-CT83, and Cos-7-A2 / Ii-CT83 cells. Figure 8C shows that A2-CT83 TCR-T cells specifically recognized 293T cells pulsed with CT83 PEP90-98 compared to 293T cells pulsed with other CT83 peptides. Figure 8D shows that A2-CT83 TCR-T cells specifically recognized MDA-MB-231 cells (expressing CT83 and HLA-A2) but did not recognize MCF7 (A2+CT83-), HTB-2 (A2+CT83-), or MDA-MB-436 (A2-CT83+) cells that expressed only one of the polypeptides. Figure 8E shows that A2-CT83 TCR-T cells were able to recognize CT83+ and HLA-A2+ lung cancer cells (HOP92 / A2, NCI-H358 / A, and NCI-H838 / A2) but were unable to recognize CT83+HLA-A2- tumor cells (HOP92, NCI-H358, and NCI-838). These results demonstrate that the A2-CT83 TCR can specifically recognize the naturally processed CT83 epitope presented by HLA-A2 molecules.

[0049] [Figures 9A-9C]Figures 9A, 9B, and 9C show the antitumor activity of A2-CT83 TCR-T cells in vivo. Figure 9A shows a diagram of the administration schedule (injections) of NCI-H838 tumor cells and A2-CT83 TCR-T cells in an NSG mouse model. Figures 9B and 9C show the inhibition of tumor growth by A2-CT83 TCR-T cells in vivo. In contrast, tumor-bearing mice treated with control T cells developed large tumor masses. These studies suggest that A2-CT83 TCR-T cells have potent antitumor activity in vivo.

[0050] [Figures 10A-10B] Figures 10A and 10B show the generation and characterization of HLA-A2-restricted HCMV (pp65 and IE-1 proteins)-specific T cell clones. Figure 10A shows that seven T cell clones (495-503) reactive to pp65 and five T cell clones (316-324) reactive to IE-1 were selected, screened with peptide-pulsed T2 cells, and expanded in vitro. Figure 10B shows the characterization of HLA-restriction of pp65 T cell clone #3 and IE-1 T cell clone #5. Both T cell clones were able to specifically recognize HLA-A2-presented pp65 and IE-1 antigens, respectively, on Cos-7 cells.

[0051] [Figures 11A-11C]Figures 11A, 11B, 11C, 11D, and 11E show the identification, cloning, and characterization of HLA-A2-restricted pp65- and IE-1-specific TCRs and their functions in TCR-transduced human T cells. Figure 11A shows the transduction efficiency of A2-pp65 TCR and A2-IE-1 TCR in human CD8+ T cells isolated from HCMV-seronegative donors. Figure 11B shows that A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells specifically recognized glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or glioblastoma cells infected with HCMV. Figure 11C shows the dose-dependent recognition of A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells against T2 cells pulsed with pp65 (495-503) or IE-1 (316-324) peptides, respectively. Figure 11D shows that A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells specifically killed glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or HCMV-infected glioblastoma cells. Figure 11E shows the dose-dependent cytotoxicity of HCMV / AD169-infected U87 tumor cells by A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells. [Figures 11D-11E]Figures 11A, 11B, 11C, 11D, and 11E show the identification, cloning, and characterization of HLA-A2-restricted pp65- and IE-1-specific TCRs and their functions in TCR-transduced human T cells. Figure 11A shows the transduction efficiency of A2-pp65 TCR and A2-IE-1 TCR in human CD8+ T cells isolated from HCMV-seronegative donors. Figure 11B shows that A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells specifically recognized glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or glioblastoma cells infected with HCMV. Figure 11C shows the dose-dependent recognition of A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells against T2 cells pulsed with pp65 (495-503) or IE-1 (316-324) peptides, respectively. Figure 11D shows that A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells specifically killed glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or HCMV-infected glioblastoma cells. Figure 11E shows the dose-dependent cytotoxicity of HCMV / AD169-infected U87 tumor cells by A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells.

[0052] [Figures 12A-12B]Figures 12A, 12B, 12C, and 12D show the antitumor activity of A2-pp65 TCR-T cells in vivo. U87 cells expressing pp65 or IE-I together with luciferase were used to establish a transplanted tumor model in immunodeficient mice. Tumor cells were grown in SCID / beige for 3 days and then treated by adoptive transfer of A2-pp65 TCR, A2-IE-1 TCR, or control TCR-transduced human T cells via intravenous injection of 2 x 10 T cells per mouse. Figure 12A shows a diagram of the procedure for in vivo functional analysis of A2-pp65 TCR-T cells. Figure 12B shows the migration of A2-pp65 TCR-T cells after injection into tumor-bearing mice. Figure 12C shows that A2-pp65 TCR-T cells specifically inhibited tumor growth of pp65-expressing U87 tumors in vivo. Figure 12D shows that tumor weight was dramatically reduced after treatment with A2-pp65 TCR-T cells, suggesting the potential antitumor activity of pp65 TCR-T cells in treating glioblastoma. [Figures 12C-12D] Figures 12A, 12B, 12C, and 12D show the antitumor activity of A2-pp65 TCR-T cells in vivo. U87 cells expressing pp65 or IE-I together with luciferase were used to establish a transplanted tumor model in immunodeficient mice. Tumor cells were grown in SCID / beige for 3 days and then treated by adoptive transfer of A2-pp65 TCR, A2-IE-1 TCR, or control TCR-transduced human T cells via intravenous injection of 2 x 10 T cells per mouse. Figure 12A shows a diagram of the procedure for in vivo functional analysis of A2-pp65 TCR-T cells. Figure 12B shows the migration of A2-pp65 TCR-T cells after injection into tumor-bearing mice. Figure 12C shows that A2-pp65 TCR-T cells specifically inhibited tumor growth of pp65-expressing U87 tumors in vivo. Figure 12D shows that tumor weight was dramatically reduced after treatment with A2-pp65 TCR-T cells, suggesting the potential antitumor activity of pp65 TCR-T cells in treating glioblastoma.

[0053] [Figures 13A-13B]Figures 13A, 13B, 13C, and 13D show the antitumor activity of A2-IE-1 TCR-T cells in vivo. Figure 13A shows a diagram of the procedure for in vivo functional analysis of A2-IE-1 TCR-T cells. Figure 13B shows the migration of A2-IE-1 TCR-T cells after injection into tumor-bearing mice. Figure 13C shows that A2-IE-1 TCR-T cells specifically inhibited tumor growth of U87 cells expressing IE-1 tumors in vivo. Figure 13D shows that tumor weight was dramatically reduced after treatment with A2-IE-1 TCR-T cells, suggesting the potential antitumor activity of A2-IE-1 TCR-T cells in treating glioblastoma. [Figures 13C-13D] Figures 13A, 13B, 13C, and 13D show the antitumor activity of A2-IE-1 TCR-T cells in vivo. Figure 13A shows a diagram of the procedure for in vivo functional analysis of A2-IE-1 TCR-T cells. Figure 13B shows the migration of A2-IE-1 TCR-T cells after injection into tumor-bearing mice. Figure 13C shows that A2-IE-1 TCR-T cells specifically inhibited tumor growth of U87 cells expressing IE-1 tumors in vivo. Figure 13D shows that tumor weight was dramatically reduced after treatment with A2-IE-1 TCR-T cells, suggesting the potential antitumor activity of A2-IE-1 TCR-T cells in treating glioblastoma.

[0054] [Figures 14A-14C]Figures 14A, 14B, 14C, 14D, 14E, 14F, and 14G show enhancement of A2-ESO-1 TCR-T cell surface expression and function in human T cells by mouse constant sequences. Figure 14A shows a schematic diagram of the conventional and modified A2-ESO-1 TCR constructs of the present disclosure. Figure 14B shows that the modified TCR of the present disclosure has higher transduction efficiency than the conventional TCR construct. Figure 14C shows that TCR cell surface expression was detected by FACS. Figure 14D shows that TCR cell surface expression was detected by confocal microscopy. Figure 14E shows the results of an LDH assay to detect the target cell-killing ability of conventional TCR-T cells and modified TCR-T cells. Figure 14F shows that cytokine secretion was detected by ELISA after co-culture with A2-ESO-1-positive breast cancer cells. FIG. 14G shows that the long-term tumor cell killing ability of a representative composition of the present disclosure was detected by in vitro co-culture. [Figure 14D-14E] Figures 14A, 14B, 14C, 14D, 14E, 14F, and 14G show enhancement of A2-ESO-1 TCR-T cell surface expression and function in human T cells by mouse constant sequences. Figure 14A shows a schematic diagram of the conventional and modified A2-ESO-1 TCR constructs of the present disclosure. Figure 14B shows that the modified TCR of the present disclosure has higher transduction efficiency than the conventional TCR construct. Figure 14C shows that TCR cell surface expression was detected by FACS. Figure 14D shows that TCR cell surface expression was detected by confocal microscopy. Figure 14E shows the results of an LDH assay to detect the target cell-killing ability of conventional TCR-T cells and modified TCR-T cells. Figure 14F shows that cytokine secretion was detected by ELISA after co-culture with A2-ESO-1-positive breast cancer cells. FIG. 14G shows that the long-term tumor cell killing ability of a representative composition of the present disclosure was detected by in vitro co-culture. [Fig. 14F-14G]Figures 14A, 14B, 14C, 14D, 14E, 14F, and 14G show enhancement of A2-ESO-1 TCR-T cell surface expression and function in human T cells by mouse constant sequences. Figure 14A shows a schematic diagram of the conventional and modified A2-ESO-1 TCR constructs of the present disclosure. Figure 14B shows that the modified TCR of the present disclosure has higher transduction efficiency than the conventional TCR construct. Figure 14C shows that TCR cell surface expression was detected by FACS. Figure 14D shows that TCR cell surface expression was detected by confocal microscopy. Figure 14E shows the results of an LDH assay to detect the target cell-killing ability of conventional TCR-T cells and modified TCR-T cells. Figure 14F shows that cytokine secretion was detected by ELISA after co-culture with A2-ESO-1-positive breast cancer cells. FIG. 14G shows that the long-term tumor cell killing ability of a representative composition of the present disclosure was detected by in vitro co-culture.

[0055] [Figures 15A-15B] Figures 15A, 15B, 15C, and 15D show that modified A2-ESO-1-specific TCR-T cells have better therapeutic efficacy in preclinical breast cancer models. Figure 15A shows a schematic diagram of the animal experiment. 1x106 cells of the NY-ESO-1-positive breast cancer line MDA-MB-231 (ESO-1+) were subcutaneously injected into the NSG fat pad. A2-ESO-1 TCR-T / A2-ESO-1 TCR-MT cells were intravenously injected into tumor-bearing mice, followed by administration of three doses of IL-2. Figure 15B shows tumor growth tracking. Figure 15C shows tumor images after sacrifice. Figure 15D shows tumor weight after sacrifice. [Figures 15C-15D]Figures 15A, 15B, 15C, and 15D show that modified A2-ESO-1-specific TCR-T cells have better therapeutic efficacy in preclinical breast cancer models. Figure 15A shows a schematic diagram of the animal experiment. 1x106 cells of the NY-ESO-1-positive breast cancer line MDA-MB-231 (ESO-1+) were subcutaneously injected into the NSG fat pad. A2-ESO-1 TCR-T / A2-ESO-1 TCR-MT cells were intravenously injected into tumor-bearing mice, followed by administration of three doses of IL-2. Figure 15B shows tumor growth tracking. Figure 15C shows tumor images after sacrifice. Figure 15D shows tumor weight after sacrifice.

[0056] [Figure 16A] Figures 16A, 16B, and 16C show in vitro tumor killing of A2-ESO-1 TCRs with amino acid substitutions and mouse TCR constant sequences. Figure 16A shows five substitutions of the A2-ESO-1 TCR and the original A2-ESO-1 TCR transduced into human T cells and tested for their ability to recognize tumor cells with or without HLA-A2 and NY-ESO-1 expression. Figure 16B shows the cytotoxicity of substituted A2-ESO-1 TCR-T cells against tumor cells with or without HLA-A2 and NY-ESO-1 expression. S2 and S5 of the A2-ESO-1 TCR-transduced T cells showed higher cytolytic activity. Figure 16C shows the replacement of the human TCR constant regions of S2 and S5 of the A2-ESO-1 TCR and the original A2-ESO-1 TCR with mouse TCR constant regions. The S2 variant of the A2-ESO-1 TCR, which has a murine TCR constant region sequence, elicited potent T cell responses. [Figures 16B-16C]Figures 16A, 16B, and 16C show in vitro tumor killing of A2-ESO-1 TCRs with amino acid substitutions and mouse TCR constant sequences. Figure 16A shows five substitutions of the A2-ESO-1 TCR and the original A2-ESO-1 TCR transduced into human T cells and tested for their ability to recognize tumor cells with or without HLA-A2 and NY-ESO-1 expression. Figure 16B shows the cytotoxicity of substituted A2-ESO-1 TCR-T cells against tumor cells with or without HLA-A2 and NY-ESO-1 expression. S2 and S5 of the A2-ESO-1 TCR-transduced T cells showed higher cytolytic activity. Figure 16C shows the replacement of the human TCR constant regions of S2 and S5 of the A2-ESO-1 TCR and the original A2-ESO-1 TCR with mouse TCR constant regions. The S2 variant of the A2-ESO-1 TCR, which has a murine TCR constant region sequence, elicited potent T cell responses.

[0057] [Figure 17A] Figures 17A, 17B, and 17C show the antitumor activity of A2-CT83 TCR-MT cells (murine constant region). Figure 17A shows the transduction efficiency of A2-CT83 TCR-M in human T cells. Figure 17B shows that A2-CT83 TCR-MT cells specifically recognized MDA-MB-231 and NCI-H1563 cells (expressing CT83 and HLA-A2), but not CAMA-1 cells (A2+CT83-). Figure 17C shows the cytotoxicity of A2-CT83 TCR-MT cells against MDA-MB-231 and NCI-H1563 cells. These results demonstrate that A2-CT83 TCR-M T cells are potent and specific against tumor cells, with reduced TCR mismatching. [Figures 17B-17C]Figures 17A, 17B, and 17C show the antitumor activity of A2-CT83 TCR-MT cells (murine constant region). Figure 17A shows the transduction efficiency of A2-CT83 TCR-M in human T cells. Figure 17B shows that A2-CT83 TCR-MT cells specifically recognized MDA-MB-231 and NCI-H1563 cells (expressing CT83 and HLA-A2), but not CAMA-1 cells (A2+CT83-). Figure 17C shows the cytotoxicity of A2-CT83 TCR-MT cells against MDA-MB-231 and NCI-H1563 cells. These results demonstrate that A2-CT83 TCR-M T cells are potent and specific against tumor cells, with reduced TCR mismatching.

[0058] [Figures 18A-18B] Figures 18A, 18B, 18C, 18D, and 18E show the novel CAR-T constructs fused with ZAP300 and ZAP327, derived from ZAP70, and their functional comparison with conventional CAR-T constructs containing the CD3-ζ signaling domain. Figure 18A shows a schematic diagram of the conventional CD19-CD28-CD3z (1928z) and the novel constructs containing CD19-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327). Figure 18B shows the T cell transduction efficiency of the three CARs in human T cells. Figures 18C and 18D show antigen-specific recognition and tumor cell lysis after CAR-T cells cocultured with Raji tumor cells. Figure 18E shows the in vivo antitumor activity of three CAR-T cells (1928z, 1928ZAP300, and 1928ZAP327). Importantly, 1928ZAP300 and 1928ZAP327 CAR-T cells outperformed 1928z CAR-T cells in in vivo experiments and significantly prolonged overall mouse survival in the Raji lymphoma tumor model. These studies suggest that replacing the CD3 ζ chain with the Zap70 kinase domain (ZAP300 and ZAP327) significantly enhanced antitumor activity in vivo. [Figures 18C-18E]Figures 18A, 18B, 18C, 18D, and 18E show the novel CAR-T constructs fused with ZAP300 and ZAP327, derived from ZAP70, and their functional comparison with conventional CAR-T constructs containing the CD3-ζ signaling domain. Figure 18A shows a schematic diagram of the conventional CD19-CD28-CD3z (1928z) and the novel constructs containing CD19-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327). Figure 18B shows the T cell transduction efficiency of the three CARs in human T cells. Figures 18C and 18D show antigen-specific recognition and tumor cell lysis after CAR-T cells cocultured with Raji tumor cells. Figure 18E shows the in vivo antitumor activity of three CAR-T cells (1928z, 1928ZAP300, and 1928ZAP327). Importantly, 1928ZAP300 and 1928ZAP327 CAR-T cells outperformed 1928z CAR-T cells in in vivo experiments and significantly prolonged overall mouse survival in the Raji lymphoma tumor model. These studies suggest that replacing the CD3 ζ chain with the Zap70 kinase domain (ZAP300 and ZAP327) significantly enhanced antitumor activity in vivo.

[0059] [Figures 19A-19B]Figures 19A, 19B, 19C, 19D, and 19E show that novel 4-1BB-containing CAR-T constructs fused with ZAP300 and ZAP327, derived from ZAP70, produced lower amounts of cytokines but stronger anti-tumor immunity. Figure 19A shows the schematic construction of 19bbz and 19bbZAP327. Figure 19B shows that 19bbZAP327 CAR-T cells produced significantly less cytokines than conventional 19bbz CAR-T cells after stimulation with tumor cells. Figure 19C shows specific lysis of tumor cells by 19bbZAP327 CAR-T cells. Figures 19D and 19E show that 19bbZAP327 CAR-T cells had superior anti-tumor activity in vivo and significantly prolonged mouse survival, suggesting that 19bbZAP327 CAR-T cells have improved safety and anti-tumor immunity compared to conventional 19bbz CAR-T cells. [Figures 19C-19E] Figures 19A, 19B, 19C, 19D, and 19E show that novel 4-1BB-containing CAR-T constructs fused with ZAP300 and ZAP327, derived from ZAP70, produced lower amounts of cytokines but stronger anti-tumor immunity. Figure 19A shows the schematic construction of 19bbz and 19bbZAP327. Figure 19B shows that 19bbZAP327 CAR-T cells produced significantly less cytokines than conventional 19bbz CAR-T cells after stimulation with tumor cells. Figure 19C shows specific lysis of tumor cells by 19bbZAP327 CAR-T cells. Figures 19D and 19E show that 19bbZAP327 CAR-T cells had superior anti-tumor activity in vivo and significantly prolonged mouse survival, suggesting that 19bbZAP327 CAR-T cells have improved safety and anti-tumor immunity compared to conventional 19bbz CAR-T cells.

[0060] [Figures 20A-20B]Figures 20A, 20B, and 20C show that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. Figure 20A shows higher in vivo persistence of 1928ZAP327 CAR-T cells in the bone marrow and spleen of T cell-transferred mice. Figure 20B shows a higher percentage of central memory 1928ZAP327 CAR-T cells compared to 1928z CAR-T cells. Figure 20C shows that 1928ZAP327 CAR-T cells expressed lower amounts of PD-1 (a marker of exhaustion) than 1928z CAR-T cells, suggesting that the ZAP327 signaling domain reduces T cell exhaustion. [Figure 20C] Figures 20A, 20B, and 20C show that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. Figure 20A shows higher in vivo persistence of 1928ZAP327 CAR-T cells in the bone marrow and spleen of T cell-transferred mice. Figure 20B shows a higher percentage of central memory 1928ZAP327 CAR-T cells compared to 1928z CAR-T cells. Figure 20C shows that 1928ZAP327 CAR-T cells expressed lower amounts of PD-1 (a marker of exhaustion) than 1928z CAR-T cells, suggesting that the ZAP327 signaling domain reduces T cell exhaustion.

[0061] [Figure 21A] Figures 21A and 21B show modulation of TCR-T cell function in vivo by knocking down the expression of metabolic genes PD1, VHL, and PPP2R2D. Figure 21A shows the transduction efficiency of A2-ESO-1 TCR constructs with or without PD1, VHL, or PPP2R2D shRNA, respectively. Figure 21B shows MDA-MB-231 / A2 / NY-ESO-1-bearing mice injected with A2-ESO-1 TCR-T cells with or without PD1, VHL, or PPP2R2D knockdown, respectively. Top: Mean tumor growth in each group. Middle: Mouse survival curves for each group. Bottom: Tumor growth in each mouse in each group. [Figure 21B]Figures 21A and 21B show modulation of TCR-T cell function in vivo by knocking down the expression of metabolic genes PD1, VHL, and PPP2R2D. Figure 21A shows the transduction efficiency of A2-ESO-1 TCR constructs with or without PD1, VHL, or PPP2R2D shRNA, respectively. Figure 21B shows MDA-MB-231 / A2 / NY-ESO-1-bearing mice injected with A2-ESO-1 TCR-T cells with or without PD1, VHL, or PPP2R2D knockdown, respectively. Top: Mean tumor growth in each group. Middle: Mouse survival curves for each group. Bottom: Tumor growth in each mouse in each group.

[0062] [Figure 22] FIG. 22 shows the enhancement of an enhanced CD44+CD62L- memory T cell population by Jmjd3 conditional knockout (cKO) in CD4+ T cells compared to wild-type (WT) cells.

[0063] [Figures 23A-23C] Figures 23A, 23B, 23C, 24D, 25E, and 25F show enhanced T cell survival and persistence in vivo and in vitro by Jmjd3 cKO T cells. Figures 23A and 23B show that CD4+ T cells from Jmjd3 cKO 2d2 transgenic mice stimulated in vivo with MOG peptide plus complete Freund's adjuvant significantly enhanced clinical scores in an EAE mouse model. Figure 23C shows a higher number of Jmjd3 cKO T cells compared to wild-type 2d2 cells after T cell transfer. Figures 23D, 23E, and 23F show a higher number of Jmjd3 cKO T cells compared to wild-type 2d2 cells after T cell transfer using T cells stimulated in vitro with MOG peptide. [Figures 23D-23F]Figures 23A, 23B, 23C, 24D, 25E, and 25F show enhanced T cell survival and persistence in vivo and in vitro by Jmjd3 cKO T cells. Figures 23A and 23B show that CD4+ T cells from Jmjd3 cKO 2d2 transgenic mice stimulated in vivo with MOG peptide plus complete Freund's adjuvant significantly enhanced clinical scores in an EAE mouse model. Figure 23C shows a higher number of Jmjd3 cKO T cells compared to wild-type 2d2 cells after T cell transfer. Figures 23D, 23E, and 23F show a higher number of Jmjd3 cKO T cells compared to wild-type 2d2 cells after T cell transfer using T cells stimulated in vitro with MOG peptide.

[0064] [Figures 24A-24C] Figures 24A, 24B, and 24C show that Jmjd3 KO enhances T cell survival and persistence by reducing T cell apoptosis. Figure 24A shows that Jmjd3 cKO T cells had reduced apoptosis-related protein levels after stimulation with anti-CD3 and CD28 antibodies. Figure 24B shows that Jmjd3 cKO T cells had much lower levels of T cell apoptosis after stimulation. Figure 24C shows that Jmjd3 cKO T cells had significantly lower levels of cleaved caspase 3 compared to WT T cells.

[0065] [Figures 25A-25B]Figures 25A, 25B, 25C, and 25D show enhanced CAR-T cell survival and in vivo persistence by Jmjd3 knockdown (KD). Figure 25A shows the experimental design using Raji tumor cells to monitor luciferase-labeled T cell survival. Figures 25B and 25C show that CAR-T cells with Jmjd3 KD (1928z-shJMJD3) had robust proliferation 4 days after T cell transfer into Raji tumor-bearing NSG mice, but maintained high levels of T cell proliferation compared to 1928z-control shRNA. Figure 25D shows that 1928z-shJMJD3 CAR-T cells significantly inhibited tumor growth and prolonged mouse survival compared to 1928z-control shRNA CAR-T cells with control shRNA. [Figures 25C-25D] Figures 25A, 25B, 25C, and 25D show enhanced CAR-T cell survival and in vivo persistence by Jmjd3 knockdown (KD). Figure 25A shows the experimental design using Raji tumor cells to monitor luciferase-labeled T cell survival. Figures 25B and 25C show that CAR-T cells with Jmjd3 KD (1928z-shJMJD3) had robust proliferation 4 days after T cell transfer into Raji tumor-bearing NSG mice, but maintained high levels of T cell proliferation compared to 1928z-control shRNA. Figure 25D shows that 1928z-shJMJD3 CAR-T cells significantly inhibited tumor growth and prolonged mouse survival compared to 1928z-control shRNA CAR-T cells with control shRNA.

[0066] [Figures 26A-26C] Figures 26A, 26B, and 26C show that forced expression of chemokine receptors enhances T cell trafficking to tumor cells in vivo. Figure 26A shows a diagram of the construction of 1928z CAR fused with CCR5. Figures 26B and 26C show that 1928z-CCR5 CAR-T cells significantly inhibited the growth of MDA-MB-231 / CD19 tumor cells in vivo, suggesting that forced expression of chemokine receptors enhances T cell trafficking to tumor cells.

[0067] [Figure 27] Figure 27 shows a strategy to enhance both T cell trafficking and T cell persistence by expression of chemokine receptors and shRNA KD in TCR or CAR constructs. DETAILED DESCRIPTION OF THE INVENTION

[0068] Detailed Description A.Definition Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, unless otherwise specified, or to particular reagents, unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0069] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0070] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each range are significant in relation to the other endpoint, as well as independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of ordinary skill in the art, when a value is disclosed, it is understood that "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to," and "greater than or equal to," It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are considered to be disclosed. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. It is also understood that whenever a series of values ​​is disclosed, any range between any two of the listed values ​​would be understood by one of ordinary skill in the art.

[0071] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0072] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0073] As used herein, the term "antibody" may include both polyclonal and monoclonal antibodies, primatized (e.g., humanized), murine, mouse-human, mouse-primate, and chimeric antibodies; may be intact molecules, fragments thereof (which may or may not include scFv, Fv, Fd, Fab, Fab', and F(ab)'2 fragments), or multimers or aggregates of intact molecules and / or fragments; may be naturally occurring or produced, for example, by immunization, synthesis, or genetic engineering; "antibody fragment," as used herein, refers to fragments derived from or related to antibodies that bind to antigens and, in some embodiments, may be derivatized to exhibit structural features that facilitate clearance and uptake, for example, by incorporation of galactose residues. "Antibodies" include, for example, F(ab), F(ab)'2, scFv, light chain variable regions (VL), heavy chain variable regions (VH), and combinations thereof.

[0074] Checkpoint inhibitors are drugs that target checkpoint proteins or their derivatives and are sometimes referred to as "checkpoint inhibitors." Checkpoint inhibitors can include or exclude proteins, polypeptides, amino acid residues, and monoclonal or polyclonal antibodies. Multivalent vaccines can include or be administered with one or more checkpoint inhibitors. Checkpoint inhibitors can bind to ligands or proteins found in any of the family of T cell regulators, such as CD28 / CTLA-4, for example. Targets of checkpoint inhibitors include, but are not limited to, receptors or coreceptors expressed on immune system effector or regulator cells (e.g., T cells) (e.g., CTLA-4; CD8), proteins expressed on the surface of antigen-presenting cells (e.g., proteins expressed on the surface of activated T cells, including PD-1, PD-2, PD-L1, PD-L2, 4-1BB, and OX40), metabolic enzymes or metabolic enzymes expressed by both tumor and tumor-infiltrating cells (e.g., indoleamine (IDO), including isoforms such as IDO1 and IDO2), proteins belonging to the immunoglobulin superfamily (e.g., lymphocyte activation gene 3, also known as LAG3), and proteins belonging to the B7 superfamily (e.g., B7-H3 or its homologs). B7 proteins can be found on both activated antigen-presenting cells and T cells.

[0075] As used herein, the term "separation" includes any means of substantially purifying one component from another (eg, by filtration, magnetic attraction, etc.).

[0076] As used herein, the term "isolate" or "isolating" includes any means of separating one species from another species.

[0077] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one aspect, the subject may be a human, non-human primate, cow, horse, pig, dog, or cat. The subject may also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0078] As used herein, the terms "preventing" or "inhibiting" the development of cancer or cancer cells refers to the development of the cancer being prevented or the delay in the onset of cancer.

[0079] As used herein, the terms "treat" or "reduce the presence of cancer or cancer cells" mean that cancer growth is inhibited, as reflected, for example, by tumor volume or the number of malignant cells. Tumor volume can be determined by various known procedures, for example, by measuring the observed image and comparing the average cross-sectional diameter of the tumor to a calibration line (e.g., as performed in ImageJ).

[0080] As used herein, "preventing or inhibiting the onset of an infectious disease" means that the onset of an infectious disease is prevented, or the onset of an infectious disease is delayed, or the spread of an existing infection is reversed.

[0081] As used herein, the term "activation" refers to the state of a cell after sufficient cell surface moiety ligation to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cell proliferation. T cell activation can also induce cytokine production and the performance of regulatory or cytolytic effector functions. In the context of other cells, the term refers to either the up- or down-regulation of specific physicochemical processes.

[0082] As used herein, the term "cancer antigen" or "tumor antigen" encompasses tissue-specific differentiation antigens, tumor-specific shared antigens, and variant tumor-specific and unique antigens, as well as any portion, peptide, or polypeptide of those antigens that can elicit a CD4+ or CD8+ T cell immune response. Tumor or cancer antigens recognized by CD8+ or CD4+ T cells can be classified into several categories (Wang, RF & Wang, H.Y. Immune targets and neoantigens for cancer immunotherapy and precision medicine. Cell research 27, 11-37, doi:10.1038 / cr.2016.155(2017)): 1) tissue-specific differentiation antigens, including MART-1 (Kawakami, Y. et al. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J. experimental medicine 180, 347-352(1994); Schneider, J., Brichard, V., Boon, T., Meyer zum Buschenfelde, KH & Wolfel, T. Overlapping peptides of melanocyte differentiation antigen Melan-A / MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1. International journal of cancer 75, 451-458 (1998), TRP-1 / gp75 (Wang, RF, Parkhurst, MR, Kawakami, Y., Robbins, PF & Rosenberg, SAUtilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen.J.experimental medicine 183,1131-1140(1996))、TRP-2(Wang,R.F.,Appella,E.,Kawakami,Y.,Kang,X.&Rosenberg,S.A.Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.J.experimental medicine 184,2207-2216(1996);Parkhurst,M.R.ら Identification of a shared HLA-A. * 0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2(TRP2).Cancer research 58,4895-4901(1998);Sun,Y.ら Identification of a new HLA-A( *)0201-restricted T-cell epitope from the tyrosinase-related protein 2(TRP2)melanoma antigen.International journal of cancer 87,399-404(2000)), and gp100(Kawakami, Y. et al. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.J.immunology 154, 3961-3968 (1995); Bakker, AB et al. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating processed lymphocytes. J. experimental medicine 179, 1005-1009 (1994); Skipper, JC et al. epitope from Pmel-17 / gp100. J. Immunol. 157, 5027-5033 (1996); Tsai, V. et al. Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells. J. Immunol. 158, 1796-1802 (1997)) has higher expression in cancer cells compared to normal cells.2) Tumor-specific shared antigens, which may include or exclude MAGE-A1 (Traversari, C. et al., A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-EJ experimental medicine 176, 1453-1457 (1992); Fujie, T. et al., A MAGE-1-encoded HLA-A24-binding synthetic peptide induces specific anti-tumor cytotoxic T lymphocytes. International journal of cancer 80, 169-172 (1999)).and NY-ESO-1 (Jager, E. et al., Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J. experimental medicine 187, 265-270 (1998); Rimoldi, D. et al., Efficient simultaneous presentation of NY-ESO-1 / LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma.J.Immunol.165,7253-7261(2000);Valmori, D. et al., Naturally occurring human lymphocyte antigen-A2 restricted CD8+T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients.Cancer research 60, 4499-4506 (2000); Wang, R.-F., Johnston, S.L., Zeng, G., Schwartzentruber, D.J. & Rosenberg, S.A. Breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames. J. Immunol. 161, 3596-3606 (1998)) are expressed in cancer and testis but not in other normal tissues. These antigens are also called cancer-testis (CT) antigens.3) CDK4 (Wolfel, T. et al., A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281-1284 (1995)), catenin (Robbins, P. F. et al., A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor-infiltrating lymphocytes. J. Experimental Medicine 183, 1185-1192 (1996)), and caspase-8 (Mandruzzato, S., Brasseur, F., Andry, G., Boon, T. & van der Bruggen, P. A., CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J. Experimental Medicine 186, 785-793 (1997)) antigens, and 4) tumor-specific and unique antigens that are mutant antigens, including overexpressed tumor antigens that are overexpressed in cancer cells compared to normal cells.

[0083] Two cancer-testis (CT) antigens, NY-ESO-1 (encoded by the CTAG1B gene) and CT83 (also known as KK-LC-1), encoded by the CT83 gene, are widely expressed in various tumors, including lung and breast cancer. Both CD8+ T cells and antibodies have been shown to recognize NY-ESO-1, and 50–80% clinical responses using NY-ESO-1-specific TCRs have been demonstrated in several solid tumors, including melanoma, sarcoma, and myeloma. Despite the importance of CD4+ T cells (HLA-DP4 is the most frequently expressed HLA II molecule in the general human population, accounting for 70% of positive cases), HLA-DP4-restricted NY-ESO-1-specific TCRs have not yet been tested in clinical settings. We previously identified the HLA-DR4- and HLA-DP4-restricted NY-ESO-1 epitope (Zeng, G. et al., Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules. J. Immunol. 165, 1153-1159 (2000); Zeng, G., Wang, X., Robbins, P.F., Rosenberg, S.A., & Wang, R.-F. CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA-DP4 allele: association with NY-ESO-1 antibody production. Proc. Natl. Acad. Sci. USA 98, 3964-3969 (2001)) and showed that the HLA-DP4-NY-ESO-1 peptide overlaps with the HLA-A2-restricted NY-ESO-1 peptide. Zeng, G. et al., Generation of NY-ESO-1-specific CD4+ and CD8+T cells by a single peptide with dual MHC class I and class II specificities: a new strategy for vaccine design. Cancer Res. 62, 3630-3635. (2002).

[0084] As disclosed herein, HLA-DP4-restricted NY-ESO-1 CD4+ T cells and TCRs were generated and tested to determine whether a combination of DP4-ESO-1 TCR-engineered T cells and A2-ESO-1 TCR-engineered T cells could generate stronger anti-tumor immunity than either alone.

[0085] In addition to the CT antigen NY-ESO-1, CT83 is highly expressed in 60-70% of breast cancers, particularly TNBC, consistent with previous reports (Fukuyama, T. et al., "Identification of a new cancer / germline gene, KK-LC-1, encoding antigen recognized by autologous CTL induced on human lung adenocarcinoma," Cancer Research 66, 4922-4928, doi:10.1158 / 0008-5472.CAN-05-3840 (2006); Paret, C. et al., "CXorf61 is a target for T cell-based immunotherapy of triple-negative breast cancer," Oncotarget 6, 25356-25367, doi:10.18632 / oncotarget.4516 (2015)). However, relatively little is known about its immunogenicity, T cell epitopes, and cognate TCRs for tumor recognition by T cells. As disclosed herein, antigen-specific CD4+ and CD8+ T cells were generated and used to identify an HLA-A2-restricted CT83-specific TCR (A2-CT83 TCR) and determine whether CT83 could serve as an attractive target for TCR-T cell immunotherapy.

[0086] It has been demonstrated that the persistence of CAR-T and TCR-T cells is closely correlated with patient survival. Therefore, modulation of TCR-T and CAR-T cell signaling can enhance T cell persistence and reduce T cell exhaustion by directly regulating CAR or TCR signaling and knocking down or knocking out negative signaling molecules, which can include or exclude epigenetic factors, which can include or exclude PD1, VHL, PPP2R2D, and Jmjd3 and LSD1.

[0087] In some embodiments, the immunogenic peptides and epitopes contained within the tumor antigens of the present disclosure are derived from NY-ESO-1 and CT83 proteins, both of which are widely expressed in various types of cancer, including, but not limited to, breast cancer, lung cancer, prostate cancer, etc. The tumor antigens of the present invention are expressed at significantly higher levels in tumor cells and testis compared to lower levels in normal cells.

[0088] In some embodiments, a "tumor antigen" or "cancer antigen" is NY-ESO-1, CT83 protein, HCMV pp65 protein, and / or HCMV IE-1 protein, and any portion, peptide, or polypeptide of NY-ESO-1, CT83 protein, HCMV pp65 protein, and / or HCMV IE-1 protein that can elicit a CD4+ or CD8+ T cell immune response, including full-length NY-ESO-1 and CT83 proteins.

[0089] "Immunogenic peptides and epitopes," as that term is used herein, include any epitope or fragment of the NY-ESO-1, CT83, HCMV pp65 and / or HCMV IE-1 proteins that act as tumor antigens.

[0090] A "fragment" or "portion," as the term is used herein, refers to any segment of a protein or gene having at least 5 or 6 amino acids in the case of a protein fragment, or at least 15-18 nucleotides in the case of a gene.

[0091] In one embodiment, the tumor antigen-specific T cell line of the present invention comprises all generated CD4+ or CD8+ T lymphocytes that immunologically recognize tumor antigens presented by antigen-presenting cells that are HLA-DP4 or HLA-A2 positive.

[0092] As used herein, "presented" includes the procedure of transfecting DNA encoding the full-length or any part of a tumor antigen into antigen-presenting cells, or the procedure of loading a peptide of the full-length or any part of a tumor antigen into antigen-presenting cells.

[0093] "Antigen-presenting cells," as that term is used herein, encompass any natural or artificial cell line or cell that expresses a particular type of HLA molecule of interest on its cell surface. As used herein, the term "antigen" refers to 1) any molecule, either in its entirety or a fragment thereof, that can be specifically recognized and bound by the "idiotypic" portion (antigen-binding region) of a mAb or its derivative; 2) any molecule that contains a peptide sequence that can be bound by MHC and, in turn, in the context of MHC presentation, can specifically engage its cognate T-cell antigen receptor.

[0094] "HLA-DP4 positive," as the term is used herein, refers to a person who is resistant to the HLA class II molecules DPA1 and DPB1 * The term "cell line" encompasses any natural or artificial cell line or cell that expresses .O4 (including all subtypes thereof on the cell surface).

[0095] "HLA-A2 positive," as the term is used herein, refers to a cell that has an HLA class I molecule A on the cell surface. *The term "Cells" encompasses any natural or artificial cell line or cell that expresses C.O2 (including all subtypes thereof).

[0096] In one embodiment of the present invention, at least two T cell receptors are derived from antigen-specific CD4+ or CD8+ T cell lines. The full-length alpha and beta chains of the TCRs are cloned separately. "Full length," as the term is used herein, refers to an alpha chain variable region fused to a human alpha chain constant region (non-limiting examples include TRAC, IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS) (SEQ ID NO: 10), or a mouse alpha chain constant region (trac) (SEQ ID NO: 13)), or a human beta chain constant region type 2 (TRBC2, DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLS beta chain variable region fused to a human beta chain constant region type 1 (TRBC1, DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF) (SEQ ID NO: 12), or human beta chain constant region type 1 (TRBC1, DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF) (SEQ ID NO: 11), or mouse beta chain constant region type 1 (trbc1) (SEQ ID NO: 14), or mouse beta chain constant region type 2 (trbc2) (SEQ ID NO: 15). In some embodiments, the constant region can have a sequence having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15).The constant region may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0097] In some embodiments, the chimeric TCR comprises a chimeric alpha chain comprising an HLA-A2-restricted CT83 TCR alpha chain variable domain fused to a murine alpha constant domain comprising SEQ ID NO:20 or a variant thereof having 85, 86, 87, 88, 89, 90, 91 92 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:20 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions (which may be conservative substitutions) to SEQ ID NO:20; and a murine beta constant domain2 having SEQ ID NO:21 or a variant thereof having 85, 86, 87, 88, 89, 90, 91 92 A CT83-specific TCR having a chimeric beta chain comprising an HLA-A2-restricted CT83 TCR beta chain variable domain fused to a variant thereof having 93, 94, 95, 96, 97, 98 or 99% sequence identity and / or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions (which may be conservative substitutions) relative to SEQ ID NO:21.

[0098] In some embodiments, the chimeric TCR comprises an HLA-A2 restricted NY-ESO-1 polypeptide fused to a mouse alpha constant domain selected from a polypeptide having SEQ ID NO:22 or a variant thereof having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:22 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions (which may be conservative substitutions) relative to SEQ ID NO:22. HLA-A2 restricted NY-ESO-1 fused to a TCR(S2) alpha chain variable domain and a murine alpha constant domain having SEQ ID NO:24 or a variant thereof having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:24 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions (which may be conservative substitutions) to SEQ ID NO:24. a chimeric alpha chain comprising a polypeptide selected from a TCR(S5) alpha chain variable domain and an HLA-A2 restricted NY-ESO-1 fused to a murine beta constant domain 2 comprising SEQ ID NO:23 or a variant thereof having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:23 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions (which may be conservative substitutions) relative to SEQ ID NO:23; NY-ESO-1 specific TCR having a chimeric beta chain selected from an HLA-A2 restricted NY-ESO-1 TCR(S5)(G50A, A51E, A97L) beta chain variable domain fused with a TCR(S2)(G50A, A51E) beta chain variable domain and a murine beta constant domain 2 having SEQ ID NO:25 or a variant thereof having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:25 and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions to SEQ ID NO:25.

[0099] In some embodiments, the chimeric TCR comprises a sequence having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15). The constant region may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0100] As used herein, the term "proliferation" means to grow or increase in number by producing new cells.

[0101] The terms "purify" or "pure" refer to a molecule that has been isolated from other reactants or cellular components. The terms "substantially pure" or "substantially purified" refer to a molecule that is 85, 86, 87, 88, 89, 90, 91, 9, 93, 94, 95, 96, 97, 98, 99, or 100% pure.

[0102] In yet another embodiment of the present invention, tumor antigen epitopes that specifically interact with CD4+ T cell lines or TCRs to elicit a T cell immune response include or exclude peptides / portions of the NY-ESO-1 protein that contain specific amino acids of the NY-ESO-1 protein (e.g., PEP 161-180 contains amino acids 161-180 of the NY-ESO-1 protein), such as NY-ESO-1 PEP161-180 (WITQCFLPVFLAQPPSGQRR, SEQ ID NO: 34), NY-ESO-1 PEP156-175 (LSLLMWITQCFLPVFLAQPP, SEQ ID NO: 35), and NY-ESO-1 PEP157-170 (SLLMWITQCFLPVF, SEQ ID NO: 1). In some embodiments, the epitope includes variants containing one, two, or three conservative substitutions. NY-ESO-1 PEP161-180 (SEQ ID NO: 34) has been identified as a peptide with high affinity for HLA-DP4. Zeng, G. et al., "Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules." J. Immunol. 165, 1153-1159 (2000). Using this peptide, peptide-stimulated CD4+ T cells that specifically recognize HLA-DP4-presented NY-ESO-1 were generated. Zeng, G., Wang, X., Robbins, P.F., Rosenberg, S.A., & Wang, RF. "CD4(+) T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA-DP4 allele: association with NY-ESO-1 antibody production." Proc. Natl Acad Sci USA 98, 3964-3969, doi:10.1073 / pnas.061507398 (2001). NY-ESO-1 PEP157-170 (SEQ ID NO: 1) has been identified as the shortest functional epitope that maintains an undiminished immune response compared to full-length NY-ESO-1. 42. Ibid. Reference. (Zeng et al., PNAS doi:10.1073 / pnas.061507398(2001)).

[0103] In yet another embodiment of the present invention, epitopes of tumor antigens that specifically interact with CD8+ T cell lines or TCRs to elicit a T cell immune response can include or exclude peptides / portions of CT83 proteins containing the designated amino acids of the CT83 protein: CT83 PEP90-98 (KLVELEHTL, SEQ ID NO:2), CT83 PEP6-14 (LLASSILCA, SEQ ID NO:36), CT83 PEP4-12 (YLLLASSIL, SEQ ID NO:37), CT83 PEP79-87 (RILVNLSMV, SEQ ID NO:38), CT83 PEP10-31 (SILCALIVFWKYRRFQRNTGEM, SEQ ID NO:39), and CT83 PEP66-76 (ILNNFPHSIAR, SEQ ID NO:40). In some embodiments, the epitopes include variants containing one, two, or three conservative substitutions.

[0104] In yet another embodiment of the present invention, the epitope of a tumor antigen that specifically interacts with a CD4+ T cell line or TCR to elicit a T cell immune response includes or excludes, but is not limited to, the pp65 peptide (495-503) (NLVPMVATV, SEQ ID NO: 26). pp65 is an HCMV protein and antigen expressed by glioblastoma cells. In some embodiments, the epitope includes a variant containing one, two, or three conservative substitutions.

[0105] In yet another embodiment of the present invention, the epitope of a tumor antigen that specifically interacts with a CD4+ T cell line or TCR to elicit a T cell immune response includes or excludes, but is not limited to, IE-1 peptide 316-324 (VLEETSVML, SEQ ID NO: 31). IE-1 is an HCMV protein and antigen expressed by glioblastoma cells. In some embodiments, the epitope includes a variant containing one, two, or three conservative substitutions.

[0106] The term "self-cleaving peptide" includes, but is not limited to, the P2A sequence (RAKRSGSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 51) located between two proteins and capable of self-cleaving to separate the two proteins. Ryan, MD, King, AM & Thomas, GP Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. General Virology 72(Pt 11), 2727-2732, doi:10.1099 / 0022-1317-72-11-2727(1991).

[0107] As used herein, the term "stimulation" refers to a primary response induced by cell surface moiety ligation. For example, in the context of a receptor, such stimulation involves receptor ligation and a subsequent signaling event. With respect to T cell stimulation, such stimulation, in one embodiment, refers to ligation of T cell surface moieties that subsequently induces a signaling event, which may include or exclude binding of the TCR / CD3 complex. Furthermore, the stimulatory event may activate the cell and upregulate or downregulate the expression or secretion of molecules, which may include or exclude downregulation of TGF-β. Thus, cell surface moiety ligation, even in the absence of a direct signaling event, may result in cytoskeletal structural rearrangements or cell surface moiety coalescence, which may serve to enhance, modify, or alter subsequent cellular responses, respectively.

[0108] As used herein, the term "vector" includes, but is not limited to, pMSGV, pMSCV, pFU3W, or any other vector that functions as a carrier for DNA inserted into living cells.

[0109] In yet another embodiment of the present invention, a vector is provided for inserting cDNA encoding a TCR alpha chain and / or a TCR beta chain. In some embodiments, the translation product of the vector comprises at least one alpha chain variable region combined with at least one alpha constant region and / or at least one beta chain variable region combined with at least one beta constant region linked by a self-cleaving peptide. The vector is useful for delivery of the insert into naive T cells by viral transduction.

[0110] As used herein, the term "viral transduction" encompasses the procedure of producing recombinant viruses, including but not limited to retroviruses, lentiviruses, adeno-associated viruses, or other suitable viruses in host cells, and using these recombinant viruses containing a gene encoding a TCR in their genome to infect, transfect, or transduce target cells. The gene encoding the TCR is integrated into the genome of the target cell and is stably expressed and replicated in the proliferating cells. The term "target cells" includes, but is not limited to, CD4+ T cells, CD8+ T cells, tumor cells, etc.

[0111] In one embodiment, the invention also provides a host cell transfected or transduced with a vector comprising DNA encoding a TCR region or chain according to any preceding aspect or any aspect or embodiment disclosed herein, for example a TCR alpha chain variable region combined with an alpha constant region and a beta chain variable region combined with a beta constant region linked by a P2A sequence (SEQ ID NO: 51) for virus production and TCR delivery to naive T cells.

[0112] In one embodiment, the TCR is a chimeric TCR comprising a TCR variable region modified or fused to a non-human constant region. In some embodiments, the chimeric TCR comprises a cancer antigen-specific TCR variable region of any of the embodiments disclosed herein fused to a non-human TCR constant region, e.g., a murine TCR constant region. For example, the chimeric TCR can comprise a variable region that may include or exclude the CT83 TCR variable region, the NY-ESO-1 TCR variable region, the pp65 TCR variable region, or the IE-1 TCR fused to a non-human, e.g., murine, TCR constant region. Among other features, chimeric TCRs reduce mispairing between the chimeric TCR and the endogenous TCR of transduced T cells. For example, a chimeric CT83 TCR (MC) reduces mispairing between the chimeric CT83 TCR (MC) and the endogenous TCR (HC) in transduced cells. For example, a chimeric CT83 TCR reduces mispairing between a chimeric CT83 TCR (MC) and an endogenous TCR (HC), or a chimeric NY-ESO-1 TCR reduces or reduces mispairing between a chimeric NY-ESO-1 (MC) and an endogenous TCR (HC). As another example, a chimeric pp65 TCR reduces or reduces mispairing between a chimeric pp65 (MC) and an endogenous TCR (HC). As another example, a chimeric IE-1 TCR reduces or reduces mispairing between a chimeric IE-1 (MC) and an endogenous TCR (HC).

[0113] The term "host cell" includes, but is not limited to, PG-13 cell line, Phoenix-Eco cell line, Phoenix-Ampho cell line, 293GP cell line, or other suitable cell line that can assemble the viral genome intracellularly, package the virus with capsule proteins, and secrete the mature virus extracellularly.

[0114] In yet another embodiment of the present invention, shRNA or antisense RNA or DNA sequences that specifically knock down metabolic genes to enhance the anti-tumor activity of TCR or CAR-based therapy in vivo are provided in the form of 21-base pair stems. TCRs can be engineered with shRNAs to knock down target genes to improve T cell trafficking and persistence in vivo and enhance anti-tumor activity. As used herein, the term "knockdown" or "" refers to reducing gene expression, for example, by causing mRNA degradation or by blocking RNA expression to reduce protein expression of the target gene. As used herein, the term "metabolic genes" includes, but is not limited to, PD1, VHL, and PPP2R2D.

[0115] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0116] The present invention encompasses CD4+ or CD8+ T lymphocytes that immunologically recognize tumor antigens by restricting them to one HLA class II or class I molecule. The present invention further encompasses at least one T cell receptor derived from the above-described CD4+ or CD8+ T lymphocytes. The T cell receptor can be delivered to naive CD4+ or CD8+ T lymphocytes that have no immune response to the above-described tumor antigen, altering the function of those CD4+ or CD8+ T lymphocytes to specifically recognize and respond to the above-described tumor antigen. This reaction between the tumor antigen and the transduced T cell receptor enables the T lymphocytes to respond to human cancers bearing the above-described tumor antigen and help prevent, eliminate, or reduce the disease. B. Methods for TCR Identification Immunoassays and Fluorescent Dyes

[0117] The steps of various useful immunodetection methods are described in the scientific literature, which may include or exclude, for example, Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety for its teachings regarding immunodetection methods. In its simplest and most straightforward sense, an immunoassay is a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, all suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunobead capture assay, Western blotting, dot blotting, gel shift assay, flow cytometry, protein array, multiplexed bead array, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching / localization (FRAP / FLAP).

[0118] Generally, immunoassays involve contacting a sample suspected of containing a molecule of interest (which may or may not include a disclosed biomarker) with an antibody to the molecule of interest, or, in some cases, contacting the antibody to the molecule of interest (which may or may not include an antibody to a disclosed biomarker) with a molecule that can be bound by the antibody, under conditions effective to allow the formation of an immune complex. Contacting a sample with an antibody to the molecule of interest or a molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of an immune complex (primary immune complex) generally involves simply contacting the molecule or antibody with the sample and incubating the mixture long enough for the antibody to form an immune complex, i.e., bind to any molecules present (e.g., antigens) to which the antibody can bind. In many forms of immunoassays, the sample-antibody composition, which may or may not include a tissue section, ELISA plate, dot blot, or Western blot, can be washed to remove any nonspecifically bound antibody species and detect only the specifically bound antibody within the primary immune complex.

[0119] Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest in a sample (which may include or exclude the disclosed biomarkers or their antibodies), which generally includes the detection or quantification of any immune complexes formed during the binding process. In general, the detection of immune complex formation is well known in the art and can be achieved by the application of numerous approaches. These methods are generally based on the detection of a label or marker, which may include or exclude any radioactive, fluorescent, biological, or enzymatic tag or any other known label.

[0120] As used herein, a label may include a fluorescent dye, a member of a binding pair that may or may not include biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, and may or may not include by producing a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorescent dyes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of this application because they can be detected in very small amounts. Furthermore, when multiple antigens are reacted on a single array, each antigen can be labeled with a separate fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorometer (the presence of a signal indicating the antigen bound to the specific antibody).

[0121] A fluorophore is a compound or molecule that emits light. Typically, a fluorophore absorbs electromagnetic energy at one wavelength and emits it at a second wavelength. Representative fluorophores include, but are not limited to, 1,5-IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-I methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; acriflavine; acriflavine feulgen SITSA; aequorin (photoprotein); AFP-autofluorescent protein (AutoFluorescent Protein) Protein) - (Quantum Biotechnologies) See sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aniline Blue; Anthrocyl Stearate stearate);APC-Cy7;APTRA-BTC;APTS;Astrazon Brilliant Red 4G;Astrazon Orange R;Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (High pH); BCECF (Low pH); Berberine sulfate; Beta-lactamase; BFP Blueshift GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X Conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green-1 Ca; 2+ Dye; Calcium Green-2 Ca 2+ Calcium Green-5N Ca 2+ Calcium Green - C18 Ca 2+Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin phalloidin; C-phycocyanin; CPM I methylcoumarin; CTC; CTC formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; Cyclic AMP Fluorescent Sensor (FiCRhR); Dabcyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl Fluoride; DAPI; Dapoxil; Dapoxil 2; Dapoxil 3'DCFDA; DCFH (Dichlorodihydrofluorescein diacetate); DDAO; DHR (Dihydrodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP; dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18(5)); DIDS; dihydrodamine 123 (DHR); Dil (DilC18(3)); Idinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; eosin; erythrosine; erythrosine ITC; ethidium bromide; ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; europium(111) chloride; EYFP; Fast Blue; FDA;Feulgen (pararosaniline); FIF (formaldehyde-induced fluorescence); FITC; Furazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoroemerald; Fluorogold (hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red-shifted (rsGFP); GFP wild-type non-UV-excited (wtGFP); GFP wild-type, UV-excited (wtGFP); GFPuv; Gloxalic acid Acid); Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (Fluorogold); Hydroxytryptamine; Indo-1, High Calcium; Indo-1, Low Calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntraWhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; calcein / ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lysotracker Blue; Lysotracker Blue-White; Lysotracker Green; Lysotracker Red; Lysotracker Yellow; Lysosensor Blue; Lysosensor Green; Lysosensor Yellow / Blue; Mag Green; Magdala Red (Phloxine B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue;Maxillon Brilliant Flavin 10 GFF; Maxillon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mithramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene P); NBD; NBD-amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Flavin Lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxine B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primulin; Procion Yellow; Propidium iodide Iodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhodamine-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B Extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine:Phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-Phycocyanin; R-Phycoerythrin (PE); rsGFP;S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Cebron Brilliant Red 2B; Cebron Brilliant Red 4G; Cebron Brilliant Red B; Cebron Orange; Cebron Yellow L; sgBFP™ (Super Glow BFP); sgGFP™ (Super Glow GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectrum Aqua; Spectrum Green; Spectrum Orange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl) quinoline); SYTO 11;SYTO 12;SYTO 13;SYTO 14;SYTO 15;SYTO 16;SYTO 17;SYTO 18;SYTO 20;SYTO 21;SYTO 22;SYTO 23;SYTO 24;SYTO 25;SYTO 40;SYTO 41;SYTO 42;SYTO 43;SYTO 44;SYTO 45;SYTO 59;SYTO 60;SYTO 61;SYTO 62;SYTO 63;SYTO 64;SYTO 80;SYTO 81;SYTO 82;SYTO 83;SYTO 84;SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ Conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolite; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC Tetramethylrhodamine isothiocyanate; Tru Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; xylene orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1; YOYO-3; Sybr Green; thiazole orange (an interchelating dye); semiconductor nanoparticles that may or may not include quantum dots; or caged fluorophores (that can be activated by light or other electromagnetic energy sources), or combinations thereof.

[0122] A modifying unit that can include or exclude a radionuclide can be directly incorporated into or attached to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, and fluorine-18. In another aspect, the radionuclide can be attached to a linking group or via a chelating group, which is then attached to the compound directly or via a linker. Examples of radionuclides useful for upsetting include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques that can include or exclude these are routinely used in the radiopharmaceutical industry.

[0123] Radiolabeled compounds are useful as imaging agents for diagnosing neurological diseases (e.g., neurodegenerative diseases) or psychiatric conditions, or for tracking the progression or treatment of such diseases or conditions in mammals (e.g., humans). The radiolabeled compounds described herein can be advantageously used in conjunction with imaging techniques, which may include or exclude positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0124] Labeling can be either direct or indirect. In direct labeling, the detection antibody (antibody against the molecule of interest) or the detection molecule (molecule that can be bound by an antibody to the molecule of interest) contains a label. Detection of the label indicates the presence of the detection antibody or detection molecule, which in turn indicates the presence of the molecule of interest or an antibody against the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety contacts the immune complex or is generated at the site of the immune complex. For example, a signal-generating molecule or moiety, which may include or exclude an enzyme, can bind to or associate with the detection antibody or detection molecule. The signal-generating molecule can then generate a detectable signal at the site of the immune complex. For example, an enzyme, when provided with an appropriate substrate, can generate a visible or detectable product at the site of the immune complex. ELISA uses this type of indirect labeling.

[0125] As another example of indirect labeling, an additional molecule (which may be referred to as a binder) capable of binding to either the molecule of interest or an antibody to the molecule of interest (primary antibody) (which may or may not include a secondary antibody to the primary antibody) can be contacted with the immune complex. The additional molecule can have a label or a signal-generating molecule or moiety. The additional molecule can be an antibody and thus referred to as a secondary antibody. Binding of the secondary antibody to the primary antibody can form a so-called sandwich between the first (or primary) antibody and the molecule of interest. The immune complex can be contacted with a labeled secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove nonspecifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can be detected. The additional molecule can also be or include one of a pair of molecules or moieties capable of binding to each other, which may or may not include a biotin / avidin pair. In this embodiment, the detection antibody or detection molecule must contain the other member of the pair.

[0126] Other modes of indirect labeling include detecting primary immune complexes using a two-step approach. For example, as described above, a molecule (which can be referred to as a first binding agent) that can include or exclude antibodies with binding affinity for the molecule of interest or the corresponding antibody can be used to form secondary immune complexes. After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent under conditions effective and for a period of time sufficient to again allow immune complex formation (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signal-generating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide signal amplification.

[0127] Immunoassays involving detection of a substance, which may or may not include a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic tools that determine the presence or absence of a specific protein or an antibody to a specific protein in a sample. Protein separation methods are more useful for evaluating the physical properties of proteins, which may or may not include size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein or an antibody to a specific protein in a sample. Finally, in vivo detection is useful for evaluating the spatial expression pattern of a substance, i.e., when the substance can be found in a subject, tissue, or cell.

[0128] At sufficient concentrations, molecular complexes ([Ab-Ag]n) generated by antibody-antigen interactions are visible to the naked eye, but due to their ability to scatter a beam of light, smaller amounts can also be detected and measured. Complex formation indicates the presence of both reactants, and in immunoprecipitation assays, a fixed concentration of reagent antibody is used to measure the specific antigen ([Ab-Ag]n), while the reagent antigen is used to detect the specific antibody ([Ab-Ag]n). When the reagent species is first coated onto cells (as in a hemagglutination assay) or very small particles (as in a latex agglutination assay), "clumping" of the coated particles is evident at much lower concentrations. A variety of assays based on these basic principles are commonly used, including the Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidimetric and nephelometric assays. The main limitations, which may be included or excluded, are limited sensitivity (detection limit) compared to assays using labels and the fact that, in some cases, very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedure more complicated. Some of these Group 1 assays date back to shortly after the discovery of antibodies, and none of them have actual "labels" (e.g., Ag-enz). Other types of label-free immunoassays rely on immunosensors, and a variety of instruments are now commercially available that can directly detect antibody-antigen interactions. Most rely on generating evanescent waves on the sensor surface using immobilized ligands, which allows continuous monitoring of binding to the ligand. Immunosensors allow for easy investigation of kinetic interactions, and with the emergence of low-cost specialized instruments, immunoassays may find widespread application in the future.

[0129] The use of immunoassays to detect specific proteins can involve separating proteins by electrophoresis. Electrophoresis is the movement of charged molecules in solution in response to an electric field. Their rate of movement depends on the strength of the magnetic field and is determined by the net charge, size, and shape of the molecule, as well as the ionic strength, viscosity, and temperature of the medium through which the molecule is moving. As an analytical tool, electrophoresis is simple, rapid, and sensitive. It is used analytically to study the properties of singly charged species and as a separation technique.

[0130] Typically, samples are run in a support matrix, which can include or exclude paper, cellulose acetate, starch gels, agarose, or polyacrylamide gels. The matrix suppresses convective mixing caused by heating and provides a record of the electrophoretic run. At the end of the run, the matrix can be stained and used for scanning, autoradiography, or archiving. Furthermore, the most commonly used support matrices (agarose and polyacrylamide) offer a means of separating molecules by size, in that they are porous gels. Porous gels can act as sieves by slowing or even completely blocking the migration of large macromolecules while allowing smaller molecules to migrate freely. Dilute agarose gels are generally more rigid and easier to handle than polyacrylamide at the same concentration, so agarose is used to separate larger macromolecules, which can include or exclude nucleic acids, large proteins, and protein complexes. Polyacrylamide, which is easier to handle and can be made at higher concentrations, is used to separate most proteins and small oligonucleotides, which require small gel pore sizes for slowing down.

[0131] Proteins are amphoteric compounds. Therefore, their net charge is determined by the pH of the medium in which they are suspended. In solutions with a pH above their isoelectric point, proteins have a net negative charge and migrate toward the anode in an electric field. Below their isoelectric point, proteins are positively charged and migrate toward the cathode. Furthermore, the net charge carried by a protein is independent of its size—i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of the molecule varies from protein to protein. Therefore, at a given pH and under non-denaturing conditions, electrophoretic separation of proteins is determined by both the size and charge of the molecule.

[0132] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "wrapping" around the polypeptide backbone; SDS binds to proteins with high specificity at a mass ratio of 1.4:1. In doing so, SDS imparts a negative charge to polypeptides proportional to their length. Furthermore, it is usually necessary to reduce disulfide bridges in proteins (denaturation) before they can adopt the random coil configuration required for size separation. This is accomplished using 2-mercaptoethanol or dithiothreitol (DTT). Thus, in denaturing SDS-PAGE separations, migration is determined by the molecular weight of the polypeptides, not their intrinsic charge.

[0133] Molecular weight determination is performed by SDS-PAGE of proteins of known molecular weight along with the protein being characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide or native nucleic acid and its Rf. Rf is calculated as the ratio of the distance migrated by the molecule to the distance migrated by the marker dye front. A simple method for determining relative molecular weight (Mr) by electrophoresis is to plot a standard curve of distance migrated vs. log10 MW for known samples and read the logMr of the samples after measuring the distance migrated on the same gel.

[0134] In two-dimensional electrophoresis, proteins are first fractionated based on one physical property, and in a second step, they are fractionated based on another physical property. For example, isoelectric focusing can be used in the first dimension, conveniently performed in a tube gel, and SDS electrophoresis in a slab gel can be used in the second dimension. An example of a procedure is that of O'Farrell, P.H., "High Resolution Two-dimensional Electrophoresis of Proteins," J. Biol. Chem. 250:4007-4021 (1975), which is incorporated herein by reference in its entirety for its teachings regarding two-dimensional electrophoresis. Other examples include, but are not limited to, Anderson, L and Anderson, NG, "High resolution two-dimensional electrophoresis of human plasma proteins," Proc. Natl. Acad. Sci. 74:5421-5425 (1977); Ornstein, L., "Disc electrophoresis," L. Ann. NY Acad. Sci. 121:321-349 (1964), each of which is incorporated herein by reference in its entirety for its teachings regarding electrophoresis. Laemmli, UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4," Nature 227:680 (1970), which is incorporated herein by reference in its entirety for its teachings regarding electrophoresis, discloses a discontinuous system for separating SDS-denatured proteins. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Thus, the resolving and stacking gels were configured in Tris-HCl buffers (of different concentrations and pH), and the tank buffer was Tris-glycine. All buffers contained 0.1% SDS.

[0135] One example of an immunoassay using electrophoresis contemplated by current methods is Western blot analysis. Western blotting, or immunoblotting, allows for the determination of protein molecular weight and the measurement of the relative amounts of proteins present in different samples. Detection methods include chemiluminescence and chromogenic detection. Standard methods for Western blot analysis are described, for example, in D. M. Bollag et al., Protein Methods (2nd edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Pat. No. 4,452,901, each of which is incorporated herein by reference in its entirety for its teachings regarding Western blotting. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. Proteins are transferred to a sheet of special blotting paper, such as nitrocellulose, although other types of paper or membranes can also be used. Proteins retain the same separation pattern as they did on the gel. The blot is incubated with a common protein (which can include or exclude milk proteins) to bind to any remaining sticky sites on the nitrocellulose. An antibody capable of binding to that specific protein is then added to the solution.

[0136] The binding of specific antibodies to specific immobilized antigens can be easily visualized by indirect enzyme immunoassay techniques, which usually use chromogenic (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include fluorescent or radioisotope labels (e.g., fluorescein, 125 Probes for detecting antibody binding can be conjugated anti-immunoglobulin, conjugated Staphylococcus aureus protein A (which binds IgG), or probes for biotinylated primary antibodies (e.g., conjugated avidin / streptavidin).

[0137] The power of this technique lies in the simultaneous detection of specific proteins by their antigenicity and their molecular mass. Proteins are first separated by mass in SDS-PAGE and then specifically detected in an immunoassay step. Thus, protein standards (ladders) can be run simultaneously to approximate the molecular mass of proteins of interest in heterogeneous samples.

[0138] Gel shift assays or electrophoretic mobility shift assays (EMSAs) can be used to detect the interaction between a DNA-binding protein and its cognate DNA recognition sequence in both qualitative and quantitative ways. Exemplary techniques are described in Ornstein L., Disc electrophoresis-I: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudaira, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. (87:386-396 (1987)), each of which is incorporated herein by reference in its entirety for its teachings regarding gel shift assays.

[0139] In a typical gel shift assay, purified proteins or crude cell extracts are labeled (e.g., 32P (radioactively labeled) DNA or RNA probes can be incubated with the probe, followed by separation of the complex from the free probe through a non-denaturing polyacrylamide gel. The complex migrates through the gel more slowly than the unbound probe. Depending on the activity of the binding protein, the labeled probe can be either double-stranded or single-stranded. For detection of DNA-binding proteins, which can include or exclude transcription factors, purified or partially purified proteins, or nuclear cell extracts, can be used. For detection of RNA-binding proteins, purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of the DNA- or RNA-binding protein for the putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides containing the binding site for the protein of interest or other unrelated sequences. Differences in the nature and strength of the complex formed in the presence of specific and nonspecific competitors allow for the identification of specific interactions.

[0140] Gel shift methods can include, for example, using a colloidal form of COOMASSIE (Imperial Chemicals Industries, Ltd.) blue stain to detect proteins in gels, which may include or exclude polyacrylamide electrophoresis gels. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985) and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is incorporated herein by reference in its entirety for its teachings regarding gel shift methods. In addition to the conventional protein assay methods described above, a combined washing and protein staining composition is described in U.S. Pat. No. 5,424,000, which is incorporated herein by reference in its entirety for its teachings regarding gel shift methods. Solutions can include phosphoric, sulfuric, and nitric acids, as well as acid violet dye.

[0141] Radioimmunoprecipitation assay (RIPA) is a highly sensitive assay that uses radiolabeled antigens to detect specific antibodies in serum. The antigen is reacted with serum and then precipitated using special reagents that can include or exclude, for example, protein A sepharose beads. The bound radiolabeled immunoprecipitate is then analyzed, typically by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test to diagnose the presence of HIV antibodies. RIPA is also known in the art as the Farr assay, Precipitin assay, Radioimmune Precipitin assay, radioimmunoprecipitation analysis, radioimmunoprecipitation analysis, and radioimmunoprecipitation analysis.

[0142] The immunoassays described above, which use electrophoresis to separate and detect specific proteins of interest, allow for the assessment of protein size but are not very sensitive for assessing protein concentration. However, immunoassays are also contemplated in which proteins or protein-specific antibodies are bound to a solid support (e.g., a tube, well, bead, or cell) and combined with a method for detecting the protein or protein-specific antibody on the support to capture the target antibody or protein from a sample, respectively. Examples of such immunoassays include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture.

[0143] Radioimmunoassay (RIA) is a classical quantitative assay for directly or indirectly detecting antigen-antibody reactions using radioactively labeled substances (radioligands) and measuring the binding of unlabeled substances to specific antibodies or other receptor systems. Radioimmunoassays are used, for example, to test hormone levels in blood without the need to use bioassays. Non-immunogenic substances (e.g., haptens) can also be measured when they are bound to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) that can induce antibody formation. RIA is advantageous in that it uses radioactive antigens (radioisotopes) because it is easy to introduce iodine atoms into tyrosine residues in proteins. 125 I or 131 This technique involves mixing a radioactive antigen (often referred to as "radioactive antigen") with an antibody against that antigen. The antibody is generally attached to a solid support, which may or may not include tubes or beads. A known amount of unlabeled or "cold" antigen is then added, and the amount of labeled antigen displaced is measured. First, radioactive antigen is bound to the antibody. When cold antigen is added, the two compete for antibody binding sites, with higher concentrations of cold antigen binding more to the antibody and displacing the radioactive variant. The bound antigen is separated from unbound antigen in solution, and a binding curve is plotted using the radioactivity of each. This technique is extremely sensitive and specific.

[0144] Enzyme-linked immunosorbent assays (ELISAs), or more commonly referred to as EIAs (enzyme immunoassays), are immunoassays capable of detecting protein-specific antibodies. In such assays, the detectable label attached to either the antibody- or antigen-binding reagent is an enzyme. When exposed to its substrate, the enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorometry, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0145] Various ELISA techniques are known to those skilled in the art. In one variation, an antibody capable of binding to a protein can be immobilized on a selected surface exhibiting protein affinity, which can include or exclude wells in a polystyrene microtiter plate. A test composition suspected of containing a marker antigen can then be added to the well. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a second antibody specific for the target protein linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second antibody followed by a third antibody with binding affinity for the second antibody, which is linked to a detectable label.

[0146] Another variation is the competitive ELISA. In a competitive ELISA, a test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive species in the sample can be determined by mixing the sample with a known labeled species before or during incubation with the coated well. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well, thus reducing the final signal.

[0147] Regardless of the format used, ELISAs share certain features that may or may not include coating, incubation or binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. One or more antigens can be linked to a solid support, which may or may not be in the form of a plate, bead, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a plate with an antigen or antibody, the wells of the plate are generally incubated overnight or for a specified period with a solution of the antigen or antibody. The wells can then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells can then be "coated" with a nonspecific protein that is antigenically neutral with respect to the test antisera. These include bovine serum albumin (BSA), casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of antisera to the surface.

[0148] Rather than a direct procedure, ELISA can also use secondary or tertiary detection means. Thus, after binding of protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control clinical or biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a secondary binding agent together with a labeled secondary binding agent or a labeled tertiary binding agent.

[0149] An enzyme-linked immunospot assay (ELISpot) is an immunoassay capable of detecting antibodies specific to a protein or antigen. In such assays, the detectable label attached to either the antibody-binding reagent or the antigen-binding reagent is an enzyme. When exposed to its substrate, the enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorometry, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In this assay, a nitrocellulose microtiter plate is coated with the antigen. The test sample is exposed to the antigen and then reacted in a manner similar to an ELISA assay. This differs from traditional ELISA in that detection is determined by counting spots on a nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. The spots can then be counted to determine the number of cells in the sample that are specific for the antigen.

[0150] By "under conditions effective to allow immune complex (antigen / antibody) formation" is meant that the conditions include diluting the antigen and antibody in a solution that may or may not include BSA, bovine gamma globulin (BGG), and phosphate buffered saline (PBS) / Tween® to reduce nonspecific binding and promote a reasonable signal-to-noise ratio.

[0151] Appropriate conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be for about 1 minute to 12 hours at a temperature of about 20° to 30° C., or overnight at about 0° to about 10° C.

[0152] After all incubation steps in an ELISA, the contacted surface can be washed to remove uncomplexed material. The washing procedure can include washing with a solution that can include or exclude PBS / Tween® or borate buffer. After the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.

[0153] To provide a means of detection, as described above, the secondary or tertiary antibody can have an associated label to allow detection. This can be an enzyme that can generate color upon incubation with an appropriate chromogenic substrate. Thus, for example, the first or second immune complex can be contacted with and incubated with a labeled antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., a 2-hour incubation at room temperature in a PBS-containing solution, which may or may not contain PBS-Tween®).

[0154] After incubation with the labeled antibody and following washing to remove unbound material, the amount of label can be quantified by incubation with a chromogenic substrate, which can include or exclude, for example, urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and HO (in the case of peroxidase as the enzyme label). Quantitation can then be achieved by measuring the extent of color development, for example, using a visible spectrum spectrophotometer.

[0155] Protein arrays are solid-phase ligand-binding assay systems that use proteins immobilized on surfaces including glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. Assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and providing a wealth of data for a single experiment. Bioinformatics support is crucial; data processing requires sophisticated software and data comparison and analysis. However, software, as well as many hardware and detection systems, can be adapted from that used for DNA arrays.

[0156] One major format is the capture array, in which ligand-binding reagents (usually antibodies, but also alternative protein scaffolds, peptides, or nucleic acid aptamers) are used to detect target molecules in a mixture that may include or exclude plasma or tissue extracts. In diagnostics, capture arrays can be used to run multiple immunoassays in parallel, for example, both testing several analytes in individual serum samples and simultaneously testing many serum samples. In proteomics, capture arrays are used to quantify and compare protein levels in different samples in health and disease, i.e., protein expression profiling. Proteins other than specific ligand binders are used in array formats for in vitro functional interaction screening, which may include or exclude protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.

[0157] For array construction, protein sources include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production using cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high-throughput production. For capture arrays and protein function analysis, it is important that proteins are correctly folded and functional. This is not always the case, for example, when recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful for screening antibodies for cross-reactivity, identifying autoantibodies, and selecting ligand-binding proteins.

[0158] Protein arrays are designed as miniaturizations of well-known immunoassay methods, often utilizing fluorescent readout and capable of including or excluding ELISA and dot blotting, and facilitated by robotics and high-throughput detection systems to allow multiple assays to be performed in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdroplets of proteins delivered onto flat surfaces are the most well-known format, alternative architectures include CD centrifugation devices based on the development of microfluidics (Gyros, Monmouth Junction, NJ), and specialized chip designs that can include or exclude engineered microchannels within plates (e.g., The Living Chip™, Biotrove, Woburn, MA) and tiny 3D posts on silicon surfaces (Zyomyx, Hayward, CA). Particles in suspension can also be used as the basis for arrays, as long as they are coded for identification. Systems include color coding of microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), and barcoding of beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).

[0159] Protein immobilization involves both the coupling reagent and the properties of the surface to be coupled. A good protein array support surface is chemically stable before and after the coupling procedure, allows for good spot morphology, exhibits minimal nonspecific binding, does not contribute to background in the detection system, and is compatible with different detection systems. The immobilization method used is reproducible, applicable to proteins with different properties (size, hydrophilicity, hydrophobicity), amenable to high throughput and automation, and compatible with preserving fully functional protein activity. The orientation of surface-bound proteins is recognized as a critical factor in presenting them to ligands or substrates in an active state; in capture arrays, the most efficient binding results are obtained using oriented capture reagents, which generally require site-specific labeling of the protein.

[0160] Both covalent and noncovalent methods of protein immobilization are used and offer various advantages and disadvantages. Passive adsorption to surfaces is methodologically simple but offers little quantitative or orientational control. It may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide stable attachment, can be applied to a range of proteins, and have good reproducibility. However, orientation can be variable, chemical derivatization may alter protein function, and require a stable interaction surface. Biological capture methods that utilize tags on proteins provide stable attachment and bind to proteins in specific and reproducible orientations, but the biological reagent must first be sufficiently immobilized, and arrays require special handling and may have variable stability.

[0161] Several immobilization chemistries and tags for the creation of protein arrays have been described. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA), reversible covalent coupling is achieved through the interaction between proteins derivatized with phenyldiboronic acid and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence, allowing immobilized proteins to retain their function. Noncovalent attachment of unmodified proteins occurs within a porous structure based on three-dimensional polyacrylamide gels, which can include or exclude HydroGel (PerkinElmer, Wellesley, MA). This substrate has been reported to provide particularly low background on glass microarrays, resulting in high capacity and retention of protein function. Widely used biological coupling methods rely on biotin / streptavidin or hexahistidine / Ni interactions with appropriately modified proteins. Biotin can be conjugated to a polylysine backbone immobilized on a surface that can include or exclude titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0162] Array fabrication methods include robotic contact printing, inkjet, piezoelectric spotting, and photolithography. Several commercially available arrayers are available (e.g., Packard Biosciences), and manual devices (V&P Scientific) are also available. Bacterial colonies can be robotically gridded onto PVDF membranes for in situ induction of protein expression.

[0163] At the limit of spot size and density are nanoarrays, which have spots on the nanometer spatial scale and can perform thousands of reactions on a single chip less than 1 mm square. BioForce Laboratories has developed a nanoarray with 1,521 protein spots on 85 square microns (equivalent to 25 million spots / cm square), at the limit of optical detection. The readout methods are fluorescence and atomic force microscopy (AFM).

[0164] Fluorescent labeling and detection methods are widely used. The same equipment used to read DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g., Cy-3, Cy-5) and mixed, resulting in color acting as a readout of changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100-fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) allows for ultrasensitive fluorescence detection with the added advantage of no intervening washing steps. High sensitivity can also be achieved with suspended beads and particles using phycoerythrin (Luminex) or the properties of semiconductor nanocrystals (quantum dots) as labels. Numerous novel alternative readouts are being developed, particularly in the commercial biotechnology field. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonant light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy [BioForce Laboratories].

[0165] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They use high-affinity capture reagents that can include or exclude traditional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers to bind and detect specific target ligands in a high-throughput manner.

[0166] Antibody arrays have the necessary properties of specificity and acceptable background, and several are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are generated by conventional immunization (polyclonal sera and hybridomas) or after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA), usually as recombinant fragments expressed in E. coli. In addition to conventional antibodies, Fab and scFv fragments, single V domains from camelids, or engineered human equivalents (Domantis, Waltham, MA) can also be useful in arrays.

[0167] The term "scaffold" refers to the ligand-binding domain of a protein engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. Variants can be generated in a genetic library format and selected against individual targets by phage, bacterial, or ribosome display. Such ligand-binding scaffolds or frameworks include "affibodies" based on Staphylococcus aureus protein A (Affibody, Bromma, Sweden), "trinectins" based on fibronectin (Phylos, Lexington, MA), and "anticalins" based on lipocalin structures (Pieris Proteolab, Freising-Weihenstephan, Germany). These can be used on capture arrays in a manner similar to antibodies and may have the advantages of robustness and ease of manufacturing.

[0168] Non-protein capture molecules, particularly single-stranded nucleic acid aptamers that bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides using the Selex™ procedure, and their interaction with proteins can be enhanced by incorporating brominated deoxyuridine and covalently linking via UV-activated crosslinking (photoaptamers). Photocrosslinking to the ligand reduces the crosslinking reactivity of the aptamer due to specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA. Photoaptamer arrays allow binding to be detected using universal fluorescent protein stains.

[0169] Protein analytes bound to antibody arrays can be detected in sandwich assays, either directly or via secondary antibodies. Direct labeling is used to compare different samples with different colors. When pairs of antibodies directed against the same protein ligand are available, sandwich immunoassays offer high specificity and sensitivity, making them the method of choice for low-abundance proteins, which can include or exclude cytokines. They also offer the possibility of detecting protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance, and atomic force microscopy, avoid ligand alteration. What is required from any method is optimal sensitivity and specificity with low background to provide high signal to noise. Analyte concentrations range widely, so sensitivity must be appropriately adjusted. Serial dilution of the sample or the use of antibodies with different affinities are solutions to this problem. Proteins of interest are often low-abundance proteins in body fluids and extracts, requiring detection in the pg range or below, which can include or exclude cytokines or low-expression products in cells.

[0170] An alternative to arrays of capture molecules is that created by "molecular imprinting" techniques, in which peptides (e.g., from the C-terminal region of a protein) are used as templates to generate structurally complementary sequence-specific cavities in a polymeric matrix, which can then specifically capture (denaturate) proteins with the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA).

[0171] Another methodology that can be used diagnostically and in expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), in which a solid-phase chromatographic surface binds proteins with similar characteristics of charge or hydrophobicity from a mixture that can include or exclude plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detect the retained proteins.

[0172] Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and have been used to assay a wide range of biochemical functions, which can include or exclude protein interactions with other proteins, drug-target interactions, enzyme-substrate interactions, etc. Generally, they require expression libraries, cloned into E. coli, yeast, or similar, from which the expressed proteins are purified and immobilized, e.g., via His tags. Cell-free protein transcription / translation is a viable alternative for the synthesis of proteins that do not express well in bacterial or other in vivo systems.

[0173] For detecting protein-protein interactions, protein arrays may be an in vitro alternative to the cell-based yeast two-hybrid system, which may be useful when the latter is lacking, and may include or exclude interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and various functions of the yeast proteome (protein-protein and protein-lipid interactions), and the majority of all yeast open reading frames have been expressed and immobilized on microarrays. Large-scale "proteome chips" hold great promise for identifying functional interactions, drug screening, and the like (Proteometrix, Branford, CT).

[0174] Protein arrays can be used to screen phage or ribosome display libraries to select specific binding partners, including antibodies, synthetic scaffolds, peptides, and aptamers, as two-dimensional displays of individual elements. In this way, "library against library" screening can be performed. Screening drug candidates in combinatorial chemical libraries against arrays of protein targets identified from genome projects is another application of this approach.

[0175] Multiplexed bead assays, which may or may not include BD™ Cytometric Bead Arrays, are a series of spectrally distinct particles that can be used to capture and quantify soluble analytes. The analytes are then measured by fluorescence-based emission detection and flow cytometry analysis. Multiplexed bead assays are comparable to ELISA-based assays, but generate data in a "multiplexed" or simultaneous manner. Concentrations of unknowns are calculated for cytometric bead arrays, similar to any sandwich-format assay: by using known standards and plotting the unknowns against a standard curve. Furthermore, multiplexed bead assays enable quantification of soluble analytes in samples that were previously not considered due to sample volume limitations. In addition to quantitative data, powerful visual images can be generated that reveal unique profiles or signatures that provide additional information to the user at a glance.

[0176] Thus, in one aspect, disclosed herein is a method of identifying a T cell receptor disclosed herein, wherein T cell activity (e.g., which may or may not include release of cytokines including, but not limited to, IFN-γ, TGF-β, lymphotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25) is measured by any immunodetection method disclosed herein, such as, but not limited to, ELISA, ELISpot, intracellular cytokine staining, or chromium release.

[0177] The disclosed T cell receptors (e.g., T cell receptors that bind to cancer antigens, which can include or exclude any of, but are not limited to, DP4-ESO-1 TCR, A2-CT83 TCR, A2-pp65-TCR, and / or A2-IE-1-TCR) are useful in treating cancers including, but not limited to, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, melanoma, It is understood and contemplated herein that the compounds can be used to treat any cancer that expresses a particular type of MHC molecule and antigen, including, but not limited to, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, breast cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma. Thus, in one aspect, also disclosed herein is a method of identifying a TCR disclosed herein, wherein the cancer is selected from the group consisting of B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, breast cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma.

[0178] C. Composition Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that, although specific reference to various individual and collective combinations and permutations of these compounds is not expressly disclosed, each is specifically contemplated and described herein. For example, when a particular TCR is disclosed and discussed, and several modifications that can be made to several molecules comprising the TCR are discussed, any and all combinations and permutations of the TCR and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C and classes of molecules D, E, and F and examples of combined molecules are disclosed, AD is disclosed, and each is individually and collectively contemplated, even if each is not individually listed, meaning combinations, and AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups of AE, BF, and CE are considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0179] In one embodiment, the methods disclosed herein detect or identify TCRs that can be used in compositions for the treatment of cancer (either as therapeutic or prophylactic treatments) as well as in the preparation of TCR T cells that can be used to treat cancer. Accordingly, in one embodiment, also disclosed are TCR T cells engineered to express receptors (which can, for example, include or exclude T cell receptors) that can recognize the antigens disclosed herein.

[0180] In one embodiment, the present disclosure also features a method for enhancing the persistence of CAR-T and TCR cells by expression of chemokine receptors and shRNA KO in TCR or CAR constructs. In one embodiment, TCR T cells specific for one of the antigens disclosed herein (e.g., NY-ESO-1, CT83, pp65, and / or IE-1) can be further engineered to knock out or down negative signaling molecules, such as, but not limited to, programmed cell death protein 1 (PD1), von Hippel-Lindau tumor suppressor (VHL), and / or protein phosphatase 2 regulatory subunit Bdelta (PPP2R2D), to enhance their function, which may include or exclude cytotoxic activity and persistence or survival in vivo after adoptive transfer into cancer patients.

[0181] In some embodiments, negative signaling molecules are, for example, indoleamine(2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death-ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3). In certain aspects, negative signaling molecules are, for example, PD-1, VHL, PPP2R2D, and epigenetic factors, which may include or exclude JMJD3 and LSD1.

[0182] In one embodiment, the disclosure also features a method of enhancing T cell trafficking and / or tumor cell location in vivo within a tumor by forced expression of a chemokine receptor. In some embodiments, expression of the chemokine receptor is forced by fusing a CAR or TCR construct with a chemokine. In some embodiments, the chemokine receptor is CXCR3, CCR5, CCR2. In some embodiments, the chemokine receptor is CCR5.

[0183] Once the sequence of a TCR has been identified, it is understood and contemplated herein that the skilled artisan will have complete knowledge of the nucleic acid encoding said amino acid TCR, and it is well within the skill set of the skilled artisan to create said nucleic acid construct. Thus, in one aspect, nucleic acids encoding the polypeptides of any of the TCRs disclosed herein are also disclosed herein. Identity / Homology

[0184] It is understood that one way to define any known or potential variants and derivatives of the genes and proteins disclosed herein is by defining the variants and derivatives in terms of identity or homology and / or identifying them against a particular known sequence. For example, SEQ ID NO: 3 sets forth a particular sequence of the TCR alpha chain variable region. Specifically disclosed are variants of these and other genes and proteins disclosed herein that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology or identity to the set forth sequence. Those skilled in the art will readily understand how to determine the homology or identity of two proteins or nucleic acids, which may include or exclude genes. For example, the homology or identity can be calculated after aligning the two sequences so that the homology is at its highest level.

[0185] Other methods for calculating homology or identity can be performed using published algorithms. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0186] The same type of homology or identity is described, for example, in Zuker, M. Science 244:48-52, 1989, Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al., Methods Enzymol. 183:281-306, 1989, which are incorporated herein by reference at least with respect to material related to nucleic acid alignment. nucleic acid

[0187] For example, there are various nucleic acid-based molecules disclosed herein, including, for example, a nucleic acid encoding SEQ ID NO: 1, or any of the nucleic acids disclosed herein or fragments thereof, as well as various functional nucleic acids. In some embodiments, the nucleic acids included herein can include or exclude cDNA encoding a TCR alpha chain and / or a TCR beta chain. As used herein, the term "cDNA" refers to a nucleic acid that spans exon-exon or exon-only coding sequences. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, when a vector is expressed intracellularly, it is understood that the expressed mRNA is typically composed of A, C, G, and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment, for example, by exogenous delivery, it is understood that it is advantageous for the antisense molecule to be composed of nucleotide analogs that reduce degradation of the antisense molecule in the cellular environment. In some embodiments, the nucleotide analog comprises one or more modifications to one or more base, sugar, or phosphate moieties in the nucleic acids further disclosed herein. Nucleotides and Related Molecules

[0188] A nucleotide is a molecule comprising a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked to each other via their phosphate and sugar moieties to form an internucleoside bond. The base moiety of a nucleotide can be adenine-9-yl (A), cytosin-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), or thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art and can be used herein.

[0189] Nucleotide analogs are nucleotides that contain some type of modification to either the base moiety, sugar moiety, or phosphate moiety. Modifications to nucleotides are well known in the art and include, for example, 5-methylcytosine (5-me-C), 2-methylcytosine (2-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications to the sugar moiety or the phosphate moiety. There are many varieties of these types of molecules available in the art and that can be used herein.

[0190] Nucleotide substitutes are molecules that have similar functional properties to nucleotides but do not contain a phosphate moiety, which may or may not include peptide nucleic acids (PNAs). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked to each other through a moiety other than a phosphate moiety. Nucleotide substitutes can adopt a double helix-type structure when interacting with an appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and applicable herein.

[0191] Other types of molecules (conjugates) can also be linked to nucleotide or nucleotide analogs, for example, to enhance cellular uptake. The conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties that can include or exclude cholesterol moieties. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and can be used herein.

[0192] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute, which includes the C2, N1, and C6 positions of a purine-based nucleotide, nucleotide analog, or nucleotide substitute, and the C2, N3, and C4 positions of a pyrimidine-based nucleotide, nucleotide analog, or nucleotide substitute.

[0193] A Hoogsteen interaction is an interaction that occurs on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face contains reactive groups (NH or O) at the N7 position and at the C6 position of purine nucleotides. Primers and probes

[0194] Disclosed are compositions comprising primers and probes capable of interacting with the disclosed nucleic acids, which may comprise the tumor antigens, epitopes, and TCRs disclosed herein. In certain embodiments, primers are used to support DNA amplification reactions. Typically, primers can be extended in a sequence-specific manner. Sequence-specific primer extension includes any method in which the sequence and / or composition of the nucleic acid molecule to which the primer hybridizes or otherwise associates directs or influences the composition or sequence of the product produced by primer extension. Thus, sequence-specific primer extension includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify primers in a sequence-specific manner are preferred. In certain embodiments, primers are used in DNA amplification reactions, which may include or exclude PCR or direct sequencing. In certain embodiments, primers can also be extended using non-enzymatic techniques, for example, nucleotides or oligonucleotides used to extend the primer are modified so that they chemically react to extend the primer in a sequence-specific manner. Typically, the disclosed primers hybridize to the disclosed nucleic acids or regions of nucleic acids, or they hybridize to the complement of the nucleic acid or the complement of a region of the nucleic acid.

[0195] The size of the primer or probe for interacting with the nucleic acid in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, which can include or exclude DNA amplification or simple hybridization of the probe or primer. Exemplary primers or probes include at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, , 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0196] In other embodiments, the primers or probes are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 12 It can be up to 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0197] In certain embodiments, the product has at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0198] In other embodiments, the product is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0199] peptide Protein variants As discussed herein, numerous variants of TCRs are known and contemplated herein. Protein variants and derivatives are well understood by those skilled in the art and can include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional variants. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions are typically smaller than those of amino- or carboxyl-terminal fusions, e.g., on the order of one to four residues. Immunogenic fusion protein derivatives, which may include or exclude those described in the Examples, are generated by fusing a polypeptide large enough to confer immunogenicity to a target sequence by in vitro crosslinking or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about two to six residues are deleted at any one site within the protein molecule. These variants are typically prepared by site-directed mutagenesis of nucleotides in the DNA encoding the protein, thereby generating DNA encoding the variant, followed by expression of the DNA in recombinant cell culture. Methods for introducing substitution mutations at predetermined sites in DNA with a known sequence, such as M13 primer mutagenesis and PCR mutagenesis, are known. Amino acid substitutions are typically single residues, but can occur at multiple different positions at once. Insertions are usually on the order of about 1 to 10 amino acid residues. Deletions range from about 1 to 30 residues. Substitutions can include or exclude one or more of the six TCR CDR regions. Deletions or insertions are preferably made in adjacent pairs, i.e., two-residue deletions or two-residue insertions. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final construct. Mutations should not place the sequence out of frame and preferably do not create complementary regions that could generate secondary mRNA structures. Substitution variants are those in which at least one residue has been removed and a different residue inserted in its place.Such substitutions are generally made in accordance with Tables 1 and 2 below, and are called conservative substitutions. [Table 1] [Table 2]

[0200] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., by selecting residues that have a more significantly different effect on (a) the structure of the polypeptide backbone, e.g., sheet or helix conformation, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains. In general, the substitutions expected to produce the greatest changes in protein properties are those in which (a) a hydrophilic residue, such as seryl or threonyl, is substituted for (or by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue with an electropositive side chain, such as lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is substituted for (or by) a residue without a side chain, such as glycine, by (e) increasing the number of sites for sulfation and / or glycosylation.

[0201] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue with another, or one polar residue with another. Substitutions include, for example, combinations that may include or exclude Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservative substitution variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

[0202] Substitutional or deletional mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteine ​​or other labile residues may also be desirable. Deletion or substitution of potential proteolysis sites, e.g., Arg, is accomplished, for example, by deleting one of the basic residues or substituting one with glutaminyl or histidyl residues.

[0203] Certain post-translational derivatizations are the result of the action of recombinant host cells on expressed polypeptides. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the O-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] ), acetylation of N-terminal amines, and in some cases amidation of C-terminal carboxyls.

[0204] It is understood that one way to define variants and derivatives of the proteins disclosed herein is by defining them in terms of homology / identity to a particular known sequence. Specifically, variants of these and other proteins disclosed herein that have at least 70%, 75%, 80%, 85%, 90%, or 95% homology / identity to the described sequence are disclosed. Those skilled in the art will readily understand how to determine the homology / identity of two proteins. For example, the homology / identity can be calculated after aligning the two sequences so that the homology / identity is at its highest level.

[0205] Other methods for calculating homology / identity can be performed using published algorithms. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0206] The same type of homology / identity can be obtained for nucleic acids by the algorithms disclosed in, for example, Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Nat. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al., Methods Enzymol. 183:281-306, 1989.

[0207] The descriptions of conservative variation and homology / identity can be combined together in any combination, which can include or exclude embodiments in which a variant has at least 70% homology / identity to a particular sequence in which it is a conservative variation.

[0208] As this specification discusses various proteins and protein sequences, it is understood that the nucleic acids capable of encoding those protein sequences are also disclosed. This includes all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that encodes a particular protein sequence, as well as all nucleic acids including degenerate nucleic acids that encode variants and derivatives of the disclosed protein sequences. Thus, while each specific nucleic acid sequence may not be described herein, it is understood that each and every sequence is, in fact, disclosed and described herein through the disclosed protein sequences.

[0209] It is understood that there are numerous amino acid and peptide analogs that can be incorporated into the disclosed compositions. For example, there are numerous D-amino acids or amino acids with different functional substituents than those shown in Tables 1 and 2. Opposite stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogs, are disclosed. These amino acids can be readily incorporated into a polypeptide chain by charging a tRNA molecule with the amino acid of choice and engineering a genetic construct that utilizes, for example, an amber codon, to site-specifically insert the analog amino acid into the peptide chain.

[0210] Molecules can be created that resemble peptides but are not linked via natural peptide bonds. For example, amino acid or amino acid analog linkages can include CHNH--, --CHS--, --CH--CH--, --CH=CH-- (cis and trans), --COCH--, --CH(OH)CH--, and --CHHSO-- (these and others are described in Spatola, A. F., In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (--CH2NH--, CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH2H2--S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH2--); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (--COCH2--); Szelke et al. European Appln., EP 45665 CA (1982):97:39405 (1982) (--CH(OH)CH--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH--); and Hruby Life Sci 31:189-199 (1982) (--CH--S--), each of which is incorporated herein by reference. A particularly preferred non-peptide bond is --CHNH--.It is understood that peptide analogs can have more than one atom between the bond atoms and can include or exclude b-alanine, g-aminobutyric acid, and the like.

[0211] Amino acid analogs and analogs, as well as peptide analogs, often have enhanced or desirable properties, which may include or exclude more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broader range of biological activity), reduced antigenicity, etc.

[0212] D-amino acids can be used to generate more stable peptides because they are not recognized by peptidases and the like. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together. This can be beneficial for constraining peptides to a particular conformation.

[0213] Pharmaceutical Carriers / Drug Delivery In one aspect, disclosed herein is a composition comprising a therapeutically effective amount of one or more TCR T cells. The TCR T cells are engineered to express a receptor for one of the tumor antigens disclosed herein. In one aspect, TCR T cells specific for one of the tumor antigens disclosed herein can be further engineered to knock out or knock down programmed cell death protein (PD1), von Hippel-Lindau tumor suppressor (VHL), and / or protein phosphatase 2 regulatory subunit Bdelta (PPP2R2D) to enhance their function, which can include or exclude cytotoxic activity and persistence or survival in vivo after adoptive transfer into a cancer patient.

[0214] As mentioned above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with a nucleic acid or vector without causing undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained. As is well known to those skilled in the art, the carrier will naturally be selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject.

[0215] The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically, etc., including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" refers to delivery of a composition to the nose and nasal passages through one or both nostrils and may include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalant can occur through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, etc. Therefore, it is not possible to specify an exact amount for each composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation, given the teachings herein.

[0216] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid before injection, or emulsions.A more recent approach to parenteral administration involves the use of sustained release or sustained release systems to maintain a constant dosage.See, for example, U.S. Patent No. 3,610,795, incorporated herein by reference.

[0217] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe et al., Br. J. Cancer, 58:700-703, (1988); Senter et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler et al., Biochem. Pharmacol, 42:2062-2065, (1991)). This vehicle may include or exclude "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors participate in either constitutive or ligand-induced endocytic pathways. These receptors aggregate in clathrin-coated pits, enter cells via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then recycle to the cell surface, be stored intracellularly, or be degraded in lysosomes.Internalization pathways perform a variety of functions that may include or exclude nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0218] Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0219] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release preparations that may or may not include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the composition being administered.

[0220] Pharmaceutical carriers are known to those skilled in the art. These will most typically be standard carriers for administering drugs to humans, including sterile water, saline, and solutions that may or may not contain buffered solutions at physiological pH. The composition can be administered intramuscularly or subcutaneously. Other compounds are administered according to standard procedures used by those skilled in the art.

[0221] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients which may include or exclude antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0222] Pharmaceutical compositions can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topically (including ophthalmically, vaginally, rectally, or intranasally), orally, by inhalation, or parenterally, for example, by intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0223] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils with or without olive oil, and injectable organic esters with or without ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (with or without those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, which may include or exclude, for example, antibacterial agents, antioxidants, chelating agents, and inert gases.

[0224] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0225] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0226] Some compositions may potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids, which may include or exclude hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, which may include or exclude formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases, which may include or exclude sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, which may include or exclude mono-, di-, trialkyl, and aryl amines and substituted ethanol amines.

[0227] therapeutic use The effective dosage and schedule for administering the composition may be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. The dosage range for administering the composition is large enough to produce the desired effect in which the symptoms of the disorder are addressed. The dosage should not be so large as to cause adverse side effects, which may include or exclude undesirable cross-reactions, anaphylactic reactions, etc. In general, the dosage will vary depending on the patient's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. The dosage can be adjusted by an individual physician in the event of any contraindications. The dosage may vary and may be administered daily, for one day, or for several days, with one or more doses administered. Guidance regarding the appropriate dosage for a given class of pharmaceuticals can be found in the literature. For example, guidance in selecting an appropriate dose of an antibody can be found in literature on the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Typical daily dosages of antibodies used alone can range from about 1 μg / kg to up to 100 mg / kg body weight or more per day, depending on the factors noted above. Pharmaceutical compositions containing the subject engineered T cells may be administered in doses ranging from 10 to 100 mg / kg body weight per day, including all integer values ​​within those ranges. 4 ~10 7 engineered T cells / kg body weight, preferably 10 5 ~10 6It can generally be stated that the engineered T cell compositions can be administered at a dose of 1000 engineered T cells / kg body weight. The engineered T cell compositions can also be administered multiple times at these doses. The cells can be administered by using injection techniques commonly known in immunotherapy (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0228] D. Methods of Using the Composition Cancer Treatment Methods The disclosed compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, which may or may not include cancer. Accordingly, in one aspect, disclosed herein is a method of stimulating an immunological response against cancer, or treating, inhibiting, and / or preventing cancer, comprising administering to a subject a composition comprising a therapeutically effective amount of T cells engineered with an antigen-specific TCR disclosed herein (e.g., which may include or exclude any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:33).

[0229] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such an amelioration need not necessarily eliminate, but only reduce or modify. The exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the condition of the patient.

[0230] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the onset of the associated disease, pathological condition, or disorder; and supportive care, i.e., treatment used to supplement another specific treatment aimed at ameliorating the associated disease, pathological condition, or disorder.

[0231] A non-limiting list of various types of cancer that can be treated by the disclosed methods is as follows: lymphoma (Hodgkin's and non-Hodgkin's), leukemia, carcinoma, cancer of solid tissue, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or cancer in general.

[0232] A representative, but non-limiting list of cancers that can be treated using the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer which can include or exclude small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, and / or rectal cancer. [Example]

[0233] E. Working Example Example 1: Identification, cloning, construction and application of DP4-ESO-1 TCR Generation of HLA-DP4-restricted NY-ESO-1-specific T cells and clones In the present invention, HLA-DP4-restricted TCRs were cloned from T cell clones that specifically recognized the HLA-DP4-presented NY-ESO-1 peptide. In vitro sensitization was performed to obtain peptide-reactive T cells. NY-ESO-1, a peptide containing an HLA-DP4-restricted epitope, was used. 161~180 was synthesized with a purity of over 95%. The peptide was pulsed onto 1088 EBV-B cells, an HLA-DP4+ cell line, as APCs and co-cultured with human PBMCs in 96-well plates for 21 days. During treatment, NY-ESO-1 161~180 The specific T cell populations can be expanded under the pressure of continuous peptide stimulation, while nonspecific T cells and other types of immune cells can be depleted. After 21 days of stimulation, T cell populations in different wells were harvested for further characterization.

[0234] NY-ESO-1 161~180 We first examined the recognition of stimulated T cells from different wells against the peptide. T cells were cocultured with mock 1088 EBV-B cells and the same APCs pulsed with the target peptide, and T cell activation was determined by cytokine release in the supernatant. T cell populations from some wells showed very high activity against the peptide. These T cell populations were collected for CD8+ T cell depletion. After CD8+ T cell depletion, the CD4+ T cell line was designated DP4 ESO-reactive T cells. It was confirmed that the T cell line recognized the HLA-DP-restricted NY-ESO-1 epitope but was not HLA-DR-restricted. Meanwhile, specific peptide recognition by DP4 ESO-1-reactive T cells was blocked only by antibodies against all HLA class II molecules and HLA-DP molecules, indicating that recognition was HLA-DP-restricted, excluding other class I and class II molecules. Further attempts were made to identify the DP4 ESO-reactive T cell epitope. The shortest truncated form that maintained high T cell recognition and activity was designated NY-ESO-1. 157~170 Limited to.

[0235] To perform molecular cloning of the DP4-restricted NY-ESO-1 TCR from the DP4 ESO-reactive T cell line, single T cell clones were generated from the population. The DP4 ESO-reactive T cell line was serially diluted and seeded into a 96-well plate at a ratio of 0.3 cells / well. Single live T cells in the wells were cultured for 14 days for expansion. The expanded T cell clones were then cloned into 1088 EBV-B (HLA-DP4+)-presenting peptide NY-ESO-1. 157~170 The recognition of these clones against the epitope was determined by measuring cytokine release. 157~170 The assay results, partially shown in Figure 1, demonstrate the production of several DP4-ESO-1-reactive T cell clones that reacted with DP4-ESO-1. These peptide-reactive T cell clones were further cultured for 14 days for expansion. However, while many DP4-ESO-1-reactive T cell clones survived expansion, some T cell clones were exhausted and stopped growing, indicating that the lifespans of these T cell clones differed. The surviving T cell clones were further used for TCR cloning.

[0236] Molecular cloning of DP4-ESO-1 TCR Using live T cell clones, mRNA was added to 1 x 10 6mRNA was extracted from 100 T cells. Reverse transcription was performed using mRNA to generate templates for the following three rounds of nested PCR. During each round of PCR, the complementarity-determining region 3 (CDR3) of the TCR alpha and beta chains was amplified separately with a different primer set, including primers targeting all types of TRAV or TRBV. After recovery of the PCR products from the third round, the subtypes of the TCR alpha and beta chains were identified by Sanger sequencing of the PCR products. The full-length TCR alpha and beta chains were then amplified according to the identified TCR subtype (TRAV34, TRBV30). The amplified full-length TCRs (TRAV-TRAJ-TRAC, TRBV-TRBD-TRBJ-TRBC) were cloned into the MSGV retroviral vector linked by a P2A sequence (Figure 2).

[0237] Transduction of DP4-TCR into naive T cells for in vitro and in vivo function A two-step transduction strategy was applied to deliver TCRs to naive T cells. The TCR constructs were transfected into the Phoenix-Eco cell line using Lipofectamine to generate primary viral supernatants. 48 hours later, the viral supernatants were collected and added to the culture medium of the PG-13 cell line for infection. After infection, the PG-13 cell line began to secrete secondary viral supernatants. The viral supernatants from the PG-13 cell line were coated onto 24-well non-tissue culture plates pre-coated with retronectin. Naive CD4+ T cells, bead-isolated from human PBMCs and pre-stimulated with CD3 antibodies, were infected in the wells with the coated retrovirus. To improve the transduction efficiency of naive T cells, the PG-13 cell line transduced with the TCR-encoding constructs was cloned by limiting dilution in 96-well plates. Single PG-13 cells were expanded in each well for 15 or more days to generate PG-13 cell lines encoding 100% TCR. The transduction efficiency of naive T cells from different PG-13 clones was determined by staining with a TRBV30-specific antibody and flow cytometry, reaching an average of 60-70% (Figure 3A). The activity of TCR-transduced CD4+ T cells was tested with 586mel, 624mel, and DP4-ESO monomer, demonstrating specific recognition of the TCR (Figure 3B).

[0238] The activity of TCR-transduced T cells was further tested in a series of assays to determine whether they had similar functionality to the DP4 ESO-reactive T cell line. First, the transduced T cells expressed the HLA-DP4-restricted epitope NY-ESO-1. 157~170The transduced TCR demonstrated specific recognition of HLA-DP4-processed NY-ESO-1 (Figure 4A). Second, the transduced T cells specifically recognized naturally HLA-DP4-processed NY-ESO-1 when assayed with either artificial APCs or HLA-DP4-positive or -negative tumor cells transfected with the plasmid (Figure 4B). To confirm that the cloned TCR functions as a CD4+ TCR, bead-isolated CD8+ and CD4+ T cells were transduced, respectively. This assay demonstrated that only CD4+ T cells transduced with this TCR were functional at the HLA-DP4-restricted epitope, consistent with expectations (Figure 4C). All results confirmed the ability of the TCR-engineered CD4+ T cells to recognize HLA-DP4, which specifically presents NY-ESO-1. In one embodiment, this HLA-DP4-restricted NY-ESO-1 TCR invention serves as a novel strategy for clinical responses in cancer immunotherapy.

[0239] Humanized mice were used to evaluate the efficacy and safety of the NY-ESO-1 TCR due to limitations in the use of transgenic mice. Humanized NSG (NOD SCID IL2γ- / -) mice were obtained and used to test the growth of human cancer cells. MDA-MB-231 / DP4 / ESO tumor cells were prepared in 50 μl of growth medium / Matrigel (50%) and orthotopically injected into the fourth fat pad of female NSG mice (n=6 per group) on day 1. After injection, it was observed that this human tumor line could grow in NSG mice. On day 5, tumor-bearing NSG mice were inoculated with 2 × 10 human T cells per mouse in four groups: 1. untransduced CD8+ and CD4+ T cells; 2. A2-ESO-1 TCR-CD8+ and untransduced CD4+ T cells; 3. untransduced CD8+ and DP4-ESO-1 TCR-CD4+ T cells; and 4. A2-ESO-1 TCR-CD8+ and DP4-ESO-1 TCR-CD4+. 6After intravenous injection of the cells, the mice were treated with three doses of IL-2 intraperitoneally to stimulate T cell proliferation. Tumor growth in each group was monitored every 3–5 days, and T cell migration was tracked by luciferase transduction in CD8+ T cells. The results showed that the injected A2-ESO-TCR-engineered CD8+ T cells gradually but significantly migrated to the tumor site after injection, demonstrating the specificity and safety of the engineered T cells in vivo (Figure 5A). A2-ESO-1 TCR-engineered CD8+ T cells also dramatically inhibited tumor growth, as expected (Figures 5B and 5C). Surprisingly, DP4-ESO-1 TCR-CD4+ T cells showed significant suppression of tumor growth, as did A2-ESO-1 TCR-engineered CD8+ T cells (Figures 5B and 5C), indicating that CD4+ cytolytic activity was activated in this case. Furthermore, the combination of A2-ESO-1 TCR-CD8+ T cells with DP4-ESO-1 TCR CD4+ T cells achieved maximal inhibition of tumor growth when mice were sacrificed (Figure 5B, C), demonstrating that the combined use of CD8+ and CD4+ T cells transduced with TCRs targeting the same antigen was superior to either transduced TCR-T cell type alone. This confirmed that CD4+ T cells provided efficient support for the antitumor activity of CD8+ T cells, providing striking synergistic results.

[0240] Six- to eight-week-old female NSG mice were purchased from the Jackson Laboratory or housed in the animal facility at Houston Methodist Research Institute. All procedures were approved by the Houston Methodist Research Institute Animal Care and Use Committee (IACUC) and performed under standard laboratory protocols. MDA-MB-231 / DP4 / ESO tumor cells in 50 μl of growth medium / Matrigel (50%) were orthotopically injected into the fourth fat pad of female NSG mice (n = 6 per group) on day 1. Four groups of human T cells (1. untransduced CD8+ and CD4+; 2. A2-ESO-1 TCR-CD8+ and untransduced CD4+; 3. untransduced CD8+ and DP4-ESO-1 TCR-CD4+; 4. A2-ESO-1 TCR-CD8+ and DP4-ESO-1 TCR-CD4+) were injected into tumor-bearing NSG mice at 2 × 10 per mouse. 6 Cells were injected intravenously on day 5, followed by three doses of IL-2 intraperitoneally. Tumor growth was monitored every 3–5 days, and T cell migration was tracked by luciferase transduction in CD8+ T cells.

[0241] Example 2: Identification, construction and application of T cells and A2-CT83 TCR-T cells Identification of a novel HLA-A2-restricted CT83 epitope recognized by T cells Because HLA-A2 is the predominant HLA class I molecule and is highly expressed in approximately 50% of the general human population, we conducted experiments to identify novel HLA-A2-restricted T cell epitopes derived from CT83. To this end, we synthesized a series of CT83 peptides containing potential HLA-A2-binding motifs (CT83 PEP66-74, PEP79-87, and PEP90-98). + T cells expressing HLA A2 +PBMCs were isolated from healthy donors and stimulated with peptide-loaded autologous dendritic cells (DCs) for 10 days. T cell medium containing IL-7 and IL-15 (5 ng / ml each) was added every 2–3 days. For the second stimulation, autologous PBMCs were irradiated (60 Gy) and pulsed with 1 μg / ml peptide for 2–4 hours. After washing, these irradiated, peptide-pulsed PBMCs were added and incubated with the first-stimulated T cells for 10 days. These T cells were fed with T cell medium containing IL-2 (30 IU / ml), IL-7 (5 ng / ml), and IL-15 (5 ng / ml) every 2–3 days. To test specific recognition of CT83, in vitro stimulated T cells were stimulated with CT83 peptide. 90~98The 293T / CT83 peptides were incubated with HLA-A2+ HEK293T cells with or without CT83. Results showed that in vitro peptide-stimulated T cells specifically recognized the 293T / CT83 peptide (PEP90-98) but did not respond to 293T cells (Figure 6A). No T cells specific for CT83 PEP66-74 or CT83 PEP79-87 were generated (data not shown). To determine whether these T cells endogenously processed and presented CT83 PEP90-98 via HLA-A2, they were tested against 293T cells transfected with either invariant chain (II)-fused CT83 or CT83-GFP (full-length CT83 linked to the P2A sequence and green fluorescent protein (GFP)). 293T / control peptide and 293T / CT83 PEP90-98 served as negative and positive controls. The results showed that CT83-specific T cells recognized 293T / Ii-CT83 and 293T / CT83-GFP cells, as well as 293T / CT83 PEP90-98, but not 293T / control peptide (Figure 6B). We next determined whether these CT83-specific T cells could recognize breast cancer cells. We found that MDA-MB-231 cells (expressing CT83 and HLA-A2), but not MDA-MB-468 cells (expressing CT83 but not HLA-A2), could stimulate CT83-specific T cells to secrete interferon-γ (IFN-γ) (Figure 6C), suggesting that T cells recognize CT83 PEP90-98 presented by HLA-A2 molecules. To further verify this point, antibody blocking experiments were performed, and the results showed that T cell recognition of MDA-MB-231 cells could be completely blocked by anti-MHC-I, but not by anti-MHC-II or control antibodies (Figure 6D), indicating that these T cells are specific and can recognize breast cancer cells that naturally express CT83 and HLA-A2 molecules.

[0242] Vaccination with CT83 PEP90-98 inhibits breast cancer growth To further test whether A2-CT83 PEP90-98 could induce antitumor immunity in HLA-A2 transgenic (Tg) mice, E0771-A2-CT83 mouse breast cancer cells (0.5 × 10 6 TAT-CT83 PEP90-98 cells / mouse) were orthotopically injected into the mammary gland pads of HLA-A2 mice on day 0. Tumor-bearing mice were treated with self-assembling nanoparticle vaccines containing TAT-CT83 PEP90-98 or TAT-CT83 PEP66-74 along with TLR ligands (CpG, MPLA, and poly(I:C), abbreviated as CMI) on days 7, 10, and 15 (Figure 7A). The results surprisingly showed that TAT-CT83 PEP90-98-CMI significantly inhibited tumor growth, whereas TAT-CT83 PEP66-74-CMI did not (Figure 7B). CT83 PEP66-74 has been reported to induce T cell responses using RNA vaccines. 35 However, no activity was induced with CT83 PEP90-98. Thus, this was the first demonstration that the CT83 PEP90-98 epitope can induce T cell responses and inhibit tumor growth in vitro and in vivo.

[0243] Identification and characterization of the A2-CT83 TCR using single-cell barcoding technology To identify CT83-specific TCRs, A2-CT83-specific T cells were stimulated with 293T / CT83 cells, intracellularly stained with anti-IFN-γ (Figure 8A), and purified by FACS sorting. Purified T cells (several thousand) were split into nanowriter-scale gel beads-in-emulsion (GEM) on a Chromium Next GEM Chip G treated with a 10x Genomics Chromium Controller. After multiple steps of cell lysis, reverse transcription (RT), PCR application, and barcoded cDNA for TCR V(D)J library construction according to the manufacturer's protocol, the final enriched TCR V(D)J library was sequenced using an Illumina sequencer (HiSeq 2500). After TCR sequence alignment and analysis, dominant and subdominant paired TCRα and TCRβ were identified, and then full-length TCRα and TCRβ were generated using TCR subtype-specific primers. These full-length TCRs were cloned into the retroviral expression vector pMSGV1 or the lentiviral expression vector pFU3W (driven by the U3 promoter) (Figure 8A). Results demonstrated that A2-CT83 TCR-T cells were able to recognize 293T cells transfected with CT83-GFP and Cos-7 cells transfected with HLA-A2 and CT83, but not 293T, Cos-7, or Cos-7 transfected with HLA-A2 alone (Figure 8B). Results further demonstrated that A2-CT83 TCR-T cells recognized 293T / CT83 PEP90-98, but not 293T cells or 293T cells pulsed with other CT83 peptides (Figure 8C). To demonstrate whether these A2-CT83 TCR-T cells can recognize HLA-A2 and CT83-expressing MDA-MB-231 cells, we co-cultured these T cells with different breast cancer cells. Indeed, A2-CT83 TCR-T cells specifically recognized MDA-MB-231 cells, but not MCF7 (A2 + CT83 - ), HTB-21(A2 + CT83 - ) or MDA-MB-436(A2- CT83 + ) cells (Figure 8D). Furthermore, when the A2-CT83 TCR-T cells were tested for their ability to recognize lung cancer cells, the results showed that these A2-CT83 TCR-T cells could specifically recognize CT83+ and HLA-A2+ lung cancer cells (HOP92 / A2, NCI-H358 / A, and NCI-H838 / A2), but could not recognize CT83+HLA-A2- tumor cells (HOP92, NCI-H358, and NCI-838) (Figure 8E). These results indicate that the A2-CT83 TCR can specifically recognize the CT83 epitope naturally processed by HLA-A2 identified in this invention in both transfected cell models and natural tumor cell lines.

[0244] Antitumor activity of A2-CT83 TCR-T cells in vivo To further demonstrate the antitumor activity of A2-CT83 TCR-T cells in vivo, immunocompromised NSG mice were used as a tumor model. NCI-H838 / A tumor cells were injected on day 0, followed by A2-CT83 TCR-T cells (5 × 10e6 per mouse, intravenously) on days 3 and 5, along with IL-2 (50,000 IU / day, intraperitoneally) on days 3–7 (Figure 9A). Results showed that A2-CT83 TCR-T cells completely inhibited tumor growth (Figures 9B and 9C). In contrast, tumor-bearing mice treated with control T cells developed large tumor masses (Figures 9B and 9C). These studies suggest that A2-CT83 TCR-T cells have potent antitumor activity in vivo. Example 3 Identification, construction and application of A2-HCMV TCR

[0245] Human cytomegalovirus (HCMV) nucleic acids and proteins could be detected in glioblastoma. Two proteins of the HCMV particle (pp65 and IE-1) were targeted for antigen-specific TCR recognition.

[0246] Selection of HLA-A2-restricted pp65- and IE-1-specific T cells and clones Stimulation of pp65- and IE-1-specific T cells was similar to that of DP4-ESO-1 T cells. CD8+ T cells isolated from PBMCs from healthy donors (with HLA-A2 typing) were pulsed and stimulated with two HLA-A2-restricted epitopes, pp65 (495-503) and IE-1 (316-324), respectively. Peptides encoding the two epitopes were synthesized with greater than 95% purity. Mature dendritic cells isolated from autologous PBMCs were resuspended in T cell culture medium and pulsed overnight with each peptide at a concentration of 10 μg / ml. Pulsed cells were irradiated with 60 Gy for 3 minutes and cocultured with CD8+ T cells at a 1:5 ratio. Primed CD8+ T cells were restimulated with 10 μg / ml of peptide on day 7 and harvested on day 14.

[0247] After in vitro stimulation, peptide-reactive CD8+ T cells (15% for pp65 and 8% for IE-1) were selected and T cell clones were generated by limiting dilution (Figures 10A and 10B). Seven T cell clones specifically recognized pp65 (495-503), and five T cell clones specifically recognized IE-1 (316-324) in contrast to the β-gal control peptide when the peptides were loaded into T2 cells (Figure 10A). T cell clone #3 reactive to pp65 and T cell clone #5 reactive to IE-1 were selected for further in vitro activity testing, respectively. Two T cell clones specifically recognized Cos-7 cells cotransfected with HLA-A2 and pp65, or HLA-A2 and IE-1, respectively, compared with transfection with other HLA molecules (Figure 10B). These two T cell clones were used for further TCR cloning.

[0248] Identification, cloning, and in vitro activity of the A2-pp65 TCR and A2-IE-1 TCR Identification of A2-pp65 TCR and A2-IE-1 TCR from T cell clones was performed using the same method as described above. After CDR3 sequencing, the TCR repertoires of both TCRs (TRAV24, TRBV6-5 from pp65 T cell clone #3; TRAV25 and TRBV5-1 from IE-1 T cell clone #5) were identified. Full-length TCRs were amplified with specific primers and then constructed into pMSGV vectors, respectively. Retroviral TCR transduction in human T cells was performed as described above. The transduction efficiency of both TCRs was confirmed by FACS after staining with TCR-specific antibodies, respectively. Both TCRs showed transduction efficiencies of over 60% (Figure 11A), indicating good candidates for in vitro assays. After incubation, A2-pp65 TCR-transduced T cells or A2-IE-1 TCR-transduced T cells were able to recognize the U87 glioblastoma line (HLA-A2+) transfected with pp65 or IE-1, respectively, but not the U118 glioblastoma line (HLA-A2-) transfected with pp65 or IE-1 (Figure 11B). Notably, both TCR-transduced T cells specifically recognized the U87 cell line infected with HCMV strain AD169 compared with the U118 cell line after infection (Figure 11B). Both TCR-transduced T cells had a dose-dependent recognition pattern by T2 cells pulsed with the pp65 (495-503) or IE-1 (316-324) peptide, respectively (Figure 11C). To determine their cytolytic potential, the cytotoxicity of A2-pp65 TCR-transduced T cells or A2-IE-1 TCR-transduced T cells was also assayed. Both TCR-transduced T cells demonstrated nearly 100% cytolysis on U87 cells transfected with pp65 or IE-1, respectively, or infected with HCMV strain AD169 (Figure 11D). The cytotoxicity of both TCR-transduced T cells against AD169-infected U87 cells also progressed in a dose-dependent manner (Figure 11E).

[0249] Antitumor activity of A2-pp65 TCR- and A2-IE-1 TCR-transduced T cells in vivo To evaluate the antitumor activity of A2-pp65 TCR-transduced T cells and A2-IE-1 TCR-transduced T cells against HCMV antigen-expressing tumors in vivo, we used a xenograft model in which transplanted tumors were established in immunodeficient mice with U87 cells expressing pp65 or IE-1 and luciferase. Tumors were established in SCID / beige mice for 3 days, and then 2 × 10 per mouse were transplanted by adoptive transfer of A2-pp65 TCR-, A2-IE-1 TCR-, or control TCR-transduced human T cells. 6 Mice were treated with A2-pp65 TCR-transduced T cells by intravenous injection (Figures 12A and 13A). Migration of the injected T cells was monitored every 3 days until the mice were sacrificed (Figures 12B and 13B). Tumor growth was efficiently suppressed in both TCR-treated groups (n = 5) compared with the control group receiving control TCR-transduced T cells (n = 3) from the same healthy donor. A reduction in tumor size was observed in all animals treated with A2-pp65 TCR-transduced T cells or A2-IE-I TCR-transduced T cells (Figures 12C, 12D, and 13C, 13D). These results demonstrate the antitumor activity of both TCRs and their further application in the treatment of glioblastoma.

[0250] Example 4 Reducing TCR mispairing with endogenous TCRs to improve TCR expression, specificity and function Because each T cell contains its own endogenous TCR, CT83-specific TCRα and TCRβ can mispair with endogenous TCRα and TCRβ, generating nonfunctional TCR(α / β)s or new TCR(α / β)s with unexpected antigen specificities. Spear, TT, Foley, KC, Garrett-Mayer, E. & Nishimura, MI. TCR modifications that enhance chain pairing in genetically modified T cells can increase cross-reactivity and alleviate CD8 dependence. J. Leukoc. Biol. 103, 973-983 (2018; Bethune, M. T. et al. Domain-swapped T cell receptors improve the safety of TCR gene therapy. Elife 5 (2016). Two strategies are useful to circumvent this problem. One approach is to knock out the endogenous TCR (α / β) using C-CRISPR / Cas9 technology (Legut, M., Dolton, G., Mian, A. A., Ottmann, O. G. & Sewell, A. K. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood 131, 311-322 (2018)). Another approach is to generate chimeric TCRs by fusing the NY-ESO-1 or CT83 TCR variable region to a non-human TCR, e.g., a murine TCR constant region, thus combining the endogenous TCR (HC) with the NY-ESO-1 TCR (MC) or CT83 TCR. The goal of this study is to reduce mismatching between the TCR (MC) and the NY-ESO-1 TCR. Using the latter approach, we demonstrated that replacing the human NY-ESO-1 TCR with a mouse constant region improved TCR expression and functional recognition of tumor cells (Figures 14A-G). Importantly, A2-ESO-1 TCR-M engineered T cells exhibited stronger antitumor activity in vivo than A2-ESO-1 TCR-M engineered T cells (Figures 15A-D).

[0251] A2-ESO-1 TCR and A2-CT83 TCR using mouse TCR constant regions Five constructs of the A2-ESO-1 TCR were engineered with different amino acid substitutions in the variable regions of the alpha or beta chains. The pMSGV-A2-ESO-1 TCR construct was used as a template for PCR amplification of TCR fragments using primers containing site-specific mutations. After overlapping of multiple PCR amplicons, the five variants of A2-ESO-1 were cloned back into the pMSGV vector (sub1: encoding the S53W mutation in the alpha chain; sub2: encoding the G50A, A51E mutations in the beta chain; sub3: encoding the G50A mutation in the beta chain; sub4: encoding the A97L mutation in the beta chain; and sub5: encoding the G50A, A51E, A97L mutations in the beta chain). The results showed that these A2-ESO-1 TCR-transduced T cells were able to recognize NY-ESO-1-expressing 624mel (HLA-A2+) and MDA-MB-231 (HLA-A2+) tumor cells, but not 586mel (HLA-A2-) tumor cells (Figure 16A). Consistently, cytotoxicity of A2-ESO-1 TCR (containing amino acid substitutions)-transduced T cells against NY-ESO-1-expressing 624mel (HLA-A2+) and MDA-MB-231 (HLA-A2+) tumor cells was demonstrated (Figure 16B).

[0252] To reduce potential mismatching between the A2-ESO-1 TCR and the endogenous human TCR (without defined antigen specificity), we replaced the human A2-ESO-1 TCR constant region with a mouse TCR constant region to generate chimeric A2-ESO-1 TCRs (human TCR alpha or beta variable regions fused with mouse TCR alpha or beta constant regions, respectively). T cells transduced with TCRs bearing mouse constant regions (A2-ESO-1 TCR-M, A2-ESO-1 TCR(S2)-M, and A2-ESO-1 TCR(S5)-M) were found to be able to recognize MDA-MB-231 / ESO cells (Figure 16C), suggesting that the mouse constant region replacement enhances TCR expression and function in human T cells.

[0253] Antitumor activity of A2-CT83 TCR-M T cells (mouse constant region) FACS analysis demonstrated that the transduction efficiency of A2-CT83 TCR-M (mouse constant region) in human T cells exceeded 70% (Figure 17A). A2-CT83 TCR-M-transduced T cells strongly recognized HLA-A2+ and CT83+ tumor cells (MDA-MB-231 and NCI-H1563) but not CT83- tumor cells (CAMA-1) (Figure 17B). Consistently, the results showed 60-80% tumor cell lysis against MDA-MB-231 and NCI-H1563 (Figure 17C). These results demonstrate that A2-CT83 TCR-M T cells are potent and specific for tumor cells, with reduced TCR mismatching. Example 4: Modulation of CAR-T cell signaling for T cell persistence by replacing CD3ζ signaling with a signaling domain derived from the ZAP70 kinase domain

[0254] TCR complex containing anti-CD19 scFv-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327) CAR constructs contain a single-chain variable fragment (ScFv) for antigen recognition, a transmembrane domain, and an intracellular T cell activation moiety (consisting of a CD28 or 4-1BB costimulatory signaling domain fused to a CD3ζ signaling domain). Sadelain, M., Brentjens, R. & Riviere, I. The basic principles of chimeric antigen receptor design. Cancer Discov 3, 388-398, doi:10.1158 / 2159-8290.CD-12-0548 (2013). The CD3ζ chain is a key component of the TCR-CD3 complex, playing a key role in signal transduction. The function of CD3ζ is to recruit Zap70 after phosphorylation at the ITAM upon activation following TCR engagement. Current CAR constructs contain the CD3ζ chain for T cell signal transduction and recruit ZAP70. Experiments were performed to demonstrate that the CD3z signaling domain can be replaced with other signaling domains to enhance CAR-T cell activation and persistence, for example, to demonstrate that CAR constructs containing signaling moieties derived from Zap70 enhance CAR-T cell signaling as well as CAR-T cell activation and persistence.

[0255] To this end, we screened signaling domains derived from ZAP70 and LAT and identified anti-CD19 scFv-CD28-ZAP300 (from amino acid residue 300 to the C-terminus) and anti-CD19-CD28-ZAP327 (from amino acid residue 327 to the C-terminus of the ZAP protein) (Fig. 11A). These Zap70 kinase domains (starting at aa 300 and 327 of Zap70) were fused to the C-terminus of the CD3 ζ chain, using CD28 as a costimulatory domain, to generate anti-CD19 scFv-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327) (Fig. 18A). Flow cytometry demonstrated comparable transduction efficiencies of 1928ZAP300 and 1928ZAP327 to conventional anti-CD19-CD28-CD3ζ (abbreviated as 1928Z) for CAR expression (Figure 18B). Furthermore, these CAR-T cells directly killed Raji tumor target cells in a non-radioactive LDH cytotoxicity assay using serial dilutions of E:T ratios (Figure 18C). Thus, Figures 18C and 18D show antigen-specific recognition and tumor cell lysis after co-culture of CAR-T cells with Raji tumor cells, demonstrating that ZAP300 and ZAP327 CAR-T cells are functional and specific for their target antigens in in vitro experimental studies.

[0256] The results further demonstrated that 1928ZAP300 and 1928ZAP327 CAR-T cells outperformed 1928z CAR-T cells in in vivo experiments and surprisingly and dramatically extended overall survival in the Raji lymphoma mouse model (Figure 18D). Raji-bearing NSG mice were treated with untransduced T cells, 1928Z, 1928ZAP300, or 1928ZAP327 CAR-T cells, and tumor-bearing NSG mice treated with control T cells were found to die within 20 days after tumor injection. Treatment of tumor-bearing mice with 1928Z CAR-T cells resulted in death around day 40. In contrast, treatment of tumor-bearing mice with 1928ZAP300 or 1928ZAP327 CAR-T cells significantly extended mouse survival to 70 days or longer after tumor injection (Figure 18E). Notably, over 60% of tumor-bearing mice treated with 1928ZAP327 CAR-T cells survived for more than 80 days. These studies demonstrate that replacement of the CD3ζ chain with the Zap70 kinase domain (ZAP300 and ZAP327) significantly enhances antitumor activity in vivo.

[0257] 19bbZAP327 CAR-T cells produce fewer cytokines and stronger anti-tumor immunity To test whether the Zap70 kinase domain could also function in 4-1BB-containing CAR constructs, we created a CAR construct in which the Zap70 kinase domain (e.g., ZAP327) was fused to the 4-1BB domain (19bbZAP327) (Figure 19A). The results showed that 19bbZAP327 CAR-T cells produced significantly less IFN-γ, IL-2, and TNF-α than 19bbz CAR-T cells after stimulation with tumor cells (Figure 19B). 19bbZAP327 and 19bbz CAR-T cells exhibited comparable specific tumor lysis in vitro (Figure 19C). Importantly, tumor-bearing mice treated with 19bbZAP327 showed superior antitumor activity compared to 19bbz CAR-T cells (Figures 19D and 19E). Mice treated with 19bbZAP327 CAR-T cells significantly prolonged survival compared with mice treated with 19bbZ CAR-T cells (Figures 19D and 19E). Collectively, our results demonstrate that 19bbZAP327 CAR-T cells produce reduced cytokines and more potent anti-tumor immunity compared with conventional 19bbz CAR-T cells.

[0258] Furthermore, ZAP327 could be fused to TCR constructs to enhance T cell signaling. These studies demonstrate that the ZAP327 signaling domain can enhance the signaling, function, and persistence of CAR-T and TCR-T cells. In other embodiments, other signaling domains derived from ZAP300 or ZAP70 can be used in CAR or TCR constructs to enhance anti-tumor activity while reducing the amount of cytokines produced.

[0259] The ZAP327 signaling domain promotes T cell memory function and persistence in vivo To understand why 1928ZAP327 CAR-T cells conferred stronger antitumor immunity, we examined T cell survival 30 days after T cell transfer. We found a higher percentage of 1928ZAP327 CAR-T cells in the bone marrow and spleen compared with 1928z-treated mice 30 days after T cell transfer (Figure 20A). Furthermore, the percentage of central memory 1928ZAP327 CAR-T cells was higher than that of 1928z CAR-T cells (Figure 20B). 1928ZAP327 CAR-T cells expressed lower amounts of PD-1 molecules (a marker of exhaustion) than 1928z CAR-T cells (Figure 20C), suggesting that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. In other embodiments, other signaling domains derived from ZAP300 or ZAP70 can be used in CAR or TCR constructs to promote T cell memory function and persistence in vivo.

[0260] Example 6 Modulation of TCR-T cell function in vivo by knocking down the expression of metabolic and epigenetic genes PD1, VHL, PPP2R2D, or JMJD3 Knockdown of PD1, VHL, and PPP2R2D enhances TCR-T cell function ShRNAs for PD1, VHL, and PPP2R2D were constructed and cloned downstream of the A2-ESO-1 TCR expression vector. Retroviral particles were generated and used to transduce naive human T cells. Transduction efficiency was determined by A2-ESO-1 TCR staining and FACS analysis. We demonstrated the transduction efficiency of T cells (Figure 21A) and used them for animal experiments. Breast cancer-bearing mice were generated by subcutaneous injection of engineered MDA-MB-231 / NY-ESO-1 / luciferase cells into NSG mice. Three days later, naive T cells, A2-ESO-1 TCR-T cells with or without knockdown of PD1, VHL, and PPP2R2D, were intratumorally injected. Tumor burden was assessed by in vivo luciferase imaging at the indicated time points, and mouse survival was monitored (Figure 21B). All A2-ESO-1 TCR-T cells with PD1, VHL, or PPP2R2D knockdown exhibited better or similar tumor suppression than A2-ESO-1 TCR-T cells alone. When combined with PD1, VHL, or PPP2R2D knockdown, TCR-T cells exhibited longer mouse survival times. In the VHL and PPP2R2D groups, some mice survived until the end of the experiment (Figure 21B).

[0261] Knockdown or knockout of JMJD3 enhances T cell function and persistence in vivo Furthermore, we show that CAR constructs containing JMJD3 shRNA or LSD1 shRNA can prolong T cell persistence (memory T cell function) and thus enhance anti-tumor immunity and mouse survival.

[0262] As described herein, Jmjd3 conditional knockout (KO) in CD4+ T cells was demonstrated to enhance the CD44+CD62L- memory T cell population compared to wild-type (WT) mice (Figure 22). Using WT and Jmjd3 cKO T cells, results further demonstrated that Jmjd3 cKO 2d2 transgenic CD4+ T cells stimulated in vivo with MOG peptide plus complete Freund's adjuvant (Figure 23A) significantly enhanced clinical scores in a closely related EAE mouse model (Figure 23B) with higher numbers of Jmjd3 cKO T cells compared to WT 2dT cells after T cell transfer (Figure 23C). Similar results were obtained using in vitro T cell stimulation (Figures 23D, 23E, and 23F). These results indicate that Jmjd3 KO significantly enhances T cell survival and persistence.

[0263] To determine the molecular mechanisms involved in enhanced T cell survival and persistence, we further demonstrated that Jmjd3 cKO T cells had significantly reduced levels of p19, p21, and p53 (key proteins regulating T cell apoptosis) after a second stimulation with anti-CD3 and anti-CD28 antibodies (Figure 24A). Indeed, the results showed that Jmjd3 cKO T cells had much lower levels of T cell apoptosis compared with WT T cells after a second stimulation with anti-CD3 and CD28 antibodies (Figure 24B). To provide direct evidence of reduced T cell apoptosis in Jmjd3 cKO T cells, we also determined that Jmjd3 cKO T cells had significantly lower levels of cleaved caspase 3 compared with WT T cells after anti-CD3 and CD28 stimulation (Figure 24C). These results indicate that Jmjd3 KO significantly enhances T cell survival and persistence by reducing caspase 3 activation.

[0264] Next, we determined whether knockdown of JMJD3 enhanced CAR-T cell survival and persistence, thus increasing antitumor immunity. Figure 25A shows the experimental design using Raji tumor cells to monitor luciferase-labeled T cell survival. 1928z-shJMJD3 CAR-T cells showed robust proliferation 4 days after T cell transfer into Raji tumor-bearing NSG mice, while maintaining high levels of T cells (Figures 25B and 25C). In contrast, 1928z-control-sh CAR-T cells significantly reduced T cell numbers 6 days after T cell transfer (Figures 25B and 25C). Consistent with these observations, the results showed that 1928z-shJMJD3 CAR-T cells significantly inhibited tumor growth and prolonged mouse survival compared with mice treated with 1928z-control-sh CAR-T cells (Figure 25D).

[0265] These studies demonstrate that knockdown or knockout of negative regulatory or epigenetic factors can modulate CAR-T and TCR-T cell function, persistence, and anti-tumor immunity in vivo. Example 7. Redirecting T cell trafficking to tumor sites by forced expression of chemokine receptors

[0266] To study the function of chemokine receptors in anti-tumor immunity, we engineered CAR constructs with chemokine receptor expression. The results showed that CCR5 could significantly enhance 1928z CAR-T cell trafficking to tumor sites compared with control tumor-specific T cells (Figure 26A). Using MDA-MB-231 / CD19 tumor cells, we demonstrated that 1928z-CCR5 CAR-T cells surprisingly significantly inhibited the growth of solid tumor cells (Figures 26B and 26C). These results suggest that forced expression of chemokine receptors enhances T cell trafficking to tumor cells.

[0267] To further enhance T cell survival once the T cells migrate to the tumor site, we could combine T cell trafficking with T cell persistence using the strategy shown in Figure 27. Chemokine receptor and shRNA KD could be inserted into the TCR or CAR construct.

[0268] F. Sequence SEQ ID NO: 1 HLA-DP4-restricted NY-ESO-1 epitope (157-170) SLLMWITQCFLPVF SEQ ID NO: 2 HLA-A2 restricted CT83 epitope (90-98) KLVELEHTL SEQ ID NO: 3 HLA-DP4-restricted NY-ESO-1 TCR alpha chain variable domain (TRAV34-TRAJ26) METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY SEQ ID NO:4 HLA-DP4-restricted NY-ESO-1 TCR beta chain variable domain (TRBV30-TRBJ 2-7) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE SEQ ID NO:5 HLA-A2-restricted CT83 TCR alpha chain variable domain (TRAV5-TRAJ28) MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN SEQ ID NO:6 HLA-A2 restricted CT83 TCR beta chain variable domain (TRBV29-1-TRBJ 1-1) MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE SEQ ID NO:7 21-nucleotide core of PD1 shRNA CCGTGTCACACAACTGCCCAA SEQ ID NO:8 The 21-nucleotide core of VHL shRNA CAGGAGCGCATTGCACATCAA SEQ ID NO:9 21-nucleotide core of PPP2R2D shRNA AAGGTCATTACTCAGAATAAA SEQ ID NO: 10 Human TCR alpha constant domain (TRAC) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 11 Human TCR beta constant domain 1 (TRBC1) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 12 Human TCR beta constant domain 2 (TRBC2) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 13 Mouse TCR alpha constant domain (trac) IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 14 Mouse TCR beta constant domain 1 (trbc1) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS SEQ ID NO: 15 Mouse TCR beta constant domain 2 (trbc2) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 16 ZAP300 TSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVH RDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA SEQ ID NO: 17 ZAP327 DKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNR HYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA SEQ ID NO: 18 ATGCTAACACAGTGCCACTTGAGTTCACATTAAGTTAGAAATTGAGAATCTAAAGGTACCTTTATTTTAACTAAAAAAATAATTTATATACTGTATATTGATTGTGACACAATTTACAAAGTCTGAGGTGTGGAAACAGTTATTTAAGCATTAGTCAACCCTGGTCCTTAAGACAGTTCTAGTAAAATGGGATTGTATATATTTGTTCAACTATTTGACCAAAAAGTTCAATAAATTTTAAAGTTTAACTGGA sequence number 19 SEQ ID NO: 20 HLA-A2-restricted CT83 TCR alpha chain variable domain fused to a mouse alpha constant domain MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIP NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 21 HLA-A2-restricted CT83 TCR beta chain variable domain fused to mouse beta constant domain 2 MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIAN KQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 22 HLA-A2-restricted NY-ESO-1 TCR(S2) alpha chain variable domain fused to a mouse alpha constant domain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYI QNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 23 HLA-A2-restricted NY-ESO-1 TCR(S2)(G50A, A51E) beta chain variable domain fused to mouse beta constant domain 2 MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAEGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 24 HLA-A2-restricted NY-ESO-1 TCR(S5) alpha chain variable domain fused to a mouse alpha constant domain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYI QNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 25 HLA-A2-restricted NY-ESO-1 TCR(S5)(G50A, A51E, A97L) beta chain variable domain fused to mouse beta constant domain 2 MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAEGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGLAGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 26 HLA-A2 restricted pp65 epitope (495-503) NLVPMVATV SEQ ID NO: 27 HLA-A2-restricted pp65 TCR (#1-15) alpha chain variable domain (TRAV21-TRAJ18) METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQGSTLGRLYFGRGTQLTVWPD SEQ ID NO: 28 HLA-A2-restricted pp65 TCR (#1-15) beta chain variable domain (TRBV13-TRBJ 1-5) MLSPDLPDSAWNTRLLCRVMLCLLGAGSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSGYHSELNMSSLELGDSALYFCASSLENNQPQHFGDGTRLSILE SEQ ID NO: 29 HLA-A2-restricted pp65 TCR (#132~3) alpha chain variable domain (TRAV24-TRAJ49) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCFSPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPN SEQ ID NO: 30 HLA-A2-restricted pp65 TCR (#132~3) beta chain variable domain (TRBV6-5-TRBJ 1-2) MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPITGTGDYGYTFGSGTRLTVVE SEQ ID NO: 31 HLA-A2-restricted IE-1 epitope (316-324) VLEETSVML SEQ ID NO: 32 HLA-A2-restricted IE-1 TCR alpha chain variable domain (TRAV25-TRAJ42) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGHIYGGSQGNLIFGKGTKLSVKPN SEQ ID NO: 33 HLA-A2-restricted IE-1 TCR beta chain variable domain (TRBV5-1-TRBJ 2-5) MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHHQGPLETQYFGPGTRLLVLE SEQ ID NO: 34 NY-ESO-1 PEP161-180 WITQCFLPVFLAQPPSGQRR SEQ ID NO: 35 NY-ESO-1 PEP156-175 LSLLMWITQCFLPVFLAQPP SEQ ID NO: 36 CT83 PEP6-14 LLASSILCA SEQ ID NO: 37 CT83 PEP4-12 YLLLASSIL SEQ ID NO: 38 CT83 PEP79-87 RILVNLSMV SEQ ID NO: 39 CT83 PEP10-31 SILCALIVFWKYRRFQRNTGEM SEQ ID NO: 40 CT83 PEP66-76 ILNNFPHSIAR SEQ ID NO: 41 DNA encoding the HLA-DP4-restricted NY-ESO-1 TCR alpha chain variable domain ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCAGCCTGGGTCAGTAGCCAAGAACTGGAGCAGAGTCCTCAGTCCTTGATCGTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACGTTATATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATCTTCTTGA TGATGCTACAGAAAGGTGGGGAAGAGAAAAGTCATGAAAAGATAACTGCCAAGTTGGATGAGAAAAAAGCAGCAAAGTTCCCTGCATATCACAGCCTCCCAGCCCAGCCATGCAGGCATCTACCTCTGTGGAGCAGACATAGTAGACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGGTTGTCCGTGCTGCCCTAT SEQ ID NO: 42 DNA encoding the HLA-DP4-restricted NY-ESO-1 TCR beta chain variable domain ATGCTCTGCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCACGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCT TCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGGCGCCGGGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG SEQ ID NO: 43 DNA encoding the HLA-A2-restricted CT83 TCR alpha chain variable domain ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGT ATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAAGAGCGGGTACTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAAT SEQ ID NO: 44 DNA encoding the HLA-A2-restricted CT83 TCR beta chain variable domain ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTG CAAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGTTCAAGACAGTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAG SEQ ID NO: 45 DNA encoding the HLA-A2-restricted pp65 TCR (#1-15) alpha chain variable domain ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTG CTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGGCCTCAGGGCTCAACCCTGGGGAGGCTATACTTTGGAAGAGGAACTCAGTTGACTGTCTGGCCTGAT SEQ ID NO: 46 DNA encoding the HLA-A2-restricted pp65 TCR (#1-15) beta chain variable domain ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTCTGCCGTGTCATGCTTTGTCTCCTGGGAGCAGGTTCAGTGGCTGCTGGAGTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCCACTCTGAAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTACCAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTCATTTCGTTTTATGAAAAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAACAGTTCAGTGGCTATCATTCTGAACTGAACATGAGCTCCTTGGAGCTGGGGGACTCAGCCCTGTACTTCTGTGCCAGCAGCTTAGAGAACAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG Sequence number 47 DNA encoding the variable domain of the HLA-A2 restricted pp65 TCR (#132 - 3) alpha chain ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTTGTACATCAAAGGATCCCAGCCTGAAGACTCAGCCACATACCTCTGTGCCCGAAACACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAAT Sequence number 48 DNA encoding the variable domain of the beta chain of HLA-A2-restricted pp65 TCR (#132~3) ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTCCTATCACCGGGACAGGGGACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG SEQ ID NO: 49 DNA encoding the variable domain of the alpha chain of HLA-A2-restricted IE-1 TCR ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGACACATTTATGGAGGAAGCCAAGGAAATCTCATCTTTGGAAAAGGCACTAAACTCTCTGTTAAACCAAAT SEQ ID NO: 50 DNA encoding the variable domain of the HLA-A2-restricted IE-1 TCR beta chain ATGGGCTCCAGGCTGCTCTGTTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTAAAGGCTGGAGTCACTCAAACTCCAAGATATCTGATCAAAACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCATAGGAGTGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTCCTCTTTGAATACTTCAGTGAGACACAGAGAAACAAAGGAAACTTCCCTGGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGATGAATGTGAGCACCTTGGAGCTGGGGGACTCGGCCCTTTATCTTTGCGCCAGCAGCCACCATCAGGGGCCGTTAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG SEQ ID NO: 51 P2A RAKRSGSGATNFSLLKQAGDVEENPGP

Claims

1. 1. A composition comprising one or more polypeptides comprising an alpha chain variable region and one or more polypeptides comprising a beta chain variable region of a T cell receptor (TCR) specific for a NY-ESO-1 peptide, wherein the TCR specific for the NY-ESO-1 peptide is an HLA-A2-restricted NY-ESO-1 TCR and / or an HLA-DP4-restricted NY-ESO-1 TCR, and wherein the C-terminus of the alpha chain or the beta chain of the TCR is fused to a signaling moiety comprising a ZAP70 kinase domain, wherein the ZAP70 kinase domain is a signaling moiety of ZAP300 (SEQ ID NO: 16) or ZAP327 (SEQ ID NO: 17).

2. The composition comprises alpha and beta chain variable regions of a TCR specific for NY-ESO-1, wherein the alpha chain variable region of the HLA-DP4-restricted NY-ESO-1 TCR comprises: a. a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; or b. A polypeptide comprising an amino acid sequence having one, two, or more amino acid substitutions in the amino acid sequence of SEQ ID NO:3, and having at least 90% identity to the amino acid sequence of SEQ ID NO:3, wherein the amino acid substitutions exclude amino acid substitutions within the CDR regions of the TCR; Including; The beta chain variable region of the HLA-DP4 restricted NY-ESO-1 TCR comprises: a. a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; or b. A polypeptide comprising an amino acid sequence having one, two, or more amino acid substitutions in the amino acid sequence of SEQ ID NO:4 and having at least 90% identity to the amino acid sequence of SEQ ID NO:4, wherein the amino acid substitutions exclude amino acid substitutions within the CDR regions of the TCR; The composition of claim 1 comprising:

3. 2. The composition of claim 1, wherein the TCR is fused to a chemokine receptor, and the chemokine receptor is selected from CCR5, CCR2, and CXCR3 or a chemokine receptor that enhances T cell trafficking.

4. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 1 and a pharmaceutically acceptable carrier.

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