T cell antigen receptor, LMP2-specific t cell, and use thereof

Abstract

Provided are a T cell antigen receptor (TCR), an LMP2-specific T cell, and a use thereof. The TCR specifically recognizes an EBV antigen and consists of a TCR α chain and a TCR β chain, wherein the TCR α chain comprises CDR1α, CDR2α and CDR3α; and the TCR β chain comprises CDR1β, CDR2β and CDR3β. The provided TCR binds to an EBV latent membrane protein LMP2, T cells transduced with the TCR can specifically recognize a corresponding pMHC complex, resulting in a high level of TCR-T cell activation, and the T cells have significant functional activity on EBV-infected target cells, thereby treating EBV-related diseases.

Description

T cell antigen receptors, LMP2-specific T cells and their applications Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a T-cell antigen receptor, LMP2-specific T cells and their applications. Background Technology

[0002] Epstein-Barr virus (EBV) is a gamma herpesvirus isolated by Epstein and Barr in 1964 from samples of Burkitt's lymphoma (BL). It is a double-stranded DNA virus. The infection rate in the population is over 90%. In immunocompetent individuals, the clinical course of infection is mostly mild, after which the virus enters a lifelong latent state, persisting in human B lymphocytes and remaining a lifelong carrier. Although EBV infection does not pose a significant threat to the vast majority of immunocompetent individuals, it is fatal to those with congenital or acquired immunodeficiency, leading to a variety of life-threatening diseases. During latent EBV infection, the virus does not proliferate in re-infected cells, but expresses a series of gene products: six EBV-determined nuclear antigens (EBNA) (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP) and three latent membrane proteins (LMP) (LMP1, LMP2A, and LMP2B). These have been proven to be directly related to the occurrence, development, and clinical prognosis of various malignant tumors, including EBV-related post-transplant lymphoproliferative diseases (EBV-PTLD), Hodgkin's lymphoma (HL), Burkitt's lymphoma, gastric cancer, and nasopharyngeal carcinoma (NPC). Based on the differential expression of latent proteins in these diseases, EBV latent phenotypes can be classified into stages I, II, and III. Currently, antiviral therapy targeting EBV has a low success rate in effectively treating the aforementioned EBV-related diseases due to issues such as drug resistance and severe side effects. Improving the efficacy for these patients is an urgent problem to be solved. Post-transplant EBV-related severe infections are caused by a lack of EBV-CTLs in the patient's body. Papadopounds et al. first discovered that infusing lymphocytes from healthy EBV carriers into PTLD patients could cure them, and subsequently, autologous EBV-CTL cell therapy emerged clinically. Currently, EBV-CTLs have been clinically applied in EBV-related lymphoma and nasopharyngeal carcinoma, showing good safety and some therapeutic effect in humans. However, in vitro culture of CTLs also has many shortcomings, thus limiting its clinical efficacy. First, the proportion of EBV antigen-specific T cells in CTLs is relatively low (<0.05%), therefore, high doses (greater than 10) are generally required. 10The treatment involves several drawbacks: firstly, the need for multiple infusions (4-6 times); secondly, the lengthy and complex preparation process leads to patients being unable to wait for treatment; and thirdly, the changes in T cell differentiation subsets during in vitro preparation further weaken the cells' function after in vivo. Therefore, there is an urgent need to develop more specific, more potent, and longer-lasting cell immunotherapy targeting EBV antigens for the treatment of EBV-related diseases.

[0003] T cell receptors (TCRs) are antigen recognition receptors expressed on the surface of T cells. T cells recognize the major histocompatibility complex (MHC) and presented antigen complexes on target cells via TCRs, triggering T cell activation and a series of subsequent cell signaling and other physiological responses, enabling antigen-specific T cells to exert an immune effect on their target cells. TCR-T cell immunotherapy involves cloning highly specific and high-affinity T cell receptors for tumor or viral antigen peptides and introducing them into autologous or allogeneic T cells through genetic engineering. This allows T cells to specifically recognize tumor or viral antigen peptides, thereby eliminating tumors or pathogens. Compared to the currently hotly researched CAR-T therapy, which utilizes antibody recognition of antigens and can only recognize viral or tumor antigens expressed on the cell surface, TCR-T therapy can recognize polypeptide fragments derived from proteins at any location on the cell after proteasome cleavage, exhibiting stronger killing effects and lower cytokine storms, showing greater potential for the treatment of solid tumors. Currently, it has been applied to adoptive cell immunotherapy for various malignant tumors and has achieved significant therapeutic effects in patients with advanced tumors who have failed other treatments. Among them, the TCR-T product targeting MAGE-A4 for the treatment of advanced synovial sarcoma has achieved positive clinical results and has been launched on the market.

[0004] The EBV latent membrane protein LMP2 possesses membrane receptor characteristics, activating lymphocyte proliferation and anti-apoptotic cell signaling systems, thereby inducing malignant transformation of normal cells. It is one of the major EBV genes expressed in EBV type II and III diseases / malignant tumors. Literature reports that the proportion of LMP2-specific CTL cells is closely related to clinical efficacy, suggesting that LMP2 could serve as a therapeutic target for EBV infection-related type II / III diseases.

[0005] Therefore, how to isolate TCRs that are specific to the short peptide of LMP2 antigen, and how to transduce these TCRs into T cells to obtain T cells that are specific to the short peptide of LMP2 antigen, so that they can play a role in cell immunotherapy, has become an urgent problem to be solved. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a T-cell antigen receptor that can specifically bind to the EBV latency protein LMP2 peptide (containing the sequence CLGGLLTMV / SLGGLLTMV), significantly activating TCR-T cells and significantly killing target cells.

[0007] A second objective of this invention is to provide a nucleic acid encoding a T-cell antigen receptor.

[0008] A third objective of this invention is to provide an expression carrier.

[0009] A fourth objective of this invention is to provide an LMP2-specific T cell.

[0010] The fifth objective of this invention is to provide a method for preparing LMP2-specific T cells.

[0011] The sixth object of the present invention is to provide an application of a T-cell antigen receptor.

[0012] To achieve the above objectives, the present invention provides a T-cell antigen receptor that specifically binds to the EBV latent membrane protein LMP2, wherein the binding epitope of the EBV latent membrane protein LMP2 is the sequence shown in Seq ID No. 1: CLGGLLTMV, or the sequence shown in Seq ID No. 2: SLGGLLTMV.

[0013] The T-cell antigen receptor is composed of an α chain and a β chain, wherein the α chain contains CDR1α, CDR2α and CDR3α; and the β chain contains CDR1β, CDR2β and CDR3β.

[0014] The sequence of the TCRα chain of this T cell antigen receptor is shown in Seq ID No. 3, and the sequence of the TCRβ chain is shown in Seq ID No. 4; the sequence of CDR1α is shown in Seq ID No. 5, the sequence of CDR2α is shown in Seq ID No. 6, the sequence of CDR3α is shown in Seq ID No. 7, the sequence of CDR1β is shown in Seq ID No. 8, the sequence of CDR2β is shown in Seq ID No. 9, and the sequence of CDR3β is shown in Seq ID No. 10; or

[0015] The sequences of the TCRα chain of this T cell antigen receptor are shown in Seq ID No. 11, the sequence of the TCRβ chain is shown in Seq ID No. 12; the sequence of CDR1α is shown in Seq ID No. 13, the sequence of CDR2α is shown in Seq ID No. 14, the sequence of CDR3α is shown in Seq ID No. 15, the sequence of CDR1β is shown in Seq ID No. 16, the sequence of CDR2β is shown in Seq ID No. 17, and the sequence of CDR3β is shown in Seq ID No. 18.

[0016] The present invention also provides a nucleic acid encoding a T-cell antigen receptor as described above.

[0017] The present invention also provides an expression vector comprising the nucleic acid as described above.

[0018] The present invention also provides an LMP2-specific T cell expressing the T cell antigen receptor as described above, comprising the nucleic acid as described above and / or the expression vector as described above.

[0019] This invention also provides a method for preparing LMP2-specific T cells, comprising the following steps:

[0020] 1) Transform the above expression vector into the Stbl3 strain to amplify the expression vector;

[0021] 2) The expression vector was mixed with psPAX2, pMD2.G and transfection reagent PEI to prepare a mixture. The mixture was added to 293T cells, cultured, and the cell culture supernatant was collected after 48 h and 72 h. The virus particles were collected by ultracentrifugation to concentrate the supernatant.

[0022] 3) T cells were infected with viral particles, cultured at 37°C for 10 hours, and then the infection was terminated. The cells were cultured for another 3 days, and then stained with anti-human CD3 and anti-mouse TCRβ flow cytometry antibodies to obtain LMP2-specific T cells.

[0023] The present invention also provides the use of the above-mentioned T cell antigen receptor, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned LMP2-specific T cell in the preparation of a medicament for treating EBV-related diseases.

[0024] The present invention also provides the above-mentioned applications, wherein the EBV-related diseases are infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary exudative lymphoma, or B / T / NK cell lymphoma.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention provides a T-cell antigen receptor, LMP2-specific T cells, and their applications. The T-cell antigen receptor binds to the EBV latent membrane protein LMP2 and can specifically recognize the corresponding pMHC complex. The resulting LMP2-specific T cells have significant functional activity against EBV-infected target cells, thereby generating high levels of TCR-T cell activation. Attached Figure Description

[0027] Figure 1 shows the HLA-A*A0201-CLGGLLTMV-tetramer staining results.

[0028] Figure 2 shows a schematic diagram of the connection of the TCR β chain and α chain in the pCDH-CMV-MCS-EF1 vector, where the connection order is promoter, β chain, FuP2A, α chain, WPRE sequence.

[0029] Figure 3 shows the affinity of the HLA-A*0201 epitope CLGGLLTMV (CLG) and SLGGLLTMV (SLG) specific TCRs for binding to the tetramer probes as detected by flow cytometry.

[0030] Figure 4 shows CD69 expression in Jurkat-TCR cells stimulated with different concentrations of CLG peptides.

[0031] Figure 5 shows CD69 expression in Jurkat-TCR cells stimulated with different concentrations of SLG peptide.

[0032] Figure 6 shows the bioluminescence intensity of Jurkat-NFAT-LUC-TCR cells stimulated with different concentrations of CLG peptides.

[0033] Figure 7 shows the bioluminescence intensity of Jurkat-NFAT-LUC-TCR cells stimulated with different concentrations of SLG peptides.

[0034] Figure 8 shows the EC50 statistics of CLG or SLG epitope-specific TCRs.

[0035] Figure 9 shows the statistical results of primary TCR-T cells killing JVM-2-Luc / GFP cells at different effector-to-target ratios.

[0036] Figure 10 shows the statistical results of primary TCR-T cells killing EBV-LCL-Luc / GFP cells at different effector-to-target ratios.

[0037] Figure 11 shows the statistical results of the killing effect of primary TCR-T cells on T2 cells under CLG peptide stimulation.

[0038] Figure 12 shows the statistical results of the killing effect of primary TCR-T cells on T2 cells under SLG peptide stimulation. Detailed Implementation

[0039] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0040] The HLA-A*0201 restriction peptide CLGGLLTMV / SLGGLLTMV of the LMP2 protein provided in this invention is a highly immunogenic antigenic epitope that can induce the body to produce specific T lymphocytes and corresponding immune responses. Therefore, this invention provides various T cell antigen receptors that can specifically bind to the EBV latent protein LMP2 peptide (containing the sequence CLGGLLTMV / SLGGLLTMV) and their application in the preparation of pharmaceutical compositions for treating EBV-related diseases. The TCRs described in this invention can specifically recognize the corresponding pMHC complex, significantly activate TCR-T cells, and significantly kill target cells.

[0041] Material:

[0042] 1. HLA-A*A0201-CLGGLLTMV-tetramer was purchased from MBL, product number: TS-M032-1.

[0043] 2. The endotoxin-free plasmid small-scale extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., product number: DP118-02.

[0044] 3.293T cells were purchased from the American Type Culture Collection (ATCC), product number: CRL-1573.

[0045] 4. T2 cells were purchased from the American Type Culture Collection (ATCC), product number: CRL-1992.

[0046] 5. Peripheral blood lymphocytes (PBMCs) of healthy volunteers with genotype HLA-A*0201 were purchased from Shanghai Miaoshun Biotechnology Co., Ltd.

[0047] 6. The short peptides CLGGLLTMV and SLGGLLTMV were synthesized by Jiangsu Genscript Biotech Co., Ltd.

[0048] 7. The antibody CD8-FITC was purchased from Biolegend, product number: 980908.

[0049] 8. The transfection reagent PEI was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product number: 40815ES03.

[0050] 9. Jurkat cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-400-0214.

[0051] 10. JVM-2 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0062.

[0052] 11. EBV-LCL was purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0202.

[0053] 12. Jurkat-NFAT-LUC cells were purchased from Nanjing Novizan Biotechnology Co., Ltd., product number: DD1302-01.

[0054] 13. The luciferase assay reagent was purchased from Nanjing Novizan Biotechnology Co., Ltd., product number: DL101-01.

[0055] Example 1: Enrichment of LMP2-specific T cells and TCR gene

[0056] Peripheral blood lymphocytes (PBMCs) from healthy volunteers with the HLA-A*0201 genotype were stimulated using the synthetic short peptide CLGGLLTMV. Antigen-specific single cells were enriched using HLA-A*A0201-CLGGLLTMV-tetramer and the antibody CD8-FITC. A certain number of antigen-specific single cells were obtained by flow cytometry sorting. The sorted specific cells were then processed using 10X Genomics Chromium... TM Single-cell sequencing platforms were used to construct and sequence TCR libraries, obtaining the nucleotide sequences of the variable regions of the TCRα and TCRβ chains, as well as information on their CDR1, CDR2, and CDR3. TCRα and TCRβ gene fragments were synthesized based on the sequence information obtained from single-cell sequencing. Two sets of TCRα and TCRβ gene fragments were obtained, named 004 and 006, respectively, i.e., 004-TCRα, 004-TCRβ and 006-TCRα, 006-TCRβ. The nucleotide sequence of 004-TCRα is shown in Seq ID No. 19; the nucleotide sequence of 004-TCRβ is shown in Seq ID No. 20; the nucleotide sequence of 006-TCRα is shown in Seq ID No. 21; and the nucleotide sequence of 006-TCRβ is shown in Seq ID No. 22.

[0057] Figure 1 shows the flow cytometry results of LMP2 A0201-CLGGLLTMV specific T cells after three rounds of antigen-induced stimulation in vitro. As can be seen from Figure 1, compared with the control group (antigen-unstimulated group), the antigen-stimulated group had a significant specific cell population, with a double positivity rate of 14.7% for CD8-FITC and EBV LMP2 pMHC tetramer-PE (Tetramer-PE).

[0058] Example 2: Construction of TCR lentiviral vector and viral packaging

[0059] (1) Construction of TCR lentiviral vector

[0060] The TCRα and TCRβ nucleotide sequences of 004 ​​and 006, respectively, were linked together using the FuP2A nucleotide sequence as shown in Seq ID No. 23 and cloned into a commercial expression plasmid based on pCDH-CMV-MCS-EF1. Two TCRβ-FuP2A-TCRα gene fragments were obtained by overlap-PCR amplification (KOD-Plus-Neo, TOYOBO), named 004-TCR and 006-TCR, respectively. The nucleotide sequence of 004-TCR is shown in Seq ID No. 24; the nucleotide sequence of 006-TCR is shown in Seq ID No. 25.

[0061] According to the Clone Express II One Step Cloning kit instructions, 004-TCR and 006-TCR were ligated with the linearized pCDH-CMV-MCS-EF1 after enzyme digestion via overlap, as shown in Figure 2. The ligation products were transformed into Stbl3 strain and inoculated onto LB agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, single colonies were picked and grown overnight at 37°C with shaking in 15 ml of LB agar containing 100 μg / ml ampicillin. The cloned plasmids were purified using an endotoxin-free plasmid mini-prep kit according to the instructions, and the plasmids were sequenced to obtain TCR plasmids containing either the 004-TCR or 006-TCR sequence fragments. These two plasmids were designated E004 and E006, respectively.

[0062] (2) Lentiviral packaging

[0063] 293T cells were cultured in 10cm cell culture dishes. Plasmid transfection was initiated when the cell volume did not exceed 80%. E004 and E006 plasmids were mixed with packaging plasmids psPAX2 and pMD2.G, and transfection reagent PEI (polyethylenimine), respectively, in serum-free DMEM medium at a mass ratio of 3:2:1. The mixture containing either E004 or E006 plasmids was then added to the 293T cells and incubated at 37°C. The cell culture supernatants were collected after 48 and 72 hours, and concentrated by ultracentrifugation to collect viral particles loaded with E004 or E006 TCRs.

[0064] Example 3: Detection of TCR membrane expression and affinity using pMHC tetramer staining method

[0065] (1) Construction of Jurkat cell line expressing TCR

[0066] Jurkat cells in logarithmic growth phase were infected with viral particles loaded with E004 or E006 TCR prepared in Example 2 (MOI = 0.3).

[0067] The infection method involved taking Jurkat cells in logarithmic growth phase, adding viral particles loaded with TCRs of E004 or E006, centrifuging the Jurkat cells at 1500 rpm for 2 hours at 32°C, removing the cells, and placing them in a 37°C cell culture incubator for 10 hours. Infection was then terminated by adding 1640 complete medium, and the cells were continued to be cultured at 37°C. Three days after infection, the cells were stained with anti-human CD3 and anti-mouse TCRβ flow cytometry antibodies. Cells with the same TCR expression level were sorted by flow cytometry based on color and then cultured to obtain two Jurkat-TCR cell lines, E004-P and E006-P.

[0068] (2) TCR membrane and affinity detection

[0069] Take 1×10 6 E004-P or E006-P samples were stained with FITC anti-mouse TCRβ (Biolegend) and the corresponding EBV LMP2 pMHC tetramer-PE (Tetramer-PE), followed by flow cytometry analysis. The results are shown in Figure 3.

[0070] As shown in Figure 3, the prepared specific TCRs targeting EBV LMP2 HLA-A*A0201 CLGGLLTMV / SLGGLLTMV all exhibited good binding to the CLGGLLTMV / SLGGLLTMV epitopes. This result not only demonstrates that both screened TCRs can be well expressed on the membrane, but also indicates that both obtained TCRs have good affinity for EBV LMP2 pMHC tetramers.

[0071] Example 4: Evaluation of TCR functional activity and EC50

[0072] Given that the pMHC tetramer assay in Example 3 detects the structural affinity of the TCR, and that the tetramer binds tetravalently to the TCR on the Jurkat surface, to further identify the functional activity of the TCR, T2 cell lines loaded with the antigen peptide were co-cultured with Jurkat-NFAT-LUC-TCR. This was used to verify the TCR-transduced T cell-specific activation response to target cells, and to quantify the half-maximal effector antigen concentration (EC50) of the TCR, thus comparing the functional activity of the TCR.

[0073] (1) Construction of Jurkat-NFAT-LUC-TCR cell line expressing TCR

[0074] Jurkat-NFAT-LUC cells (MOI = 0.3) in logarithmic growth phase were infected with viral particles loaded with TCRs of E004 or E006 prepared in Example 2. This cell line stably expressed the Lucia luciferase reporter gene under the control of NFAT (activated T cell nuclear factor) reactive elements. When the TCR binds to the specific antigen loaded on T2 cells, it activates the NFAT functional signaling pathway. The activation level of Jurkat-NFAT-LUC-TCR can be shown by measuring the bioluminescent signal generated by the NFAT-dependent luciferase reporter protein.

[0075] The infection method involved taking Jurkat-NFAT-LUC cells in logarithmic growth phase, adding viral particles loaded with E004 or E006 TCRs, centrifuging the cells at 1500 rpm for 2 hours at 32°C, then placing them in a 37°C cell culture incubator for 10 hours. Infection was then terminated by adding and replacing the culture medium, and the cells were cultured again at 37°C. Three days after infection, the cells were stained with anti-human CD3 and anti-mouse TCRαβ flow cytometry antibodies. Cells with the same TCR expression level were sorted and cultured to obtain two Jurkat-NFAT-LUC-TCR cell lines, named E004-T and E006-T, respectively.

[0076] (2) Detection of TCR-specific activation function

[0077] Two types of Jurkat-NFAT-LUC-TCR cells were co-cultured with T2 cells loaded with 1 nM, 100 nM, and 10000 nM concentrations of the peptide CLGGLLTMV (CLG peptide) or the peptide SLGGLLTMV (SLG peptide). After 24 h, the cell pellet was collected and stained with APC anti-mouse TCRβ (Biolegend), FITC-CD69 (Biolegend), and the corresponding EBV LMP2 pMHC tetramer-PE (Tetramer-PE). Flow cytometry analysis was then performed to detect the proportion of TCR and CD69 double-positive cells.

[0078] Figures 4 and 5 represent the CD69 protein expression in two types of Jurkat-NFAT-LUC-TCR cells after 24 hours of stimulation with three different concentrations of CLG and SLG peptides, respectively. As shown in Figures 4 and 5, compared with the T2 cell group alone (T2 cells without peptide loading, negative target cell group) and the Mock-T group (Jurkat-NFAT-LUC cells without TCR loading, negative control group), both E004 and E006 cells showed significant CD69 protein expression under CLG or SLG peptide loading stimulation, and this expression increased with increasing peptide concentration. This suggests that both TCRs can specifically recognize and be activated by SLG and CLG peptides.

[0079] (3) EC50 detection

[0080] The synthesized epitope peptides CLG and SLG were diluted to a storage concentration of 40 mg / mL using DMSO. Then, CLG and SLG were serially diluted with complete culture medium to obtain 2 × 10⁻⁶ peptides. -8 -2×10 -4 M CLG and SLG peptide solutions were added at a volume ratio of 1:100 to 1×10 6 In a T2 cell suspension of 100 μL / mL cells, mix well and seed the cells into 96-well plates at 100 μL per well. Simultaneously add 100 μL of a 1×10⁻⁶ mcg solution. 6 The concentration of the peptide is 1 × 10⁻⁶ cells / mL of Jurkat-NFAT-LUC-TCR cells E004-T or E006-T, after mixing. -10 -1×10 -6 The T2 incubation system of M was used. After co-culturing for 24 hours, the cell suspension was collected, luciferase assay reagent was added, and the bioluminescent signal of Jurkat-NFAT-LUC-TCR cells E004-T or E006-T was detected by microplate reader to detect the EC50 of TCR.

[0081] Figures 6 and 7 represent the bioluminescence intensities of Jurkat-NFAT-LUC-TCR cells stimulated with different concentrations of CLG and SLG peptides, respectively. The corresponding EC50 values ​​were calculated using Prim's algorithm, and the EC50 values ​​for three replicates are shown in Figure 8. Figures 6 to 8 show that all TCRs exhibited good functional activity for both the synthesized CLG and SLG epitopes, indicating that the results of E004 and E006 are consistent with tetramer staining, demonstrating strong recognition ability for the CLGGLLTMV / SLGGLLTMV epitopes.

[0082] Example 5: Construction and in vitro functional activity assessment of primary human TCR-T cells

[0083] (1) Construction of primary human TCR-T cells

[0084] To further verify the recognition and killing function of the screened TCRs against the EBV LMP2 antigen, mononuclear cells (PBMCs) were isolated from the peripheral blood of volunteers using Ficoll lymphocyte separation medium. After stimulating the T cells with anti-CD3 / CD28 magnetic beads, they were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clumping was observed. Lentiviral virus carrying the E004, E006 TCR genes of this invention, obtained in Example 2, was added at an MOI (multiple of infection) of 3 for transfection. The infection method involved mixing the virus with primary T cells, centrifuging the cells at 32°C and 1500 rpm for 2 hours, then placing them in a 37°C cell culture incubator for 10 hours. Infection was then terminated by adding and replacing the culture medium, and the cells were cultured again at 37°C. Infection efficiency was assessed by flow cytometry 3-4 days after transduction using 1640 medium containing 200 IU / ml IL-2 and 10% FBS.

[0085] (2) Target cell construction

[0086] JVM-2, EBV-LCL, and T2 cells in logarithmic growth phase were infected with lentiviral particles loaded with luciferase-GFP (Luciferas-GFP). JVM-2, EBV-LCL, and T2 cells stably expressing Luciferas-GFP were obtained through drug screening and flow cytometry sorting, and named JVM-2-Luc / GFP, EBV-LCL-Luc / GFP, and T2-Luc / GFP cells, respectively.

[0087] (3) Verify the TCR's ability to recognize and kill endogenous antigenic epitopes.

[0088] EBV-LCL refers to immortalized human B cells infected with Epstein-Barr virus (EBV), and JVM-2 refers to EBV-infected human peripheral lymphocytes; both realistically simulate the antigen levels of tumor cells in vivo. Therefore, E004-T and E006-T cells were co-cultured with JVM-2-Luc / GFP and EBV-LCL-Luc / GFP at effector-to-target ratios (E:T) of 10:1, 5:1, 1:1, and 0.5:1, respectively, with the target cells (JVM-2-Luc / GFP or EBV-LCL-Luc / GFP) fixed at 2 × 10⁻⁶ cells / year. 5 Cells. After co-incubation for 24 hours, cell suspensions were collected, and luciferase assay reagents were added. Bioluminescent signals of JVM-2-Luc / GFP and EBV-LCL-Luc / GFP cells were detected using a microplate reader to assess the killing effect of TCR-T cells on target cells expressing endogenous antigens. Primary T cells without TCR loading were used as a negative control group (Mock-T).

[0089] Figures 9 and 10 represent the specific lysis of the killing intensity of E004-T and E006-T cells against JVM-2-Luc / GFP and EBV-LCL-Luc / GFP cells, respectively. The results show that for JVM-2-Luc / GFP cells, compared with the negative control group (Mock-T group), both TCRs exhibited significant killing ability at E:T = 0.5:1 and 1:1, with E004-T showing relatively stronger killing function. For EBV-LCL-Luc / GFP cells, compared with the Mock-T group, E004-T showed strong killing ability at E:T = 5:1 and 10:1, while E006-T only showed some killing ability at E:T = 10:1. These results indicate that the TCRs prepared in this invention can effectively mediate the recognition of tumor endogenous antigens and have a strong killing ability against target cells.

[0090] (4) Verify the TCR's ability to recognize and kill exogenous antigenic epitopes.

[0091] To further demonstrate the specific cytotoxic function of TCR-transduced primary T cells against target cells, E004-T and E006-T cells were co-cultured with T2-Luc / GFP cells loaded with 5 μM CLG or SLG peptides, respectively. After 24 h, the cell suspension was collected, and luciferase assay reagent was added. The bioluminescent signal of T2-Luc / GFP cells was detected using a microplate reader to assess the cytotoxic effect of TCR-T cells on target cells loaded with exogenous antigens. Primary T cells without TCR loading served as a negative control group (Mock-T). Figures 11 and 12 show the cytotoxic intensity (specific lysis) of E004-T and E006-T cells against T2 cells after 24 h of stimulation with 5 μM CLG and SLG peptides, respectively. As shown in Figures 11 and 12, compared with the negative control Mock-T group and the T2 group alone, both TCRs exhibited strong specific cytotoxic activity against T2 cells loaded with the synthesized CLG antigen epitope and the SLG epitope, respectively. Furthermore, the cytotoxic function significantly increased with the increase of the E:T ratio. This suggests that the TCR described in this invention can bind to the EBV antigen short peptide, and T cells transduced with the TCR can be specifically activated, exhibiting a strong cytotoxic effect on target cells.

[0092] As can be seen from the above embodiments, the T-cell antigen receptor provided by the present invention can specifically bind to the EBV latency protein LMP2 peptide (containing the sequences CLGGLLTMV / SLGGLLTMV), significantly activating TCR-T cells and significantly killing target cells. This provides a basis for the effective treatment of EBV-related diseases.

[0093] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A T-cell antigen receptor, characterized in that, The T-cell antigen receptor specifically binds to the EBV latent membrane protein LMP2, wherein the binding epitope of the EBV latent membrane protein LMP2 is the sequence shown in Seq ID No.1 or Seq ID No.

2. The T-cell antigen receptor is composed of a TCRα chain and a TCRβ chain. The TCRα chain contains CDR1α, CDR2α and CDR3α; the TCRβ chain contains CDR1β, CDR2β and CDR3β. The sequence of the TCRα chain of this T cell antigen receptor is shown in Seq ID No. 3, and the sequence of the TCRβ chain is shown in Seq ID No. 4; the sequence of CDR1α is shown in Seq ID No. 5, the sequence of CDR2α is shown in Seq ID No. 6, the sequence of CDR3α is shown in Seq ID No. 7, the sequence of CDR1β is shown in Seq ID No. 8, the sequence of CDR2β is shown in Seq ID No. 9, and the sequence of CDR3β is shown in Seq ID No. 10; or The sequences of the TCRα chain of this T cell antigen receptor are shown in Seq ID No. 11, the sequence of the TCRβ chain is shown in Seq ID No. 12; the sequence of CDR1α is shown in Seq ID No. 13, the sequence of CDR2α is shown in Seq ID No. 14, the sequence of CDR3α is shown in Seq ID No. 15, the sequence of CDR1β is shown in Seq ID No. 16, the sequence of CDR2β is shown in Seq ID No. 17, and the sequence of CDR3β is shown in Seq ID No.

18.

2. A nucleic acid encoding the T-cell antigen receptor as described in claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid as described in claim 2.

4. An LMP2-specific T cell expressing the T cell antigen receptor as described in claim 1, comprising the nucleic acid as described in claim 2 and / or the expression vector as described in claim 3.

5. A method for preparing LMP2-specific T cells, characterized in that, Includes the following steps: 1) Transform the expression vector according to claim 3 into the Stbl3 strain to amplify the expression vector; 2) The expression vector was mixed with psPAX2, pMD2.G and transfection reagent PEI to prepare a mixture. The mixture was added to 293T cells, cultured, and the cell culture supernatant was collected after 48 h and 72 h. The virus particles were collected by ultracentrifugation to concentrate the supernatant. 3) T cells were infected with viral particles, cultured at 37°C for 10 hours, and then the infection was terminated. The cells were cultured for another 3 days, and then stained with anti-human CD3 and anti-mouse TCRβ flow cytometry antibodies to obtain LMP2-specific T cells.

6. The use of the T cell antigen receptor of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, or the LMP2-specific T cell of claim 4 in the preparation of a medicament for treating EBV-related diseases.

7. The application as described in claim 6, characterized in that, The EBV-related diseases mentioned are infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary exudative lymphoma, or B / T / NK cell lymphoma.

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