A novel coronavirus-specific t cell and a preparation method and application thereof

Abstract

The application provides a novel coronavirus specific T cell and a preparation method and application thereof, and belongs to the technical field of functional cells. The novel coronavirus specific T cell is enriched from peripheral blood PBMC cells, and after two rounds of in-vitro amplification, the novel coronavirus specific T cell with the order of 10 billion, an amplification multiple higher than 10,000 times and a purity higher than 90% is obtained, and the novel coronavirus specific T cell shows the characteristics of specific immune response to the novel coronavirus, thereby providing a new idea for preventing and treating the novel coronavirus pneumonia in clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional cells, and particularly relates to a novel coronavirus-specific T cell and a preparation method and application thereof. BACKGROUND

[0002] The production of neutralizing antibodies by inoculation of the novel coronavirus vaccine is a powerful guarantee for resisting novel coronavirus infection and reducing the rate of severe cases and mortality. However, the amount of neutralizing antibodies produced by vaccination decays over time, and can only provide protection for approximately half a year. Moreover, the protection obtained from vaccination is limited for some immunocompromised groups (including tumor patients and the elderly, etc.) with underlying diseases. In addition, the novel coronavirus has been in mutation, especially after the novel coronavirus infects immunocompromised people, which accelerates the mutation, and their unbalanced immune response creates opportunities for the evolution of the novel coronavirus and super strains, so additional means are needed to help them resist novel coronavirus infection.

[0003] In addition to stimulating humoral immune responses to produce neutralizing antibodies, vaccination can also stimulate cellular immune responses in some vaccinated individuals, including CD4 + helper T cells that stimulate B cells and other immune cells, and CD8 + killer T cells that target and destroy infected cells. Compared with neutralizing antibodies of the novel coronavirus, which are more susceptible to viral mutations, novel coronavirus-specific T cells can recognize multiple novel coronavirus variants. Studies have shown that the novel coronavirus-specific T cell response in recovered patients and vaccine recipients is largely unaffected by mutations in the variant strains, and provides protection against emerging variants.

[0004] Studies have shown that novel coronavirus patients with strong T lymphocyte responses have a higher survival rate in novel coronavirus infection, revealing the important role of T cell immune response in the recovery of novel coronavirus patients. Therefore, developing an in vitro expansion technology for novel coronavirus-specific T cells to culture and expand the scarce novel coronavirus-specific T cells in peripheral blood in a GMP environment cell preparation workshop, and treating immunocompromised groups infected with the novel coronavirus with virus-specific T cells to help patients recover as soon as possible and avoid them becoming a super mutant strain incubator, causing nosocomial infection and epidemic, has great medical value and social and economic value. However, the current novel coronavirus-specific T cells are only produced by in vivo immunization, which greatly hinders the progress of preventing and treating novel coronavirus pneumonia by T lymphocytes. SUMMARY

[0005] Therefore, the present application aims to provide a novel coronavirus-specific T cell and a preparation method thereof, which can obtain a large number of novel coronavirus-specific T cells with high purity through in vitro enrichment and two rounds of expansion, thereby providing a basis for the clinical prevention and treatment of novel coronavirus infection.

[0006] The present application provides a preparation method of a novel coronavirus-specific T cell, comprising the following steps:

[0007] 1) enriching novel coronavirus-specific T cells from lymphocytes;

[0008] 2) culturing the novel coronavirus-specific T cells in a T cell complete culture medium containing feeder cells, IL-2 and autologous plasma for the first time,

[0009] 3) culturing the novel coronavirus-specific T cells obtained in step 2) in a T cell complete culture medium containing feeder cells, IL-2, autologous plasma and novel coronavirus antigen peptides for the second time to obtain novel coronavirus-specific T cells.

[0010] Preferably, the number ratio of the feeder cells to the novel coronavirus-specific T cells in step 2) is 95-105:1;

[0011] The final density of the feeder cells is (1-5)×10 6 cells / ml;

[0012] The volume percentage content of the autologous plasma is 4%-6%;

[0013] The final concentration of the IL-2 is 90-110 IU / ml.

[0014] Preferably, the basic culture medium of the T cell expansion culture medium in step 2) is Stemcell ImmunoCult TM -XF T cell complete culture medium.

[0015] Preferably, in the first culture in step 2), fresh T cell complete culture medium is added every 2-3 days to control the density of living cells to 1.0-3.0×10 5 cells / mL.

[0016] Preferably, the temperature of the first culture in step 2) is 36-38℃;

[0017] The CO2 concentration of the first culture is 5%;

[0018] The time of the first culture is 10-12d.

[0019] Preferably, the final concentration of the novel coronavirus antigen peptide in step 3) is 1.8-2.2 μg / ml.

[0020] The density of the trophocyte is (1-4) x 10 6 cells / ml.

[0021] The ratio of the number of the trophocyte to the novel coronavirus specific T cell is 95-105:1.

[0022] The volume percentage of the autologous plasma is 4%-6%.

[0023] The final concentration of the IL-2 is 90-110 IU / ml.

[0024] Preferably, in the second culture in step 3), fresh T cell complete medium is added every 2-3 days to control the density of the living cells to (0.5-1.0) x 10 6 cells / mL.

[0025] Preferably, in the method for enriching the novel coronavirus specific T cell in step 1), the lymphocyte is sequentially stimulated and cultured by the novel coronavirus antigen peptide, IFN-gamma capture reagent and IFN-gamma enrichment reagent, and the novel coronavirus specific T cell is obtained by separation and enrichment.

[0026] The application provides the novel coronavirus specific T cell prepared by the preparation method, and the novel coronavirus specific T cell has a specific immune response to the novel coronavirus.

[0027] The application provides the novel coronavirus specific T cell prepared by the preparation method or the application of the novel coronavirus specific T cell in the preparation of a drug for preventing and treating novel coronavirus infection.

[0028] The application provides a preparation method of a novel coronavirus specific T cell, which enriches the novel coronavirus specific T cell from lymphocyte, then performs first culture in T cell complete medium containing trophocyte and autologous plasma, performs second culture of the cultured novel coronavirus specific T cell in T cell complete medium containing trophocyte, autologous plasma and novel coronavirus antigen peptide, and obtains the novel coronavirus specific T cell. The T cell product (experimental group) obtained by enrichment and restimulation of the novel coronavirus antigen peptide has a purity of more than 90% and an amplification multiple of 10,000-70,000, which greatly improves the number of the novel coronavirus specific T cell and meets the demand of clinical cell therapy.

[0029] Meanwhile, the application also provides the prepared novel coronavirus specific T cells, which produce specific immune response to the novel coronavirus, greatly improve the immune effect of the population, especially the population with low immunity, to the novel coronavirus, and provide a basis for the novel coronavirus specific T cell treatment and help patients recover as soon as possible. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The amplification fold results of the novel coronavirus specific T cells prepared by the application under different enrichment and stimulation conditions;

[0031] Figure 2 The optimization experiment results of the T cells in vitro amplification in the application. DETAILED DESCRIPTION

[0032] The application provides a preparation method of novel coronavirus specific T cells, comprising the following steps:

[0033] 1) Enriching novel coronavirus specific T cells from lymphocytes;

[0034] 2) Culturing the novel coronavirus specific T cells in a T cell complete culture medium containing trophoblasts, IL-2 and autologous plasma for the first time;

[0035] 3) Culturing the novel coronavirus specific T cells obtained in step 2) in a T cell complete culture medium containing trophoblasts, IL-2, autologous plasma and novel coronavirus antigen peptides for the second time, to obtain the novel coronavirus specific T cells.

[0036] The application enriches novel coronavirus specific T cells from lymphocytes.

[0037] In the application, the lymphocytes are preferably separated from peripheral blood. The separation method of the lymphocytes preferably comprises the following steps:

[0038] Mixing the peripheral blood and the lymphocyte separation medium, centrifuging, and collecting the supernatant and the white membrane layer cells respectively;

[0039] Freezing the supernatant after inactivating the complement components; and collecting the precipitated cells after washing and centrifuging the white membrane layer, as the lymphocytes.

[0040] In this invention, the volume ratio of peripheral blood to lymphocyte separation fluid is preferably 1:1. The lymphocytes are preferably peripheral blood lymphocytes. The centrifugation speed is preferably 800×g, and the centrifugation time is preferably 15–25 min, more preferably 20 min. The temperature for inactivating complement components is preferably 56–58°C, and the inactivation time is preferably 25–35 min, more preferably 30 min. After inactivating complement components, centrifugation is preferably performed, and the supernatant plasma is collected. The centrifugation speed is preferably 1100×g–1300×g, more preferably 1200×g. The centrifugation time is preferably 9–11 min, more preferably 10 min.

[0041] In this invention, the white membrane is preferably washed and centrifuged twice. After collecting the white membrane layer cells, it is preferable to add physiological saline for washing. The centrifugation is preferably 450×g to 550×g, more preferably 500×g. The centrifugation time is preferably 9 to 11 min, more preferably 10 min. After collecting the precipitated cells, it is preferable to wash with physiological saline. The concentration of the lymphocytes is (1 to 5)×10⁻⁶. 7 per ml.

[0042] In this invention, the method for enriching novel coronavirus-specific T cells preferably involves sequentially stimulating and culturing lymphocytes with a novel coronavirus antigenic peptide, an IFN-gamma capture reagent, and an IFN-γ enrichment reagent, followed by separation and enrichment to obtain novel coronavirus-specific T cells. The final concentration of the antigenic peptide is preferably 1.5–2.5 μg / mL, more preferably 2 μg / mL. The culture medium used for antigenic peptide stimulation is preferably a medium containing 10% AB serum. The stimulation and culture time is 16–20 h, more preferably 18 h. The stimulation and culture environment is preferably 37°C and 5% CO2. The amino acid sequence of the novel coronavirus antigenic peptide is YSVLYNSASFSTFKCY, CTFEYVSQPFLMD, QPFLMDLEGKQGN, AYYVGYLQPRTFLLKY, SAFSTFKCYGVSPTKL, RISNCVADYSVLY, LQIPFAMQMAYRF, KPFERDISTEIYQ, RVYSSANNCTFEY, AYTMSLGAENSVAY, FQFCNDPFLGVY, RIFTIGTVTLKQGEI, LMSFPQSAPHGVVF (SEQ ID NO: 1~SEQ ID NO: 13). In this embodiment of the invention, the novel coronavirus antigenic peptide was purchased from Mabtech. After stimulation culture, the antigenic peptide was washed with cold culture medium containing 2% AB serum. Every 10 7The cells are added with 75 μl of the IFN-gamma capture reagent. The stimulation culture environment of the IFN-gamma capture reagent is preferably incubation on ice for 5 min. The stimulation culture medium of the IFN-gamma capture reagent is preferably 1640 medium. The IFN-gamma capture reagent is purchased from Promega. After incubation on ice, the cells are preferably incubated with warm medium (37℃) containing 10% AB serum for 40-50 min. The incubated cells are treated with cold buffer. Each 10 7 The cells are added with 50-100 μl of the IFN-γ enrichment reagent, more preferably 75 μl. The stimulation culture environment of the IFN-γ enrichment reagent is preferably incubation on ice for 5 min. The stimulation culture medium of the IFN-γ enrichment reagent is preferably cold buffer containing 0.5% serum. The IFN-γ enrichment reagent is purchased from Promega. After stimulation culture of the IFN-γ enrichment reagent, the cells are preferably washed with cold buffer containing 0.5% serum. In the separation method, the separation column is placed on a magnet separator, the stimulation cultured cells are added to the separation column, the separation column is eluted with cold buffer containing 0.5% serum, the eluted separation column is removed from the magnet separator, and the magnetically labeled cells are eluted with cold buffer containing 0.5% serum to obtain enriched novel coronavirus specific T cells.

[0043] After obtaining the novel coronavirus specific T cells, the novel coronavirus specific T cells are cultured in a T cell complete medium containing feeder cells and autologous plasma for the first time.

[0044] In the present application, the ratio of the number of feeder cells to the novel coronavirus specific T cells is preferably 95-105:1, more preferably 100:1. The final density of the feeder cells is preferably (2.0-3.0) x 10 6 / ml, more preferably 2.5 x 10 6 / ml. The feeder cells are prepared from peripheral blood lymphocytes, specifically, the cells are treated with 25 μg / mL mitomycin C (2 x 10 7 cells per milliliter of 1640 medium), incubated at 37℃ for 30 min, washed with 1640 medium for 3 times, centrifuged at 300 x g for 10 min, the supernatant is discarded, the cells are resuspended with 1640 medium, an appropriate amount of cell suspension is taken for cell counting, centrifuged at 300 x g for 10 min, the supernatant is taken, the cell pellet is resuspended with freezing solution, and 1 x 10 7 cells per tube are frozen. The feeder cells provide stimulation signals and growth conditions for the in vitro expansion of T lymphocytes.

[0045] In the present application, the volume percentage content of the autologous plasma is preferably 4% to 6%, more preferably 5%. The autologous plasma is derived from a patient to be subjected to cell therapy with novel coronavirus-specific T cells. The final concentration of IL-2 is 90 to 110 IU / ml, more preferably 100 IU / ml. The T cell expansion medium is preferably Stemcell ImmunoCult TM -XFT cell complete medium. In the first culture, fresh T cell complete medium is added every 2 to 3 days to control the density of viable cells to (1.0 to 3.0) x 10 5 cells / mL. The temperature of the first culture is preferably 36 to 38°C, more preferably 37°C. The CO2 concentration of the first culture is 5%, and the time of the first culture is preferably 10 to 12 days.

[0046] After the first culture, the present application performs a second culture of the novel coronavirus-specific T cells obtained by the culture in a T cell complete medium containing feeder cells, IL-2, autologous plasma, and novel coronavirus antigen peptides, to obtain novel coronavirus-specific T cells.

[0047] In the present application, the final concentration of the novel coronavirus antigen peptide is preferably 1.8 to 2.2 μg / ml, more preferably 2 μg / ml. The density of the feeder cells is preferably (1.5 to 2.5) x 10 6 cells / mL, more preferably 2.0 x 10 6 cells / mL. The novel coronavirus antigen peptide functions to provide a stimulation signal for the activation and proliferation of novel coronavirus-specific T cells. The ratio of the feeder cells to the novel coronavirus-specific T cells is 95 to 105:1, more preferably 100:1. The final concentration of the feeder cells is (1.5 to 2.5) x 10 6 cells / mL, more preferably 2.0 x 10 6 cells / mL. The feeder cells function to provide a stimulation signal and growth conditions for the in vitro expansion of T lymphocytes. The volume percentage content of the autologous plasma is preferably 4% to 6%, more preferably 5%. The autologous plasma functions to provide essential nutrients for the in vitro expansion of T lymphocytes. The final concentration of IL-2 is 90 to 110 IU / ml, more preferably 100 IU / ml.

[0048] In the present application, in the second culture, fresh T cell complete medium is preferably added every 2 to 3 days to control the density of viable cells to 0.5 to 1.0 x 10 6 cells / mL.

[0049] The application provides a novel coronavirus specific T cell prepared by the preparation method, and the novel coronavirus specific T cell has a specific immune response to the novel coronavirus.

[0050] The application provides application of the novel coronavirus specific T cell prepared by the preparation method or the novel coronavirus specific T cell in preparation of prevention and treatment of novel coronavirus infection.

[0051] The application provides a novel coronavirus specific T cell, a preparation method and application thereof, which are described in detail in combination with examples below, but they cannot be understood as limitations to the protection scope of the application.

[0052] The reagents and configuration methods involved in the examples of the application are as follows:

[0053] 1.1 Cold buffer containing 0.5% serum (4℃): 0.85ml human AB serum is added to 169.15ml PBS.

[0054] 1.2 Cold medium (4℃): 60ml 1640 medium is prepared.

[0055] 1.3 Cold medium containing 2% AB serum (4℃): 0.8ml human AB serum is added to 39.2ml 1640 medium.

[0056] 1.4 Warm medium containing 10% AB serum (37℃): 6.6ml human AB serum is added to 60ml 1640 medium.

[0057] Example 1

[0058] Isolation method of peripheral blood lymphocytes (PBMC)

[0059] Fresh peripheral blood is slowly added to a 50ml centrifuge tube containing lymphocyte separation medium (lymphocyte separation medium:diluted blood cell suspension = 1:1), centrifuged at 800xg for 10min, and separated into upper liquid, white membrane layer, separation medium layer and red blood cell precipitate.

[0060] The upper liquid (plasma) is sucked into a 50ml centrifuge tube, inactivated in a 56℃ water bath for 30min, and then centrifuged at 1200xg for 10min, and the upper clear plasma is sucked and stored in an EP tube, and stored at -20℃.

[0061] The white membrane layer in the centrifuge tube in step 1.1 is collected in a 50ml centrifuge tube, and physiological saline is added to 50ml, and the cells are washed by centrifugation at 500xg for 10min.

[0062] The upper physiological saline is sucked and discarded, 50ml physiological saline is added, the precipitated cells are mixed by blowing, and an appropriate amount of cell suspension is taken for AO / PI cell counting.

[0063] Take an appropriate amount of cells for subsequent operations:

[0064] 1. Take 5 x 10 7 PBMC cells for novel coronavirus specific T cell enrichment;

[0065] 2. 1 x 10 7 PBMC cells as non-VST control;

[0066] 3. The remaining PBMC cells are used as raw materials for the preparation of trophoblasts, and the specific method is as follows:

[0067] 3.1 Use 25 μg / mL mitomycin C to treat cells (2 x 10 7 cells / mL 1640 medium), incubate at 37°C for 30 min.

[0068] 3.2 Wash the cells with 1640 medium 3 times, centrifuge at 300 x g for 10 min. Completely aspirate the supernatant.

[0069] 3.3 Resuspend the cells with 1640 medium, take an appropriate amount of cell suspension for cell counting.

[0070] 3.4 Centrifuge at 300 x g for 10 min, completely aspirate the supernatant.

[0071] 3.5 Resuspend the cell pellet with cryopreservation solution, 1 x 10 7 cells / ml frozen per tube.

[0072] Example 2

[0073] A method for preparing novel coronavirus specific T cells

[0074] 1. Novel coronavirus specific T cell enrichment

[0075] Transfer the cells prepared in Example 1 to a 50 ml centrifuge tube, add 1.2 cold 1640 medium to 25 ml.

[0076] Centrifuge at 200 x g for 10 min at 4°C, carefully aspirate the supernatant.

[0077] Resuspend the cells in 1 mL of 1640 medium containing 10% AB serum, and add the culture components according to Table 1:

[0078] Table 1 Antigen peptide enrichment system

[0079] Experimental group PBMC (5 x 10 7 ​ 1000 μl (5 x 10 7 ​ Antigen peptide pool (total 2 μg / ml, SEQ ID NO: 1 ~ SEQ ID NO: 13) 50 μl Culture medium containing 10% AB serum 3950 μl Total volume 5000 μl

[0080] 1.1 Incubate at 37°C in a 5% CO2 cell incubator for 18 hours.

[0081] 1.2 Transfer non-VST control and experimental cells into 15 ml and 50 ml centrifuge tubes, respectively, and add 5 ml and 25 ml of cold media containing 2% AB serum.

[0082] 1.3 Centrifuge at 300 x g for 10 minutes and carefully remove the supernatant.

[0083] 1.4 Resuspend non-VST control and experimental cells in 100 μL and 500 μL of cold 1.2 1640 media, respectively, and add 75 μL and 375 μL of IFN-gamma capture reagent. Incubate the cell suspension on ice for 5 minutes.

[0084] 1.5 Add 10 ml and 50 ml of warm media (37°C) containing 10% AB serum to the non-VST control and experimental cells, respectively, to dilute the cells to 10 cells / mL. 6

[0085] 1.6 Incubate the cells at 37°C for 45 minutes, rotating the tubes every 5 minutes to resuspend the pelleted cells.

[0086] 1.7 Add 5 ml and 25 ml of cold buffer (4°C) 1.1 to the non-VST control and experimental cells, respectively, and place on ice for 10 minutes. Centrifuge the cells at 300 x g for 10 minutes at 4°C.

[0087] 1.8 Completely remove the supernatant and resuspend the cells in 5 ml and 25 ml of cold buffer (4°C) 1.1, respectively.

[0088] 1.9 Centrifuge the cells at 300 x g for 10 minutes at 4°C and carefully remove the supernatant.

[0089] 1.10 Resuspend non-VST control and experimental cells in 100 μL and 500 uL of cold buffer 1.1, respectively, and add 75 μL and 375 μL of IFN-gamma enrichment reagent. Incubate on ice for 15 minutes.

[0090] 1.11 Add 10 ml and 50 ml of cold buffer 1.1, respectively, and centrifuge at 300 x g for 10 minutes at 4°C.

[0091] 1.12 Carefully remove the supernatant and resuspend the cells in 1 mL of cold buffer 1.1 (4°C).

[0092] 1.13 Place the separation column in a magnetic separator and rinse the column with 3 ml of cold buffer 1.1.

[0093] 1.14 Add the cell suspension to the separation column and collect the unlabeled cells that flow through.

[0094] ​1.15 Wash the column 3 times with 3 ml of 1.1 buffer.

[0095] 1.16 Remove the separation column from the magnet and place it on a 15 ml centrifuge tube.

[0096] 1.17 Add 5 ml of 1.1 buffer to the separation column and push the plunger into the column to rinse the magnetically labeled cells out.

[0097] 1.18 Take an appropriate amount for cell counting.

[0098] 2. Novel coronavirus specific cell expansion in vitro

[0099] Day 0

[0100] Prepare fresh T cell complete medium: Add IL-2 (final concentration 100 IU / ml) and 5% autologous inactivated plasma to Stemcell ImmunoCult TM -XF T cell expansion medium. The medium should be prepared and used within 7 days.

[0101] Mix the novel coronavirus VST cells with feeder cells prepared from autologous PBMC at a ratio of 1:100 and incubate in complete T cell expansion medium supplemented with 5% autologous plasma at 37°C, 5% CO2 for 3 days.

[0102] Day 3

[0103] Mix the cell suspension and take a sample for viable cell counting. Add an appropriate amount of T cell complete medium to adjust the viable cell density to 1.0-2.5 x 10 5 cells / mL.

[0104] Continue incubation of the cells in a 37°C, 5% CO2 cell incubator.

[0105] Day 5

[0106] Mix the cell suspension and take a sample for viable cell counting. Add an appropriate amount of T cell complete medium to adjust the viable cell density to 1.0-3.0 x 10 5 cells / mL.

[0107] Continue incubation of the cells in a 37°C, 5% CO2 cell incubator.

[0108] Day 7

[0109] Mix the cell suspension and take a sample for viable cell counting. Add an appropriate amount of T cell complete medium to adjust the viable cell density to 1.0-3.0 x 10 5 cells / mL.

[0110] The cells were incubated at 37°C in a 5% CO2 cell incubator.

[0111] Day 10

[0112] The cell suspension was mixed and sampled for viable cell count. The T cell complete medium was added according to the cell number to keep the viable cell density at 0.5-1.0 x 10 6 cells / mL).

[0113] The cells were incubated at 37°C in a 5% CO2 cell incubator.

[0114] Day 12

[0115] The cell suspension was mixed and sampled for viable cell count.

[0116] The cells were harvested and subjected to the second round of stimulation expansion.

[0117] The feeder cells and the harvested T cells were mixed at 10:1 in the T cell complete medium, and the novel coronavirus antigen peptide was added to stimulate the novel coronavirus specific T cells (the final concentration was 2 μg / ml). The mixture was incubated at 37°C in a 5% CO2 incubator.

[0118] The T cell complete medium was added every 2-3 days to adjust the cell density, and the method was the same as that of the previous expansion culture.

[0119] Day 22

[0120] The cells were harvested and subjected to the second round of stimulation expansion.

[0121] Comparative Example 1

[0122] A method for preparing a non-novel coronavirus specific T cell control

[0123] The novel coronavirus specific T cells were prepared according to the method of Example 1, except that in the first enrichment with antigen peptides, the antigen peptides were omitted and replaced with DMSO, and the volume of the system was only 1 / 5 of that of Example 1.

[0124] Comparative Example 2

[0125] A method for preparing a novel coronavirus specific T cell negative control

[0126] The novel coronavirus specific T cell negative control was prepared according to the method of Example 1, except that the peripheral blood lymphocytes were a cell population containing no novel coronavirus specific T cells, and in the second expansion culture, the CD3 / CD28 activator (25 μl per milliliter of culture system) was used for stimulation.

[0127] Results of Example 1, Comparative Examples 1-2 are shown in Table 1 Figure 1 Table 1: Results of Example 1, Comparative Examples 1-2 Table 1: Results of Example 1, Comparative Examples 1-2

[0128] Example 3

[0129] Optimization of T cell in vitro expansion method

[0130] To compare the difference of T cell expansion fold, cell purity and cell viability under different conditions (including IL-2 concentration, addition of autologous plasma and feeder cells), the optimization of T cell in vitro expansion experiment was carried out, and the specific method was as follows:

[0131] Day 0

[0132] 1) The seed T cells were placed in ImmunoCult TM -XFT cell culture medium with different components, and the cell concentration was adjusted to 1×10 6 cells / mL;

[0133] Sample 1: ImmunoCult TM -XFT cell culture medium + 50 IU / mL IL-2;

[0134] Sample 2: ImmunoCult TM -XFT cell culture medium + 100 IU / mL IL-2;

[0135] Sample 3: ImmunoCult TM -XFT cell culture medium + 50 IU / mL IL-2 + 5% autologous plasma;

[0136] Sample 4: ImmunoCult TM -XFT cell culture medium + 100 IU / mL IL-2 + 5% autologous plasma;

[0137] Sample 5: ImmunoCult TM -XFT cell culture medium + 100 IU / mL IL-2 + feeder cells;

[0138] Sample 6: ImmunoCult TM -XFT cell culture medium + 100 IU / mL IL-2 + 5% autologous plasma + feeder cells.

[0139] 2) Add human CD3 / CD28 T cell activator (concentration of 50 μL / ml), mix well, and incubate the cells at 37°C, 5% CO2 for 3 days;

[0140] Day 3

[0141] 3) Mix the cell suspension, take samples for viable cell counting, and supplement with appropriate amount of T cell complete medium added with corresponding components according to the number of viable cells, so as to adjust the viable cell density to (1.0-2.5) x 10 5 cells / mL);

[0142] 4) Continue to incubate the cells at 37°C, 5% CO2 in a cell incubator;

[0143] Day 5

[0144] 5) Mix the cell suspension, take samples for viable cell counting, and supplement with appropriate amount of T cell complete medium added with corresponding components according to the number of viable cells, so as to adjust the viable cell density to (1.0-3.0) x 10 5 cells / mL);

[0145] 6) Continue to incubate the cells at 37°C, 5% CO2 in a cell incubator;

[0146] Day 7

[0147] 7) Mix the cell suspension, take samples for viable cell counting, and supplement with appropriate amount of T cell complete medium added with corresponding components according to the number of viable cells, so as to adjust the viable cell density to (1.0-3.0) x 10 5 cells / mL);

[0148] 8) Continue to incubate the cells at 37°C, 5% CO2 in a cell incubator;

[0149] Day 10

[0150] 9) Mix the cell suspension, take samples for viable cell counting, and supplement with appropriate amount of T cell complete medium added with corresponding components according to the number of viable cells, so as to adjust the viable cell density to (0.5-1.0) x 10 6 cells / mL);

[0151] 10) Cells were incubated at 37℃ in 5% CO2 incubator.

[0152] Day 12

[0153] 11) The cell suspension was mixed and the viable cell staining was counted for each sample separately, and the cell expansion fold and cell viability of each sample were calculated.

[0154] Under the condition of pure cytokine culture, the decrease of IL-2 concentration from 100 IU / ml to 50 IU / ml reduced the in vitro expansion fold of T cells by about half, and the addition of autologous plasma compensated for the effect of the decrease of IL-2 concentration; under the condition of 100 IU / ml IL-2 concentration, the addition of 5% autologous plasma slightly improved the cell viability, but had no significant effect on the improvement of the expansion ability of T cells; on the contrary, the addition of feeder cells greatly improved the expansion ability of T cells, and on this basis, the additional addition of 5% autologous plasma could further slightly improve the cell viability and expansion fold (see Table 2). Figure 2

[0155] After several rounds of comparison experiments on the optimization of the culture scheme, the two key indicators of cell expansion fold and cell viability were compared, and the best in vitro expansion process of T cells was determined to be the addition of 100 IU / ml IL-2 + 5% autologous plasma + feeder cells on the basis of ImmunoCult TM -XFT cell culture medium.

[0156] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for preparing SARS-CoV-2-specific T cells, characterized in that, Includes the following steps: 1) Enriching SARS-CoV-2-specific T cells from lymphocytes; The method for preparing SARS-CoV-2-specific T cells enriched from lymphocytes involves sequentially stimulating and culturing the lymphocytes with SARS-CoV-2 antigen peptides, IFN-gamma capture reagents, and IFN-γ enrichment reagents, then separating and enriching them to obtain SARS-CoV-2-specific T cells. The amino acid sequence of the novel coronavirus antigen peptide is SEQ ID NO:1~SEQ ID NO:13; 2) The SARS-CoV-2-specific T cells were cultured for the first time in a complete T cell culture medium containing feeder cells, IL-2 and autologous plasma; The ratio of the number of trophoblast cells to the number of SARS-CoV-2-specific T cells in step 2) is 95-105:1; The final density of the trophoblast cells is (1~5)×10⁻⁶. 6 cells / ml; The trophoblast cells were prepared from peripheral blood lymphocytes. Specifically, the cells were treated with 25 μg / mL mitomycin C, incubated at 37°C for 30 min, washed three times with 1640 medium, centrifuged at 300×g for 10 min, the supernatant was discarded, and the cells were resuspended in 1640 medium. The volume percentage of the autologous plasma is 4% to 6%; The final concentration of IL-2 is 90~110 IU / ml; 3) The SARS-CoV-2-specific T cells obtained in step 2) are cultured a second time in a complete T cell culture medium containing feeder cells, IL-2, autologous plasma and SARS-CoV-2 antigen peptides to obtain SARS-CoV-2-specific T cells.

2. The preparation method according to claim 1, characterized in that, In step 2), the basal medium for T cell expansion is Stemcell Im munoCult™-XF T cell complete medium.

3. The preparation method according to claim 1, characterized in that, In step 2), during the first culture, fresh T-cell complete culture medium is added every 2-3 days to maintain a viable cell density of 1.0-3.0 × 10⁻⁶ cells / year. 5 Cells / mL.

4. The preparation method according to claim 1, characterized in that, In step 2), the temperature of the first culture is 36~38℃; The CO2 concentration in the first culture was 5%; The first culture period is 10-12 days.

5. The preparation method according to claim 1, characterized in that, The final concentration of the SARS-CoV-2 antigenic peptide described in step 3) is 1.8~2.2 μg / ml; The density of the trophoblast cells is (1~4)×10 6 cells / ml; The ratio of the number of trophoblasts to the number of SARS-CoV-2-specific T cells is 95-105:1; The volume percentage of the autologous plasma is 4% to 6%; The final concentration of IL-2 is 90~110 IU / ml.

6. The preparation method according to claim 1, characterized in that, In step 3), during the second culture, fresh T-cell complete culture medium is added every 2-3 days to maintain a viable cell density of (0.5-1.0) × 10⁻⁶ cells / year. 6 Cells / mL.

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