EBV antigen-specific t cell, and preparation method therefor and use thereof

Abstract

Provided are a method for preparing an EBV antigen-specific T cell and the use thereof. The preparation method comprises the following steps: 1) loading a polypeptide on a DC line cell; 2) isolating a peripheral blood mononuclear cell; and 3) preparing an antigen-specific T cell. The prepared EBV antigen-specific T cell is a fully autologous EBV antigen-specific T cell without gene editing.

Description

An EBV antigen-specific T cell, its preparation method and application Technical Field

[0001] This invention belongs to the field of medicine and relates to an EBV antigen-specific T cell, its preparation method, and its application. Background Technology

[0002] Epstein-Barr virus (EBV) was the first human tumor-associated virus discovered (in 1964). More than 90% of adults worldwide have been infected with EBV. B cells are the host cells for EBV, and EBV remains dormant in memory B cells throughout life, usually without producing any symptoms. However, EBV infection causes 2% of malignant tumors, including various lymphomas (NK / T-cell lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, etc.) and solid tumors (nasopharyngeal carcinoma and 10% of gastric and head and neck cancers). Similarly, EBV infection can cause infectious mononucleosis (IM), chronic active EBV infection (CAEBV), hemophagocytic lymphohistiocytosis (HLH), post-transplant lymphoproliferative disorder (PTLD), and autoimmune diseases. Globally, approximately 200,000 cancers are caused by EBV infection each year, and 140,000 patients die from it.

[0003] Cells latently infected with EBV express specific proteins such as latent membrane protein 1 (LMP-1) and latent membrane protein 2 (LMP-2). These proteins affect the cell cycle and apoptosis through multiple mechanisms. Ultimately, these molecular changes lead to potential carcinogenicity by causing B cell proliferation and transformation. Under normal circumstances, the overexpression of LMP1 and LMP-2 in latently EBV-infected B cells promotes a robust T cell response, killing proliferating EBV-infected cells, thereby maintaining a cellular homeostasis and preventing disease progression.

[0004] In different disease types, EBV expresses different gene products. For example, in nasopharyngeal carcinoma patients, EBV expresses EBERs, BART microRNAs, EBNA1, LMP1, and LMP2. However, in Burkitt lymphoma patients, only EBERs, BART microRNAs, and EBNA1 are expressed. BART microRNAs do not affect the virus's growth and transformation capabilities, the growth and transformation capabilities of EBNA1 are controversial, and the function of EBERs has not yet been clearly studied.

[0005] Immunotherapy is one of the most advanced treatments for conditions caused by EBV, and researchers have now demonstrated its clinical efficacy in treating cancer and infectious diseases. T-cell therapy is considered the most advanced in this category, including CAR-T, TCR-T, CTL, AST, and TIL.

[0006] EBV-CTL is a more targeted treatment strategy for restoring immune system function in EBV-related diseases. EBV-CTL can be used for both prevention and treatment of EBV-related diseases, showing good efficacy in PTLD resistant to rituximab or other chemotherapy regimens. EBV-CTL consists of CD3+ T cells that recognize EBV-related antigens on tumor cells. In one study, EBV-CTL demonstrated good activity as both a prevention and treatment strategy, preventing PTLD in a cohort of 101 high-risk HSCT patients and achieving complete remission in 11 of 13 documented PTLD patients. Tabelecleucel, an EBV-specific cell therapy developed by Atara, was conditionally approved by the FDA in December 2022 for adult and pediatric patients with relapsed or refractory EBV+PTLD.

[0007] Although the therapeutic efficacy of EBV-CTLs for HSCT-related PTLD has been clearly demonstrated, several obstacles remain before this important treatment method can be widely adopted in clinical practice. The production of EBV-CTLs begins with co-culturing donor-derived T cells with EBV-infected lymphoblasts (LCLs). The EBV-infected LCLs present EBV antigens to the T cells, thereby selectively expanding EBV-specific CTL cells. This poses a significant challenge to the production of EBV-specific T cells because donor EBV-CTLs are not always readily available or may be EBV-negative. Furthermore, the production of EBV-CTL cells is currently time-consuming, often requiring several weeks.

[0008] Therefore, developing safe and effective EBV-specific immunotherapy methods that enable the rapid, large-scale, and efficient preparation of EBV-CTLs, with a high proportion of specific CTL cells in the product, which targets both EBV replication and eliminates EBV-infected cells to improve patient prognosis, will have significant clinical demand and market value. Summary of the Invention

[0009] To address the aforementioned problems, the present invention aims to provide a method for preparing EBV antigen-specific T cells, which produces a high proportion of EBV antigen-specific T cells, can be mass-produced, has good safety, and is low in cost.

[0010] Another object of the present invention is to provide an EBV antigen-specific T cell.

[0011] The third objective of this invention is to provide an application of EBV antigen-specific T cells.

[0012] To achieve the above objectives, the present invention provides a method for preparing EBV antigen-specific T cells, comprising the following steps:

[0013] 1) DC line cells loaded with peptides

[0014] Take freshly cultured DC line cells, centrifuge them, resuspend them in culture medium, seed them into cell culture plates, add one or more EBV antigen peptides, with a working concentration of 10 μg / mL for each EBV antigen peptide, mix well, and continue culturing in a CO2 incubator for 2-6 hours; after culturing, transfer them to centrifuge tubes, add fresh culture medium, mix well, centrifuge at 800g for 10 min, remove the supernatant after centrifugation, then add culture medium to resuspend the DC line cells containing the antigen peptide, mix well, and set aside;

[0015] 2) Isolation of peripheral blood mononuclear cells

[0016] Collect anticoagulated blood into a 50ml centrifuge tube and centrifuge at 3500rpm for 10min at room temperature; transfer the upper light yellow plasma to a new 50ml centrifuge tube, inactivate it in a 56℃ water bath for 30min, and store it at 4℃ for later use; add PBS to the remaining blood volume to the original blood volume and mix well.

[0017] Add the blood mixed with PBS to a 50ml centrifuge tube containing 15-20ml of lymphocyte separation medium to a total volume of 45ml. Centrifuge at 1800rpm for 30min at room temperature, with the centrifuge acceleration parameter set to 2 and the deceleration parameter set to 1.

[0018] After centrifugation, the liquid surface was observed to be divided into four layers from top to bottom. After aspirating a portion of the top layer, a 10ml serum tube was gently inserted into the second leukocyte layer, and the leukocyte layer was aspirated into a new 50ml centrifuge tube.

[0019] Add 2-3 times the volume of leukocyte layer in room temperature PBS to a final volume of 50 ml, centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and resuspend the cell clumps in 1 ml of room temperature PBS.

[0020] Then, add room temperature PBS to each tube to wash the cells again, centrifuge at 1500 rpm / min for 10 min, discard the supernatant after centrifugation, gently tap the cells to disperse the clumps, and set aside to obtain peripheral blood mononuclear cells.

[0021] 3) Preparation of antigen-specific T cells

[0022] Resuspend peripheral blood mononuclear cells in fresh culture medium to a density of 1-2.5 million / ml, and then seed the peripheral blood mononuclear cells into culture flasks;

[0023] DC line cells loaded with EBV antigen peptides were added to the peripheral blood mononuclear cell suspension at a ratio of 1:20 to 1:200, and after mixing, the suspension was placed in a carbon dioxide incubator for culture.

[0024] Add interleukin-2 to the culture medium to a final concentration of 5-1000 IU / ml to prepare a replacement solution;

[0025] Afterwards, rehydration fluids should be administered every 2-3 days;

[0026] After culturing for 3-11 days, prepare 1-3 batches of DC line cells loaded with EBV antigen peptides; add the DC line cells to the cultured somatic cells at a ratio of DC line cells: peripheral blood mononuclear cells = 1:20 to 1:200, mix well, and continue culturing for 7-14 days.

[0027] After 2-3 weeks of culture, the culture is terminated, and EBV antigen-specific T cells are harvested.

[0028] Furthermore, the classification name of the DC line cells is: human dendritic cells DC0502; the depositary institution is: China General Microbiological Culture Collection Center (CGMCC); the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is: December 25, 2024; the deposit number is: CGMCC No. 46259.

[0029] Furthermore, the culture medium is Serum-free culture medium for T cells.

[0030] Furthermore, the EBV antigen peptide is selected from one of the amino acid sequences of Seq ID No. 1 to Seq ID No. 65.

[0031] The present invention also provides EBV antigen-specific T cells prepared using the above-described preparation method.

[0032] This invention also provides the application of EBV antigen-specific T cells in the preparation of pharmaceuticals for EBV infection-related diseases.

[0033] Furthermore, the EBV infection-related diseases are EBV-induced tumors, infectious mononucleosis caused by EBV infection, chronic active EBV infection, hemophagocytic lymphohistiocytosis, post-transplant lymphoproliferative disorder, or autoimmune diseases caused by EBV infection.

[0034] The EBV antigen-specific T cells prepared by this invention can kill cells that express antigens on the cell surface and cells that present intracellular antigens to the surface. Furthermore, the preparation method provided by this invention effectively solves the problem of insufficient antigen-presenting cells, selectively targeting different EBV antigens or newly generated EBV antigens. The optimized culture parameters allow for large-scale expansion of AST cells, facilitating standardized preparation and resulting in low production costs.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention provides EBV antigen-specific T cells, their preparation method, and applications. The preparation method uses readily available DC line cells to prepare EBV antigen-specific T cells, eliminating the need for patient waiting. The proportion of EBV antigen-specific T cells prepared is over 50%. This method can be used to prepare AST cells targeting specific EBV antigens or AST cells targeting newly generated EBV antigens. The prepared EBV antigen-specific T cells are entirely autologous and have not undergone gene editing, ensuring safety. Compared to CAR-T or TCR-T technologies, the cost can be reduced by approximately 50% due to nearly half the number of process steps. The preparation of EBV antigen-specific T cells can provide a new option for patients who cannot undergo surgery or have limited financial resources, greatly promoting the development of cell therapy technology and reducing the social burden. Attached Figure Description

[0037] Figure 1A shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigenic peptide Seq ID No.1 to generate AST.

[0038] Figure 1B shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigenic peptide Seq ID No.3 to generate AST.

[0039] Figure 1 shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigen peptide Seq ID No. 5 to generate AST.

[0040] Figure 1 shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigen peptide Seq ID No. 7 to generate AST.

[0041] Figure 1 shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigen peptide Seq ID No. 9.

[0042] Figure 1 shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigen peptide Seq ID No. 11 to generate AST.

[0043] Figure 1 shows the flow cytometry results of PBMCs from different sample sources stimulating DC line cells loaded with the antigen peptide Seq ID No. 13 to generate AST.

[0044] Figure 2 is a comparison of DC line cells loaded with antigen peptides cultured in different brands of culture media.

[0045] Figure 3 shows a comparison of PBMC culture at different densities.

[0046] Figure 4A shows a line graph illustrating changes in cell viability at different resting times.

[0047] Figure 4B shows a line graph illustrating the changes in cell yield at different resting times.

[0048] Figure 5 shows a comparison of the effects of DC line cell dosage and IL-2 dosage on AST amplification.

[0049] Figure 6 shows the results of the second stimulation during co-culture.

[0050] Figure 7 shows a comparison of the effects of different DC line cell addition ratios and antigen peptide concentrations on amplification results.

[0051] Figure 8A shows the effect of loading single peptides onto DC line cells on AST amplification.

[0052] Figure 8B is a statistical graph showing the effect of DC line cells loaded with a mixture of three antigenic peptides on AST amplification.

[0053] Figure 8C is a statistical graph showing the effect of DC line cells loaded with a mixture of 5 antigenic peptides on AST amplification.

[0054] Figure 8D is a statistical graph showing the effect of DC line cells loaded with a mixture of 8 antigenic peptides on AST amplification.

[0055] Figure 9 shows the expansion of AST cell numbers. Cells can be expanded approximately 60-fold after 18-19 days of culture.

[0056] Figure 10 is a statistical graph showing the change in the proportion of CD3+ cells during AST culture from a single donor.

[0057] Figure 11 is a statistical chart showing the changes in the percentage of Tetramer+ cells from a single donor.

[0058] Figure 12 is a flow cytometry plot showing the change in the percentage of Tetramer+ positive cells during AST culture from a single donor.

[0059] Figure 13 is a statistical graph showing the high expression of IFN-γ in Tetramer+ positive cells of the AST, AST+DC and AST+AGDC groups.

[0060] Figure 14 shows the flow cytometry plot of AST.

[0061] Figure 15 shows the flow cytometry diagram of AST+DC.

[0062] Figure 16 shows the flow cytometry diagram of AST+AGDC.

[0063] Figure 17 is a statistical graph of A549, which can specifically kill the loaded peptide, in AST.

[0064] Figure 18 is a statistical chart showing the validation of the cytotoxic activity of effector cells. Detailed Implementation

[0065] 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.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0067] The DC line cells used in this invention are classified and named as: human dendritic cells DC0502; the depositary institution is: China General Microbiological Culture Collection Center; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is: December 25, 2024; the deposit number is: CGMCC No. 46259, and DC line cells will be abbreviated as DC from now on.

[0068] The EBV antigen-specific T cells (EBV-AST) prepared by the method of this invention can target multiple EBV antigens, including latent membrane proteins LMP1 and LMP2, nucleocapsid antigens EBNA1, EBNA2, EBNA3, BRLF1, BMLF1, BMRF1, BZLF1, etc. EBV-AST can kill cells expressing the above antigens after EBV infection, triggering a systemic anti-EBV immune response, clearing EBV infection, or treating related diseases caused by EBV infection. Specifically, these include EBV-induced lymphomas (NK / T-cell lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, etc.), solid tumors (nasopharyngeal carcinoma, gastric cancer, head and neck cancer, etc.), as well as infectious mononucleosis (IM), chronic active EBV infection (CAEBV), hemophagocytic lymphohistiocytosis (HLH), post-transplant lymphoproliferative disorder (PTLD), and autoimmune diseases caused by EBV infection.

[0069] This invention provides a method for preparing EBV antigen-specific T cells, comprising the following steps:

[0070] 1) DC line cells loaded with peptides

[0071] Take freshly cultured DC line cells, centrifuge them, resuspend them in culture medium, seed them into a cell culture plate, add one or more EBV antigen peptides, with a working concentration of 10 μg / mL for each EBV antigen peptide, mix well, and continue culturing in a CO2 incubator for 2-6 hours. After culturing, transfer the cells to a centrifuge tube, add fresh culture medium, mix well, centrifuge at 800g for 10 minutes, remove the supernatant after centrifugation, then add culture medium to resuspend the DC line cells containing the antigen peptide, mix well, and set aside.

[0072] 2) Isolation of peripheral blood mononuclear cells (PBMCs)

[0073] Collect anticoagulated blood into a 50ml centrifuge tube and centrifuge at 3500rpm for 10min at room temperature; transfer the upper light yellow plasma to a new 50ml centrifuge tube, inactivate it in a 56℃ water bath for 30min, and store it at 4℃ for later use; add PBS to the remaining blood volume to the original blood volume and mix well.

[0074] Add the blood mixed with PBS to a 50ml centrifuge tube containing 15-20ml of lymphocyte separation medium to a total volume of 45ml. Centrifuge at 1800rpm for 30min at room temperature, with the centrifuge acceleration parameter set to 2 and the deceleration parameter set to 1.

[0075] After centrifugation, the liquid surface was observed to be divided into four layers from top to bottom: plasma layer, white blood cell layer (lymphocyte layer), separation liquid layer, and red blood cell layer. After aspirating a portion of the top layer, a 10ml serum tube was gently inserted into the second white blood cell layer, and the white blood cell layer was aspirated into a new 50ml centrifuge tube.

[0076] Add 2-3 times the volume of leukocyte layer in room temperature PBS to a final volume of 50 ml, centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and resuspend the cell clumps in 1 ml of room temperature PBS.

[0077] Then, add room temperature PBS to each tube to wash the cells again. After centrifugation, discard the supernatant, gently tap the cells to disperse the clumps, and set aside to obtain peripheral blood mononuclear cells (PBMCs).

[0078] 3) Preparation of antigen-specific T cells

[0079] Resuspend peripheral blood mononuclear cells in fresh culture medium to a density of 1-2.5 million / ml, and then seed the peripheral blood mononuclear cells into culture flasks;

[0080] DC line cells loaded with EBV antigen peptides were added to the peripheral blood mononuclear cell suspension at a ratio of 1:20 to 1:200, and after mixing, the suspension was placed in a carbon dioxide incubator for culture.

[0081] Add interleukin-2 to the culture medium to a final concentration of 5-1000 IU / ml to prepare a replacement solution;

[0082] Afterwards, rehydration fluids should be administered every 2-3 days;

[0083] After culturing for 3-11 days, prepare 1-3 batches of DC line cells loaded with EBV antigen peptides; add the DC line cells to the cultured somatic cells at a ratio of DC line cells: peripheral blood mononuclear cells = 1:20 to 1:200, mix well, and continue culturing for 7-14 days.

[0084] The number of times DC line cells loaded with EBV antigen peptide are added to the culture system can be adjusted according to the proliferation of AST cells.

[0085] After 2-3 weeks of culture, the culture is terminated, and EBV antigen-specific T cells are harvested, hereinafter referred to as EBV-AST or AST.

[0086] Material:

[0087] 1. The culture media were LONZA's X-VIVO 15 serum-free immune cell culture medium and Suzhou Ekosei Biotechnology Co., Ltd. Serum-free culture medium for T cells.

[0088] 2. The cell culture plate RTCAE-Plate was purchased from Agilent Technologies (China) Co., Ltd.

[0089] 3. The antigen peptide-specific tetramer is QuickSwitch. TM Quant HLA-A*02:01 Tetramer Kit-PE, purchased from Beijing Bomei Biotechnology Co., Ltd.

[0090] 4. The serum substitute was purchased from Gibco, product number: 10828010.

[0091] 5. AST cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0036.

[0092] 6. JVM-2 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0062.

[0093] 7. EBV-LCL cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0202.

[0094] 8. K562 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., product number: CTCC-001-0024.

[0095] Example 1: Antigen Epitope Screening

[0096] Based on the reported different antigenic epitopes of EBV, the following different antigenic epitopes of EBV were predicted using the binding procedure, as shown in Table 1.

[0097] Table 1 shows the different antigenic epitopes of EBV.

[0098] According to the different EBV antigenic epitopes listed in Table 1, corresponding antigenic epitope peptides (hereinafter referred to as EBV antigenic peptides or antigenic peptides) were artificially synthesized, with corresponding serial numbers from Seq ID No. 1 to Seq ID No. 65. Then, the different antigenic peptides were dissolved using conventional methods to prepare a stock solution with a concentration of 10 mg / mL, which was then loaded onto DC line cells. The working concentration of each antigenic peptide was 10 μg / mL during loading, and the loading time was 4 h, after which it was used to stimulate AST. In subsequent examples, the working concentration of the antigenic peptides was 10 μg / mL.

[0099] The specific steps are as follows:

[0100] Three PBMCs from different human donors were collected and named Sample 1, Sample 2, and Sample 3. After resuscitation and resting for 2 hours, the cells were removed from the incubator and 20 mL of cell washing buffer (PBS + 1% HSA) was added. The cells were mixed well and centrifuged at 600×g for 5 minutes. The supernatant was discarded, and culture medium without IL-2 was added. Resuspend cells in 20 mL of serum-free T-cell culture medium, mix well by pipetting and aspiration, and count the cells. Take 3E5 cells into 1.5 mL EP tubes for surface flow cytometry. Seed the remaining cells into 24-well plates and supplement with culture medium and IL-2 (prepare IL-2-containing culture medium later). The serum substitute concentration is 2%, and the final IL-2 concentration is 100 IU / mL. Add DC line cells loaded with antigen peptides at a ratio of T:Ag-DC = 100:1. Add PBMCs at a ratio of 1E6 cells / well / 1 mL. Mix well and incubate at 37°C in a 5% CO2 incubator.

[0101] For rehydration, between days 3 and 5 of culture, depending on the cell proliferation status and cell density, when the cell density is greater than 2E+6 cells / ml, the corresponding cells should be rehydrated: remove the 24-well plate and add 1000 μL of culture medium containing 100 IU / mL IL-2 and 1% serum substitute to a final concentration of 20 IU / mL, and control the cell density between 0.8E+6 and 1.2E+6 cells / ml.

[0102] Co-culture (round 2 / 3), Day 5 / 10, DC line cells were loaded with antigen peptides, and the Ag-DC cell suspension concentration was finally adjusted to 1E6 cells / mL, and culture continued.

[0103] For culture, between days 8 and 14, based on cell proliferation and density, when the cell density exceeds 2E+6 cells / ml, perform fluid replacement: Remove half of the supernatant from the 24-well plate (avoid washing away suspended cells), and add the other half of medium containing 2000 IU / mL IL-2 and 2% serum substitute to a final concentration of 1000 IU / mL, maintaining a cell density of 0.8E+6 to...

[0104] The culture rate should be between 1.2E+6 cells / ml. If the existing culture containers are insufficient for the desired culture volume, replace them with suitable containers promptly.

[0105] Flow cytometry analysis of AST proportions, as shown in Figures 1A to 1G, illustrates the flow cytometry results of PBMCs stimulating some antigen peptide-loaded DC line cells to generate AST. Figure 1A corresponds to antigen peptide Seq ID No. 1, Figure 1B to Seq ID No. 3, Figure 1C to Seq ID No. 5, Figure 1D to Seq ID No. 7, Figure 1E to Seq ID No. 9, Figure 1F to Seq ID No. 11, and Figure 1G to Seq ID No. 13. Figures 1A to 1G show that different antigen peptides exhibit varying tetramer positivity rates in samples from different human sources, indicating differences in the reactivity of epitope peptides across different samples.

[0106] Example 2: Comparative Screening of Different Culture Media

[0107] In this embodiment, the mixed antigen peptides used for loading DC line cells are: GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0108] Different brands of culture media were selected: X-VIVO 15 serum-free immune cell culture medium and Serum-free T-cell medium was used as the basal medium for AST-stimulated culture. DC-line cells were loaded with mixed antigen peptides and co-cultured at days 0, 5, and 10. Four hours after loading the DC-line cells, two groups were established: one with washing and one without. During culture, fluid was replenished based on cell proliferation. The results are shown in Figure 2. Figure 2 shows that AST can be cultured in different mediums and regardless of whether the DC-line cells were washed after loading the peptides, but the cell expansion fold differed. Serum-free culture medium is more conducive to the proliferation of AST cells in T cells, and subsequent use... T cells were cultured in serum-free medium.

[0109] Example 3: Comparison of different initial seeding densities of peripheral blood mononuclear cells

[0110] In this embodiment, the mixed antigen peptides used for loading DC line cells were: GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15). Peripheral blood mononuclear cells (PBMCs) were selected from samples isolated from three different human sources, designated as donor1, donor2, and donor3, respectively.

[0111] Peripheral blood mononuclear cells (PBMCs) from the three different sources were respectively cultured in complete culture medium (… Serum-free T cell culture medium was diluted with serum substitute and IL-2 (100 IU / ml) to different densities and seeded into cell culture plates. The cell densities after seeding were 0.2E6 / cm³. 2 0.4E6 / cm 2 0.6E6 / cm 2 0.8E6 / cm 2 1.0E6 / cm 2 DC LINE cells loaded with mixed antigen peptides were co-cultured after three stimulations on D0, D5, and D10. During culture, fluid replenishment was performed based on cell proliferation. Fluid replenishment was initiated when the cell density exceeded 2E+6 cells / ml. After replenishment, the cell density ranged from 0.8E+6 to 1.2E+6 cells / ml, as shown in Figure 3. Figure 3 shows that different seeding densities all stimulated AST production, but the cell proliferation folds varied with density; lower seeding densities resulted in better AST amplification.

[0112] Example 4: Comparison of different resting times after PBMC resuscitation

[0113] After resuscitation, three groups of PBMCs were resuspended in complete culture medium and placed in an incubator at 37°C and 5% CO2. Cell counts were performed at different time points to calculate cell viability and yield. The results are shown in Figures 4A and 4B, where Figure 4A shows the changes in cell viability at different resting times, and Figure 4B shows the changes in cell yield at different resting times. Figures 4A and 4B show that cells maintained good viability and yield during a resting period of 2-8 hours. Therefore, PBMCs resuscitated for 2-8 hours were selected for subsequent AST culture.

[0114] Example 5: Effects of DC line cell dosage and IL-2 dosage on AST amplification

[0115] In this embodiment, the mixed antigen peptides used for loading DC line cells are: GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0116] PBMCs were resuspended in complete culture medium and seeded into cell culture plates. DC line cells were loaded with mixed antigen peptides and co-cultured at D0, D5, and D10, with DC:PBMC ratios of 1:20, 1:50, and 1:100, respectively. The IL-2 concentrations in the culture medium were set at 5, 10, 20, 40, 80, and 160 IU / mL between D0 and D10. During culture, fluid was replenished and observed based on cell proliferation. The results are shown in Figure 5. Figure 5 shows that different DC line cell concentrations all stimulated AST production, but higher DC line cell concentrations resulted in better AST amplification. Similarly, different IL-2 concentrations all stimulated AST production, but higher IL-2 concentrations resulted in faster AST amplification. However, when the IL-2 concentration reached 50 IU / mL, AST amplification slowed down and no longer increased rapidly.

[0117] Example 6: Effect of the second stimulation time of DC line cells on AST amplification

[0118] In this embodiment, the mixed antigen peptides used for loading DC line cells are: GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0119] PBMCs were resuspended in complete culture medium and seeded into cell culture plates. DC line cells were loaded with mixed antigen peptides and subjected to two T cell stimulations. The first stimulation was performed at D0, followed by co-culturing at D5, D8, and D11. During culture, fluid was added to monitor cell proliferation. The results are shown in Figure 6, which illustrates the results of the second stimulation. Figure 6 shows that all three time points selected for the second stimulation successfully stimulated the growth of AST cells, with D9 showing the best cell expansion.

[0120] Example 7: Effects of DC line cell dosage and DC line cell loaded peptide concentration on AST amplification

[0121] In this embodiment, the mixed antigen peptides used for loading DC line cells are: GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0122] PBMCs were resuspended in complete culture medium and seeded into cell culture plates. DC line cells were loaded with mixed antigen peptides and co-cultured after two stimulations on D0 and D7. The peptide concentrations of DC line cells were set at 2, 10, and 25 μg / mL, and the DC:PBMC ratios were 1:50, 1:100, 1:200, and 1:300, respectively. During culture, fluid was replenished to observe cell proliferation, as shown in Figure 7. Figure 7 shows that different DC concentrations all stimulated the culture of AST, but the lower the DC line cell concentration, the higher the AST amplification rate. Similarly, different peptide concentrations loaded on DC line cells all stimulated the culture of AST, but the lower the peptide concentration, the higher the AST amplification rate. This is because peptide concentration affects the survival rate of AST.

[0123] Example 8: Effects of loading single peptides or mixed antigenic peptides onto DC line cells on AST amplification

[0124] The peptides loaded in this embodiment are numbered as follows:

[0125] FLYALALLL (Seq ID No. 1), QLSPLLGAV (Seq ID No. 3), GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), CLGGLITMV (Seq ID No. 9), KLLTPVTVL (Seq ID No. 11), TLAAALALL (Seq ID No. 13), IVAPYLFWL (Seq ID No. 15).

[0126] PBMCs were resuspended in complete culture medium and seeded into cell culture plates. DC line cells were loaded with the aforementioned peptides and co-cultured after two stimulations on D0 and D7. The DC line cells were loaded with single peptides, mixtures of three peptides, mixtures of five peptides, and mixtures of eight peptides, respectively. During culture, fluid was replenished and observed based on cell proliferation, as shown in Figures 8A to 8D. Figure 8A shows the statistical graph of tetramer production after stimulation with a single peptide; Figure 8B shows the statistical graph of tetramer production after stimulation with a mixture of three antigenic peptides; Figure 8C shows the statistical graph of tetramer production after stimulation with a mixture of five antigenic peptides; and Figure 8D shows the statistical graph of tetramer production after stimulation with a mixture of all eight antigenic peptides. Figures 8A to 8D show that all peptide combinations can stimulate the production of AST, with the highest AST content observed in the mixture of three peptides. No significant pattern was observed under other conditions.

[0127] Example 9: Preparation of AST using optimized conditions

[0128] The mixed antigen peptides used for loading DC line cells in this embodiment are: FLYALALLL (Seq ID No. 1), QLSPLLGAV (Seq ID No. 3), GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), CLGGLITMV (Seq ID No. 9), KLLTPVTVL (Seq ID No. 11), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0129] PBMCs from multiple donors (denoted as Donor1 to Donor4) were resuspended in complete culture medium and seeded into cell culture plates. DC line cells were loaded with the above-mentioned mixed antigen peptides, and then the PBMCs were stimulated to culture AST. Cell expansion data were collected during the culture process, as shown in Figure 9. As can be seen from Figure 9, the number of AST cells can be expanded approximately 60-fold after 18 days of cell culture.

[0130] Example 10 AST Flow Cytometry Detection

[0131] (1) After the culture in Example 9 was completed, 1 million AST cells were taken, washed twice with PBS, and then the cells were resuspended.

[0132] (2) Add the test antibody and antigen peptide specific tetramer as shown in Table 2 respectively, and incubate at 4°C in the dark for 1 hour.

[0133] Table 2

[0134] (3) After incubation, wash the cells twice with PBS, resuspend the cells, and then perform the detection.

[0135] The statistical data on the changes in the proportion of CD3+ cells are shown in Figure 10. Figure 10 shows that the lowest proportion of CD3+ cells was 89%, with an average of around 95%. The changes in the proportion of Tetramer+ cells are shown in Figure 11, with the flow cytometry clustering results of Tetramer+ cells from one donor shown in Figure 12. Figures 11 and 12 show that the proportion of Tetramer+ positive cells changed during AST culture, continuously increasing to around 15% with prolonged culture time.

[0136] As can be seen from Figures 10 to 12, this method can be used to culture cells for about 18 days, resulting in cells with a Tetramer positivity rate of 15%.

[0137] Example 11: Intracellular Cytokine Staining (ICS) Based on Flow Cytometry

[0138] The mixed antigen peptides used for loading DC line cells in this embodiment are: FLYALALLL (Seq ID No. 1), QLSPLLGAV (Seq ID No. 3), GLGTLGAAL (Seq ID No. 5), TVCGGMMFL (Seq ID No. 7), CLGGLITMV (Seq ID No. 9), KLLTPVTVL (Seq ID No. 11), TLAAALALL (Seq ID No. 13), and IVAPYLFWL (Seq ID No. 15).

[0139] To simulate the in vivo environment, antigens are used to stimulate EBV-AST cells to produce cytokines, promoting the accumulation of cytokines within the cells for easy detection by flow cytometry.

[0140] (1) Prepare DC line cells and AGDC (i.e., DC line cells loaded with mixed antigenic peptides) cells, each at a density of 1×10⁶. 6 cells / mL, keep on ice for later use;

[0141] (2) Take AST cells and add them to 96-well plates. At the same time, add DC and AGDC at T:DC = 1:100, with a final volume of 212 μL / well per well. After mixing, incubate at 37℃ for 1 h.

[0142] (3) Then take Brefeldin A Solution and Monensin Solution, add them to the well plate respectively, mix well, and incubate at 37°C for 4 hours.

[0143] (4) After incubation, wash and centrifuge, resuspend in FACS buffer, and add Human TruStain FcX. TM (Fc Receptor Blocking Solution), add tetramer-PE reagent to each well, mix well and incubate at 37°C for 30 min;

[0144] (5) After incubation, wash and centrifuge, resuspend in FACS buffer, add surface antibody CD3-AF700 and CD8-BV510 reagent, mix well and place in a 4℃ incubator for 15 min.

[0145] (6) After the procedure, resuspend the cells with Cyto-Fast™ Fix / Prem Buffer, incubate at room temperature in the dark for 20 min, add 1×Cyto-Fast™ Prem Wash solution to each well, wash and resuspend, and then the cells can be analyzed by flow cytometry.

[0146] The results are shown in Figures 13 to 16. Figure 13 is a statistical graph showing the high expression of IFN-γ in Tetramer+ positive cells of the AST, AST+DC, and AST+AGDC groups. Figure 14 shows the flow cytometry results for AST, Figure 15 shows the flow cytometry results for AST+DC, and Figure 16 shows the flow cytometry results for AST+AGDC. As can be seen from Figures 13 to 16, approximately 60% of the Tetramer+ positive cells in AST showed high expression of IFN-γ.

[0147] Example 12 AST Killing Activity Detection

[0148] (1) Target cell preparation:

[0149] HLA-A subtype molecules corresponding to AST (e.g., HLA-A0201, HLA-A1101, HLA-A2402) are inserted into target cells expressing the corresponding antigens through gene editing.

[0150] Alternatively, the antigen corresponding to AST can be inserted into target cells that express the same HLA-A subtype molecule as AST (e.g., HLA-A0201, HLA-A1101, HLA-A2402);

[0151] Collect newly cultured target cells; A549 cells were selected as the target cells. The target cells were seeded into RTCAE-Plate cell culture plates and cultured overnight. The next day, the antigen peptide corresponding to the DC line cells was added, mixed well, and then placed in a CO2 incubator for another 2-6 hours.

[0152] After the culture is complete, wash twice with PBS and immediately add AST.

[0153] (2) Lethality experiment:

[0154] Collect freshly cultured AST cells, centrifuge them, and resuspend the cells in culture medium to 1 million / ml.

[0155] Add AST to the prepared E-plat plate, E(target cells):T(AST) = 0.5:1 to 20:1, mix well, and place it on the instrument in a carbon dioxide incubator.

[0156] Data were collected at different time points, and the killing effect was calculated, as shown in Figure 17. Figure 17 shows that AST can specifically kill A549 cells (A549+Ag) loaded with antigenic peptides. Compared with the A549 group without antigenic peptides and the Mock-T group, the AST-A549+Ag group has better killing efficiency (%Lysis), indicating that the prepared AST can specifically kill target cells with the same HLA-A and antigenic peptide information.

[0157] Meanwhile, K562 (transfected with HLA), K562-Ag (transfected with HLA), JVM-2, and EBV-LCL were selected as target cells, and cytotoxic activity was verified using AST and Mock T cells as effector cells, respectively. The results are shown in Figure 18. As can be seen from Figure 18, compared with Mock T cells, AST showed better cytotoxic efficiency (%Lysis) against cells containing EBV antigen.

[0158] As can be seen from the above embodiments, the present invention provides a method for preparing EBV antigen-specific T cells (EBV-AST). The EBV-AST prepared by this method can target a variety of EBV antigens, including tumors caused by EBV, infectious mononucleosis (IM) caused by EBV infection, chronic active EBV infection (CAEBV), hemophagocytic lymphohistiocytosis (HLH), post-transplant lymphoproliferative disorder (PTLD), and autoimmune diseases. EBV-AST can kill cells containing EBV antigens for different diseases caused by EBV. EBV-AST prepared using this technology can prevent or treat a series of diseases caused by EBV.

[0159] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing EBV antigen-specific T cells, characterized in that, Includes the following steps: 1) DC line cells loaded with peptides Take freshly cultured DC line cells, centrifuge them, resuspend them in culture medium, seed them into cell culture plates, add one or more EBV antigen peptides, with a working concentration of 10 μg / mL for each EBV antigen peptide, mix well, and continue culturing in a CO2 incubator for 2-6 hours; after culturing, transfer them to centrifuge tubes, add fresh culture medium, mix well, centrifuge at 800g for 10 min, remove the supernatant after centrifugation, then add culture medium to resuspend the DC line cells containing the antigen peptide, mix well, and set aside; 2) Isolation of peripheral blood mononuclear cells Collect anticoagulated blood into a 50ml centrifuge tube and centrifuge at 3500rpm for 10min at room temperature; transfer the upper light yellow plasma to a new 50ml centrifuge tube, inactivate it in a 56℃ water bath for 30min, and store it at 4℃ for later use; add PBS to the remaining blood volume to the original blood volume and mix well. Add the blood mixed with PBS to a 50ml centrifuge tube containing 15-20ml of lymphocyte separation medium to a total volume of 45ml. Centrifuge at 1800rpm for 30min at room temperature, with the centrifuge acceleration parameter set to 2 and the deceleration parameter set to 1. After centrifugation, the liquid surface was observed to be divided into four layers from top to bottom. After aspirating a portion of the top layer, a 10ml serum tube was gently inserted into the second leukocyte layer, and the leukocyte layer was aspirated into a new 50ml centrifuge tube. Add 2-3 times the volume of leukocyte layer in room temperature PBS to a final volume of 50 ml, centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and resuspend the cell clumps in 1 ml of room temperature PBS. Then, add room temperature PBS to each tube to wash the cells again, centrifuge at 1500 rpm / min for 10 min, discard the supernatant after centrifugation, gently tap the cells to disperse the clumps, and set aside to obtain peripheral blood mononuclear cells. 3) Preparation of antigen-specific T cells Resuspend peripheral blood mononuclear cells in fresh culture medium to a density of 1-2.5 million / ml, and then seed the peripheral blood mononuclear cells into culture flasks; DC line cells loaded with EBV antigen peptides were added to the peripheral blood mononuclear cell suspension at a ratio of 1:20 to 1:200, and after mixing, the suspension was placed in a carbon dioxide incubator for culture. Add interleukin-2 to the culture medium to a final concentration of 5-1000 IU / ml to prepare a replacement solution; Afterwards, rehydration fluids should be administered every 2-3 days; After culturing for 3-11 days, prepare 1-3 batches of DC line cells loaded with EBV antigen peptides; add the DC line cells to the cultured somatic cells at a ratio of DC line cells: peripheral blood mononuclear cells = 1:20 to 1:200, mix well, and continue culturing for 7-14 days. After 2-3 weeks of culture, the culture is terminated, and EBV antigen-specific T cells are harvested.

2. The method for preparing EBV antigen-specific T cells as described in claim 1, characterized in that, The DC line cells are classified and named as: human dendritic cells DC0502; the depositary institution is: China General Microbiological Culture Collection Center (CGMCC); the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is: December 25, 2024; the deposit number is: CGMCC No. 46259.

3. The method for preparing EBV antigen-specific T cells as described in claim 1, characterized in that, The culture medium is Serum-free culture medium for T cells.

4. The method for preparing EBV antigen-specific T cells as described in claim 1, characterized in that, The EBV antigen peptide is selected from one of the amino acid sequences from Seq ID No. 1 to Seq ID No.

65.

5. EBV antigen-specific T cells prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the EBV antigen-specific T cells as described in claim 5 in the preparation of medicaments for EBV infection-related diseases.

7. The application as described in claim 6, characterized in that, The EBV infection-related diseases mentioned are tumors caused by EBV, infectious mononucleosis caused by EBV infection, chronic active EBV infection, hemophagocytic lymphohistiocytosis, post-transplant lymphoproliferative disorder, or autoimmune diseases caused by EBV infection.

8. The application as described in claim 7, characterized in that, The tumors caused by EBV are lymphomas or solid tumors.

9. The application as described in claim 8, characterized in that, The lymphoma is NK / T-cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, or Burkitt lymphoma.

10. The application as described in claim 8, characterized in that, The solid tumors mentioned are nasopharyngeal carcinoma, gastric carcinoma, and head and neck carcinoma.

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