T cell antigen receptors and methods for preparing and using same

Abstract

The present invention relates to the technical field of T cell antigen receptors, and specifically to a TCR capable of recognizing and binding to a CMVpp65 antigen complex, a nucleic acid comprising a nucleotide sequence encoding the TCR, a vector comprising the nucleic acid molecule, a cell into which the nucleic acid molecule or the vector has been introduced, a pharmaceutical composition comprising the TCR, nucleic acid molecule, vector, or cell as an active ingredient, and use of the TCR, nucleic acid molecule, vector, cell, or pharmaceutical composition in the preparation of a medicament for treating tumors or viral infections.

Description

[Technical Field]

[0001] The present disclosure is in the field of biomedicine, and specifically relates to T cell antigen receptors and methods for their preparation and use. [Background technology]

[0002] The T cell receptor (TCR) is a molecule by which T lymphocytes specifically recognize antigens and initiate immune responses. It is a heterodimeric cell surface protein belonging to the immunoglobulin superfamily and associates with unmutated proteins of the CD3 complex, which is involved in regulating signal transduction. The TCR is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC) and is crucial for the cellular immune function of the immune system. By binding to the MHC complex, the antigen-specific TCR triggers direct physical contact and interaction between the T cell and the antigen-presenting cell, resulting in a series of cell signaling and other physiological responses, which enable different antigen-specific T cells to exert their immune effects against their target cells.

[0003] Cytomegalovirus (CMV) is a DNA virus belonging to the herpesvirus family. Its name comes from the hypertrophy of infected cells. CMV is one of the most common human pathogens, deeply ingrained in the human population, with an adult infection rate of 50%–100%. While CMV infection typically does not manifest clinical symptoms in individuals with a normal immune system, asymptomatic primary infections often fail to completely eliminate the CMV virus, resulting in persistent latent infection within the patient's body. When CMV carriers become immunosuppressed, such as through AIDS or immunosuppressive therapy following organ transplantation, latent CMV infection can reactivate, causing severe illness and even death. CMV infection is also associated with the development and progression of several tumors. For example, CMV-pp65 antigen can be detected in half of glioblastoma tissues. Therefore, the prevention and treatment of CMV-associated diseases are a key focus of drug development.

[0004] A method has now been developed to prepare TCR-T cells for CMV infection using a CMV-specific TCR. Infusion of these TCR-T cells into patients provides immediate and effective antiviral protection to patients with CMV reactivation, eliminating the reactivated virus. Furthermore, patients infused with CMV-specific TCR-T cells maintain long-lasting antiviral protection, significantly reducing the rate of subsequent reactivation. Preliminary clinical studies have also shown that CMV-pp65 vaccines can extend progression-free survival and improve overall survival in patients with glioblastoma. Therefore, TCRs targeting CMV-pp65 may also be applicable to the treatment of CMV-associated malignancies.

[0005] CN113881680A and CN102656188A both disclose TCRs capable of recognizing cytomegalovirus antigens, but their selection methods do not take into account the heterogeneity of TCR recognition, making their killing effect on target cells unclear. CN106279404A discloses a stable and soluble heterodimeric TCR. This heterodimeric TCR has an artificial interchain disulfide bond between the α chain variable region and the β chain constant region, making it stable, soluble, and easily refolded, repurified, and capable of specific binding to the original ligand. However, this patent does not disclose a TCR specific for cytomegalovirus. Summary of the Invention

[0006] TCR-T therapy can effectively control acute CMV disease and induce antiviral immune responses. However, the application of TCR-T therapy remains hindered by the difficulty of effectively obtaining T cells from patients' cytotoxic T lymphocytes stimulated in vivo or ex vivo, rapidly extracting their TCR sequences, and constructing specific T cells incorporating functional TCRs. This disclosure utilizes high-throughput sequencing based on single-cell sequencing technology to determine TCR mate sequences and evaluate the expression levels of key genes in immune cells for the study of specific T cell receptors.

[0007] Specifically, in one implementation method of the present disclosure, antigen-specific cytotoxic T lymphocytes are induced and expanded in vitro using CMVpp65 short peptide antigens. Based on the TCR pair information of positive T cells obtained by single-cell sequencing and MHC tetramer sorting, TCRs are selected according to the frequency of clonal sequences, TCR-Ts are constructed, and in vitro functional testing is performed to evaluate the function of the TCR-Ts, ultimately obtaining the specific T cell receptor of the present disclosure.

[0008] HLA-A24-CMV-pp65 according to the present disclosure 341-349 The antigen complex is composed of the human leukocyte surface antigen HLA-A24 and CMV-pp65. 341-349It consists of a short peptide (native sequence: QYDPVAALF (SEQ ID NO: 13)) and is expressed on the surface of targeted target cells.

[0009] To achieve the above object, a first aspect of the present disclosure provides a TCR that recognizes the human cytomegalovirus pp65 antigen, wherein the TCR is HLA-A24-CMV-pp65. 341-349 The T cell antigen receptor has the property of binding to an antigen complex and comprises at least one α chain variable region and / or β chain variable region. Preferably, the T cell antigen receptor is an αβ heterodimer, each comprising one TCR α chain variable region and one TCR β chain variable region.

[0010] In one embodiment, the TCR α chain variable region comprises three complementarity determining regions (CDRs), CDR1α to CDR3α, of which CDR3α comprises the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:31, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:31. Preferably, the amino acid sequence of CDR3α of the TCR α chain variable region is set forth in any one of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:31.

[0011] In one embodiment, the TCR β chain variable region comprises three CDR regions, CDR1β to CDR3β, of which CDR3β comprises the sequence shown in SEQ ID NO:6, SEQ ID NO:16, or SEQ ID NO:25, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:25. Preferably, the amino acid sequence of CDR3β of the TCR β chain variable region is shown in any one of SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:25.

[0012] In one embodiment, CDR1α of the α chain variable region of the TCR comprises the amino acid sequence set forth in SEQ ID NO: 1 or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity with SEQ ID NO: 1. CDR2α comprises the amino acid sequence set forth in SEQ ID NO: 2 or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity with SEQ ID NO: 2. CDR1β of the β chain variable region of the TCR comprises the amino acid sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 14, and SEQ ID NO: 23 or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity with SEQ ID NO: 4, SEQ ID NO: 14, and SEQ ID NO: 23. CDR2β comprises an amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:24, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:24.

[0013] In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR and CDR1β to CDR3β of the β chain are shown below.

[0014] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CARNTGKLIF (SEQ ID NO: 3), CAPSASKIIF (SEQ ID NO: 22), CAPQFNKFYF (SEQ ID NO: 31) CDR1β: any one of SQVTM (SEQ ID NO: 4), SGHVS (SEQ ID NO: 14), and LNHDA (SEQ ID NO: 23) CDR2β: any one of ANQGSEA (SEQ ID NO: 5), FQNEAQ (SEQ ID NO: 15), and SQIVND (SEQ ID NO: 24) CDR3β: CSANPTGGGTEAFF (SEQ ID NO: 6), CASSLLTRTETQYF (SEQ ID NO: 16), or CASSTTGLAGGPGNEQFF (SEQ ID NO: 25) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0015] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF(SEQ ID NO:3) CDR1β: SQVTM (SEQ ID NO: 4) CDR2β: ANQGSEA (SEQ ID NO: 5) CDR3β:CSANPTGGGTEAFF(SEQ ID NO:6) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0016] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF(SEQ ID NO:3) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β: FQNEAQ (SEQ ID NO: 15) CDR3β:CASSLLTRTETQYF(SEQ ID NO:16) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0017] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CAPSASKIIF(SEQ ID NO:22) CDR1β: LNHDA (SEQ ID NO: 23) CDR2β: SQIVND (SEQ ID NO: 24) CDR3β:CASSTTGLAGGPGGNEQFF(SEQ ID NO:25) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0018] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CAPQFNKFYF(SEQ ID NO:31) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β: FQNEAQ (SEQ ID NO: 15) CDR3β:CASSLLTRTETQYF (SEQ ID NO:16).

[0019] In one embodiment, the variable region of the alpha chain of a TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:26, or SEQ ID NO:32, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:26, or SEQ ID NO:32.

[0020] Preferably, the alpha chain of the TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:34, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:34.

[0021] In one embodiment, the variable region of the beta chain of a TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:27, or SEQ ID NO:33, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:27, or SEQ ID NO:33.

[0022] Preferably, the beta chain of the TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:35, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:35.

[0023] In a specific embodiment of the present disclosure, preferably, amino acids of the α chain of the TCR of the present disclosure are linked directly or indirectly to amino acids of the β chain of the TCR, preferably indirectly, and more preferably using a 2A peptide. 2A peptides are short peptides (18-25 amino acids long) derived from viruses, called "self-cleaving" peptides, which can cause transcription products to produce multiple proteins. 2A peptides used in the present disclosure include, but are not limited to, P2A, T2A, E2A, and F2A.

[0024] In some embodiments of the present disclosure, a 2A peptide such as P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg), and a p2a ribosomal skipping peptide to form the fp2A (furin-SGSG-p2A) linked structure.

[0025] More preferably, said fp2A comprises the amino acid sequence shown in SEQ ID NO:11.

[0026] In one specific embodiment of the present disclosure, the α chain and β chain may be linked in the order of α chain, fp2A, and β chain, or β chain, fp2A, and α chain.

[0027] In one specific embodiment of the present disclosure, the TCR of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:36, or comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:36.

[0028] CMVpp65 according to the present disclosure 495-503 A short peptide (native sequence: NLVPMVATV (SEQ ID NO: 46)) is expressed on the surface of targeted target cells.

[0029] To achieve the above object, the present disclosure provides a TCR that recognizes the human cytomegalovirus pp65 antigen, wherein the TCR is CMVpp65. 495-503 The T cell antigen receptor has the property of binding to an antigen complex and comprises at least one α chain variable region and / or β chain variable region. Preferably, the T cell antigen receptor is an αβ heterodimer, each comprising one TCR α chain variable region and one TCR β chain variable region.

[0030] In one embodiment, the TCR α chain variable region comprises three complementarity determining regions (CDRs), CDR1α to CDR3α, of which CDR3α comprises a sequence set forth in any one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69. Preferably, the amino acid sequence of CDR3α of the TCR α chain variable region is set forth in any one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69.

[0031] In one embodiment, the TCR β chain variable region comprises three CDR regions, CDR1β to CDR3β, of which CDR3β comprises the sequence set forth in SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, or SEQ ID NO: 72, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, or SEQ ID NO: 72. Preferably, the amino acid sequence of CDR3β of the TCR β chain variable region is set forth in any one of SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, or SEQ ID NO: 72.

[0032] In one embodiment, CDR1α of the α chain variable region of the TCR comprises an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:47, and SEQ ID NO:58, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity to any one of SEQ ID NO:1, SEQ ID NO:47, and SEQ ID NO:58. CDR2α comprises an amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity to any one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:59.

[0033] CDR1β of the β chain variable region of the TCR comprises an amino acid sequence set forth in any one of SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:61, and SEQ ID NO:70, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:61, and SEQ ID NO:70. CDR2β comprises an amino acid sequence set forth in any one of SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:62, and SEQ ID NO:71, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:62, and SEQ ID NO:71.

[0034] In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR and CDR1β to CDR3β of the β chain are shown below.

[0035] CDR1α: SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 47), TSGFNG (SEQ ID NO: 58) CDR2α: any one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 48), and NVLDGL (SEQ ID NO: 59) CDR3α: CASINFNKFYF (SEQ ID NO: 37), CAEFTGTASKLTF (SEQ ID NO: 49), CAVTYNNARLMF (SEQ ID NO: 60), CARNYGQNFVF (SEQ ID NO: 69) CDR1β: any one of MDHEN (SEQ ID NO: 38), GTSNPN (SEQ ID NO: 50), MNHEY (SEQ ID NO: 61), and DFQATT (SEQ ID NO: 70) CDR2β: SYDVKM (SEQ ID NO: 39), SVGIG (SEQ ID NO: 51), SMNVEV (SEQ ID NO: 62), SNEGSKA (SEQ ID NO: 71) CDR3β: CASP LNGGATEAFF (SEQ ID NO: 40), CAWSDRAAFTDTQYF (SEQ ID NO: 52), CASSSVAGGRIEQFF (SEQ ID NO: 63), CSARDIKAQQWNIQYF (SEQ ID NO: 72) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0036] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CASINFNKFYF(SEQ ID NO:37) CDR1β: MDHEN (SEQ ID NO: 38) CDR2β: SYDVKM (SEQ ID NO: 39) CDR3β:CASSPLNGGATEAFF(SEQ ID NO:40) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0037] CDR1α: DSSSTY (SEQ ID NO: 47) CDR2α: IFSNMDM (SEQ ID NO: 48) CDR3α:CAEFTGTASKLTF(SEQ ID NO:49) CDR1β: GTSNPN (SEQ ID NO: 50) CDR2β: SVGIG (SEQ ID NO: 51) CDR3β:CAWSDRAAFTDTQYF(SEQ ID NO:52) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0038] CDR1α: TSGFNG (SEQ ID NO: 58) CDR2α: NVLDGL (SEQ ID NO: 59) CDR3α:CAVTYNNARLMF(SEQ ID NO:60) CDR1β: MNHEY (SEQ ID NO: 61) CDR2β: SMNVEV (SEQ ID NO: 62) CDR3β:CASSSVAGGRIEQFF(SEQ ID NO:63) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0039] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNYGQNFVF(SEQ ID NO:69) CDR1β: DFQATT (SEQ ID NO: 70) CDR2β: SNEGSKA (SEQ ID NO: 71) CDR3β:CSARDIKAQQWNIQYF(SEQ ID NO:72) In one embodiment, the variable region of the alpha chain of a TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:41, SEQ ID NO:53, SEQ ID NO:64, or SEQ ID NO:73, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to SEQ ID NO:41, SEQ ID NO:53, SEQ ID NO:64, or SEQ ID NO:73.

[0040] Preferably, the alpha chain of the TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:66, SEQ ID NO:75, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:66, SEQ ID NO:75.

[0041] In one embodiment, the variable region of the beta chain of a TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:42, SEQ ID NO:54, SEQ ID NO:65, or SEQ ID NO:74, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to SEQ ID NO:42, SEQ ID NO:54, SEQ ID NO:65, or SEQ ID NO:74.

[0042] Preferably, the beta chain of the TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:76, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:76.

[0043] In a specific embodiment of the present disclosure, preferably, amino acids of the α chain of the TCR of the present disclosure are linked directly or indirectly to amino acids of the β chain of the TCR, preferably indirectly, and more preferably using a 2A peptide. 2A peptides are short peptides (18-25 amino acids long) derived from viruses, called "self-cleaving" peptides, which can cause transcription products to produce multiple proteins. 2A peptides used in the present disclosure include, but are not limited to, P2A, T2A, E2A, and F2A.

[0044] In some embodiments of the present disclosure, a 2A peptide such as P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg), and a p2a ribosomal skipping peptide to form the fp2A (furin-SGSG-p2A) linked structure.

[0045] More preferably, said fp2A comprises the amino acid sequence shown in SEQ ID NO:11.

[0046] In one specific embodiment of the present disclosure, the α chain and β chain may be linked in the order of α chain, fp2A, and β chain, or β chain, fp2A, and α chain.

[0047] In one specific embodiment of the present disclosure, the TCR of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:68, SEQ ID NO:77, or comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:68, SEQ ID NO:77.

[0048] CMVpp65 according to the present disclosure 501-509 A short peptide (ATVQGQNLK (SEQ ID NO:89)) is expressed on the surface of targeted target cells.

[0049] To achieve the above object, the present disclosure provides a TCR that recognizes the human cytomegalovirus pp65 antigen, wherein the TCR is CMVpp65. 501-509 The T cell antigen receptor has the property of binding to an antigen complex and comprises at least one α chain variable region and / or β chain variable region. Preferably, the T cell antigen receptor is an αβ heterodimer, each comprising one TCR α chain variable region and one TCR β chain variable region.

[0050] In one embodiment, the α chain variable region of the TCR comprises three complementarity determining regions (CDRs), CDR1α to CDR3α, of which CDR3α comprises a sequence shown in any one of CVITTSGTYKYIF (SEQ ID NO:80), CAYRSFYTGANSKLTF (SEQ ID NO:92), and CVVHSGGSYIPTF (SEQ ID NO:103), or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO:80, SEQ ID NO:92, and SEQ ID NO:103.

[0051] In one embodiment, the TCR β chain variable region comprises three CDR regions, CDR1β to CDR3β, of which CDR3β comprises a sequence set forth in any one of CASTINTYEQYF (SEQ ID NO:83), CASSLYGGPGDQPQHF (SEQ ID NO:95), and CASAQTIGAYNEQFF (SEQ ID NO:106), or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more homology to any one of SEQ ID NO:83, SEQ ID NO:95, and SEQ ID NO:106.

[0052] In one embodiment, CDR1α of the α chain variable region of the TCR comprises the amino acid sequence set forth in any one of SEQ ID NO:78, SEQ ID NO:90, and SEQ ID NO:101, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity to any one of SEQ ID NO:78, SEQ ID NO:90, and SEQ ID NO:101. CDR2α comprises the amino acid sequence set forth in any one of SEQ ID NO:79, SEQ ID NO:91, and SEQ ID NO:102, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% or more identity to any one of SEQ ID NO:79, SEQ ID NO:91, and SEQ ID NO:102.

[0053] CDR1β of the β chain variable region of the TCR comprises an amino acid sequence set forth in any one of SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104. CDR2β comprises an amino acid sequence set forth in any one of SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 105, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 105.

[0054] CDR1α: VSGLRG (SEQ ID NO: 78), TSESDYY (SEQ ID NO: 90), or NSASQS (SEQ ID NO: 101) CDR2α: LYSAGEE (SEQ ID NO: 79), QEAYKQQN (SEQ ID NO: 91), or VYSSGN (SEQ ID NO: 102) CDR3α: any one of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), and CVVHSGGSYIPTF (SEQ ID NO: 103) CDR1β: any one of MNHNS (SEQ ID NO: 81), SGHDT (SEQ ID NO: 93), and MNHNY (SEQ ID NO: 104) CDR2β: any one of SASEGT (SEQ ID NO: 82), YYEEEE (SEQ ID NO: 94), and SVGAGI (SEQ ID NO: 105) CDR3β: any one of CASTINTYEQYF (SEQ ID NO: 83), CASSLIGPGDQPQHF (SEQ ID NO: 95), and CASAQTIGAYNEQFF (SEQ ID NO: 106) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0055] CDR1α: VSGLRG (SEQ ID NO: 78) CDR2α: LYSAGEE (SEQ ID NO: 79) CDR3α:CVITTSGTYKYIF(SEQ ID NO:80) CDR1β: MNHNS (SEQ ID NO: 81) CDR2β: SASEGT (SEQ ID NO: 82) CDR3β:CASTINTYEQYF(SEQ ID NO:83) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0056] CDR1α: TSESDYY (SEQ ID NO: 90) CDR2α: QEAYKQQN (SEQ ID NO: 91) CDR3α:CAYRSFYTGANSKLTF(SEQ ID NO:92) CDR1β: SGHDT (SEQ ID NO: 93) CDR2β: YYEEEE (SEQ ID NO: 94) CDR3β:CASSLYGGPGDQPQHF(SEQ ID NO:95) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0057] CDR1α: NSASQS (SEQ ID NO: 101) CDR2α: VYSSGN (SEQ ID NO: 102) CDR3α:CVVHSGGSYIPTF(SEQ ID NO:103) CDR1β: MNHNY (SEQ ID NO: 104) CDR2β: SVGAGI (SEQ ID NO: 105) CDR3β: CASAQTIGAYNEQFF (SEQ ID NO:106).

[0058] In one embodiment, the variable region of the alpha chain of a TCR of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO:84, SEQ ID NO:96, or SEQ ID NO:107, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to any one of SEQ ID NO:84, SEQ ID NO:96, or SEQ ID NO:107.

[0059] Preferably, the alpha chain of the TCR of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO:86, SEQ ID NO:98, SEQ ID NO:109, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to any one of SEQ ID NO:86, SEQ ID NO:98, SEQ ID NO:109.

[0060] In one embodiment, the variable region of the beta chain of a TCR of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO:85, SEQ ID NO:97, and SEQ ID NO:108, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more identity to any one of SEQ ID NO:85, SEQ ID NO:97, and SEQ ID NO:108.

[0061] Preferably, the beta chain of the TCR of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO:87, SEQ ID NO:99, SEQ ID NO:110, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to any one of SEQ ID NO:87, SEQ ID NO:99, SEQ ID NO:110.

[0062] In a specific embodiment of the present disclosure, preferably, amino acids of the α chain of the TCR of the present disclosure are linked directly or indirectly to amino acids of the β chain of the TCR, preferably indirectly, and more preferably using a 2A peptide. 2A peptides are short peptides (18-25 amino acids long) derived from viruses, called "self-cleaving" peptides, which can cause transcription products to produce multiple proteins. 2A peptides used in the present disclosure include, but are not limited to, P2A, T2A, E2A, and F2A.

[0063] In some embodiments of the present disclosure, a 2A peptide such as P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg), and a p2a ribosomal skipping peptide to form the fp2A (furin-SGSG-p2A) linked structure.

[0064] More preferably, said fp2A comprises the amino acid sequence shown in SEQ ID NO:11.

[0065] In one specific embodiment of the present disclosure, the α chain and β chain may be linked in the order of α chain, fp2A, and β chain, or β chain, fp2A, and α chain.

[0066] In one specific embodiment of the present disclosure, the TCR of the present disclosure comprises an amino acid sequence set forth in any one of SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:111, or comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or more homology to SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:111.

[0067] A second aspect of the present disclosure provides a nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a TCR described in the first aspect of the disclosure, or a complementary sequence thereof.

[0068] In the technical solution of the present disclosure, the nucleic acid sequence may be single-stranded or double-stranded, and may be DNA or RNA.

[0069] In a preferred technical solution of the present disclosure, the nucleic acid sequence may be codon-optimized. More preferably, the codon optimization includes changing many rare codons used in viruses, etc., to corresponding mammalian codons, and / or removing unstable motifs and / or hidden splice sites in mRNA.

[0070] A third aspect of the present disclosure provides an expression vector, said expression vector comprising a nucleic acid according to any one of the present disclosure.

[0071] In a preferred technical solution of the present disclosure, the expression vector can be expressed in vivo, in vitro or ex vivo, and more preferably, the expression vector is consistently expressed at a high level in cells in vivo.

[0072] In a preferred technical solution of the present disclosure, the expression vector may be a prokaryotic expression vector or a retroviral vector.

[0073] In a preferred technical solution of the present disclosure, the expression vector may be Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBRMSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), avian erythroblastosis virus (AEV), etc. More preferably, the expression vector is a lentivirus expression vector.

[0074] A fourth aspect of the present disclosure provides a host cell, said host cell comprising a nucleic acid or expression vector according to any one of the present disclosure.

[0075] In a preferred technical solution of the present disclosure, the host cell may be a eukaryotic cell or a prokaryotic cell. More preferably, the host cell is a eukaryotic cell, including but not limited to yeast cells, 293 cells, CHO cells, etc.

[0076] A fifth aspect of the present disclosure provides an immune cell, wherein the immune cell expresses a T cell antigen receptor according to the present disclosure.

[0077] In a preferred technical solution of the present disclosure, the immune cells comprise one or more of the nucleic acid sequences described in any one of the claims of the present disclosure.

[0078] In a preferred technical solution of the present disclosure, the immune cells include, but are not limited to, stem cells and lymphocytes (such as T cells and B cells). Furthermore, the immune cells are B cells, which express the above-mentioned antibody or its antigen-binding fragment. The immune cells are T cells, which have the above-mentioned limited T cell antigen receptor structure.

[0079] In a preferred technical solution of the present disclosure, the T cells are CD4 + T, CD8 + It may be T, etc.

[0080] In a preferred technical solution of the present disclosure, the immune cells are isolated from T cells of a subject.

[0081] In a preferred technical solution of the present disclosure, the immune cells are T cells from a CMV seronegative donor.

[0082] A sixth aspect of the present disclosure provides a method for preparing immune cells, the method comprising the step of introducing a nucleic acid sequence encoding the T cell antigen receptor into the immune cells, and expressing the nucleic acid sequence to obtain the immune cells.

[0083] In a preferred technical solution of the present disclosure, the immune cells include, but are not limited to, stem cells and lymphocytes (such as T cells and B cells). Furthermore, the immune cells are B cells, which express the above-mentioned antibody or its antigen-binding fragment. The immune cells are T cells, which have the above-mentioned limited T cell antigen receptor structure.

[0084] A preferred technical solution of the present disclosure further includes a step of knocking out endogenous TCR in cells, specifically, by introducing a guide targeting the endogenous TCR into a lentiviral vector, which is then co-transfected into T cells together with a packaging plasmid and a transfection agent.

[0085] A seventh aspect of the present disclosure provides a method for preparing a recombinant T cell, the method comprising the steps of: 1) Obtaining a nucleic acid according to any one of the second aspects of the present disclosure from a positive T cell clone. 2) Isolate and culture primary T cells. 3) The nucleic acid obtained in step 1) is presented in the primary T cells described in step 2) to obtain recombinant T cells expressing a T cell antigen receptor described in any one of the present disclosure.

[0086] In a preferred technical solution of the present disclosure, the T cells are selected from T cells derived from hematopoietic stem cells or peripheral blood lymphocytes (PBLs).

[0087] An eighth aspect of the present disclosure provides a method for preparing a T cell antigen receptor, the method comprising the steps of: (1) Obtaining a nucleic acid according to any one of the second aspects of the present disclosure from a positive T cell clone. (2) The nucleic acid obtained in step (1) is ligated into a vector backbone to obtain an expression vector. (3) The expression vector obtained in step (2) is transformed into a host cell, and expression is induced. (4) Obtain an antibody or its antigen-binding fragment, or a T cell antigen receptor.

[0088] In a preferred embodiment of the present disclosure, the positive T cells specifically bind to an MHC-presented cytomegalovirus (CMV) phosphoprotein pp65 antigenic peptide. More preferably, the MHC-presented cytomegalovirus (CMV) phosphoprotein pp65 antigenic peptide complex is a monomeric or multimeric complex.

[0089] A ninth aspect of the present disclosure provides use of a T cell antigen receptor according to any one of the first aspect, a nucleic acid according to any one of the second aspect, an expression vector according to the third aspect, a host cell according to the fourth aspect, or an immune cell according to the fifth aspect in the preparation of a product for diagnosing or treating a tumor or a disease associated with CMV.

[0090] In a preferred technical solution of the present disclosure, the CMV-related disease is selected from neonatal CMV inclusion disease, acute acquired CMV infection, or diseases caused by CMV infection in immunocompromised patients.

[0091] In a preferred technical solution of the present disclosure, the CMV-related disease is selected from diseases of the liver, spleen, central nervous system, physical disabilities, infectious mononucleosis, musculoskeletal pain, CMV retinitis, CMV gastroenteritis or encephalitis, etc.

[0092] A tenth aspect of the present disclosure provides use of a T cell antigen receptor according to any one of the present disclosure, a nucleic acid according to any one of the present disclosure, an expression vector according to the present disclosure, a host cell according to the present disclosure, or an immune cell according to the present disclosure in labeling, detection, cell sorting, or activation of T cells.

[0093] An eleventh aspect of the present disclosure provides a pharmaceutical composition, the pharmaceutical composition comprising any one of the following: 1) T cell antigen receptor according to the present disclosure 2) Nucleic acids according to the present disclosure 3) Expression vector according to the present disclosure 4) Host cells according to the present disclosure 5) Immune cells according to the present disclosure In a preferred technical solution of the present disclosure, the pharmaceutical composition may further comprise pharmaceutically acceptable auxiliary materials.

[0094] In a preferred technical solution of the present disclosure, the pharmaceutical composition can be used in combination with other therapeutic agents, and more preferably, the therapeutic agents can be immunomodulators.

[0095] A twelfth aspect of the present disclosure provides a kit, the kit comprising any one of the following: 1) A T cell antigen receptor described in any one of the present disclosure. 2) a nucleic acid according to any one of the preceding claims 3) an expression vector according to any one of the preceding claims. 4) A host cell according to any one of the preceding claims. 5) The immune cell described in any one of the present disclosure.

[0096] A thirteenth aspect of the present disclosure provides a method for detecting cytomegalovirus (CMV) phosphoprotein pp65, the method comprising the steps of contacting a test sample with a T cell antigen receptor according to the present disclosure, and detecting a complex formed by the cytomegalovirus (CMV) phosphoprotein pp65 and the T cell antigen receptor.

[0097] Preferably, detecting cytomegalovirus (CMV) phosphoprotein pp65 means detecting the presence or content of cytomegalovirus (CMV) phosphoprotein pp65. The presence indicates presence or absence, and the content may be expression level, protein concentration, etc.

[0098] Preferably, the antibody or antigen-binding fragment thereof or T-cell antigen receptor comprises a detectable tag.

[0099] In one specific embodiment of the present disclosure, the tag may be His and / or HA.

[0100] In one embodiment, the method for detecting cytomegalovirus (CMV) phosphoprotein pp65 is not a method for diagnosing a disease. First, the test sample is not a living organism or its ex vivo tissues or cells. Second, the presence of cytomegalovirus (CMV) phosphoprotein pp65 or a certain concentration or expression level of cytomegalovirus (CMV) phosphoprotein pp65 in a living organism does not identify a disease, but is merely one possibility.

[0101] A fourteenth aspect of the present disclosure provides a method for treating and / or preventing a disease associated with CMV, the method comprising administering to an individual an effective amount of a T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell, or the pharmaceutical composition according to the present disclosure.

[0102] Preferably, the method comprises adoptively transferring T cells expressing a T cell antigen receptor according to the present disclosure into a subject.

[0103] Preferably, the method includes a step of localizing a T cell antigen receptor of the present disclosure in the vicinity of a CMV-associated disease (preferably a tumor or metastatic tumor) so as to improve the efficacy of a toxin or immunostimulant.

[0104] Preferably, it is used for the treatment and / or prevention of CMV reactivation after allogeneic hematopoietic stem cell transplantation.

[0105] Preferably, it is used for the treatment and / or prevention of CMV reactivation after organ transplantation (e.g., kidney, liver, pancreas, intestine, cornea), tissue transplantation, cell transplantation (pancreatic islet cells, corneal limbal stem cells) or stem cell therapy.

[0106] Preferably, the T cells expressing the T cell antigen receptor according to the present disclosure are derived from a subject.

[0107] Preferably, the T cells expressing the T cell antigen receptor according to the present disclosure are derived from the same donor as the hematopoietic stem cells, organs, tissues, cells, or stem cells.

[0108] A fifteenth aspect of the present disclosure provides a method for diagnosing a disease associated with CMV, the method comprising the steps of obtaining a sample, contacting the sample with a T cell antigen receptor according to the present disclosure, and detecting a complex formed by CMVpp65 and the T cell antigen receptor.

[0109] Preferably, the T cell antigen receptor comprises a detectable tag.

[0110] The TCRs disclosed herein specifically recognize the corresponding CMVpp65 antigenic peptide-MHC molecule complex, activate TCR T cells, and produce high levels of the cytokines IFNγ, IL2, and TNFα, resulting in significant tumor cell killing in both in vivo and in vitro studies.

[0111] The "T cell antigen receptor" according to the present disclosure is a molecule capable of recognizing a peptide when presented by an MHC molecule or its tetramer, and is usually present on the surface of a T cell in a complex with a CD3 molecule. The TCR of most T cells consists of an α peptide chain and a β peptide chain, while the TCR of a minority of T cells consists of a γ peptide chain and a δ peptide chain.

[0112] "Tumor" according to the present disclosure includes, but is not limited to, pancreatic cancer, liver cancer, colon cancer, rectal cancer, gastric cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, malignant melanoma, myelodysplastic syndrome, and sarcoma. The leukemia is selected from acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia. The lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia. The sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. Preferably, the tumor is selected from pancreatic cancer, liver cancer, oral squamous cell carcinoma, colon cancer, ovarian cancer, and gastric cancer. In one specific embodiment of the present disclosure, the tumor is a lymphoma.

[0113] "CMV-associated diseases" according to the present disclosure include diseases caused by CMVpp65 infection, such as neonatal CMV inclusion disease, which in severe cases can impair bodily functions, affecting the liver, spleen, and central nervous system. It also includes acute acquired CMV infections, such as infectious mononucleosis, which can include fever, muscular pain, and other symptoms. It also includes CMV retinitis, CMV gastroenteritis, or encephalitis caused by infection in immunocompromised patients, such as organ transplant recipients and those infected with HIV.

[0114] In accordance with the present disclosure, "comprising," when used in the present disclosure to describe a protein or nucleic acid sequence, means that the protein or nucleic acid may consist of the sequence or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid and also have an activity in accordance with the present disclosure.

[0115] "Prevention" in the present disclosure refers to any action that suppresses symptoms or delays the onset of a particular symptom by administering a product in accordance with the present disclosure.

[0116] For purposes of this disclosure, "diagnosis" means determining whether a patient has had, is at the time of diagnosis, or will have a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression or likelihood of future progression of a disease, or assessing a patient's response to treatment.

[0117] For purposes of this disclosure, "treatment" means slowing, interrupting, preventing, controlling, halting, mitigating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily mean the complete elimination of all signs, symptoms, disorders, or disorders associated with a disease, and may also mean therapeutic intervention to ameliorate signs, symptoms, etc. of a disease or condition that has already begun to develop.

[0118] An "effective amount," according to the present disclosure, refers to a dose or dosage of a product according to the present disclosure that is capable of providing the desired treatment or prevention after administration to a patient or organ in a single dose or multiple doses.

[0119] The "products" of the present disclosure include, but are not limited to, the antibodies or antigen-binding fragments thereof of the present disclosure, the T cell antigen receptors, the nucleic acids, the expression vectors, the host cells, the immune cells, the multimeric complexes, other adjuvants of the above products, and reagents used in combination with the above products.

[0120] An "article of manufacture" according to the present disclosure may be a kit, a microarray, an antibody conjugate, a pharmaceutical composition such as a multifunctional antibody, or the like.

[0121] A "subject" according to the present disclosure includes, but is not limited to, a human or non-human mammal. Preferably, the non-human mammal includes, but is not limited to, a mouse, rat, monkey, pig, rabbit, etc.

[0122] "Homology" in the present disclosure means that, in using an amino acid sequence or a nucleotide sequence, a person skilled in the art can adjust the sequence according to the actual working needs without changing the main structure or function of the original sequence, and in comparing the sequence used in that case with the specific sequence in the present disclosure, the degree of "homology" is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, This refers to, but is not limited to, 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%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% homology. For example, the phrase "having at least 80% identity with the amino acid sequence set forth in any one of SEQ ID NOs:1-12 and 14-73" in the present disclosure refers to a sequence that retains the ability to bind to the peptide epitope / MHC complex of the cytomegalovirus (CMV) phosphoprotein pp65. As long as the sequence retains 80% or more identity and the ability to bind to the pp65 epitope / MHC complex or pp65 epitope / MHC molecule tetramer, the amino acid sequence can be adjusted according to actual operational needs. For example, the amino acid sequence may include one or more modifications, such as substitution, deletion, and / or insertion of one or more amino acids, truncation, or extension of one or both ends. The modifications may be substitutions, additions, or deletions. Examples of such modifications include substitutions between amino acids of the same polarity, substitutions between amino acids of the same charge, substitutions between uncharged amino acids, substitutions between aliphatic amino acids, substitutions between aromatic amino acids, substitutions between nonpolar amino acids, and substitutions between amino acids with related side chain properties. Basic side chains include, but are not limited to, lysine, arginine, and histidine. Acidic side chains include, but are not limited to, aspartic acid and glutamic acid.Uncharged amino acids include, but are not limited to, asparagine, glutamine, serine, threonine, and tyrosine. Nonpolar side chains include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. At least 80% includes, but is not limited to, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0123] Compared with the prior art, the present disclosure has the following beneficial technical effects:

[0124] 1. We isolated a TCR that recognizes and binds to the CMV-pp65 antigen epitope polypeptide fragment. T cells transfected with this TCR have the ability to recognize the virus and virus-infected cells, initiate an immune response, and specifically kill target cells.

[0125] 2. The TCR disclosed herein has a novel and specific CDR3α / β structure, forming a two-chain structure that naturally pairs. Furthermore, the present disclosure utilizes single-cell sequencing to construct a library of cellular VDJ fragments, perform multiplex sequencing, and compare the sequences to select effective sequences.

[0126] 3. The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding the TCR, a vector comprising the nucleic acid molecule, a cell into which the nucleic acid molecule or the vector has been introduced, a pharmaceutical composition comprising the TCR, nucleic acid molecule, vector, or cell as an active ingredient, and uses of the TCR, nucleic acid molecule, vector, cell, or pharmaceutical composition, which are of pharmaceutical and industrial value. [Brief explanation of the drawings]

[0127] [Figure 1] FIG. 1 is a schematic diagram showing the element structure of a TCR sequence. [Figure 2]1 shows the structure of the transfer plasmid in Example 2. [Figure 3] 1 shows the results of selecting TCR-introduced T cells in Example 2. [Figure 4] 1 shows the results of selecting TCR-introduced T cells in Example 5. [Figure 5] 1 shows the results of selecting TCR-introduced T cells in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0128] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as understood by those skilled in the art. Reference may be made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin, "Genes VIII"; Roitt et al., "Immunology" (8th Edition), and the general prior art cited herein. It should be noted that, unless otherwise specified, all methods, steps, techniques, and operations not described in detail can be and have been performed in a known manner as understood by those skilled in the art. Reference may also be made, for example, to standard manuals, the general prior art mentioned above, and other documents cited therein.

[0129] The present disclosure will be described below with reference to examples, but the present disclosure is not limited to the examples. [Example]

[0130] Example 1 CMV-pp65 341-349 Cultivation, detection and sequencing of specific T cells Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors (the HLA type of the healthy human donor can be any, and in this example, the HLA-A*2402 genotype of HLA-A*24 is used as an example) are stimulated in vitro with a chemically synthesized short peptide C1 (amino acid sequence QYDPVAALF (SEQ ID NO: 13)) to induce and culture CD4-CD8+ CTL cells that can recognize the antigen peptide C1 and secrete IFN-γ. Specifically, the process involves the following steps:

[0131] 1) Experimental group: X-C1 cell culture obtained by culturing PBMCs of a donor stimulated with antigen peptide C1 was used as the experimental group. Control group: A donor's PBMC cell culture cultured in parallel under the same conditions as the experimental group without stimulation with polypeptides serves as the control group. 2) For cytokine secretion detection, both groups of cells were stimulated again with the antigen peptide C1, incubated, and observed for the presence of CD8+IFN-γ+ double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN-γ. In subsequent experiments, monoclonal cell lines were isolated and selected from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN-γ.

[0132] 1. Isolation and Selection of Monoclonal Cell Lines EBV-LCL cells: Lymphoblastoid cell lines (LCL cell lines) are immortalized cell lines obtained by in vitro infection of human-derived PBMCs with EB virus. Different HLA-restricted LCL cell lines can be prepared from different HLA-restricted PBMCs.

[0133] The construction of immortalized human-derived B lymphocyte LCLs by EB virus infection specifically involves the following steps.

[0134] 1) PBMCs were isolated from peripheral blood and suspended in 2 ml of RPMI1640 medium containing 10% FBS (abbreviated as RPMI / 10%FBS; RPM1640 manufacturer: ThermoFisherScientific, catalog number: 22400-089; FBS manufacturer: ThermoFisherScientific, catalog number: 10099-141C).

[0135] 2) 10 μL of the cell solution was aspirated and diluted 10-fold with 90 μL of RPMI / 10% FBS, and the cells were counted under a microscope.

[0136] 3) Based on the counting results, calculate the required volume of B95-8 (purchased from the Cell Library of the Chinese Academy of Sciences, catalog number GNO3) supernatant (1 x 10 6 (corresponding to 500 μL of B95-8 supernatant per 100 PBMC cells).

[0137] 4) 10 ml of B95-8 cells (ATCC) were cultured at an initial density of 1 x 10 2 days before the start of the experiment. 6 After culturing at a concentration of 1000 cells / ml for 48 hours in an incubator at 37°C and 5% CO2, the supernatant of the B95-8 cells was aspirated into a centrifuge tube and centrifuged at 2000 rpm for 15 minutes.

[0138] 5) The supernatant of the B95-8 cells in the centrifuge tube was filtered through a 0.45 μm filter membrane.

[0139] 6) After centrifugation at 1000 rpm for 5 minutes, the PBMC supernatant was discarded and the PBMC cells were collected.

[0140] 7) Based on the cell counting results, the concentration of PBMC cells in the cell solution was 2 × 10 6 An appropriate amount of B95-8 cell supernatant was added to resuspend the PBMC cells to a concentration of 1 / ml. Five ml of RPMI-10 (RPMI1640 medium containing 10% FBS) containing 1 μg / ml of cyclosporine A was then added and mixed well. The cell suspension was then poured into a 25 cm 2 The cells were then transferred to a culture flask and cultured in an incubator at 37°C and 5% CO2 for 3 weeks.

[0141] 8) At the end of the 3-week culture period, the medium became acidic, the cells formed visible clusters, the cell volume increased, and the cells tended to form distinct, usually hairy, and dense clusters of varying sizes, indicating immortalization of B cells by EBV.

[0142] X-C1 cells, an experimental group of CD4-CD8+ CTL cells capable of recognizing the antigen peptide and secreting IFN-γ, obtained in step 1, were diluted to 0.3 cells / 2 μl and then plated onto Terasaki plates (0–0.3 cells / well). Furthermore, UV-irradiated EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an immortalized cell line obtained by in vitro infection of human-derived PBMCs with EB virus) were used as feeder cells to stimulate and proliferate the CTL-positive T cells. The UV-irradiated EBV-LCL cells were used as feeder cells to stimulate and proliferate the CTL-positive T cells. ... 2 After 5-7 days of static culture, monoclonal cell proliferation was confirmed under a microscope. Monoclonal cell clusters were isolated and transferred to a U-bottom 96-well plate, where they were further expanded and cultured in RPMI1640 medium containing 30 ng / ml OKT3 and 3000 IU / ml IL-2. The cell cultures were supplemented with the solution, replaced with a new solution, or replaced with a new culture vessel every 3-4 days, and irradiated EBV-LCL feeder cells were added again every 30-40 days to stimulate the cells and maintain their specificity. In this way, when the cell volume reached 10 7 We obtained T cell monoclonal cell lines with a viability of 80% to 90%. During the expansion culture, a certain amount of cells was periodically sampled and cytokine secretion was detected to confirm the presence of cytotoxic CTL cells.

[0143] 2. TCR Sequencing and Construction Samples were collected from the monoclonal cell cultures and subjected to TCR sequencing, resulting in the following TCR sequences:

[0144] The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0145] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CARNTGKLIF (SEQ ID NO: 3), CAPSASKIIF (SEQ ID NO: 22), CAPQFNKFYF (SEQ ID NO: 31) CDR1β: any one of SQVTM (SEQ ID NO: 4), SGHVS (SEQ ID NO: 14), and LNHDA (SEQ ID NO: 23) CDR2β: any one of ANQGSEA (SEQ ID NO: 5), FQNEAQ (SEQ ID NO: 15), and SQIVND (SEQ ID NO: 24) CDR3β: CSANPTGGGTEAFF (SEQ ID NO: 6), CASSLLTRTETQYF (SEQ ID NO: 16), or CASSTTGLAGGPGNEQFF (SEQ ID NO: 25) The amino acid sequences of CDR1α to CDR3α of the α chain of one of the TCRs and CDR1β to CDR3β of the β chain of the TCR are shown below.

[0146] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF(SEQ ID NO:3) CDR1β: SQVTM (SEQ ID NO: 4) CDR2β: ANQGSEA (SEQ ID NO: 5) CDR3β:CSANPTGGGTEAFF(SEQ ID NO:6) The amino acid sequences of CDR1α to CDR3α of the α chain of another TCR and CDR1β to CDR3β of the β chain of another TCR are shown below.

[0147] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF(SEQ ID NO:3) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β: FQNEAQ (SEQ ID NO: 15) CDR3β:CASSLLTRTETQYF(SEQ ID NO:16) The amino acid sequences of CDR1α to CDR3α of the α chain of another TCR and CDR1β to CDR3β of the β chain of another TCR are shown below.

[0148] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CAPSASKIIF(SEQ ID NO:22) CDR1β: LNHDA (SEQ ID NO: 23) CDR2β: SQIVND (SEQ ID NO: 24) CDR3β:CASSTTGLAGGPGGNEQFF(SEQ ID NO:25) The amino acid sequences of CDR1α to CDR3α of the α chain of another TCR and CDR1β to CDR3β of the β chain of another TCR are shown below.

[0149] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CAPQFNKFYF(SEQ ID NO:31) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β: FQNEAQ (SEQ ID NO: 15) CDR3β:CASSLLTRTETQYF (SEQ ID NO:16).

[0150] The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are specifically shown in Table 1 below.

[0151] [Table 1]

[0152] The present invention involves obtaining CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of a TCR in Groups 1 to 4 of Table 1, and then obtaining paired alpha and beta sequences of the TCR. Specifically, these can be obtained by, but are not limited to, sequencing, bioinformatics, etc.

[0153] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 1 in Table 1 are as follows:

[0154] TCR alpha sequence (amino acid sequence is SEQ ID NO: 9), TCR beta sequence (amino acid sequence is SEQ ID NO: 10) The TCR alpha sequence contains the variable region Vα (amino acid sequence SEQ ID NO:7), and the TCR beta sequence contains the variable region Vβ (amino acid sequence SEQ ID NO:8).

[0155] The variable region Vα contains the complementarity-determining regions CDR1α: SSNFYA, CDR2α: MTLNGDE, and CDR3α: CARNTGKLIF (SEQ ID NOs: 1-3), and the variable region Vβ contains the complementarity-determining regions CDR1β: SQVTM, CDR2β: ANQGSEA, and CDR3β: CSANPTGGGTEAFF (SEQ ID NOs: 4-6).

[0156] A guide sequence can also be added before the above TCR alpha sequence and TCR beta sequence, and after linking via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:12) was obtained.

[0157] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 2 in Table 1 are as follows:

[0158] TCR alpha sequence (amino acid sequence is SEQ ID NO: 19), TCR beta sequence (amino acid sequence is SEQ ID NO: 20) The variable region Vα of the TCR alpha sequence contains complementarity-determining regions CDR1α: SSNFYA (SEQ ID NO: 1), CDR2α: MTLNGDE (SEQ ID NO: 2), and CDR3α: CARNTGKLIF (SEQ ID NO: 3), while the variable region Vβ contains complementarity-determining regions CDR1β: SGHVS (SEQ ID NO: 14), CDR2β: FQNEAQ (SEQ ID NO: 15), and CDR3β: CASSLLTRTETQYF (SEQ ID NO: 16).

[0159] After linking the above TCR alpha sequence and TCR beta sequence via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:21) was obtained.

[0160] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 3 in Table 1 are as follows:

[0161] TCR alpha sequence (amino acid sequence is SEQ ID NO: 28), TCR beta sequence (amino acid sequence is SEQ ID NO: 29) The variable region Vα of the TCR alpha sequence contains complementarity-determining regions CDR1α: SSNFYA (SEQ ID NO: 1), CDR2α: MTLNGDE (SEQ ID NO: 2), and CDR3α: CAPSASKIIF (SEQ ID NO: 22), while the variable region Vβ contains complementarity-determining regions CDR1β: LNHDA (SEQ ID NO: 23), CDR2β: SQIVND (SEQ ID NO: 24), and CDR3β: CASSTTGLAGGPGNEQFF (SEQ ID NO: 25).

[0162] After the above TCR alpha sequence and TCR beta sequence were linked via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:30) was obtained.

[0163] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 4 in Table 1 are as follows:

[0164] TCR alpha sequence (amino acid sequence is SEQ ID NO: 35), TCR beta sequence (amino acid sequence is SEQ ID NO: 36) The variable region Vα of the TCR alpha sequence contains the complementarity determining regions CDR1α: SSNFYA (SEQ ID NO: 1), CDR2α: MTLNGDE (SEQ ID NO: 2), and CDR3α: CAPQFNKFYF (SEQ ID NO: 31), while the variable region Vβ contains the complementarity determining regions CDR1β: SGHVS (SEQ ID NO: 14), CDR2β: FQNEAQ (SEQ ID NO: 15), and CDR3β: CASSLLTRTETQYF (SEQ ID NO: 16).

[0165] After linking the above TCR alpha sequence and TCR beta sequence via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:36) was obtained.

[0166] We then constructed a full-length TCR containing the constant region and inserted it into a lentiviral vector. The element composition of the TCR sequence is shown in Figure 1.

[0167] For the sake of convenience, Examples 2 and 3 will be described using as an example the full-length TCR sequence obtained based on the amino acid sequence of Group 1.

[0168] Example 2 CMV-pp65 341-349 Preparation of specific TCR gene-modified T cells The TCR gene sequence obtained in Example 1 was cloned into a lentiviral vector and transfected into 293T cells, a virus packaging cell line, to prepare a viral solution. This viral solution was then introduced into T cells to obtain T cells expressing the target TCR sequence.

[0169] Transfection was carried out as follows. 293T cells were cotransfected with the gag / pol packaging plasmid, VSV-G envelope plasmid, and a transfer construct containing the following lentiviral vector sequence to prepare lentiviral supernatant. Briefly, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and then mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After 15 minutes of incubation at room temperature, the resulting mixture was added to 293T cells. The lentivirus-containing medium was collected within 24 hours after transfection. Each time, the supernatant was filtered through a 0.45 μm filter. The collected lentivirus was combined, stored at 4°C, and then ultracentrifuged at 20,000 x g for 90 minutes. The lentiviral particles were resuspended in PBS to obtain the lentiviral supernatant. The structure of the transfer plasmid is shown in Figure 2.

[0170] The introduction took place as follows: Donor blood was collected on day 0, and PBMCs were isolated. PBMCs were activated with CD3 / CD28 Dynabeads (Gibco) magnetic beads for 2 days (magnetic beads:cells = 3:1), and then cultured in X-VIVO15 serum-free medium (LONZA) containing 200 IU / mL IL-2 at 1 × 10 6 The cells were resuspended at 3 × 10 cells / ml. On day 2, the lentiviral supernatant was used to transduce T cells, which were then centrifuged at 1200 × g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed. The cells were then resuspended at 3 × 10 cells / ml in medium containing rhIL-2 (200 IU / mL). 5The cells were suspended at 1000 ng / ml and supplemented with X-VIVO15 serum-free medium containing IL-2 (200 IU / mL) every 2 to 3 days. The TCR expression level, i.e., the transduction positive rate, was detected by flow cytometry on the day before collection, and the results are shown in Figure 3. Non-transduced cells (NC) did not express specific TCRs. T cells transduced with lentiviral vectors containing the first, second, third, and fourth group TCRs of Example 1 expressed specific TCRs (CD8 + Tetramer + The positive rates for each group were as follows: Group 1 had a TCR transduction positive rate of 30.36%, Group 2 had a TCR transduction positive rate of 9.63%, Group 3 had a TCR transduction positive rate of 8.63%, and Group 4 had a TCR transduction positive rate of 3.28%, indicating that the transduction was successful.

[0171] Example 3 CMV-pp65 341-349 In vitro functional validation of specific TCR gene-modified T cells In the present invention, the function of TCR-T cells can be evaluated in vitro by constructing target cells that overexpress the CMV-PP65 protein, to which QYDPVAALF belongs. Specifically, this was performed as follows.

[0172] 1. Effector Cell Preparation Effector cells were T cells expressing the TCR sequence of interest prepared in Example 2. Cells transfected with and expressed TCR lentivirus were designated the TCR group, and untransfected cells were designated the control group (NC group).

[0173] 2. Preparation of Overexpressing Target Cells In the present invention, the target cells are tumor cells that overexpress the CMV-PP65 protein to which QYDPVAALF belongs, and are constructed by introducing a gene for a target antigen into the tumor cells.

[0174] A lentivirus containing the target antigen is introduced into tumor cells, and one day later, the medium is replaced with complete medium and cultured for 1-2 days. After that, the medium is replaced with complete medium containing puromycin and culture is continued, and the expression of the target antigen by the tumor cells in the transfected group is detected. Specifically, the steps include the following:

[0175] 1) Introducing the target antigen gene into the lentivirus Tumor cells were cultured in complete medium (RPMI 1640 medium (Gibco) containing 10% FBS, the same medium used for OS-RC-2 human renal carcinoma cells) at a density of 4–6 × 10 cells / ml. 1 ml of the medium was added to each well of a 6-well plate. 8 μl of Polybrene (1 mg / ml) was added to each well for a final concentration of 8 μg / ml. Additionally, 10–30 μl of a lentiviral vector containing the target antigen, QYDPVAALF, was added to each well. Control wells (untransfected cells) were then added with 1 ml of the tumor cells and 8 μl of Polybrene. After uniform mixing, the wells were placed in a CO2 incubator (37°C, 5% CO2) and cultured for 1 day. The supernatant was discarded, and 2 ml of complete medium was added to each well, followed by incubation in a CO2 incubator for 2 days.

[0176] 2) Puromycin selection of transfected tumor cells The supernatant was discarded, and 2 ml of complete medium containing 1 μg / ml puromycin (Solarbio) was added to each well, followed by incubation in a CO 2 incubator. The cells were observed every 2 days, the complete medium containing 1 μg / ml puromycin was replaced, and the adherent cells were passaged when they had fully grown. Selection was performed by culturing for 7 days in complete medium containing 1 μg / ml puromycin. If there were still viable cells in the control group, the concentration of puromycin should be increased and the culture continued until all cells in the control group had died.

[0177] 3) Single cloning of cells containing the antigen of interest Tumor cells containing the target antigen were cultured for 7 days or more and then seeded at 1 cell / well or 3 cells / well by limiting dilution. They were cultured in complete medium containing puromycin, and the cells in the cell wells were observed. Monoclonal wells were labeled, and the cell volume was at least 6 × 10 7 The cells were cultured until they reached more than 1000 cells, and then frozen to obtain overexpressing target cells.

[0178] 3. In Vitro Killing Experiments OS-RC-2-PP65 cells overexpressing PP65 protein prepared in step 2 were used as target cells and seeded at 5 x 104 cells per well. Effector cells from the TCR group and effector cells from the NC group in step 1 were mixed with target cells at effector / target ratios of 1:1, 5:1, 10:1, and 20:1, respectively, and co-cultured at 37°C. The killing efficiency was determined using a label-free real-time cell analyzer (xCELLigence@Real Time Cell Analyzer, RTCA, Agilent).

[0179] Killing efficiency was assessed by collecting data 4 hours after effector cell plating. Specific killing results are shown in Table 2.

[0180] [Table 2]

[0181] Example 4 CMV-pp65 495-503 Culturing and sequencing of specific T cells 1. Antigen-specific T cells Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors (the HLA type of the healthy human donor can be any; in this example, the HLA-A*02 genotype, HLA-A*0201, is used as an example) are stimulated in vitro with chemically synthesized antigen peptide C1 (amino acid sequence NLVPMVATV (SEQ ID NO: 46)) to induce and culture CD4-CD8+ CTL cells that can recognize antigen peptide C1 and secrete IFN-γ. Specifically, the process involves the following steps:

[0182] 3) Experimental group: X-C1 cell culture obtained by culturing PBMCs of a donor stimulated with antigen peptide C1 was used as the experimental group. Control group: A donor's PBMC cell culture cultured in parallel under the same conditions as the experimental group without stimulation with polypeptides serves as the control group.

[0183] 4) For cytokine secretion detection, both groups of cells were stimulated again with the antigen peptide C1, incubated, and observed for the presence of CD8+IFN-γ+ double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN-γ. In subsequent experiments, monoclonal cell lines were isolated and selected from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN-γ.

[0184] 2. Isolation and Selection of Monoclonal Cell Lines EBV-LCL cells: Lymphoblastoid cell lines (LCL cell lines) are immortalized cell lines obtained by in vitro infection of human-derived PBMCs with EB virus. Different HLA-restricted LCL cell lines can be prepared from different HLA-restricted PBMCs. The construction of immortalized human-derived B lymphocyte LCLs by EB virus infection specifically involves the following steps.

[0185] 9) PBMCs were isolated from peripheral blood and suspended in 2 ml of RPMI1640 medium containing 10% FBS (abbreviated as RPMI / 10%FBS; RPM1640 manufacturer: ThermoFisherScientific, catalog number: 22400-089; FBS manufacturer: ThermoFisherScientific, catalog number: 10099-141C).

[0186] 10) 10 μL of the cell solution was aspirated and diluted 10-fold with 90 μL of RPMI / 10% FBS, and the cells were counted under a microscope.

[0187] 11) Based on the counting results, calculate the required volume of B95-8 (purchased from the Cell Library of the Chinese Academy of Sciences, catalog number GNO3) supernatant (1 x 10 6 (corresponding to 500 μL of B95-8 supernatant per 100 PBMC cells).

[0188] 12) 10 ml of B95-8 cells (ATCC) were cultured at an initial density of 1 x 10 2 days before the start of the experiment. 6 After culturing at a concentration of 1000 cells / ml for 48 hours in an incubator at 37°C and 5% CO2, the supernatant of the B95-8 cells was aspirated into a centrifuge tube and centrifuged at 2000 rpm for 15 minutes.

[0189] 13) The supernatant of the B95-8 cells in the centrifuge tube was filtered through a 0.45 μm filter membrane.

[0190] 14) After centrifugation at 1000 rpm for 5 minutes, the PBMC supernatant was discarded and the PBMC cells were collected.

[0191] 15) Based on the cell counting results, the concentration of PBMC cells in the cell solution was 2 x 10 6 An appropriate amount of B95-8 cell supernatant was added to resuspend the PBMC cells to a concentration of 1 / ml. Five ml of RPMI-10 (RPMI1640 medium containing 10% FBS) containing 1 μg / ml of cyclosporine A was then added and mixed well. The cell suspension was then poured into a 25 cm 2 The cells were then transferred to a culture flask and cultured in an incubator at 37°C and 5% CO2 for 3 weeks.

[0192] 16) At the end of the 3-week culture period, the medium became acidic, the cells formed visible clusters, the cell volume increased, and the cells tended to form distinct, usually hairy, and tightly packed clusters of varying sizes, indicating immortalization of B cells by EBV.

[0193] X-C1 cells, an experimental group of CD4-CD8+ CTL cells capable of recognizing the antigen peptide and secreting IFN-γ, obtained in step 1, were diluted to 0.3 cells / 2 μl and then plated onto Terasaki plates (0–0.3 cells / well). Furthermore, UV-irradiated EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an immortalized cell line obtained by in vitro infection of human-derived PBMCs with EB virus) were used as feeder cells to stimulate and proliferate the CTL-positive T cells. The UV-irradiated EBV-LCL cells were used as feeder cells to stimulate and proliferate the CTL-positive T cells. ... 2 After 5-7 days of static culture, monoclonal cell proliferation was confirmed under a microscope. Monoclonal cell clusters were isolated and transferred to a U-bottom 96-well plate, where they were further expanded and cultured in RPMI1640 medium containing 30 ng / ml OKT3 and 3000 IU / ml IL-2. The cell cultures were supplemented with the solution, replaced with a new solution, or replaced with a new culture vessel every 3-4 days, and irradiated EBV-LCL feeder cells were added again every 30-40 days to stimulate the cells and maintain their specificity. In this way, when the cell volume reached 10 7 We obtained T cell monoclonal cell lines with a viability of 80% to 90%. During the expansion culture, a certain amount of cells was periodically sampled and cytokine secretion was detected to confirm the presence of cytotoxic CTL cells.

[0194] 3. TCR Sequencing and Construction Samples were collected from the monoclonal cell cultures and subjected to TCR sequencing, resulting in the following TCR sequences:

[0195] The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0196] CDR1α: SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 47), TSGFNG (SEQ ID NO: 58) CDR2α: any one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 48), and NVLDGL (SEQ ID NO: 59) CDR3α: CASINFNKFYF (SEQ ID NO: 37), CAEFTGTASKLTF (SEQ ID NO: 49), CAVTYNNARLMF (SEQ ID NO: 60), CARNYGQNFVF (SEQ ID NO: 69) CDR1β: any one of MDHEN (SEQ ID NO: 38), GTSNPN (SEQ ID NO: 50), MNHEY (SEQ ID NO: 61), and DFQATT (SEQ ID NO: 70) CDR2β: SYDVKM (SEQ ID NO: 39), SVGIG (SEQ ID NO: 51), SMNVEV (SEQ ID NO: 62), SNEGSKA (SEQ ID NO: 71) CDR3β: CASP LNGGATEAFF (SEQ ID NO: 40), CAWSDRAAFTDTQYF (SEQ ID NO: 52), CASSSVAGGRIEQFF (SEQ ID NO: 63), CSARDIKAQQWNIQYF (SEQ ID NO: 72) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0197] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CASINFNKFYF(SEQ ID NO:37) CDR1β: MDHEN (SEQ ID NO: 38) CDR2β: SYDVKM (SEQ ID NO: 39) CDR3β:CASSPLNGGATEAFF(SEQ ID NO:40) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0198] CDR1α: DSSSTY (SEQ ID NO: 47) CDR2α: IFSNMDM (SEQ ID NO: 48) CDR3α:CAEFTGTASKLTF(SEQ ID NO:49) CDR1β: GTSNPN (SEQ ID NO: 50) CDR2β: SVGIG (SEQ ID NO: 51) CDR3β:CAWSDRAAFTDTQYF(SEQ ID NO:52) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0199] CDR1α: TSGFNG (SEQ ID NO: 58) CDR2α: NVLDGL (SEQ ID NO: 59) CDR3α:CAVTYNNARLMF(SEQ ID NO:60) CDR1β: MNHEY (SEQ ID NO: 61) CDR2β: SMNVEV (SEQ ID NO: 62) CDR3β:CASSSVAGGRIEQFF(SEQ ID NO:63) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0200] CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNYGQNFVF(SEQ ID NO:69) CDR1β: DFQATT (SEQ ID NO: 70) CDR2β: SNEGSKA (SEQ ID NO: 71) CDR3β:CSARDIKAQQWNIQYF(SEQ ID NO:72) The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are specifically shown in Table 3 below.

[0201] [Table 3]

[0202] The present invention involves obtaining CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of a TCR in Groups 5 to 8 of Table 3, and then obtaining paired TCR alpha and beta sequences. Specifically, these can be obtained by, but are not limited to, sequencing, bioinformatics, etc.

[0203] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 5 in Table 3 are as follows:

[0204] TCR alpha sequence (amino acid sequence is SEQ ID NO: 43), TCR beta sequence (amino acid sequence is SEQ ID NO: 44) The TCR alpha sequence contains the variable region Vα (amino acid sequence SEQ ID NO: 41), and the TCR beta sequence contains the variable region Vβ (amino acid sequence SEQ ID NO: 42).

[0205] The variable region Vα contains the complementarity-determining regions CDR1α: SSNFYA, CDR2α: MTLNGDE, and CDR3α: CASINFNKFYF (SEQ ID NOs: 1, 2, and 37), and the variable region Vβ contains the complementarity-determining regions CDR1β: MDHEN, CDR2β: SYDVKM, and CDR3β: CASSPLANGGATEAFF (SEQ ID NOs: 38-40).

[0206] The above TCR alpha sequence and TCR beta sequence could also be preceded by guide sequences, respectively, and after linking via a linking sequence (SEQ ID NO: 11), the full-length TCR sequence (SEQ ID NO: 45) was obtained.

[0207] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 6 in Table 3 are as follows:

[0208] TCR alpha sequence (amino acid sequence is SEQ ID NO: 55), TCR beta sequence (amino acid sequence is SEQ ID NO: 56) The variable region Vα of the TCR alpha sequence contains complementarity-determining regions CDR1α: DSSSTY (SEQ ID NO: 47), CDR2α: IFSNMDM (SEQ ID NO: 48), and CDR3α: CAEFTGTASKLTF (SEQ ID NO: 49), while the variable region Vβ contains complementarity-determining regions CDR1β: GTSNPN (SEQ ID NO: 50), CDR2β: SVGIG (SEQ ID NO: 51), and CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 52).

[0209] After the above TCR alpha sequence and TCR beta sequence were linked via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:57) was obtained.

[0210] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 7 in Table 3 are as follows:

[0211] TCR alpha sequence (amino acid sequence is SEQ ID NO: 66), TCR beta sequence (amino acid sequence is SEQ ID NO: 67) The variable region Vα of the TCR alpha sequence contains the complementarity determining regions CDR1α: TSGFNG (SEQ ID NO: 58), CDR2α: NVLDGL (SEQ ID NO: 59), and CDR3α: CAVTYNNARLMF (SEQ ID NO: 60), while the variable region Vβ contains the complementarity determining regions CDR1β: MNHEY (SEQ ID NO: 61), CDR2β: SMNVEV (SEQ ID NO: 62), and CDR3β: CASSSVAGGRIEQFF (SEQ ID NO: 63).

[0212] After linking the above TCR alpha sequence and TCR beta sequence via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:68) was obtained.

[0213] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 8 in Table 3 are as follows:

[0214] TCR alpha sequence (amino acid sequence is SEQ ID NO: 75), TCR beta sequence (amino acid sequence is SEQ ID NO: 76) The variable region Vα of the TCR alpha sequence contains complementarity-determining regions CDR1α: SSNFYA (SEQ ID NO: 1), CDR2α: MTLNGDE (SEQ ID NO: 2), and CDR3α: CARNYGQNFVF (SEQ ID NO: 69), while the variable region Vβ contains complementarity-determining regions CDR1β: DFQATT (SEQ ID NO: 70), CDR2β: SNEGSKA (SEQ ID NO: 71), and CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 72).

[0215] After the above TCR alpha sequence and TCR beta sequence were linked via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:77) was obtained.

[0216] Subsequently, a full-length TCR containing the constant region was constructed and inserted into a lentiviral vector. The element structure of the TCR sequence is shown in Figure 1. For ease of explanation, Examples 5 and 6 use the full-length TCR sequence obtained based on the amino acid sequence of Group 5 as an example.

[0217] Example 5 CMV-pp65 495-503 Preparation of specific TCR gene-modified T cells The TCR gene sequence obtained in Example 4 was cloned into a lentiviral vector and transfected into 293T cells, a virus packaging cell line, to prepare a viral solution. This viral solution was then introduced into T cells to obtain T cells expressing the target TCR sequence.

[0218] Transfection was carried out as follows. 293T cells were cotransfected with the gag / pol packaging plasmid, VSV-G envelope plasmid, and a transfer construct containing the following lentiviral vector sequence to prepare lentiviral supernatant. Briefly, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and then mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After 15 minutes of incubation at room temperature, the resulting mixture was added to 293T cells. The lentivirus-containing medium was collected within 24 hours after transfection. Each time, the supernatant was filtered through a 0.45 μm filter. The collected lentivirus was combined, stored at 4°C, and then ultracentrifuged at 20,000 x g for 90 minutes. The lentiviral particles were resuspended in PBS to obtain the lentiviral supernatant. The structure of the transfer plasmid is shown in Figure 2.

[0219] The introduction took place as follows: Donor blood was collected on day 0, and PBMCs were isolated. PBMCs were activated with CD3 / CD28 Dynabeads (Gibco) magnetic beads for 2 days (magnetic beads:cells = 3:1), and then cultured in X-VIVO15 serum-free medium (LONZA) containing 200 IU / mL IL-2 at 1 × 10 6 The cells were resuspended at 3 × 10 cells / ml. On day 2, the lentiviral supernatant was used to transduce T cells, which were then centrifuged at 1200 × g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed. The cells were then resuspended at 3 × 10 cells / ml in medium containing rhIL-2 (200 IU / mL). 5 The cells were suspended at 1000 ng / ml and supplemented with X-VIVO15 serum-free medium containing IL-2 (200 IU / mL) every 2-3 days. The TCR expression level, i.e., the transduction positive rate, was detected by flow cytometry on the day before collection, and the results are shown in Figure 4. Non-transduced cells (NC) did not express specific TCRs. T cells transduced with lentiviral vectors containing the 5th, 6th, 7th, and 8th TCRs of Example 4 expressed specific TCRs (CD8 + Tetramer + The positive rates for each group were as follows: Group 5 had a TCR transduction positive rate of 25.75%, Group 6 had a TCR transduction positive rate of 23.77%, Group 7 had a TCR transduction positive rate of 17.51%, and Group 8 had a TCR transduction positive rate of 23.02%, indicating that the transduction was successful.

[0220] Example 6 CMV-pp65 495-503 In vitro functional validation of specific TCR gene-modified T cells In the present invention, target cells overexpressing the CMV-PP65 protein, to which NLVP MVATV belongs, can be constructed to evaluate the function of TCR-T cells in vitro. Specifically, this was performed as follows.

[0221] 1. Effector Cell Preparation Effector cells were T cells expressing the TCR sequence of interest prepared in Example 5. Cells transfected with and expressed TCR lentivirus were designated the TCR group, and untransfected cells were designated the NC group.

[0222] 2. Preparation of Overexpressing Target Cells In the present invention, the target cells are tumor cells that overexpress the CMV-PP65 protein to which NLVP MVATV belongs, and are constructed by introducing a gene for a target antigen into the tumor cells.

[0223] A lentivirus containing the target antigen is introduced into tumor cells, and one day later, the medium is replaced with complete medium and cultured for 1-2 days. After that, the medium is replaced with complete medium containing puromycin and culture is continued, and the expression of the target antigen by the tumor cells in the transfected group is detected. Specifically, the steps include the following:

[0224] 1) Introducing the target antigen gene into the lentivirus Tumor cells were cultured in complete medium (McCoy's 5A medium (Gibco) containing 10% FBS, the same medium used for U-2OS human osteosarcoma cells) at a density of 4–6 × 10 cells / ml. 1 ml of this medium was added to each well of a 6-well plate. 8 μl of Polybrene (1 mg / ml) was added to each well for a final concentration of 8 μg / ml. Additionally, 10–30 μl of a lentiviral vector containing the target antigen, NLVPMVATV, was added to each well. Control wells (untransfected cells) were then cultured with 1 ml of the tumor cells and 8 μl of Polybrene. After uniform mixing, the cells were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 1 day. The supernatant was discarded, and 2 ml of complete medium was added to each well, followed by incubation in a CO2 incubator for 2 days.

[0225] 2) Puromycin selection of transfected tumor cells The supernatant was discarded, and 2 ml of complete medium containing 1 μg / ml puromycin (Solarbio) was added to each well, followed by incubation in a CO 2 incubator. The cells were observed every 2 days, the complete medium containing 1 μg / ml puromycin was replaced, and the adherent cells were passaged when they had fully grown. Selection was performed by culturing for 7 days in complete medium containing 1 μg / ml puromycin. If there were still viable cells in the control group, the concentration of puromycin should be increased and the culture continued until all cells in the control group had died.

[0226] 3) Single cloning of cells containing the antigen of interest Tumor cells containing the target antigen were cultured for 7 days or more and then seeded at 1 cell / well or 3 cells / well by limiting dilution. They were cultured in complete medium containing puromycin, and the cells in the cell wells were observed. Monoclonal wells were labeled, and the cell volume was at least 6 × 10 7 The cells were cultured until they reached more than 1000 cells, and then frozen to obtain overexpressing target cells.

[0227] 3. In Vitro Killing Experiments U-2OS-PP65 cells overexpressing PP65 protein prepared in step 2 were used as target cells, seeded at 5 × 10 cells per well. Effector cells from the TCR group and effector cells from the NC group prepared in step 1 were mixed with target cells at effector / target ratios of 1:1, 5:1, 10:1, and 20:1, respectively, and co-cultured at 37°C. The killing efficiency was determined using a label-free real-time cell analyzer (xCELLigence@Real Time Cell Analyzer, RTCA, Agilent). Label-free real-time cell analysis involves incorporating a microelectronic cell sensor chip into the bottom of a cell detection plate through a special process, creating a cell impedance sensing measurement system that dynamically and quantitatively tracks changes in cell morphology, proliferation, and differentiation in real time. Adherent cells growing on the surface of the microelectrodes change the electrical resistance between the electrodes. This change correlates with the real-time functional status of the cells. Therefore, dynamic, real-time monitoring of this electrical resistance can provide biological information related to cell physiology, such as cell proliferation, elongation, morphological changes, death, and adhesion strength. Killing efficiency was evaluated by collecting data 4 hours after seeding effector cells. Specific killing results are shown in Table 4.

[0228] [Table 4]

[0229] Example 7 CMV-pp65 501-509 Culturing and sequencing of specific T cells 1. Antigen-specific T cells Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors (the HLA type of the healthy human donor can be any; in this example, the HLA-A*11 genotype, HLA-A*1101, is used as an example) are stimulated in vitro with chemically synthesized antigen peptide C1 (amino acid sequence ATVQGQNLK (SEQ ID NO:89)) to induce and culture CD4-CD8+ CTL cells that can recognize antigen peptide C1 and secrete IFN-γ. Specifically, the process involves the following steps:

[0230] 5) Experimental group: X-C1 cell culture obtained by culturing PBMCs of a donor stimulated with antigen peptide C1 was used as the experimental group. Control group: A donor's PBMC cell culture cultured in parallel under the same conditions as the experimental group without stimulation with polypeptides serves as the control group.

[0231] 6) For cytokine secretion detection, both groups of cells were stimulated again with the antigen peptide C1, incubated, and observed for the presence of CD8+IFN-γ+ double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN-γ. In subsequent experiments, monoclonal cell lines were isolated and selected from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN-γ.

[0232] 2. Isolation and Selection of Monoclonal Cell Lines EBV-LCL cells: Lymphoblastoid cell lines (LCL cell lines) are immortalized cell lines obtained by in vitro infection of human-derived PBMCs with EB virus. Different HLA-restricted LCL cell lines can be prepared from different HLA-restricted PBMCs. The construction of immortalized human-derived B lymphocyte LCLs by EB virus infection specifically involves the following steps.

[0233] 17) PBMCs were isolated from peripheral blood and suspended in 2 ml of RPMI1640 medium containing 10% FBS (abbreviated as RPMI / 10%FBS; RPM1640 manufacturer: ThermoFisherScientific, catalog number: 22400-089; FBS manufacturer: ThermoFisherScientific, catalog number: 10099-141C).

[0234] 18) 10 μL of the cell solution was aspirated and diluted 10-fold with 90 μL of RPMI / 10% FBS, and the cells were counted under a microscope.

[0235] 19) Based on the counting results, calculate the required volume of B95-8 (purchased from the Cell Library of the Chinese Academy of Sciences, catalog number GNO3) supernatant (1 x 10 6 (corresponding to 500 μL of B95-8 supernatant per 100 PBMC cells).

[0236] 20) 10 ml of B95-8 cells (ATCC) were cultured at an initial density of 1 x 10 2 days before the start of the experiment. 6 After culturing at a concentration of 1000 cells / ml for 48 hours in an incubator at 37°C and 5% CO2, the supernatant of the B95-8 cells was aspirated into a centrifuge tube and centrifuged at 2000 rpm for 15 minutes.

[0237] 21) The supernatant of B95-8 cells in the centrifuge tube was filtered through a 0.45 μm filter membrane.

[0238] 22) After centrifugation at 1000 rpm for 5 minutes, the PBMC supernatant was discarded and the PBMC cells were collected.

[0239] 23) Based on the cell count results, the concentration of PBMC cells in the cell solution was 2 × 10 6 An appropriate amount of B95-8 cell supernatant was added to resuspend the PBMC cells to a concentration of 1 / ml. Five ml of RPMI-10 (RPMI1640 medium containing 10% FBS) containing 1 μg / ml of cyclosporine A was then added and mixed well. The cell suspension was then poured into a 25 cm 2 The cells were then transferred to a culture flask and cultured in an incubator at 37°C and 5% CO2 for 3 weeks.

[0240] 24) At the end of the 3-week culture period, the medium became acidic, the cells formed visible clusters, the cell volume increased, and the cells tended to form distinct, usually hairy, and tightly packed clusters of varying sizes, indicating immortalization of B cells by EBV.

[0241] X-C1 cells, an experimental group of CD4-CD8+ CTL cells capable of recognizing the antigen peptide and secreting IFN-γ, obtained in step 1, were diluted to 0.3 cells / 2 μl and then plated onto Terasaki plates (0–0.3 cells / well). Furthermore, UV-irradiated EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an immortalized cell line obtained by in vitro infection of human-derived PBMCs with EB virus) were used as feeder cells to stimulate and proliferate the CTL-positive T cells. The UV-irradiated EBV-LCL cells were used as feeder cells to stimulate and proliferate the CTL-positive T cells. ... 2 After 5-7 days of static culture, monoclonal cell proliferation was confirmed under a microscope. Monoclonal cell clusters were isolated and transferred to a U-bottom 96-well plate, where they were further expanded and cultured in RPMI1640 medium containing 30 ng / ml OKT3 and 3000 IU / ml IL-2. The cell cultures were supplemented with the solution, replaced with a new solution, or replaced with a new culture vessel every 3-4 days, and irradiated EBV-LCL feeder cells were added again every 30-40 days to stimulate the cells and maintain their specificity. In this way, when the cell volume reached 10 7 We obtained T cell monoclonal cell lines with a viability of 80% to 90%. During the expansion culture, a certain amount of cells was periodically sampled and cytokine secretion was detected to confirm the presence of cytotoxic CTL cells.

[0242] 3. TCR Sequencing and Construction Samples were collected from the monoclonal cell cultures and subjected to TCR sequencing, resulting in the following TCR sequences:

[0243] The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0244] CDR1α: VSGLRG (SEQ ID NO: 78), TSESDYY (SEQ ID NO: 90), or NSASQS (SEQ ID NO: 101) CDR2α: LYSAGEE (SEQ ID NO: 79), QEAYKQQN (SEQ ID NO: 91), or VYSSGN (SEQ ID NO: 102) CDR3α: any one of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), and CVVHSGGSYIPTF (SEQ ID NO: 103) CDR1β: any one of MNHNS (SEQ ID NO: 81), SGHDT (SEQ ID NO: 93), and MNHNY (SEQ ID NO: 104) CDR2β: any one of SASEGT (SEQ ID NO: 82), YYEEEE (SEQ ID NO: 94), and SVGAGI (SEQ ID NO: 105) CDR3β: any one of CASTINTYEQYF (SEQ ID NO: 83), CASSLIGPGDQPQHF (SEQ ID NO: 95), and CASAQTIGAYNEQFF (SEQ ID NO: 106) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0245] CDR1α: VSGLRG (SEQ ID NO: 78) CDR2α: LYSAGEE (SEQ ID NO: 79) CDR3α:CVITTSGTYKYIF(SEQ ID NO:80) CDR1β: MNHNS (SEQ ID NO: 81) CDR2β: SASEGT (SEQ ID NO: 82) CDR3β:CASTINTYEQYF(SEQ ID NO:83) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0246] CDR1α: TSESDYY (SEQ ID NO: 90) CDR2α: QEAYKQQN (SEQ ID NO: 91) CDR3α:CAYRSFYTGANSKLTF(SEQ ID NO:92) CDR1β: SGHDT (SEQ ID NO: 93) CDR2β: YYEEEE (SEQ ID NO: 94) CDR3β:CASSLYGGPGDQPQHF(SEQ ID NO:95) In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are shown below.

[0247] CDR1α: NSASQS (SEQ ID NO: 101) CDR2α: VYSSGN (SEQ ID NO: 102) CDR3α:CVVHSGGSYIPTF(SEQ ID NO:103) CDR1β: MNHNY (SEQ ID NO: 104) CDR2β: SVGAGI (SEQ ID NO: 105) CDR3β: CASAQTIGAYNEQFF (SEQ ID NO:106).

[0248] The amino acid sequences of CDR1α to CDR3α of the TCR α chain and CDR1β to CDR3β of the TCR β chain are specifically shown in Table 5 below.

[0249] [Table 5]

[0250] The present invention obtains CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of a TCR in Groups 9 to 11 of Table 5, and then obtains paired TCR alpha and beta sequences. Specifically, these can be obtained by, but are not limited to, sequencing, bioinformatics, etc.

[0251] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 9 in Table 5 are as follows:

[0252] TCR alpha sequence (amino acid sequence is SEQ ID NO: 86), TCR beta sequence (amino acid sequence is SEQ ID NO: 87) The TCR alpha sequence contains the variable region Vα (amino acid sequence SEQ ID NO:84), and the TCR beta sequence contains the variable region Vβ (amino acid sequence SEQ ID NO:85).

[0253] The variable region Vα contains the complementarity-determining regions CDR1α: VSGLRG, CDR2α: LYSAGEE, and CDR3α: CVITTSGTYKYIF (SEQ ID NOs: 78-80), and the variable region Vβ contains the complementarity-determining regions CDR1β: MNHNS, CDR2β: SASEGT, and CDR3β: CASTINTYEQYF (SEQ ID NOs: 81-83).

[0254] The above TCR alpha sequence and TCR beta sequence could also be preceded by guide sequences, respectively, and after linking via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:88) was obtained.

[0255] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 10 in Table 5 are as follows:

[0256] TCR alpha sequence (amino acid sequence is SEQ ID NO: 98), TCR beta sequence (amino acid sequence is SEQ ID NO: 99) The variable region Vα of the TCR alpha sequence contains the complementarity-determining regions CDR1α: TSESDYY, CDR2α: QEAYKQQN, and CDR3α: CAYRSFYTGANSKLTF (SEQ ID NOs: 90-92), and the variable region Vβ contains the complementarity-determining regions CDR1β: SGHDT, CDR2β: YYEEEE, and CDR3β: CASSLYGGPGDQPQHF (SEQ ID NOs: 93-95).

[0257] After the above TCR alpha sequence and TCR beta sequence were linked via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:100) was obtained.

[0258] The alpha and beta sequences of the paired TCRs obtained according to the amino acid sequences of Group 11 in Table 5 are as follows:

[0259] TCR alpha sequence (amino acid sequence is SEQ ID NO: 109), TCR beta sequence (amino acid sequence is SEQ ID NO: 110) The variable region Vα of the TCR alpha sequence contains complementarity-determining regions CDR1α: NSASQS, CDR2α: VYSSGN, and CDR3α: CVVHSGGSYIPTF (SEQ ID NOs: 101-103), and the variable region Vβ contains complementarity-determining regions CDR1β: MNHNY, CDR2β: SVGAGI, and CDR3β: CASAQTIGAYNEQFF (SEQ ID NOs: 104-106).

[0260] After the above TCR alpha sequence and TCR beta sequence were linked via a linking sequence (SEQ ID NO:11), the full-length TCR sequence (SEQ ID NO:111) was obtained.

[0261] We then constructed a full-length TCR containing the constant region and inserted it into a lentiviral vector. The element composition of the TCR sequence is shown in Figure 1.

[0262] For the sake of convenience, Examples 8 and 9 will be described using as an example the full-length sequence of a TCR obtained based on the amino acid sequence of Group 9.

[0263] Example 8 CMV-pp65 501-509 Preparation of specific TCR gene-modified T cells The TCR gene sequence obtained in Example 7 was cloned into a lentiviral vector and transfected into 293T cells, a virus packaging cell line, to prepare a viral solution. This viral solution was then introduced into T cells to obtain T cells expressing the target TCR sequence.

[0264] Transfection was carried out as follows. 293T cells were cotransfected with the gag / pol packaging plasmid, VSV-G envelope plasmid, and a transfer construct containing the following lentiviral vector sequence to prepare lentiviral supernatant. Briefly, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and then mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After 15 minutes of incubation at room temperature, the resulting mixture was added to 293T cells. The lentivirus-containing medium was collected within 24 hours after transfection. Each time, the supernatant was filtered through a 0.45 μm filter. The collected lentivirus was combined, stored at 4°C, and then ultracentrifuged at 20,000 x g for 90 minutes. The lentiviral particles were resuspended in PBS to obtain the lentiviral supernatant. The structure of the transfer plasmid is shown in Figure 2.

[0265] The introduction took place as follows: Donor blood was collected on day 0, and PBMCs were isolated. PBMCs were activated with CD3 / CD28 Dynabeads (Gibco) magnetic beads for 2 days (magnetic beads:cells = 3:1), and then cultured in X-VIVO15 serum-free medium (LONZA) containing 200 IU / mL IL-2 at 1 × 10 6The cells were resuspended at 3 × 10 cells / ml. On day 2, the lentiviral supernatant was used to transduce T cells, which were then centrifuged at 1200 × g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed. The cells were then resuspended at 3 × 10 cells / ml in medium containing rhIL-2 (200 IU / mL). 5 The cells were suspended at 1000 cells / ml and supplemented with X-VIVO15 serum-free medium containing IL-2 (200 IU / mL) every 2 to 3 days. The TCR expression level, i.e., the transduction positive rate, was detected by flow cytometry on the day before collection, and the results are shown in Figure 5. Non-transduced cells (NC) did not express specific TCRs. T cells transduced with lentiviral vectors containing Group 9 TCR, Group 10 TCR, and Group 11 TCR, respectively, as in Example 7, expressed specific TCRs (CD8 + Tetramer + The positive rates for each group were as follows: Group 9 had a TCR transduction positive rate of 59.09%, Group 10 had a TCR transduction positive rate of 14.38%, and Group 11 had a TCR transduction positive rate of 91.67%, indicating successful transduction.

[0266] Example 9 CMV-pp65 501-509 In vitro functional validation of specific TCR gene-modified T cells In the present invention, target cells overexpressing the CMV-PP65 protein, to which ATVQGQNLK belongs, were constructed, allowing the function of TCR-T cells to be evaluated in vitro. Specifically, this was performed as follows.

[0267] 1. Effector Cell Preparation Effector cells were T cells expressing the TCR sequence of interest prepared in Example 8. Cells transfected with and expressed TCR lentivirus were designated the TCR group, and untransfected cells were designated the NC group.

[0268] 2. Preparation of Overexpressing Target Cells In the present invention, the target cells are tumor cells that overexpress the CMV-PP65 protein, to which ATVQGQNLK belongs, and are constructed by introducing a gene for a target antigen into the tumor cells.

[0269] A lentivirus containing the target antigen is introduced into tumor cells, and one day later, the medium is replaced with complete medium and cultured for 1-2 days. After that, the medium is replaced with complete medium containing puromycin and culture is continued, and the expression of the target antigen by the tumor cells in the transfected group is detected. Specifically, the steps include the following:

[0270] 1) Introducing the target antigen gene into the lentivirus Tumor cells were cultured in complete medium (McCoy's 5A medium (Gibco) containing 10% FBS, the same medium used for Caki-2 clear cell renal carcinoma cells) at a density of 4–6 × 10 cells / ml. 1 ml of the medium was added to each well of a 6-well plate. 8 μl of Polybrene (1 mg / ml) was added to each well for a final concentration of 8 μg / ml. Additionally, 10–30 μl of a lentiviral vector containing the target antigen, ATVQGQNLK, was added to each well. Control wells (untransfected cells) were then cultured with 1 ml of the tumor cells and 8 μl of Polybrene. After uniform mixing, the cells were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 1 day. The supernatant was discarded, and 2 ml of complete medium was added to each well, followed by incubation in a CO2 incubator for 2 days.

[0271] 2) Puromycin selection of transfected tumor cells The supernatant was discarded, and 2 ml of complete medium containing 1 μg / ml puromycin (Solarbio) was added to each well, followed by incubation in a CO 2 incubator. The cells were observed every 2 days, the complete medium containing 1 μg / ml puromycin was replaced, and the adherent cells were passaged when they had fully grown. Selection was performed by culturing for 7 days in complete medium containing 1 μg / ml puromycin. If there were still viable cells in the control group, the concentration of puromycin should be increased and the culture continued until all cells in the control group had died.

[0272] 3) Single cloning of cells containing the antigen of interest Tumor cells containing the target antigen were cultured for 7 days or more and then seeded at 1 cell / well or 3 cells / well by limiting dilution. They were cultured in complete medium containing puromycin, and the cells in the cell wells were observed. Monoclonal wells were labeled, and the cell volume was at least 6 × 10 7 The cells were cultured until they reached more than 1000 cells, and then frozen to obtain overexpressing target cells.

[0273] 3. In Vitro Killing Experiments Caki-2-PP65 cells overexpressing PP65 protein prepared in step 2 were used as target cells, seeded at 5 × 10 cells per well. Effector cells from the TCR group and effector cells from the NC group prepared in step 1 were mixed with target cells at effector / target ratios of 1:1, 5:1, or 10:1, respectively, and co-cultured at 37°C. The killing efficiency was determined using a label-free real-time cell analyzer (xCELLigence@RealTimeCellAnalyzer, RTCA, Agilent). Label-free real-time cell analysis involves incorporating a microelectronic cell sensor chip into the bottom of a cell detection plate through a special process, creating a cell impedance sensing measurement system that dynamically and quantitatively tracks changes in cell morphology, proliferation, differentiation, and other changes in real time. Adherent cells growing on the surface of the microelectrodes change the electrical resistance between the electrodes. This change correlates with the real-time functional status of the cells. Therefore, dynamic, real-time monitoring of this electrical resistance can provide biological information related to cell physiology, such as cell proliferation, elongation, morphological changes, death, and adhesion strength. Killing efficiency was evaluated by collecting data 4 hours after seeding effector cells. Specific killing results are shown in Table 6.

[0274] [Table 6]

[0275] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various modifications and variations according to the present invention without departing from the spirit of the present invention, and all of them fall within the scope of protection of the claims of the present invention.

Claims

1. comprising CDR1α to CDR3α of the α chain and / or CDR1β to CDR3β of the β chain; CDR3α comprises any one of the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, SEQ ID NO: 69, SEQ ID NO: 80, SEQ ID NO: 92, and SEQ ID NO: 103, or any one of the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, SEQ ID NO: 69, SEQ ID NO: 80, SEQ ID NO: 92, and SEQ ID NO:

103. and / or comprising an amino acid sequence having at least 85% or more identity to any one of SEQ ID NO:103; CDR3β comprises an amino acid sequence set forth in any one of SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO: 95, and SEQ ID NO: 106, or any one of SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO: 95, and SEQ ID NO:

106. No. 106, and Preferably, CDR1α comprises an amino acid sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 78, SEQ ID NO: 90, and SEQ ID NO: 101, or an amino acid sequence having at least 85% or more homology with any one of SEQ ID NO: 1, SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 78, SEQ ID NO: 90, and SEQ ID NO: 101, and CDR2α comprises an amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 59, SEQ ID NO: 79, SEQ ID NO: 91, or SEQ ID NO:

102. or an amino acid sequence having at least 85% homology to any one of SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 59, SEQ ID NO: 79, SEQ ID NO: 91, and SEQ ID NO: 102, and CDR1β comprises an amino acid sequence represented by any one of SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 38, SEQ ID NO: 50, SEQ ID NO: 61, SEQ ID NO: 70, SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104, or an amino acid sequence having at least 85% homology to any one of SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 91, and SEQ ID NO:

102. and CDR2β comprises an amino acid sequence having at least 85% homology with any one of SEQ ID NO: 23, SEQ ID NO: 38, SEQ ID NO: 50, SEQ ID NO: 61, SEQ ID NO: 70, SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104, and CDR2β comprises an amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 39, SEQ ID NO: 51, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 105, or NO: 15, SEQ ID NO: 24, SEQ ID NO: 39, SEQ ID NO: 51, SEQSEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 82, SEQ ID NO: 94, SEQ ID NO:

105. A T cell antigen receptor characterized by:

2. The T cell antigen receptor according to claim 1, which specifically binds to CMVpp65.

3. The amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain are CDR1α: SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 47), TSGFNG (SEQ ID NO: 58), VSGLRG (SEQ ID NO: 78), TSESDYY (SEQ ID NO: 90), NSASQS (SEQ ID NO: 101), any one of CDR2α: any one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 48), NVLDGL (SEQ ID NO: 59), LYSAGEE (SEQ ID NO: 79), QEAYKQQN (SEQ ID NO: 91), and VYSSGN (SEQ ID NO: 102); CDR3α: any one of CARNTGKLIF (SEQ ID NO: 3), CAPSASKIIF (SEQ ID NO: 22), CAPQFNKFYF (SEQ ID NO: 31), CASINFNKFYF (SEQ ID NO: 37), CAEFTGTASKLTF (SEQ ID NO: 49), CAVTYNNARLMF (SEQ ID NO: 60), CARNYGQNFVF (SEQ ID NO: 69), CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), CVVHSGGSYIPTF (SEQ ID NO: 103); CDR1β: any one of SQVTM (SEQ ID NO: 4), SGHVS (SEQ ID NO: 14), LNHDA (SEQ ID NO: 23), MDHEN (SEQ ID NO: 38), GTSNPN (SEQ ID NO: 50), MNHEY (SEQ ID NO: 61), DFQATT (SEQ ID NO: 70), MNHNS (SEQ ID NO: 81), SGHDT (SEQ ID NO: 93), and MNHNY (SEQ ID NO: 104); CDR2β: any one of ANQGSEA (SEQ ID NO: 5), FQNEAQ (SEQ ID NO: 15), SQIVND (SEQ ID NO: 24), SYDVKM (SEQ ID NO: 39), SVGIG (SEQ ID NO: 51), SMNVEV (SEQ ID NO: 62), SNEGSKA (SEQ ID NO: 71), SASEGT (SEQ ID NO: 82), YYEEEE (SEQ ID NO: 94), and SVGAGI (SEQ ID NO: 105); CDR3β: CSANPTGGGTEAFF (SEQ ID NO: 6), CASSLLTRETETQYF (SEQ ID NO: 16), CASSTTGLAGGPGNEQFF (SEQ ID NO: 25), CASSPLNGGATEAFF (SEQ ID NO: 40), CAWSDRAAFTDTQYF (SEQ ID NO: 52), CASSSVAGGRIEQFF (SEQ ID NO: 63), CSARDIKAQQWNIQYF (SEQ ID NO: 72), CASTINTYEQYF (SEQ ID NO: 83), CASSLYGGPGDQPQHF (SEQ ID No. 95), CASAQTIGAYNEQFF (SEQ ID No. 106), In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF (SEQ ID NO: 3) CDR1β:SQVTM (SEQ ID NO: 4) CDR2β: ANQGSEA (SEQ ID NO: 5) CDR3β: CSANPTGGGTEAFF (SEQ ID NO: 6) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CARNTGKLIF (SEQ ID NO: 3) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β:FQNEAQ (SEQ ID NO: 15) CDR3β: CASSLLTRTEQYF (SEQ ID NO: 16) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CAPSASKIIF (SEQ ID NO: 22) CDR1β:LNHDA (SEQ ID NO: 23) CDR2β:SQIVND (SEQ ID NO: 24) CDR3β: CASSTTGLAGGPGNEQFF (SEQ ID NO: 25) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CAPQFNKFYF (SEQ ID NO: 31) CDR1β: SGHVS (SEQ ID NO: 14) CDR2β:FQNEAQ (SEQ ID NO: 15) CDR3β: CASSLLTRTEQYF (SEQ ID NO: 16) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α:CASINFNKFYF (SEQ ID NO: 37) CDR1β: MDHEN (SEQ ID NO: 38) CDR2β: SYDVKM (SEQ ID NO: 39) CDR3β: CASSPLNGGATEAFF (SEQ ID NO: 40) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:DSSSTY (SEQ ID NO: 47) CDR2α: IFSNMDM (SEQ ID NO: 48) CDR3α: CAEFTGTASKLTF (SEQ ID NO: 49) CDR1β: GTSNPN (SEQ ID NO: 50) CDR2β: SVGIG (SEQ ID NO: 51) CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 52) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α: TSGFNG (SEQ ID NO: 58) CDR2α: NVLDGL (SEQ ID NO: 59) CDR3α: CAVTYNNARLMF (SEQ ID NO: 60) CDR1β: MNHEY (SEQ ID NO: 61) CDR2β:SMNVEV (SEQ ID NO: 62) CDR3β: CASSSVAGGRIEQFF (SEQ ID NO: 63) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α:SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CARNYGQNFVF (SEQ ID NO: 69) CDR1β: DFQATT (SEQ ID NO: 70) CDR2β: SNEGSKA (SEQ ID NO: 71) CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 72) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α: VSGLRG (SEQ ID NO: 78) CDR2α: LYSAGEE (SEQ ID NO: 79) CDR3α:CVITTSGTYKYIF (SEQ ID NO:80) CDR1β:MNHNS (SEQ ID NO:81) CDR2β: SASEGT (SEQ ID NO:82) CDR3β: CASTINTYEQYF (SEQ ID NO:83) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α: TSESDYY (SEQ ID NO: 90) CDR2α:QEAYKQQN (SEQ ID NO:91) CDR3α: CAYRSFYTGANSKLTF (SEQ ID NO: 92) CDR1β:SGHDT (SEQ ID NO:93) CDR2β: YYEEEE (SEQ ID NO: 94) CDR3β: CASSLYGGPGDQPQHF (SEQ ID NO: 95) And In a preferred embodiment, the amino acid sequences of CDR1α to CDR3α of the α chain and CDR1β to CDR3β of the β chain of the TCR are CDR1α: NSASQS (SEQ ID NO: 101) CDR2α: VYSSGN (SEQ ID NO: 102) CDR3α: CVVHSGGSYIPTF (SEQ ID NO: 103) CDR1β:MNHNY (SEQ ID NO: 104) CDR2β: SVGAGI (SEQ ID NO: 105) CDR3β: CASAQTIGAYNEQFF (SEQ ID NO: 106) becomes The T cell antigen receptor according to claim 1 .

4. The α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 41, SEQ ID NO: 53, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 107, or SEQ ID NO: 96, SEQ ID NO: 107, and an amino acid sequence having at least 85% homology with SEQ ID NO: 107; Preferably, the variable region of the β chain comprises the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 42, SEQ ID NO: 54, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 108, or No. 97, SEQ ID No. 108, and an amino acid sequence having at least 85% homology with the amino acid sequence of the present invention; Preferably, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:66, SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:98, SEQ ID NO:109, or comprising an amino acid sequence having at least 85% or more homology with ID NO: 109; Preferably, the β chain comprises the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:87, SEQ ID NO:99, SEQ ID NO:110, or No. 99, containing an amino acid sequence having at least 85% homology with SEQ ID No.

110. The T cell antigen receptor according to any one of claims 1 to 3.

5. the amino acids of the α chain are linked directly or indirectly, preferably indirectly, more preferably via fp2A, to the amino acids of the β chain; Preferably, the amino acid sequence of said fp2A is shown in SEQ ID NO: 11; Preferably, the antibody comprises the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 57, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO: 111, or the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 57, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO:

111. Contains an amino acid sequence having at least 85% homology with ID NO: 111 The T cell antigen receptor according to any one of claims 1 to 4.

6. A nucleic acid encoding the T cell antigen receptor according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the expression vector of claim 7.

9. An immune cell characterized by expressing the T cell antigen receptor according to any one of claims 1 to 5.

10. The method includes a step of introducing a nucleic acid sequence encoding the T cell antigen receptor according to claim 6 into immune cells and expressing the nucleic acid sequence to obtain the T cell antigen receptor. A method for preparing immune cells, comprising:

11. Step 1) obtaining the nucleic acid of claim 6 from a CMV-positive T cell clone; 2) isolating and culturing primary T cells; and step 3) presenting the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing the T cell antigen receptor described in any one of claims 1 to 5. A method for preparing recombinant T cells, comprising:

12. (1) obtaining the nucleic acid of claim 6 from a positive T cell clone; Step (2) of ligating the nucleic acid obtained in step (1) into a vector to obtain an expression vector; Step (3) of transforming the expression vector obtained in step (2) into a host cell and inducing expression; (4) obtaining a T cell antigen receptor. A method for preparing a T cell antigen receptor, comprising:

13. Use of a T cell antigen receptor according to any one of claims 1 to 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a host cell according to claim 8, or an immune cell according to claim 9 in the preparation of a product for diagnosing, preventing, or treating a disease associated with CMV; or use of a T cell antigen receptor according to any one of claims 1 to 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a host cell according to claim 8, or an immune cell according to claim 9 in the labeling, detection, cell sorting, or activation of T cells.

14. 1) A T cell antigen receptor according to any one of claims 1 to 5, 2) The nucleic acid according to claim 6, 3) The expression vector according to claim 7. 4) The host cell according to claim 8. 5) The immune cell according to claim 9. Contains one of the following: A pharmaceutical composition comprising:

15. 1) A T cell antigen receptor according to any one of claims 1 to 5, 2) The nucleic acid according to claim 6, 3) The expression vector according to claim 7. 4) The host cell according to claim 8. 5) The immune cell according to claim 9. Contains one of the following: A kit characterized by:

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