Antigen-specific T cell receptors and chimeric antigen receptors, and methods for their use in regulating immune signaling in cancer immunotherapy.

Abstract

This invention relates to T cell receptors (TCRs) for NY-ESO-1 and CT83, which are cancer-testicular antigens (CTAs) presented by multiple HLA molecules. The preferred TCRs of this invention are derived from human T cells and exhibit high affinity and antigen specificity in vitro and in vivo. This invention also relates to the regulation of TCR-T cell and CAR-T cell signaling in cancer immunotherapy, as well as the improvement of functional persistence.

Description

Technical Field

[0001] The present invention claims priority based on U.S. Application No. 18 / 212,127, filed on June 20, 2023, which is a continuation-in-part of U.S. Application No. 18 / 002,969, filed on December 22, 2022. Further, the U.S. Application No. 18 / 002,969 is a national stage application under 371 of International Application PCT / US2021 / 039262, filed on June 25, 2021, which claims priority based on U.S. Provisional Application No. 63 / 044,150, filed on June 25, 2020. The disclosure content of each of these applications is hereby incorporated by reference in its entirety into this specification.

[0002] The present invention relates to a method for identifying a T cell receptor (TCR) from tumor antigen-specific T cells and verifying its function. Further, the present invention relates to a regulatory method for enhancing and prolonging the persistence of TCR-T cells and CAR-T cells and reducing T cell exhaustion by directly manipulating the signal transduction domain of TCR or chimeric antigen receptor (CAR), and knocking down or knocking out negative signaling molecules. Furthermore, the present invention relates to a method for treating cancer using these TCRs, TCR-T cells, and CAR-T cells. The present invention also relates to cancer antigen-specific T cell receptors, such as NY-ESO-1 (“ESO-1 TCR”), CT83 (“CT83-TCR”), etc., and polypeptides containing one or more of the α and β chains of T cell receptors specific for viral antigens, such as human cytomegalovirus (HCMV)-derived PP65 and HCMV IE1, nucleic acids encoding them, recombinant vectors, and cells containing the nucleic acids or recombinant vectors. [Sequence Listing]

[0003] This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML sequence listing was created on June 19, 2024, is named "H3131-00110_SL.xml", and has a file size of 104,258 bytes. [Background technology]

[0004] The host immune system consists of innate and adaptive immunity, and has the function of recognizing and eliminating exogenous or endogenous antigens derived from pathogens or abnormal tissues, including cancer cells. Various immune cells are known to contribute to the recognition, suppression, and elimination of cancer cells. Although tumor-reactive T lymphocytes (T cells) have been shown to be directly involved in tumor rejection, the clinical efficacy of early cancer immunotherapy has been limited, particularly due to several factors such as immunosuppression. In recent years, the identification of immune checkpoints has led to significant progress in the development of targeted immunotherapy. For example, inhibiting or eliminating immunosuppressive checkpoints such as programmed cell death protein-1 (PD-1) and its ligand PD-L1, as well as cytotoxic T lymphocyte antigen-4 (CTLA-4), has dramatically enhanced antitumor immunity, demonstrating remarkable and sustained clinical effects in many cancer patients. Against the backdrop of these breakthroughs in cancer immunotherapy, T cell-based immunotherapy has been successfully applied to the treatment of various human cancers, including or excluding melanoma, renal cell carcinoma, and lymphoma, with the degree of tumor regression varying depending on the type of cancer.

[0005] CD8-positive T cells and CD4-positive T cells are major components of T cell-based antitumor immunity. CD8-positive T cells, also known as cytotoxic T lymphocytes (CTLs), specifically recognize complexes of epitopes bound to major histocompatibility complex (MHC) class I molecules (called human leukocyte antigen (HLA) in humans) via the T cell receptor (TCR), and have the ability to kill cells when these complexes are presented on the cell surface. On the other hand, CD4-positive T cells, primarily called helper T cells (Th cells), are another type of T cell that plays a crucial role in the immune system. CD4-positive T cells specifically recognize complexes with epitopes bound to MHC class II molecules via the TCR, releasing cytokines to regulate the immune system. CD4-positive T cells are also essential for the activation of CD8-positive T cells, B lymphocytes, and other immune cells, including or excluding macrophages.

[0006] To initiate a tumor-specific T cell response, tumor antigens are processed and degraded in tumor cells or other antigen-presenting cells (APCs) via the proteasome pathway (for MHC class I molecule binding) or the endosomal / lysosome pathway (for MHC class II molecule binding) to form peptides consisting of 9 to 13 amino acids (called epitopes). The final product of such antigen treatment binds to specific MHC class I or class II molecules of the APC and is transported to the cell surface. This allows for the activation of CD8-positive or CD4-positive T cells if the epitope-HLA complex specifically binds to the TCR on the T cell surface. While the cytotoxic activity of CD8-positive T cells can directly kill tumor cells, other studies have shown that CD4-positive T cells also play an important role in anti-tumor immunity. Furthermore, certain subsets of CD4-positive T cells (CD4 CTLs) are known to possess cytotoxic activity and are directly involved in killing tumor cells in an HLA class II-restrictive manner.

[0007] T cells modified with chimeric antigen receptors (CARs) (CAR-T cells) have shown sustained clinical efficacy against hematological malignancies, including leukemia and lymphoma. In particular, CAR-T therapies targeting CD19 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of lymphoma and leukemia. However, CAR-T cell therapy has not shown sufficient efficacy in the treatment of solid tumors. Furthermore, it has been reported that approximately 30-50% of cancer patients who achieve remission with CD19-CAR-T therapy experience relapse within 12 months after treatment. [Overview of the project]

[0008] In one embodiment, the present invention relates to a method for identifying and verifying the function of T cell receptors (TCRs) from cancer antigen-specific T cells or antigen-specific T cells associated with other diseases or conditions such as inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-associated conditions, cancer, and infectious diseases. In a non-limiting example, the present invention relates to a method for identifying and functionally verifying TCRs from NY-ESO-1 specific, CT83 specific, human cytomegalovirus (HCMV)-pp65 specific, and / or HCMV-IE-1 specific T cells.

[0009] In some embodiments, the present invention provides a method for detecting and cloning TCRs from cancer antigen-specific T cells (including, but not limited to, NY-ESO-1-specific, CT83-specific, HCMV-pp65-specific, and / or HCMV-IE-1-specific T cells) in human subjects. The method comprises at least the following steps: (a) A step of stimulating naive T cells in vitro with a cancer antigen containing a class I or class II HLA-restrictive epitope complex; (b) A step of detecting cancer antigen epitope-specific T cell populations in vitro and / or in vivo; (c) A step of separating a population of cancer antigen epitope-specific CD4-positive or CD8-positive T cells; (d) The process of isolating single T cells from the separated T cells; (e) A step to obtain the V(D)J sequence of T cells by single-cell next-generation sequencing; (f) A step of synthesizing primers for TCR cloning based on the sequence; (g) A step of amplifying the variable regions of the TCRα chain and β chain from a population of T cells stimulated by a cancer antigen; (h) A step of incorporating the amplified variable regions of the TCR α chain and β chain into a vector to prepare a complete TCR construct. Furthermore, this method may further include the steps of introducing a cloned TCR into naive CD4-positive or CD8-positive T cells, measuring the activity of the introduced cells, and screening the introduced T cells based on their binding ability, recognition ability, and / or activation ability to multiple targets (peptides, cells, cell lines, gene-modified cell lines, and / or tumor cell lines). TCRs derived from cancer antigen-specific T cells identified by the methods described herein may bind to and / or recognize any cancer antigen, fragment thereof, or epitope thereof as described herein.

[0010] In yet another embodiment, the present invention provides a method for identifying epitope-specific T cells and TCRs as described in each of the above embodiments, wherein the cells or cell lines used in the method may or may not include, for example, HEK293 cells, HEK293 T cells, Cos-7 cells, 586-mel cells, 624-mel cells, MDA-MB-231 cells, MDA-MB-436 cells, E0771 cells, and HTB-21 cells. In some embodiments, tumor cell lines may be selected from 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 cancer, lung cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, rectal cancer, AIDS-related lymphoma, or AIDS-related sarcoma.

[0011] In one embodiment, the present invention provides a method for identifying epitope-specific T cells and T cell receptors (TCRs) according to any of the above embodiments or any embodiments described herein. One or more cells or cell lines used in the method are modified to express MHC class I molecules or MHC class II molecules. The recognized epitopes may be associated with diseases or disorders selected from inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-associated conditions, cancer, or infectious diseases.

[0012] Furthermore, the present invention provides a method for identifying epitope-specific T cells and TCRs according to any of the above embodiments or any embodiment described herein, wherein the T cell activity measured in the method is measured by an immunoassay. The T cell activity may include, for example, the release of cytokines including IFN-α, TGF-β, lymphotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25. The T cell activity can be measured by ELISA, chemiluminescence, ELISPOT, intracellular cytokine staining, chromium release test, or other immunoassay.

[0013] In one embodiment, TCRs derived from cancer antigen-specific T cells or antigen-specific T cells associated with other diseases or conditions, identified by each of the above embodiments or any embodiment described herein, can bind to and / or recognize any cancer antigen or other disease antigen described herein, as well as their fragments or epitopes.

[0014] In one embodiment, the present invention provides a method for identifying epitope-specific T cells and TCRs according to any of the above embodiments or any embodiments or embodiments described herein, wherein the cancer in the method may be selected from 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 cancer, lung cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon and rectal cancer, prostate cancer, AIDS-related lymphoma, or AIDS-related sarcoma.

[0015] In one embodiment, the present invention provides a cancer epitope that is recognized or bound by cancer antigen-specific T cells and TCRs, which is identified or detected by any of the above embodiments or any embodiment described herein. The cancer epitope may include an epitope derived from any cancer antigen or tumor antigen.

[0016] In one embodiment, the cancer epitope may include an epitope derived from NY-ESO-1 identified by any of the embodiments described herein or by any embodiment or method described herein. For example, the present invention provides a polypeptide comprising the amino acid sequence SLLMWITQCFLPVF (SEQ ID NO: 1) and variants thereof.

[0017] In one embodiment, the present invention provides cancer epitopes or other epitopes associated with diseases or conditions such as inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-associated conditions, cancer, or infectious diseases. The epitopes may include NY-ESO-1, CT83, HCMV-pp65, HCMV-IE-1, and other antigens described in the present invention, as well as epitopes derived from their variants or variants, which can be recognized or bound by epitope-specific T cells or TCRs identified by any of the above embodiments or by any embodiment described herein. The epitopes may consist substantially of identified epitopes. An essential characteristic of TCR epitopes is that they are part of target proteins that can be recognized or bound by TCRs in the context of MHC class I or class II. For example, the present invention provides polypeptides comprising the amino acid sequence KLVELEHTL (SEQ ID NO: 2) and variants thereof.

[0018] In one embodiment, the present invention provides a cancer epitope that may include an epitope derived from CT83 identified by the method described above. The epitope may consist substantially of the identified epitopes. The essential characteristic of the epitope is that it is part of a target protein that can be recognized or bound by a TCR in the context of MHC class I or class II. For example, the present invention provides a polypeptide comprising the amino acid sequence KLVELEHTL (SEQ ID NO: 2) and variants thereof. In one embodiment, the present invention provides cancer epitopes that may include epitopes derived from HCMV-pp65 identified by the method described above. For example, the present invention provides polypeptides comprising the amino acid sequence NLVPMVATV (SEQ ID NO: 26) and variants thereof.

[0019] In one embodiment, the present invention provides cancer epitopes that may include epitopes derived from HCMV-IE-1 identified by the method described above. For example, the present invention provides polypeptides comprising the amino acid sequence VLEETSVML (SEQ ID NO: 31) and variants thereof.

[0020] In one embodiment, the present invention provides a pharmaceutical composition comprising one or more epitopes described herein, which is useful for treating diseases or conditions to which the epitope is pathologically related. In one embodiment, the epitope is used in a cancer treatment vaccine. Furthermore, the present invention relates to a composition comprising one or more antigens and / or epitopes conjugated by any TCR described herein.

[0021] In one embodiment, the composition comprises one or more antigens and epitopes pathologically related to a disease or condition, the epitopes being selected from NY-ESO-1-derived peptides (A2-ESO-1, DP4-ESO-1), CT83-derived peptides (A2-CT83, DR13-CT83, PEP4-12, PEP6-14, PEP10-31 TC, PEP17-31, PEP90-98), HCMV pp65, HCMV IE-1, or functional variants thereof. Functional variants can be identified, for example, by alanine scanning.

[0022] In one embodiment, the present invention provides a composition comprising the α-chain and / or β-chain of a cancer antigen-specific T cell receptor (TCR) identified or detected by the method described above. The α-chain and / or β-chain may bind to and / or recognize any cancer antigen described herein. For example, it may bind to cancer antigens such as NY-ESO-1 (ESO-1 TCR), CT83 (CT83-TCR), or virus-derived cancer antigens. Furthermore, the TCR may include embodiments in which it may bind to CT83-derived polypeptides (SEQ ID NO: 39 or 61) but not to the polypeptide shown in SEQ ID NO: 62 or 63. In addition, the TCR may bind to HCMV pp65 or HCMV IE-1, and their fragments or epitopes.

[0023] In one embodiment, the present invention relates to a composition comprising an α chain or α region and / or a β chain or β region of a cancer antigen-specific T cell receptor. The cancer antigen may be selected from NY-ESO-1, CT83, HCMV-pp65, and / or HCMV-IE-1. For example, the composition may comprise a polypeptide comprising an α chain or α region of a NY-ESO-1, CT83, pp65, or IE-1 specific TCR, and a polypeptide comprising the corresponding β chain or β region.

[0024] In one embodiment, the present invention provides an α-variable region of a cancer antigen-specific T cell receptor (TCR) detected or identified by the method described above. The α-variable region may or may not include the α-variable regions of DP4-ESO-1 TCR, A2-CT83 TCR, A2-pp65 TCR, and / or A2-IE-1 TCR. Furthermore, the α-variable region can be used in combination with any variable region sequence or epitope-specific sequence identified herein. For example, the present invention provides a polypeptide (α-variable region of DP4-ESO-1 TCR) comprising the amino acid sequence METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY shown in SEQ ID NO: 3. Similarly, polypeptides containing Sequence ID No. 5 (A2-CT83 TCR α variable region), Sequence ID No. 29 (A2-pp65 TCR α variable region), and Sequence ID No. 32 (A2-IE-1 TCR α variable region) are also provided.

[0025] The present invention provides a polypeptide or fragment thereof that has substantially the same antigen-binding ability as the reference TCR (full-length and unmodified) and antigen specificity. The aforementioned mutant may include conservative amino acid substitutions and may or may not include substitutions in the six CDR regions of the TCR. For example, mutants of the DP4-ESO-1 TCR may include D95S substitution or Q98Y substitution in the α chain, and Y98L or Y98M substitution in the β chain. Furthermore, mutants containing only the Q98Y substitution in the α chain are also provided. These variants have been shown to significantly enhance T cell function in terms of IFN-gamma release and tumor-specific lysis. For example, a single Q98Y substitution in the TCR α chain has been shown to increase T cell function based on NFAT-GFP expression by 3 to 4 times.

[0026] In one embodiment, the present invention provides a β-variable region of a cancer antigen-specific TCR identified by the method described above. The β-variable region may or may not include the β-variable regions of DP4-ESO-1 TCR, A2-CT83 TCR, A2-pp65 TCR, and / or A2-IE-1 TCR. Furthermore, the β-variable region can be used in combination with any variable region sequence or epitope-specific sequence identified herein. For example, the present invention provides polypeptides comprising SEQ ID NO: 4 (DP4-ESO-1 TCR β variable region), SEQ ID NO: 6 (A2-CT83 TCR β variable region), SEQ ID NO: 30 (A2-pp65 TCR β variable region), and SEQ ID NO: 33 (A2-IE-1 TCR β variable region). Furthermore, the present invention provides variants of the β variable region or a fragment thereof. The variants include conservative amino acid substitutions and may or may not include substitutions in the six CDR regions of the TCR.

[0027] The present invention provides variants of the polypeptide or fragment. The variants include conservative amino acid substitutions and may or may not include substitutions in one or more CDR regions of the TCR.

[0028] The present invention provides variants of the polypeptide or fragment. The variants include conservative amino acid substitutions and may or may not include substitutions in one or more CDR regions of the TCR.

[0029] In one embodiment, the present invention provides a chimeric TCR in which the variable region of the T cell receptor (TCR) is fused with a modified human-derived constant region, or a non-human-derived constant region that may or may not be modified. In one embodiment, the chimeric TCR includes a cancer antigen-specific TCR variable region, or an antigen-specific TCR variable region pathologically associated with another disease or condition, and may be fused with a non-human, for example, mouse-derived TCR constant region. In one embodiment, the TCR variable region includes an α-chain variable region and a β-chain variable region, which are fused with an α-chain TCR constant region and a β-chain TCR constant region, respectively. It may also include any α-chain and / or β-chain according to other embodiments described herein. Furthermore, in one embodiment, mispairing between the chimeric TCR and the endogenous TCR is reduced by fusing the TCR variable region with a modified human steady-state region or a non-human steady-state region. In one embodiment, the chimeric TCR may or may not include any of the variable regions of CT83 TCR, NY-ESO-1 TCR, pp65 TCR, and / or IE-1 TCR, and is fused with a non-human, for example, mouse-derived TCR constant region. Furthermore, in one embodiment, the chimeric TCR reduces mispairing between the chimeric TCR and endogenous TCR in the introduced T cells. For example, the chimeric CT83 TCR reduces mispairing between the chimeric CT83 TCR (MC) and endogenous TCR (HC). Similarly, the chimeric NY-ESO-1 TCR, chimeric pp65 TCR, and chimeric IE-1 TCR also reduce mispairing with their respective endogenous TCRs.

[0030] In one embodiment, the present invention provides a chimeric TCR in which a TCR variable region is fused with a modified human steady-state region or a non-human steady-state region. In one embodiment, the chimeric TCR includes a cancer antigen-specific TCR variable region and is fused with a non-human, for example, mouse-derived TCR constant region. In one embodiment, the TCR variable region includes an α-chain variable region and a β-chain variable region, each fused with a corresponding TCR constant region. It may also include any α-chain and / or β-chain according to other embodiments described herein. Furthermore, in one embodiment, the chimeric TCR may or may not include any of the variable regions of CT83 TCR, NY-ESO-1 TCR, pp65 TCR, or IE-1 TCR, and is fused with a non-human TCR constant region. In one embodiment, the chimeric TCR reduces mispairing between the chimeric TCR and endogenous TCR in the introduced T cells. For example, the chimeric CT83 TCR, chimeric NY-ESO-1 TCR, chimeric pp65 TCR, and chimeric IE-1 TCR each reduce mispairing with their corresponding endogenous TCRs.

[0031] The present invention provides nucleic acids that encode epitopes, receptor chains and / or polypeptides as described in each of the above embodiments, or any other polypeptides as described herein. Furthermore, recombinant nucleic acids containing the nucleic acids, vectors or constructs containing the recombinant nucleic acids, and cells into which one or more of the nucleic acids or vectors have been introduced are also provided.

[0032] In one embodiment, the present invention provides a nucleic acid encoding a polypeptide comprising the epitope described in each of the above embodiments. In one embodiment, the present invention provides an mRNA vaccine comprising mRNA encoding the epitope or a variant thereof, which is used for the treatment of a disease or condition pathologically associated with the antigen and / or antigen epitope. In one embodiment, the epitope-coding mRNA is produced by in vitro transcription and produces an epitope antigen to induce an immune response to the epitope. In one embodiment, the mRNA comprises a 5' cap, a 5' untranslated region (UTR), an open reading frame encoding an antigen, a 3' UTR, and a poly(A) tail, extending from the 5' end to the 3' end. Furthermore, in one embodiment, the mRNA vaccine comprising the mRNA described herein further comprises lipid nanoparticles. Lipid nanoparticles suitable for mRNA formulations are described, for example, in WO2020 / 146906 and US2022 / 0088221, which disclosures are incorporated herein by reference.

[0033] In one embodiment, the present invention provides a nucleic acid encoding a variable region or polypeptide of the α-chain and / or β-chain of the TCR described in each of the above embodiments. In one embodiment, the nucleic acid encodes a polypeptide corresponding to any of SEQ ID NOs: 3-6, 10-11, 12-15, 20-25, 27-30, 32, or 33. Furthermore, in one embodiment, the nucleic acid has any of the sequences of SEQ ID NOs: 41-50 and 53-55, and these sequences encode the TCR variable region shown below. [Table 1]

[0034] In one embodiment, the nucleic acid sequence may include one or more codon substitutions that do not alter the sequence of the encoded polypeptide.

[0035] In some embodiments, the present invention is characterized in that the transmembrane domain of the TCR or CAR is derived from CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, A2aR, ICAM-1, DAP10, KIR, LAG-3, LAT, Fc receptor, cytokine receptor (IL-2R, IL-4R, IL-7R, etc.), NK receptor, NOTCH, T cell receptor polypeptide, CD3, CD8, CD16, CD28, CD40, PD-L1 / PD-L2, ICOS, GITR, ZAP70, etc., or any combination thereof.

[0036] In some embodiments, the present invention comprises nucleic acids that code for a TCR region or chain as described in the present invention. These nucleic acids may further comprise signaling components. In one embodiment, the TCR or CAR has an intracellular T cell activation motif comprising a signaling domain. In another embodiment, the intracellular T cell activation motif comprises one or more co-stimulatory signaling domains fused with the signaling domain, the co-stimulatory domains being selected from or a combination thereof from CD28, 4-1BB, ICOS, CD27, OX40, MyD88, ZAP70, CD3 zeta, various interleukin receptors, TLRs, etc.

[0037] In some embodiments, the signaling domain of the TCR or CAR includes substituting CD28 and / or CD3 zeta with a ZAP70 kinase domain, or a variant or mutant thereof. In other embodiments, the TCR or CAR includes a functional ZAP70 kinase domain derived from the functional wild type of ZAP70 kinase, or a variant or mutant thereof. The ZAP70 kinase domain may include ZAP255, ZAP280, ZAP300, ZAP327, ZAP338, etc., or variants or mutants thereof. These domains can enhance T cell activation, antigen recognition, and specific lytic ability.

[0038] In some embodiments, the functional ZAP70 kinase domain has an N-terminus and a C-terminus that begin at amino acids 250-338, 281-338, or 300-338 of ZAP70 and terminate at amino acid 619. In another embodiment, the signaling component confers enhanced persistence and / or antitumor activity in cells modified with the nucleic acid.

[0039] This specification discloses compositions containing one or more of the variable regions of the TCR α-chain or β-chain described in any of the embodiments described above, in a therapeutically effective amount. In some embodiments, the composition may further contain any of the signaling components described above, for example, functional kinase domains such as ZAP327, ZAP300, ZAP338, ZAP255, ZAP280, or other signaling components derived from the ZAP70 kinase domain, or variants or mutants thereof. In some embodiments, T cells expressing the CAR or TCR exhibit reduced expression of T cell exhaustion markers such as PD-1, TIM3, and LAG3.

[0040] This specification discloses compositions containing, in therapeutically effective amounts, one or more TCR-T cells modified to express the nucleic acids described in any of the embodiments described above. In some embodiments, the TCR-T cells express receptors that recognize cancer antigens or neoantigens. In other embodiments, the TCR-T cells express one or more of the TCR α-chain and / or β-chain variable regions described in the present invention. Furthermore, they may include a functional kinase domain derived from the ZAP70 kinase domain or its variants or mutants. In some embodiments, the modified TCR-T cells have significantly improved persistence and / or antitumor activity. In another embodiment, the TCRs include 4-1BB, CD27, CD28, OX40, ICOS, MyD88, MALT-1, TLR, etc., or their variants or mutants, which enhance T cell activation, antigen recognition, and specific lytic ability.

[0041] In one embodiment, modified or transduced T cells do not express endogenous TCR(α / β). For example, endogenous TCR(α / β) is knocked out using CRISPR / Cas9 or CRISPR / Cas12a technology before introducing a cancer antigen-specific TCR construct.

[0042] The enhancement of TCR-T cell persistence and reduction of fatigue by directly manipulating modified TCRs derived from tumor antigen-specific T cells, as well as the TCR signaling domain, is described in U.S. Application No. 18 / 002,969, a national phase application of PCT / US2021 / 039262, and its entire contents are incorporated herein by reference.

[0043] The present invention includes methods for extending the persistence of CAR-T cells or TCR-T cells by directly manipulating the CAR signaling domain or by knocking down or knocking out negative signaling molecules. Furthermore, it includes methods for improving the persistence of CAR-T cells or TCR-T cells using chemokine receptor expression and shRNA knockout. In some embodiments, therapy with modified TCR-T cells of the present invention reduces relapse or cancer recurrence after initial treatment. Furthermore, it is also effective in reducing the recurrence of inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-associated conditions, infections, or age-related symptoms.

[0044] The present invention provides modified immune cells (TCR-T cells or CAR-T cells) having knockdown, knockout, or inactivating mutations in one or more endogenous genes selected from a group of negative regulatory factors. In some embodiments, the negative regulatory factors include immune checkpoint molecules, immunosuppressive molecules, or epigenetic factors, such as IDO (including IDO1 and IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-H3, VHL, PPP2R2D, JMJD3, LSD1, etc. In specific embodiments, modifications are made to PD-1, VHL, PPP2R2D and / or epigenetic factors. In some embodiments, treatment with the modified TCR-T cells of the present invention significantly reduces relapse or tumor recurrence after initial treatment.

[0045] Knockout, knockdown, or inactivating mutations of any negative signaling molecule described herein, and / or expression of chemokine receptors or shRNA knockout, may be applied to CAR-T cells or TCR-T cells or CAR constructs as described in any of the foregoing embodiments. For example, such CAR-T cells may express one or more CARs including antigen recognition sites, hinge regions, transmembrane regions, and intracellular signaling regions.

[0046] This specification discloses nucleic acids encoding gene knockdown siRNA or shRNA for enhancing the antitumor activity of TCR-transformed T cells. In some embodiments, the shRNA targets negative signaling molecules of the immune system, such as checkpoint molecules or immunosuppressive molecules.

[0047] In some embodiments, knockout or inactivation of a target gene is carried out by a CRISPR / Cas9 system including sgRNA and Cas endonuclease, reducing or eliminating the expression of negative regulatory genes of the immune system.

[0048] This specification discloses methods for inducing an immune response against cancer, or for treating, suppressing, or preventing cancer, the methods comprising administering to a subject a therapeutically effective dose of the epitope or TCR α-chain or β-chain variable region described in any of the above embodiments.

[0049] The present invention provides a chimeric antigen receptor (CAR) and T cells or other immune cells that express the CAR. In some embodiments, the CAR comprises an antigen recognition site, a transmembrane region, and an intracellular T cell activation site comprising a CD28 or 4-1BB costimulatory signaling region and a ZAP70-derived signaling domain. Furthermore, the CAR may include an antigen recognition site that specifically binds to CD19, BCMA, B7-H3, mesoserine, or HER-2.

[0050] Natural killer (NK) cells and natural killer T (NKT) cells are considered immune cells of the innate immune system. NKT cells are a subpopulation of T cells that express T cell receptor α and β chains, but possess NK cell-like properties, including several NK cell markers such as NK1.1. NKT cells recognize various foreign or self-lipid antigens presented by CD1d-presenting molecules.

[0051] In some embodiments of the present invention, a TCR, CAR, bispecific antibody, or polyspecific binding protein containing any TCR antigen-binding site described herein may be modified to be expressed on immune cells such as T cells, NK cells, NKT cells, and macrophages. This yields CAR-T, CAR-NK, CAR-NKT, and CAR-M cells. Similarly, a TCR can be expressed on T cells or NKT cells, but NK cells do not express endogenous TCR chains and therefore cannot directly express a TCR. Accordingly, NK cells are modified to express TCR complex subunits before TCR introduction. The TCR complex involved in antigen recognition comprises six CD3 subunits and a TCR heterodimer (α / β chain or gamma / δ chain). In some embodiments, the modified cells are TCR-T, TCR-NK, and / or TCR-NKT cells.

[0052] Modified immune cells lacking functional TCR and / or HLA expression can be obtained by any means, such as knockout or knockdown of one or more subunits of the TCR and / or HLA. For example, Treg cells can have their TCR and / or HLA knocked down using siRNA, shRNA, CRISPR, TALEN, ZFN, meganuclease, or megaTAL.

[0053] In some embodiments of the present invention, the nucleic acid encoding the TCR or CAR described herein is inserted into a specific locus in the immune cell genome, such as a locus to be deleted. In some embodiments, the nucleic acid is inserted into a TCR and / or HLA locus, thereby inhibiting the expression of endogenous TCR and / or HLA.

[0054] In some embodiments of the present invention, the expression of TCR and / or HLA in T cells is inhibited by siRNA and / or shRNA targeting the nucleic acids encoding them. The expression of siRNA or shRNA can be achieved using any expression system, such as a lentiviral expression system.

[0055] In another embodiment, the TCR α-chain and / or β-chain variable regions may further include other signaling motifs derived from the ZAP255, ZAP280, ZAP300, ZAP327, or ZAP338, or ZAP70 kinase domains, or variants thereof.

[0056] The present invention provides applications for using TCR-T cells or CAR-T cells, or immune cell populations, as described in any of the above embodiments, in the manufacture of pharmaceuticals for the treatment of diseases or disorders in human subjects requiring treatment. In some embodiments, the disease or disorder is selected from inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related diseases, cancer, or infectious diseases.

[0057] In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, systemic lupus erythematosus, vasculitis, osteoarthritis, gout, ankylosing spondylitis, Sjögren's syndrome, Behçet's disease, polymyalgia rheumatica, and juvenile idiopathic arthritis.

[0058] In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, type 1 diabetes, Graves' disease, Hashimoto's disease, myasthenia gravis, Addison's disease, pemphigus, scleroderma, Goodpasture syndrome, autoimmune hepatitis, and autoimmune hemolytic anemia.

[0059] 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., allergies to peanuts, shellfish, milk, eggs, and wheat), drug allergies (e.g., penicillin, sulfonamides), latex allergies, insect bite allergies, and urticaria.

[0060] In some embodiments, organ transplant-related conditions are selected from the group consisting of graft-versus-host disease (GvHD), acute rejection, chronic rejection, transplant vascular complications, post-transplant lymphoproliferative disorder (PTLD), and delayed graft function.

[0061] In some embodiments, the infection 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 hemorrhagic fever, chikungunya virus infection, Lyme disease, SARS-CoV-2 infection, long COVID, and sepsis.

[0062] While T cell recognition of target antigens is HLA-restricted, target recognition by chimeric antigen receptor (CAR) T cells is HLA-independent. As disclosed herein, the regulation of TCR-T cell and CAR-T cell signaling and function in vivo can be achieved by extending T cell persistence (reducing T cell exhaustion), direct regulation of TCR or CAR signaling domains, or by knockdown or knockout of negative signaling molecules such as PD-1, VHL, PPP2R2D, as well as epigenetic factors including JMJD3 and LSD1. Negative regulators of endogenous genes, including ANKRDJ1, ARID1A, BACH2, CTLA4, FOXP3, LAG3, TIGIT, TNFAIP3, TOX1, and TOX2, may also be targeted.

[0063] The present invention further features a method for promoting the migration of T cells to tumors in vivo by forcing the expression of chemokine receptors. In some embodiments, the chemokine receptors are expressed by fusing them to a CAR or TCR construct. The chemokine receptors may be selected from CCR5, CXCR3, and / or CCR2. Furthermore, cytokine receptors may include IL-1R, IL-2Rβ, IL-4Rα, IL-7Rα, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-21Rα, and common gamma chains.

[0064] In some embodiments, chemokine receptor expression and shRNA knockdown or CRISPR / Cas9 sgRNA knockout are applicable to any TCR or CAR construct described herein and can target endogenous genes, including negative regulators such as ANKRDJ1, ARID1A, BACH2, CTLA4, FOXP3, LAG3, TIGIT, TNFAIP3, TOX1, TOX2, and VHL. [Brief explanation of the drawing]

[0065] The drawings accompanying this specification constitute part of this specification and illustrate several embodiments, as well as compositions and methods according to the present invention.

[0066] Figure 1 shows the results of analyzing the characteristics of single T cell clones generated from T cell lines that respond to NY-ESO-1 presented by HLA-DP4. T cell clones were isolated from HLA-DP4-presenting NY-ESO-1-responsive T cell lines, and after each clone was grown, the antigen recognition ability was evaluated using a peptide (SEQ ID NO: 1) containing amino acid sequences 157-170 of NY-ESO-1 presented by HLA-DP4-positive antigen-presenting cells (APCs).

[0067] Figure 2 shows the construction map for incorporating a DP4-ESO-1 TCR derived from a single T cell clone into a pMSGV vector.

[0068] Figures 3A and 3B show the DP4-ESO-1 TCR in naive CD4 + This figure shows the results after introduction into T cells. Figure 3A shows naive CD4 measured by TCR-specific antibody staining and flow cytometry. + This shows the efficiency of DP4-ESO-1 TCR introduction into T cells. Naive CD4 cells were analyzed using retroviral supernatant produced from different DP4-ESO-1 TCR PG-13 clones. + T cells were transduced, and the average transduction efficiency was approximately 60-70%. Figure 3B shows CD4 cells transduced with DP4-ESO-1 TCR.+ Shows the functional evaluation results of T cells.

[0069] Figures 4A, 4B, and 4C are diagrams showing the functional characteristics of DP4-ESO-1 TCR-T cells. Figure 4A shows the T cell recognition ability for peptides, indicating that DP4-ESO-1 TCR-T cells recognize the peptide of amino acid sequences 157 - 170 of NY-ESO-1 presented by HLA-DP4 positive cells. Figure 4B shows the T cell recognition ability for naturally processed NY-ESO-1, indicating that DP4-ESO-1 TCR-T cells recognize 293T cells transfected with full-length NY-ESO-1, HLA-DPA1, and HLA-DP4. Figure 4C shows that DP4-ESO-1 TCR + Functions only in T cells, and CD8 + Compared with T cells, only CD4 T cells transfected with DP4-ESO-1 TCR recognize the amino acid sequences 157 - 170 of NY-ESO-1, indicating that the function of this TCR is limited to CD4 + T cells. + Indicates that it is limited to T cells.

[0070] Figures 5A, 5B, and 5C are diagrams showing the improvement of the in vivo anti-tumor function by DP4-ESO-1 TCR when A2-ESO-1 TCR and DP4-ESO-1 TCR are used in combination against MDA-MB-231 / DP4 / ESO. Figure 5A shows the results of tracking cell migration in vivo by luciferase imaging after administration of A2-ESO-1 TCR-transfected CD8 + T cells. Figure 5B shows the in vivo growth of MDA-MB-231 / DP4 / ESO tumors when treated with different T cell groups. Figure 5C shows the comparison of tumor sizes after treatment with each T cell group at the time of mouse sacrifice.

[0071] Figures 6A, 6B, 6C, and 6D show the generation and characterization of HLA-A2-restricted CT83-specific T cells. Figure 6A shows the results of evaluating the ability of T cells induced by peptide stimulation in vitro to recognize 293T cells expressing a peptide containing amino acid sequences 90-98 of CT83 (CT83 PEP90-98, SEQ ID NO: 2), compared to 293T cells presenting a control peptide. Figure 6B shows that A2-CT83-specific T cells recognize 293T cells introduced with Ii-CT83 or CT83-GFP plasmid DNA, or 293T cells loaded with CT83 PEP90-98 (positive control), but do not recognize control 293T cells. Figure 6C shows that A2-CT83-specific T cells recognize the human breast cancer cell line MDA-MB-231, which expresses HLA-A2 and CT83, but do not recognize the HLA-A2-negative, CT83-positive MDA-MB-436. Figure 6D shows that recognition of MDA-MB-231 cells is inhibited by an anti-MHC class I antibody, but not by an anti-MHC class II antibody.

[0072] Figures 7A and 7B show that vaccination with CT83-PEP90-98 suppresses the proliferation of breast cancer cells. Figure 7A shows a schematic diagram of the experimental design and schedule. Figure 7B shows that intravenous administration (iv) of TAT-CT83 PEP90-98-CMI nanoparticles to tumor-bearing mice showed a tumor-suppressing effect, but this effect was not observed with TAT-CT83 PEP66-74-CMI. Note that CMI stands for CpG, MPLA, and poly(I:C). **P value < 0.01.

[0073] Figures 8A, 8B, 8C, 8D, and 8E illustrate the construction and characterization of HLA-A2-restricted CT83-specific TCRs. Figure 8A shows a flowchart of the process for TCR sequencing, cloning, and construction using 10x single-cell barcoding technology and next-generation sequencing from an A2-CT83-specific T cell population purified by FACS. Figure 8B shows the results of functional analysis of A2-CT83 TCR-introduced T cells and vector-introduced PBMCs, comparing their reactivity to 293T, 293T / CT83-GFP, Cos-7, Cos-7 / Ii-CT83, and Cos-7-A2 / Ii-CT83 cells. Figure 8C shows that A2-CT83 TCR-T cells specifically recognize 293T cells loaded with CT83 PEP90-98 compared to 293T cells loaded with other CT83 peptides. Figure 8D shows that A2-CT83 TCR-T cells recognize MDA-MB-231 cells that express both CT83 and HLA-A2, but cells that express only one of them, i.e., MCF7(A2 + CT83 - ), HTB-2(A2 + CT83 - ) or MDA-MB-436(A2 - CT83 + Figure 8E shows that A2-CT83 TCR-T cells recognize lung cancer cells expressing CT83 and HLA-A2 (HOP92 / A2, NCI-H358 / A2, and NCI-H838 / A2), but do not recognize tumor cells that are CT83-positive and HLA-A2-negative (HOP92, NCI-H358, and NCI-H838).

[0074] Figures 9A, 9B, and 9C illustrate the in vivo antitumor activity of A2-CT83 TCR-T cells. Figure 9A shows a schematic diagram of the administration schedule of NCI-H838 tumor cells and A2-CT83 TCR-T cells in an NSG mouse model. Figures 9B and 9C show that tumor growth was suppressed in vivo by A2-CT83 TCR-T cells. In contrast, tumor-bearing mice treated with control T cells formed large tumor masses. These results suggest that A2-CT83 TCR-T cells possess potent antitumor activity in vivo.

[0075] Figures 10A and 10B show the generation and characterization of HLA-A2-restricted HCMV (pp65 and IE-1 protein) specific T cell clones. Figure 10A shows the results of screening seven T cell clones that respond to pp65 (495-503) and five T cell clones that respond to IE-1 (316-324) using peptide-loaded T2 cells and growing them in vitro. Figure 10B shows the results of HLA-restriction analysis of pp65-specific T cell clone #3 and IE-1-specific T cell clone #5, showing that both clones specifically recognize pp65 and IE-1 antigens presented by HLA-A2, respectively, on Cos-7 cells.

[0076] Figures 11A, 11B, 11C, 11D, and 11E show the identification, cloning, and characterization of HLA-A2-restricted pp65 and IE-1-specific TCRs, as well as their function in human T cells transduced by these TCRs. Figure 11A shows human CD8 derived from an HCMV seronegative donor. +Figure 11B shows the transduction efficiency of A2-pp65 TCR and A2-IE-1 TCR in T cells. Figure 11B shows that A2-pp65 TCR-transformed T cells and A2-IE-1 TCR-transformed T cells specifically recognize glioblastoma cells expressing HLA-A2 and HCMV antigens (pp65 or IE-1), or glioblastoma cells infected with HCMV. Figure 11C shows the dose-dependent recognition by A2-pp65 TCR-transformed T cells and A2-IE-1 TCR-transformed T cells against T2 cells loaded with pp65 (495-503) or IE-1 (316-324) peptides, respectively. Figure 11D shows that these cells specifically cytotoxicize glioblastoma cells expressing HLA-A2 and HCMV antigens, or glioblastoma cells infected with HCMV. Figure 11E shows dose-dependent cytotoxicity of HCMV / AD169-infected U87 tumor cells by T cells transducing A2-pp65 TCR and A2-IE-1 TCR.

[0077] Figures 12A, 12B, 12C, and 12D show 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-1 and luciferase. Tumor cells were grown in SCID / Beige mice for 3 days, after which A2-pp65 TCR, A2-IE-1 TCR, or control TCR-transformed human T cells were administered intravenously (2 × 10⁶ cells per mouse). 6 Figure 12A shows a schematic diagram of the in vivo functional analysis procedure for A2-pp65 TCR-T cells. Figure 12B shows the migration of A2-pp65 TCR-T cells after injection into tumor-bearing mice. Figure 12C shows that A2-pp65 TCR-T cells specifically suppress the growth of pp65-expressing U87 tumors. Figure 12D shows a marked reduction in tumor weight after treatment with A2-pp65 TCR-T cells, suggesting the potential antitumor activity of pp65 TCR-T cells in glioblastoma treatment.

[0078] Figures 13A, 13B, 13C, and 13D show the in vivo antitumor activity of A2-IE-1 TCR-T cells. Figure 13A shows a schematic diagram of the procedure for in vivo functional analysis of A2-IE-1 TCR-T cells. Figure 13B shows the migration of A2-IE-1 TCR-T cells after injection into tumor-bearing mice. Figure 13C shows that A2-IE-1 TCR-T cells specifically suppress the growth of IE-1-expressing U87 tumors. Figure 13D shows a marked reduction in tumor weight after treatment with A2-IE-1 TCR-T cells, suggesting the potential antitumor activity of A2-IE-1 TCR-T cells in glioblastoma treatment.

[0079] Figures 14A-14G show the improvement of cell surface expression and function of A2-ESO-1 TCR-T cells in human T cells by mouse constant-chain sequences. Figure 14A shows schematic diagrams of the conventional and modified A2-ESO-1 TCR constructs of the present invention. Figure 14B shows that the modified TCR has a higher transduction efficiency than the conventional TCR. Figure 14C shows the detection results of TCR cell surface expression by FACS. Figure 14D shows the detection results of TCR cell surface expression by confocal microscopy. Figure 14E shows the results of LDH assays detecting the target cell toxicity of conventional and modified TCR-T cells. Figure 14F shows the results of cytokine secretion detection by ELISA after co-culture with A2-ESO-1 positive breast cancer cells. Figure 14G shows the results of detecting the long-term tumor cell killing ability of a representative composition of the present invention by in vitro co-culture.

[0080] Figures 15A-15D show that modified A2-ESO-1 specific TCR-T cells exhibit high therapeutic efficacy in a preclinical breast cancer model. Figure 15A shows a schematic diagram of the animal experiment. NY-ESO-1 positive breast cancer cell line MDA-MB-231(ESO-1 + ) 1 × 10 6 After transplanting cells into the subcutaneous adipose tissue of NSG mice, A2-ESO-1 TCR-T cells or A2-ESO-1 TCR-MT cells were administered intravenously, followed by three doses of IL-2. Figure 15B shows the results of tumor growth tracking. Figure 15C shows images of the tumors after sacrificial tumors. Figure 15D shows the tumor weight after sacrificial tumors.

[0081] Figures 16A-16C show the in vitro tumor cell killing activity of A2-ESO-1 TCRs with amino acid substitutions and mouse TCR constant chain sequences. Figure 16A shows the results of introducing five types of substituted A2-ESO-1 TCRs and the original A2-ESO-1 TCR into human T cells and evaluating the tumor cell recognition ability depending on the presence or absence of HLA-A2 and NY-ESO-1 expression. Figure 16B shows the tumor cell cytotoxic activity by substituted A2-ESO-1 TCR-T cells, showing that T cells with S2 and S5 TCRs exhibited high cytotoxic activity. Figure 16C shows that when the human TCR constant chain region of the S2 and S5 A2-ESO-1 TCRs was replaced with the mouse TCR constant chain, the modified S2 TCR showed a strong T cell response.

[0082] Figures 17A, 17B, and 17C show the antitumor activity of A2-CT83 TCR-MT cells with mouse constant chain sequences. Figure 17A shows the efficiency of A2-CT83 TCR-M transduction into human T cells. Figure 17B shows that A2-CT83 TCR-MT cells specifically recognize MDA-MB-231 and NCI-H1563 cells expressing CT83 and HLA-A2, while CAMA-1 cells (A2 + CT83 - This indicates that the cells do not recognize the TCR. Figure 17C shows the cytotoxic activity of A2-CT83 TCR-MT cells against MDA-MB-231 and NCI-H1563 cells. These results suggest that A2-CT83 TCR-MT cells have high specificity and activity against tumor cells and reduce TCR mispairing.

[0083] Figures 18A-18E show a functional comparison between novel CAR-T cell constructs fused with ZAP300 and ZAP327 derived from ZAP70 and conventional CAR-T cells possessing a CD3 zeta signaling domain. Figure 18A shows schematic diagrams of the conventional CD19-CD28-CD3 zeta (1928z) and the novel CD19-CD28-ZAP300 (1928ZAP300) and CD19-CD28-ZAP327 (1928ZAP327) constructs. Figure 18B shows the translocation efficiency of the three CARs in human T cells. Figures 18C and 18D show antigen-specific recognition and tumor cell lysis after co-culture with Raji tumor cells. Figure 18E shows the in vivo antitumor activity of the three CAR-T cell types (1928z, 1928ZAP300, and 1928ZAP327). In particular, 1928ZAP300 and 1928ZAP327 CAR-T cells showed superior antitumor activity in vivo compared to 1928z CAR-T cells, significantly extending overall survival in mice in a Raji lymphoma model. These results suggest that substituting the CD3 zeta chain with the ZAP70 kinase domain (ZAP300 and ZAP327) dramatically improves in vivo antitumor activity.

[0084] Figures 19A-19E illustrate the characteristics of a novel CAR-T cell construct containing 4-1BB, fused with ZAP300 and ZAP327. Figure 19A shows schematic diagrams of 19bbz and 19bbZAP327. Figure 19B shows that 19bbZAP327 CAR-T cells produce significantly lower cytokines compared to conventional 19bbz CAR-T cells after tumor cell stimulation. Figure 19C shows specific lysis of tumor cells by 19bbZAP327 CAR-T cells. Figures 19D and 19E show that 19bbZAP327 CAR-T cells exhibit superior antitumor activity in vivo, extending mouse survival time and suggesting improved safety and antitumor immunity compared to conventional 19bbz CAR-T cells.

[0085] Figures 20A–20D show the improvement of T cell memory function and in vivo persistence by the ZAP327 signaling domain. Figures 20A and 20B show the high in vivo persistence of 1928ZAP327 CAR-T cells in the bone marrow and spleen of T cell-transferred mice. Figure 20C shows that the proportion of central memory T cells in 1928ZAP327 CAR-T cells is higher than that of 1928z CAR-T cells. Figure 20D shows that 1928ZAP327 CAR-T cells express low levels of PD-1 (a T cell exhaustion marker), suggesting that the ZAP327 signaling domain reduces T cell exhaustion.

[0086] Figures 21A-21D show the in vivo regulation of TCR-T cell function by knockdown of the expression of metabolic genes such as PD1, VHL, and PPP2R2D. Figure 21A shows the efficiency of A2-ESO-1 TCR introduction with and without PD1, VHL, and PPP2R2D shRNAs. Figures 21B-21D show the results of administering A2-ESO-1 TCR-T cells to MDA-MB-231 / A2 / NY-ESO-1 tumor-carrying mice with and without each shRNA. The top panel shows the average tumor growth for each group, the middle panel shows the survival curve for each group, and the bottom panel shows the tumor growth for each individual mouse.

[0087] Figure 22 shows CD4 + CD44 denaturation by Jmjd3 conditional knockout (cKO) in T cells + CD62L - This shows an enhancement of the memory T cell population, indicating an increase compared to wild-type (WT) cells.

[0088] Figures 23A-23F show the improved T cell survival and in vivo / in vitro persistence by Jmjd3 cKO T cells. Figures 23A and 23B show CD4 cells from Jmjd3 cKO 2d2 transgenic mice stimulated in vivo with MOG peptide and complete Freund's auxiliary. +This shows that T cells significantly improve clinical scores in the EAE mouse model. Figure 23C shows that the number of Jmjd3 cKO T cells after T cell transfer is higher than that of wild-type 2d2 cells. Figures 23D-23F show that the number of Jmjd3 cKO T cells is higher than that of wild-type cells when T cells are transferred after MOG peptide stimulation in vitro.

[0089] Figures 24A-24C demonstrate the **improvement in T cell viability and persistence (suppression of T cell apoptosis)** by Jmjd3 knockout. Figure 24A shows that Jmjd3 cKO T cells reduce apoptosis-related protein levels after anti-CD3 / CD28 stimulation. Figure 24B shows that T cell apoptosis after anti-CD3 / CD28 stimulation is significantly lower in Jmjd3 cKO T cells. Figure 24C shows that cleavage caspase 3 levels in Jmjd3 cKO T cells are significantly lower compared to WT T cells.

[0090] Figures 25A-25D show the improved in vivo viability and persistence of CAR-T cells by Jmjd3 knockdown (KD). Figure 25A shows the experimental design for T cell viability monitoring using luciferase-labeled Raji tumor cells. Figures 25B and 25C show that Jmjd3 KD (1928z-shJMJD3) CAR-T cells maintain a high level of T cell number while showing strong proliferation on day 4 after T cell transfer (compared to 1928z control shRNA CAR-T cells). Figure 25D shows that 1928z-shJMJD3 CAR-T cells significantly suppress tumor growth and extend mouse survival time (compared to 1928z control shRNA CAR-T cells).

[0091] Figures 26A, 26B, and 26C show the enhanced tumor migration of T cells by forced expression of chemokine receptors. Figure 26A shows a schematic diagram of the construction of a 1928z CAR fused with CCR5. Figures 26B and 26C show that 1928z-CCR5 CAR-T cells significantly suppress the in vivo proliferation of MDA-MB-231 / CD19 tumor cells, suggesting that forced expression of chemokine receptors enhances tumor migration of T cells.

[0092] Figure 27 illustrates a strategy for simultaneously improving T cell tumor migration and persistence. It schematically shows a method combining chemokine receptor expression with shRNA-mediated knockdown in a TCR or CAR construct.

[0093] Figure 28 shows an example of identifying the Zap70 kinase domain as an alternative to the CAR signaling domain. Figure 28A shows schematic diagrams of CARs with various signaling domains. The CAR uses anti-CD19 scFv (FMC063) as the antigen-binding domain, followed by a CD28 hinge and transmembrane domain. The CD28 tail is fused with CD3 zeta (aa52-164), ZAP300 (aa300-619), LAT (aa28-262), or full-length SLP76. Figure 28B shows the results of flow cytometry analysis of CAR cell surface expression in human T cells. Figure 28C shows cytokine secretion (ELISA) of CAR-T cells co-cultured with CD19-positive Raji cells. Figure 28D shows the results of measuring specific cytotoxicity of CAR-T cells against Raji cells using an LDH non-radioactive assay. Statistical significance is indicated by an asterisk (**: p<0.01, ***: p<0.001, ****: p<0.0001), and "ns" indicates no significant difference.

[0094] Figure 29 shows examples of CD3 zeta substitution with different lengths of the Zap70 kinase domain. Figure 29A shows schematic diagrams of CARs containing shortened Zap70 (ZAP255, ZAP280, ZAP300). Figure 29B shows flow cytometry analysis of CAR surface expression in human T cells. Figure 29C shows the results of statistical analysis of the activity of ZAP255, ZAP280, and ZAP300 CAR-T cells. Figure 29D shows the results of flow cytometry detection of GFP expression when ZAP255, ZAP280, and ZAP300 CARs were introduced into NFAT-GFP reporter Jurkat cells and co-cultured with NALM6 cells. Figure 29E shows the results of analysis of T cell cytotoxic activity by live cell imaging using Cellcyte X. Statistical significance is indicated by an asterisk (****: p<0.0001), and "ns" indicates no significant difference.

[0095] Figure 30 shows the determination of the Zap70 kinase domain required for T cell signaling and function. Figure 30A shows schematic diagrams of truncated Zap70 kinase domains of different lengths. The full-length Zap70 has an N-terminal SH2, a C-terminal SH2, an interdomain A, an interdomain B, and a kinase domain. Figure 30B shows the results of detecting CAR expression in T cells introduced with 1928ZAP300, 1928ZAP327, 1928ZAP377, 1928ZAP420, 1928ZAP540, and 1928ZAP560 CAR constructs using Protein L and Streptavidin-PE. Figure 30C shows the results of measuring IFN-gamma production in CAR-T cells with different Zap70 kinase domain lengths by ELISA and evaluating tumor cell damage by LDH assay. 1928ZAP300 CAR-T cell activity is used as a positive control. Figure 30D shows the results of measuring the tumor cytotoxic activity of 1928ZAP300 and 1928ZAP327 CAR-T cells by LDH assay at E:T ratios of 0.3:1 to 6:1. Figure 30E shows the survival curves (Kaplan-Meier) of mice administered 5 × 10^6 Raji cells on day 0 and 0.5 × 10^6 CAR-T cells on day 5. Statistical significance is (**: p<0.01, ***: p<0.001, ****: p<0.0001), and "ns" indicates no significant difference.

[0096] Figure 31 shows cytokine secretion and tumor cell-specific killing by CAR-T cells possessing the Zap70 kinase domain (ZAP327). Figure 31A shows surface expression of 1928z and 1928ZAP327 CAR-T cells using Protein L and Streptavidin-PE. Figure 31B shows the results of ELISA measurement of IFN-gamma production after co-culture with CD19-positive Raji cells using T cells derived from three healthy donors. Figure 31C shows the results of LDH assay measurement of cytotoxicity of 1928z and 1928ZAP327 CAR-T cells against Raji cells with E:T ratios of 6:1 to 0.3:1. Statistical significance is indicated by an asterisk (****: p<0.0001), and "ns" indicates no significant difference.

[0097] Figure 32 shows the in vitro antitumor response of 1928ZAP327 CAR-T cells. Figure 32A shows IFN-gamma production after co-culture with Raji and NALM6 cells. Figure 32B shows intracellular staining of IFN-gamma, IL2, and TNF-α in 1928z and 1928ZAP327 CAR-T cells after co-culture. Figure 32C shows the measurement and analysis of the T cell activation marker CD69. Figure 32D shows Western blot analysis and quantitative analysis of TCR signaling pathway-related proteins. Figure 32E shows intracellular staining of granzyme B (GZMB) and perforin A (PRF1) after co-culture. Figure 32F shows the measurement of cytotoxicity by luciferase activity, and Figure 32G shows specific killing activity against CD19-positive Raji-GFP cells under co-culture conditions with CD19-negative THP1 cells (ET ratio 5:1). Statistical significance is indicated by * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, and ns indicates no significant difference.

[0098] Figure 33 shows the superior antitumor response of 1928ZAP327 CAR-T cells. Figure 33A shows a schematic diagram of the in vivo experimental design. NSG mice were administered 5 × 10^6 Raji lymphoma cells and treated with various CAR-T cells. Figure 33B shows the results of ELISA measurement of IFN-gamma concentration in mouse serum one day after T cell administration. Figures 33C and 33D show Kaplan-Meier curves of mouse survival rates after administration of 5 × 10^5 or 2 × 10^6 1928z or 1928ZAP327 CAR-T cells. The control group consists of untreated mice. P values ​​were calculated using a one-sided log-rank Mantel-Cox test. (**: p<0.01, ***: p<0.001, ****: p<0.0001), "ns" indicates no significant difference.

[0099] Figure 34 shows the in vitro and in vivo antitumor activity of ZAP327-driven 41BB CAR-T cells. Figure 34A shows the results of detecting CAR surface expression in transviral T cells with Protein L and Streptavidin-PE. Conventional CARs were 19BBz, in which anti-CD19 scFv (FMC63) was bound to the CD8α hinge / transmembrane domain and the 41BB costimulatory domain, and the CD3 zeta-signaling domain. 19BBZAP327 CARs had the CD3 zeta-signaling domain replaced with the ZAP327 kinase domain. Figure 34B shows cytokine production of 19BBz and 19BBZAP327 CAR-T cells after co-culture with CD19-positive MM231-CD19 cells. Figure 34C shows the results of measuring cytotoxic activity against MM231-CD19. Figure 34D shows the in vivo experimental design, and Figure 34E shows the cytokine levels in mouse serum (ELISA) 4 days after T cell administration. Figure 34F shows the mouse survival rate (Kaplan-Meier curve) after administration of 5 × 10^5 CAR-T cells. The data is presented as a summary of three independent experiments. Statistical significance is indicated by * p<0.05, *** p<0.001, **** p<0.0001, and ns indicates no significant difference.

[0100] Figure 34C shows the cytotoxic activity of 19BBz and 19BBZAP327 CAR-T cells against MM231-CD19. Figure 34D shows the in vivo experimental design in which mice were administered Raji cells and then treated with CAR-T cells. Figure 34E shows the results of measuring cytokine secretion in mouse serum 4 days after T cell administration by ELISA. Figure 34F shows the survival rate of mice administered 5 × 10^5 19BBz or 19BBZAP327 CAR-T cells using Kaplan-Meier curves. Data are a summary of three independent experiments. Statistical significance is * p<0.05, *** p<0.001, **** p<0.0001, ns no significant difference.

[0101] Figure 35 shows the excellent antitumor activity of ZAP327-driven 41BB CAR-T cells. Figure 35A shows intracellular staining for IFN-gamma, IL2, and TNF-α in 19BBz and 19BBZAP327 CAR-T cells after co-culture with Raji cells. Figure 35B shows cytotoxicity of CAR-T cells against tumor cells by luciferase assay. Figure 35C shows intracellular staining for granzyme B (GZMB) and perforin A (PRF1) after co-culture. Figure 35D shows a schematic diagram of an experiment comparing the therapeutic effects of 19BBz and 19BBZAP327 in vivo. Mice were administered 2 × 10^6 CD19-positive NALM6 leukemia cells, followed by 1 × 10^6 CAR-T cells. Figure 35E shows the results of measuring the overall survival rate of mice. Statistical significance levels are * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, and no significant difference for ns.

[0102] Figure 36 shows that Zap70 kinase domain-modified T cells exhibit superior antitumor response in solid tumor models. Figure 36A shows the results of ELISA measurements of tumor growth and serum IFN-gamma in NSG mice after subcutaneous administration of 1 × 10^6 MM231-CD19 tumor cells and treatment with CAR-T cells. Figure 36B shows the results of tumor growth and serum IFN-gamma measurements after subcutaneous administration of 1 × 10^6 Mel1558 tumor cells and treatment with CAR-T cells in NSG mice. Statistical significance is * p<0.05, ** p<0.01, **** p<0.0001, and ns indicates no significant difference.

[0103] Figure 37 shows the gating strategy for human Tscm. Lymphocyte populations were identified by FSC vs. SSC gating, and single cells were obtained by FSC-A vs. FSC-H gating. Live cells were obtained as negative populations by live / dead staining. CAR-positive cells were gated. CD95 is widely expressed in post-stimulation memory and effector cells, but negative in naive cells. CD4 and CD8 cells were isolated, and CCR7 and CD127 double-positive populations were gated. Different memory populations were identified with CD45RO and CD62L markers. CD45RO-CD62L+ cells were Tscm, and CD45RO+CD62L- cells were Tcm.

[0104] Figure 38 shows that the ZAP327 signaling domain improves the proportion of Tscm and Tcm cells and T cell persistence. Figure 38A is a schematic diagram of the experimental design and analysis. Human T cells derived from healthy donors were introduced with CAR retrovirus and cultured in vitro. Memory populations were analyzed before injection. Raji cells were administered to NSG mice and treated with luciferase-expressing CAR-T cells. Luciferase signaling was measured on days 0, 1, 3, 7, and 14 after T cell injection. Mice were euthanized on day 40 after tumor injection, and tissue was collected to compare the proportion of CD3+ T cells. Memory phenotypes of Tscm and Tcm cells were also analyzed. Exhaustion markers using PD1+TIM3+LAG3+ as indicators were also evaluated. Figure 38B is a representative example of the proportion of Tscm and Tcm cells in vitro 20 days after T cell activation. Figure 38C is a representative example of luciferase signaling. Figure 38D is a statistical analysis of Figure 38C. Figure 38E shows the percentage of CD3+CAR-T cells collected from bone marrow, lung, and spleen 36 days after T cell infusion, analyzed by FACS. Figure 38F shows the analysis of CD62L+CD45RO+(Tcm) and CD62L+CD45RO-(Tscm) percentages after gating CD3+CAR+CD95+CCR7+IL7R+ cells. Statistical significance is *P < 0.05, **P < 0.01.

[0105] Figure 39 shows the suppression of exhaustion in 1928ZAP327 CAR-T cells. Figure 39A shows the results of flow cytometry analysis of exhaustion markers PD1, LAG3, and TIM3 in 1928z and 1928ZAP327 CAR-T cells. Figure 39B is a SPICE analysis plot, and Figure 39C shows flow cytometry analysis of intracellular expression of negative regulators TOX and NR4A1. Statistical significance is * p<0.05, ** p<0.01, and ns indicates no significant difference.

[0106] Figure 40 shows that the 1928ZAP327 CAR establishes a specific transcriptional signature. Figure 40A shows a volcanic map of significant gene expression changes in 1928z and 1928ZAP327 CAR-T cells 24 hours after CD19-positive target cell stimulation (n=3 replicates / group). Figure 40B shows genes involved in different biological processes as determined by biological GO analysis. Figure 40C shows a heatmap analysis of genes involved in glycolysis and mitochondrial function.

[0107] Figure 41 shows that 1928ZAP327 CAR establishes a specific metabolic pathway. Figure 41A shows the OCR measurement results of 1928z and 1928ZAP327 CAR-T cells by Seahorse. It shows quantitative analysis of basal respiration, ATP production, maximal respiration, reserve capacity, proton leak, and non-mitochondrial respiration. Figure 41B shows the ECAR measurement results. It shows quantitative analysis of basal acidification, glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Figure 41C shows the results of 2-NBDG and BODIPY-C16 uptake and mitochondrial strength measured by MitoTracker. Statistical significance is * p<0.05, ** p<0.01, **** p<0.0001.

[0108] Figure 42 shows CT83 expression in lung cancer and breast cancer samples. Figure 42A shows RT-PCR analysis of CT83 expression in lung cancer cell lines and tumor samples. Figure 42B shows Western blot analysis of CT83 protein expression in lung cancer cell lines using an anti-CT83 antibody. Figure 42C shows the results of RT-PCR analysis of CT83 expression in breast cancer cell lines and triple-negative breast cancer samples. NY-ESO-1 was used as a positive control.

[0109] Figure 43 shows the generation and characterization of HLA-DR13-restricted CT83-specific T cells. Figure 43A shows CT83 peptide-specific CD4+ T cells by intracellular staining of T cells stimulated in vitro with CT83 peptide. Figure 43B shows that T cells can recognize 293DR13 / CT83 PEP10-31 but not 293DR13 / control peptide. Figure 43C shows that T cell recognition of 293DR13 / CT83 PEP10-31 cells is inhibited by anti-MHC class II or anti-HLA-DR antibodies, but not by anti-HLA-DP, anti-HLA-DQ, and anti-MHC I antibodies. Figure 43D shows that CT83-specific T cells recognize only 293DR13 / CT83 PEP10-31, but do not recognize the same peptide when pulsed to 293DR1, DR3, DR4, DR7, and DR11. *** P < 0.001.

[0110] Figures 44A and 44B show T cell epitope mapping and tumor-specific recognition. Figure 44A shows that 293DR13 cells transformed with CT83 (aa10-31) and CT83 (aa17-31) gene fragments are recognized by T cells. Figure 44B shows that DR13-CT83 TCR-transformed CD4+ T cells from three healthy donors specifically recognize MDA-MB-231 cells (DR13+ CT83+), but not MDA-MB-436 and MDA-MB-468 (DR13- CT83+) or M1495 (DR13+ CT83-). ** P < 0.01; *** P < 0.001. The figures disclose sequence numbers s 39, 62-63, and 61, respectively, in order of appearance.

[0111] Figure 45 shows the identification and characterization of CT83-specific TCRs. Figure 45A shows a schematic diagram of the identification and cloning of CT83-specific TCRs using single-cell TCR sequencing technology. Figure 45B shows that 293DR13 cells expressing the Ii-CT83 gene or 293DR13 cells loaded with the DR13 CT83 peptide are recognized by HLA-DR13-restricted CT83-specific TCR-T cells. Figure 45C shows that CT83-TCR-transformed CD4+ T cells recognize 293DR13 / Ii-CT83 cells and MDA-MB-231 cells, but not CT83-TCR-transformed CD8+ T cells. ** P < 0.01; *** P < 0.001.

[0112] Figure 46 shows the complete inhibition or elimination of breast cancer cells by CD4+ CT83 TCR-T cells. Figure 46A shows the results when NSG mice with MDA-MB-231 tumors were administered control T cells or CT83 TCR CD4+ T cells. CD4+ CT83 TCR-T cells completely inhibited or eliminated breast cancer cells. Figure 46B shows gated FACS analysis of human CD3+ T cells isolated from the spleen of treated mice. Anti-CD62L and anti-CD45RA analysis in CD4+ T cells is shown. **** P < 0.0001.

[0113] Figure 47 shows various TCR modification designs incorporating 4-1BB-ZAP327 (BBZAP327) and knockdown / knockdown of negative regulators using shRNA or sgRNA.

[0114] Figure 47A shows the design and construction of human TCR [TCR(H)], mouse TCR with a constant region [TCR(M)], and TCR(M) (also called TCR-STEM) incorporating 4-1BB-ZAP327 (BBZAP327). All TCR constructs include shRNA or sgRNA knockdown / knockout of negative regulators for improved T cell persistence and function. In some embodiments, TCR(M) is further modified to express 4-1BB, CD28, CD27, OX40, ICOS, MyD88, or MALT-1 downstream of the TCR-TM domain. In other embodiments, TCR(M) is modified to express T2A-MyD88 or T2A-MALT-1 downstream of TCR-TM. Figure 47B shows schematic diagrams of the TCR complexes of TCR(H), TCR(M), TCR-STEM, and TCR(M) incorporating additional signaling molecules.

[0115] Figure 48 shows tumor-specific recognition, killing, and anti-tumor immune responses of A2 / CT83 TCR-T cells and A2 / CT83 TCR-BBZAP327 (also called A2-CT83 TCR-STEM) cells. Figure 48A shows schematic diagrams of A2 / CT83 TCR-T cells and A2 / CT83 TCR-BBZAP327 cells. Figure 48B shows specific cytotoxicity of A2 / CT83 TCR-T cells and A2 / CT83 TCR-BBZAP327 cells. Figure 48C shows target recognition (cytokine release). A2 / CT83 TCR-BBZAP327 cells show lower IFN-gamma production than A2 / CT83 TCR-T cells. * P < 0.05, **** P < 0.0001, ns are not statistically significant.

[0116] Figure 49 shows that A2-CT83 TCR-BBZAP327 cells exhibit superior antitumor activity compared to CT83 TCR-T cells in a challenging tumor model (PD-L1-expressing MDA-MB-231). Figure 49A shows a direct comparison of antitumor activity due to tumor suppression. Figure 49B shows a comparison of tumor weight between groups. Figure 49C shows T cell analysis in tumor tissue from each treatment group. ** 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 shows the T cell 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. CD4 TCR(MC) T cells show significantly higher IFN-gamma release compared to CD4 TCR(HC) T cells against 293DR13 / CT83 cells. Figure 50B shows a schematic diagram of pCD4 TCR-STEM (pCD4 TCR-BBZAP327). Figure 50C shows tumor recognition (IFN-gamma release) by CD4 TCR-STEM T cells. Figure 50D shows specific killing of MDA-MB-231 tumor cells by CD4 TCR-STEM T cells. * P < 0.05, ** P < 0.01.

[0118] Figure 51 shows the potent in vivo antitumor immunity of CD4 TCR-STEM T cells in breast and lung cancer models. Figure 51A shows the experimental design of a breast cancer model using CD4 TCR-STEM T cells or control CD4 T cells. Figure 51B shows a comparison of antitumor activity among four groups (control group, CD4 TCR(HC) group, CD4 TCR(MC) group, and CD4 TCR-STEM group). Figure 51C shows that CD4 TCR-STEM T cells completely inhibited or eliminated the growth of MDA-MB-231 tumors in two independent experiments using T cells from three healthy donors. Figure 51D shows that both CD4 TCR-STEM T cells and A2-CT83 TCR-STEM T cells completely inhibited or eliminated 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 shows the HLA-A2 restriction CT83 T cell epitopes and HLA-DR13 restriction CT83 T cell epitopes. Figure 52B shows CT83 expression in various cell lines. Figure 52C shows that A2-CT83 TCR-STEM T cells do not exhibit cross-reactivity. Figure 52D shows CT83 recognition by TCR-STEM T cells in the context of the HLA-A2 molecule.

[0120] Figure 53 shows the alanine scanning analysis of the HLA-DP4 restricted NY-ESO-1 TCR. Alanine substitutions of amino acid residues in the CDR region can cause loss of function. Alanine substitutions that resulted in a significant decrease in TCR function are shown. ** P < 0.01, *** P < 0.001 (WT control TCR ratio).

[0121] Figure 54 shows detailed amino acid mutations and functional screening in the HLA-DP4 NY-ESO-1 TCR. Figure 54A shows the results of introducing further mutations at each essential amino acid position for key residues identified by alanine scanning. Figure 54B shows functional screening using Jurkat NFAT-GFP reporter cells.

[0122] Figure 55 shows the functional analysis of wild-type (WT) and mutant TCR-transformed CD4+ T cells. Figure 55A shows IFN-gamma release from WT and mutant DP4 NY-ESO-1 TCR-transformed CD4+ T cells against NY-ESO-1 expressing MDA-MB-231 / DP4 cells and MDA-MB-231 cells. Figure 55B shows the cytotoxic activity of WT and mutant TCR-transformed CD4+ T cells against MDA-MB-231 / DP4 cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0123] Figure 56 shows the identification of essential amino acids for the HLA-A2 CT83 peptide (90-98) and HLA-DR13 CT83 peptide (17-31) required for the corresponding TCR-T cells. Figure 56A shows the alanine scanning analysis of the HLA-A2 CT83 peptide (90-98) (SEQ ID NO: 2) to identify the essential amino acids required for A2-CT83 TCR-T cells. Figure 56B shows the alanine scanning analysis of the HLA-DR13 CT83 peptide (17-31) (SEQ ID NO: 61) to identify the essential amino acids required for CD4 TCR-STEM T cell recognition. Figure 56C shows the truncation analysis of amino acid residues at the N-terminus and C-terminus of the HLA-DR13 CT83 peptide required for CD4 TCR-STEM T cell recognition. [Detailed explanation] Definition

[0124] Prior to disclosing and describing the compounds, compositions, articles, apparatus and / or methods of the present invention, it should be understood that, unless otherwise stated, they are not limited to specific synthesis methods or specific recombinant biotechnology techniques or specific reagents, and are naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the invention.

[0125] In this specification and the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise. Therefore, for example, a reference to "pharmaceutical carrier" includes a mixture of two or more carriers, etc.

[0126] In this specification, ranges may be expressed as "about" a particular value to "about" another particular value. Where such ranges are described, the range from one particular value to another is also included as another embodiment. Similarly, where a numerical value is expressed as an approximation using the prefix "about," the particular value itself is understood to be included as another embodiment. Furthermore, it is understood that the endpoints of each range are important both in relation to each other and independently of each other. Numerous numerical values ​​are disclosed in this specification, and each numerical value is disclosed not only as the numerical value itself but also "about" that numerical value. For example, if the numerical value "10" is disclosed, "about 10" is also disclosed. Also, where a numerical value is disclosed, "less than or equal to that numerical value," "greater than or equal to that numerical value," and possible ranges between those numerical values ​​are also disclosed to the extent that it is understood by those skilled in the art. For example, if the numerical value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. Throughout this application, data is provided in several different formats, and these data are understood to represent ranges based on endpoints, starting points, and any combination of data points. For example, if the data points "10" and "15" are disclosed, it is understood that the values ​​"greater than," "greater than or equal to," "less than," "less than or equal to," "equal to," and the values ​​between 10 and 15 are disclosed. It is also understood that the values ​​of each unit that exist between the two numbers are disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. Furthermore, if a set of numbers is disclosed, any range that falls between any two enumerated numbers will be understood by those skilled in the art.

[0127] Terms used in this specification and in the following claims shall have the following meanings unless otherwise specified.

[0128] "Optional" or "optionally" means that it includes both cases in which the event or situation described thereafter occurs and cases in which it does not occur, and encompasses both embodiments in which such event or situation occurs and embodiments in which it does not occur.

[0129] In this specification, “antibody” includes both polyclonal and monoclonal antibodies, and includes primate-like antibodies (e.g., humanized antibodies), mouse antibodies, mouse-human antibodies, mouse-primate antibodies, and chimeric antibodies. Antibodies may also be complete molecules or fragments thereof (which may or may not include scFv, Fv, Fd, Fab, Fab′, and F(ab)′2 fragments), or multimers or aggregates of complete molecules and / or fragments. Antibodies may be naturally occurring or produced by immunoassay, synthesis, or genetic engineering. “Antibody fragment” refers to a fragment derived from or related to an antibody that possesses antigen-binding ability, and in some embodiments, structural features that promote clearance and uptake may be conferred, for example, by the introduction of galactose residues. “Antibodies” include, for example, F(ab), F(ab)′2, scFv, light chain variable regions (VL), heavy chain variable regions (VH), and combinations thereof.

[0130] A checkpoint inhibitor is a drug that targets checkpoint proteins or derivatives thereof, and may also be called a "checkpoint inhibitor." Checkpoint inhibitors may or may not include proteins, polypeptides, amino acid residues, and monoclonal or polyclonal antibodies. A multivalent vaccine may contain one or more checkpoint inhibitors, or may be administered in combination with them. Checkpoint inhibitors may bind to ligands or proteins belonging to the T cell regulatory factor family, such as CD28 / CTLA-4. Targets of checkpoint inhibitors include, but are not limited to, receptors or co-receptors expressed on effector or regulatory cells of the immune system (e.g., T cells) (e.g., CTLA-4, CD8), proteins expressed on the surface of antigen-presenting cells (e.g., PD-1, PD-2, PD-L1, PD-L2, 4-1BB, and OX40 expressed on the surface of activated T cells), metabolic enzymes expressed on both tumor cells and tumor-infiltrating cells (e.g., indoleamine 2,3-dioxygenase (IDO), including isoforms such as IDO1 and IDO2), proteins belonging to the immunoglobulin superfamily (e.g., lymphocyte activation gene 3 (LAG3)), and proteins belonging to the B7 superfamily (e.g., B7-H3 or its homologs).

[0131] In this specification, "separation" includes any means of substantially purifying one component from another, such as filtration or magnetic adsorption.

[0132] In this specification, “isolation” or “isolating” includes any means of separating a species belonging to a genus from other species of the same genus.

[0133] "Subject" refers to the individual being administered or treated. The subject may be a vertebrate, such as a mammal. In one embodiment, the subject may be a human, a non-human primate, a cattle, a horse, a pig, a dog, or a cat. It may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Therefore, the subject may be a human patient or an animal being treated in veterinary medicine. "Patient" refers to a subject under the treatment of a clinician, such as a physician.

[0134] In this specification, “preventing the development of cancer or cancer cells” or “inhibiting the development of cancer” means that the development of cancer is prevented or the onset of cancer is delayed.

[0135] In this specification, “treating” or “reducing” the presence of cancer or cancer cells means that the growth of cancer is inhibited, as reflected by a reduction in tumor volume or the number of malignant cells. Tumor volume can be measured by known methods, such as measuring observational images and comparing the mean cross-sectional diameter of the tumor to a calibration curve (e.g., the ImageJ method).

[0136] In this specification, "to treat a disease or condition such as inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, infections and / or age-related conditions" means to reduce or improve the severity of the signs or symptoms of the condition.

[0137] In one embodiment, the disease or disorder to be treated is selected from the group consisting of inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related conditions, cancer, and infections.

[0138] In some embodiments, 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.

[0139] In some embodiments, the autoimmune disease to be treated is selected from the group consisting of multiple sclerosis, type 1 diabetes, Graves' disease, Hashimoto's disease, myasthenia gravis, Addison's disease, pemphigus, scleroderma, Goodpasture syndrome, autoimmune hepatitis, and autoimmune hemolytic anemia.

[0140] In some embodiments, 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.

[0141] In some embodiments, the organ transplant-related conditions to be treated are selected from the group consisting of graft-versus-host disease (GvHD), acute rejection, chronic rejection, transplant vascular complications, post-transplant lymphoproliferative disorders (PTLD), and transplant delay function.

[0142] In some embodiments, the infection to be treated 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 infection, long COVID, and sepsis.

[0143] In this specification, "preventing or inhibiting the outbreak of an infectious disease" means preventing the outbreak of an infectious disease, delaying the onset of an infectious disease, or suppressing or reversing the spread of an existing infection.

[0144] In this specification, "activation" refers to the state of a cell after significant biochemical or morphological changes have been induced by sufficient binding (ligand binding) of components on the cell surface. In the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cell proliferation. T cell activation can induce cytokine production and the exertion of regulatory or cytotoxic effector functions. In the context of other cells, this term means the upregulation or downregulation of specific physicochemical processes.

[0145] As used herein, "cancer antigen" or "tumor antigen" includes tissue-specific differentiation antigens, tumor-specific covalent antigens, and mutated tumor-specific and unique antigens, and furthermore, CD4 + or CD8 + This refers to any part of these antigens, including peptides or polypeptides, that can trigger an immune response in T cells. CD8 + or CD4 + Tumor antigens or cancer antigens recognized by T cells can be classified into several categories (Wang, RF & Wang, HY, Cell Research 27, 11-37 (2017)). That is, 1) Tissue-specific differentiation antigens, including MART-1, TRP-1 / gp75, TRP-2, and gp100, which are antigens that are highly expressed in cancer cells compared to normal cells; 2) These are tumor-specific co-antigens, which may include MAGE-A1 and NY-ESO-1. These antigens are expressed in cancer and the testes but not in other normal tissues, and are also called cancer-testicular (CT) antigens. 3) Mutant antigens that are tumor-specific and unique antigens, including antigens such as CDK4, catenin, and caspase-8; and 4) These are overexpressed tumor antigens that are overexpressed in cancer cells compared to normal cells.

[0146] Two cancer testis (CT) antigens, NY-ESO-1 (encoded by the CTAG1B gene) and CT83 (also known as KK-LC-1 and encoded by the CT83 gene), are widely expressed in various tumors, including lung cancer and breast cancer. NY-ESO-1 is CD8 + It has been shown to be recognized by both T cells and antibodies, and clinical response rates using NY-ESO-1-specific TCRs have been demonstrated to reach 50-80% in several solid tumors, including melanoma, sarcoma, and multiple myeloma. Meanwhile, CD4 + Despite the importance of T cells (HLA-DP4 is the most frequent HLA class II molecule in the general population, positive in approximately 70%), HLA-DP4-restricted NY-ESO-1 specific TCRs had not been clinically validated. The inventors previously identified HLA-DR4 and HLA-DP4-restricted NY-ESO-1 epitopes and further demonstrated that the HLA-DP4-NY-ESO-1 peptide overlaps with the HLA-A2-restricted NY-ESO-1 peptide.

[0147] As disclosed herein, HLA-DP4-restricted NY-ESO-1 specific CD4 + We generated T cells and TCRs and investigated whether the combined use of DP4-ESO-1 TCR-modified T cells and A2-ESO-1 TCR-modified T cells could induce stronger antitumor immunity than using either cell alone.

[0148] In addition to the CT antigen NY-ESO-1, CT83 is highly expressed in 60-70% of breast cancers, particularly in triple-negative breast cancer (TNBC), which is consistent with previous reports. However, little has been known about its immunogenicity, T cell epitope, and corresponding TCR involved in tumor recognition by T cells. This specification discusses antigen-specific CD4 + and CD8 + To generate T cells and determine whether CT83 could be a promising target for TCR-T cell immunotherapy, we identified HLA-A2-restricted CT83-specific TCRs (A2-CT83 TCRs).

[0149] The persistence of CAR-T and TCR-T cells in the body has been shown to be closely correlated with patient survival. Therefore, regulating TCR-T and CAR-T cell signaling can enhance T cell persistence and reduce T cell exhaustion through direct control of CAR or TCR signaling, as well as through knockdown or knockout of negative signaling molecules such as PD-1, VHL, and PPP2R2D, or epigenetic factors such as Jmjd3 and LSD1.

[0150] In some embodiments, the immunogenetic peptides and epitopes contained in the tumor antigens of the present invention are derived from the NY-ESO-1 protein and the CT83 protein, both of which are widely expressed in various types of cancer, including but not limited to breast cancer, lung cancer, and prostate cancer. The tumor antigens of the present invention are expressed at low levels in normal cells, but at significantly higher levels in tumor cells and testes.

[0151] In some embodiments, the “tumor antigen” or “cancer antigen” is NY-ESO-1, CT83 protein, HCMV pp65 protein and / or HCMV IE-1 protein, as well as CD4 + or CD8 + The material comprises any portion, peptide, or polypeptide of the NY-ESO-1, CT83, HCMV pp65, and / or HCMV IE-1 proteins that can elicit an immune response from T cells, including full-length NY-ESO-1 and CT83 proteins.

[0152] As used herein, “immunogenetic peptides and epitopes” encompass any epitopes or fragments of the NY-ESO-1, CT83, HCMV pp65, and / or HCMV IE-1 proteins that function as tumor antigens.

[0153] As used herein, "fragment" or "part" means a segment having at least 5 or 6 amino acids in the case of a protein, or a segment having at least 15 to 18 nucleotides in the case of a gene.

[0154] In one embodiment, the tumor antigen-specific T cell line of the present invention immunologically recognizes tumor antigens presented by HLA-DP4-positive or HLA-A2-positive antigen-presenting cells, and all generated CD4 + or CD8 + Includes T lymphocytes.

[0155] As used herein, “presented” includes the steps of transfecting antigen-presenting cells with DNA encoding a tumor antigen in its entirety or in any portion thereof, or loading antigen-presenting cells with a peptide of a tumor antigen in its entirety or in any portion thereof.

[0156] As used herein, “antigen-presenting cell” includes any natural or artificial cell line or cell that expresses a desired type of HLA molecule on its cell surface. Furthermore, as used herein, “antigen” means (1) a molecule whose whole or fragment can be specifically recognized and bound by the idiotype portion (antigen-binding region) of a monoclonal antibody (mAb) or a derivative thereof, and (2) a molecule comprising a peptide sequence capable of binding to MHC and capable of specifically interacting with the corresponding T cell antigen receptor in the context of MHC presentation.

[0157] As used herein, "HLA-DP4 positive" includes any natural or artificial cell line or cell that expresses the HLA class II molecules DPA1 and DPB1*04 (including all subtypes thereof) on its cell surface.

[0158] As used herein, "HLA-A2 positive" encompasses any natural or artificial cell line or cell that expresses the HLA class I molecule A*02 (including all its subtypes) on its cell surface.

[0159] As used herein, "HLA-DR13 positive" includes any natural or artificial cell line or cell that expresses the HLA class II molecule DRB1*13 (including all its subtypes) on its cell surface.

[0160] In one embodiment of the present invention, at least two T cell receptors are derived from antigen-specific CD4+ or CD8+ T cell lines. The full-length alpha and beta chains of the TCR are cloned separately. As used herein, the term “full length” refers to the human alpha chain constant region (as a non-limiting example, TRAC, IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS) (SEQ ID NO: 10), or the alpha chain variable region fused with the mouse alpha chain constant region (trac) (SEQ ID NO: 13), or the human beta chain constant region type 2 (TRBC2, DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE It includes a beta-chain variable region fused with a human beta-chain constant region type 1 (TRBC1, DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF) (SEQ ID NO: 11), a mouse beta-chain constant region type 1 (trbc1) (SEQ ID NO: 14), or a mouse beta-chain constant region type 2 (trbc2) (SEQ ID NO: 15). In some embodiments, the constant region may have sequences having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with respect to sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, or sequence number 15.The steady-state region may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0161] In some embodiments, the chimeric TCR is a CT83-specific TCR and comprises a chimeric α chain fused with an HLA-A2-restricted CT83 TCR α chain variable domain containing the mouse α chain constant domain including SEQ ID NO: 20, or a variant having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 20, and / or a sequence having 1 to 10 substitutions (which may be conservative substitutions) to SEQ ID NO: 20. Furthermore, the chimeric TCR comprises a chimeric β-chain in which the HLA-A2-restricted CT83 TCR β-chain variable domain is fused with a mouse β-chain constant domain 2 containing SEQ ID NO: 21, or a mutant having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 21, and / or a sequence having 1 to 10 substitutions (which may be conservative substitutions) to SEQ ID NO: 21.

[0162] In some embodiments, the chimeric TCR is a NY-ESO-1 specific TCR and comprises a chimeric α chain selected from a polypeptide in which the HLA-A2-restricted NY-ESO-1 TCR(S2) α-chain variable domain is fused with a mouse α-chain constant domain containing SEQ ID NO: 22, or a variant having 85-99% sequence identity to SEQ ID NO: 22, and / or a sequence having 1-10 substitutions (which may be conservative substitutions) relative to SEQ ID NO: 22, and a polypeptide in which the HLA-A2-restricted NY-ESO-1 TCR(S5) α-chain variable domain is fused with a mouse α-chain constant domain containing SEQ ID NO: 24, or a variant having 85-99% sequence identity to SEQ ID NO: 24, and / or a sequence having 1-10 substitutions (which may be conservative substitutions) relative to SEQ ID NO: 24. Furthermore, the chimeric TCR includes a polypeptide selected from the following: a polypeptide in which the HLA-A2-restricted NY-ESO-1 TCR(S2)(G50A, A51E) β-chain variable domain is fused with mouse β-chain constant domain 2 containing SEQ ID NO: 23, or a mutant having 85-99% sequence identity to SEQ ID NO: 23, and / or a sequence having 1-10 substitutions (these may be conservative substitutions) relative to SEQ ID NO: 23; and a polypeptide in which the HLA-A2-restricted NY-ESO-1 TCR(S5)(G50A, A51E, A97L) β-chain variable domain is fused with mouse β-chain constant domain 2 containing SEQ ID NO: 25, or a mutant having 85-99% sequence identity to SEQ ID NO: 25, and / or a sequence having 1-10 substitutions relative to SEQ ID NO: 25.

[0163] In some embodiments, the chimeric TCR contains a sequence having 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The constant region may further contain 1 to 10 substitutions, which may be conservative substitutions.

[0164] As used herein, "proliferation" means growth or increase in number by producing new cells.

[0165] As used herein, “purify” or “pure” refers to molecules separated from other reaction components or cellular components. “Substantially pure” or “substantially purified” refers to molecules having a purity of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0166] In yet another embodiment of the present invention, CD4 +Epitopes of tumor antigens that specifically interact with 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). These are peptides / parts of the NY-ESO-1 protein and contain the corresponding amino acid sequences (e.g., PEP161-180 are amino acids 161-180 of the NY-ESO-1 protein). In some embodiments, these epitopes include variants containing one to three conservative substitutions. NY-ESO-1 PEP161-180 (SEQ ID NO: 34) has been identified as a peptide with high affinity for HLA-DP4 (Zeng et al., J. Immunol. 165, 1153-1159 (2000)). Using this peptide, peptide-stimulated CD4 specifically recognizes NY-ESO-1 presented by HLA-DP4. + T cells are induced (Zeng et al., Proc. Natl. Acad. Sci. USA 98, 3964-3969 (2001)). In addition, NY-ESO-1 PEP157-170 (SEQ ID NO: 1) has been identified as the shortest functional epitope that maintains function without a decrease in immune response compared to full-length NY-ESO-1.

[0167] In yet another embodiment of the present invention, CD8 +The tumor antigen epitopes that specifically interact with T cell lines or TCRs to induce a T cell immune response are 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), CT83 PEP10-24 (SILCALIVFWKYRRF, SEQ ID NO: 62), and CT83 This includes, but is not limited to, PEP13-27 (CALIVFWKYRRFQRN, SEQ ID NO: 63). These are peptides / parts containing the specified amino acid sequences of the CT83 protein. In some embodiments, these epitopes include variants containing 1 to 3 conservative substitutions.

[0168] In yet another embodiment of the present invention, CD4 + Epitopes of tumor antigens that specifically interact with T cell lines or TCRs to elicit a T cell immune response include, but are not limited to, the pp65 peptide (495-503) (NLVPMVATV, SEQ ID NO: 26). pp65 is an HCMV protein and an antigen expressed on glioblastoma cells. In some embodiments, these epitopes include variants containing one to three conservative substitutions.

[0169] In yet another embodiment of the present invention, CD4 + Examples of tumor antigen epitopes that specifically interact with T cell lines or TCRs and induce a T cell immune response include epitopes containing, but not limited to, IE-1 peptide 316-324 (VLEETSVML, SEQ ID NO: 31). IE-1 is an HCMV-derived protein and an antigen expressed on glioblastoma cells. In some embodiments, the epitope includes variants containing one, two, or three conservative substitutions.

[0170] In this specification, "self-cleaving peptide" includes, but is not limited to, a P2A sequence (RAKRSGSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 51) positioned between two proteins and capable of separating both proteins by self-cleavage.

[0171] In this specification, “stimulation” refers to a primary response induced by ligand binding to a cell surface component. For example, with respect to a receptor, this includes ligand binding to the receptor and subsequent signaling events. In the case of T cell stimulation, this refers to the binding of T cell surface components, which, in some embodiments, induces signaling events that include or do not include binding to the TCR / CD3 complex. Furthermore, such stimulation may enhance or suppress the expression or secretion of molecules, including or not including a decrease in TGF-β expression. In addition, even if direct signaling does not occur, rearrangement of the cytoskeleton or assembly of cell surface components may occur, modifying or altering subsequent cellular responses.

[0172] In this specification, “vector” includes, but is not limited to, pMSGV, pMSCV, pFU3W, or any other vector having the function of delivering inserted DNA into living cells.

[0173] In yet another embodiment of the present invention, a vector for inserting cDNA encoding a TCRα chain and / or a TCRβ chain is provided. In some embodiments, the translation product of the vector comprises at least one α-chain variable region and at least one α-chain constant region, and / or at least one β-chain variable region and at least one β-chain constant region, linked by a self-cleaving peptide. The vector is used to deliver genes to naive T cells by viral introduction.

[0174] In this specification, "viral introduction" includes methods for producing recombinant viruses such as retroviruses, lentiviruses, and adeno-associated viruses in host cells, and infecting, transducing, or introducing target cells using said recombinant viruses containing a gene encoding the TCR. The gene encoding the TCR is integrated into the genome of the target cell and is stably expressed and replicated even after cell proliferation. "Target cell" refers to CD4 + T cells, CD8 + This includes, but is not limited to, T cells, tumor cells, etc.

[0175] In one embodiment, the present invention provides a host cell transduced or introduced by a vector containing DNA encoding a TCR region or chain according to any of the above embodiments. For example, it includes a TCRα chain variable region and an α chain constant region linked by a P2A sequence (SEQ ID NO: 51), as well as a TCRβ chain variable region and a β chain constant region, and is used for virus production and TCR introduction into naive T cells.

[0176] In one embodiment, the TCR is a chimeric TCR in which a TCR variable region is fused to a modified human constant region or a non-human constant region. In some embodiments, a TCR variable region specific to a cancer antigen or other disease-related antigen is fused to a non-human, for example, mouse-derived TCR constant region. Examples include chimeric TCRs in which a TCR variable region specific to CT83, NY-ESO-1, pp65, or IE-1 is fused to a mouse TCR constant region. These chimeric TCRs have the effect of reducing mispairing with endogenous TCRs in transfused T cells. For example, a chimeric CT83 TCR (MC) reduces mispairing with endogenous TCRs (HC). Similarly, chimeric NY-ESO-1 TCRs, chimeric pp65 TCRs, or chimeric IE-1 TCRs also reduce mispairing with endogenous TCRs.

[0177] In this specification, “host cell” includes, but is not limited to, the PG-13 cell line, the Phoenix-Eco cell line, the Phoenix-Ampho cell line, the 293GP cell line, or other suitable cell lines capable of assembling the viral genome within the cell, packaging the virus with a capsid protein, and secreting the mature virus extracellularly.

[0178] In one embodiment, the present invention provides methods and strategies for extending the persistence of TCR-T cells or CAR-T cells by direct modification of the CAR signaling domain or knockdown / knockout of negative signaling molecules. In some embodiments, methods are provided for improving the persistence of TCR-T cells or CAR-T cells by expressing chemokine receptors and / or knocking out shRNA in the CAR construct. Furthermore, therapy with modified TCR-T cells according to the present invention has been found to significantly reduce relapse or cancer recurrence after initial treatment and after reduction of tumor burden. It is also effective in reducing the recurrence of inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant-related diseases, infections, and / or age-related symptoms.

[0179] In some embodiments, the present invention provides modified immune cells (TCR-T or CAR-T) having knockdown, knockout, or inactivating mutations in one or more endogenous genes selected from a group of negative regulators. These negative regulators include ANKRDJ1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1 / 2 / 3, JMJD1C, JMJD3(KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1 / 2 / 3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, and PTP. The modified TCR-T cells include, or do not include, epigenetic factors such as N1 / 2 / 6 / 22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1 / 2, TIGIT, TNFAIP3, TOX1 / 2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, IDO (IDO1 / IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, and JMJD3 and LSD1. In certain embodiments, PD-1, VHL, PPP2R2D and / or the aforementioned epigenetic factors are selected. Treatment with these modified TCR-T cells contributes to a reduction in tumor recurrence.

[0180] In some embodiments, knockout, knockdown, or inactivating mutations for any negative signaling molecule, and / or expression of chemokine receptors and / or shRNA knockout, are applicable to any CAR-T or TCR-T cells or CAR constructs described herein. For example, such CAR-T cells may express one or more CARs comprising an extracellular binding domain including an antigen recognition site, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0181] In some embodiments, the present invention provides modified immune cells (TCR-T or CAR-T) having knockdown, knockout, or inactivating mutations in one or more endogenous genes selected from the negative regulatory group. In certain embodiments, epigenetic factors including PD-1, VHL, PPP2R2D, and / or JMJD3 or LSD1 are selected.

[0182] In one embodiment, the present invention provides nucleic acids encoding siRNA, such as shRNA, for gene knockdown to enhance the antitumor activity of TCR-transformed T cells in vivo. The shRNA targets negative signaling molecules such as immune checkpoint proteins or immunosuppressive proteins. The target gene may be selected from the group of negative regulators. The siRNA or shRNA contains about 20 to 30 nucleotides that bind to RNA derived from the target gene. In certain embodiments, targets for the shRNA include PD-1 (SEQ ID NO: 7), VHL (SEQ ID NO: 8), and PPP2R2D (SEQ ID NO: 9). The target sequence of PPP2R2D may include all or part of the transcript variant 1 (SEQ ID NO: 18) or variant 3 (SEQ ID NO: 19). Furthermore, antisense RNA or DNA may be used to repress the expression of genes encoding negative signaling molecules.

[0183] In some embodiments, knockout or inactivation of target gene expression is achieved by a CRISPR / Cas9 system comprising a single guide RNA (sgRNA) molecule and a Cas endonuclease. The system includes ANKRDJ1, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1 / 2 / 3, JMJD1C, JMJD3(KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1 / 2 / 3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, and PT. The expression of one or more target genes selected from PN1 / 2 / 6 / 22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1 / 2, TIGIT, TNFAIP3, TOX1 / 2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, IDO (IDO1 / IDO2), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-H3, etc. can be reduced or deleted. The sgRNA contains a target RNA sequence that binds to the target DNA sequence encoding the negative regulator.

[0184] In another embodiment, a sequence of shRNA, antisense RNA, or DNA that specifically knocks down metabolism-related genes to enhance the antitumor activity of TCR or CAR therapy in vivo is provided as a 21-base pair stem structure. TCRs can be modified in combination with shRNA to improve the migration and persistence of T cells in the body and enhance antitumor activity by knocking down target genes. As used herein, "knockdown" means reducing gene expression by degrading mRNA or inhibiting RNA expression. "Metabolic genes" include, but are not limited to, PD-1, VHL, and PPP2R2D.

[0185] This specification references several publications, the disclosures of which are incorporated herein by whole reference for the purpose of complementing the understanding of the technical field to which the present invention pertains. In particular, PCT Publication WO / 2021 / 263211 and its corresponding U.S. Patent Application No. 18 / 002,969 are incorporated herein by full text reference.

[0186] The present invention comprises CD4+ or CD8+ T lymphocytes that immunologically recognize tumor antigens under the restriction of HLA class II or class I molecules. It also comprises at least one T cell receptor (TCR) derived from the said CD4+ or CD8+ T lymphocytes. The TCR can be introduced into naive CD4+ or CD8+ T lymphocytes that do not have an immune response to tumor antigens, thereby conferring the function of specifically recognizing and responding to tumor antigens to the said T lymphocytes. This contributes to the prevention, elimination, or reduction of human cancers that express tumor antigens. method Immunoassay and fluorescent dyes

[0187] The procedures for various immunoassay methods are described in prior art literature (e.g., Maggio et al., Nakamura et al.), and the descriptions of immunoassay methods are incorporated herein by full text reference. Immunoassays are binding assays based on the binding of antibodies to antigens, and examples include ELISA, RIA, RIPA, immunobead capture, Western blotting, dot blotting, gel shift assay, flow cytometry, protein arrays, multiple bead arrays, magnetic capture, in vivo imaging, FRET, FRAP / FLAP, etc.

[0188] An immunoassay involves contacting a test sample with an antibody or a molecule capable of binding to an antibody, and reacting it under conditions that allow for the formation of an immune complex. After the reaction, nonspecifically bound antibodies are washed away, allowing only specific immune complexes to be detected.

[0189] Detection or quantification of immune complexes is performed by detecting labels attached to the complexes. Radioactive, fluorescent, biological, enzymatic, or other known labels may be used.

[0190] In this specification, "label" includes fluorescent dyes, biotin / streptavidin binding pairs, metals, or epitope tags. Fluorescent dyes are particularly preferred because they allow for detection in trace amounts. When detecting multiple antigens simultaneously, different fluorescent labels can be used, and the presence of antigens bound to specific antibodies can be confirmed by detecting the label signals on the array using a fluorometer.

[0191] A phosphor is a compound or molecule that emits luminescence (light). Typically, a phosphor absorbs electromagnetic energy at one wavelength and emits electromagnetic energy at a different, second wavelength. Representative phosphors include, but are not limited to, the following. 1,5 IAEDANS; 1,8-ANS; 4-methylambelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacrycidine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; acriflavin; acridine feulgen; SITSA; aequorin (photoprotein); AFP (autofluorescent protein: Quantum Biotechnologies' sgGFP, sgBFP, etc.); Alexa Fluor® series (350, 430, 488, 532, 546, 568, 594, 633, 647, 660, 680); Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aniline Blue; Anthrosil Stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrin; ATTO-TAG® CBQCA; ATTO-TAG® FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyl oxadiazole); BCECF (high or low pH); Berberine sulfate; Beta-lactamase; BFP (Blue-shifted GFP, Y66H); Blue fluorescent protein; BFP / GFP FRET; Bimain; Bisbenzemide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO(registered trademark)-1; BOBO(registered trademark)-3;Bodipy series (492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676); Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X Conjugate; Bodipy TMR-X SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO(registered trademark)-1; BO-PRO(registered trademark)-3; Brilliant Sulfoflavine FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green-1 (Ca2+ pigment); Calcium Green-2; Calcium Green-5N; Calcium Green-C18; Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue (registered trademark); Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyanide Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; CL-NERF; CMFDA; Coelenterazine (cp, f, fcp, h, hcp, ip, n, O); Coumarin Phalloidin; C-Phycocyanin; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2 (registered trademark); Cy3.1 8; Cy3.5 (registered trademark); Cy3 (registered trademark); Cy5.1 8; Cy5.5(registered trademark); Cy5(registered trademark); Cy7(registered trademark); Cyanide GFP; Cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansylamine; Dansylcadaverine; Dansylchloride; DansylDHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH (Dichlorodihydrofluorescein diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (Non-proportional); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein diacetate (DCFH); DiD (Lipophilic tracer);DiD(DilC18(5));DIDS;Dihydrorhodamine 123(DHR);Dil(DilC18(3));Dinitrophenol;DiO(DiOC18(3));DiR;DiR(DilC18(7));DM-NERF(High pH);DNP;Dopamine;DsRed;DTAF;DY-630-NHS;DY-635-NHS;EBFP;ECFP;EGFP;ELF 97;Eosin;Erythrosine;Erythrosine ITC; Ethidium bromide; Ethidium homodimer-1 (EthD-1); Euclicin; EukoLight; Europium(III) chloride; EYFP; Fastblue; FDA; Feulgen (pararosanilin); FIF (formaldehyde-induced fluorescence); FITC; Furazo orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoroemerald; Fluorogold (hydroxystilvamidine); Fluororuby; FluorX; FM 1-43 (registered trademark); FM 4-46; Fura Red (registered trademark) (high pH); Fura Red (registered trademark) / 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); Red-shifted GFP (rsGFP); Wild-type GFP (non-UV excited or UV excited); GFPuv; Glyoxylic acid; Granular blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilvamidine (Fluorogold); Hydroxytryptamine; Indo-1 (high-calcium or low-calcium); Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA or RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lisamin Rhodamine; Lisamin Rhodamine B; Calcein / Ethidium Homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow;Lyso Tracker series (Blue, Blue-White, Green, Red, Yellow); LysoSensor series (Blue, Green, Yellow / Blue); Mag Green; Magdalena Red (Phloxine B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromovimaine (containing mBBr-GSH); Monoclovimaine; MPS (Methylgreen Pyronin Stilbene); NBD; NBDamine; Nile Red; Nitrobenzoxadiazole; Norepinephrine; Nuclear Fast Red; Nuclear Yellow; Nylosain Brilliant Flavin E8G; Oregon Green (registered trademark) (488, 500, 514); Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxine B (Magdala Red); Phorwite series (AR, BKL, Rev, RPA); Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primurin; Procyon Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resolfin; RH 414; Rhod-2; Rhodamine (110, 123, 5 GLD, 6G, B, B 200, B Extra, BB, BG); Rhodamine Green; Rhodamine Faricidine; Rhodamine Phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-Phycocyanin; R-Phycoerythrin (PE); rsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sebron Brilliant Red (2B, 4G, B); Sebron Orange; Sebron Yellow L; sgBFP (registered trademark) (Superglow BFP); sgGFP (registered trademark) (Superglow GFP); SITS (Primulin; Stilbeni Isothiosulfonic Acid); SNAFL Calcein; SNAFL-1; SNAFL-2; SNARF Calcein; SNARF1; Sodium Green; Spectrum (Aqua, Green, Orange, Red); SPQ (6-Methoxy-N-(3-Sulfopropyl)Quinolinium); Stilbeni; Sulforodamine B and C; Sulforhodamine Extra; SYTO Series (11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 40, 41, 42, 43, 44, 45, 59, 60, 61, 62, 63, 64, 80, 81, 82, 83, 84, 85); SYTOX Series (Blue, Green, Orange); Tetracycline; Tetramethylrhodamine (TRITC); Texas Red®; Texas Red-X® Conjugate; Thiadiacarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin (5, S, TON); Thiolite; Thiazole Orange; Tinopol CBS (Chalcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (Tetramethylrhodamine isothiocyanate); True Blue; True Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1; YOYO-3; Sybr Green; Thiazole Orange (intercalate dye); Semiconductor nanoparticles, which may or may not contain quantum dots; Caged phosphors that can be activated by light or other electromagnetic energy sources; or combinations thereof.

[0192] Modification units, with or without radionuclides, can be directly incorporated into or bonded to any compound described herein by halogenation. Useful radionuclides in this embodiment include tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18, etc. Radionuclides can also be bonded to compounds via linkers or chelating groups, in which case examples include ⁹⁹mTc, ⁺¹⁸⁶Re, ⁶⁸⁸Ga, ⁺¹⁸⁸Re, ⁹⁰Y, ⁺¹⁵³Sm, ⁺²¹²Bi, ⁶⁷Cu, ⁶⁴Cu, ⁶⁰Cu, etc. These radiolabeling techniques are well known in the field of radiopharmaceuticals.

[0193] Radiolabeled compounds are useful as imaging agents for the diagnosis of neurological diseases (e.g., neurodegenerative diseases) or psychiatric disorders in mammals (e.g., humans), and for tracking the progression of such diseases or conditions or the effectiveness of treatment. These radiolabeled compounds can be used in combination with positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0194] Labeling can be performed by either direct or indirect labeling. In direct labeling, the detection antibody or detection molecule itself contains the label, and detection of the label indicates the presence of the target molecule or an antibody against that target molecule. In indirect labeling, an additional molecule or part is contacted or generated at the site of the immune complex. For example, a signal-generating molecule, with or without an enzyme, can be bound to the detection antibody or detection molecule to generate a detectable signal in the presence of a substrate. ELISA is an example of such indirect labeling.

[0195] Another example of indirect labeling involves using an additional molecule (conjugate) that can bind to the target molecule or primary antibody. This additional molecule may contain a label or signal-generating molecule and can function as a secondary antibody. The secondary antibody binds to the immune complex of the primary antibody and the target molecule, forming a so-called sandwich-type immune complex. After washing away nonspecific conjugates, any remaining labels can be detected. Alternatively, interbinding molecular pairs, such as biotin / avidin pairs, can also be used.

[0196] Furthermore, this method also includes indirect labeling using a two-step approach. Specifically, a secondary immune complex is formed using a first binder, and then a second binder containing the label is attached to form a tertiary immune complex. This method allows for amplification of the detection signal.

[0197] Immunoassays involving the detection of a specific protein or an antibody against that protein include label-free assays, protein separation methods (e.g., electrophoresis), solid-phase capture assays, or in vivo detection. Label-free assays are useful for determining presence or absence, and separation methods are useful for evaluating physical properties such as size or charge. Capture assays are suitable for quantitative evaluation, and in vivo detection is useful for evaluating the spatial expression distribution within a subject, tissue, or cell.

[0198] Molecular complexes ([Ab-Ag]n) formed by antibody-antigen interactions are observable to the naked eye at sufficient concentrations, but can also be detected and quantified at lower concentrations based on their ability to scatter light beams. The formation of these complexes indicates the presence of both reactants, and in immunoprecipitation assays, a specific antigen ([Ab-Ag]n) is measured using a certain concentration of reagent antibody, and the specific antibody ([Ab-Ag]n) is detected using the reagent antigen. If the reagent species is pre-coated to cells (e.g., hemagglutination test) or microparticles (e.g., latex agglutination test), the "aggregation" of the coated particles becomes visible even at much lower concentrations. Various assays based on these basic principles are commonly used, including Octarony immunodiffusion, rocket immunoelectrophoresis, immunoturbidimetry, and nephelometry. The main limitations of these assays are that their sensitivity (detection limit) is limited compared to assays using labeling, and in some cases, extremely high concentrations of the analyte can inhibit complex formation, requiring safety measures that complicate the procedure. Some of these Group 1 assays date back to the early days of antibody discovery and none of them have substantial "labels" (e.g., antigen-enzyme). Other immunoassays that do not use labeling are based on immunosensors, and various devices capable of directly detecting antibody-antigen interactions are currently commercially available. Many of these rely on generating evanescent waves on the surface of a sensor with an immobilized ligand and continuously monitoring the binding to that ligand. Immunosensors facilitate the analysis of kinetic interactions of binding, and with the emergence of low-cost dedicated devices, they may be widely applied in immunoassays in the future.

[0199] Immunoassays for detecting specific proteins include the separation of proteins by electrophoresis. Electrophoresis is the phenomenon in which charged molecules move in a solution in response to an electric field. The speed of this movement depends on the strength of the electric field, the net charge, size, and shape of the molecules, as well as the ionic strength, viscosity, and temperature of the medium through which the molecules move. As an analytical technique, electrophoresis is simple, rapid, and highly sensitive, and is used for the property analysis and separation of single charged species.

[0200] Generally, samples are electrophoresed in a support matrix, which may include paper, cellulose acetate, starch gel, agarose, or polyacrylamide gel. The matrix suppresses convective mixing due to heating and also serves to record the electrophoresis results. After electrophoresis, the matrix is ​​stained and can be used for scanning, autoradiography, or storage. Furthermore, agarose and polyacrylamide, the most commonly used support matrices, are porous gels, enabling separation based on molecular size. Porous gels act as sieves, delaying or inhibiting the movement of large macromolecules while allowing the movement of small molecules. Low-concentration agarose gels are more rigid and easier to handle than polyacrylamide gels of the same concentration, and are therefore used for separating large macromolecules such as nucleic acids, large proteins, and protein complexes. On the other hand, polyacrylamide is easy to prepare and handle at high concentrations and is used for separating most proteins and short-chain oligonucleotides that require retention by small gel pore sizes.

[0201] Proteins are amphoteric compounds, and their net charge is determined by the pH of the suspension medium. At pH levels above their isoelectric point, proteins become negatively charged and move towards the anode in an electric field. Conversely, at pH levels below their isoelectric point, they become positively charged and move towards the cathode. The net charge of a protein is independent of its size; the amount of charge per unit mass or unit length varies from protein to protein. Therefore, electrophoretic separation under non-denaturing conditions is determined by both the size and charge of the molecule.

[0202] Sodium dodecyl sulfate (SDS) is an anionic surfactant that binds to the polypeptide backbone and denatures proteins. SDS binds to proteins relatively specifically in a mass ratio of approximately 1.4:1, conferring a negative charge proportional to the polypeptide length. Furthermore, to form the random coil structure necessary for size separation, the disulfide bonds are usually reduced using 2-mercaptoethanol or dithiothreitol (DTT). Therefore, under denaturation conditions by SDS-PAGE, mobility is determined not by the polypeptide's inherent charge, but by its molecular weight.

[0203] Molecular weight is determined by simultaneously electrophoresing a protein with a known molecular weight and the test protein using SDS-PAGE. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide or native nucleic acid and its Rf value. The Rf value is calculated as the ratio of the molecular migration distance to the migration distance of the marker dye front. A simple method for calculating relative molecular weight (Mr) by electrophoresis is to create a standard curve from the relationship between migration distance and log10 molecular weight for known samples, and then read the logMr from the migration distance of samples measured on the same gel.

[0204] In two-dimensional electrophoresis, proteins are first fractionated based on one physical property, and then re-fractionated based on another physical property. For example, isoelectric focusing can be performed as the first dimension on a tube gel, and SDS electrophoresis can be performed as the second dimension on a slab gel. One example of this is the high-resolution two-dimensional electrophoresis method by O'Farrell (1975), and this document is incorporated herein by reference in its entirety as it provides instruction on two-dimensional electrophoresis methods. Other examples include the methods by Anderson et al. (1977) and Ornstein (1964), which are also incorporated herein by reference. Laemmli (1970) discloses a discontinuous system for separating SDS-denatured proteins. In this Laemmli buffer system, the leading ion is chloride, the trailing ion is glycine, the separation gel and concentration gel are prepared with Tris-HCl buffer at different concentrations and pH, and the electrophoresis tank buffer is Tris-glycine. All buffers contain 0.1% SDS.

[0205] One example of an immunoassay method using electrophoresis, as envisioned in this method, is Western blot analysis. Western blotting or immunoblotting allows for the determination of the molecular weight of a protein and the measurement of the relative amount of the protein present in different samples. Detection methods include chemiluminescence detection and colorimetric detection. Standard methods for Western blot analysis are described in Bollag et al. (1996), Harlow and Lane (1988), and U.S. Patent No. 4,452,901, which are incorporated herein by reference in their entirety for the purpose of teaching the Western blotting method. Generally, proteins are separated by SDS-PAGE and then transferred to blotting paper such as nitrocellulose or other membranes. The proteins are immobilized on the membrane while maintaining the separation pattern on the gel. The blot is incubated with a general-purpose protein (with or without milk protein) to block nonspecific binding sites on nitrocellulose, and then an antibody capable of specifically binding to the target protein is added.

[0206] The binding of specific antibodies to immobilized specific antigens can usually be easily visualized by indirect enzyme immunoassays using colorimetric substrates (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other detection methods include those using fluorescent labeling or radioisotope labeling (e.g., fluorescein, ¹²⁵I). Probes that can be used to detect antibody binding include labeled anti-immunoglobulins, labeled Staphylococcus aureus protein A (bound to IgG), or probes that bind to biotinylated primary antibodies (e.g., labeled avidin / streptavidin).

[0207] The usefulness of this technology lies in its ability to simultaneously detect specific proteins based on their antigenicity and evaluate their molecular weight. Specifically, proteins are first separated based on their molecular weight by SDS-PAGE, and then specifically detected in the immunoassay step. Therefore, protein standards (ladders) can be run simultaneously to estimate the molecular weight of the target protein present in a heterogeneous sample.

[0208] Gel shift assays or electrophoretic mobility shift assays (EMSA) can be used to qualitatively and quantitatively detect the interaction between DNA-binding proteins and their corresponding DNA recognition sequences. Representative techniques are described in Ornstein (1964) and Matsudaira et al. (1987), and these publications are incorporated herein by reference in their entirety for instructional purposes regarding gel shift assays.

[0209] In a typical gel shift assay, purified protein or crude cell extract is incubated with a labeled (e.g., ^32P radiolabeled) DNA or RNA probe, and then the complex and free probe are separated using a non-denaturing polyacrylamide gel. The complex moves more slowly through the gel than the unbound probe. Depending on the activity of the binding protein, the labeled probe may be double-stranded or single-stranded. For the detection of DNA-binding proteins with or without transcription factors, purified or partially purified protein or nuclear extract can be used. For the detection of RNA-binding proteins, purified or partially purified protein or nuclear or cytoplasmic extract can be used. The specificity of DNA or RNA-binding proteins to their putative binding sites is confirmed by competitive experiments using DNA or RNA fragments or oligonucleotides containing the binding site of the target protein, or unrelated sequences. Specific interactions can be identified by the differences in the properties and strength of the complexes formed in the presence of specific and non-specific competitors.

[0210] Gel shift methods include, for example, methods using colloidal Coomassie blue staining (Imperial Chemical Industries) to detect proteins in gels containing or not containing polyacrylamide electrophoresis gels. These methods are described by Neuhoff et al. (1985, 1988), and their entire contents are incorporated herein by reference for teaching purposes regarding gel shift methods. Furthermore, in addition to conventional protein assay methods, a composition combining washing and protein staining is described in U.S. Patent No. 5,424,000, which is also incorporated herein by reference. The solution may contain phosphoric acid, sulfuric acid, nitric acid, and acid violet dye.

[0211] Radioimmunoprecipitation assay (RIPA) is a highly sensitive assay that detects specific antibodies in serum using radiolabeled antigens. After reacting the antigen with serum, precipitation is performed using a specific reagent, such as one containing or not containing protein A Sepharose beads. The bound radiolabeled immunoprecipitation is typically analyzed by gel electrophoresis. RIPA is often used as a confirmatory test to diagnose the presence of HIV antibodies. RIPA is also known in the industry as a fur assay, precipitation assay, radioimmunoprecipitation assay, or radioimmunoprecipitation analysis.

[0212] While immunoassay methods that use electrophoresis to separate and detect specific proteins, as described above, are useful for evaluating protein size, they are not highly sensitive for evaluating protein concentration. On the other hand, this specification also envisions immunoassay methods in which a target protein or an antibody specific to that protein is bound to a solid support (e.g., a tube, well, beads, or cells), the antibody or protein is captured from the sample, and detected on the support. Examples of these immunoassay methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture methods.

[0213] Radioimmunoassay (RIA) is a classic quantitative assay that detects antigen-antibody reactions using radiolabeled substances (radioligands) directly or indirectly, and measures the binding of unlabeled substances to specific antibodies or receptor systems. RIA is used for applications such as measuring blood hormone concentrations without using biological assays. Substances that do not have immunogenicity (e.g., haptens) can also be measured by binding them to large carrier proteins that can induce antibody production (e.g., bovine gamma globulin or human serum albumin). In RIA, a radioactive antigen (¹²⁵I or ¹³¹I are commonly used because iodine atoms can be easily introduced into tyrosine residues in proteins) is mixed with an antibody. The antibody is often bound to a solid support such as a tube or beads. Next, a known amount of unlabeled (cold) antigen is added, and the amount of labeled antigen that is substituted is measured. Initially, the radioactive antigen is bound to the antibody, but upon addition of the cold antigen, the two compete for the binding site, and the higher the cold antigen concentration, the more the radioactive antigen is substituted from the antibody. The binding antigen and free antigen are separated, and their respective radioactivity is measured to create a binding curve. This method is extremely sensitive and specific.

[0214] Enzyme-linked immunosorbent assay (ELISA), or more generally enzyme-mediated immunoassay (EIA), is an immunoassay capable of detecting antibodies against specific proteins. In this assay, an enzyme is used as a detectable label bound to an antibody-binding or antigen-binding reagent. The enzyme reacts with a substrate to produce a chemical species that can be detected by spectrophotometric analysis, fluorescence assay, or visual observation. Enzymes that can be used as detection labels 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, glucoamylase, and acetylcholinesterase.

[0215] Modifications of ELISA techniques are well known to those skilled in the art. In one embodiment, an antibody capable of binding to a protein is immobilized on a well of a selected surface having protein affinity, such as a polystyrene microtiter plate. A test composition presumed to contain a marker antigen is then added to the well. After washing away bound and nonspecifically bound immunocomplexes, the bound antigen is detected. Detection can be performed by adding a second antibody specific to the target protein and attaching a detectable label to the second antibody. This form of ELISA is known as "sandwich ELISA". Another detection method may be employed in which, after adding the second antibody, a third antibody having binding affinity to the second antibody and attached a detectable label may be added.

[0216] Another variation is competitive ELISA. In competitive ELISA, reactive substances in the test sample compete for binding to a known amount of labeled antigen or antibody. The amount of reactive substances in the sample can be measured by mixing the sample with the known labeled substance before or during incubation with the immobilized wells. The presence of reactive substances in the sample reduces the amount of labeled substance that can bind to the wells, resulting in a decrease in the final signal.

[0217] Regardless of the specific ELISA format used, ELISAs generally share common characteristics. These characteristics may include immobilization, incubation or binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. Antigens or antibodies can be bound to solid supports in the form of plates, beads, dipsticks, membranes, or column matrices, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating plates with antigens or antibodies, the wells are typically incubated with the antigen or antibody solution, for example, overnight or for a predetermined time. The wells can then be washed to remove any incompletely adsorbed material. The remaining empty surfaces of the wells can be "blocked" with nonspecific proteins that are antigenically neutral to the sample antiserum. These include bovine serum albumin (BSA), casein, milk powder solution, etc. Coating can block nonspecific adsorption sites on the immobilized surface and reduce background noise caused by nonspecific binding of the antiserum.

[0218] In ELISA, secondary or tertiary detection methods can be used instead of the direct method. Specifically, after the protein or antibody binds to the well, the sample is coated with a non-reactive substance to reduce background noise and washed to remove unbound material. Then, the sample is brought into contact with an immobilized surface and reacted under conditions that promote immune complex (antigen / antibody) formation. For detection of immune complexes, a labeled secondary binder or a combination of a secondary binder and a labeled tertiary binder is used.

[0219] Enzyme-conjugated immunosorbent spot (ELISpot) is an immunoassay capable of detecting antibodies against specific proteins or antigens. This method uses an enzyme as a detectable label bound to an antibody-binding or antigen-binding reagent. The enzyme reacts with a substrate to produce a chemical species detectable by spectrophotometric analysis, fluorescence imaging, or visual observation. Enzymes that can be used for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In this measurement method, a nitrocellulose microtiter plate is coated with the antigen. The sample is exposed to the antigen and then reacted in the same manner as in ELISA. The difference from conventional ELISA is that detection is performed by counting the number of spots on the nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. By counting the spots, the number of cells in the sample that are specific to the antigen can be determined.

[0220] "Effective conditions that allow for immune complex (antigen / antibody) formation" means conditions that reduce nonspecific binding and ensure an appropriate signal-to-noise ratio by diluting the antigen and antibody with or without BSA, bovine gamma globulin (BGG), and phosphate-buffered saline (PBS) / Tween.

[0221] Appropriate conditions also mean that the incubation temperature and time are sufficient to allow for effective bonding. The incubation process typically ranges from about 1 minute to 12 hours, at a temperature of about 20°C to 30°C, or overnight at about 0°C to 10°C.

[0222] After the entire incubation process in ELISA is complete, the contact surface can be washed to remove non-complexed material. The washing process can be carried out with or without a PBS / Tween or borate buffer solution. Specific immunocomplexes are formed between the sample and the immobilized material, and even trace amounts of these immunocomplexes can be detected after washing.

[0223] To provide a detection method, the second or third antibody may have a detectable label as described above. The label may be an enzyme that produces color upon reaction with a suitable color-developing substrate. That is, the first or second immunocomplex can be brought into contact with the labeled antibody and incubated under conditions that promote immunocomplex formation (e.g., in a PBS-containing solution at room temperature for 2 hours).

[0224] After incubation with the labeled antibody, washing is performed to remove unbound substances, and the amount of labeling can be quantified. For example, in the case of peroxidase labeling, quantification is possible by incubation with urea and bromocresol purple, or with a color-developing substrate containing 2,2'-azidodi-(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] and H₂O₂. By measuring the degree of color development, quantification can be performed using a visible light spectrophotometer or the like.

[0225] Protein arrays are solid-phase ligand-binding assay systems that use proteins immobilized on surfaces such as glass, membranes, microtiter plate wells, mass spectrometry plates, beads, or other particles. These assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Advantages include high speed, automation capabilities, high sensitivity, excellent reagent cost-effectiveness, and the ability to acquire abundant data in a single experiment. Bioinformatics support for data analysis is also crucial, requiring advanced software and data comparative analysis. However, software developed for DNA arrays can be applied, and many hardware and detection systems are similarly applicable.

[0226] One of the representative formats is the capture array. In capture arrays, ligand-binding reagents are used. Ligand-binding reagents are usually antibodies, but alternative protein scaffolds, peptides, and nucleic acid aptamers can also be used. Capture arrays are used to detect target molecules in mixtures that contain or do not contain plasma or tissue extracts. In diagnostic applications, capture arrays allow for the parallel execution of multiple immunoassays. For example, it is possible to measure multiple analytes in individual serum samples or to simultaneously measure numerous serum samples. In proteomics, capture arrays are used for the quantification and comparison of protein levels in different samples in health and disease, i.e., protein expression profiling. In addition to specific ligand-binding proteins, proteins can also be used for in vitro functional interaction screening of proteins, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. Capture reagents themselves can be selected and screened for multiple proteins, and it is also possible to perform tests on multiple protein targets in a multiplex array format.

[0227] Protein sources used in array construction may or may not include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production using cell-free translation systems, and peptide synthesis. Many of these methods are automatable for high-throughput production. In capture arrays and protein function analysis, it is crucial that proteins are correctly folded and functional. For example, recombinant proteins extracted from bacteria under denaturation conditions are not always correctly folded and functional. Nevertheless, arrays of denatured proteins are useful for screening cross-reactive antibodies, identifying autoantibodies, and selecting ligand-binding proteins.

[0228] Protein arrays are designed as miniaturizations of known immunoassay methods such as ELISA and dot blotting, and often utilize fluorescent readouts, enabling parallel execution of multiple assays using robotics and high-throughput detection systems. Commonly used physical supports include, or do not include, glass slides, silicon, microwells, nitrocellulose or PVDF membranes, magnetic beads, and other microparticles. While the delivery of minute protein droplets onto a planar surface is the most common, alternative architectures include CD centrifuge devices based on microfluidic technology (Gyros, Monmouth Junction, NJ) and specialized chip designs with microchannels within a plate (The Living Chip). TM Examples include Biotrove (Woburn, MA) and microscopic 3D posts on silicon surfaces (Zyomyx, Hayward CA). Particles in suspension can also be used as the basis for arrays if they are coded for identification. Systems include color-coded microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (QDots). TM Quantum Dot, Hayward, CA), barcoding of beads (UltraPlex TM SmartBead Technologies Ltd, Babraham, Cambridge, UK, polymetallic microrods (Nanobarcodes) TM One example is particles (Nanoplex Technologies, Mountain View, CA). The beads can also be arranged as a planar array on a semiconductor chip (LEAPS technology, BioArray Solutions, Warren, NJ).

[0229] Protein immobilization depends on both the properties of the binding reagent and the target surface. A good protein array support surface is chemically stable before and after the binding procedure, exhibits good spot morphology, minimizes nonspecific binding, does not increase background noise in the detection system, and is compatible with different detection systems. The immobilization method used should be reproducible, applicable to proteins with different properties (size, hydrophilicity, hydrophobicity), suitable for high throughput and automation, and capable of preserving full functional activity. The orientation of surface-bound proteins is recognized as a crucial factor in presenting them in an active state to the ligand or substrate. In capture arrays, the most efficient binding results are generally obtained with oriented capture reagents that require site-specific labeling of the protein.

[0230] Covalent and non-covalent methods are used for protein immobilization, each with its own advantages and disadvantages. Passive adsorption to a surface is technically simple, but quantitative or orientation control is almost impossible, and it may alter the functional properties of the protein, with potential for variability in reproducibility and efficiency. Covalent methods provide stable binding, are applicable to a variety of proteins, and offer good reproducibility, but orientation can vary. Chemical induction may alter protein function and requires a stable interaction surface. Biological capture methods using tags attached to proteins provide stable binding and bind proteins in a specific and reproducible orientation, but require sufficient immobilization of biological reagents, careful handling of the array is necessary, and stability may vary.

[0231] Several immobilization chemistry methods and tags for protein array fabrication have been reported. Covalent bonding substrates may or may not include silane-treated glass slides containing amino or aldehyde groups. (Versalinx) TMIn the system (Prolinx, Bothell, WA), reversible covalent bonding is achieved through the interaction between phenyl diboronic acid-modified proteins and salicylic acid immobilized on the support surface. This method exhibits low background binding, low intrinsic fluorescence, and enables the preservation of the immobilized protein's function. Non-covalent bonding of unmodified proteins is achieved through a porous structure based on a three-dimensional polyacrylamide gel (HydroGel). TM This has been performed in PerkinElmer (Wellesley, MA), and particularly low background, high volume, and protein function preservation have been reported on glass microarrays. Widely used biological binding methods include biotin / streptavidin or hexahistidine / Ni interactions (SEQ ID NO: 88), which require appropriate protein modification. Biotin can or cannot be conjugated to a polylysine backbone immobilized on titanium oxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0232] Array fabrication methods include robotic contact printing, inkjet printing, piezoelectric spotting, and photolithography. Commercial arrayers (e.g., Packard Biosciences) and manual equipment (V&P Scientific) are available. Bacterial colonies are gridded onto a PVDF membrane by a robot and can be used for in-situ protein expression induction.

[0233] At the limits of spot size and density, nanoarrays exist, enabling thousands of reactions on a single chip of less than 1 square millimeter using nanometer-scale spots. BioForce Laboratories has developed a nanoarray with 1521 spots within 85 square micrometers, which corresponds to 25 million spots per square centimeter, reaching the limits of optical detection. Readout methods include fluorescence and atomic force microscopy (AFM).

[0234] Fluorescent labeling and detection methods are widely used. In this method, the equipment used for reading DNA microarrays can also be applied to protein arrays. In differential display, a capture array (e.g., an antibody array) can be probed with fluorescently labeled proteins derived from two different cellular states. In this case, cell lysates are directly bound to and mixed with different fluorophores (e.g., Cy-3, Cy-5), and the resulting fluorescence color serves as an indicator of changes in the abundance of the target molecule. The sensitivity of fluorescent readout can be amplified 10 to 100 times by tyramide signal amplification (TSA, PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultra-high-sensitivity fluorescence detection and offers the advantage of detection without a washing step. High sensitivity can also be achieved by using suspension beads or microparticles and utilizing the properties of phycoerythrin (Luminex) or semiconductor nanocrystals (Quantum Dot). Numerous other novel alternative readout methods have also been developed and are particularly used in commercial biotechnology. These may or may not include applications of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonant light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy (BioForce Laboratories).

[0235] Capture arrays form the basis for diagnostic chips and expression profiling arrays. These can use high-affinity capture reagents to bind and detect target ligands, including or excluding conventional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers, at high throughput.

[0236] Antibody arrays possess specificity and acceptable background, and some are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are produced by conventional immunization methods (polyvalent serum and hybridoma) or as recombinant fragments, usually expressed in E. coli, and selected from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, single V domains from camelids, or engineered human homologs (Domantis, Waltham, MA) are also useful in arrays.

[0237] A "scaffold" refers to the ligand-binding domain of a protein, and is designed to have antibody-like specificity and affinity, and to bind to a variety of target molecules, with multiple variants being constructed. These variants are produced in the form of a gene library and selected for individual targets by phage, bacterial, or ribosome display. Relevant ligand-binding scaffolds or frameworks may or may not include 'Affibodies' (Affibody, Bromma, Sweden) based on Staph. aureus protein A, 'Trinectins' (Phylos, Lexington, MA) based on fibronectin, and 'Anticalins' (Pieris Proteolab, Freising-Weihenstephan, Germany) based on lipocalin structures. These can be used in capture arrays like antibodies and may offer advantages in robustness and ease of production.

[0238] Non-protein capture molecules include single-chain nucleic acid aptamers, which bind to protein ligands with high specificity and affinity (SomaLogic, Boulder, CO). Aptamers include Selex. TM The oligonucleotides are selected from an oligonucleotide library by method, and their interaction with proteins can be enhanced by incorporating brominated deoxyuridine and UV-activated crosslinking (photoaptamer). Photocrosslinking to the ligand reduces aptamer cross-reactivity under specific stereochemical conditions. The aptamers can be easily produced by automated oligonucleotide synthesis and possess DNA stability and robustness. Binding detection is possible using universal fluorescent protein staining in photoaptamer arrays.

[0239] Proteins analytes bound to antibody arrays can be detected directly or via secondary antibodies in sandwich assays. Direct labeling is used when different samples need to be compared using different colors. When antibody pairs against the target protein are available, sandwich immunoassays offer high specificity and sensitivity and are preferred as a standard method for low-concentration proteins (with or without cytokines). They can also detect protein modifications. Label-free detection methods (with or without mass spectrometry, surface plasmon resonance, and atomic force microscopy) avoid ligand modification. In any method, optimal sensitivity and specificity, along with a high signal-to-noise ratio due to low background, are required. Since analyte concentrations vary widely, sensitivity needs to be appropriately adjusted; sequential dilution of samples or the use of antibodies with different affinities are solutions to this problem. Target proteins are often present at low concentrations in body fluids and extracts, and detection at subpg levels may be required (with or without cytokines or low-expression products in cells).

[0240] As an alternative to arrays of capture molecules, there is molecular imprinting technology. In this method, peptides (e.g., derived from the C-terminal region of a protein) are used as templates to form structurally complementary and sequence-specific vacancies in a polymerizable matrix. These vacancies can specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint). TM , Aspira Biosystems, Burlingame, CA).

[0241] Another method that can be used for diagnosis and expression profiling is the ProteinChip array. R (Ciphergen, Fremont, CA) is one example. In this method, a solid-phase chromatography surface binds proteins with similar charge or hydrophobicity from a mixture containing or not containing plasma or tumor extract, and the retained proteins are detected by SELDI-TOF mass spectrometry.

[0242] Large-scale functional chips are constructed by immobilizing numerous purified proteins and can assay a wide range of biochemical functions. These may or may not include protein-protein interactions, drug target interactions, enzyme-substrate interactions, etc. Generally, expression libraries are required, which are cloned into E. coli, yeast, etc., and the expressed proteins are purified and immobilized using methods such as His tagging. Cell-free protein transcription / translation systems are effective for synthesizing proteins that are not adequately expressed in bacteria or other in vivo systems.

[0243] To detect protein-protein interactions, protein arrays can be used as an in vitro alternative to cell-based yeast dimerization methods, and are useful when the latter is deficient (including or excluding interactions between secreted proteins or proteins with disulfide bonds). High-throughput analysis of biochemical activity on arrays has been described for various functions of yeast protein kinases and the yeast proteome (protein-protein and protein-lipid interactions), and the majority of all yeast open reading frames are expressed and immobilized on microarrays. Large-scale proteome chips are expected to be extremely useful for identifying functional interactions, drug screening, etc. (Proteometrix, Branford, CT).

[0244] Protein arrays, which display individual elements in two dimensions, can be used to screen phage or ribosome display libraries and select specific binding partners such as antibodies, synthetic scaffolds, peptides, and aptamers. This method enables "library-versus-library" screening. Another application of this approach is screening drug candidates in chemically synthesized libraries against arrays of protein targets identified from genome projects.

[0245] Multiplex bead assay (e.g., BD) TMCytometric bead arrays (with or without) are spectrally distinct particle sequences that can be used for the capture and quantification of soluble analytes. Analytes are detected by fluorescence-based emission and measured by flow cytometry. Multiplex bead assays produce data equivalent to ELISA-based assays, but are performed in a "multiplex" or simultaneous manner. The concentration of unknown samples is calculated based on a standard curve, similar to sandwich assays using known standards. Furthermore, multiplex bead assays enable the quantification of soluble analytes that were previously difficult to measure due to sample volume limitations. In addition to quantitative data, it is also possible to generate visual images showing unique profiles and signatures, providing users with additional information at a glance.

[0246] Accordingly, in one embodiment disclosed herein, a method for identifying disclosed T cell receptors is provided. In this method, T cell activity (e.g., including or excluding cytokine release; cytokines including or excluding IFN-gamma, TGF-β, limbophotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25, etc.) is measured by any immunodetection method disclosed herein. The measurement method is exemplified but not limited to ELISA, ELISpot, intracellular cytokine staining, or chromium release.

[0247] The T cell receptors disclosed in this specification (exemplary but not limited to T cell receptors that bind to cancer antigens, including or not including DP4-ESO-1 TCR, A2-CT83 TCR, DR13-CT83 TCR, A2-pp65-TCR and / or A2-IE-1-TCR) can be used for the treatment of cancers that express specific MHC molecules and antigens. The cancers targeted include, but are not limited to, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain tumor, 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, urogenital cancer, lung cancer, esophageal cancer, colorectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma. Therefore, a method for identifying a T cell receptor for a cancer selected from the above group is also disclosed in this specification.

[0248] Animals: Female NSG mice at 6 - 8 weeks of age were purchased from Jackson Laboratory or bred in the animal facility of Houston Methodist Research Institute. All procedures were approved by the Houston Methodist Research Institute Institutional Animal Care and Use Committee (IACUC). In the CD19 - positive B - cell lymphoma mouse model, 5×10^6 Raji - ffluc cells were administered to the mice by intravenous injection. Tumor burden was confirmed by bioluminescence imaging using Xenogen IVIS Spectrum and analyzed with Living Image software (Perkin Elmer). On the 5th day after tumor cell transplantation, CAR + T cells were administered once. The mice were observed at least once a day and were euthanized by CO2 inhalation when weakness due to leukemia progression and hind - limb paralysis occurred, or when the body weight decreased by more than 20%. For CAR - T function evaluation, 2 - 5×10^6 Raji cells were administered intravenously on day 0, and 0.5 - 2×10^6 CAR - T cells were administered intravenously on day 5. Blood was collected to detect CD3 + CAR - T cells and CD19 + tumor cells. The mice were sacrificed on day 40, and the spleens were harvested for flow - cytometry analysis of memory T cells and exhaustion markers.

[0249] CAR construct: The 19BBz CAR gene was generated by linking the scFv sequence derived from FMC63 to the extracellular, transmembrane, and intracellular regions of CD8α and the sequences of 4 - 1BB and CD3 zeta signaling domains. 1928z used the CD28 hinge, transmembrane region, and intracellular region. The ZAP70 kinase - domain - containing fragment was generated by overlap PCR and replaced the CD3 zeta signaling domain of the CAR construct. All constructs were cloned into the pMSGV1 vector and sequenced.

[0250] Human PBMCs and Transduction: Healthy donor blood was obtained from the Gulf Coast Regional Blood Center. Fresh PBMCs were isolated with Ficoll reagent according to the manufacturer's instructions. The buffy layer was collected and washed twice with PBS. PBMCs suspended in T cell medium were seeded onto anti-human CD3 antibody (OKT3) coated plates and activated. Retroviruses were packaged in HEK293 T cells using envelope plasmid RD114 and packaging plasmid Gag-pol, or a stably transfected PG13 packaging cell line. Virus particles were harvested 48 hours after transfection and filtered through a 0.45 μm filter. Activated PBMCs were transfected twice with retroviruses in the presence of RetroNectin according to the manufacturer's instructions. T cells were cultured for at least 3 days before use.

[0251] Flow cytometry: The antibody used for analysis is as follows: Pierce TM Recombinant Protein L, Biotinylated (29997, ThermoFisher), Streptavidin PE / APC (12-4317-87 / 17-4317-82, eBioscience), CD3-APC-eFluor TM 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 TM 710 (46-1271-82, eBioscience), CD45RO-SB600 (63-0457-42, eBioscience), CD62L-eFluor450 (48-0621-82, eBioscience). Labeled cells were analyzed using BD LSR II (BD Biosciences) or Attune NxT (ThermoFisher). Data analysis was performed using FlowJo software (Tree Star).

[0252] Cell lines HEK293T and MDA-231-CD19 cells were cultured in DMEM supplemented with 10% inactivated FBS, 100 units / ml penicillin, and 100 ug / ml streptomycin. THP-1, Raji, Raji-GFP, Raji-ffluc, and NALM-6 were cultured in RPMI-1640 supplemented with 10% inactivated FBS, 100 units / ml penicillin, and 100 ug / ml streptomycin. All cells were regularly confirmed to be mycoplasma-negative.

[0253] ELISA: Virus-transmitted T cells were cooled in culture medium for 1-2 days under IL-2 depletion. The cells were co-cultured with tumor cells at a different E:T ratio. The following day, the diluted supernatant was added to plates pre-coated with human IFN-gamma (1:1000, Thermo, M700A) and 1% BSA blocked, and incubated at room temperature for 1 hour with gentle shaking. After washing the plates twice, they were incubated with biotin-conjugated IFN-gamma antibody (1:1000, Thermo M700B) for 1 hour, washed twice more, and incubated with avidin-HRP (1:5000) in the dark for 30 minutes. After washing, 100 μl of TMB was added to the reaction, and the reaction was stopped with 50 μl of 2.5 N sulfuric acid.

[0254] LDH cytotoxicity release assay: Transduced T cells were co-cultured with tumor cells in 96-well plates for 6 or 24 hours at a series of E:T ratios. The supernatant containing LDH was transferred to a new enzyme reaction plate, and the cytotoxicity assay was performed according to the manufacturer's instructions. Absorbance was measured at 490 nm using a spectrophotometer (Bio-Tek). Specific lysis was calculated using the following formula: %Cytotoxicity = (Experimental value - Effector - Spontaneous value - Target spontaneous value) / (Target maximum value - Target spontaneous value) × 100

[0255] Serum cytokines: Peripheral blood samples were collected from the tail vein of mice. The collected blood was kept at room temperature for 30 minutes, then centrifuged at 4°C at 8000 rpm for 15 minutes. Serum concentrations of human IL-2, IFN-gamma, and TNF-α in Raji-transplanted mice were measured using enzyme-linked immunosorbent assay (ELISA). Concentrations were calculated using a four-parameter logistic regression (4-PL) model.

[0256] Statistical analysis: Statistical significance between two groups was evaluated using a paired / unpaired t-test. Comparisons of three or more groups were performed using ANOVA and Tukey's multiple comparison test. Differences were considered statistically significant if the p-value < 0.05. In mouse experiments, overall survival in the T-cell treatment group was represented by a Kaplan-Meier curve, and survival differences between groups were compared using the log-rank test. All statistical analyses were performed using GraphPad Prism 8 software.

[0257] This specification discloses the components used to prepare the disclosed compositions and the compositions themselves. These and other materials are also disclosed herein, and where combinations, subsets, interactions, groups, etc., of these materials are disclosed, each individual combination or recombination is considered and described specifically, even if not explicitly enumerated. For example, if a particular TCR is disclosed and further modifications of molecules containing the TCR are discussed, all combinations and substitutions of the TCR and modifications are considered unless otherwise indicated. Therefore, if molecular classes A, B, C and D, E, F are disclosed and the combination A and D is exemplified, then AE, AF, BD, BE, BF, CD, CE, and CF are also considered disclosed. Similarly, any subsets or combinations of these are also disclosed. This concept applies to all aspects of this specification, including steps for the preparation and use of the disclosed compositions.

[0258] In one embodiment of this specification, the methods disclosed include methods for detecting or identifying TCRs that can be used for cancer treatment (as therapeutic or prophylactic treatment), and methods for preparing TCR T cells that can be used for cancer treatment. Accordingly, in one embodiment, this specification also discloses TCR T cells that have been engineered to express receptors (including or not including T cell receptors) that can recognize the disclosed antigens.

[0259] In one embodiment, the present invention also features a method for enhancing the persistence of CAR-T and TCR cells by chemokine receptor expression and shRNA knockout in TCR or CAR constructs. In one embodiment, specific antigen (e.g., NY-ESO-1, CT83, pp65, and / or IE-1) specific TCR T cells disclosed herein can be further engineered to enhance their function (including or not including cytotoxic activity and in vivo persistence or survival) by knocking out or knocking down negative signaling molecules (e.g., PD1, VHL, and / or PPP2R2D).

[0260] In one embodiment, the negative signaling molecule may be, for example, indoleamine (2,3) dioxygenase (IDO) (including IDO1 and IDO2 isoforms), OX40, CTLA-4, PD-1, PD-L1, PD-L2, LAG3, and B7-H3. In certain embodiments, the negative signaling molecule may be PD-1, VHL, PPP2R2D, and epigenetic factors (including or not including JMJD3 and LSD1).

[0261] In one embodiment, the present invention also features a method for enhancing T cell migration within tumors and / or the in vivo localization of tumor cells by forced expression of a chemokine receptor. In some embodiments, the expression of the chemokine receptor is forced by fusing a CAR or TCR construct with a chemokine. The chemokine receptor may be CCR5, CCR2, or CXCR3. In some embodiments, the chemokine receptor is CCR5.

[0262] In any of the disclosed TCRs, if the TCR is NY-ESO-1 specific, the α variable region may consist of a DP4-ESO-1 TCRα variable region containing the amino acid sequence of SEQ ID NO: 3, a variant that binds to the antigen with the same specificity as the reference receptor (full length and unmodified), a polypeptide having at least 85% identity to SEQ ID NO: 3, and a polypeptide containing one 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)), or a polypeptide having a substitution in the CDR region of a sequence having at least 85% identity to SEQ ID NO: 3. The β variable region may consist of the HLA-DP4 NY-ESO-1 TCRβ variable region containing the amino acid sequence of SEQ ID NO: 4, a variant that binds to the antigen with specificity equivalent to that of the reference receptor, a polypeptide having at least 85% identity with SEQ ID NO: 4, a polypeptide having one 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)), or a polypeptide having substitutions in the CDR region of a sequence having at least 85% identity with SEQ ID NO: 4.

[0263] In any TCR embodiment, the α or β chain C-terminus of the TCR / chimeric TCR may be fused with a signaling component including a ZAP70 kinase domain or a variant / derivative that is a functional ZAP70 kinase. Furthermore, in any CAR embodiment, the CAR / chimeric CAR may include an intracellular T cell activation motif, which includes a signaling domain, and may replace the CD3 zeta with a ZAP70 kinase domain or a functional variant / derivative thereof. The ZAP70 kinase domain may be derived from functional wild-type ZAP70 or a variant / derivative thereof, and may have a functional ZAP70 kinase domain. The variant / derivative may be a fragment with the biological activity of ZAP70 kinase. The functional ZAP70 kinase domain may consist of an amino acid sequence having at least 55% identity to the amino acid sequence of SEQ ID NOs: 16, 17, 52, 64, or 65. The ZAP70 portion may include ZAP300 (SEQ ID NO: 16, 300-619 aa), ZAP327 (SEQ ID NO: 17, 327-619 aa), ZAP338 (SEQ ID NO: 52, 338-619 aa), ZAP255 (SEQ ID NO: 64), ZAP280 (SEQ ID NO: 65), or their variants / derivatives. The ZAP70 kinase domain may have an N-terminal amino acid in the range of 250-338, 256-338, 281-338, 309-338, 300-338, 250-327, 281-327, 309-327, or 300-327, and a C-terminal amino acid in the range of 610-619. In some embodiments, the C-terminus is 619. The ZAP70 kinase domain may or may not contain ZAP255 (255-619 aa), ZAP280 (280-619 aa), or ZAP308 (308-619 aa).

[0264] In any of the TCRs and CARs according to the present invention (for example, in T cells expressing a chimeric antigen receptor, a chimeric TCR receptor, or a CAR / chimeric CAR or TCR / chimeric TCR), in some embodiments, the CAR or TCR comprises an antigen recognition site, a transmembrane domain, and an intracellular T cell activation module, wherein the intracellular T cell activation module further comprises a signaling domain including a ZAP70 kinase domain or a variant thereof.

[0265] In some embodiments, the TCR or CAR is specific to cancer antigens or antigens pathogenically associated with inflammatory diseases, autoimmune diseases, allergic diseases, organ transplant status, infectious diseases, or aging. In some embodiments, the TCR or CAR is specific to cancer antigens that include, exclude, or are selected from the following group: Alpha (α)-fetoprotein (AFP), melanoma-deficient protein 2 (AIM2), T-cell-recognizing adenocarcinoma antigen 4 (ART-4), BCMA, B antigen (BAGE), CTL-recognizing antigen on melanoma (CAMEL), oncoemulsifying antigen peptide-1 (CAP-1), caspase 8 (CASP8), cell division cycle protein 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDK12, oncoemulsifying antigen (CEA), calcium-activated chloride channel 2 (CLCA2), CFTR, CMV, carcinometris antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor receptor variant III), EGFR and its isovariants, EGFR E746-A750del, EGFRVIII, Epithelial-specific antigen (ESA), Epithelial cell adhesion molecule (EpCAM), Ephrin type A receptor 2,3 (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-acetylglucosaminyltransferase V (GnT-V), Glycoprotein 100 (GP100), HCMV pp65, HCMV IE-1, helicase antigen (HAGE), H3.3K27M, carcinoembryonic antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, human epithelial receptor 2 / neuron (HER2 / neu), HBV, HERV-E, HIV-1 gag, HMI.24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, human telomerase reverse transcriptase (hTERT), V-Ki-ras2 Carsten rat sarcoma virus oncogene (KRAS),KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, L1 cell adhesion molecule (L1-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), protein melan-A (T lymphocyte-recognized melanoma antigen) MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC2, Mucin 16 (Muc-16), Myo-D1, Dimeric form of pyruvate kinase isoenzyme M2 (tumor M2-PK), Necl-2, Neurofilament, NKCSI, NKG2D, Neuron-specific enolase (NSE), NY-ESO, New York esophagus 1 (NY-ESO-1), Preferentially expressed melanoma antigen (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 cell carcinoma rejection antigens 1, 2, 3 (SART-1, -2, -3), SOX10, synovial sarcoma X rupture point 2 (SSX-2), Survivin, OVA1, HE4, DR-70, total PSA, alpha-methylacyl-CoA racemase / AMACR, CA125 / MUC16, ER-alpha / NR3A1, ER-beta / NR3A2, thymidine kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, cytokeratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF R / TNFRSF16, NM23-H1, PARP, PP4, Serpine 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, PD1, PD-L1, CTLA-4, PDGFR Alpha, VEGF, TRAG-3, T cell receptor gamma alternative leading frame protein (TARP), TGFbII, 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 (Fc gamma RIII), CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 (Fc gamma RII), 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 (integrin β) 3) CD62L, CD63, CD64 (Fc gamma RI), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (endogrin), 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 (PD1), 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, cytokeratin 8, 18+, 19+), prostate stem cell antigen (PSCA), α2β1, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAME, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), CS-I, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), estrogen receptor (ER), progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, free PSA, thyroglobulin, nuclear fission machinery protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, aldo-keto reductase 1C3 / AKR1C3, alpha-1B glycoprotein, alpha-1 microglobulin, alpha-B cristo Tallinn / CRYAB, alpha-methylacyl-CoA racemase / AMACR, AMFR / gp78, annexin A3, annexin A8 / ANXA8, APC, apolipoprotein AI / ApoA1, apolipoprotein A-II / ApoA2, apolipoprotein E / ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, attractin, aurora A, BAP1, Bcl-2, Bcl-6, beta-2-microglobulin, beta-1,3-glucuronyltransferase 1 / B3GAT1, beta-catenin,Beta-III tubulin, bicin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, cadherin-13, cardesmon / CALD1, carponin 1, calretinin, carbonic anhydrase IX / CA9, catalase, cathepsin D, caveolin-1, caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, chitinase-like 1, coresi Stokinin-BR / CCKBR, chorionic gonadotropin alpha chain (alpha-HCG), chorionic gonadotropin alpha / beta (HCG), CKAP4 / p63, claudin-18, clathrin, c-Maf, c-Myc, coactosin-like protein 1 / CotL1, COMMD1, cornulin, cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, cyclin A1, cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, Fetal Acetylcholine Receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD10 5, Endosialin / CD248, Enolase 2 / Neuron-specific enolase, EpCAM / TROP1, Eps15, ER Alpha / NR3A1, ER Beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fasin, FATP3, FCRLA, Fetuin A / AHSG, FGF Acid, FGF Basic, FGF R3, FGF R4, Fibrinogen, Folate-binding protein (FBP), Fibroblast-activating protein alpha / FAP, Follistatin-like 1 / FSTL1, FOLR1, FOLR2,FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / disadherin, FcεRIα, FITC, FLT3, GABA-A R alpha 1, GADD153, GADD45 alpha, galectin-3, galectin-3BP / MAC-2BP, galactin, ganglioside, Crude cystic fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma-glutamylcyclotransferase / CRF21, Gas1, gastrin-releasing peptide R / GRPR, gastrokine 1, gelzolin / GSN, glial fibrillary acidic protein (GFAP), GLI-2, glutathione peroxidase 3 / GPX3, gpA33, glycopeptide, glypican 2 (GPC2), glypican 3, Golgi glycoprotein 1 / GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, heparanase / HPSE, hepsin, HGF R / c-MET, HIF-2 Alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, Hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, Interleukin-13 receptor α2 chain (IL-13Rα), Interleukin-13 receptor subunit alpha 2 (IL-13Rα2), Integrin, Integrin B7, IMP dehydrogenase 1 / IMPDH1, Importin alpha 2 / KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQG AP1, Isocitrate dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, OGR1, Olig2, Osteopontin / OPN, Ovastacin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Bisfatin, PDCD4, PDCD5, PDGF R-alpha, PDGFR-beta, PDZD2, PEA-15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2, PGCP, PI 3-kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone RB / NR3C3, Progesterone R / NR3C3, Progranulin / PGRN, Prolactin, Prostaglandin E Synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADaseI, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP Beta / Zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, Secret Serpine R, Serpine A9 / Centerin, Serpine E1 / PAI-1, Serum Amyloid A1, Serum Amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostain, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCL1, SPINK1, Src, Six-pass transmembrane epithelial antigen of the prostate (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin-1, Syndecan-1 / CD138, Syntaxin 4, Synuclein-gamma, Synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CA M, Lactate dehydrogenase A / LDHA, Lamin B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matryptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin-1R / MC1R, Melanotransferrin / CD228, Melatonin, Mer, Mesothelin, Metadoherin, Metastine / KiSS1, Methionine Aminopeptidase, Methionine Aminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mindbomb 2 / MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesphatin-1 / Nucleobindin-2, Nestin, NFkB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankylase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-Beta 1, TGF-Beta 1, 2, 3, TGF-beta 1 / 1.2, TGF-beta 2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin Beta 10, Thymosin Beta 4, Thyroglobulin, TIMP Assay Kit, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoreglin-2, TMEM219, TMEM87A, TNF-Alpha, TOP2A, TopBP1, t-plasminogen activator / tPA, TRA-1-60(R), TRA-1-85 / CD147, TRAF-4, Transgerin / TAGLN, Trypsin 2 / PRSS2, Tryptase Alpha / TPS1, TSPAN1, UBE2S, uPAR, u-plasminogen activator / urokinase, Urotensin-IIR, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, CNKSR1 / CNK / KSR, or any combination thereof.

[0266] In one embodiment, the present invention provides a pharmaceutical composition containing one or more epitopes of any of the antigens described herein, which is useful for treating a disease or condition to which the antigen or antigen epitope is pathologically related. In one embodiment, a composition containing one or more epitopes described herein is used as a vaccine for treating cancer. In one embodiment, the present invention relates to a composition containing one or more antigens and / or their epitopes conjugated by any of the TCRs described herein. In some embodiments, the composition includes, but is not limited to, the epitopes of NY-ESO-1, CT83, HCMV-pp65, HCMV-IE-1, and other antigens described herein and their variants or mutations.

[0267] In some embodiments, the signaling domain of the TCR or CAR comprises a ZAP70 kinase domain or its variants, mutants, or derivatives, the intracellular T cell activation portion comprises a signaling domain, and the signaling domain further comprises a CD3 zeta replaced by a ZAP70 kinase domain or its variants or mutants, and the TCR or CAR is fused to a chemokine receptor. Here, the chemokine receptor may be optionally selected from CCR5, CCR2, CXCR3, or other chemokine receptors to promote T cell migration. In some embodiments, the cytokine receptor includes, for example, those selected from IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, and the common cytokine receptor gamma chain. Furthermore, in some embodiments, the signal transduction domain, which includes the ZAP70 kinase domain or a variant or mutant thereof, includes an amino acid sequence having at least 55% identity with any of the amino acid sequences shown in SEQ ID NOs: 16, 17, 52, 64, or 65.

[0268] In any of the TCRs or CARs described herein (including T cells expressing them), in some embodiments, the TCR or CAR comprises an antigen recognition moiety, a transmembrane domain, and an intracellular T cell activation moiety, wherein the intracellular T cell activation moiety comprises a co-stimulatory signaling domain fused with a signaling domain. In some embodiments, the co-stimulus signaling domains include CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CD2, CD4, CD5, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD29, CD30, CD40, CD48, CD49a, CD49d, CD49f, CD53, ICAM-1 (CD54), CD69, CD70, CD80, CD82, CD83, CD84, CD86, CD90, CD96, CD100, CD103, CD122, CD Selected from 132, CD150, CD160, CD162, CD223, CD226, CD229, CD244, CD270, CD273, CD274, LAT, LFA-1, LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, DAP10, DAP12, LAG-3, 2B4, CTLA-4, ZAP70, FcεRI gamma, BAFF, GITR, HVEM, OX40L, BTLA, various integrins, IL receptor, PD-1, SLP-76, TLRs, and any combination thereof, and further, the signal transduction domain includes the ZAP70 kinase domain or its variants or mutants.

[0269] In any of the CARs or TCRs described herein, the antigen recognition portion includes an antigen-specific antibody, a bispecific antibody or binding portion, a trispecific antibody or binding portion, a multivalent or multispecific binding portion, an antigen-binding fragment, an antibody mimetic, a protein receptor, a ligand for a specific receptor, or a TCR-like antibody. In some embodiments, these include scFv, Fv, Fd, Fab, Fab’, F(ab’)2, VH domain, VL domain, monoclonal antibody, polyclonal antibody, nanobody, bispecific or trispecific antibody fusion protein, or any combination thereof. In still some embodiments, the CAR, TCR or multispecific binding portion described herein includes a leucine zipper, a SYNZIP coiled coil or other peptide structure to promote oligomerization.

[0270] In some embodiments, the present invention provides a method of extending T cell persistence or reducing T cell exhaustion by modulating the signaling and function of a TCR, chimeric TCR, CAR or cell, or by directly manipulating the signaling domain of a TCR or CAR. In this method, the signaling domain of the TCR or CAR is regulated by a negative signaling molecule, and the negative signaling molecule is knocked down or knocked out. The negative signaling molecule is selected from, for example, PD-1, VHL, PPP2R2D, indoleamine 2,3-dioxygenase (including IDO1 and IDO2), OX40, CTLA-4, PD-L1, PD-L2, LAG-3, B7-H3, and epigenetic factors including JMJD3 and LSD1, or any combination thereof.

[0271] As understood herein and contemplated by the present invention, once the sequence of a TCR is identified, one of ordinary skill in the art has sufficient knowledge of the nucleic acid sequence encoding the amino acid sequence and can make the nucleic acid construct within the ordinary skill in the art. Thus, in one aspect, the present disclosure also provides a nucleic acid encoding a polypeptide encoding any of the TCRs described herein. Identity / Homology

[0272] One way to define known and future variants and derivatives of the genes and proteins described herein is to define them in terms of identity or homology and / or identity to a particular known sequence. For example, Sequence ID No. 3 represents a specific sequence of the TCR α chain variable region. This specification specifically discloses variants of these and other genes or proteins 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 to the described sequences, as well as those having a range between any two of these percentages. Those skilled in the art readily understand methods for determining the homology or identity of two proteins or nucleic acids (which may or may not contain genes). For example, homology or identity can be calculated after aligning two sequences to maximize their homology.

[0273] As an alternative method for calculating homology or identity, known algorithms can be used. Optimal alignment for sequence comparison can be performed, for example, by Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2:482, 1981), Needleman and Wunsch's homology alignment algorithm (J. Mol. Biol. 48:443, 1970), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA 85:2444, 1988), or by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA included in the Wisconsin Genetics Software Package) or by visual inspection.

[0274] Similar homology or identity can be determined for nucleic acids using algorithms disclosed, for example, by Zuker (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), and these documents are incorporated herein by reference, at least with respect to matters related to nucleic acid alignment. nucleic acid

[0275] This specification discloses a number of nucleic acid-based molecules, including, for example, the nucleic acid encoding 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 TCR β chain. In this specification, “cDNA” means a nucleic acid in which exon-exon or exon-only coding sequences are linked. The disclosed nucleic acids consist, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are described herein. Those skilled in the art will understand, for example, that when a vector is expressed in a cell, the expressed mRNA is usually composed of A, C, G, and U. Similarly, it will be understood that when an antisense molecule is introduced into a cell or cellular environment by, for example, exogenous delivery, it is advantageous that the antisense molecule consists of a nucleotide analog that reduces degradation in the cellular environment. In some embodiments, the nucleotide analog includes one or more modifications to one or more bases, sugars, or phosphate moieties in the nucleic acid, as further described herein. Nucleotides and related molecules

[0276] A nucleotide is a molecule containing a base, a sugar, and a phosphate group. Nucleotides are linked to each other via the phosphate and sugar groups, forming nucleoside bonds. The base of a nucleotide can be selected from adenine (A), cytosine (C), guanine (G), uracil (U), and thymine (T). The sugar group of a nucleotide is ribose or deoxyribose. The phosphate group of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides include 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). A wide variety of these molecules are known in the art and are also available herein.

[0277] A nucleotide analog is a nucleotide that contains some modification to either the base, sugar, or phosphate moiety. Nucleotide modifications are well known in the art and include, for example, 5-methylcytosine, 2-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, and modifications to the sugar or phosphate moiety. A wide variety of these molecules are known in the art and are also available herein.

[0278] Nucleotide substitutes are molecules that have similar functional properties to nucleotides but do not contain a phosphate group, and may include peptide nucleic acids (PNAs). Nucleotide substitutes recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked to each other via a portion other than the phosphate group. Nucleotide substitutes can form a double-helix-like structure when interacting with a suitable target nucleic acid. A variety of these molecules are known in the art and are also available herein.

[0279] Furthermore, other types of molecules (conjugates) can be attached to nucleotides or nucleotide analogs, for example, to promote intracellular uptake. These conjugates can be chemically attached to nucleotides or nucleotide analogs. Such conjugates may or may not include a lipid moiety containing a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). A wide variety of these molecules are known in the art and are also available herein.

[0280] A Watson-Crick interaction is defined as at least one interaction between a nucleotide, nucleotide analog, or nucleotide substitute and a Watson-Crick surface. The Watson-Crick surfaces of nucleotides, nucleotide analogs, or nucleotide substitutes include the C2, N1, and C6 positions in purine-based nucleotides, nucleotide analogs, or nucleotide substitutes, and the C2, N3, and C4 positions in pyrimidine-based nucleotides, nucleotide analogs, or nucleotide substitutes.

[0281] Hoogsteen interactions refer to interactions that occur at the Hoogsteen plane of nucleotides or nucleotide analogs exposed in the large groove of double-stranded DNA. The Hoogsteen plane contains reactive groups (NH2 or O) at the N7 and C6 positions of purine nucleotides. Primers and probes

[0282] This specification discloses compositions comprising primers and probes capable of interacting with nucleic acids disclosed herein (which may or may not include tumor antigens, epitopes, and TCRs disclosed herein). In some embodiments, primers are used to support DNA amplification reactions. Typically, primers are sequence-specifically extendable. Sequence-specific primer extension includes any method by which the sequence and / or composition of the nucleic acid molecule to which the primer is hybridized or otherwise bound indicates or influences the composition or sequence of the product produced by the extension of the primer. Thus, sequence-specific primer extension includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions for sequence-specific primer amplification are preferred. In some embodiments, primers are used in DNA amplification reactions which may or may not include PCR or direct sequencing. It is also understood that in some embodiments, primers may be extended using non-enzymatic methods, such as when the nucleotide or oligonucleotide used to extend the primer is modified to chemically extend the primer in a sequence-specific manner. Typically, the disclosed primers hybridize with the disclosed nucleic acid or region thereof, or with a complementary sequence or region thereof of said nucleic acid.

[0283] In one embodiment, the size of the primer or probe for interacting with nucleic acid may be any size that supports the desired enzymatic operation, which may or may not include DNA amplification or simple hybridization of the probe or primer. Typical primers or probes are 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, The nucleotide lengths may be 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 nucleotide lengths, or a range between any two of these lengths.

[0284] In other embodiments, the primer or probe is 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, The nucleotide lengths may be 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 or less, or in the range between any two of these lengths.

[0285] In one embodiment, 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 nucleotide lengths, or a range between any two of these lengths.

[0286] In other embodiments, the product is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 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 or less, or within a range between any two of these enumerated lengths. peptide. Protein variant.

[0287] As discussed herein, numerous known variants of TCR exist, all of which are assumed herein. Antigen epitopes recognized by TCR and CAR, as well as their variants and variants, are also included herein. Variants and derivatives of proteins and peptides are well known to those skilled in the art and may include modifications of amino acid sequences. For example, amino acid sequence modifications are usually classified into one or a combination of three types: substitution, insertion, or deletion. Insertions include amino-terminal and / or carboxyl-terminal fusions, as well as insertions of one or more amino acid residues into a sequence. Insertions into a sequence are usually smaller than amino-terminal or carboxyl-terminal fusions, for example, about 1 to 4 residues. Immunogenic fusion protein derivatives may or may not include those described in the examples and are prepared by crosslinking in vitro or by recombinant cell culture transformed with DNA encoding the fusion, using polypeptides large enough to confer immunogenicity to a target sequence. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence, usually in the range of about 2 to 6 residues per location within the protein molecule. These mutants are generally produced by site-directed mutagenesis of nucleotides in protein-coding DNA, and then obtained by expressing the DNA in recombinant cell culture. Techniques for introducing substitutional mutations at predetermined sites in DNA with known sequences are well known, such as the M13 primer mutagenesis method and the PCR mutagenesis method. Amino acid substitutions are usually single residues, but can occur simultaneously at multiple different sites. Insertions are usually around 1 to 10 amino acid residues, and deletions range from approximately 1 to 30 residues. Substitutions may or may not include substitutions in one or more of the six TCR CDR regions. Deletions or insertions are preferably carried out as adjacent pairs, i.e., as deletions or insertions of two residues. The final construct can be obtained by combining substitutions, deletions, insertions, or any combination thereof. Mutations must not deviate from the leading frame and, preferably, do not form complementary regions that can generate secondary mRNA structures. A substitutional mutant is one in which at least one residue is removed and replaced by a different residue.Such substitutions are generally carried out according to Tables 1 and 2 below and are referred to as conservative substitutions. [Table 2] [Table 3]

[0288] Substantial changes in functional or immunological identity can be conferred by selecting non-conservative substitutions rather than conservative ones, as shown in Table 2. Specifically, this can be achieved by selecting residues that have a greater impact on (a) the structure of the polypeptide backbone near the substitution site (e.g., β-sheet or α-helix structure), (b) the molecular charge or hydrophobicity at that site, or (c) the bulkiness of the side chain. Generally, substitutions that are predicted to bring about the greatest changes in the properties of a protein include: (a) substituting a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine or alanine), or vice versa; (b) substituting cysteine ​​or proline with any other residue, or vice versa; (c) substituting a residue with a positively charged side chain (e.g., lysine, arginine or histidine) with a negatively charged residue (e.g., glutamic acid or aspartic acid), or vice versa; (d) substituting a residue with a bulky side chain (e.g., phenylalanine) with a residue without a side chain (e.g., glycine), or vice versa; and in this case, (e) increasing the number of sulfated and / or glycosylated sites.

[0289] For example, substituting one amino acid residue with another amino acid residue that is biologically and / or chemically similar is well known to those skilled in the art as a conservative substitution. Examples include substituting one hydrophobic residue with another hydrophobic residue, or substituting one polar residue with another polar residue. Such substitutions may include, for example, combinations of Gly and Ala, Val, Ile and Leu, Asp and Glu, Asn and Gln, Ser and Thr, Lys and Arg, and Phe and Tyr. Variants of each explicitly disclosed sequence containing these conservative substitutions are included in the mosaic polypeptides described herein.

[0290] Substitutional or deletion mutations can introduce N-linked glycosylation sites (Asn-X-Thr / Ser) or O-linked glycosylation sites (Ser or Thr). Deletion of cysteine ​​or other unstable residues may also be preferable. Furthermore, deletion or substitution of potential proteolytic sites, such as arginine residues, can be achieved by deleting one of the basic residues or by substituting it with a glutamine or histidine residue.

[0291] Certain post-translational modifications can occur as a result of the recombinant host cell's action on the expressed polypeptide. Glutamine and asparagine residues are often deamidated post-translation to their corresponding glutamic acid and aspartic acid residues, respectively. These residues can also be deamidated under weakly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the ε-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp.79-86, 1983), acetylation of N-terminal amines, and, in some cases, amidation of C-terminal carboxyl groups.

[0292] One way to define the variants and derivatives of the proteins disclosed herein is to define them based on homology / identity to a known specific sequence. Specifically, for each protein disclosed herein, variants having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% homology / identity (e.g., to SEQ ID NOs: 1, 2, 26, 31, 34, 35, 36, 37, and 61) are included. Those skilled in the art will readily understand how to calculate homology / identity between two proteins. For example, homology / identity can be calculated after optimally aligning the two sequences.

[0293] Homology / identity can also be calculated using known algorithms. Optimal alignment for sequence comparison can be performed using the local homology algorithm by Smith and Waterman (Adv. Appl. Math. 2:482, 1981), the alignment algorithm by Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the similarity search method by Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, TFASTA; Wisconsin Genetics Software Package, Genetics Computer Group), or visual inspection.

[0294] Similar concepts of homology / identity can also be applied to nucleic acids, and can be calculated using algorithms described, for example, Zuker (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).

[0295] The descriptions of conservative mutations and homology / identity can be used in any combination, and include, for example, embodiments in which a variant has at least 55% homology / identity with respect to a specific sequence and is a conservative mutation.

[0296] Since various proteins and protein sequences are described in this specification, it is understood that nucleic acids capable of encoding these protein sequences are also disclosed. This includes all nucleic acid sequences encoding specific protein sequences, as well as all nucleic acid sequences (including degenerate sequences) encoding disclosed protein variants and derivatives. Therefore, even if each nucleic acid sequence is not explicitly stated individually, it is understood that such nucleic acid sequences are disclosed in this specification through the disclosure of the corresponding protein sequences.

[0297] It is understood that the compositions disclosed herein can incorporate a large number of amino acids and peptide analogs. Examples include D-type amino acids, or amino acids having functional groups different from those shown in Tables 1 and 2. Stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogs, are also disclosed. These amino acids can be introduced into polypeptide chains site-specifically by charging tRNA with selected amino acids and designing gene constructs, for example, utilizing amber codons.

[0298] It is also possible to produce molecules with peptide-like structures without relying on natural peptide bonds. For example, possible bonding patterns between amino acids or amino acid analogs include CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans forms), -COCH2-, -CH(OH)CH2-, and -CH2SO- (see Spatola, AF, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, edited by B. Weinstein, Marcel Dekker, New York, p.267 (1983); Spatola, AF, Vega Data (March 1983), Vol.1, Issue 3; Morley, Trends Pharm. Sci. (1980) pp. 463-468; Hudson et al., Int. J. Pept. Prot. Res. 14:177-185 (1979); Spatola et al., Life Sci. 38:1243-1249). (1986); Hann, J. Chem. Soc. Perkin Trans. I 307-314 (1982); Almquist et al., J. Med. Chem. 23:1392-1398 (1980); Jennings-White et al., Tetrahedron Lett. 23:2533 (1982); Szelke et al., European Patent Application EP 45665; Holladay et al., Tetrahedron Lett. 24:4401-4404 (1983); Hruby, Life Sci. 31:189-199 (1982), all of which are incorporated herein by reference). A particularly preferred non-peptide bond is -CH2NH-. The peptide analog may contain multiple atoms between the bonded atoms, including β-alanine, gamma-aminobutyric acid, etc.

[0299] Amino acid analogs and peptide analogs may possess desirable or advantageous properties such as more economical manufacturing, improved chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broader bioactivity spectrum), and reduced antigenicity.

[0300] D-type amino acids are less easily recognized by peptidases and other enzymes, and can therefore be used to produce more stable peptides. For example, by systematically substituting one or more amino acids in a consensus sequence with the same type of D-type amino acid (e.g., D-lysine instead of L-lysine), peptides with improved stability can be obtained. Furthermore, cysteine ​​residues can be used to cyclically form peptides or to link two or more peptides together, thereby constraining them to a specific three-dimensional structure. Delivery of pharmaceutical carriers / pharmaceutical products

[0301] In one embodiment, the Specified herein discloses a composition comprising a therapeutically effective amount of one or more TCR-T cells, wherein the TCR-T cells are genetically modified to express a receptor for one of the tumor antigens disclosed herein. Furthermore, the tumor antigen-specific TCR-T cells may be further modified to knock out or knock down programmed cell death protein (PD1), von Hippel-Lindau tumor suppressor (VHL), and / or protein phosphatase 2 regulatory subunit B delta (PPP2R2D) in order to improve their function, such as cytotoxic activity and / or persistence or viability in the body after adoptive transfer to cancer patients.

[0302] The composition may also be administered in vivo in a pharmaceutically acceptable carrier. In this specification, "pharmaceutically acceptable" means a material that does not have undesirable properties from a biological or other standpoint, does not cause undesirable biological effects when administered to a subject together with nucleic acids or vectors, and does not undergo harmful interactions with other components of the pharmaceutical composition. The carrier is selected according to methods well known to those skilled in the art to minimize degradation of the active ingredient and adverse effects in the subject.

[0303] The composition may be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, extracorporeally, or topically, including nasal topical administration or inhalation. In this specification, "nasal topical administration" means delivery to the nasal cavity and nasal passages through one or both nostrils by a spray mechanism, a dropper mechanism, or aerosolization of nucleic acids or vectors. Inhalation can be performed via the nose or mouth by a spray or dropper mechanism, and can also be delivered directly to the respiratory system (e.g., lungs) by intubation. The required dose cannot be uniformly specified as it will vary depending on the subject's species, age, weight, general condition, severity of the disease being treated, the nucleic acid or vector used, and the route of administration, but a person skilled in the art can determine an appropriate amount through normal experimentation based on the description herein.

[0304] Parenteral administration is generally carried out by injection. Injectable preparations can be prepared in conventionally known forms, such as liquid solutions or suspensions, solid preparations dissolved or suspended in liquid before injection, or emulsions. In recent years, methods using sustained-release or prolonged-release systems to maintain a constant dose have also been proposed (see, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference).

[0305] The materials may exist as solutions or suspensions (e.g., in the form of microparticles, liposomes, or encapsulated in cells). These can be targeted to specific cell types via antibodies, receptors, or receptor ligands. Examples of targeting techniques for tumor tissue are described by Senter et al. (Bioconjugate Chem. 2:447-451, 1991), Bagshawe (Br. J. Cancer 60:275-281, 1989), Bagshawe et al. (Br. J. Cancer 58:700-703, 1988), Senter et al. (Bioconjugate Chem. 4:3-9, 1993), Battelli et al. (Cancer Immunol. Immunother. 35:421-425, 1992), Pietersz and McKenzie (Immunol. Reviews 129:57-80, 1992), and Roffler et al. (Biochem. Pharmacol. 42:2062-2065, 1991). This includes so-called "stealth" liposomes and antibody-conjugated liposomes (including lipid-mediated drug delivery to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-targeted tumor targeting, and in vivo targeting of highly specific therapeutic retroviruses against mouse glioma cells. Generally, receptors are involved in constitutive or ligand-induced endocytosis pathways, accumulating in clathrin-coated pits and being taken up into cells as clathrin-coated vesicles. From there, they follow one of two pathways: recirculation to the cell surface via acidified endosomes, intracellular storage, or degradation in lysosomes. These internalization pathways perform various functions, including nutrient uptake, removal of activated proteins, removal of macromolecules, entry of viruses or toxins, dissociation and degradation of ligands, and regulation at the receptor level. Depending on the cell type, receptor concentration, ligand type, ligand valency, and concentration, receptors may take multiple intracellular pathways. The molecular and cellular mechanisms of receptor-mediated endocytosis are reviewed by Brown and Greene (DNA and Cell Biology 10(6):399-409, 1991). Pharmacologically acceptable carriers

[0306] The antibody-containing composition can be used for therapeutic purposes in combination with a pharmaceutically acceptable carrier.

[0307] Suitable carriers and examples of 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 to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and glucose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Furthermore, sustained-release formulations consisting of a semipermeable matrix of a solid hydrophobic polymer containing antibodies may also be included, and the matrix may take the form of molded articles such as films, liposomes, microparticles, nanoparticles, or LNPs. It will be apparent to those skilled in the art that certain carriers may be more preferable depending on the route of administration and the concentration of the administered composition.

[0308] Pharmaceutical carriers are well known to those skilled in the art, and typically, standard carriers used for drug administration to humans, such as sterile water, physiological saline, or solutions buffered to physiological pH, are used. The composition can be administered intramuscularly or subcutaneously, and other compounds are also administered according to standard methods well known to those skilled in the art. The pharmaceutical composition may contain, in addition to the target molecule, a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, and the like.

[0309] The pharmaceutical composition may further contain one or more active ingredients such as antibacterial agents, anti-inflammatory agents, and anesthetic agents.

[0310] The pharmaceutical composition can be administered by various methods depending on whether it is a local or systemic treatment and the site of treatment. Methods of administration include topical application (including ophthalmic, vaginal, rectal, and nasal administration), oral administration, inhalation administration, or parenteral administration, such as intravenous infusion, subcutaneous administration, intraperitoneal administration, or intramuscular administration. The antibodies disclosed herein can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarially, or percutaneously.

[0311] Parenteral administration formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oil (may include olive oil), and organic esters for injection (may include ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (may include physiological saline and buffer solutions). Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oil. Intravenous carriers include infusions, nutritional supplements, and electrolyte supplements (may include Ringer's dextrose systems). In addition, preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may be included.

[0312] Examples of externally administered formulations include ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, and powders. Conventionally known pharmaceutical carriers, aqueous bases, powder bases, or oily bases, thickeners, etc., can be used as needed.

[0313] Oral administration compositions include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets, or tablets. They may optionally contain thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders.

[0314] Some compositions can also be administered as pharmaceutically acceptable acid or base addition salts. These acid addition salts can be formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, or with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid. These base addition salts can be formed by reaction with inorganic bases such as sodium hydroxide, aqueous ammonia, and potassium hydroxide, or with organic bases such as mono-, di-, trialkylamines, arylamines, or substituted ethanolamines.

[0315] pharmaceutically acceptable carriers used in the therapeutic mRNA compositions described herein include lipid nanoparticles (LNPs) for delivering mRNA into cells. Adjuvants used in mRNA vaccines are described, for example, in Xie et al., "The advances of adjuvants in mRNA vaccines" (NPJ Vaccines, 2023, 8:162). In one embodiment, the LNP comprises a cationic lipid that forms a complex with a negatively charged mRNA molecule. Furthermore, in another embodiment, the LNP comprises a structural lipid including or selected from cholesterol, phospholipids, or polyethylene glycol (PEG)-bound lipids. In yet another embodiment, the lipids constituting the LNP may have immunostimulatory effects. For example, lipid-coated calcium phosphate nanoparticles deliver additional Ca to the cytoplasm. 2+ By supplying artificial nano-sized Ca, the expression of co-stimulatory molecules such as CD80 and CD86 is enhanced, promoting the maturation of dendritic cells. 2+It can function as a reservoir. Furthermore, the LNP may or may not contain lipids selected from, for example, the ionizable cationic lipid DLinDMA, the cationic lipid-like material C1, Lipidoid C12-TLRa, A2-Iso5-2DC18(A2), non-nucleotide-based STING agonist-derived aminolipids, SAL12, and dimethyldioctadecylammonium (DDA). In one embodiment, the mRNA vaccine includes mRNA-containing lipid nanoparticles (e.g., those filed as PCT / US2020 / 013417 and published as WO2020146906, and those described in US20220088221), which are incorporated herein by reference.

[0316] The effective dose and administration schedule for the composition of the present invention can be determined empirically by those skilled in the art. The dose is selected to be sufficient to produce the desired effect on the symptoms of the disease and to avoid causing adverse side effects such as unwanted cross-reactivity or anaphylactic reactions. In general, the dose may vary depending on the patient's age, sex, disease state and severity, route of administration, and presence or absence of concomitant medications, and can be determined by those skilled in the art. When the antibody is used alone, a typical daily dose may range from about 1 μg to about 100 mg or more per kg of body weight. Furthermore, the pharmaceutical composition containing the modified T cells according to the present invention may be 10 per kg of body weight. 4 ~10 7 pieces, preferably 10 5 ~10 6 Modified T cells may be administered in doses of one. These modified T cells may be administered multiple times by infusion techniques known in the field of immunotherapy. The optimal dosage and treatment regimen may be adjusted as appropriate by those skilled in the art while observing the patient's condition.

[0317] The compositions disclosed herein can be used to treat diseases involving uncontrolled cell proliferation, such as cancer. In one aspect, this specification provides a method for stimulating an immune response to cancer, or treating, suppressing, and / or preventing cancer, by administering to a subject a composition containing an effective amount of T cells modified with an antigen-specific TCR.

[0318] In this specification, “therapeutic amount” means an amount sufficient to improve at least some of the cause or symptoms of a disease or disorder, without necessarily requiring complete elimination.

[0319] In this specification, “treatment” means medical management undertaken for the purpose of curing, improving, stabilizing or preventing a disease, pathological condition or disorder, and includes active treatment, causal treatment, palliative care, preventive care and supportive care.

[0320] In this specification, “pathogenically related” means that an antigen or its epitope has a causal relationship with the associated disease or condition, and that the disease or condition is cured, improved, stabilized, or prevented by inducing an immune response to the antigen.

[0321] Examples of cancers treatable by the methods disclosed herein include, but are not limited to, lymphoma, leukemia, carcinoma, sarcoma, melanoma, neuroblastoma, glioblastoma, myeloma, and metastatic cancer.

[0322] Furthermore, specific examples of cancers that may be treatable include, but are not limited to, lymphoma, lung cancer, breast cancer, prostate cancer, pancreatic cancer, ovarian cancer, liver cancer, colorectal cancer, rectal cancer, kidney cancer, genitourinary cancer, head and neck cancer, and skin cancer.

[0323] In this invention, an HLA-DP4-restricted T cell receptor (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-reactive T cells. The NY-ESO-1(161-180) peptide containing the HLA-DP4-restricted epitope was synthesized with a purity of 95% or higher. This peptide was pulsed onto 1088EBV-B cells, an HLA-DP4-positive cell line, as antigen-presenting cells (APCs), and co-cultured with human PBMCs in a 96-well plate for 21 days. During this process, it was thought that continuous peptide stimulation amplified the NY-ESO-1(161-180)-specific T cell population, while non-specific T cells and other immune cells were depleted. After 21 days of stimulation, the T cell population from each well was collected and subjected to further analysis.

[0324] We first evaluated the recognition ability of stimulated T cells derived from different wells to NY-ESO-1 (161-180). T cells were co-cultured with simulated 1088EBV-B cells or the same APC pulsed with the target peptide, and T cell activation was evaluated by the amount of cytokine release in the supernatant. As a result, several well-derived T cell populations showed high activity to the peptide. From these T cell populations, we evaluated CD8 + T cells were removed, and the resulting CD4 + The T cell line was named "DP4 ESO-reactive T cell." It was confirmed that this T cell line recognizes the HLA-DP-restricted NY-ESO-1 epitope, rather than the HLA-DR-restricted epitope. Furthermore, it was shown that its specific peptide recognition is inhibited only by antibodies against HLA class II as a whole and HLA-DP, and not by other class I or class II molecules. In addition, attempts to identify the smallest epitope recognized by these T cells revealed that the shortest sequence maintaining high recognition ability is NY-ESO-1 (157-170).

[0325] To perform molecular cloning of a DP4-restricted NY-ESO-1 specific TCR, single T cell clones were generated from a DP4-ESO-reactive T cell line. These T cell lines were serially diluted and seeded at a rate of 0.3 cells / well in a 96-well plate. Single viable T cells in each well were cultured for 14 days to promote growth. The reactivity of the proliferated T cell clones was evaluated using 1088EBV-B (HLA-DP4 positive) cells displaying the NY-ESO-1(157-170) peptide, and epitope recognition ability was determined by cytokine release. Some of the results are shown in Figure 1, demonstrating that multiple DP4-ESO-1-reactive T cell clones reacted to the peptide compared to simulated APCs. These peptide-reactive T cell clones were cultured for a further 14 days, but some clones became depleted and stopped growing, suggesting differences in survival time. The viable T cell clones were then subjected to further TCR cloning.

[0326] From a surviving T cell clone, 1 × 10 6 mRNA was extracted from individual T cells. The obtained mRNA was reverse transcribed to prepare a three-step nested PCR template. In each PCR step, the complementarity-determining region 3 (CDR3) of the TCRα and β chains was amplified using different primer sets targeting all TRAV or TRBV. After collecting the third-round PCR product, the subtypes of the TCRα and β chains were identified by Sanger sequencing. Based on these results, the full-length sequences of the TCRα chain (TRAV34) and β chain (TRBV30) were amplified. The obtained full-length TCRs (TRAV-TRAJ-TRAC, TRBV-TRBD-TRBJ-TRBC) were ligated with a P2A sequence and cloned into an MSGV retroviral vector (Figure 2).

[0327] A two-step transduction method was used to introduce the TCR into naive T cells. The TCR construct was introduced into the Phoenix-Eco cell line using lipofectamine to prepare the primary viral supernatant. After 48 hours, the supernatant was collected and used to infect the PG-13 cell line. After infection, the PG-13 cell line secreted the secondary viral supernatant. The PG-13-derived viral supernatant was coated onto 24-well non-tissue culture plates pre-coated with retronectin. Naive CD4 cells were isolated from human PBMCs and pre-stimulated with anti-CD3 antibody. + T cells were infected with the retrovirus. To improve transduction efficiency, PG-13 cells with the TCR gene were cloned at limiting dilution in a 96-well plate and cultured in each well for more than 15 days. The transduction efficiency of naive T cells using viruses derived from different PG-13 clones was evaluated using TRBV30-specific antibodies and flow cytometry, and the results showed an average of 60-70% (Figure 3A). Furthermore, TCR-transformed CD4 cells were used with 586 mel, 624 mel, and DP4-ESO monomers. + When T cell activity was evaluated, specific recognition was confirmed (Figure 3B).

[0328] We evaluated whether TCR-introduced T cells possessed equivalent functionality to DP4 ESO-reactive T cell lines through multiple tests. First, the introduced T cells specifically recognized NY-ESO-1 (157-170), an HLA-DP4-restricted epitope (Figure 4A). Next, in a plasmid transduction system into artificial APCs and tests using HLA-DP4-positive or negative tumor cells, it was confirmed that they specifically recognized naturally treated NY-ESO-1 (Figure 4B). Furthermore, this TCR also recognized CD4 + To confirm that it functions as a TCR, CD8 + and CD4 + When T cells were isolated and introduced, CD4 + Only T cells showed function (Figure 4C). These results suggest that TCR-modified CD4 +It was confirmed that T cells have the ability to specifically recognize NY-ESO-1 presented by HLA-DP4. In one embodiment, the HLA-DP4-restricted NY-ESO-1 TCR of the present invention provides a novel clinical response strategy in cancer immunotherapy.

[0329] Considering the limitations of using transgenic mice, humanized mice were used to evaluate the efficacy and safety of NY-ESO-1 TCR. Humanized NSG (NOD SCID IL2 gamma - / - The proliferation of human cancer cells was evaluated using mice. MDA-MB-231 / DP4 / ESO tumor cells were prepared in 50 μl of growth medium / Matrigel (50%) and transplanted into the fourth mammary gland fat pad of female NSG mice (n=6 in each group). On day 5, 2 × 10⁶ human T cells from each of the four groups were transplanted into each mouse. 6 The drug was administered intravenously, followed by intraperitoneal administration of IL-2 to promote T cell proliferation. Tumor growth was measured every 3-5 days, and CD8 + T cell migration was tracked using luciferase introduced into T cells. As a result, A2-ESO-1 TCR modified CD8 + T cells specifically migrated to the tumor site and significantly suppressed tumor growth (Figures 5A-5C). Furthermore, DP4-ESO-1 TCR-modified CD4 + T cells also showed a similar tumor-suppressing effect, and the greatest suppressive effect was obtained by using both in combination. This is because CD8 cells target the same antigen. + and CD4 + This demonstrates that the combined use of TCR-modified T cells is superior to the use of CD4 + This suggests an unexpected synergistic effect mediated by T cells.

[0330] All animal experiments were conducted using 6-8 week old female NSG mice, following approved standard procedures. Tumor cell transplantation, T cell administration, tumor growth measurement, and T cell migration analysis were performed under the same conditions as described above.

[0331] HLA-A2 is a major HLA class I molecule and is expressed at a high frequency in approximately 50% of the general population. Therefore, in this invention, we conducted tests to identify novel HLA-A2-restricted T cell epitopes derived from CT83. For this purpose, we synthesized CT83 peptides (CT83 PEP66-74, PEP79-87, and PEP90-98) that are predicted to contain an HLA-A2 binding motif. CD8 +T cells were isolated and stimulated for 10 days with autologous dendritic cells (DCs) loaded with the peptide. During culture, T cell medium containing IL-7 and IL-15 (5 ng / ml each) was added every 2-3 days. For the second stimulation, autologous PBMCs were irradiated with 60 Gy, pulsed with 1 μg / ml of the peptide for 2-4 hours, washed, and co-cultured with primary stimulated T cells for a further 10 days. These T cells were supplemented with medium containing IL-2 (30 IU / ml), IL-7 (5 ng / ml), and IL-15 (5 ng / ml) every 2-3 days. To evaluate CT83-specific recognition ability, in vitro stimulated T cells were co-cultured with HLA-A2-positive HEK293 T cells in the presence or absence of CT83 PEP90-98. As a result, the T cells specifically recognized 293T / CT83 PEP90-98, but did not respond to 293 T cells alone (Figure 6A). On the other hand, T cells specific to CT83 PEP66-74 or PEP79-87 were not induced (data not shown). Furthermore, to evaluate whether these T cells recognize CT83 PEP90-98 endogenously treated and presented with HLA-A2, analysis was performed using 293T cells into which immutable chain (Ii) fused CT83 or CT83-GFP (full-length CT83 and GFP linked with a P2A sequence) was introduced. As a result, CT83-specific T cells recognized 293T / Ii-CT83 and 293T / CT83-GFP, but 293T cells presenting the control peptide did not (Figure 6B). Furthermore, when the recognition ability against breast cancer cells was evaluated, MDA-MB-231 cells expressing CT83 and HLA-A2 induced IFN-gamma secretion from CT83-specific T cells, but MDA-MB-468 cells lacking HLA-A2 did not (Figure 6C). Furthermore, antibody inhibition tests showed that this recognition was completely inhibited by an anti-MHC class I antibody, but not by an anti-MHC class II antibody or a control antibody (Figure 6D). Therefore, it was confirmed that these T cells specifically recognize CT83 PEP90-98 presented by HLA-A2.

[0332] Furthermore, we evaluated whether A2-CT83 PEP90-98 could induce antitumor immunity in HLA-A2 transgenic (Tg) mice. E0771-A2-CT83 mouse mammary cancer cells (0.5 × 10⁶) 6 TAT-CT83 PEP90-98 (1 / mouse) was transplanted into the mammary gland adipose pad of HLA-A2 mice on day 0. Tumor-carrying mice were administered a self-assembling nanoparticle vaccine containing TAT-CT83 PEP90-98 or TAT-CT83 PEP66-74 in combination with TLR ligands (CpG, MPLA, and poly(I:C), hereafter referred to as CMI) on days 7, 10, and 15 (Figure 7A). As a result, TAT-CT83 PEP90-98-CMI significantly suppressed tumor growth compared to TAT-CT83 PEP66-74-CMI (Figure 7B). While CT83 PEP66-74 has been reported to induce a T cell response via RNA vaccine, there have been no previous reports regarding CT83 PEP90-98. Therefore, this invention is the first to demonstrate that CT83 PEP90-98 induces a T cell response and suppresses tumor growth both in vitro and in vivo.

[0333] To identify CT83-specific TCRs, A2-CT83-specific T cells were purified by intracellular staining with anti-IFN-gamma antibody and FACS preparative after stimulation with 293T / CT83 cells (Figure 8A). Several thousand T cells were distributed into nanoliter-scale GEMs (Gel Beads-in-emulsion) using a 10x Genomics Chromium Controller. Following the manufacturer's protocol, TCR V(D)J libraries were prepared by cell lysis, reverse transcription, PCR, and barcoded cDNA, and sequenced using an Illumina HiSeq 2500. Sequence analysis identified dominant and subdominant TCRα and TCRβ chain pairs, and full-length TCRα and β chains were obtained using 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 8A). The obtained A2-CT83 TCR-T cells recognized 293T cells that had been transfected with CT83-GFP and Cos-7 cells that had been co-transfected with HLA-A2 and CT83, but did not recognize control cells (Figure 8B). Furthermore, these TCR-T cells recognized 293T / CT83 PEP90-98, but did not recognize 293T cells that displayed other CT83 peptides (Figure 8C). In addition, the recognition ability against breast cancer cells was evaluated, and it was found that MDA-MB-231 cells (A2 + / CT83 + It specifically recognized CT83 and did not recognize other cell lines (Figure 8D). Furthermore, it specifically recognized only lung cancer cells that co-expressed CT83 and HLA-A2 (Figure 8E). From the above, it was confirmed that the A2-CT83 TCR identified by the present invention specifically recognizes the CT83 epitope that is naturally treated and presented with HLA-A2.

[0334] To evaluate the in vivo antitumor activity of A2-CT83 TCR-T cells, immunodeficient NSG mice were used as a tumor model. NCI-H838 / A2 tumor cells were transplanted on day 0, and A2-CT83 TCR-T cells (5 × 10⁶ each) were transplanted on days 3 and 5. 6Mice were intravenously administered with [the number] cells / mouse, and from the 3rd day to the 7th day, IL-2 (50,000 IU / day) was intraperitoneally administered (Figure 9A). As a result, tumor growth was completely suppressed in the A2-CT83 TCR-T cell administration group (Figures 9B and 9C). On the other hand, significant tumor growth was observed in the control T cell administration group. From the above, it was shown that A2-CT83 TCR-T cells have strong antitumor activity in vivo.

[0335] It has been reported that nucleic acids and proteins of human cytomegalovirus (HCMV) are detected in glioblastoma. In this example, two types of proteins (pp65 and IE-1) derived from HCMV particles were selected as targets for antigen-specific TCR recognition.

[0336] The method for stimulating pp65-specific and IE-1-specific T cells is the same as that for DP4-ESO-1 T cells. CD8 + T cells were isolated from PBMCs derived from HLA-A2-positive healthy donors and stimulated with pp65 (495-503) and IE-1 (316-324), which are HLA-A2-restricted epitopes, respectively. Each peptide was synthesized with a purity of 95% or higher. Mature dendritic cells prepared from autologous PBMCs were pulsed overnight with 10 μg / ml of each peptide, irradiated with 60 Gy for 3 minutes, and co-cultured with CD8 + T cells at a ratio of 1:5. Restimulation was performed on the 7th day, and the cells were collected on the 14th day.

[0337] After in vitro stimulation, CD8 with pp65 reactivity (about 15%) and IE-1 reactivity (about 8%) +T cells were isolated, and T cell clones were generated using the limiting dilution method (Figures 10A and 10B). As a result, seven pp65 (495-503) specific clones and five IE-1 (316-324) specific clones were obtained. Of these, pp65-reactive clone #3 and IE-1-reactive clone #5 were selected and further analyzed. Both clones specifically recognized Cos-7 cells co-transfected with HLA-A2 and either pp65 or IE-1, respectively, but did not recognize cells with other HLA molecules (Figure 10B). These T cell clones were then used for further TCR cloning.

[0338] Using the same method as described above, A2-pp65 TCRs and A2-IE-1 TCRs were identified from T cell clones. After CDR3 sequence analysis, the repertoires of both TCRs (TRAV24 and TRBV6-5 from pp65 T cell clone #3, and TRAV25 and TRBV5-1 from IE-1 T cell clone #5) were identified. The full-length TCRs were amplified using specific primers and incorporated into pMSGV vectors. Retroviral TCR introduction into human T cells was performed as described above. The introduction efficiency of each TCR was confirmed by FACS analysis after staining with TCR-specific antibodies. As a result, both TCRs showed an introduction efficiency of over 60% (Figure 11A), indicating that they are suitable candidates for in vitro testing. After culturing, A2-pp65 TCR-transformed T cells and A2-IE-1 TCR-transformed T cells recognized U87 glioblastoma cell lines (HLA-A2 positive) that had been transfected with pp65 or IE-1, respectively, but did not recognize U118 glioblastoma cell lines (HLA-A2 negative) that had been transfected with pp65 or IE-1 (Figure 11B). In particular, both TCR-transformed T cells specifically recognized U87 cell lines infected with HCMV AD169 strain compared to similarly infected U118 cell lines (Figure 11B). Furthermore, both TCR-transformed T cells showed dose-dependent recognition patterns against T2 cells pulsed with pp65 (495-503) or IE-1 (316-324) peptides (Figure 11C). Furthermore, when cytotoxic activity was evaluated, both A2-pp65 TCR-transformed T cells and A2-IE-1 TCR-transformed T cells showed nearly 100% cytolytic activity against U87 cells that had been transduced to pp65 or IE-1, or that were infected with the HCMV AD169 strain (Figure 11D). In addition, cytotoxic activity against AD169-infected U87 cells progressed in a dose-dependent manner (Figure 11E). In vivo antitumor activity of A2-pp65 TCR and A2-IE-1 TCR-transformed T cells against HCMV antigen-expressing tumors

[0339] To evaluate the in vivo antitumor activity of A2-pp65 TCR-transformed T cells and A2-IE-1 TCR-transformed T cells against HCMV antigen-expressing tumors, a xenograft model was used in which U87 cells expressing pp65 or IE-1 and luciferase were transplanted into immunodeficient mice. After transplanting tumors into SCID / Beige mice, the cells were cultured for 3 days, and then 2 × 10^6 human T cells transduced with A2-pp65 TCR, A2-IE-1 TCR, or a control TCR were administered intravenously to each mouse (Figures 12A and 13A). The migration of the administered T cells was observed every 3 days until mouse sacrificial (Figures 12B and 13B). As a result, tumor growth was significantly suppressed in both TCR treatment groups (n=5) compared to the control group (n=3) administered with control TCR-transformed T cells from the same healthy donor. A reduction in tumor size was observed in all animals treated with A2-pp65 TCR-modified T cells or A2-IE-1 TCR-modified T cells (Figures 12C, 12D and 13C, 13D). These results indicate that both TCRs possess antitumor activity and have potential applications in the treatment of glioblastoma. Example 4: Improvement of TCR expression, specificity, and function by reducing mispairing with endogenous TCRs

[0340] Since each T cell possesses an endogenous TCR, CT83-specific TCRα and TCRβ chains may mispair with the endogenous TCRα and TCRβ chains, potentially forming a non-functional TCR(α / β) or a novel TCR(α / β) with unexpected antigen specificity. Strategies to circumvent this problem include knocking out the endogenous TCR(α / β) using CRISPR / Cas9 technology, or creating chimeric TCRs by fusing the variable region of NY-ESO-1 or CT83 TCRs with the constant region of a non-human, such as mouse-derived TCR, thereby reducing mispairing between endogenous and exogenous TCRs. The latter method has shown that replacing the constant region of the human NY-ESO-1 TCR with a mouse-derived constant region improves TCR expression and tumor cell recognition (Figures 14A-14G). Furthermore, T cells introduced with A2-ESO-1 TCR-M showed stronger in vivo antitumor activity than conventional A2-ESO-1 TCR-introduced T cells (Figures 15A-15D).

[0341] Five constructs of the A2-ESO-1 TCR were created, each containing different amino acid substitutions in the variable region of the α or β chain. Using the pMSGV-A2-ESO-1 TCR as a template, PCR amplification was performed using primers containing site-directed mutations. After superimposing multiple PCR products, the five variants were cloned into the pMSGV vector (Sub1-Sub5). As a result, all of these A2-ESO-1 TCR-introduced T cells recognized NY-ESO-1 expressing 624-mel cells and MDA-MB-231 cells (both HLA-A2 positive), but did not recognize HLA-A2 negative 586-mel cells (Figure 16A). Cytotoxic activity was also confirmed (Figure 16B).

[0342] To further reduce mispairing with human endogenous TCRs, we created chimeric A2-ESO-1 TCRs by replacing the constant region of human A2-ESO-1 TCRs with the constant region of mouse-derived TCRs. As a result, T cells introduced with TCRs containing the mouse constant region were able to recognize MDA-MB-231 / ESO cells, suggesting improved TCR expression and function (Figure 16C). Antitumor activity of A2-CT83 TCR-M T cells with a constant region in mice

[0343] FACS analysis revealed that the transduction efficiency of A2-CT83 TCR-M into human T cells was over 70% (Figure 17A). A2-CT83 TCR-M transduction T cells strongly recognized HLA-A2-positive and CT83-positive tumor cells (MDA-MB-231 and NCI-H1563), but did not recognize CT83-negative tumor cells (CAMA-1) (Figure 17B). Furthermore, 60-80% tumor cytolytic activity was observed (Figure 17C). These results indicate that A2-CT83 TCR-M T cells possess high specificity and antitumor activity with reduced mispairing. Example 5: Regulation of CAR-T cell signaling by substitution with a ZAP70 kinase-derived signal region.

[0344] The CAR construct includes a single-chain variable fragment for antigen recognition, a transmembrane domain, and an intracellular activation domain consisting of a CD28 or 4-1BB costimulatory signaling region and a CD3 zeta signaling region. The CD3 zeta chain is a key component of the TCR-CD3 complex, and its phosphorylation of ITAM recruits ZAP70, which is involved in signal transduction. Conventional CARs contain a CD3 zeta chain and transmit signals via ZAP70, but in this invention, experiments have shown that the CD3 zeta signaling region can be replaced with other signaling regions derived from ZAP70 to improve the activation and persistence of CAR-T cells.

[0345] For this purpose, signaling domains derived from ZAP70 and LAT were screened, and anti-CD19 scFv-CD28-ZAP300 (from amino acid residue 300 to the C-terminus of the ZAP protein) and anti-CD19-CD28-ZAP327 (from amino acid residue 327 to the C-terminus of the ZAP protein) were identified (Figure 11A). These ZAP70 kinase domains (domains starting from amino acid residue 300 or 327 of ZAP70) were fused to the C-terminus of the CD3 zeta chain using CD28 as a co-stimulatory domain to create anti-CD19 scFv-CD28-ZAP300 (1928ZAP300) and anti-CD19-CD28-ZAP327 (1928ZAP327) (Figure 18A). Flow cytometry analysis confirmed that 1928ZAP300 and 1928ZAP327 exhibited comparable CAR expression induction efficiency to conventional anti-CD19-CD28-CD3 zeta (hereinafter referred to as 1928Z) (Figure 18B). Furthermore, these CAR-T cells directly killed Raji tumor target cells in a non-radioactive LDH cytotoxicity assay with progressively altered E:T ratios (Figure 18C). Therefore, Figures 18C and 18D show antigen-specific recognition and tumor cell lysis after co-culturing CAR-T cells with Raji tumor cells, demonstrating that CAR-T cells, including ZAP300 and ZAP327, are functional and specific to the target antigen in in vitro tests.

[0346] Furthermore, in in vivo studies, 1928ZAP300 and 1928ZAP327 CAR-T cells showed superior efficacy compared to 1928Z CAR-T cells, significantly and unexpectedly extending overall survival in a Raji lymphoma mouse model (Figure 18D). When NSG mice transplanted with Raji tumors were administered unintroduced T cells, 1928Z, 1928ZAP300, or 1928ZAP327 CAR-T cells, respectively, tumor-bearing mice administered with control T cells died within 20 days after tumor injection. Mice administered with 1928Z CAR-T cells died around day 40. On the other hand, mice administered with 1928ZAP300 or 1928ZAP327 CAR-T cells showed a significantly extended survival period of more than 70 days after tumor injection (Figure 18E). In particular, more than 60% of tumor-carrying mice administered 1928ZAP327 CAR-T cells survived for more than 80 days. These results indicate that substituting the CD3 zeta chain with the ZAP70 kinase domain (ZAP300 or ZAP327) significantly enhances antitumor activity in vivo. 19bbZAP327 CAR-T cells exhibit low cytokine production and potent antitumor immunity.

[0347] To investigate whether the ZAP70 kinase domain functions in CAR constructs containing 4-1BB, a CAR construct (19bbZAP327) was created by fusing the ZAP70 kinase domain (e.g., ZAP327) to the 4-1BB domain (Figure 19A). As a result, 19bbZAP327 CAR-T cells showed significantly lower production of IFN-gamma, IL-2, and TNF-α compared to conventional 19bbZ CAR-T cells after tumor cell stimulation (Figure 19B). On the other hand, 19bbZAP327 and 19bbZ CAR-T cells showed similar specific tumor cell lysis ability in vitro (Figure 19C). Importantly, tumor-carrying mice administered with 19bbZAP327 CAR-T cells showed superior antitumor activity compared to mice administered with 19bbZ CAR-T cells (Figures 19D and 19E). Mice administered with 19bbZAP327 CAR-T cells showed a significantly extended survival time (Figures 19D and 19E). These results demonstrate that 19bbZAP327 CAR-T cells induce more potent anti-tumor immunity compared to conventional 19bbZ CAR-T cells, despite producing less cytokines.

[0348] Furthermore, ZAP327 can be fused to TCR constructs to enhance T cell signaling. These studies have shown that the ZAP327 signaling domain enhances the signaling, function, and persistence of CAR-T cells and TCR-T cells. In other embodiments, other signaling domains derived from ZAP300 or ZAP70 can be used in CAR or TCR constructs to improve antitumor activity while reducing cytokine production. The ZAP327 signaling domain promotes T cell memory function and persistence in vivo.

[0349] To elucidate the reasons why 1928ZAP327 CAR-T cells exhibit more potent antitumor immunity, T cell survival was evaluated 30 days after T cell transfer. The results showed that mice administered with 1928ZAP327 CAR-T cells had a higher percentage of CAR-T cells in the bone marrow and spleen compared to mice administered with 1928Z (Figures 20A and 20B). Furthermore, 1928ZAP327 CAR-T cells had a higher percentage of central memory T cells than 1928Z CAR-T cells (Figure 20C). Additionally, 1928ZAP327 CAR-T cells expressed less PD-1, a fatigue marker, than 1928Z CAR-T cells (Figure 20D). These results suggest that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. In other embodiments, the memory function and persistence of T cells can be enhanced by using other signaling domains derived from ZAP300 or ZAP70 in the CAR or TCR construct.

[0350] ShRNAs for PD1, VHL, and PPP2R2D were constructed and incorporated downstream of an A2-ESO-1 TCR expression vector. Retroviral particles were produced and introduced into undifferentiated human T cells. The efficiency of T cell introduction was confirmed by staining with an A2-ESO-1 TCR antibody and flow cytometry analysis (Figure 21A). The introduced T cells were used in animal experiments. Breast cancer model mice were generated by subcutaneous injection of modified MDA-MB-231 / NY-ESO-1 / luciferase cells into NSG mice. Three days later, undifferentiated T cells, A2-ESO-1 TCR-T cells (with PD1, VHL, or PPP2R2D knocked down or unmodified) were injected into the tumor. Tumor burden was evaluated by in vivo luciferase imaging, and mouse survival was also tracked (Figures 21B, 21C, and 21D). A2-ESO-1 TCR-T cells with PD1, VHL, or PPP2R2D knockdown, respectively, showed comparable or superior tumor suppression compared to A2-ESO-1 TCR-T cells alone. Combining the knockdown of PD1, VHL, or PPP2R2D extended the survival time of TCR-T cell-treated mice. In particular, some mice in the VHL and PPP2R2D groups survived until the end of the experiment (Figures 21B, 21C, and 21D). Knockdown or knockout of JMJD3 enhances T cell function and in vivo persistence.

[0351] Furthermore, CAR constructs containing shRNA for JMJD3 or LSD1 were shown to extend the persistence of T cells (memory T cell function), which was confirmed to improve antitumor immunity and mouse survival rates.

[0352] As described herein, conditional knockout (cKO) of Jmjd3 in CD4+ T cells has been shown to increase the CD44+ CD62L-memory T cell population compared to wild-type (WT) mice (Figure 22). When Jmjd3 cKO T cells were used, in vivo stimulation of Jmjd3 cKO 2d2 transgenic CD4+ T cells with MOG peptide and complete Freund's auxiliary (Figure 23A) resulted in a significant improvement in clinical scores in the EAE mouse model (Figure 23B). This was closely related to a higher number of Jmjd3 cKO T cells after T cell transfer compared to WT 2dT cells (Figure 23C). Similar results were obtained in in vitro T cell stimulation experiments (Figures 23D, 23E, and 23F). These results demonstrate that Jmjd3 knockout significantly enhances T cell viability and persistence.

[0353] To investigate the molecular mechanisms by which T cell survival and persistence are improved, Jmjd3 cKO T cells were shown to have significantly reduced levels of p19, p21, and p53, key proteins that regulate T cell apoptosis, after secondary stimulation with anti-CD3 and CD28 antibodies (Figure 24A). In fact, Jmjd3 cKO T cells showed significantly lower levels of T cell apoptosis compared to WT T cells under the same conditions (Figure 24B). Furthermore, to directly demonstrate the reduced apoptosis in Jmjd3 cKO T cells, levels of cleaved Caspase 3 after anti-CD3 and CD28 stimulation were evaluated, and these levels were significantly lower in Jmjd3 cKO T cells compared to WT T cells (Figure 24C). These results indicate that Jmjd3 knockout significantly improves T cell survival and persistence by reducing Caspase 3 activation.

[0354] Next, we investigated whether JMJD3 knockdown improves CAR-T cell survival and persistence and enhances anti-tumor immunity. Figure 25A shows the experimental design in the Raji tumor model for monitoring the survival of luciferase-labeled T cells. 1928z-shJMJD3 CAR-T cells showed strong proliferation on day 4 after T cell transfer into tumor-carrying NSG mice and maintained a high level of T cell count (Figures 25B, 25C). In contrast, 1928z-control-sh CAR-T cells showed a significant decrease in T cell count on day 6 after T cell transfer (Figures 25B, 25C). Consistent with these results, 1928z-shJMJD3 CAR-T cells significantly suppressed tumor growth and extended survival time compared to mice treated with 1928z-control-sh CAR-T cells (Figure 25D).

[0355] These results demonstrate that knockdown or knockout of negative regulators or epigenetic factors can modulate the function, persistence, and antitumor immunity of CAR-T cells and TCR-T cells in vivo. Example 7: Induction of T cell migration to tumor sites by forced expression of chemokine receptors

[0356] To investigate the function of chemokine receptors in antitumor immunity, we designed CAR constructs expressing chemokine receptors. The results showed that CCR5 significantly enhanced the migration of 1928z CAR-T cells to tumor sites compared to control tumor-specific T cells (Figure 26A). In experiments using MDA-MB-231 / CD19 tumor cells, 1928z-CCR5 CAR-T cells surprisingly significantly suppressed the proliferation of solid tumor cells (Figures 26B, 26C). These results suggest that forced expression of chemokine receptors promotes the migration of T cells to tumor sites.

[0357] Furthermore, to improve the survival of T cells after they migrate to the tumor site, strategies combining T cell migration and T cell persistence can be considered (see Figure 27). Specifically, chemokine receptor and shRNA knockdown can be incorporated into the TCR or CAR construct. Example 8: CAR-T cell signaling regulation and T cell persistence by cleaved CD3 zeta-signaling domains derived from ZAP70 kinase (ZAP255, ZAP280, ZAP300, and ZAP327)

[0358] We hypothesized that CAR-T cell signaling could be enhanced by directly substituting the CD3 zeta chain with other signaling domains involved in TCR signaling. To test this possibility, we created novel CAR constructs in which the CD3 zeta was substituted with signaling domains derived from ZAP70, LAT, and SLP76 (Figure 28A), and screened their ability to generate T cell responses against CD19-expressing tumor cells. The expression levels of these novel CARs were comparable to those of the conventional 1928z CAR (Figure 28B). Similar to 1928z CAR-T cells (positive control), T cells introduced with the 1928ZAP300 construct, which contained only the ZAP70 kinase domain (from amino acid 300 onwards of ZAP70, hereafter ZAP300), showed strong cytokine secretion in co-culture with Raji tumor cells (Figure 28C). In contrast, 1928LAT and 1928SLP76 CAR-T cells did not show T cell activity (Figure 28C). In particular, IFN-gamma and TNF-α secretion from 1928ZAP300 T cells was significantly lower than that of 1928z CAR-T cells (Figure 28C). Importantly, 1928ZAP300 was comparable to 1928z CAR-T cells in tumor cell-specific lysis. 1928LAT and 1928SLP76 CAR-T cells were unable to recognize or lyse tumor cells (Figure 28D). These results suggest that ZAP300 functions as a CD3 zeta-signaling domain substitution in CAR-T cell activation, significantly reducing cytokine secretion while maintaining comparable tumor cell-killing ability.

[0359] Since ZAP300 contains interdomain B (containing residues Tyr315 and Tyr319) and a kinase domain, we investigated whether the upstream domain is necessary for CAR-T cell signaling. ZAP255 (from amino acid 255 onwards of ZAP70), ZAP280 (from amino acid 280 onwards), and ZAP300 were constructed as CD3 zeta-substituted CAR constructs (Figure 29A). CAR expression levels were similar for ZAP255, ZAP280, and ZAP300 (Figures 29B, 29C). Evaluation of CAR-T cell activation using NFAT-GFP Jurkat cells showed no difference in T cell activity or killing ability against NAML6 tumor cells among ZAP255, ZAP280, and ZAP300 CAR-T cells (Figures 29D, 29E). These results indicate that interdomain B upstream of the kinase domain is not essential for T cell signaling and function. Example 9: The ZAP70 kinase domain is essential for CAR-T cell activation and signaling.

[0360] To investigate whether the entire kinase domain is required for T cell signaling and activation, CAR constructs containing ZAP70 cleavage-type signaling domains with varying lengths of interdomain B (ZAP327, ZAP377, ZAP420, ZAP540, and ZAP560) were constructed (Figure 30A). Expression levels were detectable in each CAR, and there were no differences based on the length of interdomain B (Figure 30B). Notably, 1928ZAP327 CAR-T cells containing the full-length ZAP70 kinase recognized target tumor cells at a level comparable to 1928ZAP300 CAR-T cells. On the other hand, CAR-T cells lacking the kinase domain at the N-terminus or C-terminus showed significantly reduced IFN-gamma secretion and tumor-specific lytic ability compared to 1928ZAP300 CAR-T cells (Figure 30C). There were no significant differences in cytokine secretion or tumor-specific lytic ability between 1928ZAP300 and 1928ZAP327 (Figures 30C, 30D). In vivo comparison of anti-tumor immunity in Raji tumor-carrying NSG mice showed that untreated mice died approximately 20 days after tumor injection, while 1928ZAP300 and 1928ZAP327 CAR-T cells extended survival to over 70 days after tumor injection (Figure 30E). In particular, the 1928ZAP327 CAR-T cell group showed slightly better survival than the 1928ZAP300 group, but the difference was not statistically significant. More than 60% of mice treated with 1928ZAP327 CAR-T cells survived up to 75 days after tumor injection, compared to all mice treated with 1928ZAP300 (Figure 30E). These results indicate that ZAP327, including its complete kinase domain, is essential for T cell signaling and activation. Therefore, the ZAP327 domain was selected as the primary T cell signaling domain in subsequent studies. Example 10:19bbZAP327 CAR-T cells exhibit lower cytokine secretion and stronger antitumor immunity.

[0361] The in vitro characteristics of 1928ZAP327 and conventional 1928z CAR-T cells were compared. Both 1928ZAP327 and 1928z were expressed in over 85% of the transduced T cells (Figure 31A). To avoid potential differences between donors, CAR-T cells were generated using fresh T cells from three healthy blood donors. 1928ZAP327 CAR-T cells showed significantly lower IFN-gamma secretion compared to 1928z CAR-T cells (Figure 31B). Similar results were obtained in different CD19-positive tumor cell lines (Figure 32A). Intracellular cytokine staining also showed lower levels of IFN-gamma, IL-2, and TNF-α in 1928ZAP327 CAR-T cells (Figure 32B). The basal expression levels of the T cell early activation marker CD69 were low and similar between 1928ZAP327 and 1928z CAR-T cells, but dramatically increased in both groups after co-culture with tumor cells (Figure 32C). In particular, CD69 expression was significantly lower in 1928ZAP327 CAR-T cells than in 1928z CAR-T cells (Figure 32C). This is thought to be due to a decrease in LAT phosphorylation and NFAT activity in 1928ZAP327 CAR-T cells after stimulation (Figure 32D). The phosphorylation ratios of p38, ERK, JNK, and NF-κB p65 did not change (Figure 32D). Although the activation level was low and cytokine secretion was small, the tumor-killing ability of 1928ZAP327 CAR-T cells was almost the same as that of 1928z CAR-T cells (Figure 32C). Similar results were obtained in the evaluation of cytotoxic granules PRF1 and GZMB (Figure 32E) and in tumor killing assays using luciferase or GFP-labeled tumor cells (Figures 32F, 32G). These results indicate that 1928ZAP327 CAR-T cells maintain antitumor capacity while significantly reducing cytokine secretion compared to conventional 1928z CAR-T cells. Example 11: ZAP327-driven CAR-T cells exhibit superior antitumor immunity compared to conventional CAR-T cells in different tumor models.

[0362] To compare the in vivo antitumor activity of 1928ZAP327 and 1928z CAR-T cells, Raji tumor-carrying NSG mice were used (Figure 33A). As expected, cytokine secretion in the serum of mice administered with 1928ZAP327 CAR-T cells was lower the day after T cell administration than in mice administered with 1928z CAR-T cells (Figure 33B). Untreated tumor-carrying mice died approximately 20 days after tumor injection. In contrast, the group administered with conventional 1928z CAR-T cells (5 × 10^5 cells / mouse) died within 40 days after tumor injection. In contrast, in the group administered with the same number of 1928ZAP327 CAR-T cells, more than 50% of mice survived for more than 75 days after tumor injection (Figure 33C). Similar results were obtained in both the 1928ZAP327 and 1928z CAR groups even when administered with a larger number of CAR-T cells (2 × 10^6 cells / mouse) (Figure 33D). These data indicate that 1928ZAP327 CAR-T cells induce robust antitumor immunity and significantly extend mouse survival compared to conventional 1928z CAR-T cells.

[0363] To support these findings, ZAP327-CAR-T cells containing 4-1BB (19BBZAP327) were generated and compared with 19BBz CAR-T cells. As shown in Figure 34A, the translocation efficiency of 19BBZAP327 and 19BBz CAR-T cells was approximately 60%. Consistent with the results for 1928ZAP327 CAR-T cells, 19BBZAP327 CAR-T cells generated using freshly isolated T cells from two independent healthy donors secreted very small amounts of IFN-gamma, IL-2, and TNF-α after co-culture with CD19-expressing tumor cells, and these secretions were significantly lower than those of 19BBz CAR-T cells. On the other hand, under conditions without tumor stimulation, 19BBZAP327 and 19BBz CAR-T cells alone showed almost no cytokine secretion (Figure 34B). Intracellular staining for cytokines also confirmed significantly lower proportions of IFN-gamma, IL-2, and TNF-α after stimulation (Figure 35A). In contrast to the cytokine secretion pattern, 19BBZAP327 CAR-T cells showed comparable or slightly superior tumor-specific killing ability compared to 19BBz CAR-T cells (Figure 34C). Similar results were obtained when NALM6-Luc tumor cells were used as the target (Figure 35B). Similar patterns were also observed between 19BBz and 19BBZAP327 CAR-T cells in the evaluation of intracellular toxic genes (PRF1 and GZMB) (Figure 35C). Furthermore, the in vivo antitumor activity of 19BBZAP327, 19BBz CAR-T cells, and control T cells was compared using NSG mice carrying Raji tumors (Figure 34D). Consistent with the in vitro cytokine secretion results, serum IFN-gamma concentrations in mice administered with 19BBZAP327 CAR-T cells were significantly lower than those in mice administered with 19BBz CAR-T cells (Figure 34E). Most importantly, 19BBZAP327 CAR-T cells showed significantly superior efficacy compared to 19BBz CAR-T cells. In the 19BBZAP327 CAR-T cell group, more than 60% of mice survived 75 days after tumor injection, whereas in the 19BBz CAR-T cell group, all mice died approximately 55 days after tumor injection (Figure 34F). Similar results were obtained using NSG mice carrying NALM6 tumors (Figures 35D, 35E). Example 12: ZAP327-driven CAR-T cells exhibit superior antitumor immunity in solid tumor models.

[0364] To further investigate the in vivo biological efficacy of ZAP327 CAR-T cells, two solid tumor models were used. Triple-negative mammary cancer cells expressing the CD19 molecule were inoculated into NSG mice, and then CAR-T cells were administered (Figure 36A). Serum IFN-gamma concentration was measured by ELISA, and it was confirmed that 1928ZAP327 CAR-T cells secreted less cytokines compared to 1928z CAR-T cells. Importantly, 1928ZAP327 CAR-T cells significantly reduced tumor size and suppressed tumor growth (Figure 36A). Next, we designed CARs derived from TCR-like antibodies incorporating ZAP327. A2 / ESO scFv CAR-T cells specifically recognize the HLA-A2 / NY-ESO-1_157-165 complex. We challenged NSG mice using the Mel1558 melanoma cell line, which endogenously expresses HLA-A2+NY-ESO-1+ (Figure 36B). Mice treated with A2 / ESO scFv-BBZAP327 CAR-T cells showed a decrease in serum IFN-gamma secretion and a reduction in tumor size, demonstrating superior results compared to the A2 / ESO scFv-BBz CAR-T cell group. Furthermore, conventional αβTCRs were modified to express BBZAP327 at the α-chain C-terminus (Figure 36B). These data collectively demonstrate that ZAP327-CAR-T cells exhibit a superior antitumor response, suggesting that ZAP327 has promising potential to enhance immunotherapy strategies. Example 13: The ZAP327 signaling domain in novel CARs promotes stem-like memory T cell function and persistence in vivo.

[0365] To understand the molecular mechanism by which 1928ZAP327 CAR-T cells exhibit superior antitumor activity compared to 1928z CAR-T cells in vivo, we investigated stem-like memory T cells (Tscm) between 1928ZAP327 and 1928z CAR-T cells (see Figure 37). Freshly generated 1928ZAP327 and 1928z CAR-T cells were analyzed by flow cytometry before and after administration (Figure 38A). 1928ZAP327 CAR-T cells had a significantly higher proportion of Tscm (CD45RO^-CD62L^+) and central memory T cells (Tcm, CD45RO^+CD62L^+) compared to 1928z CAR-T cells (Figure 38B). To investigate the in vivo scalability of 1928ZAP327 and 1928z CAR-T cells, luciferase-labeled CAR-T cells (CAR-ffluc T cells) were generated by introducing a luciferase-expressing retrovirus into CAR-T cells and administered to NSG mice carrying Raji tumors (2 × 10^6 luciferase-labeled CAR-T cells / mouse). The T cell population was monitored on day 0, day 1, day 3, day 7, and day 14. 1928ZAP327 CAR-T cells showed significant proliferation on days 3 and 7. On the other hand, 1928z CAR-T cells peaked on day 3, but their proliferation level was significantly lower (Figures 38C, 38D). On day 14, the signal intensity of both groups was thought to have decreased significantly due to the removal of tumor cells from the bloodstream (Figure 38D). To evaluate the differences in persistence between 1928ZAP327 and 1928z CAR-T cells, T cells were isolated from the spleen, bone marrow, and lung tissue of mice 36 days after T cell transfer. Mice administered with 1928ZAP327 CAR-T cells had significantly higher human CD3^+ T cell counts compared to mice administered with 1928z CAR-T cells (Figure 38E). Furthermore, Tscm and Tcm cell populations were also significantly increased in mice administered with 1928ZAP327 CAR-T cells (Figure 38F). In contrast, the expression of T cell exhaustion markers (PD-1, LAG3, TIM3) was lower in 1928ZAP327 CAR-T cells than in 1928z CAR-T cells (Figures 39A, 39B). Intracellular markers (TOX and NR4A1) were also lower in 1928ZAP327 CAR-T cells than in 1928z CAR-T cells (Figure 39C). These results strongly suggest that 1928ZAP327 CAR-T cells have the ability to induce a high proportion of memory T cells and reduce fatigue. Since the ZAP327 signaling domain promotes stem-like memory T cells, this technology is abbreviated as STEM (Synthetic TCR signaling Enhanced Memory T cell). Example 14: The ZAP327 signaling domain of a novel CAR establishes a unique transcriptional profile and metabolic pathway and possesses STEM memory properties.

[0366] To investigate the potential mechanisms behind the superior antitumor effects of ZAP327 CAR-T cells, transcriptional changes in 1928z and 1928ZAP CAR-T cells under unstimulated and stimulated conditions were analyzed using RNA-seq (Figure 40A). MYC, CISH, and SOCS2, which play negative roles in memory formation and antitumor responses, were downregulated, while BCL6, IL7R, and KLF2, which are important for T cell persistence, were upregulated (Figure 40A). Biological gene ontology (GO) and GSEA analyses revealed that most of these genes are rich in metabolic pathways associated with the memory phenotype (Figure 40B). Specifically, genes involved in glycolysis were significantly upregulated in 1928z CAR-T cells, while genes related to oxidative phosphorylation were upregulated in 1928ZAP327 CAR-T cells (Figure 40C). To verify this, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse assay (Figures 41A, 41B). 1928z CAR-T cells prioritized glycolysis, while 1928ZAP327 CAR-T cells prioritized oxidative phosphorylation to generate energy (Figure 41A). Although 1928ZAP327 CAR-T cells had limited glycolytic capacity, their maximum respiratory capacity and excess respiratory capacity were higher than those of 1928z CAR-T cells (Figures 41A, 41B). Experiments using 2-NBDG (glucose analog) also showed that 1928ZAP327 CAR-T cells had low glucose uptake (Figure 41C). Since memory cells tend to utilize endogenous fatty acids, BODIPY-C16 uptake was also low in 1928ZAP327 CAR-T cells (Figure 41C). Furthermore, staining with Mitotracker green showed that 1928ZAP327 CAR-T cells had a higher mitochondrial content (Figure 41C). These results indicate that 1928ZAP327 CAR-T cells primarily utilize oxidative phosphorylation to generate more memory T cells, resulting in a stronger antitumor response. Example 15: Generation and characterization of MHC-II restricted CT83-specific T cells

[0367] CT83 (also known as KK-LC-1) is highly expressed in lung and breast cancer (Figures 42A, 42B, and 15C), and is also expressed in other cancer types such as gastric, cervical, and pancreatic cancer. HLA-DR is a common HLA class II molecule for antigen presentation. HLA-DR13 accounts for 20-30% of the general population, with its major alleles being DRb11301 and DRb11302, which account for 85% of HLA-DR13 expression. Using a computer-based epitope prediction program, T cell epitopes that may be presented to HLA-DR13 were identified, and long-chain CT83 peptides (PEP10-31) were synthesized and pulsed into dendritic cells (DCs) or irradiated PBMCs derived from HLA-DR13-positive healthy donors. When CD4+ T cells from the same donor were stimulated for two cycles using peptide-pulsed DCs or irradiated PBMCs, the in vitro stimulated T cells strongly recognized 293DR13 cells (HLA-DR13-expressing HEK293 cells) carrying the CT83 peptide (PEP10-31), but did not recognize the control peptide (Figures 43A, 43B). Furthermore, antibody inhibition experiments were conducted to verify whether the HLA-DR13 molecule is necessary for peptide presentation. As a result, T cell recognition of 293IMDR13 / CT83(PEP10-31) was completely inhibited by anti-DR and anti-HLA class II antibodies, but not by anti-HLA class I, HLA-DP, and HLA-DQ antibodies (Figure 43C). In addition, T cells recognized CT83(PEP10-31) presentation by 293IMDR13 cells, but 293IMDR1, DR3, DR4, DR7, and DR11 cells did not, suggesting that these T cells recognize the HLA-DR13-restricted CT83 peptide (PEP10-31) (Figure 43D). Example 16: Identification of the DR13 CT83 T cell epitope

[0368] To further identify T cell epitopes, we examined transfected 293IMDR13 cells using abbreviated Ii-fusion CT83 coding fragments. The results showed that the T cells recognized 293IMDR13 / Ii-CT83(aa10-31) and 293IMDR13 / Ii-CT83(aa17-31), but not 293IMDR13 / Ii-CT83(aa10-24) or 293IMDR13 / Ii-CT83(aa13-27) (Figure 44A). Importantly, these T cells recognized breast cancer cell lines MDA-MB-231 and M1495, which spontaneously express CT83 and HLA-DR13 (Figure 44B). This indicates that these T cells recognize the CT83 PEP17-31 / DR13 complex endogenously treated by tumor cells. The amino acid sequence of the CT83 peptide is described in SEQ ID NOs: 61-63, and the DNA coding sequence is described in SEQ ID NOs: 83-87. Example 17: Identification and characterization of DR13-CT83 TCR using single cell barcoding technology

[0369] Using single-cell barcoding technology and CT83-specific T cells, multiple full-length TCRs were identified and cloned into the retroviral expression vector pMSGV1 (Figure 45A). Sequence analysis revealed the use of different TCR Vα and TCR Vβ. The amino acid sequences of these HLA-DR13-restricted CT83-specific TCRs are described in SEQ ID NOs: 53-56 and 67-82.

[0370] DR13-CT83 TCR gene-modified T cells using one of the listed TCR clones (SEQ ID NO: 53-XX) recognized 293DR13 cells transfected with Ii-CT83 and 293DR13 cells pulsed with DR13 CT83 peptide, but did not recognize control DP4 CT83 PEP10-27 pulsed 293R13 cells (Figure 45B). Importantly, DR13-CT83 TCR gene-modified CD4+ T cells recognized 293DR13 cells transfected with Ii-CT83 and MDA-MB-231 tumor cells, but did not recognize DR13-CT83 TCR gene-modified CD8+ T cells (Figure 45C). Because CD4+ T cells are required, the DR13-CT83 TCR is referred to as CD4 CT83 TCR, or simply CD4 TCR. Example 18: CD4 TCR-T cells exhibit superior antitumor immunity and suppress breast cancer cell proliferation in vivo.

[0371] To investigate whether DR13 CT83 TCR-modified CD4+ T cells could suppress tumor growth, MDA-MB-231 cells were injected into NSG mice. Five days later, tumor-carrying mice were administered either control T cells or DR13 CT83 TCR-CD4+ T cells. DR13 CT83 TCR-CD4+ T cells completely suppressed tumor growth, while control T cells did not (Figure 46A). Splenocytes were collected at the end of the experiment and analyzed for human CD4+ and CD8+ T cells. Only a small number of CD4+ and CD8+ T cells were detected in control T cell-treated mice (Figure 46B, top). In contrast, a large number of CD4+ T cells were detected in TCR-CD4+ T cell-treated mice 35 days after administration (Figure 46B, bottom). Further analysis confirmed that these CD4+ TCR-T cells were stem-like memory T cells (Tscm) with CD45RA+CD62L+ and central-type memory T cells (Tcm) with CD45RA-CD45RO+CD62L+ (Figure 46B). These results suggest that antigen-specific CD4+ T cells can induce tumor regression through their cytotoxic activity and long-term persistence associated with memory T cell (Tscm and Tcm) populations. Example 19: Antigen-specific TCRs can be modified in combination with STEM technology (BBZAP327) and knockdown / knockout of negative regulators, and the function and persistence of TCR-T cells can be improved along with STEM cell characteristics.

[0372] Antigen-specific TCRs, i.e., TCR(H) containing the original human TCR constant region and TCR(M) containing the mouse TCR constant region, can be fused with the BBZAP327 signaling domain (i.e., STEM technology) and combined with the knockdown / knockout of negative regulators using shRNA or CRISPR / CAS9 technology (Figure 47A). In some embodiments, TCR(M) is further designed to express 4-1BB, CD28, CD27, OX40, ICOS, MyD88, or MALT-1 downstream of the TCR-TM region. In some embodiments, TCR(M) is designed to express T2A-MyD88 or T2A-MALT-1 downstream of the TCR-TM region. Figure 20B shows a schematic diagram of the TCR(M) complex including TCR(H), TCR(M), TCR-STEM, and additional signaling molecules (Figure 47A). To investigate whether STEM technology (BBZAP327) can enhance TCR-T cell activity, A2 / CT83 TCR and A2 / CT83 TCR-BBZAP327 (or A2 CT83 TCR-STEM) T cells were generated (Figure 48A). A2 / CT83 TCR and A2 / CT83 TCR-BBZAP327 T cells specifically lysed target cells, but no significant difference was observed (Figure 48B). However, A2 / CT83 TCR-BBZAP327 T cells secreted significantly less IFN-gamma compared to A2 / CT83 TCR-T cells, which is an important characteristic of BBZAP327 signaling (STEM technology) (Figure 48C).

[0373] To investigate whether the BBZAP327 (BBZ327) signaling domain can enhance the antitumor immunity of HLA-A2-restrictive CT83 TCR-T cells (A2 CT83 TCRs), the BBZ327 domain was fused to the C-terminus of the α and β chains of A2-CT83 TCRs, respectively, and their in vivo tumor suppressor activity was evaluated. Tumor cells were injected into NSG mice on day 0, and various T cells were administered on day 5. As a result, A2-CT83 TCR-α-BBZ327 T cells showed superior antitumor activity compared to conventional A2-CT83 TCR-T cells based on tumor growth and tumor weight (Figures 49A, 49B). Notably, when BBZ327 was fused to the β-chain, no difference in T cell activity was observed, suggesting that TCR-α-BBZ327 CD4+ T cells exhibit superior function compared to TCR-β-BBZ327. Furthermore, the total number of T cells and CT83-specific T cells in tumor tissue of mice administered TCR-α-BBZ327 CD4+ T cells were significantly higher compared to other groups (Figure 49C). These results indicate that fusion of the BBZAP327 signaling domain to the α-chain of the TCR can further enhance T cell function and antitumor activity. Example 21: The BBZAP327 signaling domain enhances antitumor immunity by CD4 TCR-STEM T cells.

[0374] First, when the constant region of the human CD4 CT83 TCR was replaced with the constant region of the mouse TCR, it was shown that the CD4 CT83 TCR with the constant region of the mouse (TCR-MC) had significantly improved recognition ability for 293T cells expressing the CT83 gene compared to the conventional CD4 CT83 TCR (TCR-HC) (Figure 50A). Next, to investigate whether the BBZAP327 signaling domain can enhance the antitumor immunity of CD4 TCR-T cells, the BBZAP327 domain was fused to the C-terminus of the CD4 TCR-α chain to construct CD4 TCR-STEM (Figure 50B). To avoid variability among donor-derived T cells, CD4 TCR-STEM T cells were generated using T cells from three different healthy donors. As a result, CD4+ TCR-STEM T cells recognized and lysed MDA-MB-231 cells, but did not react to HEK293DR13 cells (Figures 50C, 50D).

[0375] Figure 51A shows the experimental design to verify whether the BBZAP327 signaling domain can enhance in vivo antitumor immunity by CD4 TCR-STEM T cells. The experimental results showed that CD4 TCR-STEM T cells exhibited superior antitumor efficacy compared to conventional CD4 TCR(HC) and TCR(MC) T cells. Conventional CD4 TCR(HC) and TCR(MC) T cells already demonstrated potent antitumor immunity, significantly suppressing tumor growth (Figure 51B). Furthermore, additional experiments using CD4 TCR-STEM T cells derived from three different healthy donors completely suppressed breast cancer cell growth (Figure 51C). Similar results were obtained in lung cancer models using A2-CT83 TCR-STEM and CD4 CT83 TCR-STEM T cells (Figure 51D). These results indicate that TCR-STEM T cells possessing the BBZAP327 signaling domain further enhance the in vivo antitumor immunity of TCR-STEM T cells.

[0376] Despite A2-CT83 TCR-STEM T cells exhibiting potent antitumor immunity, the TCR may cross-react with other potential targets, potentially leading to undesirable toxicity. To address this, database searches were conducted against various gene banks and databases (genes and proteins). As a result, no human proteins identical or highly homologous to the HLA-A2-restrictive CT83 peptide (PEP90-98) were identified (Figure 52A). This suggests that the CT83 epitope is specific in various databases. Consistent with this finding, no activity of CT83 TCR-STEM T cells against HLA-A*02-positive 293T cells was detected.

[0377] Furthermore, to rule out the possibility of potential cross-reactivity with unrelated peptides, T cell function assays were performed on a panel of HLA-A2-positive, CT83-negative human cancer cell lines derived from different human tissues (Figure 52B). As a result, CT83 TCR-STEM T cells showed strong activity against positive controls (CT83 PEP90-98 pulsed 293T cells, MDA-MB-231 cells, NCI-H1563 cells), but no activity was detected against negative control 293T cells or any HLA-A2-positive, CT83-negative human cancer cell lines (Figure 52C). This confirmed that CT83 TCR-STEM T cells do not exhibit cross-reactivity with other human proteins. CT83 TCR-STEM T cells recognize CT83 presented on HLA-A2, but not on other HLA alleles (Figure 52D). Example 23: Modification of HLA-DP4-restrictive NY-ESO-1 TCR for improved tumor recognition and lytic activity.

[0378] To demonstrate a method for identifying TCRs with improved affinity, we used the HLA-DP4-restricted NY-ESO-1 TCR (SLLMWITQCFLPVF, SEQ ID NO: 1) as an example. Recent experiments identified essential residues in the CDR2 and CDR3 regions of the TCR Vα and Vβ chains using alanine scanning. As a result, it was found that G52, D95, Y96, Q98, F199, and V101 of the TCR-α chain, and I52, W93, R96, Y97, E99, and Q100 of the TCR-β chain are essential (Figure 53).

[0379] Next, each essential amino acid was substituted with 18 other amino acids (Figure 54A). Jurkat cells expressing NFAT-GFP were used as reporters, and initial functional screening was performed based on NFAT-GFP expression in response to tumor target stimulation. As a result, four substitutions that improved T cell function were identified (Figure 54B). In particular, a single Q98Y substitution in the TCR-α chain increased T cell function based on NFAT-GFP expression by 3 to 4 times (Figure 54B).

[0380] Next, when human CD4+ T cells were introduced with WT DP4 TCR, TCRVα D95S, Q98Y, and TCRVβ Y97L, Y97M, these single amino acid mutations significantly improved T cell function in cytokine secretion and tumor-specific lysis (Figure 55A). In particular, TCRVα Q98Y-introduced T cells showed remarkable functional improvement in IFN-gamma secretion and tumor-specific lysis (Figure 55B). Example 24: Identification of amino acid residues essential for CT83 antigen peptide recognition by alanine scanning and abbreviated analysis.

[0381] To identify amino acid residues essential for the recognition of the CT83 antigen peptide, amino acid substitution or alanine scanning methods were used. As a result, several essential residues in the HLA-A2 CT83 peptide (90-98, SEQ ID NO: 2) were found to be important for recognition by A2-CT83 TCR-T cells (Figure 56A). Similarly, several essential residues in the HLA-DR13 CT83 peptide (17-31, SEQ ID NO: 61) were confirmed to be important for recognition by CD4 TCR-STEM T cells (Figure 56B). Furthermore, abbreviated analysis showed that essential residues at the N-terminus and C-terminus of the HLA-DR13 CT83 peptide (17-31, SEQ ID NO: 61) are also required for antigen recognition by CD4 TCR-STEM T cells (Figure 56C). These results suggest that CT83 peptides (90-98, SEQ ID NO: 2), CT83 peptides (17-31, SEQ ID NO: 61), and their variants or similar peptides can induce a T cell response and generate potent antitumor immunity.

[0382] Sequence List Sequence ID 1 HLA-DP4 exclusive NY-ESO-1 epitope (157-170) SLLMWITQCFLPVF

[0383] Sequence ID 2 HLA-A2 specific CT83 epitope (90-98) KLVELEHTL

[0384] Sequence ID 3 HLA-DP4-specific NY-ESO-1 TCR alpha chain variable region (TRAV34-TRAJ26) METVLQVLLGILGFQAAWVSSQELEQSPQSLIVQEGKNLTINCTSSKTLYGLYWYKQKYGEGLIFLMMLQKGGEEKSHEKITAKLDEKKQQSSLHITASQPSHAGIYLCGADIVDYGQNFVFGPGTRLSVLPY

[0385] Sequence ID 4 HLA-DP4-specific NY-ESO-1 TCR beta-chain variable region (TRBV30-TRBJ2-7) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWRRRGYEQYFGPGTRLTVTE

[0386] Sequence ID 5 HLA-A2-specific CT83 TCR alpha chain variable region (TRAV5-TRAJ28) MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIPN

[0387] Sequence ID 6 HLA-A2-specific CT83 TCR beta-chain variable region (TRBV29-1-TRBJ1-1) MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVE

[0388] Sequence ID 7 PD1 shRNA 21-nucleotide core CCGTGTCACACAACTGCCCAA

[0389] Sequence ID 8 VHL shRNA 21-nucleotide core CAGGAGCGCATTGCACATCAA

[0390] Sequence ID 9 PPP2R2D shRNA 21-nucleotide core AAGGTCATTACTCAGAATAAA

[0391] Sequence ID 10 Human TCR alpha chain constant region (TRAC) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0392] Sequence ID 11 Human TCR beta chain constant region 1 (TRBC1) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0393] Sequence ID 12 Human TCR beta chain constant region 2 (TRBC2) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0394] Sequence ID 13 Mouse TCR alpha chain constant region (trac) IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

[0395] Sequence ID 14 Mouse TCR beta chain constant region 1 (trbc1) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0396] Sequence ID 15 Mouse TCR beta chain constant region 2 (trbc2) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0397] Sequence ID 16 polypeptide containing ZAP300 TSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVH RDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0398] Sequence ID 17 polypeptide containing ZAP327 DKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNR HYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0399] Sequence ID 18 PPP2R2D Transcriptional Variant 1

[0400] Sequence ID 19 PPP2R2D Transcriptional Variant 3

[0401] Sequence ID 20 HLA-A2-specific CT83 TCR fusion of alpha chain variable region and mouse alpha chain constant region MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKSGYSGAGSYQLTFGKGTKLSVIP NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

[0402] Sequence ID 21 A fusion of the HLA-A2-specific CT83 TCR beta-chain variable region and mouse beta-chain constant region 2. MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQDSEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIAN KQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0403] Sequence ID 22 A fusion of the HLA-A2-limited NY-ESO-1 TCR (S2) alpha-chain variable region and the mouse alpha-chain constant region. METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYI QNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

[0404] Sequence ID 23 HLA-A2-limited NY-ESO-1 TCR (S2) (G50A, A51E) fusion of beta-chain variable region and mouse beta-chain constant region 2 MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAEGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0405] Sequence ID 24 HLA-A2-limited NY-ESO-1 TCR (S5) fusion of alpha-chain variable region and mouse alpha-chain constant region METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYI QNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

[0406] Sequence ID 25 HLA-A2-limited NY-ESO-1 TCR (S5) (G50A, A51E, A97L) fusion of beta-chain variable region and mouse beta-chain constant region 2 MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAEGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGLAGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0407] Sequence ID 26 HLA-A2 specificity pp65 epitope (495-503) NLVPMVATV

[0408] Sequence ID 27 HLA-A2 limited pp65 TCR (#1-15) alpha chain variable region (TRAV21-TRAJ18) METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQGSTLGRLYFGRGTQLTVWPD

[0409] Sequence ID 28 HLA-A2 limited pp65 TCR (#1-15) beta-chain variable region (TRBV13-TRBJ1-5) MLSPDLPDSAWNTRLLCRVMLCLLGAGSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSGYHSELNMSSLELGDSALYFCASSLENNQPQHFGDGTRLSILE

[0410] Sequence ID 29 HLA-A2 limited pp65 TCR (#132-3) alpha chain variable region (TRAV24-TRAJ49) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCFSPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPN

[0411] Sequence ID 30 HLA-A2 limited pp65 TCR (#132-3) beta-chain variable region (TRBV6-5-TRBJ1-2) MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPITGTGDYGYTFGSGTRLTVVE

[0412] Sequence ID 31 HLA-A2 exclusive IE-1 epitope (316-324) VLEETSVML

[0413] Sequence ID 32 HLA-A2-specific IE-1 TCR alpha chain variable region (TRAV25-TRAJ42) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGHIYGGSQGNLIFGKGTKLSVKPN

[0414] Sequence ID 33 HLA-A2-specific IE-1 TCR beta-chain variable region (TRBV5-1-TRBJ2-5) MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHHQGPLETQYFGPGTRLLVLE

[0415] Sequence ID 34 PEP161-180 corresponds to residues 161-180 of NY-ESO-1. WITQCFLPVFLAQPPSGQRR

[0416] Sequence ID 35 PEP156-175 corresponds to residues 156-175 of NY-ESO-1. LSLLMWITQCFLPVFLAQPP

[0417] Sequence ID 36 PEP6-14 corresponds to residues 6-14 of CT83. LLASSILCA

[0418] Sequence ID 37 PEP4-12 corresponds to residues 4-12 of CT83. YLLLASSIL

[0419] Sequence ID 38 PEP79-87 corresponds to residues 79-87 of CT83. RILVNLSMV

[0420] Sequence ID 39 PEP10-31 corresponds to residues 10-31 of CT83. SILCALIVFWKYRRFQRNTGEM

[0421] Sequence ID 40 PEP66-76 corresponds to the 66th-76th residue of CT83. ILNNFPHSIAR

[0422] Sequence ID 40 DNA encoding the HLA-DP4-specific NY-ESO-1 TCR alpha chain variable region. ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCAGCCTGGGTCAGTAGCCAAGAACTGGAGCAGAGTCCTCAGTCCTTGATCGTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACGTTATATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATCTTCTTGA TGATGCTACAGAAAGGTGGGGAAGAGAAAAGTCATGAAAAGATAACTGCCAAGTTGGATGAGAAAAAAGCAGCAAAGTTCCCTGCATATCACAGCCTCCCAGCCCAGCCATGCAGGCATCTACCTCTGTGGAGCAGACATAGTAGACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGGTTGTCCGTGCTGCCCTAT

[0423] Sequence ID 41 DNA encoding the HLA-DP4-specific NY-ESO-1 TCR beta-chain variable region. ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGGCGCCGGGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG

[0424] Sequence number 42 DNA encoding the HLA-DP4-restricted NY-ESO-1 TCR beta-chain variable region ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGGCGCCGGGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG

[0425] Sequence number 43 DNA encoding the HLA-A2-restricted CT83 TCR alpha-chain variable region ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGGCGCCGGGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG

[0426] Sequence number 44 DNA encoding the HLA-A2 restricted CT83 TCR beta chain variable region ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGTTCAAGACAGTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAG

[0427] Sequence number 45 DNA encoding the HLA-A2 restricted pp65 TCR (#1-15) alpha chain variable region ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGGCCTCAGGGCTCAACCCTGGGGAGGCTATACTTTGGAAGAGGAACTCAGTTGACTGTCTGGCCTGAT

[0428] Sequence number 46 DNA encoding the beta-chain variable region of HLA-A2-restricted pp65 TCR (#1-15) ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTCTGCCGTGTCATGCTTTGTCTCCTGGGAGCAGGTTCAGTGGCTGCTGGAGTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCCACTCTGAAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTACCAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTCATTTCGTTTTATGAAAAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAACAGTTCAGTGGCTATCATTCTGAACTGAACATGAGCTCCTTGGAGCTGGGGGACTCAGCCCTGTACTTCTGTGCCAGCAGCTTAGAGAACAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG

[0429] Sequence number 47 DNA encoding the variable region of the HLA-A2-restricted pp65 TCR (#132-3) alpha chain ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTTGTACA...

Claims

1. A composition comprising a chimeric antigen receptor (CAR), a chimeric TCR receptor, or T cells expressing a CAR or chimeric TCR, The CAR or TCR comprises an antigen recognition site, a transmembrane main, and an intracellular T cell activation site. Here: a. The antigen recognized by the antigen recognition site may be, arbitrarily, alpha (α)-fetoprotein (AFP), AIM2, ART-4, BCMA, BAGE, CAMEL, CAP-1, caspase 8 (CASP8), CDC27, cyclin-dependent kinase 4 (CDK4), CDK12, carcinoembryonic antigen (CEA), CLCA2, CFTR, CMV, carcinoembryonic antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII, EGFR and its isovariants, EGFR E746-A750del, EGFRVIII, ESA, EpCAM, EphA2,3, EGP2, EGP-40, EMA, ETA, Fibronectin (FN), FGF-5, FGF-6, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnT-V, Glycoprotein 100 (GP100), HAGE, H3.3K27M, h5T4, IP3KB, Influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, HER2 / neu, HBV, HERV-E, HIV-1 gag, HMI.24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, Human telomerase reverse transcriptase (hTERT), KRAS, KRAS G12D, KRAS G12V, LAGE1b, LMP2, LILRB2, LGR5, Ly49, Ly108, LI-CAM, MAGE, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE -A10, MAGE-A12, MAGE-C2, c-Met, MICA / B, MSA, MART-1, mesothelin (MSLN), MUC1, MUC2, Muc-16, myo-D1, tumor M2-PK, Necl-2 Neurofilament, NKCSI, NKG2D, NSE, NY-ESO, NY-ESO-1, PRAME, PSA, PSMA, RAGE, Ral-B, abnormal ras protein, ROR1, SLAMF7 / CS1, Sp17, SAGE, SART-1,-2,-3, SOX10, synovial sarcoma X rupture point 2 (SSX-2), Survivin, OVA1, HE4, DR-70, total PSA, AMACR, CA125 / MUC16, ER alpha / NR3A1, ERbeta / NR3A2, thymidine kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, cytokeratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, nestin, 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, PD1, PD-L1, CTLA-4, PDGFRalpha, VEGF, TRAG-3, TARP, TGFbII, thyroglobulin, abnormal p53 protein, TP53 (p53), TRAIL, TRP1, TRP2, TYRP1, tyrosinase, TAG-72, TALLA-1, TLR4, TRBC1, TRBC2, Trp-p8, thyroid transcription factor-1, Vα24, WT1, CD1a, CD1b, CD1c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16, CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, C D36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61, CD62L, CD63, CD64, CD66b, CD68, CD69, CD70, CD73, CD74, CD79a, CD79b, CD80, CD83, CD85k (ILT3), CD86, CD88, CD93, CD94, CD95, CD99, CD103, CD105, CD107a, CD107b, CD114, CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138, CD141, CD146, CD152, CD158 (Kir), CD161, CD163, CD183, CD191, CD193(CCR3), CD194 (CCR4), CD195 (CCR5), CD197 (CCR7), CD203c, CD205, CD207, CD209, CD223, CD235, CD235a, CD244, CD252 (OX40L), CD267, CD268 (BAFF-R), CD273, CD276 (B7-H3), CD279 (PD1), CD282 (TLR2), CD284 (TLR4), CD294, CD304, 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, OVA1, HE4, Fibrin / fibrinogen degradation product (DR-70), AFP-L3, circulating tumor cells, PSCA, α2β1, PAP, PAMA, P-cadherin, placental alkaline phosphatase, C3AR, CAIX, chromogranin, CLEC12A, antigen of cytomegalovirus (CMV) infected cells, CS-1, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), estrogen receptor (ER), progesterone receptor (PR), CA-125, CA15-3, CA27.29, free PSA, NuMA / NMP22, A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, AKR1C3, alpha 1B-Glycoprotein, alpha 1-Microglobulin, CRYAB, AMACR, AMFR / gp78, Annexin A3, ANXA8, APC, ApoA1, ApoA2, ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, attractin, Aurora A, BAP1, Bcl-2, Bcl-6, beta 2-Microglobulin, B3GAT1, beta-catenin, beta-IIITubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, Cadherin-13, CALD1, Carponin-1, Calretinin, CA9, Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, Chitinase-like 1, CCKBR, alpha HCG, HCG, CKAP4 / p63, Claudin-18, Clathrin, c-Maf, c-Myc, CotL1, COMMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, Cyclin A1, Cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cyto Keratin 19, Fetal Acetylcholine Receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, CD105, Endosialin / CD248, Neuron-Specific Enolase, EpCAM / TROP1, Eps15, ER alpha / NR3A1, ER beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fastin, FATP3, FCRLA, Fetuin A / AHSG, FGF acidic, FGF basic, FGF R3, FGF R4, Fibrinogen, FBP, FAP, FSTL1, FOLR1, FOLR2, FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / Dysadherin, FcεRIα, FITC, FLT3, GABA-AR alpha 1, GADD153, GADD45alpha, galectin-3, MAC-2BP, galactin, ganglioside, GCDFP-15, GD2, GD3, GM2, GM3, gamma-Glutamylcyclotransferase / CRF21, Gas1, GRPR, gastrokain 1, GSN, GFAP, GLI-2, GPX3, gpA33, glycopeptide, GPC2, glypican 3, GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, HPSE, hepsin, HGF R / c-MET, HIF-2 alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-IR, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, IL-13Ra, IL-13Ra2, Integrin, Integrin B7, IMPDH1, KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQGAP1, IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, OGR1, Olig2, OPN, Ovastatin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA-15, PGA5, PI16, Peroxiredoxin 2, PGCP, PI 3-Kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone RB / NR3C3, Progesterone R / NR3C3, PGRN, Prolactin, PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADase I, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP beta / zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, Secretin R, Serpin A9 / Centerin, SerpinE1 / PAI-1, serum amyloid A1, serum amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1 , SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARCL1, S PINK1, Src, STEAP1, STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin-1, Syndecan-1 / CD138, Syntaxin 4, Synuclein-Gamma, Synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, LDHA, La Min B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matryptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, MC1R, CD228, Melatonin, Mer, Mesotheline, Metadoherin, Metastin / KiSS1, Methionine Aminopeptidase, METAP2, MFAP3L, MGMT, MIA, MIF, MINA, MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesfatin-1 / Nucleobindin-2, Nestin, NFkB2, NF-L, NG2 / MCSP, NGFR / TNFRSF16, NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankirase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosine Beta 10, Thymosine Beta 4, Thyroglobulin, TIMP, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2, TMEM219, TMEM87A, TNF-alpha, TOP2A, TopBP1, tPA, TRA-1-60(R), TRA-1-85 / CD147, TRAF-4, TAGLN, Trypsin 2 / PRSS2, Tryptase alpha / TPS1, TSPAN1, UBE2S, uPAR, urokinase, Urotensin-II ScFv is a surface protein that can recognize a selection of surface proteins from the group consisting of R, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, and CNKSR1 / CNK / KSR, as well as any combination thereof. b. The composition wherein the intracellular T cell activation site further comprises a signal transduction domain containing a ZAP70 kinase domain or a variant thereof.

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

3. The composition according to claim 1, wherein the antigen recognition site comprises one or more polypeptides including an alpha variable region, or one or more polypeptides including a beta variable region, or any combination thereof, of a T cell receptor specific to NY-ESO-1 peptide (A2-ESO-1 TCR and DP4-ESO-1 TCR), CT83 peptide (A2-CT83 TCR, DR13-CT83 TCR, PEP4-12 TCR, PEP6-14 TCR, PEP10-31 TCR, PEP17-31 TCR, PEP90-98 TCR), HCMV pp65 (HCMV pp65 TCR), or HCMV IE-1 peptide (HCMV IE-1 TCR).

4. The composition according to claim 3, wherein the C-terminus of the TCR alpha or beta chain is fused to a signaling component comprising a ZAP255, ZAP280, ZAP300, ZAP327, or ZAP338 kinase domain, or a variant or variant thereof.

5. The composition according to claim 3, wherein at least one polypeptide comprising the alpha chain variable region of a CT83-specific T cell receptor, and at least one polypeptide comprising the beta chain variable region of a CT83-specific T cell receptor, is specific to a polypeptide comprising the amino acid sequence SILCALIVFWKYRRFQRNTGEM (CT83 aa 10-31, SEQ ID NO: 39) or a polypeptide comprising the amino acid sequence VFWKYRRFQRNTGEM (CT83 aa 17-31, SEQ ID NO: 61).

6. The composition comprises an alpha variable region of an HLA-A2-specific HCMV pp65 TCR comprising an alpha variable region comprising the amino acid sequence of SEQ ID NO: 27 and a beta variable region comprising the amino acid sequence of SEQ ID NO: 29, wherein the alpha variable region or a variant thereof is a polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, a polypeptide having one, two or more amino acid substitutions to 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 in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity to the sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and optionally further comprising a beta variable region of an HLA-A2-specific HCMV pp65 TCR comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30. The composition according to claim 3, wherein the beta variable region or a variant thereof is a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30, which binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide comprising a sequence having one, two or more amino acid substitutions to SEQ ID NO: 28 or SEQ ID NO: 30; or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 28 or SEQ ID NO: 30, 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 comprises alpha and beta variable regions of a TCR specific to NY-ESO-1 or CT83, and if the TCR is specific to NY-ESO-1, the alpha variable region may optionally be: a DP4-ESO-1 TCR alpha variable region containing the amino acid sequence of SEQ ID NO: 3; an alpha variable region or variant thereof that binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide containing an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 3; or within the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 3 (e.g., TCR-Vα Polypeptides having one, two, or more amino acid substitutions in the CDR3 sequence, such as D95S or Q98Y in GADIVDYGQNFV (SEQ ID NO: 89). (Amino acid position numbers are named according to the mature TCR sequence), or polypeptides having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity to the sequence of SEQ ID NO: 3, and the beta variable region of the HLA-DP4 NY-ESO-1 TCR optionally contains the amino acid sequence of SEQ ID NO: 4, a beta variable region or variant thereof that binds to the antigen with the same specificity as the reference (full-length and unmodified) receptor, a polypeptide containing an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 4, or within the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 4 (e.g., TCR-Vβ A2-CT83 is a polypeptide having one, two, or more amino acid substitutions in the CDR3 sequence AWRRRGYEQY (SEQ ID NO: 90), such as Y98L or Y98M, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 4, and the TCR is specific to CT83, in which case the alpha variable region is optionally a polypeptide containing the amino acid sequence of SEQ ID NO: 5.A2-CT83 comprises a TCR, an alpha variable region or a variant thereof that binds to the antigen with the same specificity as 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: 5, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 5, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 5, and the beta variable region of the CT83 TCR optionally contains a polypeptide containing the amino acid sequence of SEQ ID NO:

6. The composition according to claim 3, comprising: a TCR; a beta variable region or a variant thereof that 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: 6; a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 6; or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO:

6.

8. The composition comprises an alpha variable region of an HLA-A2-limited HCMV IE-1 TCR, wherein the alpha variable region of the HLA-A2-limited HCMV IE-1 TCR is 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 sequence of SEQ ID NO: 32, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of SEQ ID NO: 32, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 32, and optionally, the composition further comprises a beta variable region of an HLA-A2-limited HCMV IE-1 TCR. The composition according to claim 3, wherein the beta variable region of the TCR 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 in the CDR1, CDR2, and / or CDR3 regions of SEQ ID NO: 33, or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO:

33.

9. The composition comprises an alpha variable region of HLA-DR13-limited DR13-CT83 TCR, wherein the alpha variable region of HLA-DR13-limited DR13-CT83 TCR comprises an amino acid sequence selected from SEQ ID NOs. 54, SEQ ID NOs. 67, SEQ ID NOs. 69, SEQ ID NOs. 71, SEQ ID NOs. 73, SEQ ID NOs. 75, SEQ ID NOs. 77, SEQ ID NOs. 79, and SEQ ID NOs.

81. The composition comprises an amino acid sequence having at least 85% identity to the amino acid sequence selected from SEQ ID NOs. 54, SEQ ID NOs. 67, SEQ ID NOs. 69, SEQ ID NOs. 71, SEQ ID NOs. 73, SEQ ID NOs. 75, SEQ ID NOs. 77, SEQ ID NOs. 79, and sequence number A polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence selected from No. 81, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with 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, and optionally the composition further comprises the beta variable region of the HLA-DR13-limiting DR13-CT83 TCR.A polypeptide comprising an amino acid sequence selected from SEQ ID NOs. 56, SEQ ID NOs. 68, SEQ ID NOs. 70, SEQ ID NOs. 72, SEQ ID NOs. 74, SEQ ID NOs. 76, SEQ ID NOs. 78, SEQ ID NOs. 80, and SEQ ID NOs. 82, a polypeptide comprising an amino acid sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NOs. 56, SEQ ID NOs. 68, SEQ ID NOs. 70, SEQ ID NOs. 72, SEQ ID NOs. 74, SEQ ID NOs. 76, SEQ ID NOs. 78, sequence The composition according to claim 3, comprising a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence selected from number 80 and sequence number 82, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with an amino acid sequence selected from sequence number 56, sequence number 68, sequence number 70, sequence number 72, sequence number 74, sequence number 76, sequence number 78, sequence number 80, and sequence number 82.

10. The alpha variable region is specific to DR13-CT83 and consists of a polypeptide having identity selected from 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 85% to 100%, with respect to an amino acid sequence selected from SEQ ID NOs: 54, 67, 69, 71, 73, 75, 77, 79, and 81. The composition according to claim 9, wherein the beta variable region of the TCR comprises a polypeptide having identity selected from 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 85% to 100%, with respect to 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.

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

12. The composition according to claim 1, wherein a chimeric antigen receptor, a chimeric TCR receptor, or CAR or chimeric TCR expressed on a T cell is fused with a chemokine receptor, where the chemokine receptor is selected from CCR5, CCR2, and CXCR3, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, a common cytokine receptor gamma chain, or a chemokine receptor for enhancing T cell trafficking.

13. A chimeric TCR polypeptide comprising a cancer antigen-specific TCR variable region fused to a modified human TCR alpha or beta constant region and a non-human TCR alpha or beta constant region (optionally a mouse TCR alpha or beta constant region), wherein the alpha and beta variable regions of the TCR variable region fused to the modified or non-human alpha or beta constant chain region are as follows: a. HLA-A2-specific HCMV pp65 having the amino acid sequence of the alpha chain variable region of SEQ ID NO: 27 and the amino acid sequence of the beta chain variable region of SEQ ID NO: 29 An alpha variable region of a TCR, wherein the alpha variable region or a variant thereof is a polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, which binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide having one, two or more amino acid substitutions to 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 in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity to the sequence of SEQ ID NO: 27 or SEQ ID NO: 29; and optionally further comprising a beta variable region of an HLA-A2 limited HCMV pp65 TCR comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30. Here, the beta variable region or its variant comprises a polypeptide having an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30, which binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor; a polypeptide having a sequence having one, two or more amino acid substitutions to SEQ ID NO: 28 or SEQ ID NO: 30; or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 28 or SEQ ID NO: 30, 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; or b. Alpha and beta variable regions of a TCR specific to a cancer antigen selected from NY-ESO-1 and CT83, wherein, if the TCR is specific to NY-ESO-1, the alpha variable region optionally comprises DP4-ESO-1, which includes the amino acid sequence of SEQ ID NO:

3. A TCR polypeptide, an alpha variable region or a variant thereof that binds to the 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 within the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 3 (for example, D95S or Q98Y in GADIVDYGQNFV (SEQ ID NO: 89), which is the TCR-Vα CDR3 sequence; amino acid position numbers are named according to the mature TCR sequence), or a polypeptide having one, two, or more amino acid substitutions within the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 3, and DP4-ESO-1A2-CT83 is a polypeptide in which the alpha variable region of the TCR optionally contains the amino acid sequence of SEQ ID NO: 4, or a variant thereof that binds to the antigen with the same specificity as 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: 4, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 4 (for example, Y98L, Y98M in AWRRRRGYEQY (SEQ ID NO: 90), which is the TCR-Vβ CDR3 sequence), or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 4, and the TCR is specific to CT83. A2-CT83 comprises a TCR, or a variant thereof that binds to the antigen with the same specificity as 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: 5, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 5, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 5, and the beta variable region of CT83TCR is optionally a polypeptide containing the amino acid sequence of SEQ ID NO: 6.c. A polypeptide comprising the TCR, or a variant thereof that binds to the antigen with the same specificity as a reference (full-length and unmodified) receptor, a polypeptide comprising the amino acid sequence in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 6, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of the amino acid sequence of SEQ ID NO: 6, or a polypeptide having one, two, or more amino acid substitutions to an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 6, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 32, wherein the alpha variable region of the HLA-A2-limited HCMV IE-1 TCR is a polypeptide comprising the amino acid sequence of SEQ ID NO: 32 The alpha variable region of the TCR is a polypeptide comprising an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 32, a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of SEQ ID NO: 32, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 32, and optionally the composition further comprises the beta variable region of the HLA-A2 limited IE-1 TCR, and the HLA-A2 limited IE-1 The beta variable region of the TCR includes a polypeptide containing the amino acid sequence of SEQ ID NO: 33, a polypeptide containing 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 in the CDR1, CDR2, and / or CDR3 regions of SEQ ID NO: 33, or a polypeptide having one, two or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 33, or d. The alpha variable region of the TCR, where the HLA-DR13-specific DR13-CT83Polypeptide containing an amino acid sequence in which the alpha variable region of the TCR includes a sequence selected from SEQ ID NOs. 54, SEQ ID NOs. 67, SEQ ID NOs. 69, SEQ ID NOs. 71, SEQ ID NOs. 73, SEQ ID NOs. 75, SEQ ID NOs. 77, SEQ ID NOs. 79, and SEQ ID NOs. Polypeptide containing an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOs. 54, SEQ ID NOs. 67, SEQ ID NOs. 69, SEQ ID NOs. 71, SEQ ID NOs. 73, SEQ ID NOs. 75, SEQ ID NOs. 77, SEQ ID NOs. 79, and A polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of a sequence selected from SEQ ID NO: 81, or a polypeptide having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with a 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, and optionally the composition further comprises the beta variable region of the HLA-DR13-limiting DR13-CT83 TCR.A polypeptide in which the beta variable region of the TCR contains an amino acid sequence selected from SEQ ID NOs. 56, SEQ ID NOs. 68, SEQ ID NOs. 70, SEQ ID NOs. 72, SEQ ID NOs. 74, SEQ ID NOs. 76, SEQ ID NOs. 78, SEQ ID NOs. 80, and SEQ ID NOs. A polypeptide containing an amino acid sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NOs. 56, SEQ ID NOs. 68, SEQ ID NOs. 70, SEQ ID NOs. 72, SEQ ID NOs. 74, SEQ ID NOs. 76, SEQ ID NOs. Polypeptides having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence selected from 78, SEQ ID NO: 80, and SEQ ID NO: 82, or containing polypeptides having one, two, or more amino acid substitutions in the CDR1, CDR2, and / or CDR3 regions of an amino acid sequence having at least 85% identity with 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. The chimeric TCR polypeptide comprising the above.

14. A composition comprising a chimeric antigen receptor (CAR), a chimeric TCR, or T cells expressing a CAR or a chimeric TCR, wherein the CAR or TCR comprises an antigen recognition site, a transmembrane main, and an intracellular T cell activation site, and the intracellular T cell activation site comprises a co-stimulatory signaling domain fused to a signaling domain, wherein the co-stimulatory signaling domain comprises CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CD2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, C D18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD4 9D, 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, 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, FcERI Gamma, 4-1BBL, BAFF, G ADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TAC1, BLAME, CRACC, CD2F- 10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Ralpha, I L-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL -21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs such as TLR2),ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, ​​PAG / Cbp , PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, BTLA, ika The composition is selected from ros, LAG-3, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof, and further comprises a ZAP70 kinase domain or a variant or variant thereof.

15. The composition according to claim 14, wherein the intracellular T cell activation site comprising a ZAP70 kinase domain, or a variant thereof, comprises a substitution of CD3 zeta with a ZAP70 kinase domain, or a variant thereof, or a ZAP70 kinase domain derived from functional wild-type ZAP70, or a variant thereof, and further comprises a functional ZAP70 kinase domain, or a variant thereof, or the ZAP70 kinase domain derived from functional wild-type ZAP70, or a variant thereof.

16. The functional ZAP70 kinase domain, or its variant or variant, is: 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 fragmented, biologically active fragment of ZAP70 kinase having at least 70% identity to the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 52, SEQ ID NO: 64, and SEQ ID NO: 65, g. A variant or variant thereof that is a fragmented, biologically active fragment containing a functional ZAP70 kinase domain, or h. A biologically active variant or variant of ZAP70 kinase, The N-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, 300, 301, 302, 303, 304 The composition according to claim 15, comprising any of 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 the C-terminal amino acid being the 610th, 611, 612, 613, 614, 615, 616, 617, 618, or 619th amino acid.

17. The composition according to claim 14, wherein the antigen recognition site comprises an antigen-specific antibody, an antigen-binding fragment, an antibody mimetic, a bispecific antibody, a triplicate antibody, a polyvalent multimer-binding protein, a protein receptor or ligand for a specific receptor, a TCR-like antibody, or a bispecific binding protein comprising a TCR-like antibody and an anti-CD3 antibody or its binding fragment.

18. The composition according to claim 17, wherein the antibody, antigen-binding fragment, antibody mimetic, protein receptor or ligand for a specific receptor, or TCR-like antibody comprises a ScFv fragment, Fv fragment, Fd fragment, Fab fragment, Fab' fragment, F(ab)'2 fragment, VH domain, VL domain, monoclonal antibody, polyclonal antibody, nanobody, double or triple antibody fusion protein, or any combination thereof.

19. The antigens recognized by the aforementioned antigen recognition site include alpha (α)-fetoprotein (AFP), melanoma deficiency factor 2 (AIM2), adenocarcinoma antigen 4 (ART-4) recognized by T cells, BCMA, B antigen (BAGE), CTL-recognizing antigen on melanoma (CAMEL), oncoemulsifying antigen peptide-1 (CAP-1), caspase 8 (CASP8), cell division cycle protein 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDK12, oncoemulsifying antigen (CEA), calcium-activated chloride channel 2 (CLCA2), CFTR, CMV, carcinometris antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor receptor variant III), EGFR and its isovariants, and EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2,3 (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-acetylglucosaminyltransferase V (GnT-V), glycoprotein 100 (GP100), helicase antigen (HAGE), H3.3K27M, carcinoembryonic antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, human epithelial receptor 2 (HER2 / neu), HBV, HERV-E, HIV-1 gag, HMI. 24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, human telomerase reverse transcriptase (hTERT), KRAS, KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, LI cell adhesion molecule (LI-CAM), melanoma-related 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),Protein melan-A (melanoma antigen MART-1 recognized by T lymphocytes), mesothelin (MSLN), mucin 1 (MUC1), MUC2, mucin 16 (Muc-16), myo-D1, dimeric form of pyruvate kinase receptor M2 (tumor M2-PK), Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophagus 1 (NY-ESO-1), preferential expression antigen in 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 cell carcinoma rejection antigens 1, 2, 3 (SART-1, -2, -3), SOX10, synovial sarcoma X rupture point 2 (SSX-2), Survivin, OVA1, HE4, DR-70, total PSA, alpha-methylacyl-CoA racemase (AMACR), CA125 / MUC16, ER alpha / NR3A1,ER beta / NR3A2,thymidine kinase 1,AG-2,BRCA1,BRCA2,CA15-3 / MUC-1,caveolin-1,CD117 / c-kit,CEACAM-5 / CD66e,cytokeratin 14,HIN-1 / SCGB3A1,Ki-67 / MKI67,MKP-3,nestin,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, PD1, PD-L1, CTLA-4, PDGFRalpha, VEGF, TRAG-3, T cell receptor gamma substituted reading frame protein (TARP), TGFbII, thyroglobulin, abnormal p53 protein, TP53 (p53), TRAIL, tyrosinase-related Protein 1 (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, CDS, CD6, CD7,CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD1 6 (Fc gamma RIII), CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27 , CD28, CD29, CD30, CD31, CD32 (Fc gamma RII), CD33, CD34, CD35, CD36, CD37, C D38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD4 9b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64 (Fc gamma RI), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (endoglin), CD107a, CD107b, CD114 (G-CSFR), CD1 15, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BD CA3), CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD197 (CCR7), CD203c, C D205 (DEC-205), CD207 (Langerin), CD209 (DC-SIGN), CD223, CD235, CD235a, CD 244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF-R), CD273 (B7-DC, PD-L2), CD276 (B7-H3), CD279 (PD1), 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, cytokeratin 8, 18+, 19+), prostate stem cell antigen (PSCA), α2β1, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, antigens of cytomegalovirus (CMV) infected cells (e.g., small cells) Cellular surface antigen), CS-I, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), estrogen receptor (ER), progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, free PSA, thyroglobulin, nuclear fission apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, Aldo ketreductase 1C3 (AKR1C3), Alpha-1B glycoprotein, Alpha-1 microglobulin, Alpha-B crystallin (CRYAB), Alpha-methylacyl CoA racemase (AMACR), AMFR / gp78, Annexin A3, Annexin A8 (ANXA8), APC, Apolipoprotein A-I (ApoA1), Apolipoprotein A-II (ApoA2), Apolipoprotein E (ApoE), April (TNFSF13), ASCL1 / Mash1, ATBF1 / ZFHX3, Attractin, Aurora A , BAP1, Bcl-2, Bcl-6, Beta-2-microglobulin, Beta-1,3-glucuronyltransferase 1 (B3GAT1), Beta-catenin, Beta-III tubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, Cadherin-13, Caldesmon (CALD1), Carponin 1, Calretinin, Carbonic anhydrase IX (CA9), Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7,CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, Chitinase-like 1, Cholecystokinin B receptor (CCKBR), Chorionic gonadotropin alpha chain (alpha hCG), chorionic gonadotropin alpha / beta (HCG), CKAP4 / p63, claudin-18, clathrin, c-Maf, c-Myc, coactosin-like protein 1 (CotL1), COMMD1, cornulin, cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, cyclin A1, cyclin A2, cyclin D2, cyclin D3, CYLD, Cyr61 / CCN1, cytokeratin 14, cy Tokeratin 18, Cytokeratin 19, Fetal Acetylcholine Receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, DKK-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin (CD105), Endosialin (CD248), Enolase 2 (Neuron-Specific Enolase), EpCAM / TROP1, Eps15, ER alpha / NR3A1, ER beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fasin, FATP3, FCRLA, Fetuin A (AHSG), FGF acidic, FGF basic, FGF R3, FGF R4, fibrinogen, folate-binding protein (FBP), fibroblast-activating protein alpha (FAP), follistatin-like 1 (FSTL1), FOLR1, FOLR2, FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / disadherin, FcεRIα, FITC, FLT3, GABA-A alpha 1, GADD153, GADD45 alpha, galectin-3, galectin-3BP / MAC-2BP, galactin, ganglioside,Cystic disease gelatin-like protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma-glutamylcyclotransferase (CRF21), Gas1, gastrin-releasing peptide receptor (GRPR), gastrokain 1, gelzolin (GSN), glial fibrillary acidic protein (GFAP), GLI-2, glutathione peroxidase 3 (GPX3), gpA33, glycopeptide, glypican 2, GPC2), Glypican 3, Golgi glycoprotein 1 (GLG1), gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, Heparanase (HPSE), Hepsin, HGF R / c-MET, HIF-2 alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, Hyaluronidase 1 (HYAL1), ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, Interleukin-13 receptor α2 chain (IL-13Ra), Interleukin-13 receptor subunit α2 (IL-13Ra2), Integrin, Integrin B7, IMP dehydrogenase 1 (IMPDH1), Importin alpha 2 (KPNA2), ING1, Integrin beta 1 (CD29), Integrin beta 3 (CD61), IQGAP1, Isocitrate dehydrogenase 1 (IDH1), ITIH4, ITM2C, Jagged 1. JNK, JunB, JunD, OGR1, Olig2, Osteopontin (OPN), Ovastatin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA-15, pepsinogen A5 (PGA5), peptidase inhibitor 16 (PI16), peroxiredoxin 2, PGCP, PI3 kinase p85alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone receptor B (NR3C3), Progesterone receptor (NR3C3), Progranulin (PGRN), Prolactin, Prostaglandin E synthase 2 (PTGES2), PSAP, PSCA, PSMA / FOLH1 / NAALADase I, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP beta / zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, secretin receptor, Serpin A9 / Centrin, SerpinE1 / PAI-1, Serum Amyloid A1, Serum Amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostain, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC Protein 1 (SPARCL1), SPINK1, Src, six transmembrane epithelial antigens of the prostate (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin-1, Syndecan-1 / CD138, Syntaxin 4, Synuclein-gamma, Synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, Lactate dehydrogenase A (LDHA), Lamin B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, L YPD8, MAP2, Matryptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin-1 receptor (MC1R), Melanotransferrin (CD228), Melatonin, Mer, Mesothelin, Metadoherin, Metastine / KiSS1, Methionine aminopeptidase, Methionine aminopeptidase 2 (METAP2), MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 (MIB2), Minjin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSPR / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesphatin-1 / Nucleobingin-2, Nestin, NFκB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-methyltransferase (NNMT), NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankirase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta1, TGF-beta1,2,3, TGF-beta1 / 1.2, TGF-beta2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin Beta 10, Thymosin Beta 4, Thyroglobulin, TIMP Assay Kit, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoreglin-2, TMEM219, TMEM87A, TNF-Alpha, TOP2A, TopBP1, t-plasminogen activator (tPA), TRA-1-60(R), TRA-1-85 / CD147, TRAF-4, Transgerin (TAGLN), Trypsin 2 (PRSS2), Tri The composition according to claim 17, selected from the group of antigens recognized by ScFv, which consists of ptase alpha (TPS1), TSPAN1, UBE2S, uPAR, u-plasminogen activator (urokinase), urotensin II receptor, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, CNKSR1 / CNK / KSR, or any combination thereof.

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

21. The method according to claim 20, wherein the composition comprising CAR-T or TCR-T cells as described in claim 20 is a pharmaceutical composition.

22. A method for treating cancer, inflammatory disease, autoimmune disease, allergic disease, organ transplant condition, or infection by administering a composition comprising CAR-T or TCR-T cells according to claim 16 to a subject suffering from or suspected of having cancer, inflammatory disease, autoimmune disease, allergic disease, organ transplant condition, or infection.

23. A method for extending the persistence of T cells or reducing T cell exhaustion in a subject by administering a composition comprising TCR-T cells or CAR-T cells according to claim 14 or 20 to a subject, thereby modulating the signaling and function of TCR-T cells, Here, the signaling domains of TCR or CAR are: ANKRD11, ARID1A, BACH2, BCL2L11, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, J MJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PI K3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3H1 (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2 B3, SH2DJA, SMAD2, SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isotypes IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T lymphocyte anti- The method, wherein the patient is regulated, knocked down, or knocked out by negative signaling molecules selected from the following: PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activator gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D, and epigenetic factors (which may or may not include JMJD3 and LSD1).

24. The composition according to claim 14, wherein a CAR or TCR is fused to a chemokine receptor, where the chemokine receptor is optionally selected from CCR5, CCR2, and CXCR3, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, a common cytokine receptor gamma chain, or a chemokine receptor for enhancing T cell trafficking.

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

26. A method for stimulating an immunological response to cancer, or for treating, suppressing, and / or preventing cancer, comprising administering to a subject a therapeutically effective amount of a composition comprising the CAR or TCR described in claim 24 and an isolated nucleic acid encoding a gene for knockdown or an sgRNA for knockout in order to enhance the antitumor activity of TCR-transduced T cells in vivo, wherein the nucleic acid sequence of the targeted immune system negative signaling molecule is selected from checkpoint proteins and / or immunosuppressive proteins, and further, the target of the shRNA or sgRNA is ANKRD11, 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, NR4 A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, 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, von Hippel-Lindau tumor suppressor (VHL) (SEQ ID NO: 8), WDR6, ZC3H12A, indoleamine (2, 3) The method comprising selecting from -dioxygenase (IDO) (including isotypes IDO1 and IDO2), OX40, CTLA-4, PD-1 (SEQ ID NO: 7), PD-L1, PD-L2, LAG3, B7 homolog 3 (B7-H3), VHL (SEQ ID NO: 8), PPP2R2D (SEQ ID NO: 9), negative regulators, epigenetic factors, or transcription factors (which may or may not include JMJD3 and LSD1).

27. A composition comprising TCR, chimeric TCR, CAR, or cells expressing TCR or CAR, wherein the TCR or CAR comprises an antigen recognition site, a transmembrane main, and an intracellular T cell activation site. Here: a. The intracellular T cell activation site comprises a signal transduction domain, and further, the signal transduction domain comprises a ZAP70 kinase domain, or a variant thereof, or comprises a substitution of CD3 zeta with a ZAP70 kinase domain, or a variant thereof. b. The intracellular T cell activation site comprises a co-stimulatory signaling domain fused to a signaling domain, wherein the co-stimulatory signaling domain comprises CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CD2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD 154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD1 22, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAM1), CD223 (LAG3), C D226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, L AT, 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, TLRs (TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9), TRAFs (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), TRIM, ZAP70, FcERI Nma, 4-1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TAC1, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, I CAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, I L-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76,Toll-like receptors (TLRs such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, 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 selected. c. A TCR or CAR fused to a chemokine receptor, wherein the chemokine receptor is optionally selected from CCR5, CCR2, and CXCR3, or chemokine receptors for enhancing T cell trafficking; the composition comprising:

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

29. The composition according to claim 27, wherein the ZAP70 kinase domain, or a variant or variant thereof, is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence 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 extending the persistence of T cells or reducing T cell exhaustion by modulating the signaling and function of TCR, chimeric TCR, CAR, or cells, or by directly manipulating the signaling domain of a TCR or CAR, wherein the signaling domain of a TCR or CAR is modulated by a negative signaling molecule, wherein the negative signaling molecule is knocked down or knocked out, and the negative signaling molecule is selected from PD-1, VHL, PPP2R2D, indoleamine (2,3)-dioxygenase (IDO) (including isotypes IDO1 and IDO2), OX40, CTLA-4, PD-L1, PD-L2, LAG3, B7-H3, and epigenetic factors (which may or may not include JMJD3 and LSD1), or any combination thereof.

31. A method for extending the T cell persistence of CAR-T or TCR-T cells or reducing T cell exhaustion by substituting CD3 zeta with the ZAP255, ZAP280, ZAP300, ZAP327, or ZAP338 kinase domain, or a variant or variant thereof.

32. Transmenbrand's main targets are 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, FcRalph, FcRbeta, Fyn, GAL9, and cytokine receptor (IL-2Ralph). HA, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha), NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, pTalpha, T cell receptor polypeptide (human and mouse T CRα, TCRβ, TCRgamma, TCRδ), TIM3, TRIM, CD2, CD3D, CD3E, CD3G, CD3zeta, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG3), CD270 ( The composition according to claim 1 or 14, derived from transmembrane mains of HVEM), CD272 (BILA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD357 (GITR), PTCH2, ROR2, Ryk, SLP-76, SIRPalpha, ZAP70, or any combination thereof.

33. The composition according to claim 32, further comprising a signal transduction domain having a ZAP70 kinase domain selected from variants or variants of ZAP70 kinase whose intracellular T cell activation site is ZAP255, ZAP280, ZAP300, ZAP308, ZAP327, ZAP338, or whose N-terminal amino acid is any of 250 to 338 and whose C-terminal amino acid is the 610th, 611th, 612th, 613th, 614th, 615th, 616th, 617th, 618th, or 619th amino acid.

34. A composition comprising one or more antigens or antigenic epitopes of the present invention that are pathogenically related to a disease or condition, The composition wherein the epitope is optionally selected from A2-ESO-1 (SEQ ID NO: 2) and DP4-ESO-1 (SEQ ID NO: 1), which are NY-ESO-1 peptides, CT83 peptides (including, but not limited to, A2-CT83 peptide (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 mRNA or DNA sequences (SEQ ID NOs: 83-87, etc.) encoding an antigen or antigenic epitope of the present invention that is pathogenically associated with a disease or condition, wherein the epitope optionally includes, but is not limited to, NY-ESO-1 peptides A2-ESO-1 (SEQ ID NO: 2) and DP4-ESO-1 (SEQ ID NO: 1), CT83 peptides (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 The composition is selected from IE-1 (SEQ ID NO: 31) or its variants, and optionally further comprises LNPs (lipid nanoparticles).

36. A method for stimulating an immunological response to an antigen or antigenic epitope in order to treat, suppress and / or prevent a disease or condition in which the antigen is pathogenically associated, the method comprising administering to a subject the composition according to claim 34 or claim 35.

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