A method for in vitro expansion of antigen-specific T cells based on universal class II peptides

Abstract

The present invention discloses a method for amplifying antigen-specific T cells in vitro based on a universal class II peptide, belonging to the field of biotechnology. The present invention provides a method for amplifying antigen-specific T cells in vitro and a kit for the method. The present invention simultaneously adds a PADRE polypeptide during in vitro culture, and flow cytometry Tetramer detection can significantly increase the proportion of specific antigen-specific T cells. The present invention compares three different cytokine combinations and concentration ratios, and optimizes a group of relatively excellent factor combinations. Flow cytometry Tetramer detection can significantly increase the proportion of specific antigen-specific T cells. The present invention compares the effect of the number of polypeptide stimulations within a specific time, and the results show that the proportion of specific T cells amplified by two polypeptide stimulations is better than that by one polypeptide stimulation. The method of the present invention is suitable for the preparation of viral peptide, TAA and TSA antigen-specific T cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular 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 ubiquitous synthetic peptide-binding (MHC-II) receptor, specifically the human leukocyte antigen (HLA-DR) receptor, present on specific immune cells. It can activate antigen-specific CD4+ T cells and initiate the innate immune response (inflammatory cascade). PADRE has the ability to act as a Toll-like receptor (TLR) agonist, and therefore is incorporated into many immunotherapy vaccine designs to act as an adjuvant. However, its effectiveness in enhancing antigen-specific T cell expansion in vitro remains unclear. 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 expanding antigen-specific T cells in vitro, the method comprising:

[0005] PBMC cells are cultured using the first culture medium, then cultured using the first half culture medium for half-culture, then cultured using the second half culture medium for half-culture, then cultured using the third half culture medium for half-culture, and finally centrifuged, the supernatant is removed, and the cells are expanded using the second culture medium to obtain antigen-specific T cells;

[0006] The first half of the medium change consists of X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, wherein the peptides are equal concentrations of antigenic peptide and class II peptide PADRE;

[0007] 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;

[0008] The second half of the medium change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IU IL-2;

[0009] The third half-change medium composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10 IU IL-2;

[0010] The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

[0011] Furthermore, the amino acid sequence of the class II peptide PADRE is shown in SEQ ID NO:6.

[0012] Furthermore, the first culture medium can be used for a culture time of 0-24 h.

[0013] In some embodiments, the first culture medium can be cultured for a period of 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.

[0014] Furthermore, the first half of the medium change can be used for a culture time of 0-24 h.

[0015] In some embodiments, the first half-fluid 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, and 24 h.

[0016] Furthermore, the second half medium change can use a culture time of 0-72 h.

[0017] In some embodiments, the second half of the medium 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, 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.

[0018] Furthermore, the third half medium change can be used for a culture time of 0-72 h.

[0019] In some embodiments, the third half-exchange may use 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, 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.

[0020] Furthermore, the second culture medium can be used for a culture time of 0-72 h.

[0021] In some embodiments, the second culture medium can be cultured for a period of 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.

[0022] Furthermore, the culture time after adding the first culture solution is 16 h-30 h.

[0023] Furthermore, the culture time after adding the first culture solution is 24 h.

[0024] Furthermore, the culture time after adding the first half of the liquid is 16 h-30 h.

[0025] Furthermore, the culture time after adding the first half of the liquid is 24 hours.

[0026] Furthermore, the culture time after the second half of the liquid change is 3 days.

[0027] Furthermore, the total culture time using the third half medium change was 8 days.

[0028] Furthermore, the culture time after the second culture solution is added is not less than 72 h.

[0029] In some embodiments, the culture time after adding the second culture medium 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.

[0030] Furthermore, 5 μg / mL of polypeptide was added during the first half-fluid change of the third half-fluid change, wherein the polypeptides were equal concentrations of the antigen peptide and the class II peptide PADRE.

[0031] Furthermore, the antigen-specific T cells are antigen-specific CD8+ T cells.

[0032] Furthermore, the antigenic peptides include viral peptides, TSA-type antigens, and TAA-type antigens.

[0033] Furthermore, the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

[0034] 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 medium change.

[0035] Furthermore, the EBV corresponds to the A11:01 subtype of HLA.

[0036] Furthermore, the MAGE-A1 corresponds to the A02:01 subtype of HLA.

[0037] Furthermore, the MAGE-A3 corresponds to the A02:01 subtype of HLA.

[0038] Furthermore, the MAGE-A10 corresponds to the A02:01 subtype of HLA.

[0039] Furthermore, the KRAS-G12V corresponds to the A11:01 subtype of HLA.

[0040] Furthermore, during the half-fluid exchange culture, the total fluid volume was maintained by supplementing the half-fluid exchange in preparation for the decrease in the total fluid volume due to volatilization.

[0041] Furthermore, the amino acid sequence of the EBV antigen peptide is shown in SEQ ID NO: 1.

[0042] Furthermore, the amino acid sequence of the MAGE-A1 antigen peptide is shown in SEQ ID NO: 2.

[0043] Furthermore, the amino acid sequence of the MAGE-A3 antigen peptide is shown in SEQ ID NO: 3.

[0044] Furthermore, the amino acid sequence of the MAGE-A10 antigen peptide is shown in SEQ ID NO:4.

[0045] Furthermore, the amino acid sequence of the KRAS-G12V antigen peptide is shown in SEQ ID NO:5.

[0046] 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 exists only in certain tumor cells but not in normal cells.

[0047] In some embodiments, tumor-associated antigens (TAAs) refer to a class of antigenic molecules present on both tumor cells and normal cells. They are synthesized in trace amounts by normal cells but are highly expressed during tumor cell proliferation. TAAs are not specific to tumor cells and therefore have important application value in tumor immunotherapy. TAAs include various types, such as embryonic antigens, differentiation antigens, and overexpressed antigens.

[0048] 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.

[0049] 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 cancer transformation or progression.

[0050] In some embodiments, the MAGE-A3 is a human leukocyte antigen molecule HLA-A24 antigenic determinant encoded by the melanoma antigen gene A3 (MAGE-A3). It is a small molecule polypeptide composed of 9 amino acids with a specific sequence and structure.

[0051] In some embodiments, the MAGE-A10 is a member of the melanoma antigen A (MAGE-A) family, which is a highly tumor-specific cancer-testis antigen.

[0052] In some embodiments, the KRAS-G12V is a mutant protein closely associated with tumorigenesis and progression. The KRAS gene is one of the most common oncogenes, and its mutations are mainly concentrated at glycine 12 (G12), of which G12V is a common mutation type.

[0053] In some embodiments, a half-medium culture, also known as a half-cell culture medium change, is a cell culture procedure in which half of the old culture medium is discarded and replaced with an equal amount of new culture medium. This method is primarily used to maintain a stable cell growth environment while avoiding stress caused by drastic environmental changes.

[0054] In some embodiments, the antigenic peptide and the class II peptide PADRE are present in equal concentrations in the polypeptide, where equal concentrations refer to equal mass concentrations of the solutes in the solution.

[0055] The present invention provides a culture kit used in the aforementioned 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;

[0056] The first half of the medium change consists of X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, wherein the peptides are equal concentrations of antigenic peptide and class II peptide PADRE;

[0057] 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;

[0058] The second half of the medium change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IU IL-2;

[0059] The third half-change medium composition is X-vivo15+10% hAB+10 ng / mL IL-7+10 ng / mL IL-15+10 IU IL-2;

[0060] The second culture medium composition is X-vivo15+10% hAB+1000 IU IL-2.

[0061] Furthermore, 5 μg / mL of polypeptide is added to the third half-change medium, wherein the polypeptide is an antigen peptide and a class II peptide PADRE at equal concentrations.

[0062] Furthermore, the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

[0063] The present invention provides the use of the aforementioned method and the aforementioned culture kit in preparing antigen-specific T cells.

[0064] Furthermore, the antigen-specific T cells are antigen-specific CD8+ T cells.

[0065] In some embodiments, the human leukocyte antigens (HLA) are 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, with primary functions including antigen presentation and regulation of immune responses. HLA antigens are divided into three classes: HLA-I, HLA-II, and HLA-III.

[0066] In some embodiments, antigen-specific T cells play a key role in the immune system, including anti-infection and anti-tumor functions. 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 their T cell receptors (TCRs), thereby initiating an immune response. Based on their function, antigen-specific T cells can be divided into cytotoxic T cells (CTLs) and helper T cells (Th cells). CTLs are primarily responsible for killing virus-infected or tumorous cells, while Th cells help activate other immune cells, such as B cells and macrophages.

[0067] As used herein, the term "T cell" refers to thymus-dependent lymphocytes, which are derived from bone marrow-derived lymphoid stem cells that differentiate within the thymus. Mature T cells in the thymus migrate to peripheral lymphoid organs or tissues and remain relatively quiescent until stimulated by specific antigen molecules. These cells are called naive T cells. Upon exposure to the corresponding antigen, they transform into metabolically active large lymphocytes with a diameter of 15-20 μm and begin to proliferate and differentiate. Most of these cells differentiate into effector T cells, acquiring migration, cytokine production, and other effector functions, while a smaller number become memory T cells. Effector T cells have a short lifespan and possess the ability to kill target cells. Memory T cells can live for several years or even lifelong. Upon repeated exposure to the same antigen, they rapidly transform and proliferate to form large numbers of effector T cells, initiating a more intense immune response and maintaining immunity to the antigen for a longer period. T cells can be divided into three subpopulations based on their functions in the immune response: cytotoxic T cells, helper T cells, and regulatory T cells.

[0068] The term "B cell" as used herein refers to bone marrow-dependent lymphocytes, which originate from the bone marrow and account for 50-10% of the total lymphocyte population in the blood. Initial B cells mature in the bone marrow and migrate to peripheral lymphoid organs and tissues. Upon antigen stimulation, they proliferate and differentiate into effector B cells, or plasma cells, which synthesize and secrete antibodies and exert immune functions. A small number of B cells are converted into memory B cells and stored, which function similarly to memory T cells. B cells survive in the body for a short time, only a few days to a few weeks, but their memory counterparts can survive long-term.

[0069] The present invention provides a device, comprising a memory and a processor, wherein the processor is used to call program instructions and execute the above-mentioned method when the program instructions are called, and the memory is used to store the program instructions.

[0070] 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.

[0071] The present invention provides a computer-readable storage medium having a computer program stored thereon. When the computer program is called and executed by a processor, the method described above is implemented.

[0072] The term "processor" as used herein refers to any type of processor, including more than one processor, such as a multi-core design or multiple processors each having a multi-core design. 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 present invention.

[0073] The term "memory" as used herein refers to any type of long-term, short-term, volatile, nonvolatile or other memory and should not be limited to any particular type of memory or any particular number of memories or types of media storing memory.

[0074] 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.

[0075] As used herein, the term "computer-readable storage medium" 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 sets of instructions. The term "computer-readable storage medium" should also be understood to include any medium capable of storing or encoding a set of instructions for execution by a machine and causing 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 not be limited to, solid-state memories and optical and magnetic media.

[0076] To increase the positive rate of antigen amplification and simplify the culture process, we attempted to shorten the APC cell differentiation and maturation time and add 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.

[0077] Advantages and beneficial effects of the present invention:

[0078] The present invention uses the class II peptide PADRE as a synergistic part of antigen stimulation, which activates CD4+ T cells and thus facilitates the CD8+ T cell activation process, meeting the amplification requirements of various types of HLA antigens. At the same time, based on this, the cytokine combination and cytokine concentration during 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.

[0079] The present invention improves the positive ratio of antigen-amplified specific T cells, optimizes culture conditions and culture processes, and meets the amplification needs of various types of antigens. At the same time, because it adopts mixed culture, it gets rid of the dependence on DC cells during traditional amplification, and prolongs the specific T cell culture time through the change of cytokines, laying the foundation for subsequent use of amplified specific T cells to carry out other experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the result of Donor EBV polypeptide-specific CD8+T cell detection.

[0081] Figure 2 This is the result of Donor MAGE-A3 peptide-specific CD8+T cell detection.

[0082] Figure 3 It is a statistical chart of specific CD8+T cell detection results.

[0083] Figure 4 It is a statistical chart of the cell subset detection results of each Donor culture well.

[0084] Figure 5 This is a graph showing the detection results of Donor EBV polypeptide-specific CD8+T cells in different factor groups.

[0085] Figure 6 This is a graph showing the results of detecting CD8+T cells specific to each Donor MAGE-A3 peptide in different factor groups.

[0086] Figure 7 It is a statistical chart of the detection results of specific CD8+T cells in different factor groups.

[0087] Figure 8 This is a graph showing the specific CD8+ T cell detection results under different stimulation times of each Donor MAGE-A1 peptide.

[0088] Figure 9These are the specific CD8+ T cell detection results under different stimulation times of each Donor MAGE-A10 / KRAS-G12V peptide. DETAILED DESCRIPTION

[0089] The present invention uses experimental materials.

[0090] 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.

[0091] 2. Cell lines and peptides: PBMCs were purchased from Miaoshun Biotechnology; detailed information is shown in Table 1.

[0092] Table 1

[0093]

[0094] The peptides were synthesized by GenScript, and the detailed information is shown in Table 2.

[0095] Table 2

[0096]

[0097] 3. Antibodies for flow cytometry detection include: PerCP / Cyanine5.5 anti-human CD3 Antibody purchased from Biolegend, Catalog No. 300430; APC / Cyanine7 anti-human CD4 Antibody purchased from Biolegend, Catalog No. 344616; Brilliant Violet 510™ anti-human CD8 Antibody purchased from Biolegend, Catalog No. 344731; HLA-A*11:01 EBV LMP2 S9T Tetramer-SSCSSCPLTK-PE purchased from MBL, Catalog No. TS-M135-1; iTAg Tetramer / PE - HLA - A*02:01 MAGE-A1 (KVLEYVIKV) purchased from MBL, Catalog No. 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 at MBL with the catalog number TS2974.

[0098] Example 1 Exploring the Effect of Addition of Class II Peptides

[0099] 1. Experimental methods

[0100] Day 0: Cell thawing and plating: 1) Thaw frozen PBMCs and 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 2E5 / well / 200 μL into a 96-well U-shaped plate, and culture in an incubator.

[0101] Day 1: Add antigen: 3) Centrifuge the culture plate and perform a half-medium change with X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, then continue culturing; the NC control group does not add peptide, while the experimental groups use equal concentrations of antigen peptide and class II peptide PADRE.

[0102] Day 2:4) Centrifuge the culture plate and perform a half-medium change with X-vivo15 + 10% hAB + 20 ng / mL IL-7 + 20 ng / mL IL-15 + 20 IU IL-2 before continuing culture.

[0103] Day 5 / 8 / 10: Half-medium change: 5) Centrifuge the plate and perform a half-medium change with X-vivo15 + 10% hAB + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 10 IU IL-2, then continue culturing (add more medium to compensate for losses due to evaporation).

[0104] Day 12: Expand the culture: 6) Collect the cultured cells from each 8-well column of the same group, centrifuge, discard the supernatant, resuspend in X-vivo15 + 10% hAB + 1000 IU IL-2, and inoculate into 12-well plates for continued culture.

[0105] Day 15: Detection: 7) Collect cells from the culture wells for detection and detect antigen-specific T cells by flow cytometry.

[0106] Flow cytometry staining and detection: Resuspend cells in 1 mL of buffer (PBS + 2% FBS), centrifuge at 350 g for 5 min at room temperature, and wash cells three times. Resuspend cells in wash buffer to a density of 3-10E6 / mL. Add 100 μL of cell suspension to each tube and centrifuge at 500 g for 3 min at room temperature. Carefully aspirate the supernatant, tap gently, and add 20 μL of ClearBack to the loosened cell pellet to mix thoroughly. Incubate at room temperature in the dark for 5 min. Remove the required tetramer from the refrigerator, add 2 μL of control tetramer to the negative control tetramer tube, and add 2 μL of the corresponding tetramer to the experimental tube according to the experimental settings. Mix thoroughly and incubate at 4°C in the dark for 30 min. Remove the prepared surface antibody mixture from the refrigerator, add 100 μL to each tube, mix thoroughly, and incubate at 4°C in the dark for at least 40 min. Add 1 mL of buffer to each tube for washing, centrifuge at 350 g for 5 min at 4°C, discard the supernatant, add an appropriate volume of buffer, gently vortex the cells, and analyze within 1 hour.

[0107] 2. Experimental results

[0108] PBMCs from three healthy donors with specific HLA subtypes were cultured using the above method and then subjected to flow cytometry Tetramer analysis. Compared with the control group (NC, no peptide added), the experimental groups induced antigen-specific CD8+ T cells after adding EBV peptide or MAGE-A3 peptide. After the simultaneous addition of the class II peptide PADRE, the proportion of amplified positive specific T cells in each group was significantly increased. Although the enhancement effect varied from person to person, it was evident in all donors ( 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 ).

[0109] Example 2 Exploring the effects of different cytokines

[0110] Based on the experimental results of Example 1, the effects of three classic cytokine addition methods on specific T cell culture were compared based on the addition of class II peptides. The specific effects of the three classic cytokine addition methods are shown in Table 3.

[0111] Table 3

[0112]

[0113]

[0114] Each group is added according to the factor information in the table, and the process is the same as in Example 1.

[0115] 2. Experimental results

[0116] PBMCs from three healthy donors with specific HLA subtypes were cultured using the above method and then analyzed using flow cytometry. After adding EBV peptides, donor 1 had the highest proportion of expanded specific T cells (3.36%) under Group 1 conditions, while donors 2 and 3 also had higher proportions of expanded specific T cells under Group 2 conditions. After adding MAGE-A3 peptides, all three donors had the highest proportions of expanded specific T cells under Group 2 conditions. In summary, the cytokine combination added in Group 2 had a better expansion and culture effect ( Figure 5-Figure 7 ).

[0117] Example 3 Exploring the Effect of Number of Peptide Additions

[0118] Based on the experimental results of Examples 1 and 2, after determining the combination of adding the class II peptide PADRE and specific cytokines, we tried increasing the number of peptide stimulations during the process to see if the expansion effect could be further improved.

[0119] 1. Experimental methods

[0120] Day 0: Cell thawing and plating: 1) Thaw frozen PBMCs and 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 2E5 / well / 200 μL into a 96-well U-shaped plate, and culture in an incubator.

[0121] Day 1: Add antigen: 3) Centrifuge the culture plate and perform a half-medium change with X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, then continue culturing. No peptide was added to the NC control group. The experimental groups were treated with equal concentrations of antigen peptide and class II peptide PADRE.

[0122] Day 2:4) Centrifuge the culture plate and perform a half-medium change with X-vivo15 + 10% hAB + 20 ng / mL IL-7 + 20 ng / mL IL-15 + 20 IU IL-2 before continuing culture.

[0123] Day 5: Secondary addition of antigen: 5) According to the plate layout, add 50 μL of X-vivo15 + 10% hAB + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 10 IU IL-2 + 5 μg / mL peptide culture medium and continue culturing. The NC control group and the primary peptide stimulation group do not add peptide. The secondary stimulation experimental group uses equal concentrations of antigen peptide and class II peptide PADRE.

[0124] Day 6 / 9:6) Perform a half-medium change with X-vivo15 + 10% hAB + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 10 IU IL-2 medium and continue culturing (add more medium as needed to compensate for loss due to evaporation).

[0125] Day 12: Expand the culture: 7) Collect the cultured cells from each 8-well column of the same group, centrifuge and discard the supernatant, resuspend in X-vivo15 + 10% hAB + 1000 IU IL-2, and inoculate into 12-well plates for continued culture.

[0126] Day 15: Detection: 8) Collect cells from the culture wells for detection and detect antigen-specific T cells by flow cytometry.

[0127] Flow staining and detection: refer to the detection content in Example 1.

[0128] 2. Experimental results

[0129] Three healthy donors with specific HLA subtypes were selected and cultured with the above method for flow cytometry Tetramer testing. 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 expanded in one donor. This shows that increasing the number of peptide stimulations has a significant effect on increasing the expansion of specific T cells, and there is a significant difference. 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: culturing PBMC cells using a first culture medium, then performing a half-culture using a first half-culture medium exchange, then performing a half-culture using a second half-culture medium exchange, then performing a half-culture using a third half-culture medium exchange, and finally centrifuging and removing the supernatant and using the second culture medium for expansion culture to obtain antigen-specific T cells, wherein the antigen-specific T cells are antigen-specific CD8+ T cells; The first half of the medium change consists of X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, wherein the peptides are equal concentrations 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 medium change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IUIL-2; The third half-change medium 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; 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 medium change.

2. The method according to claim 1, wherein the culture time after adding the first culture solution is 16 h-30 h.

3. The method according to claim 1, wherein the culture time after the first half-fluid change is 16 h-30 h.

4. The method according to claim 1, wherein the culture time after the second half of the liquid is added is 3 days.

5. The method of claim 1, wherein the total culture time using the third half-fluid change is 8 days. The method according to claim 1 , wherein the culture time after the second culture solution is added is not less than 72 h.

7. 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 antigenic peptide and a class II peptide PADRE at equal concentrations.

8. The method according to claim 1, wherein the antigenic peptides include viral peptides, TSA-type antigens, and TAA-type antigens.

9. The method according to claim 1, wherein the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

10. The method according to claim 9, wherein the EBV corresponds to the A11:01 subtype of HLA. The method according to claim 9 , wherein the MAGE-A1 corresponds to the A02:01 subtype of HLA.

12. The method according to claim 9, wherein the MAGE-A3 corresponds to the A02:01 subtype of HLA. The method according to claim 9 , wherein the MAGE-A10 corresponds to the A02:01 subtype of HLA. The method according to claim 9 , wherein the KRAS-G12V corresponds to the A11:01 subtype of HLA.

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

16. A culture kit for use in the method according to any one of claims 1 to 15, wherein the culture kit comprises a first culture medium, a second culture medium, a first half culture medium change, a second half culture medium change, and a third half culture medium change; The first half of the medium change consists of X-vivo15 + 10% hAB + 20 μM R848 + 200 ng / mL LPS + 20 ng / mL IL-1β + 2 μg / mL peptide, wherein the peptides are equal concentrations 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 medium change composition is X-vivo15+10% hAB+20 ng / mL IL-7+20 ng / mL IL-15+20 IUIL-2; The third half-change medium 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. 17 . The culture kit according to claim 16 , wherein the third half-change medium further comprises 5 μg / mL of a polypeptide, wherein the polypeptide comprises an antigenic peptide and a class II peptide PADRE at equal concentrations. The culture kit according to claim 16 , wherein the antigenic peptides include EBV, MAGE-A1, MAGE-A3, MAGE-A10, and KRAS-G12V.

19. Use of the method according to any one of claims 1 to 15 or the culture kit according to any one of claims 16 to 18 in preparing antigen-specific T cells, wherein the antigen-specific T cells are antigen-specific CD8+ T cells.

20. A device comprising a memory and a processor, wherein the processor is configured to call a program instruction and execute the method according to any one of claims 1 to 15 when the program instruction is called, and the memory is configured to store the program instruction.

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