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

Abstract

This invention relates to T-cell receptors (TCRs) targeting the cancer / testis antigens NY-ESO-1 and CT83 presented by various HLA molecules. Preferred TCRs of this invention are derived from human T cells and exhibit high affinity and high antigen specificity both in vitro and in vivo. Furthermore, this invention also relates to the regulation of TCR-T and CAR-T cell signaling and their functional persistence in cancer immunotherapy.

Description

[0001] Related applications This application claims priority based on U.S. Application No. 18 / 212,127, filed June 20, 2023, and is a continuation-in-part (CIP) of U.S. Application No. 18 / 002,969, filed December 22, 2022, which is a 371 national phase application of PCT / US2021 / 039262, filed June 25, 2021, which claims priority to U.S. Provisional Application No. 63 / 044,150, filed June 25, 2020. The entire contents of the above applications are incorporated herein by reference. Technical Field

[0002] This invention relates to methods for identifying and functionally validating T cell receptors (TCRs) from tumor antigen-specific T cells; modulating TCR-T and chimeric antigen receptor (CAR)-T cells to increase and prolong T cell persistence and reduce T cell exhaustion by directly manipulating TCR or CAR signaling domains and knocking down / knockout negative signaling molecules; and methods for treating cancer using TCR-T and CAR-T cells. The invention also relates to identified polypeptides, nucleic acids encoding the polypeptides, and recombinant vectors, as well as cells containing the nucleic acids or recombinant vectors, wherein the polypeptides comprise one or more α- and β-chains of T cell receptors (“TCRs”) specific to cancer antigens (e.g., NY-ESO-1 (“ESO-1 TCR”), CT83 (“CT83-TCR”)) and viral antigens (e.g., human cytomegalovirus (HCMV) PP65 and (HCMV) IE1 TCR).

[0003] sequence list

[0004] This application contains a sequence listing (nucleic acid and amino acid) that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on June 19, 2024, and is named H3131-00110_SL.xml, with a file size of 104,258 bytes. Background Technology

[0005] The host immune system comprises innate immunity and adaptive immunity, which recognize and eliminate exogenous or endogenous antigens from pathogens or abnormal tissues (including cancer cells). Schreiber (RD), Old (LJ), and 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, and Smyth, “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 shown to play a direct role in tumor rejection, clinical responses to immunotherapy in early-stage cancer are limited due to multiple factors, particularly immunosuppression. Recently, the identification of immune checkpoints has led to the development of targeted immunotherapies. The depletion or inhibition of immunosuppressive checkpoints (e.g., programmed cell death-1 protein (PD1) and its ligand PD-L1, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and other checkpoint inhibitors) has significantly enhanced anti-tumor 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) and Kuhns (M).S.), Egen, JG, and 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., and 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, HY) and Wang (RF), "Regulatory T cells and cancer," *Current Opinion in Immunology*, 19, 217-223, doi:10.1016 / j.coi.2007.02.004 (2007); Joyce (JA) and Fearon (DT), "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 recently been successfully applied to treat human cancers, including or excluding melanoma, renal cell carcinoma, and lymphoma, producing varying degrees of tumor regression.

[0006] CD8+ and CD4+ T cells are key components of T cell-based anti-tumor immunity. CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), specifically recognize epitopes that bind to class I molecules of the major histocompatibility complex (MHC—called human leukocyte antigen, HLA in humans) via the T cell receptor (TCR) and kill cells upon presentation of the complex on the cell surface. CD4+ T cells, primarily called T helper cells (Th cells), are another type of T cell that plays a crucial role in the immune system. CD4+ T cells specifically recognize epitopes that bind to class II molecules of MHC via the TCR and release cytokines and modulate the immune system. CD4+ T cells are also essential for the activation of CD8+ T cells, B lymphocytes, and other immune cells, including or excluding macrophages.

[0007] To elicit a tumor-specific T-cell response, tumor antigens are processed and degraded in tumor cells or other antigen-presenting cells (APCs) into peptides (called antigenic determinants or epitopes) containing 9 to 13 amino acids via the proteasome pathway (for binding to major histocompatibility (MHC) class I molecules) or the endosome / lysosome pathway (for binding to MHC class II molecules). The final product of this antigen processing binds to a specific type of MHC class I or II molecule on the APC and is transported to the cell surface. When the epitope-HLA complex specifically binds to TCRs on the surface of T cells, this can activate CD8+ or CD4+ T cells. The cytotoxic activity of CD8+ T cells can directly kill tumor cells. However, other studies have shown that CD4+ T cells also play a role in anti-tumor immunity. Wang (RF) and Rosenberg (SA), “Human tumor antigens for cancer vaccine development,” *Immunological Reviews*, 170, 85-100 (1999); Wang, “The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity,” *Trends in Immunology*, 22, 269-276 (2001). Furthermore, the CD4+ T cell subset (CD4 CTL) possesses cytotoxic activity and directly participates in tumor cell killing via an HLA class II-restricted pathway.Takeuchi, A. and 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 and Wang, HY, “Immune targets and neoantigens for cancer immunotherapy and precision medicine”, Cell Research 27, 11-37, doi:10.1038 / cr.2016.155 (2017).

[0008] Chimeric antigen receptor (CAR)-engineered T cells have demonstrated durable clinical benefits for blood cancers, including leukemia and lymphoma. CD19-CAR-T products have been approved by the US Food and Drug Administration (FDA) for the treatment of lymphoma and leukemia. (June, CH. and Sadelain, M., "Chimeric Antigen Receptor Therapy," *New England Journal of Medicine*, 379, 64-73, doi:10.1056 / NEJMra1706169 (2018).) However, CAR-T cell therapy has not been very effective in treating solid tumors. Furthermore, approximately 30 to 50% of cancer patients treated with CD19-CAR-T therapy experience disease relapse within 12 months of achieving remission. Shah (NN) and Fry (TJ), “Mechanisms of resistance to CAR T cell therapy,” *Nature Reviews 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,” *The New England Journal of Medicine* 378, 449-459, doi:10.1056 / NEJMoa1709919 (2018); Maude (… SL et al., “Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia”, New England Journal of Medicine 378, 439-448, doi:10.1056 / NEJMoa1709866 (2018). Summary of the Invention

[0009] In one aspect, this disclosure relates to methods for identifying and functionally validating TCRs derived from cancer antigen-specific T cells or from T cells specific to antigens associated with other diseases and conditions, including inflammatory diseases, autoimmune diseases, allergic diseases, organ transplantation conditions, cancer, and infectious diseases. As a non-limiting example, in some aspects, this disclosure relates to methods for identifying and functionally validating TCRs derived from NY-ESO-1-specific, CT83-specific, human cytomegalovirus (HCMV)-pp65-specific, and / or HCMV-IE-1-specific T cells.

[0010] In some aspects, the present disclosure is characterized by a method for detecting 0 and clone TCRs from cancer antigen-specific T cells (as a non-limiting example, cancer antigen-specific T cells may include or exclude NY-ESO-1-specific, CT83-specific, HCMV-pp65-specific, and / or HCMV-IE-1-specific T cells) in human subjects, the method comprising: a) in vitro using a cancer antigen (e.g., a class I or class II HLA-restricted epitope complex (as a non-limiting example, including or excluding class II HLA-DP4-restricted NY-ESO-1 epitope complex) (a) Stimulation of uninfected T cells with any one of the following: (b) stimulation of uninfected T cells with a cancer antigen epitope (any one of the following: NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1); (c) detection, in vitro and in vivo, of a T cell population specific to the cancer antigen epitope (as a non-limiting example, including or excluding any one of the NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1 epitopes); (d) sorting of cancer antigen epitope-specific CD4+ or CD8+ cells. T cell populations (as a non-restrictive example, including or excluding any one of the NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1 epitope-specific CD4+ or CD8+ T cell populations); d) isolating individual T cells from the sorted T cells; e) obtaining the T cell V(D)J sequence from the T cells by single-cell next-generation sequencing; f) synthesizing primers for TCR cloning based on the sequence; g) amplifying the TCR variable regions of the α and β chains of aggregated T cells stimulated by cancer antigens (as a non-restrictive example, including or excluding any one of the NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1 epitopes); and h) constructing the amplified α and β chain TCR variable regions into a vector to form a complete TCR construct. In some aspects, the method further comprises the following steps: j) transducing a cloned TCR into uninfected CD4+ or CD8+ T cells; i) measuring the activity of the transduced T cells; and k) screening the transduced T cells for binding to, recognizing, and / or being activated 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 vitro and in vivo. In one aspect, the TCR from cancer antigen-specific T cells identified by the methods of the foregoing aspects or other aspects and examples described herein can bind to and / or recognize any cancer antigen described herein, including any cancer antigen or fragment or epitope thereof described in any aspect or example described herein, including any cancer antigen described in the discussion of “cancer antigen” and “tumor antigen”.

[0011] In one aspect, this document also discloses methods for identifying epitope-specific T cells and TCRs in any of the foregoing aspects or any aspects or embodiments disclosed herein, covering one or more of these cells or cell lines (which may or may not include, for example, HEK293 cells, HEK293T cells, Cos-7 cells, 586mel cells, 624mel cells, MDA-MB-231 cells, MDA-MB-436 cells, E0771 cells, HTB-21 cells). In one aspect, one or more tumor cell lines may include or exclude any cell line 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, squamous cell carcinoma of the head and neck, 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 oral cavity, pharynx, larynx, and lungs. Cervical cancer, breast cancer, kidney cancer, urogenital tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon and rectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma, and may be selected from: HEK293 cells, HEK293T cells, Cos-7 cells, 586mel cells, 624mel cells, MDA-MB-231 cells, MDA-MB-436 cells, E0771 cells, HTB-21 cells.

[0012] In one aspect, this document also discloses methods for identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein, covering one or more cells or cell lines engineered to express MHC class I or II molecules. In one aspect, the identified epitopes are associated with diseases or conditions selected from the group consisting of: inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, cancer, and infectious diseases.

[0013] This document also discloses methods for identifying epitope-specific T cells and TCRs in any of the foregoing aspects or any aspects or embodiments disclosed herein, covering methods for measuring T cell activity (which may or may not include, for example, the release of cytokines, including but not limited to, IFN-α, TGF-β, lymphotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25) using any of the immunoassay methods disclosed herein. In some embodiments, T cell activity may be measured, for example, but not limited to, by ELISA, chemiluminescence, ELISPOT, intracellular cytokine staining or chromium release, or any other immunoassay method disclosed herein.

[0014] In one aspect, a TCR derived from cancer antigen-specific T cells, or a TCR from T cells specific to antigens associated with other diseases or conditions, identified by the methods described in the foregoing aspects or other aspects and embodiments herein, is capable of binding to and / or recognizing any cancer antigen or other disease antigen described herein, including any cancer, disease, or condition-related antigen, fragment or epitope thereof, as described in any aspect or embodiment herein, including any cancer antigen described in the discussion of “cancer antigen” and “tumor antigen”.

[0015] In one aspect, this document also discloses methods for identifying epitope-specific T cells and TCRs in any of the foregoing aspects or any aspect or embodiment disclosed herein, wherein the cancer is selected from and may include or exclude any of the following groups: 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 oral cavity, pharynx, larynx and lung, cervical cancer, breast cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon and rectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma.

[0016] In one aspect, this document also discloses cancer epitopes that may or may not include epitopes derived from any cancer antigen or tumor antigen recognized or bound by cancer antigen-specific T cells and TCRs, which are identified by methods for detecting or identifying cancer antigen / epitope-specific T cells and TCRs in any of the foregoing aspects or any of the aspects or embodiments disclosed herein.

[0017] In one aspect, cancer epitopes may or may not include epitopes of NY-ESO-1, which are identified by methods for detecting or identifying epitope-specific T cells and TCRs in any of the foregoing aspects or any of the aspects or embodiments disclosed herein. For example, this document discloses polypeptides comprising the amino acid sequence SLLMWITQCFLPVF (Seq ID NO: 1) and variants thereof, as disclosed herein.

[0018] In one aspect, this document also discloses cancer epitopes or epitopes associated with other diseases or conditions, such as inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related diseases, cancer, and infectious diseases; the epitopes may or may not include epitopes of NY-ESO-1, CT83, HCMV-pp65, and HCMV-IE-1, epitopes of any other antigens disclosed herein, and / or their mutants and variants, as described herein, including other proteins, epitopes, and their mutants and variants identified by methods for detecting or identifying epitope-specific T cells and T cell receptors (TCRs) of any of the foregoing aspects or any aspect or embodiment disclosed herein.

[0019] In some respects, epitopes can consist essentially of identified epitopes. The fundamental property and nature of an epitope is that it is part of a target protein that can be recognized or bound by a TCR in the case of class I or class II MHC. For example, this paper discloses a polypeptide containing the amino acid sequence KLVELEHTL (Seq ID NO: 2) and its variants, as illustrated herein.

[0020] In one aspect, cancer epitopes may or may not include HCMV-pp65 epitopes, which are identified by methods for detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspects or embodiments disclosed herein. For example, polypeptides comprising the amino acid sequence NLVPMVATV (SEQ ID NO:26) and variants thereof are disclosed herein.

[0021] In one aspect, cancer epitopes may or may not include HCMV-IE-1 epitopes, which are identified by methods for detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspects or embodiments disclosed herein. For example, polypeptides comprising the amino acid sequence VLEETSVML (SEQ ID NO:31) and variants thereof are disclosed herein.

[0022] In one aspect, this disclosure relates to pharmaceutical compositions comprising one or more epitopes described herein for the treatment of a disease or condition that is pathogenesis-related to the epitopes. In one aspect, the epitopes described herein can be used to prepare vaccines for the treatment of cancer. In one aspect, this disclosure also relates to compositions comprising one or more antigens and / or epitopes thereof, said antigens and / or epitopes being capable of binding to any of the T-cell receptors (TCRs) described herein. In some aspects, the compositions comprise one or more antigens and epitopes that are pathogenesis-related to said disease or condition, said epitopes may include, exclude, or be selected from the group consisting of: NY-ESO-1 peptides (A2-ESO-1 and DP4-ESO-1), CT83 peptides (A2-CT83, DR13-CT83, PEP4-12, PEP6-14, PEP10-31, PEP17-31, PEP90-98), HCMV pp65 and HCMVIE-1, or functional variants thereof, or any epitope of any antigen described herein and its mutants and variants, as disclosed herein. In some embodiments, functional variants of epitopes can be identified by alanine scanning.

[0023] In one aspect, this disclosure relates to compositions comprising one or more α-chains and / or β-chains of a cancer antigen-specific T-cell receptor (“TCR”), which are identified or detected by methods for identifying or detecting epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein. In some aspects, the α-chain and / or β-chain of the TCR recognizes and / or binds to cancer antigens, such as, but not limited to, NY-ESO-1 (“ESO-1 TCR”) and / or CT83 (“CT83-TCR”). In some aspects, the α-chain and / or β-chain of the TCR recognizes and / or binds to viral-derived cancer antigens, such as viral antigens expressed on human or mammalian cancer cells. In some aspects, the α-chain and / or β-chain of the TCR recognizes and / or binds to cancer antigens, such as, but not limited to, proteins derived from the CT83 protein or fragments thereof or epitopes. In some aspects, the α and / or β chains of the TCR recognize and / or bind polypeptides containing the amino acid sequence SILCALIVFWKYRRFQRNTGEM (CT83 amino acids 10-31, SEQ ID NO: 39) or the amino acid sequence VFWKYRRFQRNTGEM (CT83 amino acids 17-31, SEQ ID NO: 61). In some aspects, the α and / or β chains of the TCR do not recognize and / or bind cancer antigens, such as unrestricted proteins derived from the CT83 protein or fragments or epitopes thereof, such as SILCALIVFWKYRRF (CT83 amino acids 10-24, SEQ ID NO: 62) or CALIVFWKYRRFQRN (CT83 amino acids 13-27, SEQ ID NO: 63). In some aspects, the α and / or β chains of the TCR recognize and / or bind cancer antigens, such as, but not limited to, proteins derived from human cytomegalovirus (HCMV). In some respects, the α and / or β 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 respects, the α and / or β 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 respects, the α and / or β chains of the TCR recognize and / or bind to cancer antigens, which may or may not include any cancer antigens disclosed above.

[0024] In some aspects, this disclosure relates to compositions comprising one or more α chains or regions and / or one or more β chains or regions of a T-cell receptor, for example, specific to a cancer antigen. In some aspects, this disclosure relates to compositions comprising one or more α chains / regions or β chains / regions of a T-cell receptor, for example, specific to a cancer antigen, wherein the cancer antigen may or may not include any one or any combination thereof of NY-ESO-1 (ESO-1TCR), CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), and / or HCMV-IE-1 (IEI-TCR). In some aspects, the composition comprises, for example, at least one polypeptide comprising an α chain or region of a T cell receptor specific to 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 β chain of a T cell receptor specific to NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), or HCMV-IE-1 (IE-1-TCR). In some aspects, the composition comprises, for example, a polypeptide comprising, respectively, an α chain or region of a T cell receptor specific to NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), pp65 (pp65-TCR), or IE-1 (IE-1-TCR), and a β chain or region of a T cell receptor specific to NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), HCMV-pp65 (pp65-TCR), or HCMV-IE-1 (IE-1-TCR).

[0025] In one aspect, this document also discloses the α-variable region of a cancer antigen-specific TCR detected or identified by a method for detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein. In one aspect, the α-variable region may include or exclude the α-variable region of the DP4-ESO-1 TCR, the α-variable region of the A2-CT83 TCR, the α-variable region of the A2-pp65 TCR, and / or the α-region of the A2-IE-1-TCR, which are detected or identified by a method for detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein, and / or used in conjunction with any variable region sequence or epitope-specific sequence identified herein. For example, this article discloses a polypeptide containing the amino acid sequence: METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY (SEQ ID NO: 3) (α-variable region of DP4-ESO-1TCR). The bolded amino acids represent D95, Y96, and Q98 within the CDR3 region of the mature T cell receptor (TCR) sequence. As another example, this document discloses a polypeptide comprising the amino acid sequence: MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 5) (α-variable region of A2-CT83 TCR). As another example, this document discloses a polypeptide comprising the amino acid sequence MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPN (SEQ ID NO: 29) (α-variable region of A2-pp65 TCR).Another example disclosed herein is a polypeptide containing the amino acid sequence MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGHIYGGSQGNLIFGKGTKLSVKPN (SEQ ID NO:32) (α variable region of A2-IE-1-TCR).

[0026] This document also discloses fragments or variants of any polypeptide or polypeptide fragment in any of the foregoing aspects or any of the embodiments disclosed herein, which bind antigens with the same specificity as the reference (full-length and unmodified) receptor. In some aspects, the variants contain conserved amino acid substitutions as further disclosed herein. Substitutions to any α-variable region disclosed herein may or may not include substitutions in one or more of the six CDRs of the TCR. In one aspect, variants of the DP4-ESO-1 TCR contain one or more substitutions selected from: D95S or Q98Y α-chain substitution and Y98L and Y98M β-chain substitution. In another aspect, variants of the DP4-ESO-1 TCR contain a Q98Y α-chain substitution. In one aspect, the variant TCRs are capable of significantly enhancing T cell function, such as IFN-γ release and tumor-specific lysis. For example, a single Q98Y substitution in the TCR-α-chain results in a 3-4 fold increase in T cell function based on NFAT-GFP expression. On one hand, human CD4+ T cells were transduced with wild-type HLA-DP4-restricted NY-ESO-1 TCR, mutant / variant TCRVα D95S, Q98Y and TCRVβ Y98L and Y98M, and it was demonstrated that these single amino acid mutations conferred significantly enhanced T cell function, enhanced cytokine release and specific tumor lysis.

[0027] In one aspect, this document also discloses the β-variable region of a cancer antigen-specific TCR identified by methods for identifying epitope-specific T cells and TCRs of any of the foregoing aspects, and / or its use with any variable region sequence or epitope-specific sequence identified herein. In one aspect, the β-variable region may include or exclude the β-variable region of DP4-ESO-1 TCR, the β-variable region of A2-CT83TCR, the β-variable region of A2-pp65 TCR, and / or the β-variable region of A2-IE-1-TCR, which are detected or identified by methods for detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects or any aspect or embodiment disclosed herein, and / or used with any variable region sequence or epitope-specific sequence identified herein. In one aspect, this document also discloses the β-variable region of a cancer-specific DP4-ESO-1 TCR identified by methods for identifying epitope-specific T cells and TCRs of any of the foregoing aspects, and / or its use with any variable region sequence or epitope-specific sequence identified herein. For example, this document discloses a polypeptide comprising the amino acid sequence MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE (Seq ID NO: 4). In one aspect, this document also discloses the β-variable region of the A2-CT83 TCR, detected or identified by methods for detecting or identifying epitope-specific T cells and TCRs in any of the foregoing aspects, and / or used in conjunction with any variable region sequence or epitope-specific sequence identified herein. For example, this document discloses a polypeptide comprising the amino acid sequence MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE (Seq ID NO: 6). In one aspect, this document also discloses methods for detecting or identifying the β variable region of the A2-pp65 TCR by detecting or identifying epitope-specific T cells and TCRs of any of the foregoing aspects, and / or using it in conjunction with any variable region sequence or epitope-specific sequence identified herein.For example, this document discloses a polypeptide comprising the amino acid sequence MSIGLLCCAALSLLWAGP VNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPITGTGDYGYTFGSGTRLTVVE (SEQ ID NO:30). In one aspect, this document also discloses the β-variable region of the A2-IE-1 TCR, detectable or identified by methods for detecting or identifying epitope-specific T cells and TCRs in any of the foregoing aspects, and / or used in conjunction with any variable region sequence or epitope-specific sequence identified herein. For example, this document discloses a polypeptide comprising the amino acid sequence MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHHQGPLETQYFGPGTRLLVLE (SEQ ID NO:33). This document also discloses variants of any polypeptide or polypeptide fragment from any of the foregoing aspects or any of the embodiments disclosed herein. In some aspects, variants comprise conserved amino acid substitutions as further disclosed herein. Substitutions to any β-variable region disclosed herein may include, but are not limited to, substitutions in one or more of the six CDRs of the TCR.

[0028] This document also discloses variants of any polypeptide or polypeptide fragment disclosed in any of the foregoing aspects or any of the embodiments disclosed herein, wherein such variants contain conserved amino acid substitutions. Substitutions may include, or not include, substitutions in one or more of the six CDRs of the TCR.

[0029] This document also discloses variants of any polypeptides or polypeptide fragments disclosed herein, wherein such variants contain conserved amino acid substitutions. Substitutions may include, but are not limited to, substitutions in one or more of the six CDRs of the TCR.

[0030] In one aspect, this disclosure includes a chimeric TCR comprising a TCR variable region fused to a modified human constant region or fused 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., mouse) TCR constant region. In some aspects, the TCR variable region comprises α and β chain variable regions fused to α and β TCR constant regions, respectively, and may include any α and / or β chain from any other aspect of this disclosure. In some aspects, the fusion of the TCR variable region with a modified or non-human constant region reduces mismatches between the chimeric TCR and the endogenous TCR. In some aspects, the chimeric TCR comprises a variable region that may or may not include any of the following: a CT83TCR variable region fused to a non-human (e.g., mouse) TCR constant region; a NY-ESO-1 TCR variable region; a pp65 TCR variable region; and / or an IE-1 TCR variable region. In other aspects, the chimeric TCR reduces mismatches between the chimeric TCR and the endogenous TCR of transduced T cells. For example, in some respects, chimeric CT83 TCR reduces mismatches between chimeric CT83 TCR (MC) and endogenous TCR (HC). In other respects, for example, chimeric NY-ESO-1 TCR reduces mismatches between chimeric NY-ESO-1 (MC) and endogenous TCR (HC). In other respects, for example, chimeric pp65 TCR reduces mismatches between chimeric pp65 (MC) and endogenous TCR (HC). In other respects, for example, chimeric IE-1 TCR reduces mismatches between chimeric IE-1 (MC) and endogenous TCR (HC).

[0031] In one aspect, this disclosure includes a chimeric TCR comprising a TCR variable region fused to a modified human constant region or fused to 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., mouse) TCR constant region. In some aspects, the TCR variable region comprises α and β chain variable regions fused to α and β TCR constant regions, respectively, and may comprise any α and / or β chain of any other aspect of this disclosure. In some aspects, the chimeric TCR comprises a TCR variable region that may or may not include any of the following: a CT83TCR variable region fused to a non-human (e.g., mouse) TCR constant region; a NY-ESO-1 TCR variable region; a pp65 TCR variable region; or an IE-1 TCR. In other aspects, the chimeric TCR reduces mismatches between the chimeric TCR and the endogenous TCR of transduced T cells. For example, in some aspects, the chimeric CT83TCR reduces mismatches between the chimeric CT83 TCR (MC) and the endogenous TCR (HC). In other respects, for example, chimeric NY-ESO-1 TCR reduces or minimizes mismatches between chimeric NY-ESO-1 (MC) and endogenous TCR (HC). In other respects, for example, chimeric pp65 TCR reduces or minimizes mismatches between chimeric pp65 (MC) and endogenous TCR (HC). In other respects, for example, chimeric IE-1 TCR reduces or minimizes mismatches between chimeric IE-1 (MC) and endogenous TCR (HC).

[0032] Its features also include nucleic acids encoding any of the above-described epitopes, receptor chains and / or polypeptides or any other polypeptides disclosed in any aspect or embodiment herein; recombinant nucleic acids containing the above-described nucleic acids; vectors or constructs containing the above-described recombinant nucleic acids; and cells in which one or more of the above-described nucleic acids or vectors are transduced.

[0033] In one aspect, this document also discloses nucleic acids encoding polypeptides comprising any of the epitopes described above. In some aspects, this disclosure relates to mRNAs encoding any of the epitopes described above or variants thereof, for use in preparing mRNA vaccines to treat diseases or conditions that are pathogenesis-related to the said antigens and / or antigenic epitopes. In some aspects, the mRNA encoding the epitope can be produced by in vitro transcription to generate an epitope antigen and stimulate an immune response against that epitope. In some aspects, the mRNA encoding the epitope comprises, from its 5′ end to its 3′ end, the following elements in sequence: a 5′ cap structure (5′cap), a 5′ untranslated region (UTR), an open reading frame encoding the said antigen, a 3′ untranslated region (UTR), and a poly(A) tail. In some embodiments, the mRNA vaccine comprising the mRNA described herein further comprises lipid nanoparticles. In some respects, lipid nanoparticles suitable for mRNA formulations have been reported, for example, in PCT / US2020 / 013417 (published as WO2020146906) and US20220088221, the contents of which are incorporated herein by reference.

[0034] In one aspect, this document also discloses nucleic acids encoding the peptide TCR α and / or β variable regions or chains of any of the foregoing aspects or any embodiments disclosed herein. In one aspect, the nucleic acid of this disclosure encodes any one of SEQ ID NO: 3-6, 10-11, 12-15, 20-25, 27-30, 32, or 33. In one aspect, the nucleic acid has a sequence of any one of SEQ ID No: 41-50, which encodes the following TCR variable regions:

[0035] In one respect, a nucleic acid sequence may have one or more codon substitutions that do not alter the sequence of the encoded polypeptide.

[0036] In some aspects, this disclosure also relates to transmembrane domains of T-cell receptors (TCRs) or chimeric antigen receptors (CARs), wherein said transmembrane domains are derived from any one or any combination of the following transmembrane domains: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, A2αR, ICAM-1, 2B4, BILA, DAP10, KIR, KIR2DL4, KIR2DS1, LAG-3, LCK, LAT, LPA5, LRP, FcRα, FcRβ, Fyn, GAL9, cytokine receptors (IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα), NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NO TCH3, NOTCH4, pTα, T cell receptor peptides (human and mouse TCRα, TCRβ, TCRγ, TCRδ), TIM3, TRIM, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD8α, CD8β, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG-3), CD270 (HVEM), CD272 (BILA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD357 (GITR), PTCH2, ROR2, Ryk, SLP-76, SIRPα, ZAP70, or any combination thereof.

[0037] In some aspects, this disclosure also relates to nucleic acids encoding any of the T-cell receptor (TCR) regions or chains described herein. In some aspects, the nucleic acid may further comprise a signal transduction domain. In one aspect of any TCR or chimeric antigen receptor (CAR) disclosed herein, the TCR or CAR comprises an intracellular T-cell activating motif, wherein the intracellular T-cell activating motif comprises a signal transduction domain. In some aspects, in the TCR or CAR disclosed herein, the intracellular T-cell activating motif of the TCR or CAR comprises one or more co-stimulatory signaling domains fused to the signal transduction domain.The co-stimulatory signal domains may include, exclude, or be selected from the group consisting of: CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CD8, CD8α, CD8β, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD 58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG-3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, Lymphocyte Function-Associated Antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), DAP10, DAP12, LAG-3, 2B4, CARD1, CTLA-4 (CD152), TRIM, ZAP70, FcεRIγ, 4-1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACI, BLAME, CRACC, CD2F-10, NTB-A, Integrin α4, Integrin α4β1, Integrin α4β 7. IA4, ICAM-1, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLR, such as TL R2, TLR4), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR2DS2, LTBR, ​​MALT-1, PAG / Cbp, PSGL1, SLAMF6 (NTB-A,Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, TRAFs (such as TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 and TRAF6), VLA-1, VLA-6, BTLA, Ikaros, LAG-3, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof. In one aspect disclosed herein, the intracellular T cell activation motif of the TCR or CAR includes a co-stimulatory signaling domain fused to a signal transduction domain, wherein the co-stimulatory signaling domain may include, exclude, or be selected from the following components: CD28, 4-1BB, CD3ζ, IL-2Rβ, IL-4Rα, IL-7R, IL-9R, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21R, or any combination thereof. In some aspects, the CAR described herein may or may not include a co-stimulatory signaling domain derived from the CD28, 4-1BB, CD3ζ signaling domain, or ZAP70 kinase domain.

[0038] In some aspects, the signaling domain of any T-cell receptor (TCR) or chimeric antigen receptor (CAR) described herein includes a ZAP70 kinase domain, or a mutant or variant thereof, replacing CD28 and / or CD3ζ. In some aspects, the TCR or CAR may include or exclude CD28 and / or CD3ζ. In some aspects, the TCR or CAR includes a ZAP70 kinase domain derived from a functional wild-type ZAP70 or a mutant or variant thereof, wherein the ZAP70 kinase domain derived from a functional wild-type ZAP70 or a mutant or variant thereof further includes a functional ZAP70 kinase domain. In some embodiments, the functional ZAP70 kinase domain is a truncated and biologically active fragment of the ZAP70 kinase. In some embodiments, the functional ZAP70 kinase domain or signal transduction domain may include or exclude ZAP255 (SEQ ID NO: 64), ZAP280 (SEQ ID NO: 65), ZAP327 (SEQ ID NO: 17), ZAP300 (SEQ ID NO: 16), or ZAP338 (SEQ ID NO: 52).

[0039] On the other hand, the co-stimulatory signaling domain is derived from the ZAP70 kinase domain or a mutant or variant thereof, and may include or exclude, for example, ZAP300 (SEQ ID NO: 1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO: 3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5). 5), or its mutants or variants, wherein the N-terminal amino acid of the ZAP70 kinase can be any position from 250 to 338 (including 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294). (295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338), and the C-terminal amino acid is at position 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619. In some embodiments, the ZAP70 kinase domain or its mutants or variants can enhance T cell activation, antigen recognition, and specific lysis. In some embodiments, the TCR or CAR may also contain 4-1BB, CD27, CD28, OX40, ICOS, MyD88, MALT-1, TLR, or their mutants or variants to enhance T cell activation, antigen recognition, and specific lysis.

[0040] In some embodiments, the N-terminus of the functional ZAP70 kinase domain begins at amino acids 250-338, 281-338, or 300-338 of the ZAP70 kinase, and terminates at amino acid 619 of the ZAP70 kinase, or a mutant or variant thereof. In some aspects, the signal transduction domain confers enhanced persistence and / or antitumor activity to cells engineered with the nucleic acid. This document also discloses compositions comprising a therapeutically effective amount of one or more variable regions of the T-cell receptor (TCR) α-chain or β-chain, wherein the TCR α-chain or β-chain variable region is any of the foregoing aspects or any aspect or embodiment disclosed herein.

[0041] In some aspects, the composition may also include any of the signal transduction domains described in the foregoing aspects, for example, including or excluding functional kinase domains such as ZAP327 (SEQ ID NO: 17), ZAP300 (SEQ ID NO: 16), ZAP338 (SEQ ID NO: 52), ZAP255 (SEQ ID NO: 64), or ZAP280 (SEQ ID NO: 52). 65), or other signal transduction domains derived from the ZAP70 kinase domain, or mutants or variants of the ZAP70 kinase, wherein the N-terminal amino acid of the ZAP70 kinase may be any position from 250 to 338 (including 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 29). 0, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338), and the C-terminal amino acid is at position 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619. In some embodiments, T cells expressing a CAR or TCR containing any of the functional ZAP70 kinase domains described herein (e.g., ZAP300, ZAP327, or ZAP338) have low levels of expression of T cell exhaustion markers (e.g., PD-1, TIM3, and LAG3).

[0042] This document also discloses compositions comprising a therapeutically effective amount of one or more TCR-T cells, wherein the TCR-T cells are engineered to express any of the foregoing aspects or any of the aspects or embodiments disclosed herein. In some aspects, the TCR-T cells may be engineered to express, for example, a receptor (which may or may not include a T-cell receptor) capable of recognizing one or more cancer antigens or neoantigens in any of the foregoing aspects or any of the aspects or embodiments disclosed herein. In some aspects, the TCR-T cells may express one or more TCRα chain variable regions and / or one or more TCRβ chain variable regions in any of the foregoing aspects or embodiments of this disclosure. In some aspects, the TCR-T cells may also include or exclude functional kinase domains, such as ZAP327 (SEQ ID NO: 17), ZAP300 (SEQ ID NO: 16), ZAP338 (SEQ ID NO: 52), ZAP255 (SEQ ID NO: 64), or ZAP280 (SEQ ID NO: 52). 65), or other signal transduction domains derived from the ZAP70 kinase domain, or mutants or variants of the ZAP70 kinase, wherein the N-terminal amino acid of the ZAP70 kinase may be any position from 250 to 338 (including 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 29). 0, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338), and the C-terminal amino acid is at position 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619. In some respects, engineered TCR-T cells containing and / or expressing the aforementioned signaling domains exhibit unexpectedly enhanced persistence and / or antitumor activity. In some embodiments, the engineered TCR may further comprise 4-1BB, CD27, CD28, OX40, ICOS, MyD88, MALT-1, TLR, or mutants or variants thereof to enhance T cell activation, antigen recognition, and specific lysis.

[0043] In one aspect, the engineered or transduced T cells do not express endogenous TCR (α / β). For example, in some aspects, the endogenous TCR (α / β) is knocked out using CRISPR technology, such as CRISPR / Cas9 technology (see Legut, M., Dolton, G., Mian, AA, Ottmann, OG & Sewell, AK, “CRISPR-mediated TCR replacement produces superior anti-cancer transgenic T cells,” Blood, Vol. 131, pp. 311-322, 2018) or CRISPR / Cas12a technology, prior to transduction with a cancer antigen-specific TCR construct.

[0044] Engineered T-cell receptors (TCRs) derived from tumor antigen-specific T cells have been reported to regulate TCR-T cells by directly manipulating the TCR signal transduction domain, thereby increasing and prolonging T-cell survival and reducing T-cell exhaustion. For example, this has been described in U.S. Application No. 18 / 002,969, which is the 371 national phase application of PCT International Application PCT / US2021 / 039262, the entire contents of which are incorporated herein by reference as if fully described herein.

[0045] In one aspect, this disclosure also relates to methods and strategies for prolonging the persistence of CAR-T cells or TCR-T cells by directly regulating CAR signaling domains or by knocking down / knockout negative signaling molecules. In some aspects, this disclosure also relates to methods for enhancing the persistence of TCR-T cells or CAR-T cells by expressing chemokine receptors in a CAR construct and combining them with shRNA knockout. In some aspects, treatment using any engineered TCR-T cells of this disclosure, wherein the T cells contain and / or express signaling domains and / or have negative signaling molecule knockdown, can significantly reduce relapse or cancer recurrence after initial treatment and initial reduction of cancer / tumor burden. In some aspects, treatment using any engineered TCR-T cells of this disclosure, wherein the T cells contain and / or express signaling domains and / or have negative signaling molecule knockdown, can significantly reduce relapse of treated inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related diseases, infectious diseases, and / or age-related symptoms.

[0046] In some aspects, this disclosure provides an engineered immune cell (TCR-T cell or CAR-T cell) containing a knockdown, knockout, or inactivation mutation in one, two, or more endogenous genes selected from the following negative regulatory factors: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR 4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T cells) Lymphocyte antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic regulators, which may or may not include JMJD3 and LSD1. In some aspects, negative signaling molecules / regulators selected for knockdown, knockout, or inactivation of mutations include, for example: indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-H3, VHL, PPP2R2D, and epigenetic regulators (which may or may not include JMJD3 and LSD1). In some respects, the negative signaling molecules or regulators selected for knockout, knockdown or inactivation of mutations are, for example, PD-1, VHL, PPP2R2D and epigenetic regulators (which may or may not include JMJD3 and LSD1).In some aspects, the negative signaling molecules include, for example, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, and B7-H3. In some aspects, the negative signaling molecules are, for example, PD-1, VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1). In some aspects, treatment using any of the engineered TCR-T cells of this disclosure, wherein the T cells contain and / or express signal transduction domains and / or have negative signaling molecule knockdown, can significantly reduce relapse or cancer recurrence following initial treatment and initial reduction of cancer / tumor burden.

[0047] In some respects, knockout, knockdown, or inactivation mutations of any of the negative signaling molecules described herein, and / or expression of chemokine receptors and / or shRNA knockout, can be used in CAR-T cells or TCR-T cells, or CAR constructs, in any of the foregoing aspects or embodiments described herein. For example, CAR-T cells subjected to knockout, knockdown, or inactivation mutations of any of the negative signaling molecules, and / or expression of chemokine receptors and / or shRNA knockout, can express one, two, or more CARs described herein, which, for example, comprise: at least one extracellular binding domain containing at least one antigen recognition motif (as disclosed and described herein); at least one hinge domain and at least one transmembrane domain or fragments or variants thereof (as disclosed and described herein); and at least one intracellular signaling domain (as disclosed and described herein).

[0048] In one aspect, this article also discloses nucleic acids encoding siRNAs, such as shRNAs, for the purpose of knocking down genes to enhance the antitumor activity of TCR-transduced T cells in vivo. The nucleic acid sequences of said shRNAs target negative signaling molecules of the immune system, such as immune checkpoint proteins and / or immunosuppressive proteins. In some aspects, the knockdown of target gene expression is achieved through siRNA or shRNA nucleic acid molecules, targeting one, two, or more genes; the genes may include, exclude, or be selected from the group consisting of the following endogenous negative regulatory factors: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), S EMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A.

[0049] In some respects, the knockout or inactivation of target gene expression is achieved using a CRISPR / Cas9 system, which comprises a single-stranded guide RNA (sgRNA) molecule and a Cas endonuclease, and is capable of reducing or knocking out the expression of one, two, or more negative regulatory factor target genes; the target genes may include, exclude, or be selected from the group consisting of the following endogenous negative regulatory factors: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3. (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A.

[0050] In one aspect, this document also discloses a method for stimulating an immune response against cancer or for treating, inhibiting, and / or preventing cancer, the method comprising administering to a subject a composition comprising a therapeutically effective amount of any of the foregoing aspects or any of the aspects or embodiments disclosed herein, an epitope or TCR α or β variable region, and / or a composition identified by a method for detecting or identifying any of the foregoing aspects or any of the aspects or embodiments disclosed herein, epitope-specific T cells and TCRs.

[0051] In one aspect, the features described in this disclosure further include a chimeric antigen receptor and a CAR-expressing T cell. In some aspects, the CAR construct comprises an antigen recognition portion (e.g., a single-chain variable fragment (ScFv)), a transmembrane domain, and an intracellular T cell activation portion (composed of a CD28 or 4-1BB co-stimulatory signal transduction domain fused to a signal transduction domain, such as a ZAP300 (Seq ID NO: 16) or ZAP327 (SEQ ID NO: 17) or ZAP338 (SEQ ID NO: 52) or ZAP255 (SEQ ID NO: 64) or ZAP280 (SEQ ID NO: 65) signal transduction domain or other signal transduction domains derived from ZAP 70). In some aspects, the CAR may or may not include an antigen recognition portion, such as ScFv, that specifically binds to CD19, BCMA, B7-H3, mesothelin, or HER-2. In another aspect, one or more TCRα and / or β variable regions of any of the foregoing aspects or any aspects or embodiments disclosed herein may further include portions of ZAP255, ZAP280, ZAP300, ZAP327 or ZAP338 or other signal transduction portions derived from the ZAP70 kinase domain.

[0052] Natural killer (NK) cells and natural killer T (NKT) cells are considered innate immune cells. NKT cells are a subset of T cells that express T cell receptor α and / or β chains, but they also possess the characteristics of NK cells, including the expression of multiple NK cell markers, such as NK1.1. NKT cells can recognize a variety of exogenous or autolipid antigens presented by CD1d presenting molecules.

[0053] In some aspects, TCRs, CARs, bispecific antibodies, and / or bispecific, trispecific, or multivalent, multispecific binding proteins containing the antigen-binding portion of any of the TCRs described herein are engineered for expression in immune cells, such as T cells, NK cells, NKT cells, and macrophages. The corresponding engineered cell products include CAR-T cells, CAR-NK cells, CAR-NKT cells, and CAR-M cells. Similarly, in some aspects, TCRs are engineered for expression in immune cells, such as T cells and NKT cells. However, NK cells cannot be directly engineered to express TCRs because NK cells themselves do not express their own TCR chains. Therefore, NK cells need to be modified to express subunits of the TCR complex before introducing the TCRs described herein. The TCR complex responsible for antigen recognition comprises six CD3 subunits and a TCR heterodimer (α and β chains, or γ and δ chains). In some aspects, the engineered cell products are TCR-T cells, TCR-NK cells, and / or TCR-NKT cells.

[0054] Modified immune cells, such as those lacking expression of functional T cell receptors (TCRs) and / or human leukocyte antigens (HLA), can be obtained through any suitable means involving the knockout or knockdown of one or more TCR and / or HLA subunits. For example, regulatory T cells (Treg cells) can achieve TCR and / or HLA knockdown using siRNA, shRNA, the CRISPR system, transcription activator-like effector nucleases (TALENs), zinc finger endonucleases (ZFNs), macronucleases (mn, also known as homing endonucleases), or megaTAL (which binds TAL effector proteins to mn cleavage domains).

[0055] In some aspects of this disclosure, nucleic acids encoding the T-cell receptor (TCR) or chimeric antigen receptor (CAR) described herein are inserted into specific sites in the genome of immune cells, such as into loci of genes to be deleted. In some aspects, the nucleic acid encoding the TCR or CAR is inserted into loci of the TCR and / or HLA, thereby resulting in suppression of endogenous TCR and / or HLA expression.

[0056] In some aspects of this disclosure, the expression of TCR and / or HLA in T cells is suppressed by using siRNAs and / or shRNAs that target one or more nucleic acids encoding TCR and / or HLA. The expression of siRNAs and shRNAs in T cells can be achieved using any suitable expression system, such as a lentiviral expression system. For example, siRNAs and shRNAs that downregulate the expression of HLA class I and / or HLA class II genes are described in U.S. Patent Publication 2007 / 0036773; exemplary shRNAs that downregulate the expression of TCR components are described in U.S. Patent Publication 2012 / 0321667.

[0057] In another aspect, the variable regions of one or more T-cell receptor (TCR) α and / or β chains in any of the foregoing aspects or embodiments of this disclosure may further comprise ZAP255, ZAP280, ZAP300, ZAP327, or ZAP338 motifs, or other signal transduction motifs derived from the ZAP70 kinase domain, or mutants or variants thereof, wherein the N-terminal amino acid of the ZAP70 kinase is any site from 250 to 338 (including 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 2...). 81, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 31 6, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338), and the C-terminal amino acid is at position 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619.

[0058] In one aspect, this disclosure relates to the use of TCR-T cells or CAR-T cells, or the immune cell populations described in any of the foregoing aspects or embodiments of this disclosure, for the preparation of a medicament for treating a disease or condition in a human subject in need. In one aspect, the disease or condition is selected from the group consisting of inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, cancer, and infectious diseases. In one aspect, this disclosure also relates to a method of treating cancer using any of the TCR-T cells or CAR-T cells described in this disclosure.

[0059] In some embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, systemic lupus erythematosus (SLE), vasculitis, osteoarthritis, gout, ankylosing spondylitis, Sjögren's syndrome, Behçet's disease, polymyalgia rheumatica, and juvenile idiopathic arthritis.

[0060] In some embodiments, the autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, Addison's disease, pemphigus, scleroderma, Goodpassuia syndrome, autoimmune hepatitis, and autoimmune hemolytic anemia.

[0061] In some embodiments, the allergic disease is selected from the group consisting of: asthma, allergic rhinitis (hay fever), atopic dermatitis (eczema), food allergies (e.g., peanut allergy, shellfish allergy, milk allergy, egg allergy, wheat allergy), drug allergies (e.g., penicillin allergy, sulfonamide drug allergy), latex allergy, insect sting allergy, and urticaria.

[0062] In some embodiments, the organ transplant-related conditions are selected from the group consisting of: graft-versus-host disease (GvHD), acute rejection, chronic rejection, transplant vascular disease, post-transplant lymphoproliferative disorder (PTLD), and delayed graft function.

[0063] In some embodiments, the infectious disease is selected from the group consisting of: human immunodeficiency virus (HIV) infection, hepatitis B, hepatitis C, tuberculosis, pneumococcal pneumonia, influenza, malaria, dengue fever, Zika virus infection, Ebola virus disease, chikungunya virus infection, Lyme disease, SARS-CoV-2, Long COVID, and sepsis.

[0064] Although T cell recognition of target antigens is HLA-restricted, chimeric antigen receptor (CAR)-T cell recognition of targets is HLA-independent. As described in this article, regulating the signaling and function of TCR-T cells and CAR-T cells in vivo by directly modulating the signaling domains of TCRs or CARs, or by knocking down / knockout negative signaling molecules, is crucial for prolonging T cell persistence (and reducing T cell exhaustion). The negative signaling molecules may or may not include PD-1, VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1), or may be negative regulatory factors of one, two, or more endogenous genes, which may include, exclude, or be selected from the group consisting of the following negative regulatory factors: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), L AG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQU IN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A.

[0065] In one aspect, this disclosure also relates to a method for enhancing the migration of T cells to tumor tissue in vivo by forcibly expressing a chemokine receptor. In some aspects, the forced expression of the chemokine receptor is achieved by fusing any CAR or TCR construct of this disclosure with a chemokine receptor. In some aspects, the chemokine receptor is CCR5, CXCR3, and / or CCR2. In some aspects, the cytokine receptor may comprise or be selected from, for example, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, and a common cytokine receptor γ chain. In some aspects, the chemokine receptor is CCR5.

[0066] In some aspects, chemokine receptor expression and shRNA knockdown or CRISPR / Cas9 sgRNA knockout can be used in any of the foregoing aspects or embodiments described herein for any TCR or CAR construct containing regulation of one, two or more endogenous genes by negative regulators; said negative regulators may include, exclude or select from the group consisting of the following negative regulators: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LS D1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN- 1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A. Attached Figure Description

[0067] Several embodiments are illustrated in conjunction with the accompanying drawings, which are incorporated in and form part of this specification, and the disclosed compositions and methods are described together with the specification.

[0068] Figure 1 The generation and characterization of single T cell clones from the HLA-DP4-presented NY-ESO-1 reactive T cell line are illustrated. T cell clones were isolated from the HLA-DP4-presented NY-ESO-1 reactive cell line. After expansion of each T cell clone, antigen recognition was screened using a peptide containing amino acids 157-170 of NY-ESO-1 (SEQ ID NO:1) presented by an HLA-DP4-positive APC.

[0069] Figure 2 The diagram shows the construction of the DP4-ESO-1 TCR from a T cell clone into the pMSGV vector.

[0070] Figure 3A and 3BThe transduction of DP4-ESO-1 TCR in uninfected CD4+ T cells is shown. Figure 3A The transduction efficiency of DP4-ESO-1 TCR in uninfected CD4+ T cells is shown, measured by TCR-specific antibody staining and flow cytometry. Uninfected CD4+ T cells were transduced with supernatant of retroviruses generated from different DP4-ESO-1TCRPG-13 clones. DP4-ESO-1 TCR expression was detected after transduction, with an average transduction efficiency of 60–70%. Figure 3B Functional assays of CD4+ T cells transduced by DP4-ESO-1 TCR are shown.

[0071] Figure 4A , 4B 4C and 4C show the functional characterization of DP4-ESO-1 TCR-T cells. Figure 4A This demonstrates T cell recognition of peptides. DP4-ESO-1 TCR-T cells recognize the NY-ESO-1 157-170 peptide presented by HLA-DP4+ cells. Figure 4B The recognition of naturally processed NY-ESO-1 by T cells is shown. DP4-ESO-1 TCR-T cells recognize 293T cells transfected with full-length NY-ESO-1, HLA-DPA1, and HLA-DP4. Figure 4C The results showed that the DP4-ESO-1 TCR functions only in CD4+ T cells. Compared to CD8+ T cells, only CD4+ T cells transduced by the DP4-ESO-1 TCR recognize NY-ESO-1 157-170, indicating that TCR function is specific in CD4+ T cells.

[0072] Figure 5A , 5B 5C showed improved in vivo antitumor function when DP4-ESO-1 TCR was combined with A2-ESO-1 TCR to fight MDA-MB-231 / DP4 / ESO. Figure 5A The study demonstrates the in vivo migration of CD8+ T cells transduced with injected A2-ESO-1 TCRs (labeled with luciferase) by luciferase imaging. Figure 5B The growth of MDA-MB-231 / DP4 / ESO in vivo was monitored using different T cell groups. Figure 5C The comparison of tumor size at the time of mouse sacrifice is shown.

[0073] Figure 6A , 6B Images 6C and 6D illustrate the generation and characterization of HLA-A2-restricted CT83-specific T cells. Figure 6AThe ability of peptide-stimulated T cells generated in vitro to recognize 293T / CT83 PEP90-98 (a peptide containing amino acids 90-98 of CT83 (SEQ ID NO:2)) was demonstrated compared to that of the 293T / control peptide. Figure 6B The results show that A2-CT83-specific T cells recognize 293T cells transfected with Ii-CT83, CT83-GFP plasmid DNA, or treated with CT83 PEP9-98 (positive control), but do not recognize control 293T cells. Figure 6C The ability of A2-CT83-specific T cells to recognize human breast cancer cell line MDA-MB-231 (expressing HLA-A2 and CT83) was demonstrated, but not to recognize MDA-MB-436 (HL-A2-CT83+). Figure 6D It was shown that the recognition of MDA-MB-231 cells can be blocked by anti-MHC-I antibodies, but not by anti-MHC-II antibodies.

[0074] Figure 7A and 7B The study demonstrated the use of CT83-PEP90-98 to suppress breast cancer cells. Figure 7A A schematic diagram of the experimental design and schedule is shown. Figure 7B This demonstrates the treatment of tumor-bearing mice by inoculation (iv) with TAT-CT83 PEP90-98-CMI nanoparticles instead of TAT-CT83 PEP66-74-CMI. (CMI stands for CpG, MPLA, and poly(I:C)). P-value < 0.01.

[0075] Figure 8A , 8B Images 8C, 8D, and 8E illustrate the construction and characterization of HLA-A2-restricted CT83-specific TCRs. Figure 8A A flowchart is shown for TCR sequencing, cloning, and construction of an A2-CT83-specific T cell population purified from FACS using 10x single-cell barcoding technology and next-generation sequencing. Figure 8B Functional analysis of 293T, 293T / CT83-GFP, Cos-7 and Cos-7 / Ii-CT83, and Cos-7-A2 / Ii-CT83 cells was presented using A2-CT83 TCR-transduced T cells and vector-transduced PBMCs. Figure 8C This study demonstrated that A2-CT83 TCR-T cells specifically recognize 293T cells treated with CT83 PEP90-98 compared to 293T cells treated with other CT83 peptides. Figure 8DThe results show that A2-CT83 TCR-T cells specifically recognize MDA-MB-231 cells (expressing CT83 and HLA-A2), but do not recognize cells that express only one of the following peptides: MCF7 (A2+ CT83-), HTB-2 (A2+ CT83-), or MDA-MB-436 (A2- CT83+). Figure 8E The results show that A2-CT83 TCR-T cells can recognize CT83+ and HLA-A2+ lung cancer cells (HOP92 / A2, NCI-H358 / A, and NCI-H838 / A2), but cannot recognize CT83+ HLA-A2- tumor cells (HOP92, NCI-H358, and NCI-838). These results demonstrate that A2-CT83 TCRs can specifically recognize naturally processed CT83 epitopes presented by HLA-A2 molecules.

[0076] Figure 9A , 9B 9C showed the in vivo antitumor activity of A2-CT83 TCR-T cells. Figure 9A The timeline of administration (injection) of NCI-H838 tumor cells and A2-CT83 TCR-T cells in the NSG mouse model is shown. Figure 9B and 9C This study demonstrates the inhibitory effect of A2-CT83 TCR-T cells on tumor growth in vivo. In contrast, tumor-bearing mice treated with control T cells produced large tumor masses. These findings suggest that A2-CT83 TCR-T cells possess potent antitumor activity in vivo.

[0077] Figure 10A and 10B The generation and characterization of HLA-A2-restricted HCMV (pp65 and IE-1 proteins) specific T cell clones are shown. Figure 10A The study shows the selection of seven pp65-responsive T cell clones (495-503) and five IE-1-responsive T cell clones (316-324), followed by selection and in vitro expansion of T2 cells treated with peptides. Figure 10B Characterization of HLA restriction in pp65 T cell clone #3 and IE-1 T cell clone #5 is shown. Both T cell clones can specifically recognize HLA-A2-presented pp65 and IE-1 antigens in Cos-7 cells, respectively.

[0078] Figure 11A , 11B 11C, 11D, and 11E illustrate the identification, cloning, and characterization of HLA-A2-restricted pp65 and IE-1 specific TCRs, as well as their function in TCR-transduced human T cells. Figure 11AThe transduction efficiency of A2-pp65 TCR and A2-IE-1 TCR in human CD8+ T cells isolated from HCMV serum-negative donors is shown. Figure 11B The images show that T cells transduced by A2-pp65 TCR and A2-IE-1 TCR specifically recognize glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or glioblastomas infected with HCMV. Figure 11C The study demonstrated dose-dependent recognition of T2 cells treated with pp65 (495-503) or IE-1 (316-324) peptides by A2-pp65 TCR and A2-IE-1 TCR transduced T cells, respectively. Figure 11D The study demonstrates how A2-pp65 TCR-transduced and A2-IE-1 TCR-transduced T cells specifically kill glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1) or glioblastomas infected with HCMV. Figure 11E The dose-dependent cytotoxicity of T cells transduced with A2-pp65 TCR and A2-IE-1 TCR against HCMV / AD169-infected U87 tumor cells was demonstrated.

[0079] Figure 12A , 12B 12C and 12D indicate the in vivo antitumor activity of A2-pp65 TCR-T cells. A transplanted tumor model was established in immunodeficient mice using U87 cells expressing pp65 or IE-I and luciferase. Tumor cells were grown in SCID / Beige for 3 days, and then injected intravenously with 2 × 10⁻⁶ cells per mouse. 6 One T cell was processed by adoptive transfer of human T cells transduced with A2-pp65 TCR, A2-IE-1 TCR, or control TCR. Figure 12A A schematic diagram of the in vivo functional analysis process of A2-pp65 TCR-T cells is shown. Figure 12B The migration of A2-pp65 TCR-T cells after injection into tumor-bearing mice is shown. Figure 12C This demonstrates that A2-pp65 TCR-T cells specifically inhibit tumor growth in pp65 tumors expressing U87 in vivo. Figure 12D The study showed a significant reduction in tumor weight after treatment with A2-pp65 TCR-T cells, demonstrating the antitumor activity of pp65 TCR-T cells in the treatment of glioblastoma.

[0080] Figure 13A , 13B 13C and 13D show the in vivo antitumor activity of A2-IE-1 TCR-T cells. Figure 13AA schematic diagram of the in vivo functional analysis process of A2-IE-1 TCR-T cells is shown. Figure 13B The migration of A2-IE-1 TCR-T cells after injection into tumor-bearing mice is shown. Figure 13C This demonstrates that A2-IE-1 TCR-T cells specifically inhibit tumor growth in IE-1 tumors expressing U87 in vivo. Figure 13D The study showed a significant reduction in tumor weight after treatment with A2-IE-1 TCR-T cells, demonstrating the antitumor activity of A2-IE-1 TCR-T cells in the treatment of glioblastoma.

[0081] Figure 14A , 14B 14C, 14D, 14E, 14F, and 14G demonstrate the enhancement of A2-ESO-1TCR-T cell surface expression and function in human T cells by mouse constant sequences. Figure 14A Schematic diagrams of the conventional and modified A2-ESO-1 TCR constructs of this disclosure are shown. Figure 14B The modified TCR of this disclosure is shown to have higher transduction efficiency than conventional TCR constructs. Figure 14C The expression of TCR on the cell surface was detected by FACS. Figure 14D The image shows the detection of TCR cell surface expression using confocal microscopy. Figure 14E The results of an LDH assay for detecting the target cell killing ability of conventional and modified TCR-T cells are shown. Figure 14F The study demonstrates the detection of cytokine secretion by ELISA after co-culturing with A2-ESO-1 positive breast cancer cells. Figure 14G The long-term tumor cell killing ability of representative compositions of this disclosure was demonstrated by in vitro co-culture assay.

[0082] Figure 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 A schematic diagram of an animal experiment is shown. 1×10⁻⁶ cells from the NY-ESO-1 positive breast cancer cell line MDA-MB-231(ESO-1+) were used. 6 Cells were subcutaneously injected into the NSG fat pad. A2-ESO-1 TCR-T / A2-ESO-1TCR-MT cells were intravenously injected into tumor-carrying mice, followed by three doses of IL-2. Figure 15B The tumor growth was tracked. Figure 15C Images of the tumor after the euthanasia are shown. Figure 15D The tumor weight after euthanasia is shown.

[0083] Figure 16A , 16B 16C showed the in vitro tumor killing effect of A2-ESO-1 TCR with amino acid substitutions and a constant mouse TCR sequence. Figure 16A Five substitutions of A2-ESO-1 TCR transduced in human T cells and the original A2-ESO-1 TCR were shown, and their ability to recognize tumor cells with or without HLA-A2 and NY-ESO-1 expression was tested. Figure 16B The cytotoxicity of substituted A2-ESO-1 TCR-T cells against tumor cells with or without HLA-A2 and NY-ESO-1 expression was demonstrated. T cells transduced with A2-ESO-1TCR showed higher cytolytic activity at S2 and S5. Figure 16C The human TCR constant regions of S2 and S5 of the A2-ESO-1 TCR and the original A2-ESO-1 replaced by the mouse TCR constant region are shown. The S2 of the A2-ESO-1 TCR with the mouse TCR constant region sequence exhibits an effective T cell response.

[0084] Figure 17A , 17B 17C showed antitumor activity in A2-CT83 TCR-MT cells (mouse constant region). Figure 17A The transduction efficiency of A2-CT83 TCR-M in human T cells is shown. Figure 17B The results showed that A2-CT83 TCR-MT cells specifically recognize MDA-MB-231 and NCI-H1563 cells (expressing CT83 and HLA-A2), but do not recognize CAMA-1 cells (A2+ CT83-). Figure 17C The cytotoxicity of A2-CT83 TCR-MT cells against MDA-MB-231 and NCI-H1563 cells was demonstrated. These results indicate that A2-CT83 TCR-MT cells are effective and specific against tumor cells and reduce TCR mismatch.

[0085] Figure 18A , 18B Figures 18C, 18D, and 18E illustrate 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 signal transduction domain. Figure 18A A schematic diagram is shown of a conventional CD19-CD28-CD3z (1928z) and a new construct containing CD19-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327). Figure 18B The T cell transduction efficiency of three CARs in human T cells is shown. Figure 18C and 18D The study demonstrates antigen-specific recognition and tumor cell lysis after co-culturing CAR-T cells with Raji tumor cells. Figure 18E The in vivo antitumor activity of three CAR-T cells (1928z, 1928ZAP300, and 1928ZAP327) was demonstrated. Importantly, 1928ZAP300 and 1928ZAP327 CAR-T cells outperformed 1928z CAR-T cells in in vivo and significantly prolonged overall mouse survival in a Raji lymphoma tumor model. These studies indicate that replacing the CD3ζ chain with the Zap70 kinase domain (ZAP300 and ZAP327) significantly enhances in vivo antitumor activity.

[0086] Figure 19A , 19B Figures 19C, 19D, and 19E demonstrate that novel CAR-T constructs containing 4-1BB, fused with ZAP300 and ZAP327 derived from ZAP70, produce fewer cytokines but generate more effective anti-tumor immunity. Figure 19A Schematic structures of 19bbz and 19bbZAP327 are shown. Figure 19B The results showed that, compared with conventional 19bbz CAR-T cells, 19bbZAP327 CAR-T cells produced significantly lower levels of cytokines after stimulation with tumor cells. Figure 19C This demonstrates the specific lysis of tumor cells by 19bbZAP327CAR-T cells. Figure 19D and 19E The study demonstrated that 19bbZAP327 CAR-T cells exhibited excellent in vivo anti-tumor activity and significantly prolonged mouse survival, indicating that 19bbZAP327 CAR-T cells improved safety and anti-tumor immunity compared to conventional 19bbz CAR-T cells.

[0087] Figure 20A , 20B The results from 20C show that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. Figure 20A The study demonstrated that 1928ZAP327 CAR-T cells exhibited greater in vivo persistence in the bone marrow and spleen of T-cell-transferred mice. Figure 20B The study showed a higher percentage of central memory 1928ZAP327 CAR-T cells compared to 1928z CAR-T cells. Figure 20C The results showed that 1928ZAP327 CAR-T cells expressed lower levels of PD-1 (exhaustion marker) than 1928z CAR-T cells, indicating that the ZAP327 signaling domain reduces T cell exhaustion.

[0088] Figure 21A and 21B This study demonstrates how knocking down the expression of metabolic genes PD1, VHL, and PPP2R2D can regulate TCR-T cell function in vivo. Figure 21A The transduction efficiency of A2-ESO-1 TCR constructs with or without PD1, VHL, or PPP2R2D shRNA is shown. Figure 21B The diagram shows the injection of A2-ESO-1 TCR-T cells with or without PD1, VHL, or PPP2R2D knockdown into mice carrying MDA-MB-231 / A2 / NY-ESO-1. Top panel: Mean tumor growth for each group. Middle panel: Survival curves for each group of mice. Bottom panel: Tumor growth per mouse in each group.

[0089] Figure 22 This study demonstrates the enhancement of the CD44+ CD62L-memory T cell population in CD4+ T cells by Jmjd3 conditional knockout (cKO) compared to wild-type (WT) cells.

[0090] Figure 23A , 23B 23C, 24D, 25E, and 25F demonstrate the enhancement of T cell survival and persistence by Jmjd3 conditional knockout (CKO) T cells in vivo and in vitro. Figure 23A and 23B The study demonstrated that in vivo stimulation of CD4+ T cells from Jmjd3 CKO 2d2 transgenic mice with MOG peptide plus complete Freund's adjuvant significantly improved clinical scores in an EAE mouse model. Figure 23C The results showed that, after T cell transfer, the number of Jmjd3 conditional knockout T cells was higher than that of wild-type 2d2 cells. Figure 23D , 23E 23F showed that after T cell transfer using T cells stimulated in vitro with MOG peptides, the number of Jmjd3 CKO T cells was higher than that of wild-type 2d2 cells.

[0091] Figure 24A , 24B 24C studies have shown that Jmjd3 knockout (KO) enhances T cell survival and persistence by reducing T cell apoptosis. Figure 24A The study showed that Jmjd3 cKO T cells had reduced levels of apoptosis-related proteins after stimulation with anti-CD3 and CD28 antibodies. Figure 24B The results showed that Jmjd3 cKO T cells had a much lower level of T cell apoptosis after stimulation. Figure 24C The results showed that Jmjd3 cKO T cells had very low levels of cleaved caspase 3 compared to WT T cells.

[0092] Figure 25A , 25B 25C and 25D demonstrate enhanced CAR-T cell survival and persistence in vivo via Jmjd3 knockdown (KD). Figure 25A An experimental design for monitoring the survival of luciferase-labeled T cells using Raji tumor cells is shown. Figure 25B and 25C The study showed that CAR-T cells with Jmjd3 KD (1928z-shJMJD3) exhibited strong proliferation on day 4 after T cell transfusion into NSG mice carrying Raji tumors, compared to the 1928z control shRNA, while maintaining high levels of T cells. Figure 25D The study showed 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.

[0093] Figure 26A , 26B 26C showed that enhanced T cell transport to tumor cells in vivo was achieved through forced expression of chemokine receptors. Figure 26A A construction diagram of the 1928z CAR fused with CCR5 is shown. Figure 26B and 26C The study showed that 1928z-CCR5CAR-T cells significantly inhibited the growth of MDA-MB-231 / CD19 tumor cells in vivo, indicating that forced chemokine receptor expression enhanced the transport of T cells to tumor cells.

[0094] Figure 27 This study describes a strategy to enhance T cell transport and T cell persistence by expressing chemokine receptors and shRNA KD in TCR or CAR constructs.

[0095] Figure 28 shows an alternative for identifying the ZAP70 kinase domain as a CAR signaling domain. Figure 28A A schematic diagram of a CAR with different signal transduction domains is shown. The CAR uses the FMC063 anti-CD19 single-stranded variable fragment (scFv) as the antigen-binding domain, followed by the hinge domain and transmembrane domain of CD28. The intracellular tail of CD28 is fused to CD3ζ (amino acids 52–164), ZAP300 (amino acids 300–619), LAT (amino acids 28–262), or the full-length SLP76, respectively. Figure 28B The results of flow cytometry analysis of CAR surface expression in human T cells are shown. Figure 28CThe levels of cytokine secretion produced by CAR-T cells co-cultured with CD19-expressing Raji cells overnight were shown, and these levels were determined by ELISA. Figure 28D The study demonstrated specific lytic activity of CAR-T cells against Raji cells, which was detected by an LDH non-radioactive cytotoxicity assay. Statistical significance is indicated by an asterisk (*). p<0.01, p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0096] Figure 29 shows the results of replacing CD3ζ with ZAP70 kinase domains of different lengths. Figure 29A A schematic diagram of a CAR containing truncated ZAP70 kinase domains (ZAP255, ZAP280, and ZAP300) is shown. Figure 29B The results of flow cytometry analysis of CAR surface expression in human T cells are shown. Figure 29C Statistical analysis of CAR-T cell activity among ZAP255, ZAP280, and ZAP300 CAR-T cells is presented. Figure 29D The NFAT-GFP reporter gene Jurkat cells were transduced to express ZAP255, ZAP280, or ZAP300CAR, and co-cultured with NALM6 cells. GFP expression was detected by flow cytometry. Figure 29E Live-cell imaging performed using CellcyteX to investigate T cell cytotoxicity. Statistical significance is indicated by an asterisk (...). (p<0.0001), “ns” indicates no statistically significant difference.

[0097] Figure 30 shows the ZAP70 kinase domain required to determine T cell signaling and function. Figure 30A A schematic diagram of truncated ZAP70 kinase domains of different lengths is shown. The full-length ZAP70 contains an N-terminal SH2 domain and a C-terminal SH2 domain, which are connected by an interdomain A, and further includes an interdomain B and a kinase domain. Figure 30BThe results show the detection of CAR expression in T cells transduced by CAR constructs of 1928ZAP300, 1928ZAP327, 1928ZAP377, 1928ZAP420, 1928ZAP540 and 1928ZAP560 using Protein L and Streptavidin-PE staining. Figure 30C The levels of interferon-γ (IFN-γ) produced by CAR-T cells expressing different lengths of ZAP70 kinase domains were shown by ELISA. The specific tumor-killing effect mediated by CAR-T cells expressing different lengths of ZAP70 kinase domains was also shown by LDH cytotoxicity assay, with the activity of 1928ZAP300 CAR-T cells used as a positive control for comparison. Figure 30D The study demonstrated the cytotoxicity of 1928ZAP300 and 1928ZAP327 CAR-T cells to tumor cells under different effector cell to target cell ratios (E:T ratio) ranging from 0.3:1 to 6:1, using LDH assay. Figure 30E Kaplan-Meier curves are shown, representing the overall survival of mice treated with 5 million Raji cells on day 0 and 500,000 CAR-T cells on day 5. Statistical significance is indicated by an asterisk (...). p<0.01, p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0098] Figure 31 shows the cytokine release from CAR-T cells containing the ZAP70 kinase domain (ZAP327) and their specific killing effect on tumor cells. Figure 31A The results of CAR surface expression detection in virus-transduced 1928z and 1928ZAP327 CAR-T cells using Protein L and Streptavidin-PE are shown. Figure 31B The levels of interferon-γ (IFN-γ) produced by 1928z and 1928ZAP327 CAR-T cells from three different healthy donors after co-culturing with CD19-positive Raji cells are shown. The IFN-γ levels were measured by ELISA of the overnight co-culture supernatant. Figure 31CThe cytotoxicity of 1928z and 1928ZAP327 CAR-T cells against Raji cells was demonstrated using the LDH cytotoxicity assay under a range of effector cell to target cell ratios (E:T ratio) from 6:1 to 0.3:1. Statistical significance is indicated by an asterisk (*). (p<0.0001), “ns” indicates no statistically significant difference.

[0099] Figure 32 shows the in vitro antitumor response of 1928ZAP327CAR-T cells. Figure 32A The levels of interferon-γ (IFN-γ) produced by 1928z and 1928ZAP327 CAR-T cells during co-culture with Raji and NALM6 cells are shown. Figure 32B The results show the intracellular staining of IFN-γ, IL-2, and TNF-α in 1928z and 1928ZAP327 CAR-T cells after overnight co-culture with Raji cells. Figure 32C The results of the detection and analysis of CD69, a marker of T cell activation, are shown. Figure 32D The results of Western blot analysis of proteins related to the TCR signaling pathway are shown, and the data from... Figure 32D Quantitative analysis. Figure 32E The results of intracellular staining of granzyme B (GZMB) and perforin A (PRF1) in 1928z and 1928ZAP327 CAR-T cells after overnight co-culture with Raji cells are shown. Figure 32F The results of cytotoxicity assays using luciferase activity are shown. Figure 32G This study demonstrates the cytotoxic effects of Raji-GFP cells expressing CD19 and THP1 cells not expressing CD19, co-cultured overnight with CAR-T cells (effective cell to target cell ratio (E:T) 5:1), with the cytotoxic activity based on GFP. + The reduction in Raji cell count was assessed. Statistical significance is indicated by an asterisk (...). p<0.05 p<0.01, p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0100] Figure 33 shows the superior antitumor response of 1928ZAP327 CAR-T cells. Figure 33A A schematic diagram of the in vivo experimental design is shown. NSG mice were inoculated with 5 million Raji lymphoma tumor cells and treated with different types of CAR-T cells. Figure 33B The levels of interferon-γ (IFN-γ) cytokines in mouse serum collected 1 day after T-cell injection are shown by ELISA. Figure 33C and Figure 33D The results of the Kaplan-Meier survival analysis are shown, corresponding to 5×10⁻⁶ trials. 5 (F) or 2×10 6 (G) Survival rate of mice treated with 1928z or 1928ZAP327 CAR-T cells; the control group represents untreated mice. P-values ​​were determined by a one-sided log-rank Mantel–Cox test. Statistical significance is indicated by an asterisk (G). p<0.01, p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0101] Figure 34 shows that ZAP327-driven 4-1BB CAR-T cells exhibit superior antitumor activity in vitro and in vivo. Figure 34A The results show the detection of CAR surface expression in virus-transduced T cells using Protein L and Streptavidin-PE. The conventional CAR uses a single-stranded variable fragment (scFv, FMC63) against CD19, and sequentially links the hinge domain, transmembrane domain, intracellular co-stimulatory domain of 4-1BB, and CD3ζ signaling domain; this CAR is named 19BBz. The 19BBZAP327 CAR construct replaces the CD3ζ signaling domain with a ZAP70 kinase domain starting from amino acid 327. Figure 34B The study showed the cytokine levels produced by 19BBz and 19BBZAP327 CAR-T cells after co-culturing with the CD19-expressing triple-negative breast cancer cell line MDA-MB-231 (MM231-CD19). Figure 34C The results show the cytotoxicity assays of 19BBz and 19BBZAP327 CAR-T cells against MM231-CD19 cells. Figure 34DThe diagram shows an in vivo experimental design in which mice were inoculated with Raji cells and then treated with CAR-T cells. Figure 34E The results show the detection of cytokine release levels in mouse serum by ELISA on day 4 after T-cell injection. Figure 34F The display shows that 5×10 were given respectively 5 Kaplan-Meier survival analysis results of mice treated with 19BBz or 19BBZAP327 CAR-T cells. The data are a pooled result from three independent experiments. Statistical significance is indicated by an asterisk (*). p<0.05 p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0102] Figure 35 shows the superior antitumor activity of ZAP327-driven 4-1BB CAR-T cells. Figure 35A The results show the intracellular staining of interferon-γ (IFN-γ), IL-2, and TNF-α in 19BBz and 19BBZAP327 CAR-T cells after overnight co-culture with Raji cells. Figure 35B The results show the cytotoxicity of CAR-T cells against tumor cells detected by luciferase assay. Figure 35C The results show the intracellular staining of granzyme B (GZMB) and perforin A (PRF1) in 19BBz and 19BBZAP327 CAR-T cells after overnight co-culture with Raji cells. Figure 35D A schematic diagram of the in vivo experimental design is shown, used to compare the therapeutic effects of 19BBz and 19BBZAP327. Mice were inoculated with 2 million CD19-positive NALM6 leukemia cells, followed by 1 million CAR-T cells. Figure 35E The results of the overall survival assessment of mice are shown. Statistical significance is indicated by an asterisk (...). p<0.05 p<0.01, p<0.001 (p<0.0001), “ns” indicates no statistically significant difference.

[0103] Figure 36 shows that T cells engineered with the ZAP70 kinase domain exhibited a superior antitumor response in a solid tumor model. Figure 36A The study showed the results of subcutaneous (sc) inoculation of NSG mice with 1 million MM231-CD19 tumor cells and subsequent treatment with CAR-T cells; the levels of interferon-γ (IFN-γ) cytokines collected from mouse serum were measured by ELISA, and the tumor growth of tumor-bearing mice was assessed. Figure 36B This study demonstrates the results of subcutaneous (sc) inoculation of NSG mice with 1 million Mel1558 tumor cells followed by CAR-T cell therapy. The levels of interferon-γ (IFN-γ) cytokines collected from mouse serum were measured by ELISA, and tumor growth in tumor-bearing mice was assessed. Statistical significance is indicated by an asterisk (*). p<0.05 p<0.01, (p<0.0001), “ns” indicates no statistically significant difference.

[0104] Figure 37 This study demonstrates the gating strategy for human T memory stem cells (Tscm). FSC and SSC gating were used to identify lymphocyte populations; FSC-A and FSC-H gating were used to obtain single-cell populations. Live cells were obtained by selecting live / dead dye-negative populations. CAR-positive cells were subsequently gating. CD95 was widely expressed in memory cells and effector cells after stimulation, while naïve T cells were negative. CD4 and CD8 T cell populations were differentiated. Further gating was performed on CCR7 and CD127 double-positive populations. Different memory T cell subsets were distinguished using CD45RO and CD62L markers, with CD45RO… - CD62L + The population is Tscm, while CD45RO + CD62L - The population is Tcm.

[0105] Figure 38 shows the proportion of T memory stem cells (Tscm) and central memory T cells (Tcm) enhanced by the ZAP327 signaling domain in vivo, as well as T cell persistence. Figure 38AThe experimental design and analysis flowchart is shown. Human T cells were isolated from healthy donors and transduced using CAR retroviruses, and CAR-T cells were cultured in vitro. The memory phenotype of CAR-T cells was characterized before injection. NSG mice were inoculated with Raji cells and treated with luciferase-expressing CAR-T cells. Luciferase signals were measured on days 0, 1, 3, 7, and 14 after T cell injection. Mice were sacrificed on day 40 after tumor injection, and tissues were collected to compare CD3 levels. + T cell proportion; simultaneous detection of Tscm and Tcm memory phenotypes, and analysis of the exhaustion marker PD1. + TIM3 + LAG3 + . Figure 38B Representative images of the ratio of Tscm and Tcm in vitro on day 20 after T cell activation are shown. Figure 38C A representative image of the luciferase signal is shown. Figure 38D Showing Figure 38C The results of the statistical analysis. Figure 38E The results of T cell analysis on day 36 after T cell transfer into NSG mice are shown; bone marrow, lung, and spleen tissues were collected, and human CD3 was detected by FACS analysis. + The presence of CAR-T cells. Figure 38F Displaying CD3 + CAR + CD95 + CCR7 + IL7R + After T cell gating, further analysis of CD62L was performed. + CD45RO + (Tcm) and CD62L + CD45RO - (Tscm) T cell population. Statistical significance: p<0.05 p < 0.01.

[0106] Figure 39 shows that the 1928ZAP327 CAR-T cells have a lower degree of exhaustion. Figure 39A The results of flow cytometry analysis comparing T cell exhaustion markers PD1, LAG3, and TIM3 in CAR-T cells from 1928z and 1928ZAP327 are shown. Figure 39B The SPICE (Simplified Presentation of Incredibly Complex Evaluations) analysis diagram is displayed. Figure 39CThe results show the intracellular expression analysis of the negative regulators TOX and NR4A1 by flow cytometry. Statistical significance: p<0.05 p<0.01, “ns” indicates no statistically significant difference.

[0107] Figure 40 shows that the 1928ZAP327 CAR established different transcriptional signatures. Figure 40A Shown in CD19 + Volcano plots of significantly upregulated and downregulated genes in 1928z and 1928ZAP327 CAR-T cells 24 hours after target cell stimulation (n=3 replicates per group). Figure 40B Biological Gene Ontology (GO) analysis was shown, indicating that these genes are involved in different biological processes. Figure 40C A heatmap analysis of genes involved in glycolysis and mitochondrial function is shown.

[0108] Figure 41 shows that the 1928ZAP327 CAR established different metabolic pathway characteristics. Figure 41A The study shows a comparison of oxygen consumption rate (OCR) between 1928z and 1928ZAP327 CAR-T cells using Seahorse analysis, and quantitative analysis of OCR for basal respiration, ATP production, maximal respiration, reserve respiration capacity, proton leakage, and non-mitochondrial respiration. Figure 41B The study compared the extracellular acidification rate (ECAR) of 1928z and 1928ZAP327 CAR-T cells using Seahorse analysis and quantified the ECAR of basal glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Figure 41C The uptake of 2-NBDG and BODIPY-C16 is shown, as well as mitochondrial strength as determined by the mitochondrial tracer dye (MitoTracker). Statistical significance: p<0.05 p<0.01, p < 0.0001.

[0109] Figure 42 shows the expression of CT83 in lung cancer and breast cancer samples. Figure 42A The expression of CT83 in lung cancer cell lines and tumor samples was detected by RT-PCR analysis. Figure 42B The results show that the expression of CT83 protein in lung cancer cell lines was detected by Western blot analysis using an anti-CT83 antibody. Figure 42CThe expression of CT83 in breast cancer cell lines and triple-negative breast cancer samples was detected by RT-PCR analysis, with NY-ESO-1 as a positive control.

[0110] Figure 43 shows the preparation and characterization of HLA-DR13-restricted CT83-specific T cells. Figure 43A The results showed that CT83 peptide-specific CD4+ was detected in T cells stimulated in vitro and then stained intracellularly. + T cells. Figure 43B The results showed that T cells could recognize 293DR13 / CT83 PEP10-31, but could not recognize 293DR13 / control peptide. Figure 43C The recognition of 293DR13 / CT83 PEP10-31 cells by T cells can be blocked by anti-MHCII or anti-HLA-DR antibodies, but not by anti-HLA-DP, anti-HLA-DQ or anti-MHC I antibodies. Figure 43D The results showed that CT83-specific T cells only recognize 293DR13 / CT83PEP10-31, but not the same peptide loaded on 293DR1, DR3, DR4, DR7 or DR11 cells. p < 0.001.

[0111] Figure 44A and Figure 44B It demonstrates T cell epitope localization and tumor-specific recognition. Figure 44A 293DR13 cells transfected with either the CT83 (amino acid 10–31) or CT83 (amino acid 17–31) gene fragments can be recognized by T cells. Figure 44B Display of engineered CD4 from DR13-CT83 TCRs of three healthy donors + T cells can specifically recognize MDA-MB-231 (DR13) + CT83 + ) cells, but cannot recognize MDA-MB-436 and MDA-MB-468 (DR13) - CT83 + ) cells or M1495 (DR13) + CT83 - )cell. p<0.01; p < 0.001. This figure is disclosed in the order of appearance of SEQ ID NOs: 39, 62–63 and 61.

[0112] Figure 45 shows the identification and characterization of CT83-specific TCRs. Figure 45A This diagram illustrates the identification and cloning of CT83-specific TCRs using single-cell TCR sequencing technology. Figure 45B HLA-DR13-restricted CT83-specific TCR-T cells can recognize 293DR13 cells expressing the Ii-CT83 gene or loaded with the DR13-CT83 peptide. Figure 45C Display of CT83-TCR engineered CD4 + T cells, rather than CT83-TCR engineered CD8 + T cells can recognize 293DR13 / Ii-CT83 cells and MDA-MB-231 cells. p<0.01; p < 0.001.

[0113] Figure 46 shows CD4 + CT83 TCR-T cells can completely suppress or eliminate breast cancer cells. Figure 46A The results showed that in MDA-MB-231 tumor-bearing NSG mice, control T cells or CT83TCRCD4 were administered, respectively. + T-cell therapy, including CD4 + CT83 TCR-T cells can completely suppress or eliminate breast cancer cells. Figure 46B Human CD3 isolated from the spleen of treated mice. + FACS analysis was performed on T cells and on human CD4 cells. + After T-cell gating, CD62L and CD45RA were analyzed. p < 0.0001.

[0114] Figure 47 shows the engineered design and construction of various TCRs carrying 4-1BB-ZAP327 (BBZAP327) and bound to shRNA or sgRNA-mediated knockdown or knockout of negative regulators. Figure 47AThis section showcases the design and construction of human TCRs [TCR(H)], TCRs containing the constant region of mouse TCRs [TCR(M)], and TCRs (M) containing 4-1BB-ZAP327 (BBZAP327) (also known as TCR-STEM). All TCR constructs incorporate knockdown or deletion of negative regulatory factors mediated by shRNA or sgRNA to enhance T cell persistence and function. In some respects, TCRs (M) can be further engineered to express 4-1BB, CD28, CD27, OX40, ICOS, MyD88, or MALT-1 downstream of the TCR-TM domain; in others, T2A-MyD88 or T2A-MALT-1 can also be expressed. Figure 47B A schematic diagram showing TCR(H), TCR(M), TCR-STEM, and the TCR complex containing an additional signaling molecule (TCR(M)).

[0115] Figure 48 shows the tumor-specific recognition, killing, and anti-tumor immune response of A2 / CT83 TCR-T cells and A2 / CT83 TCR-BBZAP327 (also known as A2-CT83 TCR-STEM) T cells in vitro and in vivo. Figure 48A A schematic diagram showing A2 / CT83TCR-T cells and A2 / CT83 TCR-BBZAP327 T cells. Figure 48B The specific lytic activity of A2 / CT83 TCR-T cells and A2 / CT83TCR-BBZAP327 T cells was demonstrated. Figure 48C The study showed the target recognition (cytokine release) of A2 / CT83 TCR-T cells and A2 / CT83 TCR-BBZAP327 T cells, with A2 / CT83 TCR-BBZAP327 T cells producing lower levels of IFN-γ than A2 / CT83 TCR-T cells. p<0.05; p < 0.0001; ns indicates no statistically significant difference.

[0116] Figure 49 shows that A2-CT83 TCR-BBZ327 T cells exhibited superior antitumor activity compared to CT83 TCR-T cells in a refractory tumor model (PD-L1-expressing MDA-MB-231). Figure 49A This demonstrates a direct comparison of the three groups of antitumor activities based on tumor suppression. Figure 49B This shows a comparison of tumor weight between groups. Figure 49C This shows the T-cell analysis in tumor tissues from different treatment groups. p<0.01; p < 0.001; p < 0.0001.

[0117] Figure 50 shows the T cell activity of CD4 TCR (HC), CD4 TCR (MC), and CD4 TCR-STEM T cells. Figure 50A The study showed the activity of HLA-DR13-restricted CT83-specific CD4 TCR(HC) (containing the human constant region) and CD4 TCR(MC) (containing the mouse TCR constant region), with CD4 TCR(MC) T cells releasing significantly more IFN-γ than CD4 TCR(HC) T cells against 293DR13 / CT83 cells. Figure 50B A schematic diagram of pCD4 TCR-STEM (pCD4 TCR-BBZAP327) is shown. Figure 50C This demonstrates tumor recognition (IFN-γ release) of CD4 TCR-STEM T cells. Figure 50D This demonstrates the specific killing effect of CD4 TCR-STEM T cells on MDA-MB-231 tumor cells. p<0.05; p < 0.01.

[0118] Figure 51 shows the potent in vivo anti-tumor immunity of CD4 TCR-STEM T cells in breast cancer and lung cancer models. Figure 51A This demonstrates the experimental design of breast cancer models using CD4 TCR-STEM T cells or control CD4 T cells. Figure 51B This study compares the antitumor activity among four treatment groups (control group, CD4 TCR(HC) group, CD4 TCR(MC) group, and CD4 TCR-STEM group). Figure 51C The results showed that in two independent experiments involving T cells from three healthy donors, CD4 TCR-STEM T cells were able to completely suppress or eliminate MDA-MB-231 tumor growth. Figure 51D The results showed that both CD4 TCR-STEM T cells and A2-CT83 TCR-STEM T cells could completely inhibit or eliminate the growth of NCI-H838 / A2-DR13 lung cancer cells, while control T cells failed to inhibit tumor growth. p<0.05; p < 0.001; p < 0.0001.

[0119] Figure 52 shows the peptide epitopes and recognition specificity of A2-CT83 TCR-STEM T cells. Figure 52A Displays HLA-A2CT83 T cell epitopes and HLA-DR13CT83 T cell epitopes. Figure 52B This shows the expression of CT83 in different cell lines. Figure 52C The results show that A2-CT83 TCR-STEM T cells do not exhibit cross-reactivity. Figure 52D shows the recognition of CT83 by TCR-STEM T cells in the HLA-A2 molecular background.

[0120] Figure 53 Alanine scanning analysis of the HLA-DP4-restricted NY-ESO-1 TCR was performed. Alanine substitutions in the CDR region may lead to loss of function; alanine substitution sites that result in significant reductions in TCR function were marked. p<0.01; p < 0.001, relative to WT control TCR.

[0121] Figure 54 shows the deep amino acid mutation and functional screening of HLA-DP4 NY-ESO-1 TCR. Figure 54A The key amino acid residues identified by alanine scanning are shown, and each key site is further mutated. Figure 54B The results of functional screening using Jurkat NFAT-GFP reporter cells are shown.

[0122] Figure 55 shows the CD4 transduced by WT and mutant TCR. + Functional analysis of T cells. Figure 55A CD4+ transduced by WT and mutant DP4NY-ESO-1 TCR is shown. + T cells release IFN-γ in MDA-MB-231 / DP4 and MDA-MB-231 cells expressing NY-ESO-1. Figure 55B CD44 cells showing WT and mutant TCR transduction were observed. + T cell cytotoxicity against MDA-MB-231 / DP4 cells. p<0.05; p<0.01; p < 0.001.

[0123] Figure 56 shows the identification of amino acid residues in HLA-A2 CT83 peptide (90–98) and HLA-DR13 CT83 peptide (17–31) that are crucial to their respective TCR-T cells. Figure 56A The alanine scanning analysis of the HLA-A2 CT83 peptide (90–98) (SEQ ID NO: 2) is shown to identify key amino acids required for A2-CT83 TCR-T cell recognition. Figure 56B The image shows an alanine scan analysis of the HLA-DR13 CT83 peptide (17–31) (SEQ ID NO: 61) to identify key amino acids required for CD4 TCR-STEM T cell recognition. Figure 56C The analysis shows the truncation of amino acid residues at the N-terminus and C-terminus of the HLA-DR13 CT83 peptide to determine the sequence range required for CD4 TCR-STEM T cell recognition. Detailed Implementation

[0124] definition Before disclosing and describing the compounds, compositions, articles, apparatuses, and / or methods of the present invention, it should be understood that, unless otherwise stated, they are not limited to specific synthetic methods or specific recombinant biotechnology methods, or, unless otherwise stated, they are not limited to specific reagents, as these can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0125] As used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, references to “drug carrier” include mixtures of two or more such carriers, etc.

[0126] A range herein may be expressed as from “about” a particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment. It should also be understood that the endpoints of each range are significant relative to and independent of the other endpoint. It should also be understood that numerous values ​​are disclosed herein, and each value is disclosed herein as “about” in addition to being the value itself. For example, if the value “10” is disclosed, “about 10” is also disclosed. It should also be understood that when a numerical value is disclosed, as will be appropriately understood by those skilled in the art, “less than or equal to” the numerical value, “greater than or equal to” the numerical value, and possible ranges between the numerical value are also disclosed. For example, if the value “10” is disclosed, “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and this data represents endpoints and starting points, as well as ranges of any combination of data points. For example, if specific data point "10" and specific data point 15 are disclosed, it should be understood that values ​​greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as values ​​between 10 and 15, are disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed. It should also be understood that whenever a series of values ​​is disclosed, those skilled in the art should also understand that any range falling between any two of the stated values ​​is acceptable.

[0127] In this specification and the following claims, many terms will be used, which should be defined to have the following meanings: "Optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the possibility that the event or situation occurs and the possibility that it does not occur.

[0128] As used herein, the term "antibody" includes polyclonal and monoclonal antibodies; primate-derived (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and can be an intact molecule, a fragment thereof (which may include or exclude scFv, Fv, Fd, Fab, Fab', and F(ab)'2 fragments), or a polymer or aggregate of an intact molecule and / or fragments; and can be naturally occurring or generated, for example, through immunization, synthesis, or genetic engineering; as used herein, "antibody fragment" refers to a fragment derived from or associated with an antibody that binds to an antigen and, in some embodiments, can be derivatized to exhibit structural features that promote clearance and uptake, for example, by incorporating galactose residues. "Antibody" includes, for example, F(ab), F(ab)'2, scFv, light chain variable region (VL), heavy chain variable region (VH), and combinations thereof.

[0129] A checkpoint inhibitor is an agent that targets checkpoint proteins or their derivatives, and may be referred to as a "checkpoint inhibitor." Checkpoint inhibitors may or may not include proteins, peptides, amino acid residues, and monoclonal or polyclonal antibodies. Multivalent vaccines may include one or more checkpoint inhibitors or be administered in combination with one or more checkpoint inhibitors. Checkpoint inhibitors can bind to ligands or proteins found, for example, in any family of T-cell regulatory factors such as CD28 / CTLA-4. Targets of checkpoint inhibitors include, but are not limited to, receptors or co-receptors expressed on immune system effector cells or regulatory cells (e.g., T cells) (e.g., CTLA-4; CD8); proteins expressed on the surface of antigen-presenting cells (e.g., 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 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 activated antigen-presenting cells and T cells.

[0130] As used herein, the term “separation” includes any method of essentially purifying one component from another (e.g., by filtration, magnetic attraction, etc.).

[0131] As used herein, the terms “isolation” or “isolating” include any way of classifying a species of one type of genus with a species of another type of genus.

[0132] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, such as a mammal. In one respect, a subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Therefore, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., an internist).

[0133] The term “prevention” or “inhibition” used in this article refers to preventing the occurrence of cancer or delaying the onset of cancer.

[0134] As used in this article, the term "treatment" or "reduction of the presence of cancer or cancer cells" refers to the suppression of cancer growth, which is reflected, for example, by tumor volume or the number of malignant cells. Tumor volume can be determined by various known methods, such as measuring observed images and comparing the average cross-sectional diameter of the tumor to a calibration line (e.g., as done in ImageJ).

[0135] As used in this article, “treatment of a disease or condition, such as an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplant condition, an infectious disease, and / or symptoms of aging” means reducing or improving the severity of the signs or symptoms of the condition.

[0136] In one respect, the diseases or conditions treated are selected from the following groups: inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant conditions, cancer, and infectious diseases.

[0137] In some implementation schemes, the inflammatory disease to be treated is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, systemic lupus erythematosus (SLE), vasculitis, osteoarthritis, gout, ankylosing spondylitis, Sjögren's syndrome, Behçet's disease, polymyalgia rheumatica, and juvenile idiopathic arthritis.

[0138] In some implementation schemes, the autoimmune disease to be treated is selected from the group consisting of: multiple sclerosis, type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, Addison's disease, pemphigus, scleroderma, Goodpasture syndrome, autoimmune hepatitis, and autoimmune hemolytic anemia.

[0139] In some implementations, the allergic disease to be treated is selected from the group consisting of: asthma, allergic rhinitis (hay fever), atopic dermatitis (eczema), food allergies (e.g., peanut allergy, shellfish allergy, milk allergy, egg allergy, wheat allergy), drug allergies (e.g., penicillin allergy, sulfonamide allergy), latex allergy, insect bite allergy, and urticaria.

[0140] In some implementations, the organ transplant condition to be treated is selected from the group consisting of: graft-versus-host disease (GvHD), acute rejection, chronic rejection, transplant vascular disease, post-transplant lymphoproliferative disorder (PTLD), and delayed recovery of graft function.

[0141] In some implementation schemes, the infectious disease to be treated is selected from the group consisting of: human immunodeficiency virus (HIV) infection, hepatitis B, hepatitis C, tuberculosis, pneumococcal pneumonia, influenza (flu), malaria, dengue fever, Zika virus infection, Ebola virus disease, Chikungunya virus infection, Lyme disease, SARS-CoV-2, long COVID, and sepsis.

[0142] The term “prevention or suppression of the development of infectious diseases” as used in this article refers to preventing the occurrence of infectious diseases or delaying the onset of infectious diseases, or reversing the spread of existing infections.

[0143] As used herein, the term "activation" refers to a cellular state following sufficient cell surface junctions to induce significant biochemical or morphological changes. In the context of T cells, this activation refers to the state of T cells that have been adequately stimulated to induce cell proliferation. T cell activation can also induce the production and regulation of cytokines or the expression of cytolytic effector functions. In the context of other cells, the term implies the upregulation or downregulation of specific physicochemical processes.

[0144] As used herein, the term “cancer antigen” or “tumor antigen” encompasses tissue-specific differentiation antigens, tumor-specific common antigens and mutated tumor-specific and unique antigens, as well as any part, peptide or polypeptide of those antigens that can elicit an immune response from CD4+ or CD8+ T cells. Tumor antigens or cancer antigens recognized by CD8+ or CD4+ T cells can be classified into several categories (Wang, RF and Wang, HY, "Immune Targets and Neoantigens in 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", Journal of Experimental Medicine). Medicine) 180, 347-352 (1994); Schneider, J., Brichard, V., Boon, T., Meyer zum Buschenfelde, KH, and 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, Robbins (PF) and Rosenberg (SA), “Utilization of an alternative open reading frame of a normal gene ingenerating a novel human cancer antigen”, J. experimentalmedicine 183, 1131-1140 (1996); TRP-2 (Wang, Appella, E., Kawakami, Kang, X. and Rosenberg, “Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes”, J. experimental medicine 184, 2207-2216 (1996); Parkhurst et al., “Common HLA-A from melanoma antigen tyrosinase-associated protein 2 (TRP2)”. 0201 Identification of a shared HLA-A restriction epitope 0201-restricted T-cell epitope from themelanoma antigen tyrosinase-related protein 2 (TRP2), Cancer Research, 58, 4895-4901 (1998); Sun, Y. et al., "A novel HLA-A (0201-restricted T-cell epitope from themelanoma antigen tyrosinase-related protein 2 (TRP2) melanoma antigen" )0201 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 et al., Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression), Journal of Immunology 154, 3961-3968 (1995); Bakker, AB et al., Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. "Tumor-Infiltrating Lymphocytes", *J. Experimental Medicine*, 179, 1005-1009 (1994); Skipper, JC et al., "Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17 / gp100", *J. Immunology*, 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. Immunology*, 158, 1796-1802 (1997); 2) tumor-specific common antigens, which may or may not include 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-E", *J. Experimental Medicine*.) 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. Immunology*, 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, SL., Zeng, G., Schwartzentruber, DJ., and Rosenberg, SA.The study, titled "A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames," in the Journal of Immunology, 161, 3596-3606 (1998), found that this antigen is expressed in cancer and the testis, but not in other normal tissues. These antigens are also called cancer-testis (CT) antigens; 3) tumor-specific and unique antigens, which are mutant antigens, including CDK4 (Ap16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma), Science 269, 1281-1284 (1995) and catenin (Amutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes), Journal of Experimental Medicine (J. Experimental). (1996) , CASP-8 (Mandruzzato, S., Brasser, F., Andry, G., Born and 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)) antigen; and 4) overexpressed tumor antigens that are overexpressed in cancer cells compared to normal cells.

[0145] Both NY-ESO-1, encoded by the CTAG1B gene, and CT83 (also known as KK-LC-1), encoded by the CT83 gene, are cancer-testis (CT) antigens widely expressed in a variety of tumors, including lung and breast cancer. CD8+ T cells and antibodies have shown recognition of NY-ESO-1, and NY-ESO-1-specific TCRs have demonstrated clinical responses of 50 to 80% in several solid tumors, including melanoma, sarcoma, and myeloma. Although CD4+ T cells are important (HLA-DP4 is the most frequently expressed HLA II molecule in the general population, accounting for 70% positivity), HLA-DP4-restricted NY-ESO-1-specific TCRs have not yet been tested in a clinical setting. The inventors previously identified HLA-DR4 and HLA-DP4 restricted NY-ESO-1 epitopes (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, PF, Rosenberg, SA, and Wang, R.-F., "CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by common HLA-DP4 alleles: association with NY-ESO-1 antibody production"). NY-ESO-1 presented by a prevalent HLA-DP4 allele:association with NY-ESO-1 antibody production), Proceedings of the National Academy of Sciences of the United States of America (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 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 Research, 62, 3630-3635 (2002).

[0146] As disclosed in this paper, HLA-DP4-restricted NY-ESO-1 CD4+ T cells and TCRs were generated, and it was tested whether the combination of DP4-ESO-1 TCR-engineered T cells and A2-ESO-1 TCR-engineered T cells could produce stronger anti-tumor immunity than either of them alone.

[0147] Besides the CT antigen NY-ESO-1, CT83 (KK-LC-1) is highly expressed in 60-70% of breast cancers, particularly in TNBC, consistent with previous reports. Fukuyama et al., “Identification of a new cancer / germline gene, KK-LC-1, encoding an 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 et al., “CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer.” "Cancer", *Oncotarget* 6, 25356-25367, doi:10.18632 / oncotarget.4516 (2015). However, little is known about its immunogenicity, T cell epitopes, and homologous TCRs for T cell recognition of tumors. As disclosed in this article, antigen-specific CD4+ and CD8+ T cells were generated and used to identify HLA-A2-restricted CT83-specific TCRs (A2-CT83 TCRs) to determine whether CT83 can serve as an attractive target for TCR-T cell immunotherapy.

[0148] It has been demonstrated that CAR-T and TCR-T cell persistence is closely related to patient survival. Therefore, the regulation 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 may or may not include PD1, VHL, PPP2R2D, and epigenetic factors (which may or may not include Jmjd3 and LSD1)).

[0149] In some embodiments, the immunogenic peptides and epitopes contained in the tumor antigens of this disclosure are derived from the NY-ESO-1 and CT83 proteins, which are widely expressed in various types of cancer, including but not limited to breast cancer, lung cancer, and prostate cancer. Compared to low levels in normal cells, the tumor antigens of this invention are expressed at significantly high levels in tumor cells and testes.

[0150] In some embodiments, "tumor antigen" or "cancer antigen" is any part, 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.

[0151] As used in this article, the term "immunogenic peptides and epitopes" encompasses any epitope or fragment of the NY-ESO-1, CT83, HCMVpp65, and / or HCMV IE-1 proteins that serve as tumor antigens.

[0152] As used herein, the terms “fragment” or “part” mean any segment of a protein or gene that has at least 5 or 6 amino acids in the case of a protein fragment and at least 15 to 18 nucleotides in the case of a gene.

[0153] In one embodiment, the tumor antigen-specific T cell line of the present invention comprises all generated CD4+ or CD8+ T lymphocytes that immunely recognize tumor antigens presented by HLA-DP4 or HLA-A2 positive antigen-presenting cells.

[0154] As used in this article, the term "presentation" encompasses the procedure of transfecting DNA encoding the full length or any portion of a tumor antigen into antigen-presenting cells or loading a peptide encoding the full length or any portion of a tumor antigen onto antigen-presenting cells.

[0155] As used herein, the term “antigen-presenting cell” encompasses any natural or artificial cell line or cell that expresses a certain HLA molecule of interest on its cell surface. As used herein, the term “antigen” means 1) any molecule that can be specifically recognized in its whole or in fragments and is bound by the “idiotype” portion (antigen-binding region) of an mAb or its derivative; 2) contains a peptide sequence that can be bound by MHC and then, in the case of MHC presentation, can specifically bind to its homologous T-cell antigen receptor.

[0156] The term "HLA-DP4 positive" as used in this article encompasses the expression of HLA class II molecules DPA1 and DPB1 on the cell surface. Any natural or artificial cell line or cell of 04 (including all its subtypes).

[0157] The term "HLA-A2 positive" as used in this article encompasses the expression of HLA class I molecules A on the cell surface. Any natural or artificial cell line or cell of 02 (including all its subtypes).

[0158] The term "HLA-DR13 positive" as used in this article encompasses the expression of the HLA class II molecule DRB1 on the cell surface. 13 (including all its subtypes) any natural or artificial cell line or cell.

[0159] In one embodiment of the invention, at least two T cell receptors are derived from antigen-specific CD4+ or CD8+ T cell lines. The full-length α and β chains of the TCRs are cloned, respectively. The term "full-length" as used herein encompasses the human α-chain constant region (as a non-limiting example, TRAC, IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS) (Seq ID NO: 10), or the mouse α-chain constant region (trac) (SEQ ID NO: 10). NO:13)) fused α-chain variable region, or with human β-chain constant region type 2 (TRBC2, DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG)(Seq ID NO: 12), or a β-chain variable region fused from human β-chain constant region type 1 (TRBC1, DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF) (SEQ ID NO:11), or mouse β-chain constant region type 1 (trbc1) (SEQ ID NO:14), or mouse β-chain constant region type 2 (trbc2) (SEQ ID NO:15). In some embodiments, the constant region may have a sequence having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with 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 contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0160] In some embodiments, the chimeric TCR is a CT83-specific TCR having a chimeric α chain and a chimeric β chain. The chimeric α chain comprises a variable region of an HLA-A2-restricted CT83TCR α chain fused with the mouse α constant region or a variant thereof comprising SEQ ID NO:20, the variant having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:20 and / or having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions to SEQ ID NO:20, these substitutions may be conservative substitutions; the chimeric β chain comprises a variable region of an HLA-A2-restricted CT83TCR β chain fused with the mouse β constant region 2 or a variant thereof comprising SEQ ID NO:21 ...99% sequence identity with SEQ ID NO:20 and / or having 10 substitutions to SEQ ID NO:20, these substitutions may be conservative substitutions NO:21 has 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity and / or has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions to SEQ ID NO:21, which may be conservative substitutions.

[0161] In some embodiments, the chimeric TCR is a NY-ESO-1 specific TCR having a chimeric α chain and a chimeric β chain, the chimeric α chain comprising a polypeptide selected from: an HLA-A2-restricted NY-ESO-1 TCR (S2) α chain variable region fused to a mouse α constant region or a variant thereof of a polypeptide having SEQ ID NO:22, the variant having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:22 and / or having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions to SEQ ID NO:22, these substitutions may be conservative substitutions; and an HLA-A2-restricted NY-ESO-1 TCR (S5) α chain variable region fused to a mouse α constant region or a variant thereof of a polypeptide having SEQ ID NO:24 ... or 99% sequence identity with SEQ ID NO:22 and / or having 1, 2, 3, 4 SEQ ID NO:24 has 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity and / or has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions to SEQ ID NO:24, which may be conservative substitutions; the chimeric β chain is selected from the following: HLA-A2 restricted NY-ESO-1 TCR(S2)(G50A, A51E) β chain variable region fused with the mouse β constant region 2 containing SEQ ID NO:23 or a variant thereof, which variant has 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 has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions to SEQ ID NO:24. NO:23 has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, which may be conservative substitutions, as well as HLA-A2-restricted NY-ESO-1 TCR(S5)(G50A, A51E, A97L) β-chain variable regions fused with the mouse β-constant region 2 of SEQ ID NO:25 or its variants, which have 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:25 and / or have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions to SEQ ID NO:25.

[0162] 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 with 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 contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0163] The term “proliferation” as used in this article refers to growth or reproduction through the production of new cells.

[0164] The terms “purified” or “pure” refer to molecules isolated from other reactions or cellular components. The terms “substantially pure” or “substantially purified” refer to molecules with a purity of 85, 86, 87, 88, 89, 90, 91, 9, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0165] In another embodiment of the invention, the epitopes of tumor antigens that specifically interact with CD4+ T cell lines or TCRs to elicit a T cell immune response include, but are not limited to, 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), which are peptides / parts of the NY-ESO-1 protein containing 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). In some embodiments, the epitopes comprise variants containing one, two, or three conserved substitutions. NY-ESO-1 PEP161-180 (Seq ID NO:34) has been identified as a peptide with high affinity for HLA-DP4. Zeng et al., "Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules," *Journal of Immunology*, 165, 1153-1159 (2000). This peptide was used to generate peptide-stimulated CD4+ T cells that specifically recognized HLA-DP4-presented NY-ESO-1. Zeng, Wang, Robbins, Rosenberg, and Wang, "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," Proceedings of the National Academy of Sciences of the United States of America (PNAS) 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 maintaining an impaired immune response compared to full-length NY-ESO-1.(Zeng et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), doi:10.1073 / pnas.061507398 (2001)).

[0166] In another embodiment of the invention, the epitopes of tumor antigens that specifically interact with CD8+ T cell lines or TCRs to elicit a T cell immune response may include, but are not limited to, 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), CT83 PEP66-76 (ILNNFPHSIAR, Seq ID NO:40), CT83 PEP17-31 (VFWKYRRFQRNTGEM, SEQ ID NO:61), and CT83 PEP10-24 (SILCALIVFWKYRRF, SEQ ID NO:61). SEQ ID NO: 62), and CT83 PEP13-27 (CALIVFWKYRRFQRN, SEQ ID NO: 63), which are peptides / parts of the CT83 protein containing specific amino acids of the CT83 protein. In some embodiments, the epitope comprises a variant containing one, two, or three conserved substitutions.

[0167] In another embodiment of the invention, the tumor antigen epitope that specifically interacts with CD4+ T cell lines or TCRs to elicit a T cell immune response includes, but is not limited to, the pp65 peptide (495-503) (NLVPMVATV, SEQ ID NO:26). pp65 is an HCMV protein and an antigen expressed by glioblastoma cells. In some embodiments, the epitope comprises a variant containing one, two, or three conserved substitutions.

[0168] In another embodiment of the invention, the tumor antigen epitope that specifically interacts with CD4+ T cell lines or TCRs to elicit a T cell immune response includes, but is not limited to, IE-1 peptides 316-324 (VLEETSVML, SEQ ID NO:31). IE-1 is an HCMV protein and an antigen expressed by glioblastoma cells. In some embodiments, the epitope comprises a variant containing one, two, or three conserved substitutions.

[0169] The term "self-cleaving peptide" includes, but is not limited to, a P2A sequence (RAKRSGSGATNFSLLKQAGDVEENPGP, Seq ID NO:51) located between two proteins, and which can self-cleave to separate the two proteins. Ryan, MD; King, AM; and Thomas, GP, "Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence," *Journal of General Virology*, 72(Pt 11), 2727-2732, doi:10.1099 / 0022-1317-72-11-2727 (1991).

[0170] As used herein, the term "stimulation" refers to a primary response induced by the connection of cell surface portions. For example, in the case of receptors, such stimulation requires receptor connection and subsequent signal transduction events. Regarding stimulation of T cells, this stimulation refers to the connection of T cell surface portions, which in one embodiment subsequently induces signal transduction events, which may or may not include binding to the TCR / CD3 complex. Furthermore, the stimulating event can activate the cell and upregulate or downregulate the expression or secretion of molecules, which may or may not include downregulation of TGF-β. Therefore, even in the absence of direct signal transduction events, the connection of cell surface portions can lead to reorganization of the cytoskeleton or aggregation of cell surface portions, each of which can be used to enhance, modify, or alter subsequent cellular responses.

[0171] As used herein, the term "vector" includes, but is not limited to, pMSGV, pMSCV, pFU3W, or any other vector used for inserting DNA into living cells.

[0172] In another embodiment of the invention, a vector is provided for inserting cDNA encoding a TCR α chain and / or a TCR β chain. In some embodiments, the translation product of the vector comprises at least one α-chain variable region binding to at least one α-constant region and / or at least one β-chain variable region binding to at least one β-constant region, linked by a self-cleaving peptide. These vectors are used to deliver the insert to uninfected T cells via viral transduction.

[0173] As used herein, the term "viral transduction" encompasses methods of generating recombinant viruses (including but not limited to retroviruses, lentiviruses, adeno-associated viruses, or other suitable viruses) in host cells and using these recombinant viruses, which contain 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 proliferating cells. The term "target cell" includes, but is not limited to, CD4+ T cells, CD8+ T cells, tumor cells, etc.

[0174] In one embodiment, the invention also provides a host cell transfected or transduced with a vector comprising DNA encoding a TCR region or strand according to any of the foregoing aspects or any aspect or embodiment disclosed herein, such as a TCR α-strand variable region binding to an α-constant region and a β-strand variable region binding to a β-constant region, which are linked by a P2A sequence (SEQ ID NO: 51) for use in viral generation and TCR delivery to uninfected T cells.

[0175] In one embodiment, the TCR is a chimeric TCR comprising a TCR variable region fused with a modified or nonhuman constant region. In some embodiments, the chimeric TCR comprises a cancer antigen-specific TCR variable region of any embodiment disclosed herein fused with a nonhuman (e.g., mouse) TCR constant region. For example, the chimeric TCR may comprise a variable region that may or may not comprise a CT83 TCR variable region fused with a nonhuman (e.g., mouse) TCR constant region, such as an NY-ESO-1 TCR variable region, a pp65 TCR variable region, or an IE-1 TCR. Among other features, the chimeric TCR reduces mismatch between the chimeric TCR and the endogenous TCR in transduced T cells. For example, the chimeric CT83 TCR (MC) reduces mismatch between the chimeric CT83 TCR (MC) and the endogenous TCR (HC) in transduced cells. For example, chimeric CT83 TCR reduces mismatches between chimeric CT83 TCR (MC) and endogenous TCR (HC), or chimeric NY-ESO-1 TCR reduces or minimizes mismatches between chimeric NY-ESO-1 (MC) and endogenous TCR (HC). As another example, chimeric pp65 TCR reduces or minimizes mismatches between chimeric pp65 (MC) and endogenous TCR (HC). And as yet another example, chimeric IE-1 TCR reduces or minimizes mismatches between chimeric IE-1 (MC) and endogenous TCR (HC).

[0176] The term "host cell" includes, but is not limited to, cells from the PG-13 cell line, Phoenix-Eco cell line, Phoenix-Ampho cell line, 293GP cell line, or other suitable cell lines that can assemble the viral genome intracellularly, package the virus with capsular proteins, and secrete mature viruses extracellularly.

[0177] In one embodiment, this disclosure also relates to protocols and strategies for prolonging the persistence of TCR-T cells or CAR-T cells by directly manipulating CAR signaling domains or by knocking down / removing negative signaling molecules. In some embodiments, this disclosure also relates to methods for enhancing the persistence of TCR-T cells or CAR-T cells by expressing chemokine receptors and / or knocking down shRNA in a CAR construct. In some embodiments, treatment with any engineered TCR-T cells of this disclosure, wherein the T cells contain and / or express signaling domains and / or have negative signaling molecule knockdown, can unexpectedly reduce relapse or cancer recurrence following initial treatment and initial reduction of cancer / tumor burden. In some embodiments, treatment with any engineered TCR-T cells of this disclosure, wherein the T cells contain and / or express signaling domains and / or have negative signaling molecule knockdown, can unexpectedly reduce relapse of treated inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, infectious diseases, and / or age-related symptoms.

[0178] In some embodiments, this disclosure provides an engineered immune cell (TCR-T cell or CAR-T cell) containing knockdown, knockout, or inactivation mutations in one or more endogenous genes, wherein the endogenous genes are selected from the group consisting of the following negative regulatory factors (which may include or exclude): ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1 NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T cells) Lymphocyte antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic regulators (which may or may not include JMJD3 and LSD1). In some embodiments, negative signaling molecules / regulators selected for knockdown, knockout, or inactivation of mutations include, for example, IDO (including IDO1 and IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-H3, VHL, PPP2R2D, and epigenetic regulators (which may or may not include JMJD3 and LSD1). In some embodiments, the negative signaling molecules or regulatory factors selected for knocking down, knocking out, or inactivating the mutation are, for example, PD-1, VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1).

[0179] In some embodiments, knockout, knockdown, or inactivation mutations of any negative signaling molecule, and / or expression of chemokine receptors and / or shRNA knockout, can be used in CAR-T cells or TCR-T cells, or CAR constructs, in any of the foregoing aspects or embodiments described herein. For example, CAR-T cells undergoing the knockout, knockdown, or inactivation mutations and / or chemokine receptor expression and shRNA knockout can express one or more CARs described herein, comprising: at least one extracellular binding domain containing at least one antigen recognition motif; at least one hinge domain and at least one transmembrane domain or fragments or variants thereof; and at least one intracellular signaling domain.

[0180] In some embodiments, this disclosure provides an engineered immune cell (TCR-T cell or CAR-T cell) containing knockdown, knockout, or inactivation mutations in one or more endogenous genes selected from the group consisting of the following negative regulatory factors (which may include, exclude, or select from): ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR 4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T cells) Lymphocyte antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic regulators (which may or may not include JMJD3 and LSD1). In some aspects, negative signaling molecules / regulators selected for knockdown, knockout, or inactivation of mutations include, for example, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-H3, VHL, PPP2R2D, and epigenetic regulators (which may or may not include JMJD3 and LSD1). In some embodiments, the negative signaling molecules or regulatory factors selected for knocking down, knocking out, or inactivating the mutation are, for example, PD-1, VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1).

[0181] In one embodiment, this document also discloses a nucleic acid encoding siRNA, such as shRNA, for knocking down genes to enhance the antitumor activity of TCR-transduced T cells in vivo. The nucleic acid sequence of the shRNA targets negative signaling molecules of the immune system, such as immune checkpoint proteins and / or immunosuppressive proteins. In some embodiments, one or more genes are targeted via siRNA or shRNA nucleic acid molecules to achieve knockdown of target gene expression, wherein the one or more genes encode negative regulatory factors, which may include, exclude, or be selected from the group consisting of: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2. NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T cells) Lymphocyte antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1). The siRNA or shRNA molecule comprises approximately 20–30 nucleotides and can bind to the RNA sequence encoded by the DNA sequence of the selected gene. In some embodiments, the shRNA targets include, but are not limited to, programmed death protein PD-1 (SEQ ID NO: 7), von Hippel-Lindau tumor suppressor VHL (SEQ ID NO: 8), and / or protein phosphatase 2 regulatory subunit Bδ (PPP2R2D) (SEQ ID NO: 9).In some aspects, the targetable PPP2R2D mRNA sequence may include or exclude some or all of the following sequences: PPP2R2D transcript variant 1 (SEQ ID NO: 18) and / or PPP2R2D transcript variant 3 (SEQ ID NO: 19). In some embodiments, nucleic acids encoding antisense RNA or antisense DNA may also be used to knock down genes or reduce the expression of genes encoding negative signaling molecules. In some embodiments, the nucleic acids in any of the foregoing aspects or embodiments described herein may further comprise any of the aforementioned nucleic acids encoding siRNA / shRNA.

[0182] In some embodiments, the knockout or inactivation of target gene expression is achieved using a CRISPR / Cas9 system. This system includes a single-stranded guide RNA (sgRNA) molecule and a Cas endonuclease, and is capable of reducing or knocking out the expression of one or more target genes. The target genes may include, exclude, or be selected from the group consisting of: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), L AG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQU IN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A.The sgRNA molecule contains a target RNA sequence that can bind to target DNA sequences selected from the group consisting of the following negative regulatory factors: ANKRD1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRP1, PBRM1. PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T cells) Lymphocyte antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1).

[0183] In another embodiment of the invention, a sequence of shRNA, antisense RNA, or DNA specifically knocking down a metabolic gene is provided, existing in a 21-base-pair stem structure, for enhancing the antitumor activity of TCR-based or CAR-based therapies in vivo. The TCR can be engineered in conjunction with the shRNA to improve the migration and persistence of T cells in vivo and enhance antitumor activity by knocking down the target gene. As used herein, "knock down" refers to reducing gene expression, for example, by inducing mRNA degradation or blocking RNA expression to reduce the protein expression level of a target gene. "Metabolic genes" as used herein include, but are not limited to, PD-1, VHL, and PPP2R2D.

[0184] This application cites numerous references. The entire contents of these references are incorporated herein by reference in their entirety to more fully describe the prior art in the field to which this disclosure pertains. Each of these references is also incorporated herein by reference individually and verbatim. Furthermore, the entire contents of PCT Publication No. WO / 2021 / 263211 and its corresponding U.S. Patent Application 18 / 002,969 are also incorporated herein by reference as if reproduced verbatim.

[0185] This invention encompasses CD4+ or CD8+ T lymphocytes that immunely recognize tumor antigens under the restriction of an HLA class II or I molecule. The invention further encompasses at least one T-cell receptor derived from the aforementioned CD4+ or CD8+ T lymphocytes. The T-cell receptor can be delivered to uninfected CD4+ or CD8+ T lymphocytes that do not have an immune response to the aforementioned tumor antigens, and alter the function of those CD4+ or CD8+ T lymphocytes to specifically recognize and react with the aforementioned tumor antigens. This reaction between the tumor antigen and the transduced T-cell receptor leads to a T-cell response against human cancers containing the aforementioned tumor antigens, and contributes to the prevention, elimination, or reduction of human cancers.

[0186] method Immunoassay and fluorescent dyes Various useful immunoassay procedures have been described in the scientific literature, which may or may not include, 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, pp. 27.1-27.20 (1986), each incorporated herein by reference in its entirety, particularly for their teachings on immunoassay methods. An immunoassay, in its simplest and most direct sense, is a binding assay involving the interaction between an antibody and an antigen. Many types and forms of immunoassays are known and are suitable for detecting publicly known biomarkers. Examples of immunoassays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunobead capture assay, Western blotting, dot blot, gel migration assay, flow cytometry, protein array, multiple bead array, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery / localization after photobleaching (FRAP / FLAP).

[0187] Typically, immunoassays involve contacting a sample suspected of containing a molecule of interest (which may or may not include the disclosed biomarker) with an antibody against the molecule of interest, or contacting an antibody against the molecule of interest (which may or may not include the disclosed biomarker) with a molecule that can be bound by the antibody, as appropriate, under conditions that effectively allow for the formation of immune complexes. Contacting the sample with the antibody against the molecule of interest or with a molecule that can be bound by the antibody against the molecule of interest for a sufficient time under effective conditions to allow for the formation of immune complexes (primary immune complexes) typically involves simply contacting the molecule or antibody with the sample and incubating the mixture for a sufficient time to allow the antibody to form an immune complex with (i.e., bind to) any molecule that the antibody can bind to (e.g., an antigen). In many forms of immunoassays, the sample-antibody composition may or may not include tissue sections, ELISA plates, dot blots, or protein blots, which can then be washed to remove any non-specifically bound antibody species, allowing only the detection of those antibodies that specifically bind within the primary immune complex.

[0188] Immunoassays may include methods for detecting or quantifying amounts of molecules of interest in a sample (which may or may not include disclosed biomarkers or antibodies thereof), and these methods typically involve detecting or quantifying any immune complexes formed during the binding process. Typically, the detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are typically based on the detection of a label or marker, which may or may not include any radioactive, fluorescent, biological, or enzyme-labeled tag or any other known marker.

[0189] As used herein, labels may include fluorescent dyes, members of binding pairs that may or may not include biotin / streptavitin, metals (e.g., gold), or epitope tags that can specifically interact with a detectable molecule, which may or may not be included by generating a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also referred to herein as fluorescent pigments and fluorophores) and enzymes (e.g., horseradish peroxidase) that react with a colorimetric substrate. Fluorescent dyes are generally preferred in the practice of this application because they can be detected in very low amounts. Furthermore, in the case where multiple antigens react with a single array, each antigen may be labeled with a different fluorescent compound for simultaneous detection. The labeled spots on the array are detected using a fluorometer, and the presence of a signal indicates that the antigen has bound to a specific antibody.

[0190] A fluorophore is a luminescent compound or molecule. 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-carboxynaphthalenefluorescein; 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-4-1-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acrid Orange; Acrid Red; Acrid Yellow; Acriflavin; Acriflavin Folgen's SITSA; Jellyfish luminescent protein (photoprotein); AFP - Autofluorescent protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350 TM Alexa Fluor 430 TM Alexa Fluor 488 TM Alexa Fluor 532 TMAlexa Fluor 546 TM Alexa Fluor 568 TM Alexa Fluor 594 TM Alexa Fluor 633 TM Alexa Fluor 647 TM Alexa Fluor 660 TM Alexa Fluor680 TM Alizarin aminocarboxylic acid complexing agent; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aniline Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG TM CBQCA; ATTO-TAG TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; β-lactamase; BFP blue-shifted GFP (Y66H); Blue fluorescent protein; BFP / GFP FRET; Bimane; Bisbendiamide; Bisbendiamide (Hoechst); BisBTC; Blancophor FFG; Blancophor SV; BOBO TM -1; BOBO TM -3; Fluoroboropyrrole 492 / 515; Fluoroboropyrrole 493 / 503; Fluoroboropyrrole 500 / 510; Fluoroboropyrrole; 505 / 515; Fluoroboropyrrole 530 / 550; Fluoroboropyrrole 542 / 563; Fluoroboropyrrole 558 / 568; Fluoroboropyrrole 564 / 570; Fluoroboropyrrole 576 / 589; Fluoroboropyrrole 581 / 591; Fluoroboropyrrole 630 / 650-X; Fluoroboropyrrole 650 / 665-X; Fluoroboropyrrole 665 / 676; Fluoroboropyrrole Fl; Fluoroboropyrrole FL ATP; Fluoroboropyrrole Fl-ceramide; Fluoroboropyrrole R6G SE; Fluoroboropyrrole TMR; Fluoroboropyrrole TMR-X conjugate; Fluoroboropyrrole TMR-X, SE; Fluoroboropyrrole TR; Fluoroboropyrrole TR ATP; Fluoroboropyrrole TR-XSE; BO-PRO TM-1; BO-PRO TM -3; Brilliant Yellow FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Deep Red; 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; calcium fluorescent white; carboxyl-X-rhodamine (5-ROX); cascade blue TM ; Cascade yellow; Catecholamines; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromin A; Chromin A; CL-NERF; CMFDA; Coelentrin; Coelentrin cp; Coelentrin f; Coelentrin fcp; Coelentrin h; Coelentrin hcp; Coelentrin ip; Coelentrin n; Coelentrin O; Coumarin phalloidin; C-phycocyanin; CPM I methylcoumarin; CTC; CTC formazan; Cy2 TM Cy3.1 8; Cy3.5 TM Cy3 TM Cy5.1 8; Cy5.5 TM Cy5 TM Cy7 TM; Cyan GFP; Ring AMP fluorescent sensor (FiCRhR); Dabcyl; Dansyl; Dansyl sulfonamide; Dansyl cadaverine; Dansyl chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DilC18(3)); I-dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DilC18(7)); DM-NERF (high pH); DNP; Dopamine; Red fluorescent protein; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erysmine; Erysmine ITC; Ethidium bromide; Ethylphenidium bromide dimer-1 (EthD-1); Basic orange (Euchrysin); EukoLight; Europium (111) chloride; EYFP; Solid Blue; FDA; Folgen (pararosaniline); FIF (formaldehyde-induced fluorescence); FITC; Flazo orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluorescent emerald; Fluorescent gold (hydroxystilbene); Fluorescent ruby; FluorX; FM 1-43 TM FM 4-46; Fura Red TM (High pH); Fura Red TM / 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 Redshift (rsGFP); GFP Wild-type 'Non-UV Excited' (wtGFP); GFP Wild-type, UV Excited (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hearst 33258; Hearst 33342; Hearst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (Fluorescent Gold); Serotonin; Indo-1, High Calcium; Indo-1, Low Calcium; Indo-dicarbonylcyanine (DiD); Indo-tricarbonylcyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Ricofos Fluorescent Whitening Agent PAF; Ricofos Fluorescent Whitening Agent SF; Ricofos Fluorescent Whitening Agent WS; Rhodamine; Rhodamine B; Calcein / Ethylphenidate Dimer; LOLO-1; LO-PRO-1; Fluorescent Yellow; Lysosome Blue; Lysosome Blue-White; Lysosome Green; Lysosome Red; Lysosome Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Naphthalene Red (Root Bark Red B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Dock Blue; Maxilon Brilliant Yellow 10 GFF; Methylsiloxane Brilliant Yellow 8GFF; Phenylene Cyanide; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange FM; Mitotracker Red FM; Mitotracker Red FM; Mitramycin; Monobromodiphenylmethane; mBBr-GSH; Monochlorobimane; MPS (Methyl Green Phenoyl Chloride); NBD; NBD Amine; Nile Red; Nitrobenzoxadiazole; Norepinephrine; Nucleotide Red; Riboflavin; Nylosan E8G; Oregon Green TM Oregon Green TM 488; Oregon Green TM 500; Oregon Green TM514; Pacific Blue; Pararosaniline (Folgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Root Bark Red B (Naphthalene Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Floating Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primrose Extract; Propidium Iodide (Pl); PyMPO; Pyrene; Pyrozine; Pyrozine B; Pyrozine Brilliant Yellow 7GF; QSY 7; Nitrogen Mustard Quinacrine; Halogen Resin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Premium Rhodamine B; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidin; Rhodamine Red; Rhodamine WT; Rose Red; R-phycocyanin; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red I B; Sevron Orange; Sevron Yellow L; sgBFP TM (Superluminescent BFP); sgGFP TM(Superluminescent GFP); SITS (Primula styrax; Styrax 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); Styrax; Azorhodamine B and C; Extra grade azorhodamine; 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 isothiocyanate (TRITC); Texas Red TM Texas Red-X TM Conjugates; Thiazolamine iodide (DiSC3); Thiazide Red R; Thiazol Orange; Thiamine 5; Thiamine S; Thiamine TON; Thiolysis; 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; Pure Blue; Tru Red; Ultralite; Sodium Fluorescein B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylenol Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1; YOYO-3; Sybr Green; Thiazole Orange (chelating dye); Semiconductor nanoparticles, which may or may not include quantum dots; or cage-like fluorophores (which can be activated by light or other electromagnetic energy sources), or combinations thereof.

[0191] Modifier units, which may or may not include radionuclides, may be incorporated into or directly attached to any compound described herein by halogenation. Examples of radionuclides that may be used 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. Alternatively, radionuclides may be attached to a linker group or bound via a chelating group, and then attached to the compound directly or via a linker. Examples of radionuclides that may be used in this respect 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, which may or may not include these, are commonly used in the radiopharmaceutical industry.

[0192] Radiolabeled compounds can be used as imaging agents to diagnose neurological disorders (e.g., neurodegenerative diseases) or psychotic symptoms in mammals (e.g., humans) or to track the progression or treatment of such diseases or symptoms. The radiolabeled compounds described herein can be readily used in conjunction with imaging techniques, which may or may not include positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0193] Labeling can be direct or indirect. In direct labeling, the detection antibody (an antibody against the molecule of interest) or the detection molecule (a molecule that can be bound to the molecule of interest by the antibody) includes the 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 the antibody against the molecule of interest, respectively. In indirect labeling, an additional molecule or portion is brought into contact with or generated at a site on the immune complex. For example, a signal-generating molecule or portion, which may or may not include an enzyme, may attach to or bind to the detection antibody or detection molecule. The signal-generating molecule can then generate a detectable signal at a site on the immune complex. For example, when provided with a suitable substrate, an enzyme can produce a visible or detectable product at a site on the immune complex. ELISA uses this type of indirect labeling.

[0194] As another example of indirect labeling, an additional molecule (which may be referred to as a binding agent) can be contacted with the immune complex. This additional molecule may bind to the molecule of interest or an antibody (primary antibody) against the molecule of interest. The molecule of interest may or may not include a secondary antibody against the primary antibody. The additional molecule may have a label or signal-generating molecule or portion thereof. The additional molecule can be an antibody, and therefore may be referred to as a secondary antibody. The binding of the secondary antibody to the primary antibody can form a so-called sandwich between the primary antibody and the molecule of interest. The immune complex can be contacted with the labeled secondary antibody for a sufficient time under effective conditions to allow the formation of a secondary immune complex. The secondary immune complex can then typically be washed to remove any non-specifically bound labeled secondary antibody, and the remaining label in the secondary immune complex can then be detected. The additional molecule may also be or include one of a pair of molecules or portions that can bind to each other, which may or may not include a biotin / avidin pair. In this mode, the detecting antibody or detecting molecule should include the other member of that pair.

[0195] Other modes of indirect labeling include the detection of primary immune complexes via a two-step process. For example, a molecule (referred to as a first binder) that may or may not include an antibody with binding affinity for the molecule of interest or its corresponding antibody can be used to form a secondary immune complex, as described above. After washing, the secondary immune complex can be brought into contact again with another molecule (referred to as a second binder) with binding affinity for the first binder under effective conditions for a period sufficient to allow for the formation of another immune complex (thus forming a tertiary immune complex). The second binder can be linked to a detectable label or signal-generating molecule or portion, allowing for the detection of the thus formed tertiary immune complex. This system can provide signal amplification.

[0196] Immunoassays involving the detection of substances, which may or may not include proteins or antibodies against specific proteins, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo assays. Label-free assays are generally diagnostic tools for determining the presence or absence of specific proteins or antibodies against specific proteins in a sample. Protein separation methods can also be used to assess the physical properties of proteins, which may or may not include size or net charge. Capture assays are generally more suitable for quantitatively assessing the concentration of specific proteins or antibodies against specific proteins in a sample. Finally, in vivo assays can be used to assess the spatial expression patterns of substances, i.e., substances that can be found in subjects, tissues, or cells.

[0197] If the concentration is sufficient, molecular complexes ([Ab-Ag]n) formed through antibody-antigen interactions are visible to the naked eye, but smaller amounts of these complexes can also be detected and measured due to their ability to scatter light beams. The formation of these complexes indicates the presence of two reactants, and in immunoprecipitation assays, a constant concentration of reagent antibody is used to measure the specific antigen ([Ab-Ag]n), and a reagent antigen is used to detect the specific antibody ([Ab-Ag]n). If the reagent is pre-coated onto cells (as in hemagglutination assays) or onto very small particles (as in latex agglutination assays), “clumps” of coated particles can be seen at much lower concentrations. Various assays based on these fundamental principles are commonly used, including Ouchterlony immunodiffusion assays, rocket immunoelectrophoresis, and immunoturbidimetric and turbidity assays. The main limitations compared to assays using markers may include, but are not limited by, sensitivity limitations (lower detection limits), and in some cases, very high concentrations of analyte can actually inhibit complex formation, requiring more complex safety measures. Some of these Group 1 assays trace back to the discovery of antibodies, and none of them have a practical "label" (e.g., Ag-enz). Other types of label-free immunoassays rely on immunosensors, and various instruments that can directly detect antibody-antigen interactions are now commercially available. Most rely on generating evanescent waves on the sensor surface with immobilized ligands, which allows for continuous monitoring of ligand binding. Immunosensors facilitate the study of kinetic interactions and, with the advent of lower-cost dedicated instruments, may find widespread application in immunoassays in the future.

[0198] Detecting specific proteins using immunoassays may involve separating proteins via electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their migration rate depends on the strength of the field; on the net charge, size, and shape of the molecules; and also on the ionic strength, viscosity, and temperature of the medium in which the molecules move. Electrophoresis is a simple, rapid, and highly sensitive analytical tool. It is used to analytically study the properties of individual charged substances and as a separation technique.

[0199] Typically, samples are run in a supporting matrix that may or may not include paper, cellulose acetate, starch gel, agarose, or polyacrylamide gel. The matrix inhibits 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 storage. Additionally, the most commonly used supporting matrices, agarose and polyacrylamide, provide methods for separating molecules by size because they are porous gels. Porous gels act as sieves, impeding or, in some cases, completely blocking the movement of large molecules while allowing smaller molecules to migrate freely. Because diluted agarose gels are generally more rigid and easier to handle than polyacrylamide gels of the same concentration, agarose is used to separate larger macromolecules, which may or may not include nucleic acids, large proteins, and protein complexes. Polyacrylamide, which is easily handled and prepared at higher concentrations, is used to separate most proteins and small oligonucleotides that require impedimentation by small gel pore sizes.

[0200] 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 carry a net negative charge and migrate towards the anode in an electric field. Below their isoelectric point, proteins carry a positive charge and migrate towards the cathode. Furthermore, the net charge carried by a protein is independent of its size; that is, the charge carried per unit mass (or length, given that proteins and nucleic acids are linear macromolecules) varies from protein to protein. Therefore, at a given pH, under non-denaturing conditions, the electrophoretic separation of proteins is determined by both molecular size and charge.

[0201] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "coating" their backbone, and SDS binds to proteins with considerable specificity at a mass ratio of 1.4:1. This imparts a negative charge to the peptide proportional to its length. Furthermore, the reduction of disulfide bonds in proteins (denaturation) is typically required before the proteins can assume the random coil conformation desired for size separation; this is accomplished using 2-mercaptoethanol or dithiothreitol (DTT). Therefore, in denaturing SDS-PAGE separation, migration is determined not by the intrinsic charge of the peptide, but by its molecular weight.

[0202] Molecular weight determination is accomplished by SDS-PAGE of proteins with known molecular weights and the protein to be characterized. There is a linear relationship between the logarithm of the molecular weight of SDS-denatured peptides or native nucleic acids and their relative molecular weight (Rf). Rf is calculated as the ratio of the distance the molecule migrates to the distance the labeled dye front migrates. A simple method for determining relative molecular weight (Mr) by electrophoresis is to plot a standard curve of the migration distance of a known sample against log10MW, and then read the logMr of the sample after measuring the migration distance on the same gel.

[0203] In two-dimensional electrophoresis, proteins are first fractionated based on one physical property, and then fractionated again based on another physical property. For example, isoelectric focusing can be used for the first dimension, conveniently performed in tubular gels, while SDS electrophoresis in slab gels can be used for the second dimension. An example of the procedure is O'Farrell, PH, "High Resolution Two-Dimensional Electrophoresis of Proteins," *J. Biol. Chem.* 250:4007-4021 (1975), whose teachings on two-dimensional electrophoresis methods are incorporated herein by reference in their entirety. Other examples include, but are not limited to, those found in Anderson (L.) and Anderson (NG.), “High-resolution two-dimensional electrophoresis of human plasma proteins,” *Proceedings of the National Academy of Sciences* 74:5421-5425 (1977); Ornstein (L.), “Discelectrophoresis,” *Annals of the New York Academy of Sciences* 121:321349 (1964), each of whose teachings on electrophoretic methods are incorporated herein by reference in their entirety; and Laemmli (UK), “Cleavage of structural proteins during the assembly of the head of bacteriophage.” The paper "T4", published in *Nature* 227:680 (1970), discloses a discontinuous system for the degradation of proteins denatured by SDS, the teachings of which on electrophoretic methods are incorporated herein by reference in their entirety. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Therefore, degradation gels and stacking gels are prepared in Tris-HCl buffers (of varying concentrations and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.

[0204] An example of an immunoassay using electrophoresis as anticipated in current methods is Western blotting. Western blotting, or immunoblotting, allows for the determination of the molecular weight of proteins and the measurement of the relative amounts of proteins present in different samples. Detection methods include chemiluminescence and colorimetric detection. Standard methods for Western blotting analysis can be found, for example, in DMBollag et al., *Protein Methods* (2nd edition, 1996), and E. Harlow and D. Lane, *Antibodies, a Laboratory Manual* (1988), and U.S. Patent 4,452,901, each of which contains teachings on Western blotting methods that are incorporated herein by reference in their entirety. Typically, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a special absorbent paper, such as nitrocellulose, although other types of paper or membranes may be used. The proteins retain the same separation pattern on the gel. The blot is incubated with a plain protein (which may or may not include milk proteins) to bind any remaining sticky sites on the nitrocellulose. Then, an antibody that can bind to its specific protein is added to the solution.

[0205] Indirect enzyme immunoassay techniques, typically using chromogenic substrates (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates, allow for easy observation of the attachment of specific antibodies to specific fixed antigens. Other detection possibilities include the use of fluorescent or radioactive isotope labeling (e.g., fluorescein, etc.). 125 I). Probes used to detect antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal protein A (binding IgG), or probes against biotinylated primary antibodies (e.g., conjugated avidin / streptomycin).

[0206] The power of this technology lies in its ability to simultaneously detect specific proteins through both their antigenicity and molecular weight. Proteins are first separated by mass separation in SDS-PAGE, followed by specific detection in the immunoassay step. Therefore, protein standards (stepwise) can be run simultaneously to approximate the molecular weight of the protein of interest in heterogeneous samples.

[0207] Gel migration assays, or electrophoretic mobility shift analysis (EMSA), can be used to qualitatively and quantitatively detect the interaction between DNA-binding proteins and their homologous DNA recognition sequences. Exemplary techniques are described in Ornstein, “Disc electrophoresis - I: Background and theory,” *Annals of the New York Academy of Sciences*, 121:321-349 (1964), and Matsutia, PT., and D.R. Burgess, “SDS microslab linear gradient polyacrylamide gel electrophoresis,” *Analytical Biochemistry*, 87:386-396 (1987), each of whose teachings on gel migration assays are incorporated herein by reference in their entirety.

[0208] In a typical gel migration assay, purified proteins or crude cell extracts can be mixed with labeled (e.g., ) 32 P-labeled DNA or RNA probes are incubated together, followed by separation of the complex from the free probe using a non-denaturing polyacrylamide gel. The complex migrates more slowly through the gel compared to unbound probes. Depending on the activity of the binding protein, the labeled probes can be double-stranded or single-stranded. To detect DNA-binding proteins, which may or may not include transcription factors, purified or partially purified proteins or nuclear cell extracts can be used. To detect RNA-binding proteins, purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of DNA or RNA-binding proteins to a putative binding site is determined by a competition experiment using DNA or RNA fragments or oligonucleotides containing the binding site of the protein of interest, or other unrelated sequences. Differences in the nature and strength of the complexes formed in the presence of specific and non-specific competitors allow for the identification of specific interactions.

[0209] Gel migration methods may include detecting proteins in a gel using, for example, COOMASSIE (Imperial Chemicals Industries, Ltd) blue staining in colloidal form, which may or may not include a polyacrylamide electrophoresis gel. 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 incorporates its teachings on gel migration methods in their entirety by reference. In addition to the conventional protein assay methods mentioned above, U.S. Patent 5,424,000 describes a combined cleaning and protein staining composition, the teachings of which on gel migration methods are incorporated herein by reference in their entirety. Solutions may include phosphoric acid, sulfuric acid, and nitric acid, as well as an acid purple dye.

[0210] Radioimmunoprecipitation assay (RIPA) is a sensitive assay for detecting specific antibodies in serum using radiolabeled antigens. The antigen is reacted with serum, and then precipitated using a special reagent, which may or may not include agarose beads such as protein A. The bound radiolabeled immunoprecipitate is then typically analyzed by gel electrophoresis. RIPA is commonly used as a confirmatory test for the presence of HIV antibodies. RIPA is also known in the art as a farin assay, precipitin assay, radioimmunoprecipitin assay; radioimmunoprecipitation analysis; and radioimmunoprecipitation assay.

[0211] While the aforementioned immunoassays utilizing electrophoresis to separate and detect specific proteins of interest allow for the assessment of protein size, they are not very sensitive for assessing protein concentration. However, immunoassays combined with methods for detecting proteins on a support or antibodies specific to proteins are also considered, wherein the protein or protein-specific antibody is bound to a solid support (e.g., tubes, wells, beads, or cells) to capture the antibody or protein of interest from the sample, respectively. Examples of such immunoassays include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture.

[0212] Radioimmunoassay (RIA) is a classic quantitative assay for detecting antigen-antibody reactions. It uses radiolabeled substances (radioligands) to measure the binding of unlabeled substances to specific antibodies or other receptor systems, either directly or indirectly. RIA is used, for example, to detect hormone levels in the blood without the need for bioassays. It can also measure non-immunogenic substances (e.g., haptens) if conjugated to a larger carrier protein capable of inducing antibody formation (e.g., bovine gamma globulin or human serum albumin). RIA involves mixing a radioactive antigen (typically a radioactive isotope, 125I or 131I, is used because iodine atoms are readily introduced into tyrosine residues in proteins) with an antibody targeting that antigen. The antibody is typically 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 determined. Initially, the radioactive antigen binds to the antibody. When cold antigen is added, the two antigens compete for antibody binding sites—at higher concentrations of cold antigen, more cold antigen binds to the antibody, displacing the radioactive variant. The bound antigen in solution is separated from the unbound antigen, and binding curves are plotted using their respective radioactivity. This technique is both highly sensitive and specific.

[0213] Enzyme-linked immunosorbent assay (ELISA), or more generally EIA (enzyme immunoassay), is an immunoassay that detects antibodies specific to proteins. In this assay, the detectable label bound to an antibody-binding reagent or an antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts by producing a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or visual means. Enzymes that can be used to detectably label reagents 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, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucosyl amylase, and acetylcholinesterase.

[0214] Variations of ELISA technology 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 may or may not include wells in a polystyrene microtiter plate. A test composition suspected of containing a labeled antigen can then be added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a second antibody specific to the target protein, which is conjugated 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 to the second antibody, wherein the third antibody is conjugated to a detectable label.

[0215] Another variation is competitive ELISA. In competitive ELISA, the test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive material in the sample can be determined by mixing the sample with the known labeled material before or during incubation with the coated wells. The presence of reactive material in the sample reduces the amount of labeled material available for binding to the wells, thus reducing the final signal.

[0216] Regardless of the form used, ELISA shares certain common characteristics, including or excluding coating, incubation or conjugation, washing to remove non-specifically bound material, and detection of bound immune complexes. Antigens or antibodies can be attached to a solid support, which may or may not be in the form of a plate, beads, test strip, membrane, or column matrix, and the sample to be analyzed is applied to immobilize the antigen or antibody. When coating a plate with antigen or antibody, the wells of the plate are typically incubated with a solution of the antigen or antibody overnight or for a specific number of hours. The wells can then be washed to remove any incompletely adsorbed material. Any remaining usable surface of the wells can then be “coated” with a non-specific protein that is antigenically neutral for the antiserum being tested. These include bovine serum albumin (BSA), casein, and milk powder solutions. This coating allows for blocking non-specific adsorption sites on the immobilized surface and thus reduces background caused by non-specific binding of the antiserum to the surface.

[0217] In ELISA, secondary or tertiary detection methods can also be used instead of a direct procedure. Therefore, after the protein or antibody binds to the well, it is coated with a non-reactive material to reduce background and washed to… Except for The material is bound together to allow for the formation of immune complexes (antigen / antibody) by bringing the immobilized surface into contact with a control clinical or biological sample to be tested. The detection of the immune complexes then requires a labeled secondary binder or a secondary binder bound to a labeled tertiary binder.

[0218] Enzyme-linked immunospot (ELISpot) assays are immunoassays that detect antibodies specific to proteins or antigens. In this assay, the detectable label bound to the antibody-binding reagent or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts by producing a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or visual means. Enzymes that can be used to detectably label reagents 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, α-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucosyl amylase, and acetylcholinesterase. In this assay, a nitrocellulose microtiter plate is coated with an antigen. The test sample is exposed to the antigen and then reacted similarly to an ELISA assay. The detection differs from a traditional ELISA because detection is determined by counting the spots on the nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. The spots can be counted to determine the number of antigen-specific cells in the sample.

[0219] "Under conditions that effectively allow the formation of immune complexes (antigen / antibody)" means that such conditions include diluting the antigen and antibody with a solution that may or may not contain BSA, bovine gamma globulin (BGG), and phosphate-buffered saline (PBS) / Tween to reduce nonspecific binding and promote a reasonable signal-to-noise ratio.

[0220] Suitable conditions also mean that incubation is carried out at a temperature and duration sufficient to allow for effective bonding. Incubation steps can typically be carried out at a temperature of about 20°C to 30°C for about 1 minute to 12 hours, or overnight at about 0°C to about 10°C.

[0221] Following all incubation steps in an ELISA assay, the contacted surfaces can be washed to remove uncombined material. Washing procedures may include washing with a solution that may or may not include PBS / Tween or borate buffer. Specific immune complexes form between the test sample and the initially bound material, and the presence of even trace amounts of these immune complexes can be measured after subsequent washing.

[0222] To provide a detection method, the second or third antibody may have an associated label to allow detection, as described above. This can be an enzyme capable of generating a colorimetric reaction upon incubation with a suitable chromogenic substrate. Thus, for example, the first or second immune complex may be contacted with the labeled antibody and incubated for a period of time under conditions favorable to further immune complex formation (e.g., incubation for 2 hours at room temperature in a solution containing PBS, which may or may not include PBS-Tween).

[0223] After incubation with the labeled antibody, followed by washing to remove unbound material, the amount of labeling can be quantified, for example, by incubation with a chromogenic substrate in the case of peroxidase as the enzyme label. This chromogenic substrate may or may not include urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzothiazoline-6-sulfonic acid [ABTS]) and H₂O₂. Quantification can then be achieved by measuring the degree of color formation, for example, using a visible spectrophotometer.

[0224] Protein arrays are solid-phase ligand binding assay systems that immobilize proteins on surfaces including glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. These assays are highly parallel (multiplexed) and typically miniaturized (microarrays, protein chips). Their advantages include speed and automation, high sensitivity, reagent economy, and the provision of abundant data for a single experiment. Bioinformatics support is important; data processing requires sophisticated software and comparative analysis. However, software can be adapted from software used for DNA arrays, as can many hardware and detection systems.

[0225] One primary form is the capture array, where the ligand-binding reagent is typically an antibody, but can also be an alternative protein scaffold, peptide, or nucleic acid aptamer, used to detect target molecules in a mixture that may or may not include plasma or tissue extracts. In diagnostics, capture arrays can be used for parallel multiplex immunoassays, testing, for example, several analytes in a single serum sample, or simultaneously testing many serum samples. In proteomics, capture arrays are used to quantify and compare protein levels in different samples from healthy and diseased individuals, i.e., protein expression profiles. Proteins, excluding specific ligand-binding agents, are used in array form for in vitro screening of functional interactions, which may or may not include protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagent itself selects and screens for many proteins, and can also be used in multiplex array form targeting multiple protein targets.

[0226] For array construction, protein sources include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production via cell-free translation systems, and peptide synthesis methods. Many of these methods can be automated for high-throughput production. For capture arrays and protein functional analysis, it is important that the protein is correctly folded and functions; this is not always the case, for example, when recombinant proteins are extracted from bacteria under denaturing conditions. However, denatured protein arrays can be used to screen for cross-reactive antibodies, identify autoantibodies, and select ligand-binding proteins.

[0227] Protein arrays have been designed for miniaturization of common immunoassays, which may or may not include ELISA and Western blot assays, typically utilizing fluorescence readings and facilitated by robotics and high-throughput detection systems to enable parallel multiplex assays. Common physical supports include glass slides, silica, micropores, nitrocellulose or PVDF membranes, as well as magnetic microbeads and other microbeads. While delivery of protein droplets to planar surfaces is the most common form, alternative architectures include CD centrifuge devices based on microfluidics (Gyros, Monmouth, Junction, NJ) and dedicated chip designs that may or may not include engineered microchannels in plates (e.g., The LivingChip™, BioTrove, Woburn, MA) and tiny 3D pillars on silica surfaces (Zyomyx, Hayward, CA). Particles in suspension can also be used as the basis for arrays, provided they are encoded for identification; systems include color-coded microbeads (Luminex, Austin, Texas; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, California), barcodes on beads (UltraPlex™, SmartBead Technologies, Babraham Institute, Cambridge, UK) and multi-metal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, California). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS Technologies, BioArray Solutions, Warren, NJ).

[0228] Protein immobilization involves the properties of both the coupling reagent and the coupling surface. A good protein array support surface is chemically stable before and after the coupling procedure, allowing for good spot morphology, exhibiting minimal non-specific binding, contributing no background to the detection system, and being compatible with various detection systems. The immobilization methods used are reproducible, applicable to proteins of different properties (size, hydrophilicity, hydrophobicity), suitable for high-throughput and automation, and compatible with preserving the full functional activity of proteins. The orientation of the surface-bound protein is considered a crucial factor in presenting it in an active state to the ligand or substrate; for capture arrays, the use of oriented capture reagents yields the most efficient binding results, which typically requires site-specific labeling of the protein.

[0229] Both covalent and non-covalent methods for protein immobilization have been used, each with its own advantages and disadvantages. Passive adsorption to surfaces is methodologically simple but offers little to no quantitative or directional control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide stable linkages, can be applied to a range of proteins, and exhibit good reproducibility; however, orientation can be variable, chemical derivatization may alter protein function, and stable interacting surfaces are required. Biocapture methods utilizing protein tags provide stable linkages and bind proteins specifically with reproducible orientation, but the biological reagents must first be adequately immobilized, and the arrays may require special treatment and exhibit variable stability.

[0230] Several immobilization chemicals and tags for fabricating 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, .W.), reversible covalent coupling is achieved through the interaction between proteins derived from phenyl diboronic acid and salicylhydroxamic acid immobilized on a support surface. This also exhibits low background binding and low intrinsic fluorescence, allowing the immobilized proteins to retain function. Based on three-dimensional polyacrylamide gels, nonvalent binding of unmodified proteins occurs within a porous structure that may or may not include HydroGel™ (PerkinElmer, Wellesley, .M.); this substrate has been reported to produce particularly low background on glass microarrays with high capacity and retention of protein function. Widely used bioconjugation methods involve appropriate modification of proteins via biotin / streptavitin or hexahistine / Ni interactions (SEQ ID NO: 88). Biotin can be conjugated to a polylysine backbone fixed on a surface, which may or may not include titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Wittsville, Switzerland).

[0231] Array fabrication methods include robotic contact printing, inkjet printing, piezoelectric spotting, and photolithography. Many commercially available arrays (e.g., Packard Biosciences) and manual equipment (V&P Technologies) are available. Bacterial colonies can be automatically meshed onto PVDF membranes for in situ induction of protein expression.

[0232] The limits of spot size and density are nanoarrays, where spots are on a nanoscale, enabling thousands of reactions to be performed on a single chip of less than 1 square millimeter. BioForce Laboratories has developed nanoarrays with 1,521 protein spots within 85 square micrometers, equivalent to 25 million spots per square centimeter at the limits of optical detection; these are readout using fluorescence and atomic force microscopy (AFM).

[0233] Fluorescent labeling and detection methods are widely used. The same instruments used to read DNA microarrays are applicable to protein arrays. For differential display, arrays can be captured (e.g., antibodies) using fluorescently labeled proteins from two different cell states, where cell lysates are directly conjugated and mixed with different fluorophores (e.g., Cy-3, Cy-5), so that color serves as a reading of target abundance changes. Fluorescent readout sensitivity can be amplified 10 to 100 times using tyramine signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultrasensitive fluorescence detection with the added advantage of requiring no interventional washing procedures. High sensitivity can also be achieved using phycoerythrin as a label (Luminex) or semiconductor nanocrystals (Quantum Dots) with the properties of suspension beads and particles. Many novel alternative readouts have been developed, particularly in the commercial biotechnology field. These include surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, Arizona), rolling circular DNA amplification (Molecular Staging, New Haven, Connecticut), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, California), resonant light scattering (Genicon Sciences, San Diego, California), and atomic force microscopy [BioForce Laboratories].

[0234] Capture arrays form the basis of diagnostic chips and arrays used for expression profiling analysis. They employ high-affinity capture reagents, which may or may not include conventional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers, to bind and detect specific target ligands in a high-throughput manner.

[0235] The antibody arrays possess the required specificity and acceptable background characteristics, and some are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, Missouri). Antibodies used for the capture arrays are prepared via routine immunization (polyclonal serum and hybridoma) or by selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA) as recombinant fragments typically 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 used for the arrays.

[0236] The term "scaffold" refers to the ligand-binding domain of a protein, engineered into various variants capable of binding to different target molecules with specific and affinity antibody-like properties. These variants can be generated as genetic libraries and exhibit selectivity for individual targets via phages, bacteria, or ribosomes. Examples of such ligand-binding scaffolds or frameworks include "Affibodies" based on Staphylococcus aureus protein A (Affibody, Brommapojkarna, Sweden), "Trinectins" based on fibronectin (Phylos, Lexington, Massachusetts), and "Anticalins" based on lipid transport protein structures (Pieris Proteolab, Weinstef, Freising, Germany). These can be used in capture arrays in a manner similar to antibodies and can offer advantages such as robustness and ease of production.

[0237] Non-protein capture molecules, particularly single-stranded nucleic acid aptamers that bind to protein ligands with high specificity and affinity, are also used in the array (SomaLogic, Boulder, Colorado). Aptamers are selected from oligonucleotide libraries using the Selex™ method, and their interactions with proteins can be enhanced through covalent attachment, incorporation of deoxyuridine bromide, and UV-activated crosslinking (photoaptamers). Photocrosslinking with ligands reduces the cross-reactivity of aptamers due to specific spatial requirements. Aptamers offer the advantages of easy production via automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein staining can be used to detect binding.

[0238] Protein analytes bound to antibody arrays can be detected directly or via secondary antibodies in sandwich assays. Direct labeling is used to compare different samples with different colors. Sandwich immunoassays offer high specificity and sensitivity where antibody pairs against the same protein ligand are available, and are therefore a preferred method for low-abundance proteins, with or without 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 for any method is optimal sensitivity and specificity with low background to provide a high signal-to-noise ratio. Due to the wide range of analyte concentrations, sensitivity must be appropriately tuned; serial dilution of samples or use of antibodies with different affinities are solutions to this problem. Proteins of interest are often those in low concentrations in body fluids and extracts, requiring detection in the pg range or lower, which may or may not include cytokines or low-expressed products in cells.

[0239] An alternative to capture molecular arrays is capture molecular arrays made by “molecular imprinting” technology, 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 polymerizable matrix; the cavities can then specifically capture (denatured) proteins with appropriate primary amino acid sequences (ProteinPrint™, Aspira Biosystems, Burlingame, CA).

[0240] Another method that can be used for diagnostic and expression profiling analysis is the ProteinChip® array (Severge, Fremont, California), in which a solid-phase chromatography surface binds proteins from a mixture that have similar charge or hydrophobic characteristics, which may or may not include plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detect the retained proteins.

[0241] Large-scale functional chips have been constructed by immobilizing large quantities of purified proteins and used to determine a wide range of biochemical functions, which may or may not include protein-protein interactions, drug-target interactions, enzyme-substrate interactions, etc. Typically, these require cloning into expression libraries in *E. coli*, yeast, or similar organisms, followed by purification (e.g., via His-tag) and immobilization of the expressed proteins. Cell-free protein transcription / translation is a viable alternative for synthesizing proteins that are not well expressed in bacteria or other in vivo systems.

[0242] To detect protein-protein interactions, protein arrays can be in vitro alternatives to cell-based yeast two-hybrid systems and can be used in cases where the latter is deficient, including or excluding interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of the biochemical activity of various functions (protein-protein and protein-lipid interactions) of yeast protein kinases and the yeast proteome on arrays has been described, where the majority of all yeast open reading frames are expressed and immobilized on microarrays. Large-scale “proteome chips” promise to be very useful for the identification of functional interactions, drug screening, and more (Proteometrix, Branford, Connecticut).

[0243] As a two-dimensional display of individual elements, protein arrays can be used to screen phage or ribosome display libraries to select specific binding partners, including antibodies, synthetic scaffolds, peptides, and aptamers. This allows for "library-to-library" screening. Another application of this approach is screening combinatorial chemistry libraries for drug candidates targeting a range of protein targets identified from genome projects.

[0244] Multiplex bead assays, which may or may not include, such as BD™ cell counting bead arrays, are a series of spectrally discrete particles used to capture and quantify soluble analytes. The analytes are then measured by detecting fluorescence-based emission and flow cytometry analysis. Multiplex bead assays generate data equivalent to ELISA-based assays, but in a “multiplex” or simultaneous manner. For cell counting bead arrays, the concentration of unknowns is calculated, as with any sandwich assay, i.e., by using known standards and plotting the unknowns against a standard curve. Know Furthermore, multiplex bead assays allow for the quantification of soluble analytes in samples, something previously unthinkable due to sample volume limitations. In addition to quantitative data, powerful visual images can be generated to reveal unique profiles or characteristics, providing users with readily available additional information.

[0245] Therefore, in one aspect, this document discloses methods for identifying the T cell receptors disclosed herein, wherein T cell activity (which may include or exclude, for example, the 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 of the immunoassays disclosed herein, such as, but not limited to, ELISA, ELISpot, intracellular cytokine staining, or chromium release.

[0246] It should be understood and anticipated that the T-cell receptors disclosed herein (e.g., but not limited to T-cell receptors that bind to cancer antigens, which may or may not include DP4-ESO-1 TCR, A2-CT83 TCR, DR13-CT83) are... TCR, A2-pp65-TCR, and / or A2-IE-1-TCR (any one of these) can be used to treat any cancer expressing certain types of MHC molecules and antigens, 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, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the oral cavity, pharynx, larynx, and lungs, cervical cancer, breast cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma. Therefore, in one aspect, this paper also discloses a method for identifying the TCRs disclosed herein, wherein the cancers 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 oral cavity, pharynx, larynx and lungs, cervical cancer, breast cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon and rectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma.

[0247] Animals: Six- to eight-week-old female NSG mice were purchased from Jackson Laboratory or bred at the Houston Methodist Research Institute animal facility. All procedures were approved by the Houston Methodist Research Institute Animal Care and Use Committee (IACUC). In a CD19-positive B-cell lymphoma mouse model, mice were intravenously injected with 5 million Raji-fluc cells. Tumor burden was confirmed by Xenogen IVIS Spectrum bioluminescence imaging and analyzed using Living Image software (Perkin Elmer). Mice received a single infusion of CAR on day 5 post-tumor cell transplantation. +T cells. Mice were monitored at least daily and euthanized by CO2 inhalation when leukemia progression led to hind limb paralysis or weight loss exceeding 20%. For CAR-T function assessment, 2-5 million Raji cells were intravenously injected on day 0, and 500,000-2 million CAR-T cells were intravenously injected on day 5. Blood samples were collected for CD3 testing. + CAR-T cells and CD19 + Tumor cells. Mice were sacrificed on day 40. Spleen was used for flow cytometry to detect memory T cells and exhaustion markers.

[0248] CAR Construction: The 19BBz CAR encoding gene was generated by linking an FMC63-derived scFv sequence with the extracellular, transmembrane, and 4-1BB cytoplasmic domains of CD8α and the CD3ζ signaling domain. 1928z used the CD28 hinge, transmembrane, and intracellular domains. A fragment containing the ZAP70 kinase domain was generated via overlap PCR and replaced the CD3ζ signaling domain in the CAR construction. All constructs were cloned into the pMSGV1 vector and sequenced for verification.

[0249] Human peripheral blood mononuclear cells (PBMCs) and transduction: Blood from healthy donors was obtained from the Gulf Coast Regional Blood Center. Fresh PBMCs were isolated using Ficoll reagent according to the manufacturer's instructions. White membrane layers were collected and washed twice with phosphate-buffered saline (PBS). PBMCs suspended in T cell culture medium were activated using plates coated with anti-human CD3 antibody (OKT3). Virus was packaged in HEK 293T cells using the envelope plasmid RD114 and packaging plasmid Gag-pol or a stably transduced PG13 packaging cell line. Viral particles were harvested 48 hours post-transfection and filtered through a 0.45 μm filter. Activated PBMCs were transduced twice with retrovirus in the presence of recombinant human fibronectin fragment (RetroNectin). T cells were cultured for at least 3 days before use.

[0250] Flow cytometry: The following antibodies are used for flow cytometry analysis. Biotin-labeled recombinant protein L (29997, Thermo Fisher Scientific); phycoerythrin / allophycoerythrin-labeled streptavidin PE / APC (12-4317-87 / 17-4317-82, eBioscience); CD3-APC-eFluor™ 780 (47-0037-42, eBioscience), CD69-PE (11-0699-42, eBioscience), CD95-PECY7 (25-0959-42, eBioscience), CCR7-PE (12-1979-42, eBioscience), CD127-PerCP-eFluor™ 710 (46-1271-82, eBioscience), CD45RO-SB600 (63-0457-42, eBioscience) And CD62L-eFluor450 (48-0621-82, eBioscience). Stained cells were analyzed using a BD LSR II instrument (BD Biosciences) or Attune NxT (ThermoFisher). Data analysis was performed using FlowJo software (Tree Star).

[0251] Cell lines: HEK293T and MDA-231-CD19 cells were cultured in DMEM containing 10% inactivated FBS, 100 units / mL of penicillin, and 100 μg / mL of streptomycin. THP-1, Raji, Raji-GFP, Raji-fluc, and NALM-6 cells were cultured in RPMI-1640 containing 10% inactivated FBS, 100 units / mL of penicillin, and 100 μg / mL of streptomycin. All cells were negative for mycoplasma according to routine tests.

[0252] Enzyme-linked immunosorbent assay (ELISA): Virus-transduced T cells were cooled in IL-2-free medium for 1-2 days. Cells were co-cultured with tumor cells at different effector-target ratios. The next day, the diluted supernatant was added to a plate pre-coated with human IFN-γ (1:1000, Thermo, M700A) and blocked with 1% BSA, and incubated gently at room temperature for 1 hour. The plate was then washed twice, incubated with biotin-conjugated IFN-γ antibody (1:1000, Thermo M700B) for 1 hour, washed twice more, and incubated in the dark with avidin-HRP (1:5000) for 30 minutes. After washing, 100 μL of TMB was added for the reaction, and the reaction was stopped with 50 mL of 2.5 N sulfuric acid.

[0253] LDH cytotoxicity assay: Transduced T cells and tumor cells were co-cultured in 96-well plates for 6 or 24 hours at a series of effector-target ratios. The supernatant was transferred to a new ELISA plate for cytotoxicity assay according to the manufacturer's instructions. Absorbance readings were taken at 490 nm using a spectrophotometer (Bio-Tek). Specific lysis percentage was calculated using the formula: % Cytotoxicity = (Experimental group value - Effector group spontaneous value - Tumor group spontaneous value) / (Tumor group maximum value - Tumor group spontaneous value) × 100. All values ​​were subtracted from the values ​​of the culture medium blank control group.

[0254] Serum cytokines: Peripheral blood samples were collected from the tail vein of mice. Blood was incubated at room temperature for 30 minutes and then centrifuged at 8000 rpm for 15 minutes at 4°C. Serum concentrations of human interleukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor-α (TNF-α) in Raji tumor-bearing mice were determined using enzyme-linked immunosorbent assay (ELISA). Concentrations were calculated using a four-parameter logistic regression (4-PL) model.

[0255] Statistical analysis: Statistically significant differences between two groups were assessed using paired or unpaired two-tailed t-tests. ANOVA with Tukey's multiple comparison test was used for comparisons of more than two groups. A p-value <0.05 was considered statistically significant. In mouse experiments, Kaplan-Meier curves were used to plot the overall survival of mice treated with T-cell therapy, and log-rank tests were used to compare survival differences between groups. All statistical analyses were performed using GraphPad Prism 8 software.

[0256] Composition The components used to prepare the disclosed compositions and the compositions themselves used in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each is specifically considered and described herein, although specific references to every different individual and collective combination and arrangement of these compounds may not be explicitly disclosed. For example, if a particular TCR is disclosed and discussed, and many modifications that can be made to many molecules, including TCRs, are discussed, then every combination and arrangement of TCRs and possible modifications are specifically considered, unless specifically stated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and examples of combinations of molecules A through D are disclosed, then each is considered individually and collectively, even if each is not individually enumerated; this means that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, a subgroup of AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods for preparing and using the disclosed compositions. Therefore, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the disclosed method.

[0257] In one embodiment, the methods disclosed herein detect or identify compositions that can be used to treat cancer (as a therapeutic or preventative treatment) and prepare TCRs for use in treating cancer. Therefore, in one embodiment, TCRs engineered to express receptors that recognize the antigens disclosed herein (which may or may not include, for example, T cell receptors) are also disclosed.

[0258] In one embodiment, this disclosure is further characterized by a method for enhancing the persistence of CAR-T and TCR cells by expressing chemokine receptors and shRNA KO in a TCR or CAR construct. In one embodiment, the TCR T cells disclosed herein that are specific to an antigen (e.g., NY-ESO-1, CT83, pp65, and / or IE-1) may be further engineered to knock out or knock down negative signaling molecules, such as, but not limited to, programmed cell death protein (PD1), von Hippel-Lindau tumor suppressor (VHL), and / or protein phosphatase 2 regulatory subunit Bδ (PPP2R2D) to enhance their function, which may or may not include cytotoxic activity and in vivo persistence or survival after adoptive transfer to a cancer patient.

[0259] In some embodiments, negative signaling molecules are, for example, indoleamine (2,3)-dioxygenases (IDO) (including isotypes 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 some aspects, negative signaling molecules are, for example, PD-1, VHL, PPP2R2D, and epigenetic factors that may or may not include JMJD3 and LSD1.

[0260] In one embodiment, this disclosure is further characterized by a method for enhancing T cell transport to tumors and / or in vivo tumor cell localization through forced expression of chemokine receptors. In some embodiments, chemokine receptor expression is promoted by fusing a CAR or TCR construct with a chemokine. In some embodiments, the chemokine receptor is CCR5 or CCR2 of CXCR3. In some embodiments, the chemokine receptor is CCR5.

[0261] In any TCR disclosed herein, such as a specific TCR for NY-ESO-1, the α-variable region optionally comprises the DP4-ESO-1TCR α-variable region containing the amino acid sequence SEQ ID NO: 3, the α-variable region or a variant thereof, the variant specifically binding antigen with the same characteristics as the reference (full-length and unmodified) receptor, a polypeptide comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 3, a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions (e.g., D95S or Q98Y in the TCR-Vα CDR3 sequence GADIVDYGQNFV (SEQ ID NO: 89), amino acid position numbering according to the mature TCR sequence) to the amino acid sequence of SEQ ID NO: 3, or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions to the amino acid sequence having at least 85% homology with the sequence of SEQ ID NO: 3, and HLA-DP4 NY-ESO-1 The β-variable region of the TCR may optionally comprise a polypeptide of the amino acid sequence SEQ ID NO: 4, a β-variable region or a 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 at least 85% homology with the amino acid sequence of SEQ ID NO: 4, a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions (e.g., Y98L, Y98M in the TCR-Vβ CDR3 sequence AWRRRGYEQY (SEQ ID NO: 90)) to the amino acid sequence of SEQ ID NO: 4, or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions to the amino acid sequence having at least 85% homology with the sequence of SEQ ID NO: 4.

[0262] In any TCR implementation of some embodiments, the C-terminus of the TCR / chimeric TCR α or β chain is fused to a signaling domain comprising a ZAP70 kinase domain or a mutant or variant thereof. Furthermore, in any CAR implementation herein or some other implementations, the CAR / chimeric CAR comprises an intracellular T cell activation portion, wherein the intracellular T cell activation portion comprises a signaling domain. This signaling domain comprises replacing CD3ζ with a ZAP70 kinase domain or a mutant or variant thereof, which comprises a functional ZAP70 kinase. In some embodiments, any TCR or CAR characterized herein comprises a ZAP70 kinase domain derived from functional wild-type ZAP70 or a mutant or variant thereof, wherein the ZAP70 kinase domain derived from functional wild-type ZAP70 or a mutant or variant thereof further comprises a functional ZAP70 kinase domain. In some embodiments, the mutant or variant of ZAP70 is a functional ZAP70 kinase domain, which is a truncated biologically active fragment of the ZAP70 kinase. In some embodiments, the functional ZAP70 kinase domain comprises an amino acid sequence having at least 55% amino acid sequence identity with SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 52, SEQ ID NO: 64, or SEQ ID NO: 65. In any TCR disclosed herein, the ZAP70 moiety can be any of the fragments ZAP300 (SEQ ID NO: 16, amino acid sequence from 300-619), ZAP327 (SEQ ID NO: 17, amino acid sequence from 327-619), ZAP338 (SEQ ID NO: 52, amino acid sequence from 338-619), ZAP255 (SEQ ID NO: 64), ZAP280 (SEQ ID NO: 65), or a mutant or variant thereof. In some embodiments, the TCR may comprise a ZAP70 kinase domain, wherein the N-terminal amino acid is any amino acid between or including 250 and 338, between or including 256 and 338, between or including 281 and 338, between or including 309 and 338, between or including 300 and 338, between or including 250 and 327, between or including 281 and 327, between or including 309 and 327, or between or including 300 and 327, and the C-terminal amino acid is between or including amino acids 610 and 619. In some of the foregoing embodiments, the C-terminal amino acid is 619.In some implementations, the terminal amino acids are 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299. The C-terminal amino acid is any one of 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, and is 610, 611, 612, 613, 614, 615, 616, 617, 618, or 619 amino acids. In some implementations, the ZAP70 kinase domain includes or excludes ZAP255 (255-619 amino acids), ZAP280 (280-619 amino acids), or ZAP308 (308-619 amino acids).

[0263] In any of the TCRs and CARs described herein (e.g., in a CAR containing a chimeric antigen receptor, a TCR containing a chimeric TCR receptor, or T cells expressing a CAR / chimeric CAR or a TCR / chimeric TCR), in some embodiments, the CAR or TCR includes an antigen recognition portion, a transmembrane domain, and an intracellular T cell activation portion, wherein the intracellular T cell activation portion further includes a signal transduction domain comprising a ZAP70 kinase domain or a variant thereof.

[0264] In some embodiments, the T-cell receptor (TCR) or chimeric antigen receptor (CAR) is specifically targeted at cancer antigens, or antigens that are pathogenesis-related to inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, infectious diseases, or aging. In some embodiments, the cancer antigen specifically targeted by the TCR or CAR may include, exclude, or be selected from the group consisting of: alpha-fetoprotein (AFP), interferon-inducible protein 2 (AIM2) missing in melanoma, adenocarcinoma antigen 4 (ART-4) recognized by T cells, BCMA, B antigen (BAGE), antigen recognized by CTLs in melanoma (CAMEL), carcinoembryonic antigen peptide-1 (CAP-1), caspase 8 (CASP8), cell cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDK12, carcinoembryonic antigen (CEA), calcium-activated chloride channel 2 (CLCA2), CFTR, cytomegalovirus (CMV), cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, Epstein-Barr virus (EBV), EGFRvIII (epidermal growth factor receptor type III variant), EGFR and its isoforms (including EGFR) E746-A750del, EGFRVIII), Epithelial-specific antigen (ESA), Epithelial cell adhesion molecule (EpCAM), EphA2 / 3, Epithelial glycoprotein 2 (EGP2), Epithelial glycoprotein-40 (EGP-40), Epithelial membrane protein (EMA), Epithelial tumor antigen (ETA), Fibronectin (FN), FGF-5, FGF-6, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, N-acetylglucosamine transferase V (GnT-V), Glycoprotein 100 (GP100), HCMV pp65, HCMV IE-1, helicase antigen (HAGE), H3.3K27M, tumor embryonic antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, human epidermal growth factor receptor 2 (HER2 / neu), HBV, HERV-E, HIV-1 gag, HMI.24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16E7, human telomerase reverse transcriptase (hTERT), KRAS and its mutants (such as KRAS G12D, KRAS...G12V), LAGE1b, LMP2, LILRB2, LGR5, Ly49, Ly108, L1-CAM, melanoma-associated antigen (MAGE) and its family members (including MAGE-A1, A2, A3, A4, A6, A10, A12, MAGE-C2), c-Met, MICA / B, muscle-specific actin (MSA), Melan-A / MART-1, mesothelin (MSLN), mucin 1 (MUC1), MUC2, mucin 16 (MUC-16), myo- D1, tumor-type M2-PK, Necl-2, neurofilament protein, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophagus 1 (NY-ESO-1), PRAME, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), renal antigen (RAGE), Ral-B, abnormal ras protein, ROR1, SLAMF7 / CS1, spermin 17 (Sp17), sarcoma antigen (SAGE), SART-1 / -2 / -3, SOX10, synovial sarcoma X Breakpoint 2 (SSX-2), Survivin, OVA1, HE4, DR-70, Total PSA, α-Methylacyl-CoA Racemicase (AMACR), CA125 / MUC16, Estrogen Receptor α / β (ERα / ERβ), Thymidine Kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Keratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF-R / TNFRSF16, NM23-H1, PARP, PP4, SerpinE1 / PAI-1, 14-3-3 family, 5T4, TIM-3, TROP-2, Nectin-4, PD-1, PD-L1, CTLA-4, PDGFRα, VEGF, TRAG-3, TARP, TGFβII, thyroglobulin, abnormal p53 protein (TP53), TRAIL, TRP1 / gp75, TRP2, TYRP1, tyrosinase, TAG-72, TALLA-1, TLR4, TRBC1, TRBC2, Trp-p8, thyroid transcription factor-1, Vα24, WT1, and CD series molecules (such as CD1a / b / c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD19, CD20, CD22, C). D23, CD25, CD28, CD30, CD44, CD44V6, CD45, CD45RO, CD52, CD56, CD62L, CD69, CD70, CD73, CD79a / b, CD80, CD83, CD86, CD94, CD95, CD103, CD105, CD107a / b, CD117, CD 122. CD133, CD138, CD146, CD152, CD161, CD163, CD197, CD223, CD244, CD273, CD276, CD279, CD282, CD284, CD294, CD304, CD314, CD319, CD326, CD335, etc.), HLA-DR, κ / λ Light chain, PAX-5, BCL-2, MPO, TdT, FMC-7, ROMA (HE4+CA-125), circulating tumor cell markers (EpCAM, CD45, keratin 8 / 18 / 19), PSCA, PAP, P-cadherin, placental alkaline phosphatase, CAIX, CS-I, CSPG4, AC133, c-Kit, Lewis A / Y, ER, PR, CA-125, CA15-3, CA27.29, free PSA, NuMA / NMP22, A33, ABCB5 / 6 / G2, ACE / CD143, Akt, APC, ApoA1 / A2 / E, APRIL, Aurora A, BAP1, B-Raf, BRCA1 / 2, C4.4A, CA9, CathepsinD. CCR1 / 4 / 7 / 9, CEACAM-19 / -20 / -4, CHD1L, DLL3, DNMT1, E-Cadherin, EGFR / ErbB family, ERCC1, ERK, EZH2, FAP, FOLR1 / 2 / 3 / 4, FoxM 1. GPC2 / 3, GRP78, HGF-R / c-MET, HIF-2α, HOXB13, HSP70 / 90, IDH1, IGF / IGFR, IL-1β, IL-2, IL-6, ICAM-1, Integrins, JNK, KiSS1 R, LDHA, LEF1, LIN-28A / B, LKB1, MCAM, MDM2, Mesothelin, MITF, MMPs, MUC-4, NCAM-1, NDRG1, NEK2, Notch-3, OPN, PARP, PDGF-R, PI3K, PKM2, PLK1, PTEN, PTK7, Rab25, RAS, RET, S100 family, SCUBE3, SOX2 / 11 / 17, SPARC, STEAP1 / 2 / 3, Survivin, Syndecan-1, TGF-β family, Thymidine Kinase 1, TIMP-1 / -2 / -3 / -4, TMEM219, TNF-α, TOP2A, TRAF-4, Trypsin-2, TSPAN1, uPAR, VCAM-1, VEGFR1 / 2, YAP1, ZAP70, and any combination thereof.

[0265] In one embodiment, this disclosure relates to pharmaceutical compositions comprising one or more epitopes of any of the antigens described herein, for the treatment of diseases or conditions that are pathogenesis-related to said antigen or epitope. In one aspect, compositions comprising one or more epitopes described herein can be used to prepare vaccines for the treatment of cancer. In another aspect, this disclosure also relates to compositions comprising one or more antigens and / or epitopes thereof, wherein said antigen and / or epitope are capable of binding to any of the T-cell receptors (TCRs) described herein. In some aspects, the compositions comprise antigens and their epitopes (as non-limiting examples), such as, but not limited to, epitopes of NY-ESO-1, CT83, HCMV-pp65, and HCMV-IE-1, and any other antigens described herein, and their mutants and variants, as disclosed herein.

[0266] In some embodiments, the signal transduction domain of the T cell receptor (TCR) or chimeric antigen receptor (CAR) includes a ZAP70 kinase domain or a mutant, variant, or derivative thereof; the intracellular T cell activating motif includes a signal transduction domain, wherein the signal transduction domain includes replacing CD3ζ with a ZAP70 kinase domain or a mutant or variant thereof; and the TCR or CAR is fused with a chemokine receptor, wherein the chemokine receptor may optionally be selected from and may include or exclude CCR5, CCR2, and CXCR3 or other chemokine receptors to enhance T cell migration. In some aspects, the cytokine receptor may include, exclude, or be selected from, for example, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Ra, IL-17RC, IL-21Rα, and the common cytokine receptor γ chain. In some embodiments, the signal transduction domain comprising the ZAP70 kinase domain or a mutant or variant thereof further comprises an amino acid sequence having at least 55% identity with the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 52, SEQ ID NO: 64 or SEQ ID NO: 65.

[0267] In any T-cell receptor (TCR) or chimeric antigen receptor (CAR) described herein (including T cells expressing any TCR or CAR described herein), in some embodiments, the TCR or CAR includes an antigen recognition motif, a transmembrane domain, and an intracellular T-cell activation motif, wherein the intracellular T-cell activation motif includes a co-stimulatory signaling domain fused to a signal transduction domain. In some embodiments, the co-stimulatory signaling domain is selected from the group consisting of: CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CD8, CD8α, CD8β, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (C D154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG-3), CD226, CD229, CD 244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, Lymphocyte Function-Associated Antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), DAP10, DAP12, LAG-3, 2B4, CARD1, CTLA-4 (CD152), TRIM, ZAP70, FcεRIγ, 4-1BBL, BAFF, GADS, G ITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACI, BLAME, CRACC, CD2F-10, NTB-A, integrin a4, integrin a4b1, integrin a4b7, IA4, ICAM-1, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptor (TLR),For example, TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR2DS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA-1, VLA-6, BTLA, Ikaros, LAG-3, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof. Further, in some embodiments, the signal transduction domain comprises the ZAP70 kinase domain or a mutant or variant thereof.

[0268] In any CAR or TCR described herein, the antigen recognition motif may comprise: an antigen-specific antibody, a bispecific antibody or binding motif, a trispecific antibody or binding motif, or a multivalent, multispecific binding motif, as well as an antigen-binding fragment, an antibody mimic, a protein receptor or a ligand for a specific receptor, or a TCR-like antibody. In some embodiments, the antibody, antigen-binding fragment, antibody mimic, protein receptor or a ligand for a specific receptor, or TCR-like antibody includes, but is not limited to: scFv fragment, Fv fragment, Fd fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, VH domain, VL domain, monoclonal antibody, polyclonal antibody, nanobody, bispecific or trispecific antibody fusion protein, or any combination thereof. In some embodiments, the CAR, TCR, or multispecific binding motif described herein may also comprise a leucine zipper, a coiled-coil SYNZIP structure, or other peptide structures to promote oligomerization. In some embodiments, the leucine zipper derived from human B-ZIP protein and other related aspects are described, for example, in Vinson et al., "Classification of human B-ZIP proteins based on dimerization characteristics," *Molecular and Cellular Biology*, September 2002, pp. 6321–6335; and Moll et al., "With different isoelectric points and up to 10 - ¹ 5"Designed Heterodimeric Leucine Zippers for M-Stability," *Protein Science* (2001), 10:649–655, the contents of which are incorporated herein by reference as if reproduced verbatim. In other embodiments, SYNZIPs capable of heteroassociative interactions are employed to oligomerize biantigen CARs or multivalent-multispecific binding proteins. For example, Thompson et al., "SYNZIP Protein Interaction Toolkit: In Vitro and In Vivo Applications," *ACS Synthetic Biology*, 2012, 1:118–129, the contents of which are also incorporated herein by reference as if reproduced verbatim.

[0269] In some embodiments, this disclosure relates to methods for prolonging T cell persistence and / or reducing T cell exhaustion by regulating the signal transduction and function of T cell receptors (TCRs), chimeric TCRs, chimeric antigen receptors (CARs), or cells; or to achieving the above effects by directly manipulating the signal transduction domains of TCRs or CARs, wherein the signal transduction domains of TCRs or CARs are regulated by negative signaling molecules, and signal transduction is modulated by knocking down or knocking out the negative signaling molecules. In some embodiments, the negative signaling molecules are selected from the group consisting of molecules associated with the following co-stimulatory signaling domains: CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CD8, CD8α, CD8β, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40 , CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG3), CD226, CD2 29. CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, Lymphocyte Function-Associated Antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), DAP10, DAP12, LAG-3, 2B4, CARD1, CTLA-4 (CD152), TRIM, ZAP70, FcεRIγ, 4-1BBL, BAFF, GAD S, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACI, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptor (TLR),For example, TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR2DS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA-1, VLA-6, BTLA, Ikaros, LAG-3, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof. In some respects, the negative signaling molecules are further selected from the group consisting of: PD-1, VHL, PPP2R2D, indoleamine-2,3-dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4, PD-L1, PD-L2, LAG3, B7-H3, and epigenetic regulatory factors (which may or may not include JMJD3 and LSD1), or any combination thereof.

[0270] It should be understood and anticipated herein that once the TCR sequence is identified, those skilled in the art will have full knowledge of the nucleic acids encoding the TCR amino acids, and the preparation of the nucleic acid constructs is entirely within the capabilities of those skilled in the art. Therefore, in one aspect, this document also discloses nucleic acids encoding any TCR polypeptides disclosed herein.

[0271] Identity / Homology It should be understood that one way to define any known variants and derivatives, or possible variants and derivatives, of the genes and proteins disclosed herein is by defining variants and derivatives based on their identity or homology with and / or identification of a particular known sequence. For example, Seq ID NO: 3 describes a specific sequence of the variable region of the TCRα chain. Variants of these and other genes and proteins disclosed herein are specifically disclosed that have at least 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%, or 99% homology or identity with the stated sequence. Those skilled in the art will readily understand how to determine the homology or identity of two proteins or nucleic acids that may or may not contain genes. For example, homology or identity can be calculated after aligning two sequences, such that homology is at its highest level.

[0272] Another way to calculate homology or identity is through publicly available algorithms. The 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, Journal of Molecular Biology 48: 443 (1970), the similarity search method of Pearson and Lipman, Proceedings of the National Academy of Sciences 85: 2444 (1988), the computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin, 575 PhD), or by inspection.

[0273] Homology or identity of nucleic acids of the same type can be obtained by algorithms published, for example, in Zuker, M. Science 244:48-52, 1989; Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989; and Jaeger et al. Methods Enzymol. 183:281-306, 1989, at least the material related to nucleic acid alignment in these documents is incorporated herein by reference.

[0274] Nucleic acid Various nucleic acid-based molecules disclosed herein exist, including, for example, nucleic acids encoding, for example, SEQ ID NO: 1, or any nucleic acid or fragment thereof disclosed herein, as well as various functional nucleic acids. In some embodiments, the nucleic acids included herein may or may not include cDNA encoding the TCR α chain and / or the TCR β chain. The term “cDNA” as used herein refers to a nucleic acid that links exon-exon or exon-only coding sequences. The disclosed nucleic acids consist of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It will be understood, for example, that when a vector is expressed in a cell, the expressed mRNA typically consists of A, C, G, and U. Similarly, it will be understood that if an antisense molecule is introduced into a cell or cellular environment, for example, by, for example, exogenous delivery, it is advantageous for the antisense molecule to consist of nucleotide analogs that reduce the 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 acid as further disclosed herein.

[0275] Nucleotides and related molecules A nucleotide is a molecule containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together by their phosphate and sugar moieties, forming nucleoside linkages. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymine-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 are 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). Many of these types of molecules are available in the art and herein.

[0276] Nucleotide analogs are nucleotides containing certain types of modifications to their base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and include, for example, modifications to 5-methylcytosine (5-me-C), 2-methylcytosine (2-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications to the sugar or phosphate moieties. Many of these types of molecules are available in the art and herein.

[0277] Nucleotide substitutes are molecules that have similar functional properties to nucleotides but lack a phosphate moiety; they 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 together by a portion other than the phosphate moiety. When interacting with a suitable target nucleic acid, nucleotide substitutes can conform to a double helix structure. Many of these types of molecules are available in the art and herein.

[0278] Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties that may or may not include a cholesterol moiety (Letsinger et al., Proceedings of the National Academy of Sciences of the United States of America, 1989, 86, 6553 6556). Many of these types of molecules are available in this field and herein.

[0279] The Watson-Crick interaction is an interaction with at least one Watson-Crick facet of a nucleotide, nucleotide analogue, or nucleotide substitution. The Watson-Crick facet of a nucleotide, nucleotide analogue, or nucleotide substitution includes the C2, N1, and C6 positions of purine-based nucleotides, nucleotide analogues, or nucleotide substitutions, and the C2, N3, and C4 positions of pyrimidine-based nucleotides, nucleotide analogues, or nucleotide substitutions.

[0280] Husstan interactions are interactions that occur on the Husstan face of nucleotides or nucleotide analogs exposed in the major groove of double-stranded DNA. The Husstan face includes the reactive groups (NH2 or O) at the N7 and C6 positions of the purine nucleotide.

[0281] Primers and probes Compositions comprising primers and probes are disclosed, which are capable of interacting with disclosed nucleic acids, which may or may not include the tumor antigens, epitopes, and TCRs disclosed herein. In some embodiments, primers are used to support DNA amplification reactions. Typically, primers are capable of sequence-specific extension. Sequence-specific extension of primers includes any method in which the sequence and / or composition of the nucleic acid molecule hybridized with or otherwise associated with the primer guides or influences the composition or sequence of the product generated by primer extension. Thus, sequence-specific extension of primers includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Preferred techniques and conditions for sequence-specific primer amplification are specified. In some embodiments, primers are used for DNA amplification reactions, which may or may not include PCR or direct sequencing. It should be understood that in some embodiments, non-enzymatic techniques may also be used to extend primers, wherein, for example, the nucleotides or oligonucleotides used for primer extension are modified such that they extend the primers in a sequence-specific manner via chemical reactions. Typically, the disclosed primers hybridize with disclosed nucleic acids or regions of nucleic acids, or they hybridize with complements of nucleic acids or complements of nucleic acid regions.

[0282] In some embodiments, the size of the primers or probes used to interact with nucleic acids can be any size that supports the desired primer enzymatic operation, and may or may not include DNA amplification or simple hybridization of the probes or primers. Typical primers or probes will be 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. 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.

[0283] In other embodiments, the primers or probes may be less than or equal to 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, 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.

[0284] In some embodiments, the product is 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.

[0285] In other embodiments, the product is less than or equal to 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.

[0286] peptides, protein variants As discussed herein, there are various known and contemplated TCR variants. Protein variants and derivatives are well known to those skilled in the art and may include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three categories: substitution, insertion, or deletion variants. Insertions include amino and / or carboxyl terminus fusions and intra-sequence insertions of one or more amino acid residues. Insertions are typically smaller than amino or carboxyl terminus fusions, for example, about one to four residues. Immunogenic fusion protein derivatives, which may or may not include those described in the examples, are prepared by fusing a sufficiently large polypeptide to confer immunogenicity to the target sequence by in vitro crosslinking or by recombinant cell cultures transformed with DNA encoding the fusion protein. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any site within the protein molecule. These variants are typically prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, resulting in DNA encoding the variant, which is then expressed in recombinant cell cultures. Techniques for generating substitution mutations at predetermined sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically single residues, but can occur simultaneously at multiple different positions; insertions are typically about 1 to 10 amino acid residues; deletions range from about 1 to 30 residues. Substitutions may or may not include substitutions in one or more of the six TCR CDR regions. Preferably, deletions or insertions are performed in adjacent pairs, i.e., deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final construct. Mutations must not place the sequence outside the reading frame and preferably do not produce 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 has been inserted at its position. Such substitutions are typically performed according to Tables 1 and 2 below and are referred to as conserved substitutions.

[0287] Table 1. Amino Acid Abbreviations

[0288] Table 2 Amino acid substitutions

[0289] By selecting substitutions with lower conservation than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the substituted region (e.g., sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain, substantial changes in functional or immune identity can be achieved. Substitutions that typically produce the greatest changes in protein properties are those in which (a) a hydrophilic residue (e.g., serine or threonyl) replaces a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valine, or alanyl) (or is replaced by it); (b) cysteine ​​or proline replaces any other residue (or is replaced by it); (c) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidine) replaces a negatively charged residue (e.g., glutamine or aspartate) (or is replaced by it); or (d) a residue with a large side chain (e.g., phenylalanine) replaces a residue without a side chain (e.g., glycine) (or is replaced by it), in which case (e) by increasing the number of sites for sulfation and / or glycosylation.

[0290] For example, replacing one amino acid residue with another biologically and / or chemically similar amino acid residue is a conserved substitution known to those skilled in the art. For example, a conserved substitution replaces one hydrophobic residue with another, or one polar residue with another. Substitutions may include, or may not include, combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conserved substitution variants of each explicitly disclosed sequence are included in the chimeric polypeptides provided herein.

[0291] Substitution or deletion mutagenesis can be used to insert N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr) sites. Deletion of cysteine ​​or other unstable residues is also required. Deletion or substitution of potential proteolytic sites (e.g., Arg) can be accomplished, for example, by deleting a basic residue or replacing it with a glutamine acyl or histidine residue.

[0292] Some post-translational derivatizations are the result of the recombinant host cell's action on the expressed peptide. Glutamine acyl and asparagyl residues are typically deamidated post-translationally to their corresponding glutamine and asparagyl residues. Alternatively, these residues are deamidated under mild acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonyl residues, methylation of the ortho-amino groups of the lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., pp. 79–86

[1983] ), acetylation of the N-terminal amine, and in some cases, amidation of the C-terminal carboxyl group.

[0293] It should be understood that one way to define variants and derivatives of the proteins disclosed herein is by defining variants and derivatives based on their homology / identity with a particular known sequence. Specifically disclosed are variants of these and other proteins disclosed herein that have at least 70% or 75% or 80% or 85% or 90% or 95% homology / identity with the stated sequences (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 61). Those skilled in the art will readily understand how to determine the homology / identity of two proteins. For example, homology / identity can be calculated after aligning two sequences such that the homology / identity is at its highest level.

[0294] Another method for calculating homology / identity can be performed using publicly available algorithms. The optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 (1981), the homology alignment algorithm of Nedlerman and Winsley, Journal of Molecular Biology 48: 443 (1970), the similarity search method of Pearson and Lippmann, Proceedings of the National Academy of Sciences 85: 2444 (1988), the computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, Madison, Wisconsin, 575, PhD in Science), or by inspection.

[0295] Homology or identity of nucleic acids of the same type can be obtained through algorithms published, for example, in Zuckerman's *Science* 244:48-52, 1989; Jaeger et al.'s *Proceedings of the National Academy of Sciences of the United States of America* 86:7706-7710, 1989; and Jaeger et al.'s *Methods Enzymol.* 183:281-306, 1989.

[0296] The descriptions of conserved mutations and homology / identity can be combined in any combination, which may or may not include embodiments that have at least 70% homology / identity with a particular sequence, wherein the variant is a conserved mutation.

[0297] When this specification discusses various proteins and protein sequences, it should be understood that nucleic acids that can encode these protein sequences are also disclosed. This will include all degenerate sequences associated with a specific protein sequence, i.e., all nucleic acids having a sequence encoding a particular protein sequence, as well as all nucleic acids, including degenerate nucleic acids, that encode variants and derivatives of the disclosed protein sequence. Therefore, although every specific nucleic acid sequence may not be written herein, it should be understood that each sequence is actually disclosed and described herein by way of the disclosed protein sequences.

[0298] It should be understood that many amino acids and peptide analogs can be incorporated into the disclosed compositions. For example, many D amino acids or amino acids having functional substituents different from those shown in Tables 1 and 2 are present. Opposite stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogs, are disclosed. These amino acids can be readily incorporated into polypeptide chains by loading tRNA molecules with selected amino acids and engineered genetic constructs that utilize, for example, amber codons to insert analog amino acids into the polypeptide chain in a site-specific manner.

[0299] It can produce peptide-like molecules that are not linked by natural peptide bonds.For example, the bonds in amino acids or amino acid analogs can include CH2NH--, --CH2S--, --CH2--CH2--, --CH=CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CHH2SO-- (these and others can be found in Spatola, AF, *Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins*, edited by B. Weinstein, Marcel Dekker, New York, p. 267 (1983); Spatola, *Vega Data* (March 1983), Vol. 1, No. 3, "Peptide Backbone Modifications" (review); Morley, *Trends Pharmacology*. (Sci) (1980) pp. 463-468; Hudson, D. et al., International Journal of Peptide Research and Therapeutics (Int J Pept Prot Res) 14:177-185 (1979) (--CH2NH--, CH2CH2--); Spatola et al., Life Sci 38:1243-1249 (1986) (--CH H2--S); Hann, Journal of Chemistry (J. Chem. Soc Perkin Trans.) I 307-314 (1982) (--CH--CH--, cis and trans); Almquist et al., Journal of Medicinal Chemistry (J. Med. Chem.) 23:1392-1398 (1980) (--COCH2--); Jennings-White et al. Tetrahedron Letters 23:2533 (1982) (--COCH2--); Szelke et al. European application, EP 45665 CA (1982): 97:39405 (1982) (--CH(OH)CH2--); Holladay et al. Tetrahedron Letters 24:4401-4404 (1983) (--C(OH)CH2--); and Hruby Life Sciences Sci) 31:189-199(1982)(--CH2--S--); each of which is incorporated herein by reference.A particularly preferred non-peptide bond is --CH2NH--. It should be understood that peptide analogs may have more than one atom between bond atoms, and may or may not include β-alanine, γ-aminobutyric acid, etc.

[0300] Amino acid analogs and analogues, as well as peptide analogs, typically possess enhanced or desired properties, which may or may not include more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad-spectrum biological activity), reduced antigenicity, etc.

[0301] D-amino acids can be used to generate more stable peptides because they are not recognized by peptidases. Systematically replacing one or more amino acids in a common sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine) can generate more stable peptides. Cysteine ​​residues can be used to cyclize or attach two or more peptides together. This is advantageous for restricting peptides to specific conformations.

[0302] Drug carrier / drug product delivery In one aspect, this document discloses compositions comprising therapeutically effective amounts of one or more TCR T cells; wherein the TCR T cells have been engineered to express a receptor for a tumor antigen disclosed herein. In another aspect, the TCR T cells disclosed herein that are specific to a tumor antigen can be further engineered to knock out or knock down programmed cell death protein (PD1), von Hippel-Lindau tumor suppressor factor (VHL), and / or protein phosphatase 2 regulatory subunit Bδ (PPP2R2D) to enhance their function, which may or may not include cytotoxic activity and in vivo persistence or survival after adoptive transfer to a cancer patient.

[0303] As described 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; that is, the material can be administered to a subject together with the nucleic acid or carrier without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing it. As is well known to those skilled in the art, carriers can be naturally selected to minimize any degradation of the active ingredient and any adverse side effects on the subject.

[0304] The composition can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, percutaneously, in vitro, topically, including top nasal administration or inhalation. As used herein, “top nasal administration” means delivery of the composition into the nose and nasal passages through one or both nostrils, and may include delivery via a spray or droplet mechanism, or via aerosolization of a nucleic acid or carrier. Inhalation administration of the composition can be via a spray or droplet mechanism through the nose or mouth. It can also be delivered directly to any area 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 subject’s species, age, weight and general condition, the severity of the allergic condition being treated, the specific nucleic acid or carrier used, its mode of administration, etc. Therefore, it is not possible to specify an exact amount for each composition. However, those skilled in the art can determine the appropriate amount using only the routine experiments given the teachings herein.

[0305] If used, parenteral administration of the composition is typically characterized by injection. Injectable formulations can be prepared in conventional forms, as liquid solutions or suspensions, as solid suspensions suitable for dissolving in a liquid prior to injection, or as emulsions. Recently revised methods of parenteral administration involve the use of sustained-release or continuous-release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0306] Materials can be solutions or suspensions (e.g., incorporating microparticles, liposomes, or cells). These can be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of using this technique to target specific proteins to tumor tissues (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 Chemistry, 4:3-9, (1993); Battelli et al., Cancer Immunology and Immunotherapy). 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). The vector may include, but is not limited to, "stealthy" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting via cell-specific ligands of DNA, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of using this technique to target specific proteins to tumor tissues (Hughes et al., *Cancer Research*, 49:6214-6220, (1989); and Litzinger and Huang, *Biochimica et Biophysica Acta*, 1104:179-187, (1992)). Typically, receptors participate in constitutive or ligand-induced endocytosis pathways. These receptors accumulate in clathrin-coated pits, enter the cell through clathrin-coated vesicles, pass through acidified endosomes that sort the receptors, and then recirculate to the cell surface for intracellular storage or degradation in lysosomes. Internalization pathways have multiple functions, which may or may not include nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor-level regulation.Many receptors follow more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valence, 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)).

[0307] Pharmaceutically acceptable carriers Compositions including antibodies can be used therapeutically in combination with pharmaceutically acceptable carriers.

[0308] Suitable carriers and their formulations are described in *Remington: The Science and Practice of Pharmacy* (19th edition), edited by AR Gennaro, Mack Publishing Company, Easton, PA, 1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and glucose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Other carriers include sustained-release formulations that may or may not include a semi-permeable matrix of a solid hydrophobic polymer containing antibodies, in the form of a molded article, such as a film, liposome, or microparticle. It will be apparent to those skilled in the art that certain carriers may be preferred, depending, for example, on the route of administration and the concentration of the composition administered.

[0309] Drug carriers are known to those skilled in the art. These are most typically standard carriers used for administering drugs to humans, including solutions that may or may not include sterile water, saline, and buffers of physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard methods used by those skilled in the art.

[0310] In addition to the selected molecules, the pharmaceutical composition may include a carrier, thickener, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, which may or may not include antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0311] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is required, and the area to be treated. Administration can be local (including ocular, vaginal, rectal, and nasal), oral, inhalation, or parenteral, such as by intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, or percutaneously.

[0312] 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 that may or may not include olive oil, and injectable organic esters that may or may not include ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral media include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (which may or may not include those based on Ringer's glucose), etc. Preservatives and other additives may also be present, which may or may not include, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0313] Formulations intended for topical application may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oil-based bases, thickeners, etc., may be necessary or desirable.

[0314] Compositions intended for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, pouches, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be required.

[0315] Some compositions may be administered as pharmaceutically acceptable acid addition salts or base addition salts, formed by reacting with inorganic acids, which may or may not include hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanate, sulfuric acid, and phosphoric acid, and organic acids, which may or may not include 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 reacting with inorganic bases, which may or may not include sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases, which may or may not include monoalkyl, dialkyl, trialkyl, and arylamines and substituted ethanolamines.

[0316] Pharmaceutically acceptable carriers for the therapeutic mRNA compositions described herein include lipid nanoparticles (LNPs) for delivering mRNA to cells. Reports on mRNA vaccine adjuvants can be found, for example, by Xie et al., "Research Progress on mRNA Vaccine Adjuvants," NPJ Vaccines, 2023, 8:162. In some embodiments, the LNP comprises cationic lipids that can form complexes with negatively charged mRNA molecules. In some embodiments, the LNP further comprises structural lipids, which may include, exclude, or be selected from cholesterol, phospholipids, or polyethylene glycol (PEG)-coupled lipids. In some embodiments, the lipids constituting the LNP have immunostimulatory effects; for example, lipid-coated calcium phosphate nanoparticles can serve as an artificial nanoscale Ca²⁺. + The reservoir provides additional Ca² to the cytoplasm of cells in contact with LNPs. + This upregulates the expression of co-stimulatory molecules (such as CD80 and CD86), thereby promoting the maturation of dendritic cells (DCs). In some embodiments, the LNP may include, exclude, or include lipids selected from: for example, ionizable cationic lipid DLinDMA, cationic lipid material C1, liposome-like molecule C12-TLRa (composed of epoxidized C12 and an amine-containing TLR7 / 8 agonist), A2-Iso5–2DC18 (A2), amino lipids derived from non-nucleotide STING agonists, SAL12, and dimethyloctadecylammonium (DDA). In some embodiments, the mRNA vaccine may comprise lipid nanoparticles composed of mRNA (e.g., lipid nanoparticles described in PCT / US2020 / 013417 (disclosed as WO2020146906) and US20220088221), the contents of which are incorporated herein by reference as if recorded verbatim.

[0317] Therapeutic uses The effective dosage and schedule for administering the composition can be determined empirically, and such determination is within the scope of the art. The dosage range of the composition administered is a range large enough to produce the desired effect, affecting the symptoms of the condition. The dosage should not be too high to avoid adverse side effects, which may or may not include unwanted cross-reactions, allergic reactions, etc. Typically, the dosage will vary depending on the patient's age, condition, sex, and severity of the disease, the route of administration, or whether other medications are included in the regimen, and can be determined by someone skilled in the art. In the event of any contraindications, the dosage may be adjusted by an individual physician. The dosage can be varied and may be administered once or multiple times daily for one or several days. Guidance on appropriate dosages for a given class of pharmaceutical products can be found in the literature. For example, guidance on selecting appropriate dosages of antibodies can be found in literature concerning the therapeutic use of antibodies, such as the *Monoclonal Antibody Handbook*. Handbook of Monoclonal Antibodies ), Ferrone et al. Edited by Smith, Noges Publications, Parkridge, NJ, (1985), Chapter 22 and pp. 303–357; et al. Applications of Antibodies in Human Diagnosis and Treatment ( Antibodies in Human Diagnosis and Therapy ), Haber et al. Edited by Raven Press, New York (1977), pp. 365-389. Typical daily doses of antibodies used alone can range from approximately 1 µg / kg to up to 100 mg / kg body weight or higher, depending on the factors mentioned above. Generally speaking, it can be said that it can be administered in doses of 10... 4 Up to 10 7 engineered T cells / kg body weight, preferably 10 5 Up to 10 6 The pharmaceutical composition comprising the engineered T cells of the present invention shall be administered at doses of engineered T cells per kg body weight, including all integer values ​​within these ranges. The engineered T cell composition may also be administered multiple times at these doses. The cells may be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg). et al. (New England Journal of Medicine, 319:1676, 1988). Medical professionals can easily determine the optimal dosage and treatment plan for a specific patient by monitoring their disease signs and adjusting treatment accordingly.

[0318] Method of using the composition Methods of treating cancer The disclosed compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, which may or may not include cancer. Therefore, in one aspect, this document discloses methods for stimulating an immune response against cancer or for treating, suppressing, and / or preventing cancer, comprising administering to a subject a composition comprising a therapeutically effective amount of T cells engineered with the antigen-specific TCRs disclosed herein (e.g., these may include or exclude (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, SEQ ID NO: 33, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 7 ... Any one of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82).

[0319] The term "therapeuticly effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or condition. This improvement only requires reduction or alteration, not necessarily elimination. The precise amount of the composition of the invention applied can be determined by a physician taking into account individual differences in patients regarding age, weight, tumor size, degree of infection or metastasis, and symptoms.

[0320] The term "treatment" refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological symptom, or condition. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological symptom, or condition, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological symptom, or condition. Additionally, the term includes palliative treatment, which is treatment aimed at relieving symptoms rather than curing a disease, pathological symptom, or condition; preventative treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological symptom, or condition; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological symptom, or condition.

[0321] The term "pathology-related" refers to an antigen (e.g., a variant or mutated antigen, or an overexpressed antigen) that is causally related to a pathologically related disease or condition, such that stimulating an immune response against that antigen or epitope can cure, improve, stabilize, or prevent the disease, pathological condition, or disorder that is pathologically related to that antigen or epitope.

[0322] The following is a non-limiting list of different types of cancer that can be treated by the disclosed methods: lymphoma (Hodgkin and non-Hodgkin), leukemia, carcinoma, solid tissue carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, advanced glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer or general cancer.

[0323] The following is a representative, but non-limiting, list of cancers for which the disclosed compositions may be used to treat: 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 including or excluding small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the oral cavity, pharynx, larynx and lungs, colon cancer, cervical cancer, breast cancer and epithelial cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon cancer and / or rectal cancer.

[0324] 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 this invention, the HLA-DP4-restricted TCR was cloned from a T cell clone that specifically recognizes the NY-ESO-1 peptide presented by HLA-DP4. In vitro sensitization was performed to obtain peptide-responsive T cells. NY-ESO-1 with >95% purity was synthesized. 161-180 A peptide containing an HLA-DP4 restriction epitope. The peptide was used to treat 1088EBV-B cells (APCs) in the HLA-DP4+ cell line and co-cultured with human peripheral blood mononuclear cells (PBMCs) in 96-well plates for 21 days. During this process, NY-ESO-1... 161-180 Specific T cell populations can expand under the stress of continuous peptide stimulation, while non-specific T cells and other types of immune cells may be depleted. T cell populations from different wells were harvested 21 days after stimulation for further characterization.

[0325] First, the response of T cells stimulated from different pores to NY-ESO-1 was examined. 161-180 T cells were co-cultured with simulated 1088EBV-B cells, treated with the same APCs using the target peptide, and T cell activation was then measured by the release of cytokines in the supernatant. T cell populations from several wells showed considerably high anti-peptide activity. These T cell populations were collected to deplete CD8+ T cells. After CD8+ T cell depletion, the CD4+ T cell line was named DP4 ESO-responsive T cells. It was confirmed that the T cell line recognized the HLA-DP-restricted NY-ESO-1 epitope but not the HLA-DR-restricted epitope. Simultaneously, the specific peptide recognition of DP4 ESO-1-responsive T cells was blocked only by antibodies targeting all HLA class II molecules and HLA-DP molecules, indicating that the recognition was HLA-DP-restricted and excluded other class I and II molecules. Further attempts were made to identify the DP4 ESO-responsive T cell epitope. The shortest truncated variant maintaining high T cell recognition and activity was then limited to NY-ESO-1. 157-170 .

[0326] To molecularly clone DP4-restricted NY-ESO-1 TCRs from the DP4 ESO-responsive T cell line, single T cell clones were generated from this population. The DP4 ESO-responsive T cell line was serially diluted and seeded in 96-well plates at a ratio of 0.3 cells / well. The single live T cells in the wells were cultured and expanded for 14 days. The peptide NY-ESO-1, presented with 1088EBV-B (HLA-DP4+), was then... 157-170 Expanded T cell clones were analyzed, and their recognition of epitopes was determined by measuring cytokine release. The results are partially shown in [the table / section]. Figure 1 This indicates that, compared to simulated APCs, several DP4-ESO-1 reactive T cell clones showed improved reactivity with the peptide NY-ESO-1.157-170 The peptide-responsive T cell clones were then cultured and expanded for 14 days. However, a significant number of DP4-ESO-1-responsive T cell clones survived after expansion, while a portion of the T cell clones depleted and ceased growth, indicating that these T cell clones had different lifespans. The surviving T cell clones were further used for TCR cloning.

[0327] Molecular cloning of DP4-ESO-1 TCR Live T cell clones from 1×10 6 mRNA was extracted from T cells. The mRNA was reverse transcribed to generate templates for subsequent three rounds of nested PCR. In each round of PCR, the complementarity-determining region 3 (CDR3) of the TCRα and β chains was amplified using different primer sets containing primers targeting all types of TRAV or TRBV. After recovering the PCR products from the third round, the TCRα and β chain subtypes were identified by Sanger sequencing. The full-length TCRα and β chains were then amplified according to the identified TCR subtypes (TRAV34, TRBV30). The amplified full-length TCRs (TRAV-TRAJ-TRAC, TRBV-TRBD-TRBJ-TRBC) were cloned into an MSGV retroviral vector, which was ligated via the P2A sequence (…). Figure 2 ).

[0328] DP4-TCR transduction into uninfected T cells is used for in vitro and in vivo functional trials. A two-step transduction strategy was employed to deliver the TCR to uninfected T cells. The TCR construct was transfected into the Phoenix-Eco cell line using lipofectamine to generate primary viral supernatant. Forty-eight hours later, the viral supernatant was collected and added to the culture medium of the PG-13 cell line to infect them. Following infection, the PG-13 cell line began secreting secondary viral supernatant. The viral supernatant from the PG-13 cell line was coated onto 24-well non-tissue culture plates pre-coated with retrovirus. Uninfected CD4+ T cells isolated from human PBMCs and pre-stimulated with CD3 antibody were then infected in the wells via the coated retrovirus. To improve transduction efficiency to uninfected T cells, the PG-13 cell line transduced with the TCR-encoding construct was cloned in 96-well plates by limiting dilution. Single PG-13 cells were expanded in each well for 15 days or more to generate a PG-13 cell line encoding 100% TCR. The transduction efficiency of uninfected T cells from different PG-13 clones was determined by TRBV30-specific antibody staining and flow cytometry, and the average efficiency was 60-70%. Figure 3AThe activity of TCR-transduced CD4+ T cells was tested using 586 mel, 624 mel, and DP4-ESO monomers, demonstrating TCR-specific recognition. Figure 3B ).

[0329] The activity of TCR-transduced T cells was further tested using a series of assays to examine whether they possessed similar functions to the DP4ESO-responsive T cell line. First, the transduced T cells showed activity against the HLA-DP4-restricted epitope NY-ESO-1. 157-170 Specific identification ( Figure 4A Secondly, when measured using plasmids transfected into artificial APCs or HLA-DP4-positive or negative tumor cells, transduced T cells specifically recognize NY-ESO-1 processed from native HLA-DP4. Figure 4B To confirm that the cloned TCR possessed the function of a CD4+ TCR, CD8+ and CD4+ T cells isolated from beads were transduced, respectively. The assay showed that only CD4+ T cells transduced with this TCR possessed the function of the HLA-DP4 restrictive epitope, which was consistent with expectations. Figure 4C All results confirm the ability of TCR-engineered CD4+ T cells to specifically recognize HLA-DP4-presented NY-ESO-1. In one embodiment, this invention of an HLA-DP4-restricted NY-ESO-1 TCR serves as a novel strategy for clinical response in cancer immunotherapy.

[0330] Due to the limitations of using transgenic mice, humanized mice were used to evaluate the efficacy and safety of the NY-ESO-1 TCR. 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 / Matrix gel (50%) and injected orally into the fourth fat pad of female NSG mice (n=6 per group) on day 1. Following injection, the human tumor line was observed to grow in NSG mice. On day 5, mice were given four groups of human T cells (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; 4. A2-ESO-1 TCR-CD8+ and DP4-ESO-1 TCR-CD4+) at a dose of 2 × 10⁻⁶ per mouse. 6A2-ESO-TCR-engineered CD8+ T cells were administered intravenously followed by intraperitoneal injection of three doses of IL-2 to promote T cell proliferation in tumor-bearing NSG mice. Tumor growth in each group was monitored every 3 to 5 days, and T cell migration was tracked by luciferase transduced in CD8+ T cells. 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 significantly inhibited tumor growth as expected. Figure 5B , 5C Surprisingly, DP4-ESO-1 TCR-CD4+ T cells and A2-ESO-1 TCR-engineered CD8+ T cells showed significant tumor growth inhibition. Figure 5B , 5C This indicates that CD4+ cytolytic activity is activated under these conditions. Furthermore, when mice were sacrificed, the combination of A2-ESO-1 TCR-CD8+ T cells and DP4-ESO-1 TCRCD4+ T cells achieved maximum inhibition of tumor growth. Figure 5B , 5C This indicates that the combination of CD8+ and CD4+ T cells transduced with TCRs targeting the same antigen is superior to either TCR-T cell type alone. It confirms that CD4+ T cells effectively enhance the antitumor activity of CD8+ T cells, providing a surprising synergistic effect.

[0331] Female NSG mice aged six to eight weeks were purchased from Jackson Laboratory or housed in the animal facility of the Houston Methodist Research Institute. All procedures were approved by the Houston Methodist Research Institute Animal Care and Use Committee (IACUC) and performed according to laboratory standard protocols. On day 1, 50 μl of MDA-MB-231 / DP4 / ESO tumor cells in growth medium / Matrix gel (50%) were injected orally into the fourth fat pad of female NSG mice (n=6 per group). 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 administered to mice at a dose of 2 × 10⁻⁶ cells per mouse on day 5. 6 Cells were administered intravenously to tumor-bearing NSG mice, followed by three doses of IL-2 via intraperitoneal injection. Tumor growth was monitored every 3 to 5 days, and T cell migration was tracked using luciferase transduced in CD8+ T cells.

[0332] Example 2: Identification, Construction, and Application of T Cells and A2-CT83 TCR-T Cells A novel approach for identifying T cell recognition... HLA-A2-restricted CT83 epitope Because HLA-A2 is a major HLA class I molecule and is highly expressed in approximately 50% of the general population, experiments were conducted to identify novel HLA-A2-restricted T-cell epitopes from CT83. To this end, a series of CT83 peptides containing potential HLA-A2 binding motifs (CT83 PEP66-74, PEP79-87, and PEP90-98) were synthesized. + CD8 isolated from PBMCs of healthy donors +T cells were stimulated with autologous dendritic cells (DCs) loaded with peptides for 10 days. T cell culture medium containing IL-7 and IL-15 (5 ng / mL each) was added every 2 to 3 days. For the second stimulation, autologous PBMCs were irradiated (60 Gy) and treated with 1 ng / mL peptide for 2 to 4 hours. After washing, these irradiated peptide-treated PBMCs were added and incubated with the first-stimulated T cells for 10 days. These T cells were fed T cell culture medium containing IL-2 (30 IU / mL), IL-7 (5 ng / mL), and IL-15 (5 ng / mL) every 2 to 3 days. To test their specific recognition of CT83, in vitro stimulated T cells were compared with those containing or without the CT83 peptide. 90-98 HLA-A2 + HEK293T cells were incubated together. 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 to CT83 PEP66-74 or CT83 PEP79-87 were generated (data not shown). To determine whether these T cells recognize CT83 PEP90-98, which is endogenously processed and presented by HLA-A2, they were tested against 293T cells transfected with CT83 or CT83-GFP (full-length CT83 and green fluorescent protein (GFP) linked to the P2A sequence) with invariant strand (Ii). 293T / control peptide and 293T / CT83 PEP90-98 were used as negative and positive controls, respectively. 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 did not recognize 293T / control peptide (Ii-CT83-74 or CT83-GFP-98). Figure 6B Next, it was determined whether these CT83-specific T cells could recognize breast cancer cells, and it was 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 This indicates that T cells recognize CT83PEP90-98 presented by the HLA-A2 molecule. To further verify this, an antibody blocking experiment was performed, and the results showed that T cell recognition of MDA-MB-231 cells could be completely blocked by anti-MHC-I antibody, but not by anti-MHC-II or control antibody. Figure 6D This indicates that these T cells are specific and capable of recognizing breast cancer cells that naturally express CT83 and HLA-A2 molecules.

[0333] Injection of CT83 PEP90-98 inhibits breast cancer growth. To further test whether A2-CT83 PEP90-98 can induce anti-tumor immunity in HLA-A2 transgenic (Tg) mice, E0771-A2-CT83 mouse breast cancer cells (0.5 × 10⁻⁶) were introduced on day 0. 6 (1 cell / mouse) was injected orally into the mammary fat pad of HLA-A2 mice. Tumor-bearing mice were treated on days 7, 10, and 15 with self-assembled nanoparticles containing TAT-CT83 PEP90-98 or TAT-CT83 PEP66-74 and TLR ligands (CpG, MPLA, and poly(I:C, abbreviated as CMI)). Figure 7A Surprisingly, the results showed that TAT-CT83 PEP90-98-CMI significantly inhibited tumor growth, while TAT-CT83 PEP66-74-CMI did not. Figure 7B It has been reported that CT83 PEP66-74 uses an RNA vaccine to induce a T-cell response. 35 However, CT83 PEP90-98 did not exhibit inductive activity. Therefore, this is the first time that the CT83 PEP90-98 epitope has been demonstrated to induce T cell responses and inhibit tumor growth in vitro and in vivo.

[0334] A2-CT83 TCR was identified and characterized using single-cell barcoding technology. To identify TCRs specific to CT83, A2-CT83-specific T cells were stimulated with 293T / CT83 cells and intracellularly stained with anti-IFN-γ. The A2-CT83-specific T cells were then purified by FACS sorting. Figure 8A Purified T cells (thousands) were partitioned into nanoscale gel emulsion beads (GEMs) on a Chromium Next GEM chip G processed in a 10x Genomics Chromium controller. Following multiple steps of cell lysis, reverse transcription (RT), PCR application, and barcoded cDNA construction of TCR V(D)J libraries according to the manufacturer's protocol, the final enriched TCR V(D)J libraries were sequenced using an Illumina sequencer (HiSeq 2500). After TCR sequence alignment and analysis, dominant and subdominant paired TCRα and TCRβ were identified, and 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 (U3 promoter driven). Figure 8AThe results showed that A2-CT83 TCR-T cells could recognize 293T cells transfected with CT-83-GFP and Cos-7 cells tra...

Claims

1. A composition comprising a chimeric antigen receptor (CAR), a chimeric T-cell receptor (chimeric TCR), or a T cell expressing a CAR or a chimeric TCR, wherein the CAR or TCR comprises an antigen recognition component, a transmembrane domain, and an intracellular T-cell activation component, wherein: (a) the antigen recognized by the antigen recognition component can be a surface protein that can be recognized by a single-chain antibody fragment (scFv), which can be selected from the group consisting of: Alpha (a)-fetoprotein (AFP), Interferon-inducible protein absent in melanoma 2 (AIM2), Adenocarcinoma antigen recognized by T cells 4 (ART-4), BCMA, B antigen (BAGE), CTL-recognized antigen on melanoma (CAMEL), Carcinoembryonic antigen peptide-1 (CAP-1), Caspase 8 (CASP8), Cell division cycle 27 (CDC27), Cyclin-dependent kinase 4 (CDK4), CDK12, Carcinoembryonic antigen (CEA), Calcium-activated chloride channel 2 (CLCA2), CFTR, Cytomegalovirus (CMV), Cancer-testis antigen 83 (CT83), Desmin, DLK1, DLL3, Epstein-Barr virus (EBV), Epidermal growth factor receptor type III variant (EGFRvIII), EGFR and isoforms thereof, EGFR E746-A750del, EGFRVIII, Epithelial-specific antigen (ESA), Epithelial cell adhesion molecule (EpCAM), Ephrin type-A receptor 2 and 3 (EphA2, EphA3), Epithelial glycoprotein 2 (EGP2), Epithelial glycoprotein-40 (EGP-40), Epithelial membrane protein (EMA), Epithelial tumor antigen (ETA), Fos-related antigen 1 (Fos-related antigen 1, FRa1), Ganglioside GD3, G protein-coupled receptor class C group 5, member 1 (GPRC5D), Her-2 / neu, Human telomerase reverse transcriptase (hTERT), Human epidermal growth factor receptor 2 (HER2), Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (HLA)-A2, Human leukocyte antigen (antigen, ETA), Fibronectin (FN), Fibroblast growth factor-5 (FGF-5), Fibroblast growth factor-6 (FGF-6), G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, N-Acetylglucosaminyltransferase V (GnT-V), glycoprotein 100 (GP100), Helicase antigen (HAGE), H3.3K27M, oncofetal antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, Human epidermal receptor 2 / neu (HER2 / neu), Hepatitis B virus (HBV), Human endogenous retrovirus-E (HERV-E), HIV-1 gag, HMI.24, HMB-45 antigen, Human papillomavirus E6 (HPV E6), HPV E7, HPV-16 E6, HPV-16 E7, human telomerase reverse transcriptase (hTERT), Kirsten rat sarcoma viral oncogene homolog (KRAS), KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, L1 cell adhesion molecule (LI-CAM), melanoma-associated antigen (MAGE), Melanoma antigen A1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, c-Met, MICA / B, muscle-specific actin (MSA), Melanoma antigen Melan-A (also MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC1 core peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide, MUC1* peptide,1, MUC1), MUC2, Mucin 16 (MUC-16), myo-D1, Pyruvate Kinase M2 Type Isoenzyme Dimer (Tumor Type M2-PK), Necl-2, Neurofilament protein, NKCSI, NKG2D, Neuron-specific enolase (NSE), NY-ESO, New York esophageous 1 (NY-ESO-1), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific antigen (PSA), Prostate-specific membrane antigen (PSMA), Renal antigen (RAGE), Ral-B, Abnormal ras protein, ROR1, SLAMF7 / CS1, Sperm protein 17 (Sp17), Sarcoma antigen (SAGE), Squamous antigen rejection tumor 1, 2, 3 (SART-1, SART-2, SART-3), SOX10, Synovial sarcoma-associated antigen, X breakpoint 2 (SSX-2), Survivin, OVA1, HE4, DR-70, Total PSA, alpha-Methylacyl-CoA Racemase (AMACR), CA125 / MUC16, Estrogen receptor alpha (ERa / NR3A1), Estrogen receptor beta (ERb / NR3A2), Thymidine kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Keratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF receptor (NGF R / TNFRSF16), NM23-H1, PARP, PP4, Serpin E1 / PAI-1, 14-3-3 beta, 14-3-3 sigma, 14-3-3 zeta, 15-PGDH / HPGD, 5T4, TIM-3, TROP-2, Nectin-4, PD-1, PD-L1, CTLA-4, PDGFRa, VEGF, TRAG-3, T cell receptor gamma alternate readingframe protein, TARP), TGFβII, thyroglobulin, abnormal p53 protein, TP53 (p53), TRAIL, tyrosinase-related protein 1 or gp75 (TRP1), TRP2, TYRP1, tyrosinase, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TLR4, TRBC1, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, Vα24, Wilms tumor gene (WT1), CD1a, CD1b, CD1c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16 (FcyRIII), CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 (FcyRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrinCD62L, CD63, CD64 (FcyRI), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Ig a), CD79b (Ig b), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (Endoglin), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BDCA3), CD146, CD152 (CTLA-4), CD158 (KIR), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC-SIGN), CD223, CD235, CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF-R), CD273 (B7-DC, PD-L2), CD276 (B7-H3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), HLA-DR, kappa light chain, lambda light chain, Pax-5, BCL-2, Ki-67, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, fibrin / fibrinogen degradation products (DR-70), AFP-L3, circulating tumor cells (EpCAM, CD45, keratin 8, 18, 19), prostate stem cell antigen (PSCA), a2b1, prostatic acid phosphatase (PAP), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromagranin, CLEC12A, cytomegalovirus-infected cell antigens (e.g., cell surface antigens), CS-I, CSPG4, keratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), LewisY (LeY), estrogen receptor (ER), progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, free PSA, thyroglobulin, nuclear mitotic apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, aldehyde-ketone reductase 1C3 (AKR1C3), α1B-glycoprotein, α1-microglobulin, αB-crystallin (CRYAB), AMACR, AMFR / gp78, Annexin A3, Annexin A8, APC, apolipoprotein AI, A-II, E, APRIL / TNFSF13, ASCL1, ATBF1 / ZFHX3, Attractin, Aurora A, BAP1, Bcl-2, Bcl-6, β2-microglobulin, B3GAT1, β-catenin, β-III tubulin, Bikunin, BMI-1, B-Raf, Brk, C4.4A, c-Abl, Cadherin-13, Caldesmon, Calponin-1, Calretinin, CA9, Catalase, Cathepsin D, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, Chitinase-3-like-1, CCKBR, Human Chorionic Gonadotropin Alpha Chain and Alpha / β, CKAP4 / p63, Claudin-18, Clusterin, c-Maf, c-Myc, CotL1, COMMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1, CXCL17, CXCL8, CXCL9, CXCR4, CyclinA1, A2, D2, D3, CYLD, CCN1, Cytokeratin 14, 18, 19, Fetal Acetylcholine Receptor, ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2, DC-LAMP, Dkk-1, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD105, Endosialin / CD248, Enolase-2, EpCAM / T ROP1, Eps15, ERBB, ERBB2, ErbB3, ErbB4, ERCC1, ERK1, ERK5, Ets-1, Exostosin-1, EZH2, Ezrin, FABP5, Fascin, FATP3, FCRLA, Fetuin-A, Acid / Alkaline FGF, FGFR3, FGFR4, Fibrinogen, Folate-binding protein, FAP, FSTL1, FOLR1-4, FosB, FoxM1, FoxO3, FRAT2, FXYD5, FcεRIα, FLT3, GABA-A receptorα1, GADD153, GADD45α, Galectin-3, Galectin-3BP, gangliosides, GCDFP-15, GD2, GD3, GM2, GM3, CRF21, Gas1, GRPR, Gastrokine-1, Gelsolin, GFAP, GLI-2, GPX3, gpA33, glycopeptides, GPC2, GPC3, GLG1, gp96 / HSP90B1, G PR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, Heparanase, Hepsin , HGF-R / c-MET, HIF-2α, HOXB13, HOXB7, HSP70, HSP90, HYAL1, ID1, IgE, IGFBP-2, -3, -4, -6, IGF-I, I GF-IR, IGF-II, IGFL-3, IGFLR1, IL-1β, IL-17E, IL-2, IL-6, ICAM-1, immunoglobulin, IL-13Ra / IL-13Ra2, integrin , IMPDH1, KPNA2, ING1, CD29, CD61, IQGAP1, IDH1, ITIH4, ITM2C, Jagged-1, JNK, JunB, JunD, OGR1, Ol ig2, Osteopontin, Ovastacin, OXGR1, p130Cas, CDKN2B, CDKN2A, CDKN2C, CDKN1A, p27, P2RX5, PARP, PAUF, Visfatin, PDCD4, PDCD5, PDGFRα / β, PDZD2, PEA-15, PGA5, PI16, Peroxiredoxin-2, PGCP, PI3Kp85α, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin-1, progesterone receptor, PGRN, Prolactin, PTGES2, PSAP, PSCA, PSMA, PSMA1 / 2, PSMB7, PSP94, PTEN, PTH1R, PTK7, PTPRZ, Rab25, RARRES1 / 3, Ras, Reg4, Ret, RNF2, RNF43 S100 family, SART1, SCUBE3, Secretin-R, Serpin-A9, Serpin-E1, serum amyloid, SEZ6L, SEZ6L2, Skp2, SLC16A3, SLC45A3, SLC5A5, SLC5A8, SLC7A7, Smad4, SMAGP, SOCS-1 / -2 / -6, Mn-SOD, DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC L1, SPINK1, Src, STEAP1 / 2 / 3, STRO-1, STYK1, Survivin, Synaptotagmin-1, Syndecan-1, Syntaxin-4, Synuc lein-γ, Synaptophysin, Kallikrein-2, Kallikrein-6, SLC12A5, KiSS1R, KLF10, KLF17, L1CAM, LDHA, Lamin -B1, LEF1, Leptin, LIN-28A / B, NGAL, LKB1, LPAR3, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / 3, Ly6K, LYPD1, LYPD 8. MAP2, ST14, MCAM, M-CSF, MDM2, MC1R, CD228, Melatonin, Mer, Mesothelin, Metadherin, KiSS1, Methionineaminopeptidase, METAP2, MFAP3L, MGMT, MIA, MIF, MINA, MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1 / -2 / -3 / -8 / -9 / -10 / -13, MRP1, ABCC4, MS4A12, MSH2, Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin-A, NCAM-1, NC OA3, NDRG1, NEK2, NELL1 / 2, Nucleobindin-2, Nestin, NF-κB2, NF-L, NG2, NNMT, NKX2.2, NKX3.1, NM23-H1 / H 2. Notch-3, NPDC-1, NTSR1 / 2, Tankyrase-1, Tau, TCF-3, TCL1A / B, PLXDC1, ANTXR1, Tenascin-C, TFF1, TGF-β Family of thyroxine, ALK-5, THRSP, Thymidine kinase-1, Thymosin β10 / β4, thyroglobulin, TIMP-1 / -2 / -3 / -4, TLE1 / 2, TM4SF1, TMEFF2, TMEM219, TMEM87A, TNF-α, TOP2A, TopBP1, tPA, TRA-1-60, CD147, TRAF-4, Transgelin, PRSS2, TPS1, TSPAN1, UBE2S, uPAR, Urokinase, Urotensin-II receptor, VAP-1, VCAM-1, VEGFR1 / 2, VEGF / PlGF heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1, SGK, CNKSR1, or any combination thereof; (b) The intracellular T cell activation component further comprises a signal transduction domain, wherein the signal transduction domain comprises a ZAP70 kinase domain or a mutant or variant thereof.

2. The composition according to claim 1, wherein the ZAP70 kinase domain or its mutants or variants comprise ZAP255 (SEQ ID NO:64, amino acid sequence 255-619), ZAP280 (SEQ ID NO:65, amino acid sequence 280-619), ZAP300 (SEQ ID NO:1, amino acid sequence 300-619), ZAP327 (SEQ ID NO:17, amino acid sequence 327-619), ZAP338 (SEQ ID NO:52, amino acid sequence 338-619), or a polypeptide having at least 70% identity with SEQ ID NO:16, 17, 52, 64 or 65, or its mutants or variants.

3. The composition according to claim 1, wherein the composition comprises: a) at least one polypeptide comprising an α-chain variable region of a T-cell receptor specific to NY-ESO-1 (A2-ESO-1 TCR) and at least one polypeptide comprising a β-chain variable region of a T-cell receptor specific to NY-ESO-1 (A2-ESO-1 TCR). b) At least one polypeptide containing an α-chain variable region of a T-cell receptor specific to NY-ESO-1 (DP4-ESO-1 TCR) and at least one polypeptide containing a β-chain variable region of a T-cell receptor specific to NY-ESO-1 (DP4-ESO-1 TCR). c) At least one polypeptide comprising an α-chain variable region of a T-cell receptor specific to CT83 (A2-CT83 TCR) and at least one polypeptide comprising a β-chain variable region of a T-cell receptor specific to CT83 (A2-CT83 TCR); d) At least one polypeptide containing an α-chain variable region of a T-cell receptor specific to CT83 (DR13-CT83 TCR) and at least one polypeptide containing a β-chain variable region of a T-cell receptor specific to CT83 (DR13-CT83 TCR); e) at least one polypeptide comprising an α-chain variable region of a T-cell receptor specific to CT83 (PEP4-12 TCR, PEP6-14 TCR, PEP10-31 TCR, PEP17-31 TCR, PEP90-98 TCR) and at least one polypeptide comprising a β-chain variable region of a T-cell receptor specific to CT83 (PEP4-12 TCR, PEP6-14 TCR, PEP10-31 TCR, PEP17-31 TCR, PEP90-98 TCR); f) at least one polypeptide comprising an α-chain variable region of a T-cell receptor specific to HCMV pp65 (HCMV pp65 TCR) and at least one polypeptide comprising a β-chain variable region of a T-cell receptor specific to HCMV pp65 (HCMV pp65 TCR). g) At least one polypeptide containing an α-chain variable region of a T-cell receptor specific to HCMV IE-1 (HCMV IE-1-TCR) and at least one polypeptide containing a β-chain variable region of a T-cell receptor specific to HCMV IE-1 (HCMV IE-1-TCR).

4. The composition of claim 3, wherein the C-terminus of the α-chain or β-chain of the T-cell receptor (TCR) is fused with a signaling component comprising a ZAP255, ZAP280, ZAP300, ZAP327 or ZAP338 kinase domain, or fused with a mutant or variant thereof.

5. The composition of claim 3, wherein at least one polypeptide comprises an α-chain variable region of a T-cell receptor specific to CT83 and at least one polypeptide comprises a β-chain variable region of a T-cell receptor specific to CT83. The TCR-specific polypeptide comprises the amino acid sequence SILCALIVFWKYRRFQRNTGEM (CT83 amino acids 10–31, SEQ ID NO: 39) or comprises the amino acid sequence VFWKYRRFQRNTGEM (CT83 amino acids 17–31, SEQ ID NO: 61).

6. The composition of claim 3, wherein the composition comprises an α-chain variable region and a β-chain variable region of an HLA-A2-restricted HCMV pp65 T-cell receptor (TCR), wherein the variable region of the HLA-A2-restricted HCMV IE-1 TCR comprises: the amino acid sequence of the α-chain variable region SEQ ID NO: 27 and the amino acid sequence of the β-chain variable region SEQ ID NO: 29; wherein, The α-variable region or a variant thereof binds the antigen with the same specificity as the reference (full-length and unmodified) receptor; a polypeptide having an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, or having one, two, or more amino acid substitutions relative to SEQ ID NO: 27 or SEQ ID NO: 29, or having one, two, or more amino acid substitutions with SEQ ID NO: 27 or SEQ ID NO: 29 in the CDR1, CDR2, and / or CDR3 regions, and having at least 85% identity with the amino acid sequence; and further optionally, the composition further comprises a β-chain variable region of an HLA-A2-restricted HCMV pp65 TCR, comprising a polypeptide having the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30, wherein the β-chain variable region or a variant thereof binds the antigen with the same specificity as the reference (full-length and unmodified) receptor; and the variant comprising an amino acid sequence having the same amino acid sequence as SEQ ID NO: 28 or SEQ ID NO:

30. NO:30 is a polypeptide having at least 85% amino acid sequence identity; or a polypeptide having one, two, or more amino acid substitutions relative to SEQ ID NO: 28 or SEQ ID NO: 30; or a polypeptide having one, two, or more amino acid substitutions relative to SEQ ID NO: 28 or SEQ ID NO: 30 in the CDR1, CDR2, and / or CDR3 regions and having at least 85% identity with the sequence; wherein the TCR further optionally comprises SEQ ID NO: 27 and SEQ ID NO: 28, or further optionally comprises SEQ ID NO: 29 and SEQ ID NO:

30.

7. The composition of claim 3, wherein the composition comprises α- and β-chain variable regions of a T-cell receptor (TCR) specific to NY-ESO-1 or CT83 antigen, wherein if the TCR is specific to NY-ESO-1, the α-variable region of the HLA-DP4 NY-ESO-1 (DP4-ESO-1) TCR optionally comprises the following: The following are listed: A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 DP4-ESO-1 TCR, wherein the α-chain variable region or a variant thereof has the same specific binding antigen as a reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 3; a polypeptide comprising a peptide having one, two, or more amino acid substitutions relative to the sequence of SEQ ID NO: 3 in the CDR1, CDR2, and / or CDR3 regions (e.g., D95S or Q98Y substitutions occurring in the CDR3 sequence GADIVDYGQNFV (SEQ ID NO: 89) of TCR-Vα, wherein the amino acid site number is determined based on the mature TCR sequence), or a polypeptide having one, two, or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 3 in the CDR1, CDR2, and / or CDR3 regions; and a polypeptide comprising a peptide having one, two, or more amino acid substitutions relative to the .... If the TCR is specific for NY-ESO-1, the β-chain variable region of the HLA-DP4 NY-ESO-1 (DP4-ESO-1) TCR optionally comprises the following: a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, wherein the β-chain variable region or a variant thereof has the same specific binding antigen as a reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 4; or a polypeptide comprising one, two, or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 4 within the CDR1, CDR2, and / or CDR3 regions (e.g., Y98L or Y98M substitution in the CDR3 sequence AWRRGYEQY (SEQ ID NO: 90) of TCR-Vβ), or a polypeptide comprising one, two, or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 4 within the CDR1, CDR2, and / or CDR3 regions; If the TCR is specific for CT83, the α-variable region optionally comprises an A2-CT83 TCR polypeptide, wherein the α-chain variable region of the A2-CT83 TCR optionally comprises: an A2-CT83 TCR polypeptide comprising the amino acid sequence of SEQ ID NO:5, wherein the α-variable region or a variant thereof binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:5; or a polypeptide comprising, within the CDR1, CDR2, and / or CDR3 regions, having one, two, or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:5, or having one, two, or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:5 within the CDR1, CDR2, and / or CDR3 regions. The β-chain variable region of the T-cell receptor (TCR) specific for the CT83 antigen optionally comprises: an A2-CT83 TCR polypeptide comprising the amino acid sequence of SEQ ID NO: 6, wherein the β-chain variable region or a variant thereof binds the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 6; or a polypeptide comprising, within the CDR1, CDR2, and / or CDR3 regions, having one, two, or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 6, or having one, two, or more amino acid substitutions within the CDR1, CDR2, and / or CDR3 regions relative to an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:

6.

8. The composition of claim 3, wherein the composition comprises an α-chain variable region of an HLA-A2-restricted HCMV IE-1 T-cell receptor (TCR), wherein the α-chain variable region of the HLA-A2-restricted HCMV IE-1 TCR optionally comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 32; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 32; a polypeptide having one, two, or more amino acid substitutions relative to SEQ ID NO: 32 in the CDR1, CDR2, and / or CDR3 regions, or a polypeptide having two or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with SEQ ID NO: 32 in the CDR1, CDR2, and / or CDR3 regions; Optionally, the composition further comprises a β-chain variable region of an HLA-A2-restricted HCMV IE-1 TCR, wherein the β-chain variable region of the HLA-A2-restricted IE-1 TCR optionally comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 33; a polypeptide comprising an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 33; a polypeptide having one, two, or more amino acid substitutions relative to SEQ ID NO: 33 in the CDR1, CDR2, and / or CDR3 regions, or a polypeptide having one, two, or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with SEQ ID NO: 33 in the CDR1, CDR2, and / or CDR3 regions.

9. The composition of claim 3, wherein the composition comprises an α-chain variable region of an HLA-DR13-restricted DR13-CT83 T cell receptor (TCR), wherein the α-chain variable region of the HLA-DR13-restricted DR13-CT83 TCR optionally comprises: a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79 and SEQ ID NO: 81; a polypeptide comprising an amino acid sequence having at least 85% identity with any of the above sequences; a polypeptide comprising having one, two or more amino acid substitutions relative to any of the above sequences in the CDR1, CDR2 and / or CDR3 regions, or a polypeptide comprising two or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with any of the above sequences in the CDR1, CDR2 and / or CDR3 regions; Optionally, the composition further comprises a β-chain variable region of an HLA-DR13-restricted DR13-CT83 TCR, wherein the β-chain variable region of the HLA-DR13-restricted DR13-CT83 TCR optionally comprises: a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 82; a polypeptide comprising an amino acid sequence having at least 85% identity with any of the above sequences; or a polypeptide comprising a CDR1, CDR2, and / or CDR3 region having one, two, or more amino acid substitutions relative to any of the above sequences, or a polypeptide comprising a CDR1, CDR2, and / or CDR3 region having one, two, or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with any of the above sequences.

10. The composition of claim 9, wherein the α-chain variable region specific to DR13-CT83 comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the following SEQ ID NO: 54, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81, or having 85%–100% identity with the amino acid sequences thereof; and the β-chain variable region of the TCR comprises a polypeptide having 85%–100% identity with the following SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 81, SEQ ID NO: 9 ... The amino acid sequences of NO: 78, SEQ ID NO: 80 and SEQ ID NO: 82 have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, or are polypeptides with amino acid sequences having 85%–100% identity.

11. A nucleic acid, vector, or cell comprising a nucleic acid or vector encoding any polypeptide sequence in the composition of claim 1 or 2.

12. The composition of claim 1, wherein the chimeric antigen receptor (CAR), chimeric T cell receptor (chimeric TCR), or CAR / chimeric TCR expressed in T cells is further fused with a chemokine receptor to enhance T cell migration. The chemokine receptor is selected from the group consisting of CCR5, CCR2, CXCR3, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, common cytokine receptor γ chain, or other chemokine receptors.

13. A chimeric T-cell receptor polypeptide comprising a cancer antigen-specific TCR variable region fused to a constant region, said constant region being selected from modified human TCR α or β constant regions and non-human TCR α or β constant regions, optionally from mouse TCR α or β constant regions, wherein the α and β variable regions of said TCR variable region fused to a modified or non-human α or β constant chain region comprise: a. An α-chain variable region of an HLA-A2-restricted HCMV pp65 TCR comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 27, and a polypeptide containing the β-chain variable region of SEQ ID NO: 29, wherein the α-chain variable region or a variant thereof has the same specific binding antigen as a reference (full-length and unmodified) receptor; comprising a polypeptide having at least 85% identity with the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, or a polypeptide having one, two or more amino acid substitutions relative to said sequence, or a polypeptide having one, two or more amino acid substitutions relative to an amino acid sequence having at least 85% identity with said sequence in the CDR1, CDR2 and / or CDR3 regions; and Further optionally, the composition comprises a β-variable region of an HLA-A2-restricted HCMV pp65 TCR containing a polypeptide of the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30, wherein the β-variable region or a variant thereof is a specific binding antigen identical to that of a reference (full-length and unmodified) receptor; a polypeptide containing an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30; or a polypeptide having one, two, or more amino acid substitutions for the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30; or a polypeptide having one, two, or more amino acid substitutions for the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30 within the CDR1, CDR2, and / or CDR3 regions. The TCR further optionally comprises SEQ ID NO:27 and SEQ ID NO:28 or further optionally comprises SEQ ID NO:29 and SEQ ID NO:30; or b. α and β variable regions of a TCR specific to cancer antigens selected from NY-ESO-1 and CT83, wherein if the TCR is specific to NY-ESO-1, the α variable region optionally comprises a DP4-ESO-1 TCR polypeptide containing the amino acid sequence of SEQ ID NO: 3, the α variable region or a variant thereof binding antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide containing amino acids having at least 85% identity with the amino acid sequence of SEQ ID NO: 3; a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions (e.g., D95S or Q98Y substitutions occurring in the CDR3 sequence GADIVDYGQNFV (SEQ ID NO: 89) of TCR-Vα, the amino acid numbering determined according to the mature TCR sequence), or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions of SEQ ID NO:

3. The sequence of NO:3 has at least 85% amino acid identity, and contains polypeptides with one, two, or more amino acid substitutions. The β variable region of the DP4-ESO-1 TCR optionally comprises a polypeptide or a variant thereof of the amino acid sequence of SEQ ID NO: 4, which binds the antigen with the same specificity as the reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence thereof; or a polypeptide comprising having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions (e.g., Y98L or Y98M substitution in the CDR3 sequence AWRRRRGYEQY (SEQ ID NO: 90) of TCR-Vβ). If the TCR is specific for CT83, the α variable region optionally comprises: an A2-CT83 TCR polypeptide or a variant thereof comprising the amino acid sequence of SEQ ID NO: 5, which binds the antigen with the same specificity as the reference (full-length and unmodified) receptor; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 5; a polypeptide having one, two, or more amino acid substitutions for the amino acid sequence of SEQ ID NO: 5 in the CDR1, CDR2, and / or CDR3 regions, or a polypeptide comprising one, two, or more amino acid substitutions for an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 5 in the CDR1, CDR2, and / or CDR3 regions, and The β-variable region of the CT83 TCR optionally comprises: an A2-CT83 TCR polypeptide or a variant thereof comprising the amino acid sequence of SEQ ID NO: 6, which binds the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide comprising the amino acid sequence within the CDR1, CDR2, and / or CDR3 regions of SEQ ID NO: 6, a polypeptide having one, two, or more amino acid substitutions for the amino acid sequence of SEQ ID NO: 6 within the CDR1, CDR2, and / or CDR3 regions, or a polypeptide having one, two, or more amino acid substitutions for an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 6; or c. The α-variable region of an HLA-A2-restricted HCMV IE-1 TCR, wherein the α-variable region of the HLA-A2-restricted HCMV IE-1 TCR optionally comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 32; a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 32; a polypeptide comprising having one, two, or more amino acid substituents for SEQ ID NO: 32 in the CDR1, CDR2, and / or CDR3 regions; or a polypeptide comprising one, two, or more amino acid substituents for an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 32 in the CDR1, CDR2, and / or CDR3 regions; and The composition thereof comprises a β-variable region of an HLA-A2-restricted IE-1 TCR, wherein the β-variable region of the HLA-A2-restricted IE-1 TCR optionally comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 33; a polypeptide comprising an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 33; a polypeptide having one, two, or more amino acid substitutions for the sequence of SEQ ID NO: 33 in the CDR1, CDR2, and / or CDR3 regions; or a polypeptide having one, two, or more amino acid substitutions for the amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 33 in the CDR1, CDR2, and / or CDR3 regions; or d. The α-chain variable region of an HLA-DR13-restricted DR13-CT83 TCR, wherein the α-chain variable region of the HLA-DR13-restricted DR13-CT83 TCR optionally comprises: a polypeptide comprising the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81, or a polypeptide comprising an amino acid sequence having at least 85% identity with any of the above sequences, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions; and, The chimeric TCR may optionally further comprise a β-chain variable region of an HLA-DR13-restricted DR13-CT83 TCR, wherein the β-chain variable region optionally comprises: a polypeptide comprising the amino acid sequences of SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 and SEQ ID NO: 82, or a polypeptide comprising at least 85% identity with any of the above sequences, or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2 and / or CDR3 regions.

14. A composition comprising a chimeric antigen receptor (CAR), a chimeric T-cell receptor (chimeric TCR), or a T cell expressing a CAR or a chimeric TCR, wherein the CAR or TCR comprises an antigen recognition component, a transmembrane domain, and an intracellular T-cell activation component, wherein the intracellular T-cell activation component comprises a co-stimulatory signaling domain fused to a signal transduction domain, and the co-stimulatory signaling domain may be optionally selected from CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CD8α, CD8β, CD11a, CD4, CD5, CD7, CD8α, CD8β, CD11a, CD4, CD5, CD7, CD8α, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD8β, CD9 ... 11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD5 4), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG-3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, Lymphocyte Function-Associated Antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), DAP10, DAP12, LAG-3, 2B4, CARD1, CTLA-4 (CD152), TRIM, ZAP70, FcεRI γ, 4-1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACI, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptor (TLR),For example, TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR2DS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, TNFR2, TRANCE / RANKL, VLA-1, VLA-6, BTLA, Ikaros, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, TIM-4, TSLP, or any combination thereof, and the signal transduction domain further comprises a ZAP70 kinase domain or a mutant or variant thereof.

15. The composition of claim 14, wherein the intracellular T cell activating component comprises a ZAP70 kinase domain or a mutant or variant thereof, and the activating component is constituted by replacing CD3ζ with a ZAP70 kinase domain or a mutant or variant thereof, or is derived from a functional wild-type ZAP70 or a ZAP70 kinase domain derived from a mutant or variant thereof; wherein, The ZAP70 kinase domain derived from a functional wild-type ZAP70 or its mutant or variant further comprises a functional ZAP70 kinase domain or its mutant or variant.

16. The composition of claim 15, wherein the functional ZAP70 kinase domain or a mutant or variant thereof comprises: a. ZAP300 (SEQ ID NO: 16); b. ZAP327 (SEQ ID NO: 17); c. ZAP338 (SEQ ID NO: 52); d. ZAP255 (SEQ ID NO: 64); e. ZAP280 (SEQ ID NO: 65); f. A truncated but biologically active fragment of ZAP70 kinase, comprising an amino acid sequence having at least 70% identity with any of the amino acid sequences in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 52, SEQ ID NO: 64, and SEQ ID NO: 65; or g. Mutants or variants thereof, wherein the mutants or variants are truncated but biologically active fragments containing a functional ZAP70 kinase domain; or h. Biologically active mutants or variants of ZAP70, wherein the biologically active mutants or variants comprise N-terminal amino acids starting at 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 299 of the ZAP70 kinase. 5, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338, and their C-terminal amino acid ends at amino acid 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 of the ZAP70 kinase.

17. The composition of claim 14, wherein the antigen recognition component comprises an antigen-specific antibody, an antigen-binding fragment, an antibody mimic, a bispecific antibody, a trispecific antibody, a multivalent polymeric binding protein, a ligand of a protein receptor or a specific receptor, a TCR-like antibody, or a bispecific binding protein comprising a TCR-like antibody and an anti-CD3 antibody or a binding fragment thereof.

18. The composition of claim 17, wherein the antibody, antigen-binding fragment, antibody mimic, protein receptor or ligand of a specific receptor, or TCR-like antibody comprises scFv fragment, Fv fragment, Fd fragment, Fab fragment, Fab′ fragment, F(ab′)2 fragment, VH domain, VL domain, monoclonal antibody, polyclonal antibody, nanobody, bispecific or triple antibody fusion protein, or any combination thereof.

19. The composition of claim 17, wherein the antigen recognizable by the antigen recognition component is selected from the group of antigens comprising the following components recognizable by single-stranded variable fragments: Alpha (α)-fetoprotein (AFP), interferon-induced protein 2 (AIM2) missing in melanoma, adenocarcinoma antigen 4 (ART-4) recognized by T cells, BCMA, B antigen (BAGE), melanoma antigen (CAMEL) recognized by CTLs, carcinoembryonic antigen peptide-1 (CAP-1), caspase 8 (CASP8), cyclin 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDK12, carcinoembryonic antigen (CEA), calcium-activated chloride channel protein 2 (CLCA2), CFTR, cytomegalovirus (CMV), cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, Epstein-Barr virus (EBV), epidermal growth factor receptor type III variant (EGFRvIII), EGFR and its isoforms, EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), Ephrin Human epidermal growth factor receptor 2 and / or 3 (EphA2, EphA3), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), fibronectin (FN), fibroblast growth factor 5 and 6 (FGF-5, FGF-6), G antigen 1 and its family members (GAGE-1 to GAGE-7), N-acetylglucosamine transferase V (GnT-V), glycoprotein 100 (GP100), helicase antigen (HAGE), H3.3K27M, tumor embryonic antigen h5T4, IP3KB, influenza hemagglutinin (HA, HA-1, HA-1H, HA-2), human epidermal growth factor receptor 2 (HER2 / neu), hepatitis B virus (HBV), HERV-E, HIV-1 gag, HMI.24, HMB-45 antigen, human papillomavirus E6 / E7 (including HPV-16) E6 / E7), human telomerase reverse transcriptase (hTERT), KRAS and its mutants (including KRAS)G12D and G12V), L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, L1 cell adhesion molecule (LI-CAM), melanoma-associated antigen (MAGE and its subtypes), c-Met, MICA / B, muscle-specific actin (MSA), Melan-A / MART-1, mesothelin (MSLN), mucin 1, 2 and 16 (MUC1, MUC2, MUC16), myo-D1, tumor-type M2-PK, Necl-2, neurofilament protein, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO-1, melanoma preferentially expressed antigen (PRAME), prostate-specific antigen (PSA). A) Prostate-specific membrane antigen (PSMA), renal antigen (RAGE), Ral-B, abnormal ras protein, ROR1, SLAMF7 / CS1, spermin 17 (Sp17), sarcoma antigen (SAGE), squamous cell antitumor antigens (SART-1, SART-2, SART-3), SOX10, SSX-2, Survivin, OVA1, HE4, DR-70, α-methylacyl-CoA racemic enzyme (AMACR), CA125, estrogen receptor α or β, thymidine kinase 1, BRCA1, BRCA2, Caveolin-1, CEACAM-5, keratin 14, Ki-67, Nestin, NM23-H1, PARP, Serpin E1, 14-3-3 protein family, 5T4, TIM-3, TROP-2, Nectin-4, PD-1, PD-L1, CTLA-4, PDGFRα, VEGF, WT1, HLA-DR, CD series molecules, immunoglobulin light chain, Pax-5, MPO, TdT, FMC-7, ROMA, circulating tumor cell-associated markers, prostate stem cell antigen (PSCA), carbonic anhydrase IX (CAIX), CLEC12A, cytomegalovirus-infected cell antigen, CSPG4, AC133, c-Kit, Lewis A or Lewis Y antigen, estrogen receptor, progesterone receptor, nuclear mitotic apparatus protein (NuMA / NMP22), ABCB5, ABCB6, ABCG2, ACE / CD143, AKR1C3, αB-lens protein, AMFR, Annexin family, apolipoproteins, APRIL, ASCL1, Aurora A, BAP1, β2-microglobulin, β-catenin, BMI-1, B-Raf, Cadherin-13, Calponin-1, CA9, Catalase, CathepsinD, CCR family, CHD1L, Chitinase-3-like-1, Claudin-18, COX-2, CTCF, CXCR4, Cyclin family, CYLD, Ezrin, FABP5, FAP, FOLR family, FoxM1, GFAP, GPC2, GPC3, HGF-R / c-MET, HIF-2α, HOXB13, HSP70, HSP90, IDH1, IL-2, IL-6, ICAM-1, IGF axonin, Integrin family, IQGAP1, Jagged-1, JNK, Osteopontin, PARP, PDCD4, PDGF receptor, PI3K, PKM2, PLK1, PSMA, PTEN, Rab25, Ras, Ret, S100 protein family, SCUBE3, SOX family, SPARC, STEAP1, Survivin, TGF-β family, Thymosin β, TIMP family, TNF-α, TOP2A, TRAF-4, uPAR, VEGFR1 / 2, YAP1, ZAG, ZAP70, ZMIZ1, or any combination thereof.

20. A method for expressing the CAR or TCR of claim 1 in immune cells to achieve functional activation of the immune cells, wherein the immune cells may be selected from T cells, CD4 cells, etc. + T cells, CD8 + A cell group consisting of T cells, NK cells, NKT cells, and macrophages.

21. The method of claim 20, wherein the composition of CAR-T or TCR-T cells of claim 20 is a pharmaceutical composition.

22. A method for treating cancer, inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related diseases, or infectious diseases, comprising administering to a subject suffering from or suspected of suffering from the aforementioned diseases a composition comprising the CAR-T or TCR-T cells of claim 16.

23. A method for prolonging T cell persistence or reducing T cell exhaustion in a subject by modulating TCR-T cell signaling and function, comprising administering to the subject a composition containing TCR-T cells or CAR-T cells according to claim 14 or 20, wherein said modulation is achieved by regulating the signal transduction domain of the TCR or CAR, or by knocking down or knocking out a negative signaling molecule selected from the following molecular group: Contains: ANKRDJ1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PD CDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine-2,3-dioxygenase (2,3)-dioxygenase (IDO, including IDO1 and IDO2 isoforms), OX40, CTLA-4 (programmed cytotoxic T lymphocyte-associated antigen 4), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic factors, wherein the epigenetic factors may or may not include JMJD3 and LSD1.

24. The composition of claim 14, wherein the CAR or TCR is fused with a chemokine receptor, the chemokine receptor being selected from CCR5, CCR2, CXCR3, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, common cytokine receptor γ chain, or other chemokine receptor groups.

25. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 12 or 14 and a pharmaceutically acceptable carrier.

26. A method for stimulating an immune response against cancer or for treating, suppressing, and / or preventing cancer, comprising administering to a subject a composition comprising a therapeutically effective amount of the CAR or TCR composition of claim 24 and an isolated nucleic acid encoding shRNA or siRNA (for knocking down a target gene) or sgRNA (for knocking out a target gene) for enhancing the antitumor activity of TCR-transduced T cells in vivo, wherein the nucleic acid targets a negative signaling molecule of the immune system selected from the group consisting of immune checkpoint proteins and / or immunosuppressive proteins, and the target gene of the shRNA or sgRNA is further selected from the group consisting of molecules... Contains: ANKRDJ1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FO XP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SER PINA3, SETD5, SH2B3, SH2DJA, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, von Hippel-Lindau tumor suppressor (VHL, SEQ ID NO: 8), WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO, including IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T lymphocyte-associated antigen 4), PD-1 (programmed death receptor 1, SEQ ID NO: 7), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL (SEQ ID NO: 8), PPP2R2D (SEQ ID NO: 9), and negative regulators, epigenetic factors, or transcriptional regulators, wherein the epigenetic factors may or may not include JMJD3 and LSD1.

27. A composition comprising a T-cell receptor (TCR), a chimeric T-cell receptor (chimeric TCR), a chimeric antigen receptor (CAR), or a cell expressing a TCR or CAR, wherein the TCR or CAR comprises an antigen recognition component, a transmembrane domain, and an intracellular T-cell activation component, wherein: a. The intracellular T cell activation unit comprises a signal transduction domain, and the signal transduction domain comprises a ZAP70 kinase domain or a mutant or variant thereof, or is formed by replacing CD3ζ with a ZAP70 kinase domain or a mutant or variant thereof; b. The intracellular T cell activation unit includes a co-stimulatory signaling domain fused with a signal transduction domain, wherein the co-stimulatory signaling domain is selected from the group consisting of: CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CD8α, CD8β, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49b, CD49c. D. CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, C D103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG-3), CD226, CD229, CD244, CD270 (HVE M), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, Lymphocyte Function-Associated Antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), DAP10, DAP12, LAG-3, 2B4, CARD1, CARD11, CTLA-4 (CD152), MALT-1, MyD88, Toll-like receptors (TLR1, TLR2, TLR3, TLR4 ...

3. TLR4, TLR6, TLR7, TLR8 and TLR9), TRAF family (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 and TRAF6), TRIM, ZAP70, FcεRIγ, 4-1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACI, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrinα4β7, IA4, ICAM-1, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (e.g., TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2 ITGB7, KIR2DS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, TNFR2, TRANCE / RANKL, VLA-1, VLA-6, BTLA, Ikaros, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, TIM-4, TSLP, or any combination thereof; c. Among them, The TCR or CAR is fused with a chemokine receptor, which may optionally be selected from a group consisting of CCR5, CCR2, CXCR3 or other chemokine receptors, to enhance the migration and homing ability of T cells.

28. The composition of claim 27, wherein the ZAP70 kinase domain or a mutant or variant thereof is selected from the group consisting of: ZAP300 (SEQ ID NO: 16), ZAP327 (SEQ ID NO: 17), ZAP338 (SEQ ID NO: 52), ZAP255 (SEQ ID NO: 64), or ZAP280 (SEQ ID NO: 65), or a mutant or variant thereof.

29. The composition of claim 27, wherein the ZAP70 kinase domain or a mutant or variant thereof is a polypeptide comprising an amino acid sequence having at least 70% identity with any of the amino acid sequences of SEQ ID NO: 16 (ZAP300), SEQ ID NO: 17 (ZAP327), SEQ ID NO: 52 (ZAP338), SEQ ID NO: 64 (ZAP255) or SEQ ID NO: 65 (ZAP280).

30. A method for achieving T cell persistence or reducing T cell exhaustion by regulating the composition of claim 27, by regulating the signal transduction and function of a TCR, chimeric TCR, CAR, or cell, or by directly manipulating the signal transduction domains of a TCR or CAR, wherein the TCR or CAR signal transduction domains are regulated by negative signal transduction molecules, wherein the negative signal transduction molecules are knocked down or knocked out, wherein the negative signal transduction molecules are selected from the group consisting of: PD-1, VHL, PPP2R2D, indoleamine (2,3)-dioxygenase (IDO, including IDO1 and IDO2), OX40, CTLA-4, PD-L1, PD-L2, LAG3, B7-H3, and epigenetic factors, wherein the epigenetic factors may or may not include JMJD3 and LSD1, or any combination thereof.

31. A method for prolonging CAR-T or TCR-T cell persistence or reducing T cell exhaustion by replacing CD3ζ with a ZAP255, ZAP280, ZAP300, ZAP327 or ZAP338 kinase domain, or a mutant or variant thereof.

32. The composition according to claim 1 or 14, wherein the transmembrane domain source is selected from the transmembrane domain of any of the following molecules or any combination thereof, Includes: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, A2aR, ICAM-1, 2B4, BILA, DAP10, KIR, KIR2DL4, KIR2DS1, LAG-3, LCK, LAT, LPA5, LRP, FcRα, FcRβ, Fyn, GAL9, cytokine receptors (including IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-21Rα), NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, pTα, T cell receptor peptides (human or mouse TC). Rα, TCRβ, TCRγ, TCRδ), TIM3, TRIM, CD2, CD3D, CD3E, CD3G, CD3ζ, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BILA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD357 (GITR), PTCH2, ROR2, Ryk, SLP-76, SIRPα, ZAP70 or any combination thereof.

33. The composition of claim 32, wherein the intracellular T cell activating component further comprises a signal transduction domain comprising a ZAP70 kinase domain selected from ZAP255, ZAP280, ZAP300, ZAP308, ZAP327, and ZAP338, or a mutant or variant containing ZAP70, wherein the N-terminal amino acid of the mutant or variant begins with 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 3 19, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 or 338, and their C-terminal amino acid terminates at amino acid 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 of ZAP70 kinase.

34. A composition comprising one or more antigens or epitopes described herein that are pathogenically associated with the disease or condition; wherein, The epitope may be selected from the group of epitopes comprising: NY-ESO-1 peptides, including A2-ESO-1 (SEQ ID NO: 2) and DP4-ESO-1 (SEQ ID NO: 1); CT83 peptides, including but not limited to A2-CT83 peptides (90–98) (SEQ ID NO: 2), A2-CT83 PEP4-12 (SEQ ID NO: 37), A2-CT83 PEP6-14 (SEQ ID NO: 36), DR13-CT83 PEP10-31 (SEQ ID NO: 39), DR13-CT83 PEP17-31 (SEQ ID NO: 61); HCMV pp65 (SEQ ID NO: 26); and HCMV IE-1 (SEQ ID NO: 31), or variants thereof.

35. A composition comprising a therapeutically effective amount of an mRNA or DNA sequence (e.g., SEQ ID NO: 83–87), said mRNA or DNA sequence encoding an antigen or epitope as described herein, wherein said antigen or epitope is pathogenically associated with said disease or condition; wherein, The epitope may optionally be selected from the group consisting of: NY-ESO-1 peptides, including A2-ESO-1 (SEQ ID NO: 2) and DP4-ESO-1 (SEQ ID NO: 1); CT83 peptides, including but not limited to A2-CT83 peptides (90–98) (SEQ ID NO: 2), A2-CT83 PEP4-12 (SEQ ID NO: 37), A2-CT83 PEP6-14 (SEQ ID NO: 36), DR13-CT83 PEP10-31 (SEQ ID NO: 39), DR13-CT83 PEP17-31 (SEQ ID NO: 61); HCMV pp65 (SEQ ID NO: 26); and HCMV IE-1 (SEQ ID NO: 31), or variants thereof; and the composition may optionally further comprise lipid nanoparticles (LNPs).

36. A method of stimulating an immune response against an antigen or antigenic epitope to treat, suppress, and / or prevent a disease or condition that is pathogenically associated with said antigen, comprising administering to a subject the composition of claim 34 or claim 35.

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