Method for in-vitro amplification of antigen-specific T cells based on general type II peptide

Abstract

The invention discloses a method for in-vitro amplification of antigen-specific T cells based on general type II peptides, belongs to the technical field of biology, and provides the method for in-vitro amplification of the antigen-specific T cells and a kit used for the method. Flow Tetramer detection can obviously improve the proportion of specific antigen-specific T cells, three different cell factor matching modes and concentration ratios are compared, a group of relatively excellent factor combinations is optimized, flow Tetramer detection can obviously improve the proportion of specific antigen-specific T cells, the influence of polypeptide stimulation times in specific time is compared, and the specific antigen-specific T cells can be more accurately detected. Results show that the proportion of the specific T cells amplified through twice polypeptide stimulation is superior to that of the specific T cells amplified through once polypeptide stimulation, and the method is suitable for preparing the virus peptide, TAA and TSA antigen specific T cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for amplifying antigen-specific T cells in vitro based on universal class II peptides. Background Art

[0002] Pan DR-binding epitope (PADRE) is a commonly synthesized peptide-binding (MHC-II) receptor, in which the human leukocyte antigen HLA-DR receptor is present on specific immune cells. It can activate antigen-specific CD4+T cells and initiate innate immune responses (inflammatory cascades). PADRE has the ability to act as a Toll-like receptor (TLR) agonist, so it can be added to the design process of many immunotherapy vaccines to play a similar adjuvant role. However, in in vitro experiments, especially in the expansion of in vitro antigen-specific T cells, it is not clear whether PADRE has an auxiliary enhancement effect. Summary of the invention

[0003] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0004] The present invention provides a method for amplifying antigen-specific T cells in vitro, the method comprising: The PBMC cells are cultured with the first culture medium, then half-cultured with the first half-culture medium, then half-cultured with the second half-culture medium, then half-cultured with the third half-culture medium, and finally centrifuged, the supernatant is removed, and the second culture medium is used for expansion culture to obtain antigen-specific T cells; The components in the first half of the liquid change are X-vivo15+10% hAB+20 μM R848+200 ng / mL LPS+20ng / mL IL-1β+2 μg / mL polypeptide, wherein the polypeptide is an equal concentration of antigenic peptide and class II peptide PADRE; The first culture medium composition is X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50 ng / mL Flt3-L; The second half of the fluid change consists of X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20IU IL-2; The third half-change solution composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10IU IL-2; The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

[0005] Furthermore, the amino acid sequence of the class II peptide PADRE is shown in SEQ ID NO:6.

[0006] Furthermore, the first culture medium can be used for a culture time of 0-24 h.

[0007] In some embodiments, the first culture medium can be used for a culture time including 0 h, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, and 24 h.

[0008] Furthermore, the first half medium change may use a culture time of 0-24 h.

[0009] In some embodiments, the first half fluid change can use a culture time including 0 h, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h.

[0010] Furthermore, the second half medium change can use a culture time of 0-72 h.

[0011] In some embodiments, the second half of the medium replacement can use a culture time including 0 h, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h h, 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h, 70 h, 71 h, 72 h.

[0012] Furthermore, the third half medium change can use a culture time of 0-72 h.

[0013] In some embodiments, the third half-liquid exchange can use a culture time including 0 h, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h h, 57 h, 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h, 70 h, 71 h, 72 h.

[0014] Furthermore, the second culture medium can be used for a culture time of 0-72 h.

[0015] In some embodiments, the second culture medium can be used for a culture time of 0 h, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h h, 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h, 70 h, 71 h, 72 h.

[0016] Furthermore, the culture time after adding the first culture solution is 16 h-30 h.

[0017] Furthermore, the culture time after adding the first culture solution is 24 h.

[0018] Furthermore, the culture time after adding the first half of the liquid is 16 h-30 h.

[0019] Furthermore, the culture time after adding the first half of the liquid change is 24 h.

[0020] Furthermore, the culture time after the second half of the liquid change is 3 days.

[0021] Further, the total culture time using the third half medium change was 8 days.

[0022] Furthermore, the culture time after adding the second culture solution is not less than 72 h.

[0023] In some embodiments, the culture time after the second culture medium is added is not less than 72 hours, and can be extended for an unlimited period of time until the end of the experiment or cell death.

[0024] Furthermore, 5 μg / mL of polypeptide was added during the first half-fluid change of the third half-fluid change, wherein the polypeptide was an antigen peptide and a class II peptide PADRE at equal concentrations.

[0025] Furthermore, the antigen-specific T cells are antigen-specific CD8+T cells.

[0026] Furthermore, the antigenic peptides include viral peptides, TSA-type antigens, and TAA-type antigens.

[0027] Furthermore, the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

[0028] Furthermore, the PBMC cells are PBMC cells of HLA subtype, wherein the HLA subtype corresponds to the type of antigen peptide used in the first half of the fluid change.

[0029] Furthermore, the EBV corresponds to the A11:01 subtype of HLA.

[0030] Furthermore, the MAGE-A1 corresponds to the A02:01 subtype of HLA.

[0031] Furthermore, the MAGE-A3 corresponds to the A02:01 subtype of HLA.

[0032] Furthermore, the MAGE-A10 corresponds to the A02:01 subtype of HLA.

[0033] Furthermore, the KRAS-G12V corresponds to the A11:01 subtype of HLA.

[0034] Furthermore, during the half-liquid replacement culture, the total liquid volume was maintained by supplementing the half-liquid replacement culture in order to prevent the decrease in the total liquid volume due to volatilization.

[0035] Furthermore, the amino acid sequence of the EBV antigen peptide is shown in SEQ ID NO:1.

[0036] Furthermore, the amino acid sequence of the MAGE-A1 antigen peptide is shown in SEQ ID NO:2.

[0037] Furthermore, the amino acid sequence of the MAGE-A3 antigen peptide is shown in SEQ ID NO:3.

[0038] Furthermore, the amino acid sequence of the MAGE-A10 antigen peptide is shown in SEQ ID NO:4.

[0039] Furthermore, the amino acid sequence of the KRAS-G12V antigen peptide is shown in SEQ ID NO:5.

[0040] In some embodiments, the TSA-type antigen, ie, tumor-specific antigen (Tumor Specific Antigen), is a new antigen that is unique to tumor cells or only exists in certain tumor cells but not in normal cells.

[0041] In some embodiments, tumor-associated antigens (TAAs) refer to a class of antigen molecules present on both tumor cells and normal cells. They can be synthesized in trace amounts in normal cells, but are highly expressed when tumor cells proliferate. TAAs are not specific to tumor cells, so they have important application value in tumor immunotherapy. TAAs include multiple types, such as embryonic antigens, differentiation antigens, and overexpressed antigens.

[0042] In some embodiments, the EBV refers to a polypeptide encoded by Epstein-Barr Virus, wherein LMP2 polypeptide is the abbreviation of Epstein-Barr Virus Latent Membrane Protein 2, which is an important viral antigen.

[0043] In some embodiments, the MAGE-A1 is a tumor-specific antigen belonging to the human melanoma antigen family, which is expressed in a variety of tumors and is associated with gene transcription regulation and the transformation or progression of cancer.

[0044] In some embodiments, the MAGE-A3 is a human leukocyte antigen molecule HLA-A24 antigenic determinant cluster encoded by melanoma antigen gene A3 (MAGE-A3). It is a small molecule polypeptide composed of 9 amino acids and has a specific sequence and structure.

[0045] In some embodiments, the MAGE-A10 is a member of the melanoma antigen A (MAGE-A) family, which is a cancer-testis antigen with high tumor specificity.

[0046] In some embodiments, the KRAS-G12V is a mutant protein closely related to tumor occurrence and development. The KRAS gene is one of the most common oncogenes, and its mutations are mainly concentrated on glycine 12 (G12), among which G12V is a common mutation type.

[0047] In some embodiments, the half-medium replacement culture, also known as half-cell medium replacement, is a cell culture operation, the core step of which is to discard half of the old culture medium and then add an equal amount of new culture medium. This method is mainly used to maintain a stable cell growth environment while avoiding stress on cells caused by drastic changes in the environment.

[0048] In some embodiments, the antigenic peptide and the class II peptide PADRE are present in equal concentrations in the polypeptide used, where equal concentrations refer to equal mass concentrations of the solutes in the solution.

[0049] The present invention provides a culture kit used in the above method, wherein the culture kit comprises a first culture solution, a second culture solution, a first half culture solution change, a second half culture solution change, and a third half culture solution change; The components in the first half of the liquid change are X-vivo15+10% hAB+20 μM R848+200 ng / mL LPS+20ng / mL IL-1β+2 μg / mL polypeptide, wherein the polypeptide is an equal concentration of antigenic peptide and class II peptide PADRE; The first culture medium composition is X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50 ng / mL Flt3-L; The second half of the fluid change consists of X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20IU IL-2; The third half-change solution composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10IU IL-2; The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

[0050] Furthermore, 5 μg / mL of polypeptide is added to the third half-change solution, wherein the polypeptide is an antigen peptide and a class II peptide PADRE at equal concentrations.

[0051] Furthermore, the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

[0052] The present invention provides the use of the aforementioned method and the aforementioned culture kit in preparing antigen-specific T cells.

[0053] Furthermore, the antigen-specific T cells are antigen-specific CD8+T cells.

[0054] In some embodiments, the human leukocyte antigen (HLA) is a group of highly polymorphic cell surface proteins located on the short arm of human chromosome 6. They play a key role in the immune system, and their main functions include antigen presentation and regulation of immune responses. HLA antigens are divided into three categories: HLA-I, HLA-II, and HLA-III.

[0055] In some embodiments, the antigen-specific T cells play a key role in the immune system and function in anti-infection and anti-tumor. Antigen-specific T cells are immune cells that can specifically recognize and respond to specific antigens. They recognize antigenic peptides bound to the major histocompatibility complex (MHC) through the T cell receptor (TCR), thereby initiating an immune response. According to their function, antigen-specific T cells can be divided into cytotoxic T cells (CTL) and helper T cells (Th cells). CTL is mainly responsible for killing virus-infected or tumor-transformed cells, while Th cells help activate other immune cells such as B cells and macrophages.

[0056] The term "T cell" used in the present invention refers to a thymus-dependent lymphocyte, which is formed by differentiation of lymphoid stem cells derived from bone marrow in the thymus. T cells that mature in the thymus are transferred to peripheral lymphoid organs or lymphoid tissues, and remain relatively quiescent before being stimulated by specific antigen molecules, which are called initial T cells. Once stimulated by the corresponding antigen, they are transformed into large lymphocytes with active metabolism and a diameter of 15 to 20 μm, and proliferate and differentiate. Most of them differentiate into effector T cells, which acquire the functions of migration, production of cytokines and other effects, and a small part forms memory T cells. Effector T cells have a short lifespan and have the function of killing target cells. The lifespan of memory T cells can be as long as several years or even lifelong. When the body encounters the same antigen stimulation again, it can rapidly transform and proliferate to form a large number of effector T cells, initiate a stronger immune response, and keep the body immune to the antigen for a longer period of time. According to the different functions of T cells in immune response, T cells can be divided into the following three subgroups: cytotoxic T cells, helper T cells, and regulatory T cells.

[0057] The term "B cell" used in the present invention refers to bone marrow-dependent lymphocytes, which are derived from the bone marrow and account for 50-10% of the total number of blood lymphocytes. Initial B cells that mature in the bone marrow leave the bone marrow and migrate to the surrounding lymphoid organs and lymphoid tissues. After being stimulated by antigens, they proliferate and differentiate into effector B cells, i.e. plasma cells, which synthesize and secrete antibodies and exert immune functions; a small amount of them are converted into memory B cells and stored, and their role is the same as that of memory T cells. B cells survive in the body for a short time, only a few days to a few weeks, but their memory cells can survive in the body for a long time.

[0058] The present invention provides a device, which includes a memory and a processor. The processor is used to call program instructions and execute the above-mentioned method when the program instructions are called. The memory is used to store the program instructions.

[0059] The present invention provides a system, which includes using a computer program to adjust a processor so that the processor executes the above-mentioned method without artificial factors.

[0060] The present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is called and executed by a processor, the method described above is implemented.

[0061] The term "processor" used in the present invention refers to any type of processor, including more than one processor, such as a multi-core design or multiple processors with a multi-core design for each processor. The processor can be configured to execute a sequence of computer program instructions, such as a sequence of those instructions stored in a memory, to perform various operations, processes and methods according to the exemplary embodiments of the content of the present invention.

[0062] The term "memory" as used in this disclosure refers to any type of long-term, short-term, volatile, non-volatile or other memory, and should not be limited to any specific type of memory or any specific number of memories or types of media storing memory.

[0063] In some embodiments, the memory includes read-only memory (ROM), phase-change random access memory (PRAM), static random access memory (SRAM), flash memory, random access memory (RAM), dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM), electrically erasable programmable read-only memory (EEPROM), static memory, and other types of random access memory, cache memory, registers, compact disk read-only memory (CD-ROM), digital versatile disk (DVD) or other optical storage devices, cassettes, other non-transitory media.

[0064] The term "computer-readable storage medium" used in the present invention should be understood to include a single medium or multiple media (e.g., a centralized database or distributed database, and / or associated caches and servers) that store one or more instruction sets. The term "computer-readable storage medium" should also be understood to include any medium capable of storing or encoding an instruction set that is used to be executed by a machine and causes the machine to perform any one or more of the methods of the present invention. The term "computer-readable storage medium" should accordingly be understood to include, but is not limited to, solid-state memories and optical and magnetic media.

[0065] In order to improve the positive rate of antigen amplification and simplify the culture process, we tried to shorten the APC cell differentiation and maturation time, and added other effective factors on the basis of classic differentiation culture cytokines to improve the antigen presentation ability of APC cells; at the same time, we adopted the method of adding antigen peptide + class II peptide (PADRE) together for 2 rounds to enhance the antigen stimulation induction effect, and used different cytokine concentrations and combinations in subsequent culture to ensure a higher proportion of specific T cells and a longer culture duration.

[0066] Advantages and beneficial effects of the present invention: The present invention adopts the class II peptide PADRE as the synergistic part of antigen stimulation, which can activate CD4+T cells and thus facilitate the CD8+T cell activation process, and can meet the amplification requirements of various types of HLA antigens; at the same time, based on this, the cytokine combination and cytokine concentration in the culture process are changed, and specific operations are performed at fixed time nodes, thereby generating a new set of antigen in vitro amplification culture processes with better amplification effects and a wider range of applicable antigens.

[0067] The present invention improves the positive ratio of antigen-amplified specific T cells, optimizes the culture conditions and culture process, and meets the amplification needs of various types of antigens. At the same time, because mixed culture is adopted, it gets rid of the dependence on DC cells in traditional amplification, and prolongs the specific T cell culture time through the change of cytokines, laying a foundation for subsequent use of amplified specific T cells to carry out other experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is the result of Donor EBV polypeptide-specific CD8+T cell detection.

[0069] Figure 2 This is the result of Donor MAGE-A3 peptide-specific CD8+T cell detection.

[0070] Figure 3 It is a statistical chart of specific CD8+T cell detection results.

[0071] Figure 4 It is a statistical chart of the cell subset detection results of each Donor culture well.

[0072] Figure 5 This is a graph showing the results of the detection of EBV polypeptide-specific CD8+T cells in each Donor in different factor groups.

[0073] Figure 6 This is a graph showing the results of the Donor MAGE-A3 polypeptide-specific CD8+T cell detection in different factor groups.

[0074] Figure 7 It is a statistical chart of the detection results of specific CD8+T cells in different factor groups.

[0075] Figure 8 This is a graph showing the results of specific CD8+T cell detection under different stimulation times of each Donor MAGE-A1 peptide.

[0076] Fig. 9These are the results of specific CD8+T cell detection under different stimulation times of each Donor MAGE-A10 / KRAS-G12V peptide. DETAILED DESCRIPTION

[0077] The present invention uses experimental materials.

[0078] 1. Cell lines and reagents and consumables: RPMI-1640 medium was purchased from Gibco with a catalog number of 11875-093; fetal bovine serum was purchased from Cytiva with a catalog number of SV30208.02; hAB serum was purchased from Gemini Bio with a catalog number of 100-512; IL-2 was purchased from Perprotech with a catalog number of 200-02-1MG; IL-4 was purchased from RD with a catalog number of 204-GMP-010; IL-7 was purchased from Perprotech with a catalog number of 200-07-10ug; IL-15 was purchased from Perprotech with a catalog number of 200-15-10ug; GM-CSF was purchased from RD with a catalog number of 7954-GM-010 / C F; Flt3-L was purchased from Bio-Techne with the catalog number 308-FKN-005 / CF; IL-1β was purchased from Bio-Techne with the catalog number 201-LB-005 / CF; R848 was purchased from InvivoGen with the catalog number tlrl-r848-1; LPS was purchased from InvivoGen with the catalog number tlrl-smlps; 96-well U-shaped plate was purchased from Aijin Biotechnology with the catalog number P-0.36-96V-S.

[0079] 2. Cell lines and peptides: PBMCs were purchased from Miaoshun Biotechnology; the specific information is shown in Table 1.

[0080] Table 1

[0081] The peptides were synthesized by GenScript, and the specific information is shown in Table 2.

[0082] Table 2

[0083] 3. Antibodies for flow cytometry detection include: PerCP / Cyanine5.5 anti-human CD3 Antibody purchased from Biolegend, catalog number 300430; APC / Cyanine7 anti-human CD4 Antibody purchased from Biolegend, catalog number 344616; Brilliant Violet 510™ anti-human CD8 Antibody purchased from Biolegend, catalog number 344731; HLA-A*11:01 EBV LMP2 S9T Tetramer-SSCSSCPLTK-PE purchased from MBL, catalog number TS-M135-1; iTAg Tetramer / PE - HLA - A*02:01 MAGE-A1 (KVLEYVIKV) purchased from MBL, catalog number TB-M070-1; HLA-A*02:01 MAGE-A3271-279 Tetramer-FLWGPRALV-PE was purchased from MBL with the catalog number TB-M076-1; HLA-A*02:01 MAGE-A10 Tetramer-GLYDGMEHL-PE was purchased from MBL with the catalog number TS-M078-1; KRAS-G12V- Tetramer -PE was customized from MBL with the catalog number TS2974.

[0084] Example 1 Exploring the Effect of Addition of Class II Peptides 1. Experimental methods Day 0: Cell recovery and plating: 1) After thawing frozen PBMC cells, centrifuge at 1500 rpm for 5 min; 2) Discard the supernatant, add X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50 ng / mL Flt3-L, resuspend and count, inoculate 96-well U-shaped plates at 2E5 / well / 200 μL, and place incubator for culture.

[0085] Day 1: Add antigen: 3) Centrifuge the culture plate and replace half of the medium with X-vivo15+10% hAB+20 μM R848+200ng / mL LPS+20 ng / mL IL-1β +2 μg / mL peptide and continue culturing; No peptide was added to the NC control group, and the antigen peptide and class II peptide PADRE were added at equal concentrations in the experimental group.

[0086] Day 2:4) Centrifuge the culture plate and replace half of the medium with X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mLIL-15+ 20 IU IL-2 and continue culturing.

[0087] Day 5 / 8 / 10: Half medium change: 5) Centrifuge the culture plate and use X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+ 10 IU IL-2 for half medium change and continue culturing (add more medium appropriately to compensate for the loss caused by volatilization).

[0088] Day 12: Expanded culture: 6) Collect the cultured cells from 8 wells in each column of the same group, discard the supernatant after centrifugation, resuspend with X-vivo15+10% hAB+1000 IU IL-2, and inoculate into 12-well plates for continued culture.

[0089] Day 15: Detection: 7) Collect cells from the culture wells for detection and detect antigen-specific T cells by flow cytometry.

[0090] Flow staining and detection: resuspend cells with 1mL buffer (PBS + 2% FBS), centrifuge at 350g for 5min at room temperature, wash cells 3 times; resuspend cells with washing buffer to a density of 3-10E6 / mL; add 100 μL of cell suspension to each tube, centrifuge at 500g for 3min at room temperature, carefully aspirate supernatant, tap and add 20 μL of ClearBack to the loosened cell pellet to mix evenly, incubate at room temperature in the dark for 5 min; take out the required Tetramer from the refrigerator, add 2 μL of Control Tetramer to the negative control Tetramer tube, add 2 μL of the corresponding Tetramer to the experimental tube according to the experimental settings, mix evenly in each tube, incubate at 4℃ in the dark for 30 min; take out the prepared surface antibody mixture from the refrigerator, add 100 μL to each tube, mix evenly, incubate at 4℃ in the dark for at least 40 min; add 1 mL of buffer to each tube for washing, centrifuge at 350g, 4℃ for 5 min, discard the supernatant, add an appropriate volume of buffer, vortex the cells gently, and detect on the machine within 1 hour.

[0091] 2. Experimental results PBMCs of three healthy donors with specific HLA subtypes were cultured with the above method and then tested by flow cytometry. Compared with the control group (NC, no peptide added), the experimental group could induce antigen-specific CD8+T cells after adding EBV peptide or MAGE-A3 peptide. After adding class II peptide PADRE at the same time, the proportion of positive specific T cells amplified in each group was significantly increased. Although the enhancement effect was individual, it was reflected in each donor ( Figure 1-Figure 3 ); At the same time, statistical analysis of cell subsets in the culture system showed that compared with the control group and the experimental group, the addition of class II peptides did not lead to a significant increase in the proportion of CD4+T cells, which is more conducive to the expansion of antigen-specific CD8+T cells ( Figure 4 ).

[0092] Example 2 Exploring the effects of different cytokines Based on the experimental results of Example 1, on the basis of adding class II peptides, the effects of three classic cytokine addition methods on the culture of specific T cells were compared. The three classic cytokine addition methods are shown in Table 3.

[0093] Table 3

[0094] Each group is added according to the factor information in the table, and the process is the same as in Example 1.

[0095] 2. Experimental results PBMCs of three healthy donors with specific HLA subtypes were cultured with the above method and then tested by flow cytometry. After adding EBV peptide, the proportion of specific T cells amplified by donor 1 under Group 1 was the highest (3.36%), while the proportion of specific T cells amplified by donors 2 and 3 under Group 2 was higher. After adding MAGE-A3 peptide, the proportion of specific T cells amplified by all three donors under Group 2 was the highest. In summary, the cytokine combination addition method of Group 2 has a better amplification and culture effect ( Figure 5-Figure 7 ).

[0096] Example 3 Exploring the influence of the number of times the polypeptide is added Based on the experimental results of Example 1 and Example 2, after determining to add the class II peptide PADRE and the specific cytokine for use in combination, we tried to increase the number of peptide stimulations during the process to see if the amplification effect could be further improved.

[0097] 1. Experimental methods Day 0: Cell recovery and plating: 1) After thawing frozen PBMC cells, centrifuge at 1500 rpm for 5 min; 2) Discard the supernatant, add X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50 ng / mL Flt3-L, resuspend and count, inoculate 96-well U-shaped plates at 2E5 / well / 200 μL, and place incubator for culture.

[0098] Day 1: Add antigen: 3) After centrifugation of the culture plate, half of the medium was replaced with X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide and culture was continued. No peptide was added to the NC control group, and the antigen peptide and class II peptide PADRE were added at equal concentrations in the experimental group.

[0099] Day 2:4) Centrifuge the culture plate and replace half of the medium with X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mLIL-15+ 20 IU IL-2 and continue culturing.

[0100] Day 5: Second addition of antigen: 5) According to the plate layout, add 50 μL X-vivo15+10% hAB+10 ng / mLIL-7+10 ng / mL IL-15+10 IU IL-2+5 μg / mL peptide culture medium and continue culturing. No peptide was added to the NC control group and the first peptide stimulation group. The antigen peptide and class II peptide PADRE were added at equal concentrations in the second stimulation experimental group.

[0101] Day 6 / 9:6) Use X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10 IUIL-2 medium for half medium change and continue culturing (add more medium appropriately to compensate for the loss caused by volatilization).

[0102] Day 12: Expanded culture: 7) Collect the cultured cells from 8 wells in each column of the same group, discard the supernatant after centrifugation, resuspend with X-vivo15+10% hAB+1000 IU IL-2, and inoculate into 12-well plates for continued culture.

[0103] Day 15: Detection: 8) Collect cells from the culture wells for detection and detect antigen-specific T cells by flow cytometry.

[0104] Flow staining and detection: refer to the detection content in Example 1.

[0105] 2. Experimental results Three healthy donor PBMCs of specific HLA subtypes were selected and cultured with the above method for flow cytometry Tetramer method detection. After adding MAGE-A1, MAGE-A10, KRAS-G12V and PADRE peptides, the test results showed that compared with the control group and the group stimulated with peptides once, the proportion of specific T cells increased significantly after two peptide stimulations, and KRAS-G12V-specific T cells were amplified in one donor, indicating that increasing the number of peptide stimulations has a significant effect on improving the amplification of specific T cells, and there is a significant difference, and the optimized method is also applicable to TSA antigens ( Figure 8-Figure 9 ).

Claims

1. A method for expanding antigen-specific T cells in vitro, the method comprising: The PBMC cells are cultured with the first culture medium, then half-cultured with the first half-culture medium, then half-cultured with the second half-culture medium, then half-cultured with the third half-culture medium, and finally centrifuged, the supernatant is removed, and the second culture medium is used for expansion culture to obtain antigen-specific T cells; The components in the first half of the liquid change are X-vivo15+10% hAB+20 μM R848+200 ng / mL LPS+20 ng / mL IL-1β+2 μg / mL polypeptide, wherein the polypeptide is an equal concentration of antigenic peptide and class II peptide PADRE; The first culture solution composition is X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50ng / mL Flt3-L; The second half of the fluid change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IUIL-2; The third half-change solution composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10 IUIL-2; The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

2. According to the method of claim 1, the first culture medium can be used for a culture time of 0-24 h; Preferably, the first half of the medium change can be used for a culture time of 0-24 h; Preferably, the second half medium change can be used for a culture time of 0-72 h; Preferably, the third half-fluid change can be used for a culture time of 0-72 h; Preferably, the second culture medium can be used for a culture time of 0-72 h; Preferably, the culture time after adding the first culture solution is 16 h-30 h; Preferably, the culture time after the first half of the liquid change is 16 h-30 h; Preferably, the culture time after the second half of the liquid change is 3 days; Preferably, the total culture time using the third half-fluid change is 8 days; Preferably, the culture time after adding the second culture solution is not less than 72 h.

3. The method according to claim 1, wherein 5 μg / mL of polypeptide is added during the first half-fluid change of the third half-fluid change, wherein the polypeptide comprises an equal concentration of the antigenic peptide and the class II peptide PADRE.

4. The method according to claim 1, wherein the antigenic peptides include viral peptides, TSA-type antigens, and TAA-type antigens; Preferably, the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V; Preferably, the PBMC cells are PBMC cells of HLA subtype, wherein: The HLA subtype corresponds to the type of antigenic peptide used in the first half of the fluid change; Preferably, the EBV corresponds to the A11:01 subtype of HLA; Preferably, the MAGE-A1 corresponds to the A02:01 subtype of HLA; Preferably, the MAGE-A3 corresponds to the A02:01 subtype of HLA; Preferably, the MAGE-A10 corresponds to the A02:01 subtype of HLA; Preferably, the KRAS-G12V corresponds to the A11:01 subtype of HLA.

5. The method according to claim 1, wherein during the half-liquid replacement culture, the total liquid volume is maintained by supplementing the half-liquid replacement culture in order to prevent the decrease in the total liquid volume due to volatilization.

6. A culture kit for use in the method according to any one of claims 1 to 5, wherein the culture kit comprises a first culture solution, a second culture solution, a first half culture solution change, a second half culture solution change, and a third half culture solution change; The components in the first half of the liquid change are X-vivo15+10% hAB+20 μM R848+200 ng / mL LPS+20 ng / mL IL-1β+2 μg / mL polypeptide, wherein the polypeptide is an equal concentration of antigenic peptide and class II peptide PADRE; The first culture medium composition is X-vivo15+10% hAB+1000 IU / mL GM-CSF+500 IU / mL IL-4+50ng / mL Flt3-L; The second half of the fluid change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IUIL-2; The third half-change solution composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10 IUIL-2; The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

7. The culture kit according to claim 6, wherein 5 μg / mL polypeptide is further added to the third half-change medium, wherein the polypeptide is an antigen peptide and a class II peptide PADRE at equal concentrations.

8. The culture kit according to claim 6, wherein the antigen peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

9. Use of the method according to any one of claims 1 to 5 or the culture kit according to any one of claims 6 to 8 in preparing antigen-specific T cells.

10. A device, comprising a memory and a processor, wherein the processor is used to call a program instruction, and when the program instruction is called, the method according to any one of claims 1 to 5 is executed, and the memory is used to store the program instruction.

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