Application of tyrosine kinase inhibitor

Abstract

The invention discloses application of a tyrosine kinase inhibitor. According to the method for increasing the proportion of the CD8 + Tetramer + specific T cells, the proportion of the CD8 + Tetramer + specific T cells can be stably increased by adding dasatinib into a specific T cell culture system, and the obtained antigen-specific T cells have a better tumor killing effect and are wide in application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of tyrosine kinase inhibitors. Background Art

[0002] T lymphocytes, or T cells for short, are derived from lymphoid stem cells. After differentiation and maturation in the thymus, they are distributed to immune organs and tissues throughout the body through lymphatic and blood circulation to exert immune functions. T cells have a variety of biological functions, such as directly killing target cells, assisting other lymphocytes in functioning, responding to specific antigens or mitogens, and producing cytokines. They are one of the main immune cells in the body to resist disease infection and prevent tumor formation. Enriching, activating, and amplifying these immune cells in vitro can be used for transfusion to treat a variety of diseases, including malignant tumors, infections, autoimmune diseases, etc.

[0003] Adoptive T-cell therapy (ACT) refers to a treatment method that uses the patient's own or donor's T lymphocytes, which are induced and screened in vitro or genetically modified to have killing activity, and then amplified to a certain number before being infused back into the patient's body to exert a killing effect. The field of adoptive immunotherapy ACT is developing rapidly, and how to optimize T cell proliferation, improve T cell killing efficiency, reduce T cell exhaustion, reduce clinical toxicity, and avoid tumor microenvironment immunosuppression are still issues that need to be urgently addressed in this field. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides the use of tyrosine kinase inhibitors (TKI).

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides a method for increasing the proportion of antigen-specific T cells, the method comprising co-culturing T cells with a tyrosine kinase inhibitor and an antigen peptide.

[0007] Furthermore, the antigen is selected from viral antigens.

[0008] Furthermore, the viral antigen is selected from CMV.

[0009] Furthermore, the tyrosine kinase inhibitor is selected from dasatinib.

[0010] Furthermore, the increase in the proportion of antigen-specific T cells is to increase CD8 + Tetramer + The proportion of specific T cells.

[0011] Furthermore, the method specifically comprises: (1) culturing T cells using a first culture medium; (2) Supplementing the second culture medium and adding antigen peptides and tyrosine kinase inhibitors; (3) Adjust the culture medium to the third culture medium and add antigen peptides and tyrosine kinase inhibitors.

[0012] Furthermore, the T cells in step (1) are derived from PBMCs.

[0013] Furthermore, the first culture medium includes serum-free culture medium, autologous inactivated plasma, interleukin, and GM-CSF.

[0014] Furthermore, the second culture medium includes interleukin and Poly I:C.

[0015] Furthermore, the third culture medium includes serum-free culture medium, autologous inactivated plasma, interleukin, and Poly I:C.

[0016] Furthermore, the serum-free culture medium is X-VIVO 15.

[0017] Furthermore, the interleukin contained in the first culture medium is selected from IL-4.

[0018] Furthermore, the interleukin of the second culture medium is selected from IL-7.

[0019] Furthermore, the interleukin in the third culture medium is selected from IL-7, IL-15, and IL-2.

[0020] Furthermore, the concentration of dasatinib is 5-500 nM.

[0021] Furthermore, the concentration of dasatinib is 5-30 nM.

[0022] Furthermore, the concentration of CMV is 1-10 μg / ml.

[0023] Furthermore, the concentration of CMV is 5 μg / ml.

[0024] Furthermore, the concentration of IL-4 was 10-20 ng / ml.

[0025] Furthermore, the concentration of IL-4 was 20 ng / ml.

[0026] Furthermore, the concentration of IL-7 was 5-10 ng / ml.

[0027] Furthermore, the concentration of IL-7 was 5 ng / ml.

[0028] Furthermore, the concentration of IL-15 was 5-10 ng / ml.

[0029] Furthermore, the concentration of IL-15 was 5 ng / ml.

[0030] Furthermore, the concentration of IL-2 is 100-1000 U / ml.

[0031] Furthermore, the concentration of IL-2 was 100 U / ml.

[0032] Furthermore, the autologous inactivated plasma is 1-10% autologous inactivated plasma.

[0033] Furthermore, the autologous inactivated plasma is 10% autologous inactivated plasma.

[0034] Furthermore, the concentration of GM-CSF is 50-200 ng / ml.

[0035] Furthermore, the concentration of GM-CSF was 100 ng / ml.

[0036] Furthermore, the concentration of Poly I:C is 10-20 μg / ml.

[0037] Furthermore, the concentration of Poly I:C was 20 μg / ml.

[0038] Furthermore, the dasatinib is added on day 2-7.

[0039] The second aspect of the present invention provides the use of a tyrosine kinase inhibitor in increasing the proportion of antigen-specific T cells.

[0040] Furthermore, the tyrosine kinase inhibitor is selected from dasatinib.

[0041] Furthermore, the antigen is selected from CMV.

[0042] Furthermore, the T cells are CD8 + Tetramer + Specific T cells.

[0043] The third aspect of the present invention provides antigen-specific T cells prepared by the method described in the first aspect of the present invention.

[0044] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the antigen-specific T cells described in the third aspect of the present invention.

[0045] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0046] The fifth aspect of the present invention provides the use of the antigen-specific T cells described in the third aspect of the present invention in the preparation of a pharmaceutical composition for preventing / treating CMV-related diseases.

[0047] Advantages and beneficial effects of the present invention: The present application provides a method for increasing CD8 + Tetramer + The method of specific T cell ratio can stably increase CD8 by adding dasatinib to the specific T cell culture system. + Tetramer + The ratio of specific T cells can be increased, and the obtained antigen-specific T cells have better tumor killing effect and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a flow cytometry result diagram of the ratio of specific T cells induced by different donors by loading antigen peptides and adding Das, wherein 1A is a diagram of the ratio of specific T cells induced by samples from donor one, and 1B is a diagram of the ratio of specific T cells induced by samples from donor two; Figure 2 2A is a flow cytometry result graph of specific T cells induced by a donor sample loaded with antigen peptide and added with 5nM, 15nM, and 30nM Das, wherein 2A is a flow cytometry result graph of specific T cell ratio induced by a donor sample, and 2B is a bar graph of specific T cell ratios at different concentrations; Figure 3 3A is a flow cytometry result graph of specific T cells induced by samples from donor 2 by loading antigen peptides and adding 5nM, 15nM, and 30nM Das, respectively, wherein 3A is a flow cytometry result graph of specific T cell ratio induced by samples from donor 2, and 3B is a bar graph of specific T cell ratios at different concentrations; Figure 4 4A is a flow cytometry result diagram of specific T cells induced by three donor samples by loading antigen peptides and adding 5nM, 15nM, and 30nM Das, respectively, wherein 4A is a flow cytometry result diagram of specific T cell ratio induced by three donor samples, and 4B is a bar graph of specific T cell ratios at different concentrations; Figure 5 5A is a flow cytometry result graph of specific T cells induced by a donor sample loaded with antigen peptide and adding 5 nM Das on Day 2 to Day 14 and Day 2 to Day 7, wherein 5A is a flow cytometry result graph of specific T cell ratio induced by a donor sample, and 5B is a bar graph of specific T cell ratios at different concentrations; Figure 66A is a flow cytometry result graph of specific T cells induced by samples from donor 2 loaded with antigen peptides and adding 5 nM Das on Day 2 to Day 14 and Day 2 to Day 7, wherein 6A is a flow cytometry result graph of specific T cell ratio induced by one sample from donor 1, and 6B is a bar graph of specific T cell ratios at different concentrations; Figure 7 Figure 7 is a flow cytometry result graph of specific T cells induced by three donor samples loaded with antigen peptides and adding 5nM Das on Day 2 to Day 14 and Day 2 to Day 7, respectively. Figure 7A is a flow cytometry result graph of the specific T cell ratio induced by three donor samples, and Figure 7B is a bar graph of the specific T cell ratio at different concentrations. DETAILED DESCRIPTION

[0049] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] The present invention provides a method for increasing the proportion of antigen-specific T cells, which comprises co-culturing a tyrosine kinase inhibitor and an antigen peptide with T cells.

[0051] In some embodiments, tyrosine kinase inhibitors (TKI) are a class of compounds that can inhibit the activity of tyrosine kinases, including but not limited to dasatinib, trastuzumab, rituximab, lapatinib, neratinib, pyrotinib, afatinib, gefitinib, erlotinib, imatinib, nilotinib, and osimertinib.

[0052] In a specific embodiment, the tyrosine kinase inhibitor is selected from dasatinib.

[0053] The method specifically comprises: (1) culturing T cells using a first culture medium; (2) Supplementing the second culture medium and adding antigen peptides and tyrosine kinase inhibitors; (3) Adjust the culture medium to the third culture medium and add antigen peptides and tyrosine kinase inhibitors.

[0054] The first culture medium includes serum-free culture medium, autologous inactivated plasma, interleukin, and GM-CSF.

[0055] The second culture medium includes interleukin and Poly I:C.

[0056] The third culture medium includes serum-free culture medium, autologous inactivated plasma, interleukin, and Poly I:C.

[0057] In some embodiments, serum-free culture medium includes but is not limited to X-VIVO 15, OpTmizer, OptiVitro.

[0058] In a specific embodiment, the serum-free medium is selected from X-VIVO 15.

[0059] In some embodiments, interleukins include but are not limited to one or more of IL-2 (interleukin 2), IL-4 (interleukin 4), IL-7 (interleukin 7), IL-10 (interleukin 10), IL-12 (interleukin 12), IL-15 (interleukin 15), IL-21 (interleukin 21), and IL-23 (interleukin 23), and the interleukins include wild types or mutants / modified forms thereof.

[0060] In a specific embodiment, the interleukin in the first culture medium is selected from IL-4; the interleukin in the second culture medium is selected from IL-7; and the interleukin in the third culture medium is selected from IL-7, IL-15, and IL-2.

[0061] In some embodiments, the antigens include tumor-associated antigens and viral antigens.

[0062] In some embodiments, tumor-associated antigens refer to antigenic substances that are produced / expressed on tumor cells and trigger an immune response in the host. Exemplary tumor antigens include at least the following antigens: carcinoembryonic antigen (CEA) for colorectal cancer; CA-125 for ovarian cancer; MUC-1 or epithelial tumor antigen (ETA) or CA15-3 for breast cancer; tyrosinase or melanoma-associated antigen (MAGE) for malignant melanoma; and abnormal products of ras, p53 for various types of tumors; alpha-fetoprotein for liver cancer, ovarian cancer or testicular cancer; beta subunit of hCG for testicular cancer patients; prostate-specific antigen for prostate cancer; beta2 microglobulin for multiple myeloma and in some cases lymphoma; CA19-9 for colorectal cancer, bile duct cancer and pancreatic cancer; chromogranin A for lung cancer and prostate cancer; TA90 for melanoma, soft tissue sarcoma, and breast cancer, colon cancer and lung cancer.

[0063] Specific examples of tumor antigens include at least, for example, CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-α, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, Melan A / MART1, Ras mutant, gp 100, p53 mutant, proteinase 3 (Pr1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2), and TNF-α. ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.

[0064] In some embodiments, viral antigens refer to antigens that are protein in nature and closely associated with viral particles. Specific examples of viral antigens include at least one virus selected from EBV, CMV (cytomegalovirus), adenovirus, BK, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus, LCMV, mumps virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HBV, HIV, HTLV1, HHV8 and West Nile virus, Zika virus, Ebola virus.

[0065] In a preferred embodiment, the antigen is selected from viral antigens.

[0066] In a specific embodiment, the viral antigen is selected from CMV.

[0067] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antigen-specific T cells.

[0068] In some embodiments, routes of administration of the pharmaceutical composition include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, topical, intradermal, transdermal, subcutaneous, parenteral, rectal, spinal, or epidermal administration.

[0069] In some embodiments, the pharmaceutical composition can be prepared as an injectable, either as a liquid solution or suspension, or as a solid form suitable for solution or suspension in liquid vehicles prior to injection.

[0070] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0071] In some embodiments, a pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable, e.g., a material that can be incorporated into a pharmaceutical composition administered to a subject without causing any significant undesirable biological effects, or interacting in a deleterious manner with any other components of the composition in which it is contained.

[0072] Suitable pharmaceutically acceptable carriers include physiological saline (e.g., normal saline), glucose, glycerol, platelet-rich plasma (PRP) or the like, and combinations thereof. In addition, the pharmaceutically acceptable carrier may contain a small amount of auxiliary substances, such as a wetting agent or emulsifier, a pH buffer or an adjuvant. A pharmaceutically acceptable carrier may contain a physiologically acceptable compound, the effect of which is, for example, to stabilize or increase or reduce the absorption or clearance rate of the pharmaceutical composition of the present application. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, surfactants, liposome carriers, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants or preservatives. See, for example, Remington's Pharmaceutical Science, 21st edition, Mack Publishing Company, Easton, Pa. ("Remington's"). The pharmaceutical composition of the present application may also include auxiliary substances, such as pharmaceutical agents, cytokines or other biological response modifiers.

[0073] In some embodiments, whether for preventive or therapeutic purposes, the pharmaceutical composition of the present application can be administered for a period of time in the form of single-dose treatment or multiple-dose treatment according to a schedule, according to the age, weight and condition of the subject, the specific composition used and the route of administration. For example, in one embodiment, the pharmaceutical composition of the present application is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week (qiw), five times a week, six times a week, every other day (qod), every day (qd), twice a day (bid), three times a day (tid) or four times a day (qid).

[0074] In some embodiments, the duration of treatment according to the pharmaceutical composition of the present application, such as the time period for administering the pharmaceutical composition, can vary according to a variety of factors (e.g., subject response, etc.). For example, the pharmaceutical composition can be administered over a period of time ranging from about one second or several seconds to one minute or several minutes, from one hour or several hours to one day to about one week, from about two weeks to about four weeks, from about 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or longer.

[0075] Formulating parenteral pharmaceutical compositions in dosage unit form is advantageous for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as single doses for subjects to be treated; each unit contains a predetermined amount of modified NK cells and antigen-specific T cells, which are combined with the required pharmaceutical carrier to produce the desired therapeutic effect.

[0076] In some embodiments, the pharmaceutical composition can be administered alone or in combination with another therapeutic agent, such as chemotherapy, radiation therapy, or targeted therapy or a cancer vaccine.

[0077] The present invention provides the use of the above antigen-specific T cells in preparing a pharmaceutical composition for preventing / treating CMV-related diseases.

[0078] In some embodiments, CMV-related diseases include, but are not limited to, cytomegalovirus pneumonia, cytomegalovirus gastroenteritis, cytomegalovirus esophagitis, cytomegalovirus hepatitis, cytomegalovirus retinochoroiditis, cytomegalovirus radiculitis, and cytomegalovirus encephalitis.

[0079] In some embodiments, treatment is a way to obtain beneficial or desired results (including clinical results). Beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the symptoms caused by another disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the occurrence or recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) relief of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0080] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0081] Example 1: Amplification of CMV-specific T cells Step 1: Resuscitation of PBMCs and culture and expansion of T cells HLA-A*02:01; A*11:01 cryopreserved PBMCs were purchased from Miaoshun Biotechnology to simulate the state of samples with / without pre-existing immunity. The resuscitated PBMCs were resuspended in X-VIVO 15 medium, and the cell count was completed and the cell viability was recorded. After that, they were centrifuged at 350g for 5 minutes, and the supernatant was fully discarded. According to the counting results, X-VIVO 15+10% autologous inactivated plasma+20ng / ml IL-4+100ng / ml GM-CSF was used at a rate of 2×10 6 / ml cells were inoculated in a six-well plate and placed in a 37°C carbon dioxide incubator for culture. After 2 days of cell culture, the culture medium was adjusted to supplement 5ng / ml IL-7+20μg / ml PolyI:C, 5μg / ml antigen peptide, and 5nM Das; after 4 days of cell culture, the culture medium was adjusted to X-VIVO 15+10% autologous inactivated plasma+5ng / ml IL-7+20μg / ml Poly I:C+5ng / ml IL-15+100U / ml IL-2, 5μg / ml antigen peptide was added, and 5nM Das was supplemented again. No peptide was added to the control group, and only antigen peptide was added to the control group (Peptide group) throughout the whole process.

[0082] Step 2: Flow cytometry After the two batches of CTL cells were cultured for 14 days, the specific T cells induced by each group of samples were detected by flow cytometry.

[0083] Figure 1The results show the ratio of CMV-specific T cells induced by different donors, and the ratio of specific T cells induced by antigen peptide loading and Das in the control group, control group and experimental group. The ratios of donor HLA-A*02:01 group were 0.34%, 18.15% and 41.55%, respectively, and those in the HLA-A*11:01 group were 0.00%, 0.06% and 1.15% respectively. Figure 1 A). The HLA-A*02:01 group of donor 2 was 0.15%, 9.9% and 19.35%, and the HLA-A*11:01 group was 0.00%, 0.15% and 0.73% ( Figure 1 B). The experimental results show that whether the sample has pre-existing immunity or not, after peptide stimulation, the use of Das can stably increase CD8 + Tetramer + The proportion of specific T cells.

[0084] Example 2: Different concentrations of Das were added to culture PBMC-derived specific T cells Step 1: Resuscitation of PBMCs and culture and expansion of T cells HLA-A*02:01; A*11:01 cryopreserved PBMCs were purchased from Miaoshun Biotechnology. The resuscitated PBMCs were resuspended in X-VIVO 15 medium, and the cell count was completed and the cell viability was recorded. Then, the cells were centrifuged at 350g for 5 minutes, and the supernatant was fully discarded. According to the counting results, X-VIVO 15+10% autologous inactivated plasma+20ng / ml IL-4+100ng / ml GM-CSF was used at a rate of 2×10 6 / ml cells were inoculated in six-well plates and placed in a 37°C carbon dioxide incubator for culture. After 2 days of cell culture, the culture medium was adjusted to supplement 5ng / ml IL-7+20μg / ml Poly I:C, 5μg / ml antigen peptide, and 5nM, 15nM, and 30nM Das respectively; after 4 days of cell culture, the culture medium was adjusted to X-VIVO 15+10% autologous inactivated plasma+5ng / ml IL-7+20μg / ml Poly I:C+5ng / ml IL-15+100U / ml IL-2, 5μg / ml antigen peptide was added, and 5nM, 15nM, and 30nM Das were supplemented again. The control group (Peptide group) only added antigen peptide throughout the whole process.

[0085] Step 2: Flow cytometry After the two batches of CTL cells were cultured for 14 days, the specific T cells induced by each group of samples were detected by flow cytometry.

[0086] Figure 2 , 3Figure 4 shows the ratio of CMV-specific T cells induced by different donors, and the ratio of specific T cells induced by loading different antigens and adding different concentrations of Das. Donor 1 was HLA-A*02:01, and the ratios of Das 5nM, 15nM, and 30nM were 41.55%, 28.29%, and 42.41%, respectively. Figure 2 ); Donor type II was HLA-A*02:01, and the addition of Das 5nM, 15 nM, and 30nM were 20.36%, 14.51%, and 24.3%, respectively ( Figure 3 ); The HLA-A*11:01 donor group had 5nM, 15nM, and 30nM Das added, which were 0.73%, 0.36%, and 0.71%, respectively ( Figure 4 ). The experimental results showed that the addition of 5nM Das was sufficient for CD8 + Tetramer + Specific T cell expansion requirements.

[0087] Example 3: PBMC-derived specific T cells were cultured with Das during D2-D7 or D2-D14 Step 1: Resuscitation of PBMCs and culture and expansion of T cells HLA-A*02:01; A*11:01 cryopreserved PBMCs were purchased from Miaoshun Biotechnology. The resuscitated PBMCs were resuspended in X-VIVO 15 medium, and the cell count was completed and the cell viability was recorded. Then, the cells were centrifuged at 350g for 5 minutes, and the supernatant was fully discarded. According to the counting results, X-VIVO 15+10% autologous inactivated plasma+20ng / ml IL-4+100ng / ml GM-CSF was used at a rate of 2×10 6 / ml cells were inoculated in six-well plates and placed in a 37°C carbon dioxide incubator for culture. After 2 days of cell culture, the culture medium was adjusted to supplement 5ng / ml IL-7+20μg / ml Poly I:C, 5μg / ml antigen peptide, and 5nM Das respectively; after 4 days of cell culture, the culture medium was adjusted to X-VIVO 15+10% autologous inactivated plasma+5ng / ml IL-7+20μg / ml Poly I:C+5ng / ml IL-15+100U / ml IL-2, 5μg / ml antigen peptide was added, and 5nMDas was supplemented again. The control group (Peptide group) only added antigen peptide. After 7 days of cell culture, one group was removed from 5nM Das, and the other groups continued to be cultured.

[0088] Step 2: Flow cytometry After the two batches of CTL cells were cultured for 14 days, the specific T cells induced by each group of samples were detected by flow cytometry.

[0089] Figure 5 , 6 Figure 7 shows the CMV-specific T cells induced by different donors, and the proportion of specific T cells induced by different antigen peptides in the control group, D2-D14 and D2-D7 added Das experimental group. The proportion of donor-specific T cells was 18.15%, 27.05% and 41.55% respectively ( Figure 5 ); the proportions of donor-specific T cells were 7.51%, 9.9% and 19.47% respectively ( Figure 6 ); the proportions of donor trispecific T cells were 0.15%, 0.22% and 0.73% respectively ( Figure 7 ). The experimental results showed that only when Das was added from D2 to D7, the proportion of antigen-specific T cells was higher.

[0090] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for increasing the proportion of antigen-specific T cells, characterized in that: The method comprises co-culturing a tyrosine kinase inhibitor and an antigenic peptide with T cells.

2. The method according to claim 1, characterized in that The antigen is selected from viral antigens; Preferably, the viral antigen is selected from CMV; Preferably, the tyrosine kinase inhibitor is selected from dasatinib; Preferably, the increase in the proportion of antigen-specific T cells is to increase CD8 + Tetramer + The proportion of specific T cells.

3. The method according to claim 2, characterized in that The method specifically comprises: (1) culturing T cells using a first culture medium; (2) Supplementing the second culture medium and adding antigen peptides and tyrosine kinase inhibitors; (3) Adjusting the culture medium to the third culture medium and adding antigen peptides and tyrosine kinase inhibitors; Preferably, the T cells in step (1) are derived from PBMCs.

4. The method according to claim 3, characterized in that The first culture medium comprises serum-free culture medium, autologous inactivated plasma, interleukin, and GM-CSF; Preferably, the second culture medium comprises interleukin and Poly I:C; Preferably, the third culture medium comprises serum-free culture medium, autologous inactivated plasma, interleukin, and Poly I:C; Preferably, the serum-free medium is X-VIVO 15; Preferably, the interleukin contained in the first culture medium is selected from IL-4; Preferably, the interleukin of the second culture medium is selected from IL-7; Preferably, the interleukin of the third culture medium is selected from IL-7, IL-15, and IL-2.

5. The method according to claim 3, characterized in that: The concentration of dasatinib is 5-30 nM; Preferably, the concentration of CMV is 5 μg / ml; Preferably, the dasatinib is added on day 2-7.

6. Application of tyrosine kinase inhibitors in increasing the proportion of antigen-specific T cells.

7. The use according to claim 6, characterized in that: The tyrosine kinase inhibitor is selected from dasatinib; Preferably, the antigen is selected from CMV; Preferably, the T cells are CD8 + Tetramer + Specific T cells.

8. Antigen-specific T cells prepared by the method according to any one of claims 1 to 5.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antigen-specific T cells according to claim 8: Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

10. Use of the antigen-specific T cells according to claim 8 in the preparation of a pharmaceutical composition for preventing / treating CMV-related diseases.

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