Cells for treatment and / or prevention of SARS-cov-2 infection and production method therefor

Abstract

The purpose of the present disclosure is to provide highly versatile, ready-to-deliver allogenic T cells for novel coronavirus infection, and a production method therefor. The present invention provides: a method for producing a cell population for the treatment and / or prevention of SARS-CoV-2 infection and including T cells or precursor T cells that express a human T cell receptor (TCR) specific to SARS-CoV-2, said method comprising a step for causing the expression of one or more human TCRs specific to SARS-CoV-2 in T cells or precursor T cells in vitro; and a cell population for the treatment and / or prevention of SARS-CoV-2 infection and produced via said method, said cell population comprising allogenically derived T cells or precursor T cells.

Description

Cells for treating and / or preventing SARS-CoV-2 infection and their production method

[0001] The present disclosure relates to cells for treating and / or preventing SARS-CoV-2 infection and methods for making same.

[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 in the 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 development of T-cell preparations for treatment or prevention.

[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. Meanwhile, killer T cells act against viral infections from the early to late stages of infection by killing infected cells and eliminating pathogens.

[0004] A cell therapy for viral infections has been previously performed in which viral antigen-specific T cells are expanded ex vivo using third-party T cells as the source material and then administered. This treatment has been shown to be effective against reactivation of cytomegalovirus and EB virus after hematopoietic stem cell transplantation (Non-Patent Document 1). However, it is difficult to completely match the HLA of a third-party donor with that of the recipient, resulting in the administration of partially HLA-mismatched T cells. Therefore, this treatment can only be used on patients who have become immunocompromised due to treatment such as transplantation.

[0005] There have been efforts to prepare a similar treatment for SARS-CoV-2 infection (Non-Patent Document 2), but it has rarely been used. When using T cells from a third party, there is the problem of difficulty in matching HLA. Patients who have undergone hematopoietic stem cell transplantation are immunocompromised, and it is possible to administer T cells with partially mismatched HLA to treat viral reactivation. However, it is difficult to use the same method on patients with general viral infections.

[0006] In April 2020, Bertoletti et al. in Singapore proposed the idea of ​​introducing a SARS-CoV-2-specific foreign T cell receptor (TCR) into autologous T cells, rather than allogeneic T cells, to produce a T cell preparation and then return it to the patient (Non-Patent Document 3). However, there have been no further reports, and it is believed that this idea was not realized. When there are a large number of patients, producing a custom-made T cell preparation is not realistic.

[0007] To the applicant's knowledge, there have been no reports of projects using allogeneic pluripotent stem cell-derived T cells for the treatment or prevention of COVID-19. Currently, there is no effective treatment leading to a cure for patients with severe SARS-CoV-2 infection or for patients with moderate symptoms that are progressing to severe disease. Antibody therapy is only effective in the early stages and may not be effective against new mutant strains. Vaccines have made great progress in prevention, but they do not provide a cure.

[0008] While progress is being made in the development of symptomatic treatments for the severe cytokine storm caused by SARS-CoV-2 infection, such as the administration of steroids and anti-IL-6R antibodies, there is a need for the development of a treatment for the infection itself.

[0009] Blood, 121: 5113, 2013Front Immunol,12:751869, 2021Duke-NUS Medical School press release dated April 21, 2020

[0010] The present disclosure aims to provide versatile, readily available allogeneic T cells for the treatment and / or prevention of COVID-19, as well as methods for producing the same.

[0011] The present application provides the following: (1) A method for producing a cell population for treating and / or preventing SARS-CoV-2 infection, comprising T cells or T cell precursors expressing a SARS-CoV-2-specific human TCR, the method comprising the step of expressing a single or multiple SARS-CoV-2-specific human TCR in T cells or T cell precursors in vitro. (2) The method according to (1), wherein the T cells or T cell precursors expressing a SARS-CoV-2-specific human TCR express CD8αβ. (3) The method for producing a T cell according to (1) or (2), comprising T cells expressing a human TCR specific to SARS-CoV-2, the T cells having killer activity. (4) The method for producing a T cell or a T cell precursor expressing a human TCR specific to SARS-CoV-2 is a cell lacking any or all of HLA. (5) The method for producing a T cell according to any of (1) to (4), comprising the steps of introducing a human T cell receptor (TCR) into a cell capable of differentiating into a T cell, and differentiating the cell introduced with the human T cell receptor (TCR) into a T cell or a T cell precursor. (6) The method for producing a T cell according to (5), wherein the cell capable of differentiating into a T cell is a pluripotent stem cell. (7) The method for producing a T cell according to (6), wherein the pluripotent stem cell is a pluripotent stem cell lacking any or all of HLA. (8) The method of production according to (6) or (7), wherein the pluripotent stem cells are ES cells derived from the human ES cell line SEES3. (9) The method of production according to any one of (1) to (8), wherein the SARS-CoV-2-specific TCR is specific to any epitope of SEQ ID NOs: 1 to 7. (10) The method of production according to (9), wherein the CDR of the SARS-CoV-2-specific TCR is selected from the following combinations:

[0012] (11) The method according to (10), wherein the amino acid sequence of the SARS-CoV-2-specific TCR is selected from the following combinations:

[0013] (12) A cell population for treating and / or preventing SARS-CoV-2 infection, comprising allogeneic T cells or T cell precursors expressing a SARS-CoV-2-specific human TCR, prepared by the method of any one of (1) to (11). (13) A cell population for treating and / or preventing SARS-CoV-2 infection, comprising an allogeneic T cell or T cell precursor population expressing the same human TCR specific to SARS-CoV-2 and lacking any or all HLAs. (14) The cell population of (13), comprising a combination of multiple allogeneic T cell or T cell precursor populations expressing the same human TCR specific to the SARS-CoV-2 S protein and lacking any or all HLAs. (15) The cell preparation of (13), wherein the T cells or T cell precursors express CD8αβ. (16) The cell population according to any one of (13) to (15), wherein the T cells are T cells with killer activity. (17) The cell population according to any one of (13) to (16), wherein the SARS-CoV-2-specific TCR is specific for any epitope of SEQ ID NOs: 1 to 7. (18) The cell population according to (17), wherein the CDR of the SARS-CoV-2-specific TCR is selected from the following combinations:

[0014] (19) The cell population according to (18), wherein the SARS-CoV-2-specific TCR amino acid sequence is selected from the following combinations:

[0015] (20) A method for treating and / or preventing SARS-CoV-2, comprising the step of expressing a human T cell receptor (TCR) specific to SARS-CoV-2 in T cells of a patient in need thereof.

[0016] Reference Example 1: Outline of a method for inducing differentiation from ES cells to T cells. Reference Example 2: Outline of a method for producing T cells derived from HLA-I / II double knockout ES cells. Reference Example 2: Mutations introduced into the B2M / CIITA locus for HLA-I / II double knockout. Reference Example 2: DP cells were obtained by inducing differentiation into T cells from HLA-I / II double knockout and NY-ESO-1-TCR gene-introduced ES cells. Reference Example 2: CD8SP cells obtained by inducing differentiation into T cells from HLA-I / II double knockout and NY-ESO-1-TCR gene-introduced ES cells were confirmed to not express HLA-I / II. Example 1: Outline of cloning of SARS-CoV-2-specific TCR gene. Example 1: Confirmation of antigen specificity of SARS-CoV-2-specific TCR. The binding affinity of the SARS-CoV-2-specific TCR obtained in Example 1 to an antigen peptide was confirmed. The binding affinity of the SARS-CoV-2-specific TCR obtained in Example 1 to an antigen peptide was confirmed. Example 2: Outline of a method for cloning the SARS-CoV-2-specific TCR gene. Example 2: Confirmation of epitope-specific activation of the obtained TCR. Example 4: Outline of a method for producing target cells for the killing assay. Example 4: Outline of the killing assay. Example 4: Outline of the killing assay (production of effector cells and measurement of cytotoxic activity). Results of the killing assay in Example 4 (QYI A1, QYI_B1, QYI H5, RLQ 3, and YLQ 28). Results of the killing assay in Example 4 (10BA, CE2AB). Results of the killing assay in Example 4 (2BA). Results of the peptide-specific killing assay in Example 4 (10BA, CE2AB). Production of A24:02-expressing A549+ACE2 cells used in the assays of Examples 5 and 6. Overview of the killing assay of Example 5. Results of the killing assay of Example 5 (CE2AB, 10BA). Results of a peptide-specific killing assay using the xCELLigence system of Example 6 (10BA). Overview of the killing assay against SARS-CoV-infected cells of Example 6. Results of the killing assay against SARS-CoV-infected cells of Example 6 (CE2AB, QYI_B1). Example 7: Overview of obtaining SARS-CoV-2-specific TCR-single-expressing regenerated CTLs.Example 7: Double-positive cells were obtained as regenerative CTLs expressing only SARS-CoV-2-specific TCR (10BA, 2BA). Killing assay results for the regenerative CTLs expressing only SARS-CoV-2-specific TCR (2BA) in Example 7. Killing assay results for the regenerative CTLs expressing only SARS-CoV-2-specific TCR (QY1 B1) in Example 7.

[0017] The present disclosure provides a method for producing a cell population for treating and / or preventing SARS-CoV-2 infection, comprising T cells or T cell precursors expressing a human TCR specific for SARS-CoV-2, the method comprising the step of expressing a single or multiple SARS-CoV-2-specific human TCR in T cells or T cell precursors in vitro.

[0018] SARS-CoV-2-specific human TCRs can be obtained from donors who have a history of SARS-CoV-2 infection or SARS-CoV-2 vaccination. Examples of SARS-CoV-2 vaccines include, but are not limited to, inactivated vaccines, recombinant protein vaccines, peptide vaccines, messenger RNA (mRNA) vaccines, DNA vaccines, and viral vector vaccines. SARS-CoV-2-specific TCRs can be obtained from T cells obtained from donors.

[0019] Human T cells can be isolated from human tissues by known techniques. Human tissues are not particularly limited as long as they contain the T cells, but examples include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and diseased tissue. Among these, peripheral blood and umbilical cord blood are preferred because they are less invasive to humans and easier to prepare. Known techniques for isolating human T cells include flow cytometry using antibodies against cell surface markers such as CD4 and a cell sorter. Furthermore, desired T cells can also be isolated using cytokine secretion or functional molecule expression as indicators. T cells with killer activity can be isolated using secretion or production of granzymes, perforins, or CD8 expression as indicators.

[0020] Human TCRs specific for SARS-CoV-2 can be obtained by stimulating a biological sample containing T cells, such as peripheral blood mononuclear cells, obtained from a donor with inactivated SARS-CoV-2, a protein or peptide derived from SARS-CoV-2.

[0021] As an indicator for screening activated T cells, activation markers such as CD69, CD137, and CD154 can be used alone or in combination.

[0022] When isolating a specific antigen-specific human T cell, a method can be employed in which the T cell is purified from cultured human cells or human tissues containing SARS-CoV-2-specific T cells using an affinity column or the like on which the antigen of interest is immobilized. Alternatively, a method can be employed in which T cells specific for SARS-CoV-2 are purified from human tissues using a tetramer of MHC (major histocompatibility complex) bound to a desired antigen (so-called "MHC tetramer").

[0023] Furthermore, to confirm that the obtained TCR has the desired specificity, the TCR can be introduced into NFAT-GFP reporter cells, stimulated with a peptide of an amino acid length that can be recognized by HLA in the presence of donor B cells, and TCRs that show activation can be selected. A method for confirming TCR specificity using NFAT-GFP reporter cells is described in (Matsumoto et al., Biochem. Biophys. Res. Commun. 534:680-686.2021).

[0024] Examples of proteins derived from SARS-CoV-2 include the S protein and the N protein. Examples of peptides derived from SARS-CoV-2 include peptides derived from the S protein and the N protein. Peptides that are preferably used are those of 7 to 15 mers, 8 to 12 mers, for example, 8 to 10 mers.

[0025] Mutations in SARS-CoV-2 occur repeatedly, and various mutant strains and variants have been reported. Proteins and peptides derived from SARS-CoV-2 can be obtained from the mutant strain or variant of SARS-CoV-2 that is the target of treatment.

[0026] In the present disclosure, nine types of SARS-CoV-2-specific TCRs have been identified. The epitopes recognized by each TCR, the antigenic sites from which each epitope is derived, and the HLA types to which each epitope is restricted are shown in Table 5. To obtain a SARS-CoV-2-specific TCR, a biological sample obtained from a subject with a history of SARS-CoV-2 infection or vaccination may be stimulated with such an epitope peptide to obtain T cells that exhibit specific activation to any of the peptides, thereby obtaining a specific TCR.

[0027]

[0028] The sequences of the TCRα and TCRβ chains of each TCR identified in this disclosure are as follows: 10BA TCRα (SEQ ID NO: 8) MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNHLGGSNYKLTFGKGTLLTVNPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 9) MSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASSEGEGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0029] 14AB TCRα (SEQ ID NO: 10) MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNNGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 11) MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASTTLNTLEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0030] CE2AB TCRα (SEQ ID NO: 12) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFVPLSDGQKLLFARGTMLKVDLNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 13) MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSPGQGILEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISH TQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0031] 2BA TCRα (SEQ ID NO: 14) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAVDYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 15) MSLGLLCCAAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASILTGNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0032] QYI-A1 TCRα (SEQ ID NO: 16) MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCASSWGKLQFGAGTQVVVTPDIQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 17) MSTRLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLVGANTGELFFGEGSRLTVLEDLKNVFPPEVSLFEPSKAEIANK QKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0033] QYI-B1 TCRα (SEQ ID NO: 18) MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVSDIFEGGFKTIFGAGTRLFVKANIQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 19) MLLLLLLLGPGSGLGAVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDRDKAYEQYFGPGTRLTVTEDLKNVFPPEVSLFEPSKAEIANKQ KATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0034] QYI-H5 TCRα (SEQ ID NO: 20) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFPNGGSNYKLTFGKGTLLTVNPNIQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 21) MFWYRQFPKKSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDVGTGGHYEQYFGPGTRLTVTEDLKNVFPPEVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWV NGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0035] YLQ28 TCRα (SEQ ID NO: 22) MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNNNNDMRFGAGTRLTVKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 23) MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCATLDENTGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAEIANK QKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0036] RLQ3 TCRα (SEQ ID NO: 24) MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAGSSNDYKLSFGAGTTVTVRANIQNPDPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS TCRβ (SEQ ID NO: 25) MGPGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASTLRDSETQYFGPGTRLLVLEDLKNVFPPEVSLFEPSKAEIANKQ KATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRSGSG

[0037] 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.

[0038]

[0039] In the present disclosure, examples of TCRs specific to SARS-CoV-2 include those having the TCRs identified in the present disclosure. In the present disclosure, examples of TCRs specific to SARS-CoV-2 include TCRs having any of the CDR3 sequences identified in Table 6.

[0040] The method comprises expressing, in vitro, one or more human T cell receptors (TCRs) specific for SARS-CoV-2 in T cells or T cell precursors.

[0041] The T cells expressing a SARS-CoV-2-specific TCR may be T cells or T cell precursors isolated from a human. The method for expressing a TCR in T cells or T cell precursors in vitro is not limited, and any known method may be used.

[0042] The T cells are T cells that express CD3 and at least one molecule selected from the group consisting of CD4 and CD8. Examples of such human T cells include helper / regulatory T cells, which are CD4-positive cells, cytotoxic T cells, which are CD8-positive cells, and naive T cells (CD45RA ＋ CD62L ＋ cells), central memory T cells (CD45RA － CD62L ＋ cells), effector memory T cells (CD45RA － CD62L － cells), and terminal effector T cells (CD45RA ＋ CD62L － cells).

[0043] T cell precursors include CD34, CD43, and CD45-positive, CD2, CD5, CD7, and CD1a-positive, and CD4, CD8, and CD3-positive cells according to their differentiation and maturation stages, and can be typed or sorted using these markers alone or in combination. When selecting T cell lineage-specific T cell precursors at a specific differentiation and maturation stage, examples include cells expressing CD1a, CD3, CD8, CD4, etc.

[0044] The T cells or T cell precursors are preferably T cells that express CD8αβ. Furthermore, T cells having killer activity or T cells that can be differentiated into T cells having killer activity are preferably used.

[0045] In the present disclosure, T cells or T cell precursors expressing SARS-CoV-2-specific TCR are preferably cells lacking any or all of HLA. By deleting any or all of HLA, the cell population for treating and / or preventing SARS-CoV-2 infection provided by the present disclosure can be used in patients who are not immunocompromised.

[0046] HLA knockout can be performed by known methods. For example, both HLA-I and HLA-II can be knocked out by introducing mutations into the B2M and CIIT genes of cells using gene editing, e.g., CRISPR / Cas9 (Thongsin et al., Stem Cell Research, Volume 71, September 2023, 103138).

[0047] In the present disclosure, T cells or T cell precursors expressing a SARS-CoV-2-specific TCR may be cells induced to differentiate from pluripotent stem cells. Examples of pluripotent stem cells include ES cells and iPS cells. For example, an ES cell line derived from the human ES cell line SEES3 is exemplified. The human ES cell line SEES3 is available from the RIKEN BioResource Research Center (Ibaraki Prefecture, Japan) under cell number HES0008 and cell name SEES3. Here, cell lines derived from the human ES cell line SEES3 include SEES3 itself.

[0048] T cells with desired antigen specificity can be obtained from human pluripotent stem cells by any method known to those skilled in the art, such as those disclosed in WO2017 / 159087, WO2016 / 010155, WO2016 / 010154, WO2016 / 010153, WO2017 / 159088, WO2017 / 179720, and WO2018 / 124207.

[0049] Human pluripotent stem cells can be differentiated from T cells bearing SARS-CoV-2-specific TCRs. For example, T-iPS cells can be obtained by introducing Yamanaka factors into such T cells (Takahashi and Yamanaka, Cell 126, 663-673 (2006), Takahashi et al., Cell 131, 861-872 (2007), and Grskovic et al., Nat. Rev. Drug Dscov. 10, 915-929 (2011)).

[0050] The pluripotent stem cells may be an established pluripotent stem cell line, pluripotent stem cells derived from sources other than T cells, or pluripotent stem cells having a TCR other than the SARS-CoV-2-specific TCR of interest. When inducing differentiation of such pluripotent stem cells into T cells, T cells having a SARS-CoV-2-specific TCR can be obtained by introducing a SARS-CoV-2-specific TCR into the pluripotent stem cells before inducing differentiation and then inducing differentiation into T cells.

[0051] To introduce a TCR into a cell in vitro, a nucleic acid encoding the TCR may be carried on an expression vector and then introduced into the cell. Examples of vectors that can be suitably used to express the TCRα chain and TCRβ chain include those that contain a nucleic acid encoding TCRβ-P2A-TCRα 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.

[0052] In the present disclosure, a vector used for genetic recombination may be appropriately selected and used to introduce a TCR, 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.

[0053] Examples of promoters that can be used include an EF1α promoter, a CAG promoter, a ubiquitin promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, an MMLV (Moloney murine leukemia virus) LTR, an HSV-TK (herpes simplex virus thymidine kinase) promoter, a TCR Vα gene promoter, and a TCR Vβ gene promoter.

[0054] In the present disclosure, the pluripotent stem cells may have a cassette deck structure in which a cassette tape gene containing a gene encoding a marker protein is contained in the genome so as to be capable of expressing the marker protein. Cells having such a cassette deck structure are disclosed in WO2020 / 022512 and WO2022 / 065444. An example of the marker protein is a known TCR gene.

[0055] Pluripotent stem cells can be cultured with cells containing a cassette deck structure containing a known TCR gene, and then induced to differentiate into T cells or T cell precursors. Then, a cassette tape containing an allogeneic TCR gene specific to SARS-CoV-2 can be used to replace and express the known TCR contained in the cassette deck structure with the SARS-CoV-2-specific TCR gene. This method may avoid mispairing of TCRα and TCRβ.

[0056] When T cells or T progenitor cells are obtained by inducing differentiation from pluripotent stem cells, T cells in which part or all of HLA has been knocked out can be obtained by using pluripotent stem cells in which part or all of HLA has been knocked out. Examples of pluripotent stem cells that can be induced to differentiate into T cells or T progenitor cells include pluripotent stem cells in which part or all of HLA has been knocked out, and which have a cassette deck structure in which a cassette tape gene containing a gene encoding a marker protein is contained in the genome so that the marker protein can be expressed.

[0057] The present disclosure also provides a cell population for treating and / or preventing SARS-CoV-2 infection, comprising a population of allogeneic T cells or T cell precursors that express one or more identical human TCRs specific to SARS-CoV-2 and lack any or all HLAs. Here, "allogeneic T cells or T cell precursors" refers to T cells or T cell precursors obtained by manipulating cells obtained from a donor who is allogeneic to the SARS-CoV-2 infected patient being treated. The cell population for treating and / or preventing SARS-CoV-2 infection of the present disclosure may be a population of T cells or T cell precursors that each express the same one or more human TCRs, or may be a cell population that includes a combination of multiple types of T cells or T cell precursors, each expressing the same one or more human TCRs, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 types.

[0058] The cell population for treating and / or preventing SARS-CoV-2 infection provided in the present disclosure can be suitably used in treating or preventing SARS-CoV-2 infection in human subjects. In the present disclosure, "treatment" means curing or alleviating various symptoms caused by SARS-CoV-2 infection, or suppressing the progression of symptoms. In the present disclosure, "prevention" means suppressing the onset of symptoms caused by SARS-CoV-2 infection or attenuating the symptoms that do develop in a subject who has not yet developed symptoms caused by SARS-CoV-2 infection but is predicted to have a high probability of developing such symptoms.

[0059] Here, subjects who are predicted to have a high probability of developing symptoms caused by SARS-CoV-2 infection include, for example, subjects who have had close contact with an infected person, subjects who have not developed symptoms but have been found to be infected with SARS-CoV-2 through testing, subjects who have not yet been induced into an immunodeficient state through the administration of anticancer drugs or pretreatment for hematopoietic stem cell transplantation, and medical professionals working in wards where infected people are present.

[0060] The cell population for treating and / or preventing SARS-CoV-2 infection of the present disclosure may be produced from autologous cells or from polygenic cells. For broad use in treating and / or preventing SARS-CoV-2 infection, cells produced from allogeneic cells are preferably used.

[0061] The cell population for treating and / or preventing SARS-CoV-2 infection of the present disclosure is effective in terms of HLA restriction, and therefore can be used for treating and / or preventing a subject who has an HLA type recognized by the SARS-CoV-2-specific TCR carried by the cell population. The HLA of the subject can be confirmed by standard methods.

[0062] The cell population of the present application is administered to a subject by suspending it in an appropriate medium, such as physiological saline, PBS, or other known medium used in cell therapy. Administration to a subject may be performed intravenously.

[0063] The number of cells to be administered is not particularly limited and may be determined appropriately depending on the age, sex, height, weight, target disease, symptoms, etc. of the subject. The optimal number of cells to be administered may be determined appropriately through clinical trials. For example, 10 ６ -10 ７ For example, cells / kg may be administered intravenously to a subject one or more times.

[0064] The present disclosure also provides a method for producing cells, including a step of expressing a SARS-CoV-2-specific human T cell receptor (TCR) in T cells of a human subject in need thereof, and a method for treating and / or preventing SARS-CoV-2 infection. To express a SARS-CoV-2-specific human TCR in T cells of a human subject, for example, mRNA encoding the TCR can be encapsulated in a delivery vehicle, such as a lipid nanoparticle, and then administered to the subject after providing a mechanism for targeting T cells. Furthermore, if necessary, a mechanism for avoiding mispairing of TCRα and TCRβ may be provided.

[0065] The specificity of the cells for treating and / or preventing SARS-CoV-2 infection provided in the present disclosure can be confirmed by the assays shown in the examples of the present disclosure. Examples of such assays include killing assays against target cells to which a target antigen peptide or protein has been added or which express the target antigen peptide or protein. In killing assays, it is preferable to use cells expressing the target HLA as the target cells. Furthermore, cell characteristics can also be confirmed by measuring the proliferation of T cells themselves in response to stimulation from the target cells or the expression of T cell activation markers. This method also makes it possible to determine which TCR type of cells is compatible with a biological sample of interest, as well as their strength and effectiveness, which can be used as an indicator for treatment or prevention. The sample may be collected from a subject in the acute or recovery phase after infection, or from a subject in the chronic phase suspected of suffering from sequelae, etc.

[0066] The present disclosure will be described in more detail below with reference to reference examples and examples. The following examples are provided for understanding the present invention, but are not intended to limit the present invention. Reference example 1

[0067] Selection of ES cell lines capable of differentiating into T cells. Several ES cell lines established at the Institute of Medical Biology, Kyoto University, and the Center for Child Health and Development were used (JMA J. 2020 Oct 15; 3(4): 287-294. doi: 10.31662 / jmaj.2018-0029). Table 7 lists the names of each cell line. ES cells were induced to differentiate into T cells based on a previous report ("New Generation Flow Cytometry Utilization Standard" (J. Kiyota, T. Yamamoto, eds.), Yodosha, pp. 141-157, 2021). Figure 1 shows an outline of the process. To induce differentiation, embryoid bodies were first formed from ES cells and then induced into CD34 / 43+ hematopoietic progenitor cells. The hematopoietic progenitor cells contained within the embryoid bodies were then seeded onto OP9 / DLL1 to induce T cells. To confirm that the desired cells were obtained at each stage of the differentiation induction process, expression of CD34 / 43 on Day 14, CD5 / 7 on Day 30, and CD4 / 8 bi-positive on Day 37 was confirmed. The expression of these differentiation markers varied depending on the ES cell line. Among them, SEES-3-10, derived from the human ES cell line SEES3, was confirmed to stably produce CD34 / 43+ blood progenitor cells, CD5 / 7 T progenitor cells, and CD4 / 8 bi-positive (DP) immature T cells during the differentiation induction process, and furthermore, was able to produce the largest number of CD4 / 8 DP cells. Based on these results, SEES3-10 was used as the T cells into which SARS-CoV-2-specific TCRs were introduced in the following examples.

[0068] ND: No Data Reference example 2

[0069] 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. An outline is shown in Figure 2. First, B2M / CIITA at SEES3-10 was knocked out using CRISPR / Cas9. For the B2M knockout, a gRNA was designed to target the interior of exon 1 of the B2M locus (GGCCGAGATGTCTCGCTCCG). For the CIITA knockout, a gRNA was designed to target the interior of exon 3 of the CIITA locus (TCAACTGCGACCAGTTCAGC).

[0070] The knockout-manipulated ES cells were subjected to single-cell cloning, and the base sequences of B2M and CIITA were confirmed. As a result, ES cell clones were obtained in which a single-base insertion mutation occurred in both alleles of the B2M / CIITA locus (Figure 3). 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 again subjected to single-cell cloning.

[0071] Four ES cell clones (SEES3-10-DKO-NY1 / 3 / 5 / 6) were obtained by HLA-I / II knockout and NY-ESO-1-TCR gene transfer. 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 could be obtained from all of these ES cell clones (Figure 4). CD4+ cells were then isolated by magnetic cell sorting.

[0072] CD8SPT cells (CD8 single positive T cells) were expanded by co-culturing the 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 have a TCR specific for NY-ESO-1 and not express HLA-I / II (Figure 5). Hereinafter, CD8SPT cells induced from pluripotent stem cells are referred to as "regenerated CTLs."

[0073] Cloning of SARS-CoV-2 antigen-specific TCR genes (1) (1) Isolation from vaccine recipients using antigen peptides as indicators (QYI_A1, QYI_B1, QYI_H5, YLQ_28, RLQ_3) An outline is shown in Figure 6. HLA typing was performed on healthy volunteer donors who had received the novel coronavirus (SARS-CoV-2) vaccine, and donors who were positive for HLA-A24 and HLA-A02 were selected. Peripheral blood mononuclear cells collected from each donor were stained with PE fluorescent-labeled MHC tetramers carrying the HLA-A24-restricted SARS-CoV-2 spike protein antigen peptide QYIKWPWYI (QYI), the HLA-A02-restricted SARS-CoV-2 spike protein antigen peptide YLQPRTFLL (YLQ), and RLQSLQTYV (RLQ), and the frequency of spike protein antigen-specific T cells was measured by flow cytometry. CD8+ T cell populations that tested positive with the MHC tetramers were sorted into single cells per well of a 96-well PCR plate using a cell sorter, and cDNA was synthesized and amplified from single T cells in each well by multiplex RT-PCR.

[0074] Next, the TCR α chain gene and the 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.

[0075] 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 6. Single-cell analysis identified four distinct clonotypes with TCRαβ paired sequences from the T cell fraction that tested positive for HLA-A24-restricted antigens using the QYI-MHC tetramer. For HLA-A02-restricted antigens, a total of four distinct TCRαβ paired sequences were obtained for YLQ and a total of 13 distinct TCRαβ paired sequences for RLQ.

[0076] Subsequently, to perform functional analysis of the obtained TCRs, three types of TCR gene expression retroviral vectors were constructed for QYI, one type for YLQ, and one type for RLQ from the identified TCRαβ paired genes. The sequence information of the CDR3 of each TCRαβ is shown in Table 8.

[0077]

[0078] (2) Examination of the antigen specificity of the cloned TCR genes [Method] TCR-expressing reporter cells were prepared by transfecting each of the cloned TCRαβ paired genes into a T cell line (Jurkat cells) expressing the NFAT-ZsGreen-1 reporter gene. To examine the antigen specificity of the TCR-expressing reporter cells, a cell line expressing both HLA-A02 and HLA-A24, prepared by transfecting HLA-A24 into NALM6 cells, an HLA-A02-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 each of the QYI, YLQ, and RLQ peptides, and reporter activity was observed under a fluorescence microscope 8 hours after the start of culture.

[0079] The results are shown in Figure 7. Each TCR-expressing reporter cell co-cultured with antigen-presenting cells was activated only under peptide addition conditions, confirming that each cloned TCR exhibits reactivity to epitope peptides derived from SARS-CoV-2 antigens.

[0080] (3) Examination of the Binding Affinity of the Cloned TCR to the Antigen Peptide To examine the binding affinity of the cloned TCR to the antigen peptide, serial dilutions of QYI, YLQ, and RLQ peptides were added to TCR reporter cells cocultured with antigen-presenting cells, and the frequency of activated cells was measured by flow cytometry 8 hours after the start of coculture. 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, and the peptide concentration showing 50% of the maximum activity (EC50) was calculated to evaluate the binding affinity of each TCR to the antigen peptide. The results are shown in Figures 8 and 9.

[0081] In each TCR-expressing reporter cell, the expression level of NFAT-ZsGreen-1 was confirmed to increase with increasing peptide concentration (Figure 8). Stimulation indices were calculated from the frequency of ZsGreen-1-positive cell fractions measured by flow cytometry, and the dose-response curves obtained by plotting these values ​​for each peptide concentration are shown in Figure 9. The EC50 values ​​for each TCR antigen peptide obtained from these dose-response curves are shown in Table 9.

[0082] 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 be effective against not only current epidemic strains but also newly emerging variants.

[0083] The sequences of the TCRα and TCRβ of the resulting TCRs are shown below.

[0084] Cloning of SARS-CoV-2 Antigen-Specific TCR Genes (2) (1) Isolation from COVID-19-Infected Patients (10BA, 2BA) A schematic diagram is shown in Figure 10. Peripheral blood mononuclear cells (PBMCs) collected from recovered COVID-19 patients were stimulated for 20 hours with inactivated SARS-CoV-2, SARS-CoV-2 S protein, SARS-CoV-2 spike glycoprotein S peptide pool (JPT Peptide Technologies, PM-WCPV-S-1), SARS-CoV-2 membrane glycoprotein M peptide pool (Miltenyi Biotec, 130-126-702), or SARS-CoV-2 nucleoprotein or nucleocapsid phosphoprotein N peptide pool (Miltenyi Biotec, 130-126-698). T cells highly positive for the T cell activation markers CD69, CD137, or CD154 were sorted and subjected to single-cell TCR / RNA sequence (scTCR / RNA-seq) analysis.

[0085] Among the TCR clonotypes clustered in CD8+ T cells, approximately 65 clonotypes that were found to be more prevalent in COVID-19 patients than in healthy controls were selected and cloned. These TCR clonotypes were transfected into NFAT-GFP reporter cells (Matsumoto et al., Biochem. Biophys. Res. Commun. 534:680-686.2021) and stimulated with S and N peptide pools (JPT Peptide Technologies, PM-WCPV-S-1 and Miltenyi Biotec, 130-126-698) in the presence of donor B cells. 10BA and 2BA were identified as activated clonotypes (Figure 11, left).

[0086] We stimulated reporter cells transfected with the S peptide pool library and identified the activating peptides as TCR epitopes. Furthermore, we identified the HLAs that restrict the TCR by co-culturing the cells with donor HLA class I transfectants containing candidate HLAs A24:02 or B35:01.

[0087] (2) Isolated from mRNA vaccine recipients (14AB, CE2AB). Although they had no history of SARS-CoV-2 infection, peripheral blood samples were collected from recipients of the mRNA vaccine Comirnaty intramuscular BNT162b2 before vaccination and 3, 6, and 24 weeks after the first vaccination (3 and 21 weeks after the second vaccination, respectively). The resulting T cells were stimulated with an S peptide pool, and the resulting T cells were sorted and subjected to scTCR / RNA-seq analysis. Among the TCR clonotypes that proliferated after vaccination in each donor, the 16 most proliferating clonotypes were selected and cloned. After transfection into NFAT-GFP reporter cells, the cells were stimulated with the S peptide pool in the presence of the donor's autologous B cells, and 14AB and CE2AB were identified as activated clonotypes.

[0088] Identification of epitopes and restrictive HLAs was performed in the same manner as above (Figure 11, right). The target proteins, epitopes, and HLA-I restriction of each identified TCR clonotype are shown in Table 11 below.

[0089]

[0090] The amino acid sequences of the resulting TCRα chain and TCRβ chain are shown below.

[0091]

[0092] Expression of SARS-CoV-2-specific TCR in ES cell-derived regenerated CTLs (1) Preparation of retroviral vectors Retroviral vectors were constructed to transduce the SARS-CoV-2-specific TCR obtained in Examples 1 and 2. Table 13 shows each construct. To confirm gene transduction, the fluorescent protein BFP was incorporated into the construct. Retrovirus was produced in packaging cells, PLAT-E cells, and PG13 cells, and this procedure yielded PG13 cells that produce high-titer retrovirus.

[0093]

[0094] (2) Expression of SARS-CoV-2-Specific TCR in DKO-Regenerated CTLs Among the ES cell-derived DKO-regenerated CTLs obtained in Reference Example 2, SEES-10-dKO-2-NY5 was used. These cells pre-express an endogenous TCR (NY5) that recognizes the cancer antigen NY-ESO-I. Regenerated CTLs expressing both NY5 and the foreign TCR were generated by genetically introducing a foreign TCR. Note that the possibility cannot be ruled out that genetically introducing the foreign TCR may result in a mixture of TCRs with mispaired α and β chains. Mispaired TCRs pose a risk of immune responses to unknown antigens. However, since this example was an in vitro verification, a negative control (CTLs expressing only NY5 as a TCR) was used for verification.

[0095] The DKO-regenerated CTLs obtained in Reference Example 2 were infected with a retrovirus carrying a SARS-CoV-2-specific TCR gene. Fluorescence due to the expression of BFP contained in the TCR vector was used to confirm gene transfer using a flow cytometer. The regenerated CTLs confirmed to have been transferred with the TCR gene were used in the killing assays of Examples 4 to 6.

[0096] Killing Assay Using ES Cell-Derived Regenerated CTLs (1) Preparation of Target Cells Target cells were prepared to perform a killing assay using regenerated CTLs. The SARS-CoV-2-specific TCR obtained in Example 2 is expected to recognize peptides whose S protein is mounted on A2402 or A0201 and whose N protein is mounted on B3501. Therefore, the SARS-CoV-2 virus S protein gene was introduced into an HLA-A24:02 / 02:01-expressing lymphoblastoid cell line (LCL), and the SARS-CoV-2 virus N protein gene was introduced into an HLA-B35:01-expressing LCL using a retroviral vector. A schematic diagram is shown in Figure 12.

[0097] (2) HLA-restricted killing activity of regenerated CTLs expressing TCRs. A schematic diagram is shown in Figures 13 and 14 . Regenerated CTLs expressing each of the five TCRs (QYI A1, QYI B1, QYI H5, RLQ 3, and YLQ 28) obtained in Example 3 were used. A24 / A02+LCLs expressing S protein were co-cultured at an ET ratio of 1:1, and the percentage of viable LCL cells was measured by flow cytometry 18 hours later. LCLs were labeled with CTV (CeLLTrace Violet) before co-culture to distinguish them from regenerated CTLs. The results are shown in Figure 15 and Table 14. Regenerated CTLs bearing all five S protein-specific TCRs exhibited killing activity when co-cultured with S protein-expressing CTLs.

[0098] (3) HLA-restricted killing activity of TCR-expressing regenerated CTLs. The outline is shown in Figures 13 and 14. SARS-CoV-2-specific TCR (10BA, CE2AB) SARS-CoV-2-specific TCR (10BA, CE2AB)-expressing regenerated CTLs obtained in Example 3 were co-cultured with S protein-expressing LCLs, and the percentage of viable LCL cells was measured by flow cytometry after 18 hours. LCLs were labeled with CTV (CeLLTrace Violet) before co-culture to distinguish them from regenerated CTLs. The E / T ratio was calculated by dividing the number of LCL cells by 5.0 x 10 4 The results are shown in Figure 16 and Table 15.

[0099]

[0100] The 10BA-TCR-expressing regenerated CTLs and CE2AB-TCR-expressing regenerated CTLs exhibited killing activity that was dependent on the proportion of regenerated CTLs against targets in which S protein was expressed by HLA-A24:02 / 02:01-expressing LCLs.

[0101] SARS-CoV-2-specific TCR (2BA) was performed according to the methods shown in Figures 13 and 14. Regenerated CTLs expressing SARS-CoV-2-specific TCR (2BA) obtained in Example 3 were co-cultured with LCLs carrying HLA-B35:01 that had expressed N protein, and the percentage of viable LCL cells was measured by flow cytometry after 18 hours. The E / T ratio was calculated by dividing the number of LCL cells by 3.0 x 10 4 The cells were fixed to each well, and the proportion of BFP-positive regenerative CTL cells was varied. The results are shown in Figure 17 and Table 16. 2BA-TCR-expressing regenerative CTLs exhibited killing activity dependent on the proportion of regenerative CTLs against targets in which the N protein was expressed on HLA-B35:01-expressing LCLs.

[0102]

[0103] These results revealed that the S and N proteins processed within LCLs are presented to HLA and are recognized and destroyed by SARS-CoV-2-specific TCRs on regenerating CTLs.

[0104] (4) Epitope Peptide-Specific Killing Assay of Regenerated CTLs Expressing TCR Regenerated CTLs expressing 10BA-TCR and CE2AB-TCR obtained in Example 3 were used. The epitope peptides used were the S448 peptide recognized by 10BA-TCR and the S1209 peptide recognized by CE2AB-TCR. Regenerated CTLs were co-cultured with HLA-A24:02 and the amounts of each peptide listed in the table so that the E / T ratio was 1.0, and the percentage of viable cells was measured 16 hours later using a flow cytometer. The results are shown in Figure 18 and Table 17.

[0105] In co-culture with regenerated CTLs expressing TCR specific to each peptide, a peptide concentration-dependent killing activity was observed.

[0106] Killing Assay for SARS-CoV-2-Infected Cells (1) Target Cells: Generation of A24:02-Expressing A549+ACE2 Cells For SARS-CoV-2 to infect cells, membrane fusion after binding to ACE2 on the cell membrane is crucial. To investigate whether SARS-CoV-2-specific TCR-expressing regenerated CTLs can kill infected cells, we generated the A549+ACE2+A24 cell line by transfecting the ACE2-expressing human lung cancer-derived epithelial cell line A549 (HLA-A26:03 / 30:01) with HLA-A24:02 using a retroviral vector (Figure 19).

[0107] (2) Killing activity of regenerated CTLs expressing TCR against target cells. Regenerated CTLs expressing CE2AB and 10BA-TCR obtained in Example 3 were co-cultured with A549+ACE2+A24:02 in the presence of S1209 (CE2AB) or S448 (10BA) peptide, and the percentage of viable cells was measured using a flow cytometer after 16 hours. An outline is shown in Figure 20, and the results are shown in Figure 21. As a result, peptide concentration-dependent killing activity due to SARS-CoV-2-specific TCR was observed, similar to the results with LCL.

[0108] Assays using the xCELLigence system (1) Epitope peptide-specific killing assay SARS-CoV-2 infection requires analysis under P3 conditions. However, because flow cytometers cannot be used under P3 conditions, an alternative measurement device is required. Therefore, we investigated the killing activity of reconstituted CTLs against A549 cells using the xCELLigence RTCA SP system (Agilent Technologies Japan, Ltd., CA, USA #380601030), which can be used under P3 conditions.

[0109] Killing activity measurements were performed according to the instrument's manual. A549+ACE2+A24 cells were seeded onto a dedicated plate, and after the cells adhered to the plate, approximately 3 hours after addition of each SARS-CoV-2 epitope peptide, regenerated CTLs (NY5) expressing the NY-ESO-1-specific TCR obtained in Reference Example 2 or regenerated CTLs expressing the 10BA-TCR obtained in Example 3 were added at an E / T ratio of 1.0, and resistance values ​​were monitored. The results are shown in Figure 22. A SARS-CoV epitope peptide-dependent decrease in resistance value was confirmed for the 10BA-TCR-expressing regenerated CTLs, but no change was observed for the NY-ESO-1-TCR-expressing regenerated CTLs.

[0110] (2) Killing assay for SARS-CoV-2-infected cells The target cells (A24:02-expressing A549 + ACE2) obtained in Example 5(1) were seeded on xCELLigence plates. After 24 hours, the cells were contact-infected with SARS-CoV-2 Wuhan strain (UT-NCGM02) at an MOI of 10 for 1 hour.

[0111] Twenty-four hours after the completion of contact infection, regenerated CTLs (NY5) expressing the NY-ESO-1-specific TCR obtained in Reference Example 2, regenerated CTLs expressing the CE2AB-specific TCR obtained in Example 3, and regenerated CTLs expressing the QYI_B1-specific TCR were added at an E / T ratio of 1.0, and resistance values ​​were monitored. A summary is shown in Figure 23, and the results are shown in Figure 24. A decrease in antigen-specific resistance value due to infection was confirmed in regenerated CTLs expressing the CE2AB-specific TCR and regenerated CTLs expressing the QYI_B1-specific TCR. This confirmed that regenerated CTLs expressing the SARS-CoV2-specific TCR are capable of killing SARS-CoV2-infected cells.

[0112] (1) Establishment of ES cells expressing only a SARS-CoV-2-specific TCR Introducing a SARS-CoV-2-specific TCR gene into the HLA-DKO ES cells obtained in Reference Example 1 The regenerated CTLs used in Examples 3 to 5 may express four types of TCRs: the endogenous NY-ESO1-specific TCR, the exogenous SARS-CoV-2-specific TCR, and TCRs resulting from mispairing of TCRα and TCRβ. Although SARS-CoV-2 antigen-specific killing activity was observed, considering the risk of mispaired TCRs recognizing unknown antigens, it is preferable to use regenerated CTLs that express only one type of SARS-CoV-2-specific TCR, which is free from the risk of mispairing.

[0113] SARS-CoV-2-specific TCR-expressing ES cells were established. To introduce a SARS-CoV-2-specific TCR gene into the HLA-DKO-ed ES cells obtained in Reference Example 1, the gene was recombined into a lentiviral vector to prepare lentivirus-producing packaging cells. Subsequently, DKO-ES cells were infected with a lentivirus containing the SARS-CoV-2-specific TCR gene, and colonies expressing each TCR were collected. A schematic diagram is shown in Figure 25.

[0114] (2) Differentiation of SARS-CoV-2-specific TCR-expressing ES cells into regenerative CTLs The obtained ES cell clones transfected with SARS-CoV-2-specific TCR were induced to differentiate into regenerative CTLs using the same method as in Reference Example 1. As a result, on day 13, day 13+16, and day 13+23, they showed a differentiation pattern similar to that of the positive control NY-ESO1-specific TCR-transfected ES cells, and it was revealed that they had differentiated into CD4 / CD8 double-positive cells ( FIG. 26 ).

[0115] (3) HLA-restricted killing assay of TCR-expressing regenerated CTLs. Regenerated CTLs expressing the 2BA-TCR obtained in (2) above were co-cultured with HLA-B35:01+B35 LCLs expressing the N protein. Regenerated CTLs expressing the QYI_B1-TCR obtained in the same manner as above were co-cultured with A24 / A02+ LCLs expressing the S protein. After 16 hours, the percentage of viable LCL cells was measured by flow cytometry. LCLs were labeled with CTV (CeLLTrace Violet) before co-culture to distinguish them from regenerated CTLs. The E / T ratio was determined by fixing the number of LCL cells and varying the percentage of BFP-positive regenerated CTL cells. The results are shown in Figures 27 and 28.

[0116] In both cases, the proportion-dependent killing activity of regenerated CTLs was observed only in LCLs expressing the N protein or the S protein.

Claims

1. A method for producing a cell population for treating and / or preventing SARS-CoV-2 infection, comprising T cells or T cell precursors expressing a human TCR specific for SARS-CoV-2, the method comprising the step of expressing a single or multiple human T cell receptors (TCRs) specific for SARS-CoV-2 in T cells or T cell precursors in vitro.

2. The method of claim 1, wherein the T cells or T cell precursors expressing a human TCR specific for SARS-CoV-2 express CD8αβ.

3. The method of claim 1 or 2, which comprises T cells expressing a human TCR specific to SARS-CoV-2, and the T cells have killer activity.

4. The method of any one of claims 1 to 3, wherein the T cells or T cell precursors expressing a human TCR specific to SARS-CoV-2 are cells lacking any or all of HLA.

5. A method for production according to any one of claims 1 to 4, comprising the steps of introducing a human T cell receptor (TCR) into a cell capable of differentiating into a T cell, and differentiating the cell into which the human T cell receptor (TCR) has been introduced into a T cell or a T cell precursor.

6. The method of claim 5, wherein the cells capable of differentiating into T cells are pluripotent stem cells.

7. The method of claim 6, wherein the pluripotent stem cells are pluripotent stem cells lacking any or all of HLA.

8. The method of claim 6 or 7, wherein the pluripotent stem cells are derived from the human ES cell line SEES3.

9. The method of any one of claims 1 to 8, wherein the SARS-CoV-2-specific TCR is specific for an epitope of any one of SEQ ID NOs: 1 to 7.

10. The method of claim 9, wherein the CDRs of the SARS-CoV-2-specific TCR are selected from the following combinations:

11. The method of claim 10, wherein the SARS-CoV-2-specific TCR amino acid sequence is selected from the following combinations:

12. A cell population for treating and / or preventing SARS-CoV-2 infection, comprising allogeneic T cells or T cell precursors expressing a human TCR specific for SARS-CoV-2, produced by the method of any one of claims 1 to 11.

13. A cell population for treating and / or preventing SARS-CoV-2 infection, comprising a population of allogeneic T cells or T cell precursors expressing an identical human TCR specific for SARS-CoV-2 and lacking any or all HLA.

14. The cell population of claim 13, comprising a combination of multiple allogeneic T cell or T cell precursor populations that express the same human TCR specific for the S protein of SARS-CoV-2 and lack any or all HLA.

15. The cell population of claim 13, wherein the T cells or T cell precursors express CD8αβ.

16. The cell population according to any one of claims 13 to 15, wherein the T cells are T cells with killer activity.

17. The cell population of any one of claims 13 to 16, wherein the SARS-CoV-2-specific TCR is specific for an epitope of any one of SEQ ID NOs: 1 to 7.

18. The cell population of claim 17, wherein the CDRs of the SARS-CoV-2-specific TCR are selected from the following combinations:

19. The cell population of claim 18, wherein the SARS-CoV-2-specific TCR amino acid sequence is selected from the following combination:

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