TCR-T cell, preparation method and application

Abstract

The invention provides a TCR-T cell as well as a preparation method and application thereof, and belongs to the technical field of cell therapy. The preparation method comprises the following steps: stimulating a T cell by virtue of a carrier containing an SARS-CoV-2 antigen, so as to obtain the TCR-T cell specifically targeting the SARS-CoV-2 antigen. The specific TCR-T cell for recognizing the SARS-COV-2 antigen, which is obtained by stimulating T cells through the SARS-CoV-2 antigen, can be used as a universal TCR-T cell, and due to the fact that the specific TCR-T cell does not target an antigen from a receptor (patient), GVHD reaction cannot be caused, and GVHD caused by heterologous T cells can be reduced to the greatest extent.

Description

[0001] This application is a divisional application of the invention application with the application number of 202310553745.X, the application name of "TCR-T cell, preparation method and application", and the filing date of May 17, 2023. TECHNICAL FIELD

[0002] The present application belongs to the technical field of cell therapy, and particularly relates to a TCR-T cell, a preparation method and application. BACKGROUND

[0003] Adoptive T cell therapy, i.e., the transfer of antigen-specific T cells to a patient, is an effective treatment for cancer and viral infections. T cells used for adoptive cell therapy generally require in vitro expansion of antigen-specific T cells, and / or genetic engineering editing of T cells. The transfer of virus antigen-specific T cells for the treatment of transplantation-related viral infections has been a relatively mature treatment scheme [(Riddell et al., 1992)]. The isolation and transfer of tumor-specific T cells have been successfully used to treat melanoma.

[0004] CAR-T cells, i.e., chimeric antigen receptor T cells, are a type of adoptive T cell therapy. CAR-T cells are obtained by modifying T cells of a patient (wherein the modification is usually achieved by introducing a receptor gene capable of recognizing tumor-specific antigens and various gene fragments that help T cell activation to form CAR-T cells), and then the modified T cells are expanded in vitro and then transferred back to the patient. Once encountering tumor cells expressing the corresponding antigen, the T cells will be activated and expanded again to exert their great specific killing power. For example, a personalized or universal CAR-T cell disclosed in patent WO2019052577A1 is obtained by using the CRISPR / Cas9 system to knockout single genes (TRAC, B2M or PD-1), double genes (TRAC and B2M), and triple genes (TRAC, B2M and PD-1) of T cells. These genetically edited T cells can provide universal T cells for CAR or TCR targeting different targets, so that the genetically modified T cells can be used as drugs for patients in need at any time. However, the prior art is a CAR-T cell targeting cancer cell surface antigens prepared by introducing a chimeric antigen receptor into T cells based on previously prepared T cell materials. However, there are still some disadvantages: the targeted antigens are limited to be expressed on the surface of tumor cells; CAR-T cells are usually more effective for hematological tumors, but the effect on solid tumors needs to be further improved; CAR-T introduces artificially modified genes, and the body's rejection of CAR will be stronger, which may shorten the survival time of CAR-T.

[0005] TCR-T, or T-cell receptor (TCR) chimeric T cells, involves introducing pre-selected TCRs with high antigen recognition capabilities into T cells, enhancing their tumor-attacking ability and achieving therapeutic effects. TCR-T's recognition of tumor target cells is independent of specific surface antigens; it specifically recognizes the HLA complex antigen presented on the cell surface after cleavage and processing by HLA. In January 2022, the FDA approved tebentafusp-tebn (Kimmtrak, Immunocore Limited), a bispecific cell conjugate targeting the gp100 peptide-HLA and CD3, for the treatment of HLA-A*02:01-positive unresectable or metastatic uveal melanoma. This was the world's first approved TCR-T therapy for solid tumors.

[0006] Currently, the manufacturing of commercially engineered T-cell products is highly personalized, with all cell products derived from autologous peripheral blood αβ-TCR-T cells [(Chow et al., 2018; Mo et al., 2021)]. However, the preparation of autologous TCR-cells is time-consuming and expensive, typically requiring 2-5 weeks. Patients' conditions progress rapidly, making it difficult to bear the consequences of preparation failure. Therefore, the main disadvantages of T-cell products prepared from autologous cells are: early treatments such as radiotherapy and chemotherapy can affect the quality and yield of the patient's autologous cell starting material; differences between patients are uncontrollable; the waiting period for patient access to medication is long due to the limited preparation cycle; and personalized products are expensive, resulting in insufficient patient accessibility.

[0007] "Off-the-shelf" TCR-T cells are prepared using T cells derived from allogeneic healthy blood donors, also known as "off-the-shelf" or universal cells. Typically, T cells are collected from healthy donors or directly from stem cell-derived T cells, HLA-matched, and then the designed and successfully validated TCRs are loaded onto the surface of the T cells. The use of universal TCR-T cell therapy provides simplified engineered cell manufacturing and even allows for the creation of "off-the-shelf" products, facilitating faster and cheaper treatments. Realizing the "off-the-shelf" concept, TCR-T cells could theoretically be available immediately, like "medicines," without waiting. However, a potential risk of allogeneic TCR-T cells is that incomplete HLA matching between donor and recipient can trigger immune recognition, leading to graft rejection, graft-versus-tumor reactions (non-specific), and graft-versus-host disease (GVHD). GVHD is a major factor in the adverse reactions of cell transplantation therapy, with αβ T cells playing a dominant role in both acute and chronic GVHD.

[0008] Therefore, there is a need in the art for a universal TCR-T cell that can reduce graft rejection and graft versus host disease. SUMMARY

[0009] To solve the problems existing in the prior art, the present application provides a TCR-T cell, a preparation method and an application. The present application uses a TCR-T cell containing a SARS-COV-2 antigen recognition TCR as a universal TCR-T cell, which can effectively reduce the graft rejection and graft versus host disease (GVHD) generated by the recipient. The specific content is as follows:

[0010] In a first aspect, the present application provides a preparation method of a TCR-T cell. The method comprises: stimulating a T cell by a SARS-CoV-2 antigen-containing vector to obtain a TCR-T cell specifically targeting a SARS-CoV-2 antigen.

[0011] In some embodiments, the preparation method further comprises: infecting the TCR-T cell specifically targeting the SARS-CoV-2 antigen with a tumor antigen-specific TCR lentivirus to obtain a TCR-T cell specifically targeting both the SARS-CoV-2 antigen and the tumor antigen.

[0012] In some embodiments, the method further comprises: knocking out the B2M gene in the TCR-T cell specifically targeting both the SARS-CoV-2 antigen and the tumor antigen to obtain a TCR-T cell specifically targeting both the SARS-CoV-2 antigen and the tumor antigen with a knocked-out B2M gene.

[0013] In some embodiments, the tumor is derived from any one of lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, large intestine cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, and prostate cancer.

[0014] In some embodiments, the T cell is a PBMC of an allogeneic healthy blood donor.

[0015] In some embodiments, the vector is one or more of a peptide segment, a plasmid, a presentation cell containing SARS-CoV-2 antigen information, a virus, and nano-artificial antigen presentation magnetic beads.

[0016] In some embodiments, the SARS-CoV-2 antigen is presented by any one or more HLA-I type molecules of the following types:

[0017] HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-A*07:01, HLA-A*08:01, HLA-A*07:02, HLA-A*40:01.

[0018] In some embodiments, the SARS-CoV-2 antigen is derived from any one or more of the following proteins: ORF1, ORF2 spi, ORF6, ORF3, ORF9 nuc, ORF7, ORF4 env, ORF5 mem, ORF8.

[0019] In a second aspect, the present application provides a TCR-T cell prepared by the method of the first aspect.

[0020] In a third aspect, the present application provides a TCR-T cell for use in treating and / or preventing a tumor disease. The TCR-T cell is prepared by the method of the first aspect.

[0021] The present application is based on the fact that almost all humans in the world have SARS-COV-2 antigen-specific T cells in their bodies. The use of specific TCR-T cells that recognize SARS-COV-2 antigens can effectively reduce the rejection reaction and graft-versus-host disease (GVHD) of the recipient in subsequent use.

[0022] The TCR-T cells provided by the present application can be further processed into double-targeted TCR-T cells for different tumors according to the needs of tumor suppression, so that they have tumor suppression effect. For example, double-targeted TCR-T cells targeting MART-1 and SARS-COV-2 at the same time are prepared. The obtained double-targeted TCR-T cells can be used for all patients expressing MART-1 tumor antigens, and can be mass-produced for storage as spot goods in actual application.

[0023] Since the SARS-COV-2 polypeptide can be presented by a variety of different HLA restrictions, the TCR-T cells provided by the present application have the advantage of wider source of healthy blood donor screening in preparation.

[0024] In summary, the TCR-T cells and double-targeted TCR-T cells provided by the present application are both universal and spot goods, which have the advantages of low cost and wide audience. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The preparation flowchart of the TCR-T cells prepared by the embodiment 1 of the present application is shown;

[0026] Figure 2The results of detecting the HLA-I knockout efficiency of TCR-T cells in Example 1 of the present application are shown;

[0027] Figure 3 The results of detecting the proportion of antigen-specific TCR-T cells at different time points in the preparation process of Example 1 of the present application are shown;

[0028] Figure 4 The results of TCR-T cells on target cell lysis in Example 2 of the present application are shown;

[0029] Figure 5 The results of detecting the secretion of cytokines by TCR-T cells after stimulation in Example 3 of the present application are shown;

[0030] Figure 6 The experimental results of tumor weights in each group in Example 4 of the present application are shown;

[0031] Figure 7 The experimental results of tumor inhibition rates in each group in Example 4 of the present application are shown;

[0032] Figure 8 The survival results of mice in each group in Example 4 of the present application are shown. DETAILED DESCRIPTION

[0033] In order to make the above objectives, features and advantages of the present application more apparent and easy to understand, the present application will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments of the present application will be described in detail below, and the embodiments are implemented on the premise of the technical scheme of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0034] Each aspect of the present application is described in detail in the following embodiments. The purpose of the embodiments is not to limit the present application. Each embodiment can be applied to any aspect of the present application. In this application, unless otherwise stated, the use of "or" means "and / or".

[0035] Nelde et al. confirmed that the T cell immune response is an important means for the body to control viral infection, and different polypeptides derived from SARS-COV-2 can be presented by different subtypes of HLA restriction, thereby activating antigen-specific T cells (Nelde et al., 2021). Therefore, antigen-specific TCR-T cells targeting SARS-COV-2 are widely present in healthy populations, and the present application selects antigen-specific TCR-T cells targeting SARS-COV-2 as donor-derived antigen-specific T cells, thereby providing a universal TCR-T cell. The specific content is as follows:

[0036] In a first aspect, the embodiments of the present application provide a preparation method of TCR-T cells. The method comprises: stimulating T cells by a carrier containing SARS-CoV-2 antigens to obtain TCR-T cells specifically targeting SARS-CoV-2 antigens.

[0037] In order to obtain more TCR-T cells specifically targeting SARS-CoV-2 antigens with good specificity, the concentration of the carrier containing SARS-CoV-2 antigens can be in the range of 1-20 μM during stimulation. For example, the concentration can be 1 μM, 2 μM, 3 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 15 μM, 17 μM, or 20 μM.

[0038] The present application is based on the fact that almost all humans in the world have SARS-COV-2 antigen-specific TCR-T cells in their bodies. The specific TCR-T cells recognizing SARS-COV-2 antigens are used as universal TCR-T cells. On this basis, tumor antigen-specific TCRs are introduced, and after knocking out the B2M gene, the graft rejection and graft-versus-host disease (GVHD) of the recipients can be effectively reduced during application. At the same time, the TCR-T cells provided by the embodiments of the present application also have the following advantages:

[0039] 1. Since SARS-COV-2 polypeptides can be presented by a variety of different HLA restrictions (this conclusion has been confirmed in the literature Nelde, A., et al. (2021).), the TCR-T cells provided by the present application have the advantage of wider source of healthy blood donor screening during preparation.

[0040] 2. Compared with autologous TCR-T cells, the universal TCR-T cells provided by the present application have lower manufacturing cost and are off-the-shelf, which is highly accessible and has a wide audience.

[0041] 3. COVID19 poses a huge threat to the health of cancer patients. The TCR-T cells provided by the present application can target both SARS-COV-2 antigens and tumor-specific antigens during application, so the TCR-T cells can not only kill tumors in the recipient, but also target cells infected with SARS-COV-2.

[0042] In some embodiments, the preparation method further comprises: infecting the TCR-T cells specifically targeting SARS-CoV-2 antigens with a tumor antigen-specific TCR lentivirus to obtain TCR-T cells specifically targeting both SARS-CoV-2 antigens and tumor antigens.

[0043] In the infection, the MOI (multiplicity of infection) value of the tumor antigen-specific TCR lentivirus is greater than 1.

[0044] In this embodiment, the antigen used by the TCR-T in cell immunotherapy is a tumor-specific antigen, which is not limited by cell surface expression, can be an intracellular antigen, has stronger precision targeting for tumor cells, and has a wider selection of target points. Compared with CAR-T, TCR-T is more likely to penetrate into the interior of a solid tumor. Moreover, TCR-T introduces a completely humanized structure, which is less likely to cause immune rejection of the body and has a low probability of producing anti-antibodies.

[0045] In some embodiments, the method further comprises knocking out a B2M gene in the TCR-T cell that simultaneously targets the SARS-CoV-2 antigen and the tumor antigen, to obtain a TCR-T cell that simultaneously targets the SARS-CoV-2 antigen and the tumor antigen and in which the B2M gene is knocked out.

[0046] In this embodiment, the TCR in the provided T cell is antigen-specific and the B2M gene has been knocked out. Therefore, the TCR-T cell provided in the embodiments of the present application effectively weakens the rejection reaction of the recipient to the graft, increasing the sustained survival of the transplanted cells in the recipient. Moreover, compared with the prior art in the background art which requires the knockout of two or more genes, the technology provided in this embodiment only requires the knockout of one gene, which can greatly reduce the risk and difficulty of gene editing.

[0047] Meanwhile, compared with the method disclosed in the literature (Chapuis et al., 2019) (since the B2M gene is not knocked out in the literature, the donor screening requirements are higher, the risk of rejection is greater, and MHC matching needs to be screened in actual operation, so the raw materials that can be used are relatively less), the B2M gene is knocked out in the embodiments of the present application, so MHC matching does not need to be screened in actual operation, thereby increasing the proportion of available raw materials.

[0048] It should be noted that: donor-derived T cells present donor antigens in an MHC-restricted manner, which can be recognized by recipient T cells, thereby causing host immune rejection. MHC class I molecules are composed of two subunits: a transmembrane heavy chain with high polymorphism and a small constant polypeptide, β2-microglobulin (β2-m) encoded by the B2M gene. The expression of MHC class I heavy chain on the cell surface requires its binding with β2-m. Therefore, the elimination of β2-m expression in TCR-T cells can inhibit the expression of MHC class I molecules and weaken the killing effect of host cytotoxic T lymphocytes (CTL) on donor cells.

[0049] In some embodiments, the tumor is derived from any one of lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, and prostate cancer.

[0050] In some embodiments, the T cells are PBMCs of allogeneic healthy blood donors.

[0051] In some embodiments, the vector is one or more of a peptide stretch, a plasmid, a presenting cell comprising SARS-CoV-2 antigen information, a virus, and nano-artificial antigen presenting magnetic beads.

[0052] In some embodiments, the SARS-CoV-2 antigen is presented by any one or more of the following HLA-I type molecule restricted typing: HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-A*07:01, HLA-A*08:01, HLA-A*07:02, HLA-A*40:01.

[0053] The above typing provided in this embodiment is mainly referenced to the literature Nelde, A., Bilich, T., Heitmann, J. S., Maringer, Y., Salih, H. R., Roerden, M., Lubke, M., Bauer, J., Rieth, J., Wacker, M., Peter, A., Horber, S., Traenkle, B., Kaiser, P. D., Rothbauer, U., Becker, M., Junker, D., Krause, G., Strengert, M., Schneiderhan-Marra, N., Templin, M. F., Joos, T. O., Kowalewski, D. J., Stos-Zweifel, V., Fehr, M., Rabsteyn, A., Mirakaj, V., Karbach, J., Jager, E., Graf, M., Gruber, L. C., Rachfalski, D., Preuss, B., Hagelstein, I., Marklin, M., Bachhoul, T., Gouttefangeas, C., Kohlbacher, O., Klein, R., Stevanovic, S., Rammensee, H. G., & Walz, J. S. (2021). SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition. Nat Immunol, 22(1), 74-85.

[0054] In some embodiments, the SARS-CoV-2 antigen is derived from any one or more of the following proteins: ORF1, ORF2 spi, ORF6, ORF3, ORF9 nuc, ORF7, ORF4 env, ORF5 mem, ORF8.

[0055] In particular, it can be any one or more of the following in combination:

[0056] TTDPSFLGRY, LTDEMIAQY, RTFKVSIWNLDY, ALSKGVHFV, LLLLDR LNQL, KLFAAETLK, QLRARSVSPK, KTFPPTEPKK, ASMPTTIAK, ATEGALNTPK, VYIGDPAQL, QYIKWPWYI, VYFLQSINF, FYVYSRVKNL, DYKHWPQIAQF, FPRGQGVPI, NPANNAAIVL, TPKYKFVRI, FVKHKHAFL, DLKGKYVQI, EAFEKMVSL, SELVIGAVIL, YEGNSPFHPL, LEYHDVRVVL, MEVTPSGTWL, IEYPIIGDEL, YYQLYSTQL, NRFLYIIKL, QRNAPRITF.

[0057] The antigen peptide sequence provided in the present embodiment is mainly referred to the literature Nelde, A., Bilich, T., Heitmann, J. S., Maringer, Y., Salih, H. R., Roerden, M., Lubke, M., Bauer, J., Rieth, J., Wacker, M., Peter, A., Horber, S., Traenkle, B., Kaiser, P. D., Rothbauer, U., Becker, M., Junker, D., Krause, G., Strengert, M., Schneiderhan-Marra, N., Templin, M. F., Joos, T. O., Kowalewski, D. J., Stos-Zweifel, V., Fehr, M., Rabsteyn, A., Mirakaj, V., Karbach, J., Jager, E., Graf, M., Gruber, L. C., Rachfalski, D., Preuss, B., Hagelstein, I., Marklin, M., Bachhoul, T., Gouttefangeas, C., Kohlbacher, O., Klein, R., Stevanovic, S., Rammensee, H. G., & Walz, J. S. (2021). SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition. Nat Immunol, 22(1), 74-85.

[0058] In some embodiments, the TCR-T cell is a TCR-T cell with a knockout of a B2M gene.

[0059] In particular implementation, the TCR-T cell is a TCR-T cell with a knockout of a B2M gene.

[0060] In the second aspect, the present embodiment provides a TCR-T cell prepared by the preparation method of the first aspect.

[0061] In the third aspect, the present embodiment provides a use of a TCR-T cell in treating and / or preventing a tumor disease. The TCR-T cell is prepared by the method of the first aspect.

[0062] The present application also provides the following embodiments:

[0063] Embodiment 1. A method for preparing a TCR-T cell, the method comprising: stimulating a T cell by a carrier containing a SARS-CoV-2 antigen to obtain a TCR-T cell specifically targeting a SARS-CoV-2 antigen.

[0064] Embodiment 2. The method of any one of embodiments 1, wherein the carrier is one or more of a peptide segment, a plasmid, a presenting cell containing information of a SARS-CoV-2 antigen, a virus, and a nano-artificial antigen presenting magnetic bead.

[0065] Embodiment 3. The method of any one of embodiments 1-2, wherein the method further comprises: infecting the TCR-T cell specifically targeting a SARS-CoV-2 antigen with a tumor antigen specific TCR lentivirus to obtain a TCR-T cell specifically targeting both a SARS-CoV-2 antigen and a tumor antigen.

[0066] Embodiment 4. The method of embodiment 3, wherein the method further comprises: knocking out a B2M gene in the TCR-T cell specifically targeting both a SARS-CoV-2 antigen and a tumor antigen to obtain a TCR-T cell specifically targeting both a SARS-CoV-2 antigen and a tumor antigen with a knocked out B2M gene.

[0067] Embodiment 5. The method of any one of embodiments 3-4, wherein the tumor is any one of a lung cancer, a hepatocellular carcinoma, a lymphoma, a colon cancer, a colorectal cancer, a breast cancer, an ovarian cancer, a cervical cancer, a gastric cancer, a cholangiocarcinoma, a gallbladder cancer, an esophageal cancer, a renal cancer, a glioma, a melanoma, a pancreatic cancer, and a prostate cancer.

[0068] Embodiment 6. The method of any one of embodiments 1-5, wherein the T cell is a PBMC of an allogeneic healthy blood donor.

[0069] Embodiment 7. The method of any one of embodiments 1-6, wherein the SARS-CoV-2 antigen is presented by one or more of the following HLA-I type molecule restricted: HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-A*07:01, HLA-A*08:01, HLA-A*07:02, HLA-A*40:01.

[0070] Embodiment 8. The preparation method according to any one of embodiments 1-7, characterized in that the SARS-CoV-2 antigen is derived from any one or more of the following proteins: ORF1, ORF2 spi, ORF6, ORF3, ORF9 nuc, ORF7, ORF4 env, ORF5 mem, ORF8.

[0071] Embodiment 9. A TCR-T cell prepared by the preparation method according to any one of embodiments 1-8.

[0072] Embodiment 10. Use of the TCR-T cell according to embodiment 9 in the treatment and / or prevention of a tumor disease.

[0073] In order to make the skilled in the art better understand the TCR-T cell provided by the embodiments of the present application, the following is described in detail through specific embodiments. Among them, the reagents and instruments used, if not specified, are all commercial products that can be directly purchased. And, in order to facilitate the skilled in the art to understand the inventive concept of the present application and facilitate the applicant to express, the tumor in each of the following specific embodiments is selected as melanoma, but the present application is not limited to only this kind of cancer.

[0074] The donor screening and PBMC collection involved in this embodiment are carried out on healthy blood donors, and both parties have signed the informed consent form.

[0075] Embodiment 1. Preparation of TCR-T cell

[0076] Since the SARS-COV-2 derived polypeptide can be presented by different HLA subtypes, different polypeptides are selected to stimulate PBMC according to the different HLA typing of healthy blood donors in this embodiment. It should be pointed out that each antigen peptide used in this embodiment is first determined according to the sequence listed in Table 1 to determine the sequence template, and then the third party is commissioned to prepare the required antigen peptide based on the determined sequence template.

[0077] Figure 1 The preparation process schematic diagram of the TCR-T cell prepared in embodiment 1 of the present application is shown. The specific steps of preparing the TCR-T cell are described in detail below with reference to the process shown in Figure 1

[0078] ​Peripheral blood 2ml of healthy blood donors was collected for HLA-I typing after high-throughput sequencing, and 8 blood donors who met the HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-B*08:01, HLA-B*40:01, HLA-C*07:02 typing were screened. The 8 healthy blood donors were subjected to physical examination. The physical examination should meet the following requirements:

[0079] The blood routine and blood biochemistry of the healthy blood donors were basically normal, among which the white blood cells, platelets and blood calcium should be within the normal range; the coagulation function and electrocardiogram of the healthy blood donors were basically normal; the healthy blood donors were negative for hepatitis B, hepatitis C, AIDS, syphilis and new coronavirus detection; the healthy blood donors had no ongoing infection; the blood vessel access of the healthy blood donors was good after evaluation by the collection nurse.

[0080] PBMC of the healthy blood donors meeting the above physical examination conditions was collected, and the circulating blood volume was ≥2000mL (2000mL in this embodiment). The collected PBMC was resuspended to 5E5 / mL with culture medium (45% RPMI-1640, 45% AIM-V, 10% FBS; Protamine sulfate (10μg / ml), IL-2 (50IU / ml), IL-21 (30ng / ml), IL-7 (5ng / ml) and IL-15 (1ng / ml));

[0081] According to the HLA-I typing results of the healthy blood donors, the PBMC was stimulated with S protein antigen peptides according to the following Table 1, and the final concentration of S protein antigen peptides was 10μM;

[0082] Table 1. HLA and polypeptide information

[0083] Polypeptide sequence Protein HLA restriction ALSKGVHFV ORF3 A*02:01

[0084] The specific operation of antigen-specific TCR expression and B2M knockout was as follows: after 1 day and 2 days of S protein antigen peptide stimulation, the culture medium was removed by centrifugation at 300g for 10 minutes, MART-1 antigen-specific TCR alpha-P2A-TCR beta (lentivirus, MOI≤3) was added (only MART1 specific TCR was used as an example in this embodiment, and antigen-specific TCRs selected and verified according to HLA-A typing and targeting can be selected), and centrifugation was performed at 900g for 90 minutes at 30°C. After 5 days of culture at 37°C, the PBMC was collected by centrifugation at 300g for 10 minutes, and B2M-sgRNA and Cas9 RNA were electroporated, washed by centrifugation, and then PBMC was stimulated with the corresponding S protein antigen peptide (final concentration 10μM) and cultured for 7 days.

[0085] In this embodiment, the electroporation conditions were: Pulse: Square Wave, Voltage: 500V, Time: 5ms, Cuvette: 4mm, Volume: 400ul, RNA: 10ug / 106 cells; B2M-sgRNA: cas9 RNA = 4:1.

[0086] The specific procedures for HLA-I- / COV2- tetramer+ / antigen tetramer+ sorting are as follows: a. After culturing for 12 days, take 2×10 6 HLA-I (W6 / 32, FITC) expression in CD8+ T (BV421) cells was detected by PBMC. Knockout efficiency was assessed on day 5 after knockout. Figure 2 The results of HLA-I knockout efficiency detection in TCR-T cells in Example 14 of this invention are shown, as follows: Figure 2 As shown, the HLA-I knockout efficiency is greater than 80%; b. The target cell population (i.e., B2M knockout cells that specifically target both SARS-CoV-2 and MART-1 antigens) is labeled with MART-1 antigen-specific tetramer (APC), donor HLA-restricted SARS-CoV-2 polypeptide tetramer (PE), and anti-HLA-I (W6 / 32, FITC). The FITC- / APC+ / PE+ target cell population is sorted on a clinical-grade flow cytometry platform and cGMP-grade cell preparation is performed. The purity of the sorted cells should be greater than [a certain value]. 95%; c. Following the reference [Ichikawa, J., et al. (2020).], prepare nano-aAPCs (nano-aAPCs). Load MART-1 antigen peptide and SARS-CoV-2 antigen peptide onto the corresponding HLA nano-aAPCs, respectively. Mix the HLA nano-aAPCs loaded with MART-1 antigen peptide and SARS-CoV-2 antigen peptide in equal proportions according to the amount used, and adjust the concentration to 10 OD / mL; d. Adjust the concentration of antigen-specific CD8+ T cells sorted in step b to 1×10⁻⁶. 8 Cells / mL, add nano-aAPC and CD8+ T cells at a ratio of 1:5, add the nano-aAPC mixed in a moderate proportion as in step c, incubate at 4°C for 1 hour, then resuspend the cells in culture medium, centrifuge at 300g for 10 minutes, discard the supernatant and resuspend for culture (culture medium); e, after 14 days of culture (culture medium: 45% RPMI-1640, 45% AIM-V, 10% FBS; 4 ng / mL IL2, 0.3 ng / mL IL4, 0.4 ng / mL IL6, 0.2 ng / mL IL1β, and 1 ng / mL IFNγ), collect cell counts, and decide whether to repeat step d based on cell demand.

[0087] Product preparation: after the operation of step e above, the culture cells are collected after the culture is completed, centrifuged at 300 g for 10 minutes, and then the supernatant is discarded to count the viable cells, resuspended in freezing solution to 1 x 10 7 cells / mL, and then divided into freezing bags, 20 mL (2 x 10 8 cells) per bag, and then transferred to a programmed temperature controller to be cooled to about -195°C, and then stored in a liquid nitrogen gas phase for use.

[0088] Quality inspection release: the quality inspection items include: sterility, endotoxin, HLA-I (negative proportion >; 85%), MART-1 antigen peptide tetramer specific T cells (positive proportion >; 80%), SARS-CoV-2 antigen peptide tetramer specific T cells (positive proportion >; 80%), MART-1 antigen peptide tetramer specific TCR copy number (≤ 5 copies per cell), lentivirus G protein DNA copy number (≤ 5 copies per cell), and the like.

[0089] In the preparation process, the proportion of antigen-specific TCR-T cells is detected at different time points. Figure 3 The results of detecting the proportion of antigen-specific TCR-T cells at different time points in the preparation process of Example 1 of the application are shown. As Figure 3 shown, the proportion of MART-1 or SARS-COV-2 antigen-specific CD8+ T cells in PBMCs is small before sorting at 12 days of culture; the proportion of MART1 and SARS-COV-2 antigen bispecific T cells (i.e., double antigen-specific T cells) obtained after flow sorting is more than 90%; and the proportion of MART-1 and SARS-COV-2 antigen bispecific T cells can still be maintained above 50% after 14 days of continuous expansion. Figure 3 MART-1-tetramer: PE; SARS-COV-2-tetramer: FITC).

[0090] Example 2. Verification of MART-1 (HLA-A*02:01 restricted) specific TCR function of prepared TCR-T cells

[0091] After overexpression of TCR in CD8+ T cells, the TCR-T cells were mixed with target cells at different proportions for co-culture, and the specific killing efficiency of TCR-T cells was detected. Among them, OVCAR8 cells naturally lacking HLA-I were used as negative controls, and OVCAR8 and OVCAR8-HLA*02:01 cells were incubated with MART-1 polypeptide (10 μM) for 4 hours in advance as killing target cells.

[0092] Figure 4 The results of the lysis of target cells by TCR-T cells in Example 2 of the application are shown. Figure 4In the present embodiment, E represents TCR-T effector cells, and T represents OVCAR8 target cells. As shown in FIG. 1, MART-1 (HLA-A*02:01 restricted) specific TCR-T cells have no obvious killing function for OVCAR8 cells naturally lacking HLA-I, but have obvious killing function for OVCAR8-HLA*02:01 cells. Thus, it can be known that the MART-1 (HLA-A*02:01 restricted) specific TCR-T cells prepared in the present embodiment have killing effect (such as HLA*02:01 OVCAR8 cells). Figure 4

[0093] Example 3. Cytokine secretion detection of TCR-T cells

[0094] The TCR-T cells prepared in Example 1 (i.e., MART-1 and SARS-COV-2 antigen bispecific T cells) were stimulated by nano-aAPCs respectively loaded with MART-1 antigen and SARS-COV-2 antigen, and the percentages of IFN-γ, TNF-α and IL-2 secreting cells were detected, respectively.

[0095] Figure 5 The results of the cytokine secretion detection of TCR-T cells in Example 3 of the present application are shown in FIG. 2. As shown in FIG. 2, on the one hand, the secretion amounts of IFN-γ and TNF-α of the TCR-T cells prepared in Example 1 after being stimulated by nano-aAPCs loaded with MART-1 antigen both exceeded 60%, and the secretion amount of IL-2 also obviously increased relative to the control group, about 10%; on the other hand, the secretion amount of IFN-γ of the TCR-T cells prepared in Example 1 after being stimulated by nano-aAPCs loaded with SARS-COV-2 antigen was about 50%, the secretion amount of TNF-α was about 25%, and the secretion amount of IL-2 was about 8%, and the secretion amounts of IFN-γ, TNF-α and IL-2 all increased relative to the control group. That is, the TCR-T cells prepared in Example 1 can effectively respond to antigen stimulation of MART1 and SARS-COV-2, and have killing function. Figure 5 Example 4. In vivo efficacy evaluation of TCR-T cells

[0096]

[0097] ​​We selected OVCAR8 (expressing HLA-A*02:01 and MART-1 antigen) to simulate MART-1 tumor cells, and subcutaneously inoculated immunodeficient mice (NOG-dKO) to construct a tumor-bearing model, and then treated with different T cells and observed the survival of mice. Among them, the T cells of the experimental group were TCR-T cells prepared in Example 1, the T cells of the control group were CD8+ T cells without expressing MART-1 TCR, and the blank group was the PBS group. 5 mice in each group, and each mouse was treated with 5x10 6 T cells.

[0098] Experimental animals: species & strain: MHC class I-and class II-deficient NOG (referred to as NOG-dKO), and the NOG background is NOD / Shi-PrkdcscidIl2rγtm1Sug / Jic.

[0099] Figure 6 The experimental results of the tumor weights of each group in Example 4 of the application are shown. Figure 6 As shown by the experimental results shown in

[0100] Figure 7 The experimental results of the tumor inhibition rates of each group in Example 4 of the application are shown. Figure 7 Among them, the tumor inhibition rate detection at the experimental endpoint is shown; wherein, taking the control group as the reference point, the tumor inhibition rate of the experimental group is calculated. As shown by the experimental results shown in Figure 7 As shown by the experimental results shown in

[0101] Figure 8 The survival results of the mice in each group in Example 4 of the application are shown. Figure 8 As shown by the experimental results shown in As shown by the experimental results shown in

[0102] The embodiment of the present application provides a "dual-targeting" TCR-T cell simultaneously targeting MART-1 and SARS-COV-2. According to the experimental results, the "dual-targeting" TCR-T cell has a certain tumor inhibition effect and does not cause serious graft rejection and graft-versus-host disease (GVHD). Moreover, in the actual scene, the "dual-targeting" TCR-T cell can be used for all patients (universal type) against MART-1 tumor, has the advantage of wide audience, and thus can be prepared in large quantities, stored as a ready stock, and used by patients at any time.

[0103] In addition, the skilled person in the art can make a dual-targeting TCR-T cell targeting other tumors from the TCR-T cell targeting SARS-COV-2 according to specific needs, and it is not limited to the MART-1 tumor in the embodiment.

[0104] For the method embodiment, in order to simply describe, all are expressed as a series of action combinations, but the skilled person in the art should know that the present application is not limited by the order of the described actions, because according to the present application, certain steps can be performed in other order or simultaneously. Secondly, the skilled person in the art should know that the embodiments described in the specification all belong to preferred embodiments, and the actions and components involved are not necessarily necessary for the present application.

[0105] The above describes in detail the TCR-T cell, the preparation method and the application provided by the present application. The principle and implementation mode of the present application are described by applying specific examples. The above embodiment description is only used to help understand the method of the present application and its core idea; at the same time, for the skilled person in the art, according to the idea of the present application, the specific implementation mode and application range will be changed; in conclusion, the content of the specification should not be understood as the limitation of the present application.

Claims

1. A method for preparing TCR-T cells, characterized in that, The method includes: stimulating T cells with a vector containing SARS-CoV-2 antigen to obtain TCR-T cells that specifically target SARS-CoV-2 antigen.

2. The preparation method according to claim 1, further comprising the step of introducing tumor antigen-specific TCRs into TCR-T cells specifically targeting SARS-CoV-2 antigen.

3. The preparation method according to claim 1 or 2, further comprising: TCR-T cells that specifically target SARS-CoV-2 antigen were infected with tumor antigen-specific TCR lentiviruses to obtain TCR-T cells that simultaneously and specifically target both SARS-CoV-2 antigen and tumor antigen.

4. The preparation method according to any one of claims 1-3, wherein the method further comprises: Knock out the B2M gene in TCR-T cells that simultaneously and specifically target SARS-CoV-2 antigen and tumor antigen to obtain TCR-T cells that simultaneously and specifically target SARS-CoV-2 antigen and tumor antigen with the B2M gene knocked out.

5. The preparation method according to any one of claims 2-4, wherein the tumor originates from any one of lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer, and prostate cancer.

6. The preparation method according to any one of claims 1-5, wherein the T cells are PBMCs from allogeneic healthy blood donors.

7. The preparation method according to any one of claims 1-6, wherein the SARS-CoV-2 antigen is presented in a restricted manner by any one or more HLA-I type molecules from the following subtypes: HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-A*07:01, HLA-A*08:01, HLA-A*07:02, HLA-A*40:

01.

8. The preparation method according to any one of claims 1-7, wherein the SARS-CoV-2 antigen is derived from any one or more of the following proteins: ORF1, ORF2 spi, ORF6, ORF3, ORF9 nuc, ORF7, ORF4 env, ORF5 mem, ORF8.

9. A TCR-T cell, obtained by the preparation method according to any one of claims 1-8.

10. The use of the TCR-T cells as described in claim 9 in the preparation of medicaments for treating and / or preventing tumor diseases.

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