SARS-cov-2 specific tcrs

Abstract

To provide a T cell receptor (TCR) specific to SARS-CoV-2.SOLUTION: A T-cell receptor (TCR) specifically recognizing an S-protein of SARS-CoV-2 having a combination of CDR3 α and CDR β, each comprising a specific amino acid sequence, excluding TCRs for therapeutic purposes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to SARS-CoV-2 specific TCRs.

[0002] Four years have passed since the World Health Organization (WHO) declared COVID-19 a "pandemic" in March 2020. The WHO declared the end of the state of emergency in May 2023, and Japan also upgraded the disease to Category 5 under the Infectious Diseases Control Act that same month, but the COVID-19 outbreak continues. In addition to antiviral drugs, methods using serum / plasma collected from recovered patients and monoclonal antibodies created by cloning antibody molecules have been developed to treat COVID-19. However, there have been few reports on the use of T cells for treatment or testing.

[0003] It is generally known that antibodies and killer T cells act as two pillars of the immune response to viral infections. Antibodies bind to and neutralize viruses, preventing infection and alleviating symptoms, primarily acting in the early stages of infection. On the other hand, killer T cells are known to act against viral infections from the early to late stages of infection by killing infected cells and eliminating pathogens.

[0004] If a TCR specific to SARS-CoV-2 can be obtained, T cells expressing this TCR may be used for cellular immunotherapy, as well as for detecting SARS-CoV-2 and quantifying SARS-CoV-2-infected cells. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to provide a T cell receptor (TCR) specific for SARS-CoV-2. [Means for solving the problem]

[0006] The present disclosure provides a T cell receptor (TCR) that specifically recognizes the S protein of SARS-CoV-2 and has any of the following combinations of CDR3α and CDR3β, excluding TCRs used for therapeutic purposes. [Table 1]

[0007] Examples of T cell receptors of the present disclosure include T cell receptors comprising any of the following combinations of TCR α chain and TCR β chain: [Table 2]

[0008] The present disclosure further discloses a nucleic acid encoding the TCR receptor of the present disclosure. The nucleic acid encoding the TCR receptor of the present disclosure may be DNA or mRNA. Examples of DNA include the following:

[0009] [Table 3] The present disclosure further discloses vectors comprising the nucleic acids of the present disclosure. [Brief explanation of the drawings]

[0010] [Figure 1] Example 1. Overview of SARS-CoV-2-specific TCR gene cloning. [Figure 2] Example 1: The antigen specificity of SARS-CoV-2-specific TCR was confirmed. [Figure 3] The binding affinity of the SARS-CoV-2-specific TCR obtained in Example 1 to the antigen peptide was confirmed. [Figure 4] The binding affinity of the SARS-CoV-2-specific TCR obtained in Example 1 to the antigen peptide was confirmed. [Figure 5] Example 2: Outline of a method for producing HLA-I / II double knockout ES cell-derived T cells. [Figure 6] Example 2, Mutations introduced into the B2M / CIITA locus for HLA-I / II double knockout. [Figure 7] In Example 2, HLA-I / II double knockout and NY-ESO-1-TCR gene-transduced ES cells were induced to differentiate into T cells to obtain DP cells. [Figure 8] In Example 2, it was confirmed that the CD8SP cells obtained by inducing the differentiation of HLA-I / II double knockout and NY-ESO-1-TCR gene-transduced ES cells into T cells have a TCR specific to NY-ESO-1 and do not express HLA-I / II. [Figure 9] 1 is an outline of the killing assay of Example 2. [Figure 10] Overview of the killing assay in Example 2 (preparation of effector cells and measurement of cytotoxic activity) [Figure 11] Killing assay results of Example 2 (QYI A1, QYI B1, QYI H5, RLQ 3 and YLQ 28). DETAILED DESCRIPTION OF THE INVENTION

[0011] The TCRs disclosed herein are HLA-A*02:01-restricted (YLQ_28 and RLQ_3) and HLA-A*24:02-restricted (QYI_A1, QYI_B1, and QYI_H5) TCRs that have high specificity for antigens derived from the S protein of SARS-CoV-2, as confirmed in the Examples, and exhibit cytotoxic activity when gene-transferred into regenerated T cells.

[0012] The antigen sequence specifically recognized by the TCR of the present disclosure has not been found to have mutations in the spike region of previously reported epidemic variants (from the Alpha strain to the Omicron strain) in database analysis, indicating that this region is highly conserved among strains. Therefore, the TCR of the present disclosure is expected to have affinity not only for current epidemic strains but also for future emerging variants. It is also expected to be a TCR common to many individuals (public TCR). If the TCR of the present disclosure is a public TCR, it can be used in immunological tests to determine the extent to which SARS-CoV-2-specific TCRs are present in HLA-A24- or HLA-A2-positive individuals, and to what extent they change before and after vaccination.

[0013] The TCR of the present disclosure can be produced in or expressed on cells by introducing a nucleic acid encoding the TCR into an expression vector and then introducing the vector into cells. Examples of vectors suitable for expressing the TCR α chain and TCR β chain include those containing a nucleic acid encoding TCR β-P2A-TCR α downstream of a promoter. The vector may also contain, as desired, transcriptional and translational regulatory sequences, ribosome binding sites, enhancers, replication origins, poly(A) addition signals, selectable marker genes, and the like.

[0014] The present disclosure also provides a nucleic acid encoding the TCR of the present disclosure. The nucleic acid encoding the TCR may be DNA or RNA. The present disclosure further provides a vector comprising a nucleic acid encoding the TCR of the present disclosure. The vector may be appropriately selected from vectors used in genetic recombination, and examples thereof include vectors such as viruses, plasmids, and artificial chromosomes. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and Sendai viral vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Plasmids for mammalian cells may be used as the plasmid. Commercially available vectors may be appropriately selected and used depending on the purpose.

[0015] Examples of promoters that can be used include the EF1α promoter, CAG promoter, ubiquitin promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MMLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR Vα gene promoter, and TCR Vβ gene promoter.

[0016] The TCR of the present disclosure can be suitably used in testing and research for SARS-CoV-2. For example, the TCR of the present disclosure can be used as a probe for detecting SARS-CoV-2-infected cells. To use the TCR as a probe, for example, reporter cells that emit fluorescence upon receiving a signal from the TCR can be used, such as SARS-CoV-2-specific TCR-expressing reporter cells obtained by transfecting a TCR of the present disclosure into a T cell line expressing the NFAT-ZsGreen-1 reporter gene, as used in Example 1 (Landry et al., (2021). Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System, Bio-protocol 11 (2): e3883. DOI: 10.21769 / BioProtoc.3883.).

[0017] In this method, cells from a biological sample are co-cultured with reporter cells expressing SARS-CoV-2-specific TCRs. If the cells in the biological sample express the SARS-CoV-2 S protein, the TCR binds to them and emits fluorescence. If fluorescence is detected, the cells are determined to express the SARS-CoV-2 S protein and therefore to be infected with SARS-CoV-2. Furthermore, the intensity of this fluorescence can be used to quantify SARS-CoV-2-infected cells.

[0018] Another example of using the TCR of the present disclosure as a probe is a method in which the TCR is solubilized and bound to a fluorescent dye or radioisotope.

[0019] Examples of biological samples for detecting SARS-CoV-2 infected cells include, but are not limited to, bronchoalveolar lavage fluid, blood, throat swabs, sputum, cerebrospinal fluid, pleural effusion, ascites, nasal discharge, saliva, urine, and feces.

[0020] In addition, the TCR disclosed herein may be used to detect SARS-CoV-2 infected cells and the SARS-CoV-2 virus using an ELISA method using the TCR as an antigen. If cells obtained from a human subject's biological sample express the TCR of the present disclosure, the subject is considered to have a history of SARS-CoV-2 infection or vaccination. Measuring the increase or decrease in white blood cell count in such a subject infected with coronavirus can also be used to predict the coronavirus infection rate, onset and remission rates, and vaccination efficacy.

[0021] The TCRs disclosed herein do not include TCRs for therapeutic purposes. "TCRs for therapeutic purposes" refers to cases in which the TCRs disclosed herein are expressed in patient-derived or allogeneic T cells and administered to a patient for use in treating or preventing SARS-CoV-2 infection, or cases in which nucleic acids encoding TCRs, such as mRNA encoding TCRs, are administered to a patient and expressed in the patient's T cells to treat or prevent SARS-CoV-2 infection.

[0022] The present disclosure will be described in more detail below with reference to examples, but the contents of the present disclosure are not limited to the examples. [Example]

[0023] (1) Cloning of SARS-CoV-2 antigen-specific TCR genes An overview is shown in Figure 1. Healthy volunteer donors who had received the SARS-CoV-2 (SARS-CoV-2) vaccine were HLA-typed to select donors positive for HLA-A24 and HLA-A2. Peripheral blood mononuclear cells collected from each donor were stained with PE-fluorescently labeled MHC tetramers carrying the HLA-A24-restricted SARS-CoV-2 spike protein antigen peptide QYIKWPWYI (QYI), the HLA-A2-restricted SARS-CoV-2 spike protein antigen peptide YLQPRTFLL (YLQ), and the HLA-A2-restricted SARS-CoV-2 spike protein antigen peptide RLQSLQTYV (RLQ), and the frequency of spike protein antigen-specific T cells was measured by flow cytometry. The CD8+ T cell population positive for the MHC tetramers was sorted using a cell sorter and placed in each well of a 96-well PCR plate. cDNA synthesis and amplification were performed from single T cells in each well using multiplex RT-PCR.

[0024] Next, the TCR α chain gene and TCR β chain gene were amplified using primers designed from the leader peptide sequence obtained from the IMGT database, and the CDR3 sequences of the TCR α chain and β chain were identified by Sanger sequencing, followed by frequency analysis of TCR clonotypes.

[0025] The frequencies of QYI-, YLQ-, and RLQ-specific CD8+ T cells detected in donor peripheral blood mononuclear cells and the TCR clonotypes obtained by single-cell sequencing are shown in Figure 1. Single-cell analysis identified four distinct clonotypes with TCRαβ paired sequences from the T cell fraction that tested positive for the QYI-MHC tetramer against HLA-A24-restricted antigens. For HLA-A2-restricted antigens, four distinct TCRαβ paired sequences were identified for YLQ and 13 distinct TCRαβ paired sequences for RLQ.

[0026] To further analyze the function of the isolated TCRs, we constructed three retroviral vectors expressing the QYI, YLQ, and RLQ TCR genes. The amino acid sequences of the TCRα and TCRβ of each TCR are shown below.

[0027] QYI-A1 TCRα (SEQ ID NO: 11) MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCASSWGKLQFGAGTQVVVTPDIQ NPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 12) MSTRLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLVGANTGELFFGEGSRLTVLEDLKNVFPPEVSLFEPSKAEIANK QKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0028] QYI-B1 TCRα (SEQ ID NO: 13) MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVSDIFEGGFKTIFGAGTRLFVKAN IQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 14) MLLLLLLLGPGSGLGAVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDRDKAYEQYFGPGTRLTVTEDLKNVFPPEVSLFEPSKAEIANKQ KATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0029] QYI-H5 TCRα (SEQ ID NO: 15) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCFSPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFPNGGSNYKLTFGKGTLLTVNPN IQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 16) MFWYRQFPKKSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDVGTGGHYEQYFGPGTRLTVTEDLKNVFPPEVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWV NGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0030] YLQ28 TCRα (SEQ ID NO: 17) MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNNNNDMRFGAGTRLTVKPNIQN PEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 18) MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCATLDENTGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAEIANK QKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0031] RLQ3 TCRα (SEQ ID NO: 19) MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAGSSNDYKLSFGAGTTVTVRANI QNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 20) MGPGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASTLRDSETQYFGPGTRLLVLEDLKNVFPPEVSLFEPSKAEIANKQ KATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0032] The V region, J region, and CDR3 for each TCR β chain and TCR α chain obtained by analyzing each TCR sequence information based on IMGT / V-QUEST provided by IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark), are as follows.

[0033] [Table 4]

[0034] The cDNA sequences of each TCR chain are as follows. QYI_A1 TCRα (SEQ ID NO: 21) ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCCAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACCAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTATTTCTGTGCAAGTAGCTGGGGGAAATTGCAGTTTGGAGCAGGGACCCAGGTTGTGGTCACCCCAGATATCCAGAACCCTGACCCTGCCGTgtaccagctgaaggacccaagatctcagGATAGTACACTCTGTTTGTTTACGGACTTTGACTCACAAATCAACGTCCCGAAGACTATGGAAAGTGGTACGTTCATCACAGATAAGTGCGTTCTGGACATGAAGGCTATGGACTCAAAGAGCAACGGGGCAATTGCTTGGTCCAACCAGACAAGCTTTACCTGTCAGGACATTTTTAAGGAGACTAATGCTACTTATCCCTCCAGCGACGTTCCGTGTGATGCGACTCTTACCGAGAAGTCTTTTGAGACCGATATGAATCTCAACTTCCAGAATCTGCTGGTGATCGTTCTGCGGATCCTGCTTCTGAAGGTTGCAGGATTCAATCTTCTTATGACTCTCCGGCTCTGGTCTTCA TCRβ (SEQ ID NO: 22) ATGAGCACCAGGCTTCTCTGCTGGGCACTGCTTTGTCTCCTGGGACAGGCTTAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGAATCAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTAAGTCTGGGCATGACACTGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGGAGGAAGAGAGACAGAGGAGGCAACT TCCCTGATCGATTCTCAGGTCACCAGTTCCCTAACTATAGCTCTGAGCTGAATGTGAACGCCTTGTTGCTGGGGGACTCGGCCCTCTATCTCTGTCCAGCAGCTTGGTAGGGGCGAACCGGGGAGCTGTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAACGTGTTCCCacccgaggtcagtttgtttgagccatcaaaggCGGAGATCGCCAACAAA CAGAAAGCTACGCTCGTGTTTGGCTCGGGGCTTCTTCCCAGACCACGTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATTCCGGAGTGTGCACTGATCCCCAAGCGTACAAGGAATCCAACTATAGCTACTGTCTCTCATCTCGGCTCCGGTGAGTGCGACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAGTGCAGTTCATGGGTTGAGCGAGGAAG ACAAGTGGCCCGAGGGCAGTCCTAAACCAGTCACTCAAAACATAAGCGCCGAGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTCATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAAATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGTCTACTCTCGTGTTATGGCAATGGTGAACGGAAAAACAGCAGAGCCAAAAAGATGGTTCTGGC

[0035] QYI_B1 TCRα(SEQ ID NO:23) ATGCTCCTGCTGCTCGCCAGCGTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAAGACCCCTGTGGAGCTGAGGTGCAACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAA GTATTTATCAGGATCCACCCTGGTTGAAAGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAACCCTCAGTCCATATAAGCGACAGGGCTGAGTACTTCTGTGCTGTGAGTGATATTTTTTGAAGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATA TCCAGAACCCTGACCCTGCCGTgtaccagctgaaggacccaagatctcagGATAGTACACTCTGTTTGTTTACGGACTTTGACTCACAAATCAACGTCCCGAAGACTATGGAAAGTGGTACGTTCACAGATAAGTGCGTTCTGGACATGAAGGCTATGGACTCAAAGAGCAACGGGGCAATTGCTTGGTCCAACCAGACA AGCTTTACCTGTCAGGACATTTTTAAGGAGACTAATGCTACTTATCCCTCCAGCGACGTTCCGTGTGATGCGACTCTTACCGAGAAGTCTTTTGAGACCGATATGAATCTCAACTTCCAGAATCTGCTGGTGATCGTTCTGCGGATCCTGCTTCTGAAGGTTGCAGGATTCAATCTTCTTATGACTCTCCGGCTCTGGTCTTCA TCRβ(sequence number 24) ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCCAACATCCGAGCAGGGTTATCTGTAAGATGGAACCTCTGTGAAGATCGAGTGCCGTTCCTGGACTTTCAGGCCACAACTATGTTTTGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAA GGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGAGACGGGACAAGGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCacccgaggtcagtttgtttgagccatcaaaggCGGAGATCGCCAACAAACAGA AAGCTACGCTCGTGTTTTGGCTCGGGGCTTCTTCCCAGACCACGTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATTCCGGAGTGTGCACTGATCCCCAAGCGTACAAGGAATCCAACTATAGCTACTGTCTCTCATCTCGGCTCCGGTGAGTGCGACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAGTGCAGTTTCATGGGTTGAGCGAGGAAGAC AAGTGGCCCGAGGGCAGTCCTAAACCAGTCACTCAAAACATAAGGCCCGAGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTCATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAAATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGTCTACTCTCGTGTTATGGCAATGGTGAAACGGAAAACAGCAGAGCCAAAAAGATGGTTCTGGC

[0036] QYI_H5 TCRα(SEQ ID NO:25) ATGGAGAAGAATCCTTTGGCAGCCCATTACTAATCCTCTGGTTTCCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCC TTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAAGAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTGTACATCAAAGGATCCCAGCCTGAAGACTCAGCCACATACCTCTGTGCCTTCCAAATGGGGGTAGCAACTATAAACTGACATTTGGAAAAGGAACTCTCTTAACCGTGAATCCAAAT ATCCAGAAACCCTGACCCTGCCGTgtaccagctgaaggacccaagatctcagGATAGTACACTCTGTTTGTTTACGGACTTTGACTCACAAATCAACGTCCCGAAGACTATGGAAAGTGGTACGTTCACAGATAAGTGCGTTCTGGACATGAAGGCTATGGACTCAAAGAGCAACGGGGCAATTGCTTGGTCCAACCAGACA AGCTTTACCTGTCAGGACATTTTTAAGGAGACTAATGCTACTTATCCCTCCAGCGACGTTCCGTGTGATGCGACTCTTACCGAGAAGTCTTTTGAGACCGATATGAATCTCAACTTCCAGAATCTGCTGGTGATCGTTCTGCGGATCCTGCTTCTGAAGGTTGCAGGATTCAATCTTCTTATGACTCTCCGGCTCTGGTCTTCA TCRβ(sequence number 26) ATGTTTTGGTATCGTCAGTTCCCGAAAAAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGAGATGT GGGGACAGGGGGCCACTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCacccgaggtcagtttgtttgagccatcaaaggCGGAGATCGCCAACAACAGAAAGCTACGCTCGTGTGTTTGGCTCGGGGCTTCTTCCCAGACCACGTAGAACTTTCCTGGTGGGTCA ATGGAAAGGAGGTTCATTCCGGAGTGTGCACTGATCCCCAAGCGTACAAGGAATCCAACTATAGCTACTGTCTCTCATCTCGGCTCCGGGTGATGCGACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAGTGCAGTTTCCATGGGTTGAGCGAGGAACAAGTGGCCCGAGGGCAGTCCTAAACCAGTCACT CAAACATAAGCGCCGAGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTCATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAAATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGTCTACTCTCGTGTTATGGCAATGGTGAAACGGAAAAAACAGCAGAGCCAAAAAGATGGTTCTGGC

[0037] YLQ_28 TCRα(SEQ ID NO:27) ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGT TGCTGATGTCCGTATACTCCAGTGGTAATGAAGATGGAAGGTTTACAGCACAGCTATAGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGGTGAACAATAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATTCAGAAC CCCGAACCAGCCGTATATCAGTTGAAGGACCCAAGATCTCAGGATAGTACACTCTGTTTGTTTACGGACTTTGACTCACAAATCAACGTCCCGAAGACTATGGAAAGTGGTACGTTCACACAGATAAGTGCGTTCTGGACATGAAGGCTATGGACTCAAAGAGCAACGGGGCAATTGCTTGGTCCAACCAGACAAGCT TTACCTGTCAGGACATTTTTAAGGAGACTAATGCTACTTATCCCTCCAGCGACGTTCCGTGTGATGCGACTCTTACCGAGAAGTCTTTTGAGACCGATATGAATCTCAACTTCCAGAATCTGCTGGTGATCGTTCTGCGGATCCTGCTTCTGAAGGTTGCAGGATTCAATCTTCTTATGACTCTCCGGCTCTGGTCTTCA TCRβ(sequence number 28) ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGCACACAGCAGGAGGACTCCGCCGTGTATCTCTGTGCCACCCTGGATGAGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGCGCAACGTCACCCCACCAAAGGTCAGTTTGTTTGAGCCATCAAAGGCGGAGATCGCCAACAAACAGAAAGCTACGCTCGTGTGTTTGGCTCGGGGCTTCTTCCCAGACCACGTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATTCCGGAGTGTGCACTGATCCCCAAGCGTACAAGGAATCCAACTATAGCTACTGTCTCTCATCTCGGCTCCGGGTGAGTGCGACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAGTGCAGTTTCATGGGTTGAGCGAGGAAGACAAGTGGCCCGAGGGCAGTCCTAAACCAGTCACTCAAAACATAAGCGCCGAGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTCATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAAATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGTCTACTCTCGTGGTTATGGCAATGGTGAAACGGAAAAACAGCAGAGCCAAAAGAAGTGGTTCTGGC

[0038] RLQ_3 TCRα(SEQ ID NO:29) ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAG TTGATAATGTTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGGGAGTTCTAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAGCAAATATCC AGAACCCTGACCCTGCCGTgtaccagctgaaggacccaagatctcagGATAGTACACTCTGTTTGTTTACGGACTTTGACTCACAAATCAACGTCCCGAAGACTATGGAAAGTGGTACGTTCACAGATAAGTGCGTTCTGGACATGAAGGCTATGGACTCAAAGAGCAACGGGGCAATTGCTTGGTCCAACCAGACAAG CTTTACCTGTCAGGACATTTTTAAGGAGACTAATGCTACTTATCCCTCCAGCGACGTTCCGTGTGATGCGACTCTTACCGAGAAGTCTTTTGAGACCGATATGAATCTCAACTTCCAGAATCTGCTGGTGATCGTTCTGCGGATCCTGCTTCTGAAGGTTGCAGGATTCAATCTTCTTATGACTCTCCGGCTCTGGTCTTCA TCRβ(sequence number 30) ATGGGCCCTGGGCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCACCCTCCGGGACTCAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCacccgaggtcagtttgtttgagccatcaaaggCGGAGATCGCCAACAAACAGAAAGCTACGCTCGTGTGTTTGGCTCGGGGCTTCTTCCCAGACCACGTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATTCCGGAGTGTGCACTGATCCCCAAGCGTACAAGGAATCCAACTATAGCTACTGTCTCTCATCTCGGCTCCGGGTGAGTGCGACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAGTGCAGTTTCATGGGTTGAGCGAGGAAGACAAGTGGCCCGAGGGCAGTCCTAAACCAGTCACTCAAAACATAAGCGCCGAGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTCATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAAATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGTCTACTCTCGTGGTTATGGCAATGGTGAAACGGAAAAACAGCAGAGCCAAAAGAAGTGGTTCTGGC

[0039] (2) Examination of the antigen specificity of the cloned TCR gene TCR-expressing reporter cells were generated by transfecting each of the cloned TCRαβ pair genes into a T cell line (Jurkat cells) expressing the NFAT-ZsGreen-1 reporter gene (Landry et al., (2021). Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System, Bio-protocol 11 (2): e3883. DOI: 10.21769 / BioProtoc.3883., Matsumoto et al., Biochem. Biophys. Res. Commun. 534:680-686.2021). To examine the antigen specificity of the TCR-expressing reporter cells, a cell line expressing both HLA-A2 and HLA-A24, created by transfecting HLA-A24 into NALM6 cells, an HLA-A2-positive cell line, was used as an antigen-presenting cell for the reporter cells. TCR-expressing reporter cells and antigen-presenting cells were co-cultured in a culture plate with the addition of QYI, YLQ, or RLQ peptides, and reporter activity was observed under a fluorescence microscope 8 hours after the start of culture.

[0040] The results are shown in Figure 2. Each TCR-expressing reporter cell co-cultured with antigen-presenting cells was activated only under peptide-added conditions, confirming that each cloned TCR exhibited reactivity to epitope peptides derived from SARS-CoV-2 antigens.

[0041] (3) Examination of the binding affinity of the cloned TCR to antigen peptides To evaluate the binding affinity of the cloned TCRs to the antigenic peptides, serial dilutions of QYI, YLQ, and RLQ peptides were added to TCR reporter cells cocultured with antigen-presenting cells. The frequency of activated cells was measured 8 hours after the start of coculture by flow cytometry. The stimulation index (% ZsGreen-1 positive frequency in peptide-stimulated cells / % ZsGreen-1 positive frequency in unstimulated cells) was used as an index of reporter activity. The peptide concentration showing 50% of maximal activity (EC50) was calculated, and the binding affinity of each TCR to the antigenic peptide was evaluated. The results are shown in Figures 3 and 4.

[0042] In each TCR-expressing reporter cell, we confirmed that the expression level of NFAT-ZsGreen-1 increased as the peptide concentration increased (Figure 3). Figure 4 shows the dose-response curves obtained by calculating the stimulation index from the frequency of ZsGreen-1-positive cell fractions measured by flow cytometry and plotting these values ​​for each peptide concentration. Table 5 shows the EC50 values ​​for each TCR antigen peptide obtained from the dose-response curves.

[0043] [Table 5]

[0044] Among QYI-specific TCRs that recognize the same antigen epitope, it was confirmed that QYI_B1, QYI_H5, and QYI_A1 have the highest binding affinity in this order. Database analysis revealed that the antigen peptide sequences recognized by the obtained TCRs have not been found to have mutations in the spike region of previously reported epidemic variants (from alpha to omicron strains), and this region is highly conserved among strains. Therefore, these TCRs are expected to have affinity not only for current epidemic strains but also for future emerging variants. [Example]

[0045] Killing assay using ES cell-derived regenerated CTLs (1) Preparation of target cells To perform a killing assay using regenerated CTLs, target cells were prepared. The SARS-CoV-2-specific TCR obtained in Example 1 is expected to recognize peptides mounted on A2402 or A0201 of the donor's HLA. Therefore, HLA-A24:02 / 02:01-expressing lymphoblastoid cell line (LCL) was used as the target cell by introducing the SARS-CoV-2 S protein into the LCL using a retroviral vector.

[0046] (2) Generation of regenerated CTLs Generation of HLA-I / II double knockout ES cell-derived T cells We generated ES cell-derived T cells that do not express HLA-I / II. The ES cells used were SEES3-10, established at the Center for Child Health and Development. The outline of this process is shown in Figure 5. First, we used CRISPR / Cas9 to knock out B2M / CIITA in SEES3-10. For B2M knockout, we designed a gRNA targeting exon 1 of the B2M locus (GGCCGAGATGTCTCGCTCCG). For CIITA knockout, we designed a gRNA targeting exon 3 of the CIITA locus (TCAACTGCGACCAGTTCAGC).

[0047] The knockout-manipulated ES cells were single-cell cloned, and the nucleotide sequences of B2M and CIITA were confirmed. As a result, ES cell clones were obtained in which a single-nucleotide insertion mutation occurred in both alleles of the B2M / CIITA locus (Figure 6). The NY-ESO-1-TCR-IRES-Venus gene was introduced into the obtained ES cell clones via lentivirus, and colonies of Venus-expressing cells were obtained. The obtained colonies were then single-cell cloned again.

[0048] Four ES cell clones (SEES3-10-dKO-NY1 / 3 / 5 / 6) were obtained by HLA-I / II knockout and NY-ESO-1-TCR gene transduction. These cells were induced to differentiate into T cells using the same method as in Reference Example 1. It was confirmed that CD4 / 8 double positive (DP) cells were obtained from all of these ES cell clones (Figure 7). CD4+ cells were then isolated by magnetic cell sorting.

[0049] CD8SPT cells (CD8 single-positive T cells) were expanded by co-culturing isolated CD4+ cells, including CD4 / 8DP cells, with lymphoblastoid cell lines (LCLs) pulsed with NY-ESO-1 peptide every 7 days. The expanded CD8SPT cells were confirmed to possess a TCR specific for NY-ESO-1 and not express HLA-I / II (Figure 8). Hereafter, CD8SPT cells induced from pluripotent stem cells are referred to as "regenerated CTLs."

[0050] (3) HLA-restricted killing activity of regenerated CTLs The outline is shown in Figures 9 and 10. The five types of TCRs isolated in Example 1 (QYI A1, QYI B1, QYI H5, RLQ 3, and YLQ 28) were expressed in regenerated CTLs and used as effector cells.

[0051] S protein-expressing A24 / A02+ LCLs were co-cultured at an ET ratio of 1:1, and the percentage of viable LCLs was measured by flow cytometry 16 hours later. LCLs were labeled with CTV (CeLLTrace Violet) before co-culture to distinguish them from repopulating CTLs. The results are shown in Figure 11 and Table 6.

[0052] Regenerated CTLs bearing all five S protein-specific TCRs exhibited killing activity when co-cultured with S protein-expressing CTLs.

[0053] [Table 6]

Claims

1. A T cell receptor (TCR) that specifically recognizes the S protein of SARS-CoV-2 and has any of the following combinations of CDR3α and CDRβ, excluding TCRs for therapeutic purposes.

2. The TCR of claim 1, comprising any of the following combinations of TCR alpha chain and TCR beta chain:

3. A nucleic acid encoding the TCR of claim 1 or 2.

4. 4. The nucleic acid of claim 3, which is a DNA strand having the following sequence:

5. A vector comprising the nucleic acid of claim 3 or 4.

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