Antigen-specific T cell as well as preparation method and application thereof

Abstract

The invention relates to an antigen-specific T cell as well as a preparation method and application thereof. The method comprises the following steps: performing activation culture and multiplication culture on immune cells separated from peripheral blood, tumor tissues or peripheral immune organs to obtain the antigen-specific T cell, or performing multiplication culture on the immune cells obtained by a gene editing technology to obtain the antigen-specific T cells, wherein the step of activation culture and / or multiplication culture is to culture the separated cells in the presence of one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ultrasound, ray irradiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction. The antigen-specific T cell obtained by the method has an excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and in particular to an antigen-specific T cell and a preparation method and application thereof. Background Art

[0002] Immune cells refer to cells that participate in or are related to immune responses, including innate lymphocytes, various phagocytes, and lymphocytes that can recognize antigens and produce specific immune responses, such as T cells, B cells, NK cells, DC cells, macrophages, granulocytes, mast cells, etc. Autoimmune cell therapy is used to fight tumors by isolating and culturing immune cells from one's own blood. By supplementing highly active immune cells, the number of immune cells in the body is increased, and the original immune cells in the body are activated, thereby greatly improving the ability of immune cells to kill tumor cells, bacteria, viruses, etc., achieving the purpose of cancer prevention and anti-cancer.

[0003] In the prior applications CN202011027741.0 and CN202310600906.6 of the invention team of this application, a method for detecting tumor antigen-specific T cells and sorting and amplifying tumor antigen-specific T cells for treating cancer using T cells activated by nanoparticles loaded with cancer cell lysate components was disclosed. However, the detection method and the sorting and amplification method described in the application are both carried out under conventional culture conditions. It is generally believed that cells need to be cultured under conventional conditions of a temperature of 37.0°C, a CO2 volume content of 5.0%, and an oxygen volume content of 20.0%-21.0%. If cells are cultured under unconventional cell culture conditions, the cell growth state and the like will be significantly affected, resulting in slow cell growth and decreased activity. The present invention finds that by activating and / or amplifying antigen-specific T cells under certain pressure conditions, the resulting antigen-specific T cells have unexpectedly high pharmacological activity and efficacy. Summary of the invention

[0004] The object of the present invention is to provide an in vitro method for preparing antigen-specific T cells with excellent anti-tumor effect.

[0005] The present invention adopts the following technical solution:

[0006] In one aspect, the present invention provides a method for preparing antigen-specific T cells, the method comprising activating and amplifying immune cells separated from peripheral blood, tumor tissue or peripheral immune organs to obtain the antigen-specific T cells; or,

[0007] amplifying and culturing the immune cells obtained by gene editing technology to obtain the antigen-specific T cells;

[0008] Among them, the activation culture and / or expansion culture step is to culture the separated cells or gene-edited cells under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

[0009] Preferably, the high temperature is controlled to be 37.5°C to 42.0°C.

[0010] Preferably, the low temperature is controlled to be a temperature of 4.0°C to 32.0°C.

[0011] Preferably, the hypoxia is controlled to have an oxygen volume content of 3.0% to 16.0%.

[0012] Preferably, the low pH is controlled to be in the range of 4.0 to 6.8.

[0013] Preferably, the high pH is controlled to be pH 7.6-8.0.

[0014] Preferably, the high CO2 is controlled to have a CO2 volume content of 5.1% to 15.0%.

[0015] Preferably, the condition for controlling the ultrasound is that the ultrasound frequency is greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 ;

[0016] Preferably, the electromagnetic field dose is controlled to be 20 μSv-1000 mSv;

[0017] Preferably, the electron beam dose is controlled to be 0.2-20 Gy.

[0018] Preferably, the irradiation includes irradiation with ultraviolet rays, infrared rays, X-rays, α rays, β rays or γ rays.

[0019] Preferably, the intensity of the ultraviolet light is controlled to be 5 to 200 μW / cm 2 , wavelength is 100~400nm.

[0020] More preferably, the X-ray dose is 0.2 to 20 Gy.

[0021] Preferably, the terahertz wavelength of the terahertz electromagnetic wave is controlled to be 30 to 3000 μm.

[0022] Preferably, the wavelength of the microwave is controlled to be 1 mm to 1 m.

[0023] Preferably, the oxidation is an activation culture and / or an expansion culture in the presence of an oxidant.

[0024] Preferably, the reduction is activation culture and / or expansion culture in the presence of a reducing agent.

[0025] Further preferably, the oxidant is selected from one or more of peroxide, hypochlorous acid, sulfate, dichromate, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, KIO3, KBrO3, ClO3-, ClO4-, Na2O2, K2O2, MgO2, CaO2, BaO2, NO3-, MnO4-, F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0026] More preferably, the oxidant is peroxide and / or hypochlorous acid.

[0027] Still further preferably, the peroxide is selected from hydrogen peroxide, peracetic acid and sodium peroxide.

[0028] Preferably, the concentration of the oxidant is controlled to be 0.0001-1000 μM.

[0029] Preferably, the reducing agent is selected from at least one of a sulfur-containing reducing agent, a ferrous salt, and a copper-containing reducing agent.

[0030] Preferably, the sulfur-containing reducing agent is selected from one or more of glutathione, thiourea dioxide, sodium sulfide (Na2S), thiourea, thioacetic acid, TCEP, DTT, and mercaptoethanol.

[0031] Preferably, the reducing agent is selected from glutathione.

[0032] Preferably, the concentration of the reducing agent is controlled to be 0.0001-1000 μM.

[0033] More preferably, the concentration of the reducing agent is controlled to be 20-100 μM.

[0034] Preferably, the antigen-specific T cells are one or more of tumor antigen-specific T cells, bacterial antigen-specific T cells, viral antigen-specific T cells or autoantigen-specific T cells;

[0035] Further preferably, the tumor antigen-specific T cells include monoclonal tumor antigen-specific T cells and polyclonal tumor antigen-specific T cells.

[0036] Preferably, the gene-edited cells include one or more of CAR-T, TCR-T, and TILs.

[0037] Preferably, the activation culture time is 0.05 hours to 168 hours, preferably 4 hours to 96 hours.

[0038] Preferably, the amplification culture time is 0.05 hours to 42 days, preferably 1 hour to 30 days.

[0039] Preferably, the antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises:

[0040] (1) activating and culturing the isolated immune cells, and then sorting the activated and cultured cells to obtain positive cells;

[0041] (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells;

[0042] Wherein, the activation culture is carried out under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

[0043] Preferably, the step of activating the culture is selected from any one or more of the following:

[0044] (1-1) co-culturing the separated immune cells and the particles loaded with the antigen components at 37.5° C. to 42.0° C. and pH 4.0 to 6.8;

[0045] (1-2) co-culturing the isolated immune cells and the particles loaded with the antigen components under the conditions of pH 4.0 to 6.8 and an oxidant concentration of 0.0001 to 1000 μM;

[0046] (1-3) The separated immune cells and the particles loaded with the antigen components are co-cultured at 37.5°C to 42.0°C and in an oxidant concentration of 0.0001 to 1000 μM.

[0047] Preferably, the antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises:

[0048] (1) activating and culturing the isolated immune cells, and sorting the activated and cultured cells to obtain positive cells;

[0049] (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells;

[0050] The amplification culture is carried out under one or more of the following conditions: high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

[0051] The step of activating the culture is selected from any one or more of the following:

[0052] (1-4) co-culturing the isolated immune cells, particles loaded with antigen components, and antigen presenting cells;

[0053] (1-5) co-culturing the isolated immune cells and particles loaded with antigen components;

[0054] (1-6) The particles loaded with antigen components are first contacted with antigen presenting cells, and the antigen presenting cells after contact are then co-cultured with immune cells.

[0055] Preferably, the step of amplification and culturing is specifically selected from one or more of the following:

[0056] (2-1) culturing the positive cells at 37.5°C to 42.0°C or 4.0°C to 32.0°C for 1 hour to 42 days, preferably 1 hour to 21 days;

[0057] (2-2) culturing the positive cells in the presence of an oxidant or a reducing agent for 1 hour to 42 days, preferably 1 day to 21 days;

[0058] (2-3) culturing the positive cells at pH 4.0 to 6.8 or pH 7.6 to 8.0 for 1 hour to 42 days, preferably 1 day to 21 days;

[0059] (2-4) culturing the positive cells under ultraviolet light, infrared light, X-ray, α-ray, β-ray or γ-ray irradiation for 0.05 hours to 42 days; preferably 1 hour to 21 days; preferably, controlling the intensity of the ultraviolet light to be 5 to 200 μW / cm 2 , with a wavelength of 100 to 400 nm; preferably, the X-ray dose is 0.2 to 20 Gy, and the irradiation time is 0.05 hours to 24 hours;

[0060] (2-5) culturing the positive cells under conditions of an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0061] (2-6) culturing the positive cells under conditions of a CO2 volume content of 5.1% to 15.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0062] (2-7) Make positive cells at an ultrasonic frequency greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 Under the conditions, culture for 1 hour to 42 days, preferably 1 day to 21 days;

[0063] (2-8) culturing the positive cells under the conditions of a terahertz intensity of 0.1 to 10 THz and a wavelength of 30 to 3000 μm for 1 hour to 42 days, preferably 1 day to 21 days;

[0064] (2-9) culturing the positive cells under microwave conditions with a wavelength of 1 mm to 1 m or a frequency of 300 MHz to 300 GHz for 5 min to 600 min;

[0065] (2-10) culturing the positive cells under conditions of pH 4.0 to 6.8 and oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0066] (2-11) culturing the positive cells at 37.5°C to 42.0°C, pH 4.0 to 6.8, and in the presence of an oxidant at a concentration of 0.0001 to 1000 μM for 1 hour to 42 days, preferably 1 day to 21 days;

[0067] (2-12) The positive cells are cultured at 37.5°C to 42.0°C and an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days.

[0068] Preferably, the expansion culture is carried out in the presence of interleukin, antibody and / or granulocyte-macrophage colony stimulating factor;

[0069] Preferably, the interleukin is selected from one or more of IL-2, IL-7, IL-5, IL-12, IL-15, IL-17, and IL-21, preferably one or more of IL-2, IL-15, and IL-7;

[0070] Preferably, the antibody is selected from anti-CD3 antibody and / or anti-CD28 antibody.

[0071] Preferably, the antigen component is selected from one or more of the following:

[0072] (a) Whole cell lysate fraction of cancer cells / tumor tissues; (b) A portion of the whole cell fraction consisting of the protein and peptide components of the whole cell lysate fraction of cancer cells / tumor tissues; (c) The protein and peptide components of the whole cell fraction of cancer cells / tumor tissues plus the mRNA component; (d) Tumor specific antigen (TSA); (e) Tumor associated antigen (TAA).

[0073] Preferably, the concentration of the particles loaded with antigen components is 2.5 ng / mL to 50 mg / mL.

[0074] Preferably, the particles are nanoparticles and / or microparticles.

[0075] Preferably, the concentration of the antigen presenting cells is 1 to 50 million / mL.

[0076] Preferably, the antigen presenting cells are selected from one or more of B cells, DC cells and macrophages.

[0077] In another aspect, the present invention provides antigen-specific T cells obtained according to any one of the preparation methods described above.

[0078] Preferably, the antigen-specific T cells are selected from CD3 + CD69 + 、CD3 + CD8 + CD69 + 、CD3 + CD4 + CD69+、CD3 + CD137 + 、CD3 + CD4 + CD137 + 、CD3 + CD8 + CD137 + 、CD3 + CD25 + 、CD3 + CD8 + CD25 + 、CD3 + CD4 + CD25 + 、CD3 + CD134 + 、CD3 + CD8 + CD134 + 、CD3 + CD4 + CD134 + 、CD3 + IL-2R + 、CD3 + CD8 + IL-2R + 、CD3 + CD4 + IL-2R + 、CD3 + HLA-DR + 、CD3 + CD8 + HLA-DR + 、CD3 + CD4 + HLA-DR +、CD3 + FASL + 、CD3 + CD8 + FASL + 、CD3 + CD4 + FASL + 、CD3 + OX40 + 、CD3 + CD8 + OX40 + 、CD3 + CD4 + OX40 + 、CD3 + TCF-1 + 、CD3 + CD8 + TCF-1 + 、CD3 + CD4 + TCF-1 + 、CD3 + PD-1 + 、CD3 + CD8 + PD-1 + 、CD3 + CD4 + PD-1 + 、CD3 + CD39 + 、CD3 + CD8 + CD39 + 、CD3 + CD4 + CD39 + 、CD3 + CD38 + 、CD3 + CD8 + CD38 + 、CD3 + CD4 + CD38 + 、CD3 + CD28 + 、CD3 + CD8 + CD28 + 、CD3 + CD4 + CD28 + 、CD3 + CD71 + 、CD3 + CD8+ CD71 + 、CD3 + CD4 + CD71 + 、CD3 + CD44 + 、CD3 + CD8 + CD44 + 、CD3 + CD4 + CD44 + 、CD3 + CXCR3 + 、CD3 + CD8 + CXCR3 + 、CD3 + CD4 + CXCR3 + 、CD3 + CXCR1 + 、CD3 + CD8 + CXCR1 + 、CD3 + CD4 + CXCR1 + 、CD3 + ICAM-1 + 、CD3 + CD8 + ICAM-1 + 、CD3 + CD4 + ICAM-1 + 、CD3 + CD70 + 、CD3 + CD8 + CD70 + 、CD3 + CD4 + CD70 + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD62L + 、CD3 + CD8 + CD62L + 、CD3 +CD4 + CD62L + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + ICOS + 、CD3 + CD8 + ICOS + 、CD3 + CD4 + ICOS + 、CD3 + CD27 + 、CD3 + CD8 + CD27 + 、CD3 + CD4 + CD27 + 、CD3 + CD107A + 、CD3 + CD8 + CD107A + and CD3 + CD4 + CD107A + Any one or more of the above T cells.

[0079] In another aspect, the present invention provides antigen-specific T cells obtained according to any of the preparation methods described above, or use of the antigen-specific T cells described above in the preparation of drugs for treating or preventing diseases, wherein the diseases include cancer, autoimmune diseases, diseases caused by bacterial infections, and diseases caused by viral infections.

[0080] Preferably, the tumor is selected from solid tumors and / or hematological tumors;

[0081] Preferably, the tumor includes lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood tumor (such as leukemia), brain tumor, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system One or more of carcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, metastatic cancer, circulating tumors.

[0082] Effects of the Invention

[0083] Compared with antigen-specific T cells obtained under conventional cell expansion or activation culture conditions (37.0°C, containing 5.0% CO2 by volume), the present invention finds that antigen-specific T cells prepared by culturing under certain pressure conditions during cell activation, sorting and / or expansion have unexpectedly better preventive or therapeutic effects, especially in the prevention or treatment of cancer. The antigen-specific T cells obtained by the present invention can better prevent tumor recurrence, inhibit tumor growth rate, prevent cancer metastasis, and thus prolong the survival period of living organisms; at the same time, the method of the present invention has the advantages of simple operation, controllable quality, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is a schematic diagram of the effect of using antigen-specific T cells obtained by amplification and culture under high temperature and pressure conditions in Example 1 of the present invention for the prevention or treatment of melanoma, wherein a is a diagram showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a diagram showing the experimental results of mouse survival period;

[0085] Figure 2 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture in the presence of an oxidant (hydrogen peroxide) in Example 2 of the present invention for the prevention or treatment of lung cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0086] Figure 3This is a schematic diagram of the monitoring results of the number of cancer metastases when the antigen-specific T cells obtained by expansion and culture under low oxygen pressure conditions in Example 3 of the present invention are used to prevent cancer metastasis;

[0087] Figure 4 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under X-ray irradiation pressure conditions in Example 4 of the present invention for the prevention or treatment of lung cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0088] Figure 5 This is a schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under ultraviolet irradiation pressure conditions in Example 5 of the present invention for the prevention or treatment of colorectal cancer, wherein a is a graph showing the experimental results of tumor growth rate during the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0089] Figure 6 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under acidic conditions in Example 6 of the present invention for the prevention or treatment of breast cancer, wherein a is a diagram showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a diagram showing the experimental results of mouse survival period;

[0090] Figure 7 A schematic diagram of the monitoring results of the number of cancer metastases when antigen-specific T cells obtained by expansion and culture under reducing agent (glutathione) conditions are used to prevent cancer metastasis in Example 7 of the present invention;

[0091] Figure 8 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under high CO2 concentration and pressure conditions in Example 8 of the present invention for the prevention or treatment of pancreatic cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0092] Fig. 9 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under ultrasonic pressure conditions in Example 9 of the present invention for the prevention or treatment of esophageal cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0093] Fig.10 Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under hypoxic and acidic conditions in Example 10 of the present invention for the prevention or treatment of liver cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0094] Fig.11Schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under terahertz electromagnetic wave pressure conditions for the prevention or treatment of melanoma in Example 11 of the present invention, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0095] Fig.12 This is a schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under microwave pressure conditions in Example 12 of the present invention for the prevention or treatment of breast cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0096] Fig.13 This is a schematic diagram of the use of antigen-specific T cells obtained by amplification and culture under high temperature, acidic and oxidant conditions in Example 13 of the present invention for the prevention or treatment of lung cancer, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0097] Fig.14 Schematic diagram of the use of antigen-specific T cells obtained by amplifying and culturing CAT cells under high temperature and low oxygen pressure conditions for the prevention or treatment of gastric cancer in Example 14 of the present invention, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0098] Fig.15 Schematic diagram of the use of antigen-specific T cells obtained by activation and culture under high temperature and acidic conditions and conventional amplification and culture conditions for the prevention or treatment of lung cancer in Example 15 of the present invention, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0099] Fig.16 Schematic diagram of the use of antigen-specific T cells obtained by activation and culture under acidic and oxidant conditions and conventional amplification culture conditions for the prevention or treatment of pancreatic cancer in Example 16 of the present invention, wherein a is a graph showing the experimental results of tumor growth rate in the prevention or treatment of cancer, and b is a graph showing the experimental results of mouse survival period;

[0100] Among them, in each figure, the significant difference of the tumor growth inhibition experiment in a was analyzed by ANOVA method, and the significant difference in b was analyzed by Kaplan-Meier and log-rank test;

[0101] Unless otherwise specified, in the above figures, *** indicates that compared with the PBS blank control group, p < 0.005, there is a significant difference; ** indicates that compared with the PBS blank control group, p < 0.01, there is a significant difference; * indicates that compared with the PBS blank control group, p < 0.05; # indicates that p < 0.05, there is a significant difference; ## indicates that p < 0.01, there is a significant difference; ### indicates that p < 0.005, there is a significant difference. DETAILED DESCRIPTION

[0102] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0103] Since cancer patients, especially cancer patients with poor physical function, have a small number of tumor antigen-specific T cells in their bodies and low activity, their tumor antigen-specific T cells have problems such as difficulty in activation, slow amplification, long culture cycle, and low cell viability that affect anti-tumor immunotherapy. It is generally believed that cells, especially immune cells, are better cultured under conventional conditions such as 37°C and 5.0% CO2 by volume, and the amplification speed is faster and the cell viability is better. However, after extensive and in-depth research, the present invention further stimulates, sorts, and amplifies tumor antigen-specific T cells with the function of recognizing cancer cells and killing cancer cells in vitro tumor antigens, especially giving certain pressure conditions (such as high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction) during the stimulation and / or large-scale amplification process, and obtains antigen-specific T cells with excellent anti-tumor effects, and then the antigen-specific T cells are returned to the patient's body, realizing its effective method for preventing cancer occurrence, preventing cancer metastasis and treating cancer.

[0104] The technical contents such as nanoparticle activation and sorting of tumor-specific T cells disclosed in Chinese patent applications (202011027741.0; 202310600906.6) are introduced into the present invention as an indispensable component of the present invention.

[0105] In one aspect, the present invention provides a method for preparing antigen-specific T cells, the method comprising activating and amplifying immune cells separated from peripheral blood, tumor tissue or peripheral immune organs to obtain the antigen-specific T cells; or,

[0106] amplifying and culturing the immune cells obtained by gene editing technology to obtain the antigen-specific T cells;

[0107] Among them, the activation culture and / or expansion culture step is to culture the separated cells or gene-edited cells under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

[0108] In the present application, the conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction are all within the range that cells can tolerate.

[0109] In certain embodiments, the elevated temperature is controlled to be a temperature between 37.5°C and 42.0°C (eg, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C).

[0110] In certain embodiments, the low temperature is controlled to a temperature of 4.0°C to 32.0°C (e.g., 5°C, 6°C, 7°C, 7°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C).

[0111] In certain embodiments, the hypoxia is controlled to have an oxygen volume content of 3.0% to 16.0% (e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, etc.).

[0112] In certain embodiments, the low pH is controlled to be a pH of 4.0 to 6.8 (eg, 4, 4.5, 5, 5.5, 6, 6.5, etc.).

[0113] In the exemplary embodiment of the present invention, lactic acid is used as the acidic substance for preparing acidic conditions. In practical applications, any other substance that can prepare acidic conditions can also be used, preferably an acidic substance present in the human body. The acidic substance that can be used includes, but is not limited to, at least one of lactic acid, carbonic acid, phosphoric acid, pyruvic acid, citric acid, eicosanoids, sialic acid, fatty acids, and succinic acid, preferably at least one of lactic acid or carbonic acid.

[0114] In certain embodiments, the high pH is controlled to be pH 7.6 to 8.0 (eg, 7.7, 7.8, 7.9, 8, etc.).

[0115] In some embodiments, the high CO2 is controlled to have a CO2 volume content of 5.1% to 15.0% (e.g., 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, etc.).

[0116] In some embodiments, the ultrasonic frequency is greater than 2×10 4 Hz (e.g. 2×10 5 Hz), the ultrasonic intensity is greater than 0.003W / cm 2 .

[0117] In some embodiments, the ultrasonic condition is controlled to be an ultrasonic frequency of 2×10 5 Hz (e.g. 2×10 5 Hz), the ultrasonic intensity is 0.3W / cm 2 .

[0118] In some embodiments, the electromagnetic field dose is controlled to be 20 μSv-1000 mSv (e.g., 50 μSv, 100 μSv, 150 μSv, 200 μSv, 250 μSv, 300 μSv, 400 μSv, 500 μSv, 600 μSv, 700 μSv, 800 μSv, 900 μSv, 1000 μSv, 2 mSv, 3 mSv, 4 mSv, 5 mSv, 6mSv, 7mSv, 8mSv, 9mSv, 10mSv, 50mSv, 100mSv, 150mSv, 200mSv, 250mSv, 300mSv, 350mSv , 400mSv, 450mSv, 500mSv, 550mSv, 600mSv, 650mSv, 700mSv, 750mSv, 800mSv, 900mSv, etc.).

[0119] In certain embodiments, the electron beam dose is controlled to be 0.2-20 Gy (e.g., 0.2 Gy, 0.3 Gy, 0.5 Gy, 0.8 Gy, 1 Gy, 1.5 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 15 Gy, 20 Gy, etc.).

[0120] In certain embodiments, the radiation irradiation comprises irradiation with ultraviolet rays, infrared rays, X-rays, alpha rays, beta rays, or gamma rays.

[0121] In some embodiments, the intensity of the ultraviolet light is controlled to be 5 to 200 μW / cm 2 (e.g. 5μW / cm 2 、10μW / cm2 、15μW / cm 2 , 20μW / cm 2 , 25μW / cm 2 、30μW / cm 2 、35μW / cm 2 , 40μW / cm 2 , 45μW / cm 2 、60μW / cm 2 、65μW / cm 2 、70μW / cm 2 , 75μW / cm 2 、80μW / cm 2 、85μW / cm 2 , 90μW / cm 2 , 100μW / cm 2 , 120μW / cm 2 , 140μW / cm 2 、150μW / cm 2 、180μW / cm 2 , 200μW / cm 2 ), the wavelength is 100~400nm (for example, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, etc.).

[0122] In some embodiments, the intensity of the ultraviolet light is controlled to be 50 μW / cm 2 , wavelength is 300nm.

[0123] In certain embodiments, the X-ray dose is 0.2 to 20 Gy (e.g., 0.2 Gy, 0.3 Gy, 0.5 Gy, 0.8 Gy, 1 Gy, 1.5 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 15 Gy, 20 Gy, etc.), and the irradiation time is 0.05 to 24 h (e.g., 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 10 h, 15 h, 20 h).

[0124] In certain embodiments, the X-ray dose is 6 Gy and the irradiation time is 1.5 h.

[0125] In some embodiments, the wavelength of the terahertz electromagnetic wave is controlled to be 30-3000 μm (for example, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 200 μm, 3000 μm, etc.).

[0126] In certain embodiments, the terahertz wavelength of the terahertz electromagnetic wave is controlled to be 300 μm.

[0127] In certain embodiments, the wavelength of the microwave is controlled to be 1 mm to 1 m, for example, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800 mm, 900 mm, 1000 mm, etc.

[0128] In certain embodiments, the wavelength of the microwave is controlled to be 1 mm to 20 mm.

[0129] In certain embodiments, the wavelength of the microwaves is controlled to be 10 mm.

[0130] In certain embodiments, the oxidation is an activation culture and / or an expansion culture in the presence of an oxidant.

[0131] In certain embodiments, the oxidant is selected from one or more of peroxide, hypochlorous acid, sulfate, dichromate, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, KIO3, KBrO3, ClO3-, ClO4-, Na2O2, K2O2, MgO2, CaO2, BaO2, NO3-, MnO4-, F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0132] In certain embodiments, the oxidizing agent is peroxide and / or hypochlorous acid.

[0133] In certain embodiments, the peroxide is selected from hydrogen peroxide (hydrogen peroxide, H2O2), peracetic acid, and sodium peroxide.

[0134] In certain embodiments, the concentration of the oxidant is controlled to be 0.0001-1000 μM (e.g., 0.0001, 0.0002, 0.0003 μM, 0.0004 μM, 0.0005 μM, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 μM, 0.2 μM, 0.3 μM). M, 0.4μM, 0.5μM, 0.7μM, 0.8μM, 0.9, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μ M, 50μM, 100μM, 200μM, 300μM, 400μM, 500μM, 600μM, 700μM, 800μM, 900μM, 1000μM).

[0135] In certain embodiments, the oxidant is hydrogen peroxide, and the concentration of the hydrogen peroxide is 0.0001-500 μM.

[0136] In a preferred embodiment, the concentration of hydrogen peroxide is 0.0001-10 μM.

[0137] In certain embodiments, the oxidant is hypochlorous acid, and the concentration of the hypochlorous acid is 0.0001-500 μM.

[0138] In a preferred embodiment, the concentration of the perchlorous acid is 0.0001-10 μM.

[0139] In a specific and preferred embodiment, the concentration of the perchlorous acid is 0.0001 to 0.1 μM.

[0140] In certain embodiments, the reduction is an activation culture and / or an expansion culture performed in the presence of an oxidant.

[0141] In certain embodiments, the reducing agent is selected from at least one of a sulfur-containing reducing agent, a ferrous salt, and a copper-containing reducing agent.

[0142] In certain embodiments, the sulfur-containing reducing agent includes, but is not limited to, one or more of glutathione, ferrous salts, thiourea dioxide, sodium sulfide (Na2S), thiourea, sulfide reducing agents, thioacetic acid, TCEP, DTT, and mercaptoethanol.

[0143] In certain embodiments, the reducing agent is selected from glutathione.

[0144] In certain embodiments, the concentration of the reducing agent is controlled to be 0.0001-1000 μM (e.g., 0.0001 μM, 0.0002 μM, 0.0003 μM, 0.0004 μM, 0.0005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM). ,0.5μM, 0.7μM, 0.8μM, 0.9, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μMμ M, 60μM, 70μM, 80μM, 90μM, 100μM, 200μM, 300μM, 400μM, 500μM, 600μM, 700μM, 800μM, 900μM, 1000μM).

[0145] In certain embodiments, the concentration of the reducing agent is controlled to be 20-100 μM.

[0146] In a specific and preferred embodiment, the concentration of the reducing agent is controlled to be 20-60 μM.

[0147] In certain embodiments, the antigen-specific T cells are one or more of tumor antigen-specific T cells, bacterial antigen-specific T cells, viral antigen-specific T cells, or self-antigen-specific T cells.

[0148] In certain embodiments, the tumor antigen-specific T cells include monoclonal tumor antigen-specific T cells and polyclonal tumor antigen-specific T cells.

[0149] In the present application, there is no limitation on the cells obtained by gene programming. In certain embodiments, the immune cells obtained by gene editing technology include one or more of CAR-T, TCR-T, and TILs. In a specific and preferred embodiment, CAR-T cells include but are not limited to CAR-T cells that recognize Claudin18.2.

[0150] In certain embodiments, the activation culture time is 0.05 hours to 168 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 25 hours, 30 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, 85 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 140 hours, 150 hours, etc.

[0151] In certain embodiments, the activation culture time is 4 hours to 96 hours.

[0152] In certain embodiments, the expansion culture time is 0.05 hours to 42 days, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 day, 42 days, etc.

[0153] In certain embodiments, the expansion culture time is 1 hour to 30 days.

[0154] In certain embodiments, the antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises:

[0155] (1) activating and culturing the isolated immune cells, and then sorting the activated and cultured cells to obtain positive cells;

[0156] (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells;

[0157] Wherein, the activation culture is carried out under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

[0158] In certain embodiments, the step of activating the culture is selected from any one or more of the following:

[0159] (1-1) co-culturing the separated immune cells and the particles loaded with the antigen components at 37.5° C. to 42.0° C. and pH 4.0 to 6.8;

[0160] (1-2) co-culturing the isolated immune cells and the particles loaded with the antigen components under the conditions of pH 4.0 to 6.8 and an oxidant concentration of 0.0001 to 1000 μM;

[0161] (1-3) The separated immune cells and the particles loaded with the antigen components are co-cultured at 37.5°C to 42.0°C and in an oxidant concentration of 0.0001 to 1000 μM.

[0162] In certain embodiments, the antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises:

[0163] (1) activating and culturing the isolated immune cells, and sorting the activated and cultured cells to obtain positive cells;

[0164] (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells;

[0165] Wherein, the amplification culture is carried out under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction;

[0166] The step of activating the culture is selected from any one or more of the following:

[0167] (1-4) co-culturing the isolated immune cells, particles loaded with antigen components, and antigen presenting cells;

[0168] (1-5) co-culturing the isolated immune cells and particles loaded with antigen components;

[0169] (1-6) The particles loaded with antigen components are first contacted with antigen presenting cells, and the antigen presenting cells after contact are then co-cultured with immune cells.

[0170] In the present invention, nanoparticles and / or microparticles loaded with tumor antigen components can be co-incubated with antigen presenting cells and T cells (immune cells) under conventional conditions or under the pressure conditions to activate cancer cell-specific T cells; or,

[0171] Nanoparticles and / or microparticles loaded with tumor antigen components are first co-incubated with antigen presenting cells to activate the antigen presenting cells, and then the activated antigen presenting cells are co-incubated with T cells alone under conventional conditions or under the pressure conditions to activate cancer cell-specific T cells; or,

[0172] Nanoparticles and / or microparticles loaded with tumor antigen components are first co-incubated with antigen presenting cells under conventional conditions or under the pressure conditions to activate the antigen presenting cells, and then the antigen presenting cells can be co-incubated with T cells under conventional conditions or under the pressure conditions to activate specific T cells without special treatment;

[0173] In the present invention, antigen presenting cells can be fixed, irradiated, illuminated, modified, inactivated, mineralized, etc., and then co-incubated with T cells under conventional conditions or the stress conditions to activate specific T cells.

[0174] In the present invention, before the immune cells (T cells) are co-incubated with the nanoparticles / microparticles and antigen-presenting cells, the immune cells (T cells) can be cultured alone for a period of time or appropriately sorted; or before the T cells are co-incubated with the activated antigen-presenting cells, the T cells can be cultured alone for a period of time or appropriately sorted.

[0175] When step (1-4) or (1-6) is used for activation culture, the ratio of the number of antigen presenting cells to T cells used is greater than 1:1; nanoparticles or microparticles are used to activate tumor antigen-specific T cells in peripheral immune cells in vitro under conventional conditions or under the certain pressure conditions after presentation by antigen presenting cells.

[0176] In certain embodiments, the step of amplification and culturing is specifically selected from one or more of the following:

[0177] (2-1) culturing the positive cells at 37.5°C to 42.0°C or 4.0°C to 32.0°C for 1 hour to 42 days, preferably 1 hour to 21 days;

[0178] (2-2) culturing the positive cells in the presence of an oxidant or a reducing agent for 1 hour to 42 days, preferably 1 day to 21 days;

[0179] (2-3) culturing the positive cells at pH 4.0 to 6.8 or pH 7.6 to 8.0 for 1 hour to 42 days, preferably 1 day to 21 days;

[0180] (2-4) culturing the positive cells under ultraviolet light, infrared light, X-ray, α-ray, β-ray or γ-ray irradiation for 0.05 hours to 42 days; preferably 1 hour to 21 days; preferably, controlling the intensity of the ultraviolet light to be 5 to 200 μW / cm 2 , with a wavelength of 100 to 400 nm; preferably, the X-ray dose is 0.2 to 20 Gy, and the irradiation time is 0.05 hours to 24 hours;

[0181] (2-5) culturing the positive cells under conditions of an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0182] (2-6) culturing the positive cells under conditions of a CO2 volume content of 5.1% to 15.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0183] (2-7) Make positive cells at an ultrasonic frequency greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 Under the conditions, culture for 1 hour to 42 days, preferably 1 day to 21 days;

[0184] (2-8) culturing the positive cells under the condition of a terahertz wavelength of 30 to 3000 μm for 1 hour to 42 days, preferably 1 day to 21 days;

[0185] (2-9) culturing the positive cells under microwave conditions with a wavelength of 1 mm to 1 m or a frequency of 300 MHz to 300 GHz for 5 min to 600 min;

[0186] (2-10) culturing the positive cells under conditions of pH 4.0 to 6.8 and oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days;

[0187] (2-11) culturing the positive cells at 37.5°C to 42.0°C, pH 4.0 to 6.8, and in the presence of an oxidant at a concentration of 0.0001 to 1000 μM for 1 hour to 42 days, preferably 1 day to 21 days;

[0188] (2-12) The positive cells are cultured at 37.5°C to 42.0°C and an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days.

[0189] In certain embodiments, the expansion culture is: culturing the positive cells at 37.5°C to 42.0°C (e.g., 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C) for 1 hour to 21 days.

[0190] In certain embodiments, the expansion culture is: culturing the positive cells in the presence of an oxidant (such as hydrogen peroxide or hypochlorous acid) at a concentration of 0.0001 to 1000 μM for 1 hour to 2 days, and then continuing to culture in a normal culture medium (such as RPMI1640 culture medium) for 15 to 25 days.

[0191] In certain embodiments, the expansion culture is: culturing the positive cells in the presence of a reducing agent (eg, glutathione) at a concentration of 20 to 100 μM for 10 to 21 days.

[0192] In certain embodiments, the expansion culture is: culturing the positive cells at a pH of 4.0 to 6.8 (eg, 4, 4.5, 5, 5.5, 6, 6.5, etc.) for 5 to 15 days.

[0193] In some embodiments, the expansion culture is: culturing the positive cells under ultraviolet irradiation for 5 to 15 days, and sorting to obtain the antigen-specific T cells, the intensity of which is 5 to 100 μW / cm 2 , wavelength is 100~400nm.

[0194] In certain embodiments, the expansion culture is: culturing the positive cells under X-ray irradiation for 0.05 hours to 24 hours, sorting and obtaining the antigen-specific T cells, and the X-ray dose is 0.2 to 20 Gy.

[0195] In certain embodiments, the expansion culture is: culturing the positive cells under X-ray irradiation for 0.125 hours to 5 hours, sorting and obtaining the antigen-specific T cells, and the X-ray dose is 0.5 to 10 Gy.

[0196] In a preferred embodiment, the expansion culture is: after culturing the positive cells under 0.5-10Gy X-ray irradiation for 0.5 hour to 5 hours, continue culturing for 5 to 10 days (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days) without X-ray irradiation.

[0197] In certain embodiments, the expansion culture is: culturing the positive cells under the condition of an oxygen volume content of 3.0% to 16.0% for 10 to 21 days.

[0198] In certain embodiments, the expansion culture is: culturing the positive cells under the condition of CO2 volume content of 5.1% to 15.0% for 10 to 21 days.

[0199] In certain embodiments, the expansion culture is: the positive cells are cultured at an ultrasonic frequency greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 The culture was carried out under the same conditions for 1 to 10 days.

[0200] In certain embodiments, when ultrasonic conditions are selected for expansion culture, the expansion culture is performed using gradient-changing ultrasonic conditions. For example, the expansion culture is as follows: positive cells are cultured under a first ultrasonic condition for 1 to 5 days, and then cultured under a second ultrasonic condition for 1 to 5 days to sort out the antigen-specific T cells. The ultrasonic frequency and intensity of the first ultrasonic condition are both greater than those of the second ultrasonic condition.

[0201] In some embodiments, the frequency of the first ultrasound is 2×10 5 Hz; the frequency of the first ultrasound is 0.5×10 5 Hz.

[0202] In certain embodiments, the amplification culture is: culturing the positive cells under the conditions of a terahertz intensity of 0.1 to 10 THz and a wavelength of 30 to 500 μm for 1 to 10 days.

[0203] In certain embodiments, the expansion culture is: culturing the positive cells under microwave conditions with a wavelength of 1 mm to 10 mm for 5 min to 20 min.

[0204] In certain embodiments, the expansion culture is: after culturing the positive cells under microwave conditions with a wavelength of 1 mm to 10 mm for 5 min to 20 min, continue culturing under conventional conditions without microwaves for 1 to 10 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days).

[0205] In certain embodiments, the expansion culture is: culturing the positive cells under the conditions of pH 4.0-6.8 (e.g. 4, 4.5, 5, 5.5, 6, 6.5, etc.) and oxygen volume content of 3.0%-16.0% (e.g. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, etc.) for 5-15 days (e.g. 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days).

[0206] In certain embodiments, the expansion culture is as follows: the positive cells are cultured at 37.5°C to 42.0°C (e.g., 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C), a pH of 4.0 to 6.8 (e.g., 4, 4.5, 5, 5.5, 6, 6.5, etc.), and a concentration of 0.0001 to 1000 μM (e.g., 0.0001 μM, 0.0002 μM, 0.0003 μM, 0.0004 μM, 0.0005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0 .05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.7μM, 0.8μM, 0.9, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, etc.) hypochlorous acid or hydrogen peroxide is cultured under the conditions for 5 to 20 days (for example, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days).

[0207] In certain embodiments, the expansion culture is: culturing the positive cells at 37.5°C to 42.0°C (for example, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C) and an oxygen volume content of 3.0% to 16.0% (for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, etc.) for 5 to 15 days (for example, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days).

[0208] In certain embodiments, the isolated immune cells are derived from one or more of T cells, peripheral blood mononuclear cells, and spleen cells.

[0209] In certain embodiments, the expansion culture is performed in the presence of interleukins, antibodies and / or granulocyte-macrophage colony stimulating factor (GM-CSF).

[0210] In certain embodiments, the concentration of the interleukin is 1 to 1000 ng / mL, for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, etc.

[0211] In certain embodiments, the concentration of the antibody is 1 to 9000 ng / mL, for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL. g / mL, 900ng / mL, 1000ng / mL, 1500ng / mL, 2000ng / mL, 2500ng / mL, 3000ng / mL, 3500ng / mL, 4000ng / mL, 4500ng / m L, 5000ng / mL, 5500ng / mL, 6000ng / mL, 6500ng / mL, 7000ng / mL, 7500ng / mL, 8000ng / mL, 8500ng / mL, 9000ng / mL, etc.

[0212] The concentration of GM-CSF is 1 to 1000 ng / mL, for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL and the like.

[0213] In certain embodiments, the interleukin is selected from one or more of IL-2, IL-7, IL-5, IL-12, IL-15, IL-17, and IL-21.

[0214] In certain embodiments, the interleukin is one or more of IL-2, IL-15, and IL-7.

[0215] Preferably, the antibody is selected from anti-CD3 antibody and / or anti-CD28 antibody.

[0216] In certain embodiments, the expansion culture is performed in the presence of anti-CD3 antibody, anti-CD28 antibody, and IL-2.

[0217] In certain embodiments, the expansion culture is performed in the presence of IL-2, IL-15, and IL-7.

[0218] In certain embodiments, the concentrations of IL-2, IL-15, and IL-7 are each and independently 1-100 ng / mL.

[0219] In certain embodiments, the concentrations of IL-2, IL-15, and IL-7 are respectively and independently 1 to 50 ng / mL.

[0220] In certain embodiments, the concentrations of anti-CD3 antibody and anti-CD28 antibody are respectively and independently 1 to 5000 ng / mL.

[0221] In the present invention, the culture medium for the activation culture or expansion culture is not limited, and any medium that can accomplish the present invention is acceptable. For example, in the present invention, the culture medium is any one of DMEM high-glucose complete culture medium, RPM1640 culture medium, LONZAX-VIVO 15 culture medium, and AIMV serum-free culture medium.

[0222] In certain embodiments, the antigenic component is selected from one or more of the following:

[0223] (a) Whole cell lysate fraction of cancer cells / tumor tissues; (b) A portion of the whole cell fraction consisting of the protein and peptide components of the whole cell lysate fraction of cancer cells / tumor tissues; (c) The protein and peptide components of the whole cell fraction of cancer cells / tumor tissues plus the mRNA component; (d) Tumor specific antigen (TSA); (e) Tumor associated antigen (TAA);

[0224] The tumor antigen component is a cell lysis component of tumor tissue and / or cancer cells, and the cell lysis component contains one or both of a water-soluble component and a water-insoluble component produced after cell lysis of tumor tissue and / or cancer cells.

[0225] In the present invention, the method for preparing the antigen component and the particles loaded with the antigen component adopts the conventional method in the art. For example, in the present application, the method for preparing the antigen component and the particles loaded with the antigen component refers to one of the disclosed patent documents CN112114129A, CN116942802A, CN117122678A, CN117618382A, and CN117653720A.

[0226] In the present invention, the whole cell components can be subjected to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment and the like before or (and) after lysis to prepare nanoparticles or micron particles; or can be directly prepared without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment before or (and) after cell lysis.

[0227] In the preferred technical scheme of the present invention, the tumor tissue cells are inactivated or (and) denatured before lysis, and may be inactivated or (and) denatured after cell lysis, or may be inactivated or (and) denatured both before and after cell lysis.

[0228] In the preferred technical scheme of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis includes any one or a combination of ultraviolet irradiation, high temperature heating, radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, and freeze drying.

[0229] In certain embodiments, the concentration of the particles loaded with antigen components is 2.5 ng / mL to 50 mg / mL.

[0230] In a preferred technical solution of the present invention, when the particles loaded with antigen components are incubated with antigen presenting cells alone, the concentration of the particles loaded with antigen components is 10 ng / mL to 5 mg / mL.

[0231] In a preferred technical solution of the present invention, when the particles loaded with antigen components are co-incubated with antigen presenting cells and immune cells (such as T cells), the concentration of nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL.

[0232] In a preferred technical solution of the present invention, the content of protein and polypeptide components in the antigen components loaded by the nanoparticles / microparticles is higher than 10 ng / mL.

[0233] In a preferred technical solution of the present invention, the content of protein and polypeptide components in the antigen component loaded by the nanoparticles / microparticles per milligram is 10 to 1000 μg, for example, 10 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg.

[0234] In certain embodiments, the particles are nanoparticles and / or microparticles.

[0235] In certain embodiments, the particle size of the nanoparticles is 1 nm to 1000 nm, for example, 100 nm, 150 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 320 nm, 340 nm, 350 nm, 360 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, etc.

[0236] In certain embodiments, the particle size of the nanoparticles is 30 nm to 1000 nm, preferably 100 nm to 600 nm.

[0237] In certain embodiments, the particle size of the micronized particles is 1 μm to 1000 μm, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, etc.

[0238] In certain embodiments, the particle size of the micronized particles is 1 μm-100 μm, preferably 1 μm-10 μm, and more preferably 1 μm-5 μm.

[0239] In a preferred technical solution of the present invention, the shape of the nanoparticles or micron particles includes any one of sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly or disc.

[0240] In the present invention, the particles are prepared by organic synthetic polymer materials, and the organic synthetic polymer materials are biocompatible or degradable polymer materials, including but not limited to any one or a combination of PLGA, PLA, PGA, PLGA-PEG, PLA-PEG, PGA-PEG, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, and synthetic lipids.

[0241] In a preferred technical solution of the present invention, the natural polymer material is a biocompatible or degradable polymer material, including but not limited to any one of lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, and polypeptides, or a combination thereof.

[0242] In a preferred technical solution of the present invention, the surface of the nanoparticles and / or microparticles for activating cancer-specific T cells is connected with a target head that actively targets antigen-presenting cells.

[0243] In the present application, the target head may be commonly used target heads such as mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc., leading the particle system to be delivered to antigen presenting cells in a targeted manner.

[0244] In a preferred technical solution of the present invention, the interior and / or surface of the nanoparticles or microparticles may also include an immunopotentiating adjuvant, which includes but is not limited to immunopotentiators of microbial origin, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and at least one of other categories.

[0245] In the present invention, the immunopotentiating adjuvant includes but is not limited to pattern recognition receptor agonists, BCG (BCG), manganese-related adjuvants, BCG cell wall skeletons, BCG methanol extraction residues, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immunopotentiating reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, miquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly At least one of the effective ingredients of ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, various cytokines, interleukins, interferons, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, cytokines, vegetable oils, endotoxins, liposome adjuvants, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvants, CAF01, ginseng, and astragalus. Those skilled in the art will appreciate that the enumeration here is not exhaustive, and the immune enhancing adjuvant may also use other substances that can enhance the immune response.

[0246] In the preferred technical solution of the present invention, the immune enhancing adjuvant is preferably a Toll-like receptor agonist.

[0247] In a preferred technical solution of the present invention, the immune enhancing adjuvant is preferably a combination of two or more Toll-like receptor agonists.

[0248] In a preferred technical solution of the present invention, the immune adjuvant and the cell components are co-loaded into nanoparticles or microparticles, and after the nanoparticles or microparticles are phagocytosed by antigen presenting cells, they can better activate cancer-specific T cells.

[0249] In a preferred technical solution of the present invention, the microparticles or nanoparticles are loaded with substances that increase the escape of the nanoparticles and / or microparticles or the antigens loaded thereon from the lysosomes to the cytoplasm. The substances that increase the lysosomal escape include amino acids, polypeptides, sugars, lipids, and inorganic salts that can produce a proton sponge effect. Preferably, the amino acids in the substances that increase the lysosomal escape include positively charged amino acids, and preferably, the polypeptides that increase the lysosomal escape include positively charged amino acids.

[0250] In the preferred technical solution of the present invention, the nanoparticles or micron particles may not be modified during the preparation process, or appropriate modification techniques may be used to increase the antigen loading capacity of the nanoparticles or micron particles. Modification techniques include but are not limited to biomineralization (such as silicification, calcification, magnesium), gelation, cross-linking, chemical modification, and addition of charged substances.

[0251] In the present invention, the preparation method of particles loaded with antigen components is as follows:

[0252] First, the water-soluble components and the water-insoluble components are collected separately and nano- or micro-particles are prepared separately, and the water-insoluble components are dissolved using a dissolving solution containing a dissolving agent; or a dissolving solution containing a dissolving agent can be directly used to directly lyse cancer cells or tumor tissues and dissolve the whole cell components to prepare nano- or micro-particles, and the water-insoluble components are dissolved by the dissolving solution containing a dissolving agent.

[0253] In certain embodiments, when the antigen component is a whole cell lysate component of tumor tissue / cancer cells, its preparation method is: (1) first lysing the cancer cells / tumor tissue, and then separately preparing a water-soluble component and a water-insoluble component, and then dissolving the water-insoluble component using a specific dissolving agent containing a dissolving solution; (2) using a dissolving solution containing a dissolving agent to lyse the cells, and then using a dissolving solution containing a dissolving agent to dissolve the whole cell component after lysis.

[0254] In certain embodiments, when the tumor antigen component is a part of a whole cell lysate component containing tumor tissue and / or cancer cells, the preparation method thereof is:

[0255] (1) first preparing a lysate of tumor tissue / cancer cells, and then preparing a water-soluble component and a water-insoluble component separately, dissolving the water-insoluble component using a specific dissolving agent and then using it, separating and extracting the protein and polypeptide components in the water-soluble component using an appropriate method, and using the separated and extracted protein and polypeptide components together with all the water-insoluble components as antigen components; or,

[0256] (2) first preparing a lysate of tumor tissue / cancer cells, and then preparing a water-soluble component and a water-insoluble component separately, dissolving the water-insoluble component using a specific dissolving agent and then using it, separating and extracting the protein and polypeptide components in the water-soluble component from the water-insoluble component using an appropriate method, and using the separated and extracted protein and polypeptide components together with all water-soluble components as antigen components; or,

[0257] (3) first preparing a lysate of tumor tissue / cancer cells, and then preparing a water-soluble component and a water-insoluble component respectively, dissolving the water-insoluble component using a specific dissolving agent and then using it, and using an appropriate method to separate and extract the protein and polypeptide components in the water-soluble component from the water-soluble component and the water-insoluble component respectively, and then using the protein and polypeptide components separated and extracted from the water-soluble component and the water-insoluble component together as an antigen component; or,

[0258] (4) directly using a lysing solution containing a lytic agent to directly lyse cells or tissues and dissolve the whole cell components for use as antigen components; or

[0259] (5) Directly using a dissolving solution containing a dissolving agent to directly lyse cells or tissues and dissolve the whole cell components, and then preparing the protein / polypeptide components therein by appropriate methods for use as antigen components.

[0260] The above preparation method can also be added with a step of isolating and extracting whole cell mRNA, and the whole cell mRNA is used as part of the antigen component. The appropriate treatment method includes but is not limited to salting out, heating and enzymatic hydrolysis; the water-insoluble component or the precipitate produced after salting out, heating and enzymatic hydrolysis is dissolved using a dissolving solution containing a dissolving agent.

[0261] Suitable methods for separating and extracting protein and polypeptide components include, but are not limited to, salting out, heating and enzymatic hydrolysis.

[0262] After the above protein and polypeptide components are separated and extracted, they are redissolved in a dissolving solution containing a dissolving agent.

[0263] The method for preparing the protein and polypeptide components plus the mRNA components in the whole cell components of cancer cells / tumor tissues is as follows: (1) first lysing the cancer cells / tumor tissues to prepare water-soluble components and water-insoluble components respectively, and dissolving the water-insoluble components with a solvent containing a specific dissolving solution before use; then, separately separating and extracting the protein and polypeptide components and the mRNA components in the water-soluble components and / or the water-insoluble components, and then mixing the protein and polypeptide components with the mRNA component and using them as antigen components.

[0264] The dissolving agent is selected from one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0265]

[0266] R1 is C, N, S or O, and R2 to R5 are independently selected from at least one of hydrogen, alkyl, amino, carboxyl, and substituted or unsubstituted guanidine.

[0267] Compounds containing structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, and the like containing compounds containing structural formula 1.

[0268] Then the antigen component is loaded on nanoparticle or micron particle.The nanoparticle or micron particle of the load cell component can adopt any preparation method that antigen component is loaded on nano / micron particle to prepare, include but not limited to any in methods such as solvent evaporation method, dialysis method, microfluidics method, extrusion method, hot melt method.In the present invention, nanoparticle and / or micron particle loaded with antigen component utilize multiple emulsion method to prepare, can also be any other usable method that antigen is loaded on nanoparticle / micron particle in practical application.All or part of the antigen component that the aforesaid method is prepared utilizes multiple emulsion method to prepare nanoparticle and / or micron particle loaded with tumor antigen component.

[0269] The antigen component can be loaded inside the nanoparticle / microparticle, or loaded on the surface of the nanoparticle / microparticle, or loaded inside and on the surface of the nanoparticle / microparticle at the same time.

[0270] In the preferred technical scheme of the present invention, the cell components or their mixtures are loaded on the surface of nanoparticles or microparticles in ways including but not limited to adsorption, covalent bonding, charge interaction (such as adding positively charged substances, adding negatively charged substances), hydrophobic interaction, one-step or multiple-step solidification, mineralization, encapsulation, etc.

[0271] In the preferred technical scheme of the present invention, the water-soluble components and / or water-insoluble components loaded on the surface of nanoparticles or micron particles are one or more layers after loading. When the vaccine surface is loaded with multiple layers of water-soluble components and / or water-insoluble components, there are modifiers between the layers.

[0272] For the convenience of explanation, in some embodiments of the present invention, the double emulsion method in the solvent evaporation method is used to prepare nanoparticles / microparticles loaded with tumor antigens.

[0273] In the present invention, the water-soluble component or the original water-insoluble component of the cancer cell lysate or tumor tissue lysate contains poly(I:C), manganese adjuvant, BCG (BCG) or CpG, and the concentration of poly(I:C), calcium adjuvant, BCG or CpG is greater than 0.01 ng / mL.

[0274] Furthermore, when preparing the nanoparticles or microparticles of the present invention, when activating cancer-specific T cells in vitro, nanoparticles and / or microparticles that only load water-soluble components and nanoparticles and / or microparticles that only load water-insoluble components can be used simultaneously, nanoparticles and / or microparticles that only load water-soluble components can be used, nanoparticles and / or microparticles that only load water-insoluble components can be used, or nanoparticles and / or microparticles that simultaneously load water-soluble components and water-insoluble components can be used.

[0275] In certain embodiments, the concentration of the antigen presenting cells is 1 to 50 million / mL.

[0276] In certain embodiments, the antigen presenting cells are selected from one or more of B cells, DC cells and macrophages.

[0277] In the present invention, in step (1) and step (2), when sorting cancer cell-specific T cells after activation or amplification, one T cell marker can be used, or a combination of more than one different markers can be used as a marker for sorting after activation or amplification.

[0278] Molecules that can be used as surface markers include, but are not limited to, CD69, CD137 (4-1BB), CD39, CD25, CD134, CD80, CD86, OX40L, OX40, CD28, FAS-L, IL-2R, HLA-DR, CD127 (IL-7R), CD150, CD107A, FOXP3, CD83, CD166, CD39, CD178, CD212, CD229, CD100, CD107b, CD108, CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70, CTLA-4 (CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1 (CD274), CD258, CD261, CD154, ICAM-1, LFA-1, LFA-2, VLA-4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95L, CD44, CXCR 1, CD103, CD45RO, ICOS (CD278), VTCN1, HLA2, LGAL59, CCR7, CD357, BCL6, TCF-1, CD38, CD27, etc., or any combination thereof.

[0279] In certain embodiments, the molecule used as a surface marker is selected from any one of CD137, CD134, FAS-L, HLA-DR, CD127 (IL-7R), CD39, or a combination thereof.

[0280] In the preferred technical solution of the present invention, the sorting methods in step (1) and step (2) include but are not limited to any one of flow cytometry, magnetic bead method, chemiluminescence method, mass spectrometry flow cytometry and the like or a combination thereof. When sorting activated cancer cell-specific T cells, one activation marker of activated T cells can be used, or a combination of more than one different markers can be used as an activation marker.

[0281] In another aspect, the present invention provides antigen-specific T cells obtained according to any one of the preparation methods described above.

[0282] In certain embodiments, the antigen-specific T cells are selected from CD3 + CD69 + 、CD3 + CD8 + CD69 + 、CD3+ CD4 + CD69+、CD3 + CD137 + 、CD3 + CD4 + CD137 + 、CD3 + CD8 + CD137 + 、CD3 + CD25 + 、CD3 + CD8 + CD25 + 、CD3 + CD4 + CD25 + 、CD3 + CD134 + 、CD3 + CD8 + CD134 + 、CD3 + CD4 + CD134 + 、CD3 + IL-2R + 、CD3 + CD8 + IL-2R + 、CD3 + CD4 + IL-2R + 、CD3 + HLA-DR + 、CD3 + CD8 + HLA-DR + 、CD3 + CD4 + HLA-DR + 、CD3 + FASL + 、CD3 + CD8 + FASL + 、CD3 + CD4 + FASL + 、CD3 + OX40 + 、CD3 + CD8 + OX40 + 、CD3 + CD4 + OX40 + 、CD3 +TCF-1 + 、CD3 + CD8 + TCF-1 + 、CD3 + CD4 + TCF-1 + 、CD3 + PD-1 + 、CD3 + CD8 + PD-1 + 、CD3 + CD4 + PD-1 + 、CD3 + CD39 + 、CD3 + CD8 + CD39 + 、CD3 + CD4 + CD39 + 、CD3 + CD38 + 、CD3 + CD8 + CD38 + 、CD3 + CD4 + CD38 + 、CD3 + CD28 + 、CD3 + CD8 + CD28 + 、CD3 + CD4 + CD28 + 、CD3 + CD71 + 、CD3 + CD8 + CD71 + 、CD3 + CD4 + CD71 + 、CD3 + CD44 + 、CD3 + CD8 + CD44 + 、CD3 + CD4 + CD44 + 、CD3 + CXCR3 + 、CD3 + CD8 + CXCR3+ 、CD3 + CD4 + CXCR3 + 、CD3 + CXCR1 + 、CD3 + CD8 + CXCR1 + 、CD3 + CD4 + CXCR1 + 、CD3 + ICAM-1 + 、CD3 + CD8 + ICAM-1 + 、CD3 + CD4 + ICAM-1 + 、CD3 + CD70 + 、CD3 + CD8 + CD70 + 、CD3 + CD4 + CD70 + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD62L + 、CD3 + CD8 + CD62L + 、CD3 + CD4 + CD62L + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 +CD160 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + ICOS + 、CD3 + CD8 + ICOS + 、CD3 + CD4 + ICOS + 、CD3 + CD27 + 、CD3 + CD8 + CD27 + 、CD3 + CD4 + CD27 + 、CD3 + CD107A + 、CD3 + CD8 + CD107A + and CD3 + CD4 + CD107A + Any one or more of the above T cells.

[0283] In another aspect, the present invention provides antigen-specific T cells obtained according to any of the preparation methods described above, or use of the antigen-specific T cells described above in the preparation of drugs for treating or preventing diseases, wherein the diseases include cancer, autoimmune diseases, diseases caused by bacterial infections, and diseases caused by viral infections.

[0284] In certain embodiments, the tumor is selected from a solid tumor and / or a hematological tumor.

[0285] In certain embodiments, the tumors include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood tumors such as leukemia, brain tumors, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, One or more of systemic cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, metastatic cancer, circulating tumors.

[0286] In a preferred technical solution of the present invention, the specific T cells are administered multiple times before cancer occurs, after cancer occurs, or after surgical resection of tumor tissue.

[0287] In a preferred technical solution of the present invention, the administration method of the immunotherapy is any one of intravenous injection, subcutaneous injection, intratumor injection, intraperitoneal injection, intramuscular injection, and intradermal injection, or a combination thereof.

[0288] Another object of the present invention is to provide an application of the antigen-specific T cells of the present invention in combination with any one of radiotherapy, chemotherapy, targeted therapy, surgical therapy or immunotherapy in anti-tumor or tumor immunotherapy.

[0289] Another object of the present invention is to provide the use of antigen-specific T cells in preparing drugs for enhancing antiviral ability.

[0290] Another object of the present invention is to provide the use of antigen-specific T cells in preparing drugs for enhancing the treatment of autoimmune diseases.

[0291] In a preferred technical solution of the present invention, the autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytic purpura, autoimmune hemolytic anemia, ulcerative colitis, any one of skin diseases or their complications.

[0292] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise specified, when the present invention relates to the percentage between liquids and liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0293] Unless otherwise specified, the specific methods used in the embodiments of the present invention are all conventional methods; the materials, reagents, etc. used can be obtained from commercial channels. For the convenience of explanation, most of the pressure conditions in the exemplary embodiments of the present invention are fixed pressures. In practical applications, the pressure can be adjusted appropriately over time, or gradient pressure conditions can be used. In the embodiments, only one process of activation, sorting, and amplification culture is listed in which the pressure conditions are used. In practical applications, the pressure conditions can also be used only in a certain process or in multiple processes. The application of the present invention in some cancers is only listed in the embodiments, but the present invention can also be used in other cancers. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacement choices based on the existing technology on the basis of the technical ideas of the present invention, and are not limited to the specific records of the embodiments of the present invention.

[0294] Example 1 Antigen-specific T cells cultured and expanded under high temperature and pressure conditions are used to prevent melanoma

[0295] In this embodiment, the tumor antigen-specific T cells obtained by culturing mouse melanoma as a cancer model under special pressure conditions have excellent effects in preventing and or treating cancer. In practical applications, any other cancer may also be used. In this embodiment, immune cells in mouse peripheral spleen cells are used. In practical applications, immune cells in peripheral blood, immune cells in tumor tissue, or peripheral lymph node cells may be directly used.

[0296] (1) Preparation of antigen components

[0297] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000mm 3The mice were killed and the tumor tissue was removed. The tumor tissue was cut into pieces and ground, and a single-cell suspension of tumor tissue (containing cancer cells) was prepared after passing through a cell filter. Then an appropriate amount of pure water was added to the single-cell suspension of tumor tissue and it was repeatedly frozen and thawed 5 times, and ultrasound could be used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component; 8M urea aqueous solution (containing 500mM sodium chloride) was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution. The above is the antigen component for preparing nanoparticles.

[0298] (2) Preparation of Nanoparticles

[0299] In this embodiment, the nanoparticles are prepared by the double emulsion method in the solvent evaporation method. During the preparation, the nanoparticles loaded with the water-soluble components in the whole cell components and the nanoparticles loaded with the water-insoluble components in the whole cell components are prepared separately, and then used together. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa. The preparation method is as described above. During the preparation process, the antigen component is first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. The average particle size of the nanoparticles is about 200nm, and the surface potential of the nanoparticles is about -23mV; each 1mg of PLGA nanoparticles is loaded with about 50μg of protein or polypeptide components.

[0300] (3) Isolation of tumor antigen-specific T cells

[0301] Each C57BL / 6 mouse was inoculated subcutaneously with 0.5 × 10 5 B16F10 cells, when the tumor grows to about 1000mm 3 The mice were killed and their spleens were removed to prepare a single cell suspension of mouse spleen cells, and red blood cell lysis was performed to remove the red blood cells in the single cell suspension. The above cells were firstly mixed with T cell magnetic bead sorting reagent (CD3 + Magnetic bead sorting reagent, CD8 + Magnetic bead sorting reagent) or B cell magnetic bead sorting reagent, and then CD3 + CD8 + T cells and CD19 + B cells.

[0302] The 50 million CD8 +T cells, 90 million B cells and nanoparticles loaded with all components of tumor tissue antigens (250 μg of nanoparticles loaded with water-soluble components + 250 μg of nanoparticles loaded with water-insoluble components) were co-incubated in 50 mL of DMEM high-glucose complete medium for 96 hours, and then the incubated cells were separated by magnetic bead sorting method using CD3 + T cell magnetic bead sorting reagent, CD8 + T cell magnetic bead sorting reagent and CD137 magnetic bead sorting reagent were co-incubated and sorted accordingly, and CD3 + CD8 + CD137T cells (cell viability 60%) are tumor antigen-specific T cells activated by cancer antigens.

[0303] (4) Cultivation and expansion of tumor antigen-specific T cells

[0304] The tumor antigen-specific T cells (500,000) obtained by the above sorting were co-cultured with IL-2 (20 ng / mL) and αCD3 / αCD28 (10 ng / mL) in RPMI1640 complete medium at 37.5°C (5.0% CO2) for 14 days (the medium containing the above cytokines and antibodies was replaced every three days) to expand specific T cells (cell viability 80%) to obtain T cells 1 (Tcells1);

[0305] The tumor antigen-specific T cells (500,000) obtained by the above sorting were co-incubated with IL-2 (20 ng / mL) and αCD3 / αCD28 (10 ng / mL) at 39.5°C (5.0% CO2) for 14 days (the medium containing the above cytokines and antibodies was replaced every three days) to expand the specific T cells (cell viability 70%) to obtain T cells 2;

[0306] The tumor antigen-specific T cells (500,000) obtained by the above sorting were co-incubated with IL-2 (20 ng / mL) and αCD3 / αCD28 (10 ng / mL) at 42.0°C (5.0% CO2) for 1 hour to culture the sorted cancer cell-specific T cells (cell viability 40%), and T cells 3 were obtained;

[0307] The tumor antigen-specific T cells (500,000) obtained by sorting above were co-incubated with IL-2 (20 ng / mL) and αCD3 / αCD28 (10 ng / mL) at 37.0°C (5% CO2) for 42 days to amplify the sorted cancer cell-specific T cells (cell viability 85%) to obtain T cells 4.

[0308] (5) Tumor antigen-specific T cells for cancer prevention

[0309] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. One day before adoptive transfer of cells into the mice, the recipient mice were intraperitoneally injected with 100 mg / kg of cyclophosphamide to eliminate immune cells in the recipient mice. Then, 500,000 tumor antigen-specific T cells cultured or amplified under high temperature conditions in step (3) were intravenously injected into the recipient mice. The next day, 1.5×10 5 B16F10 cells. Monitor the tumor growth rate and survival of mice. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival experiment, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0310] (6) Experimental results

[0311] like Figure 1 As shown in a and b, the tumor growth rate of mice in the PBS control group was very fast and the survival period of the mice was very short. The tumor growth rate and survival period of mice treated with T cells were significantly improved, and the T cells cultured or amplified under high temperature and pressure conditions had better effects. This shows that the cell system after sorting and amplification described in the present invention has a good preventive effect on cancer.

[0312] In this embodiment, a surface marker is used as a marker of T cell activation to sort tumor antigen-specific T cells. In practical applications, a combination of more than one different markers can also be used as an activation marker.

[0313] This embodiment uses high-temperature pressure stimulation to culture tumor antigen-specific T cells. In practical applications, other stress conditions such as radiation, electromagnetic fields, high temperatures, low oxygen, low pH, ultrasound, terahertz electromagnetic waves, microwaves, electron beams, high CO2, oxidation, reduction, etc. can also be used to culture or expand antigen-specific T cells.

[0314] Example 2 Cultivation of tumor antigen-specific T cells under oxidative stress conditions for the treatment of lung cancer

[0315] (1) Preparation of antigen components

[0316] When collecting tumor tissues, 2.0×10 6 LLC cells, and when the tumor grows to a volume of approximately 1000 mm3 The mice were killed and the tumor tissues were removed. The tumor tissues were lysed with an 8M urea aqueous solution, and the tumor tissue lysate components were dissolved with an 8M urea aqueous solution to obtain the antigen components for preparing the nanoparticles.

[0317] (2) Preparation of Nanoparticles

[0318] Nanoparticles are prepared by the emulsion method in the solvent evaporation method in the present embodiment. The PLGA molecular weight of the nanoparticle 1 preparation material is 7KDa-17KDa, and the immunoadjuvants adopted are poly(I:C) and CpG2006(CpG7909). The preparation method is as previously mentioned, first in nanoparticle 1 internal load antigen component and adjuvant, then 100mg nanoparticles are centrifuged at 12000g for 25 minutes, and lyophilized 48h after the ultrapure water resuspending of 10mL containing 4% trehalose. This nanoparticle 1 mean particle size is about 300nm, and every 1mg PLGA nanoparticle is about loaded with 0.9mg protein or polypeptide component, and each 0.02mg of poly(I:C) and CpG2006 is loaded.

[0319] The control nanoparticle 2 is prepared with the same materials and method, but only loaded with antigen components without any adjuvant. The particle size is about 300 nm, and each 1 mg of PLGA nanoparticle is loaded with about 0.9 mg of protein or polypeptide components.

[0320] (3) Isolation of tumor antigen-specific T cells

[0321] Each C57BL / 6 mouse was subcutaneously inoculated with 2.0 × 10 5 LLC cells were added to the mice, and 150 μg of αPD-1 antibody was injected intraperitoneally into the mice on days 6, 8, 10, 12, and 14. The mice were killed on day 18, and peripheral blood was collected from the mice. Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of the mice using gradient centrifugation. 500,000 PBMC cells and 500 μg of nanoparticles 1 or nanoparticles 2 were co-incubated in 1 mL of DMEM complete medium for 16 hours. After the incubation, tumor antigen-specific T cells were labeled with CD3 antibodies and CD137 antibodies, and then CD3 was sorted out using flow cytometry. + CD137 + T cells are tumor antigen-specific T cells activated by antigens (cell viability 70%).

[0322] (4) Cultivation and expansion of tumor antigen-specific T cells

[0323] The 50,000 tumor-specific T cells obtained by the above sorting were co-incubated with IL-2 (10 ng / mL), IL-15 (10 ng / mL), and IL-7 (10 ng / mL) in 10 mL RPMI1640 complete medium (37.0°C, 5.0% CO2) for 1 day to obtain T cells 1;

[0324] The 50,000 tumor-specific T cells obtained by the above sorting were co-incubated with IL-2 (10 ng / mL), IL-15 (10 ng / mL), and IL-7 (10 ng / mL) in 10 mL RPMI1640 complete medium (37.0°C, 5.0% CO2) containing 0.1 nM hydrogen peroxide for 1 day to obtain T cells 2.

[0325] The 50,000 tumor-specific T cells obtained by the above sorting were co-incubated with IL-2 (10 ng / mL), IL-15 (10 ng / mL), and IL-7 (10 ng / mL) in 10 mL RPMI1640 complete medium (37.0°C, 5.0% CO2) containing 200 μM hydrogen peroxide for 1 day to obtain T cells 3.

[0326] The 50,000 tumor-specific T cells obtained by the above sorting were co-incubated with IL-2 (10 ng / mL), IL-15 (10 ng / mL), and IL-7 (10 ng / mL) in 10 mL RPMI1640 complete medium (37.0°C, 5.0% CO2) containing 1000 μM hydrogen peroxide for 1 day. Among them, T cells 4 were obtained after activation and auxiliary sorting using nanoparticles 1, and T cells 5 were obtained after activation and auxiliary sorting using nanoparticles 2.

[0327] Then, the T cells of each group cultured under oxidative stress for 1 day were cultured in 10 mL RPMI1640 complete medium (37.0° C., 5.0% CO 2 ) for further 20 days to expand the tumor antigen-specific T cells of each group.

[0328] (5) Tumor antigen-specific T cells for cancer treatment

[0329] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, 2.0×10 6 LLC cells. On the 6th and 18th day after tumor inoculation, each mouse was intravenously injected with 2 million tumor antigen-specific T cells (and 5000U IL-2 was injected every two days from the 6th day). The tumor volume and survival period of mice were monitored in the same way as above.

[0330] (6) Experimental results

[0331] like Figure 2 As shown, the tumor growth rate of mice in the PBS control group was the fastest and the survival period of mice was the shortest. The tumor growth rate and survival period of mice treated with several groups of T cells were significantly improved. The tumor antigen-specific T cells sorted and expanded using nanoparticles 1 were better than those sorted and expanded using nanoparticles 2. Moreover, the antigen-specific T cells cultured and expanded under oxidative stress conditions using hydrogen peroxide were better than those cultured and expanded without oxidative stress conditions. This shows that antigen-specific T cells cultured and expanded under oxidative stress conditions are more effective.

[0332] Example 3 Tumor antigen-specific T cells cultured and expanded under hypoxic pressure conditions for the prevention of melanoma lung metastasis

[0333] (1) Preparation of antigen components

[0334] The cultured B16-F10 melanoma cells were collected, and the cancer cells were lysed with 6M guanidine hydrochloride, and then the cancer cell lysate was dissolved. Then, a 20% ammonium sulfate aqueous solution was added to salt out the protein and polypeptide components, and then centrifuged at 12000rpm for 30min. After the supernatant was discarded, the precipitate was dissolved with 6M guanidine hydrochloride to obtain the antigen component.

[0335] (2) Preparation of Nanoparticles

[0336] In this embodiment, the nanoparticles are prepared by the double emulsion method in the solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the immune adjuvant used is poly(I:C). The antigen component and adjuvant are first loaded into the nanoparticles by the double emulsion method, and then the precipitate is resuspended with 10mL of ultrapure water containing 4% mannitol after centrifugation at 10000g for 40min, and then freeze-dried for 48h to obtain the nanoparticles. The average particle size of the nanoparticles is about 350nm; each 1mg of PLGA nanoparticles is loaded with about 300μg of protein or polypeptide components and 0.1mg of poly(I:C).

[0337] (3) Preparation of dendritic cells

[0338] This example takes the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDCs).

[0339] First, a 6-8 week old C57 mouse was killed by cervical dislocation, and the tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissue around the bone was removed with scissors and forceps. Cut the two ends of the bone with scissors, and then extract the PBS solution with a syringe. The needle was inserted into the bone marrow cavity from both ends of the bone, and the bone marrow was repeatedly flushed into the culture dish. The bone marrow solution was collected, centrifuged at 400g for 3min, and 1mL of red blood cell lysis solution was added to lyse the red blood cells. 3mL of RPMI 1640 (10% FBS) culture medium was added to terminate the lysis, centrifuged at 400g for 3min, and the supernatant was discarded. The cells were placed in a 10mm culture dish and cultured using RPMI 1640 (10% FBS) culture medium, and recombinant mouse GM-CSF (20ng / mL) was added at the same time. The cells were cultured at 37 degrees and 5% CO2 for 7 days. On the third day, the culture bottle was gently shaken and supplemented with the same volume of RPMI 1640 (10% FBS) culture medium containing GM-CSF (20ng / mL). On the 6th day, the medium was replaced by half. On the 7th day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. + CD80 + CD11c + When the ratio of BMDC in cells is between 15-20%, the induced cultured BMDC can be used for the next experiment.

[0340] (4) Isolation of antigen-specific T cells

[0341] Each C57BL / 6 mouse was inoculated subcutaneously with 0.5 × 10 5 On the 16th day, the mice were killed, the spleens of the mice were collected, and the single cell suspension of mouse spleen cells was prepared. The mouse spleen cells were incubated with CD3 antibodies and then CD3 in the single cell suspension of spleen cells was sorted by flow cytometry. + The BMDCs (1 million) prepared in step 3 were mixed with nanoparticles loaded with tumor tissue full component antigens (40 μg) and incubated in 3 mL RPMI1640 complete medium (37°C, 5% CO2) for 48 hours. The BMDCs were then collected by centrifugation at 400 g for 5 minutes and then separated from the CD3 T cells. + T cells were co-incubated for 24 hours, and then flow cytometry was used to sort the CD3 + CD134 + T cells (cell viability 75%) are antigen-specific T cells activated by antigens.

[0342] (5) Cultivation and expansion of tumor antigen-specific T cells

[0343] The 500,000 tumor antigen-specific T cells obtained by the above sorting were cultured in 10 mL of RPMI1640 complete medium under hypoxic conditions with an oxygen volume content of 3% with IL-2 (20 ng / mL) and αCD3 / αCD28 for 14 days (37°C, 5% CO2, medium was changed every two days) to expand T cells (cell viability 10%), and T cells 1 were obtained;

[0344] The 500,000 tumor antigen-specific T cells obtained by the above sorting were cultured in 10 mL of RPMI1640 complete medium under hypoxic conditions with an oxygen volume content of 16% with IL-2 (20 ng / mL) and αCD3 / αCD28 for 14 days (37°C, 5% CO2, medium was changed every two days) to expand T cells (cell viability 30%) to obtain T cells 2;

[0345] The 500,000 tumor antigen-specific T cells obtained by the above sorting were cultured with IL-2 (20 ng / mL) and αCD3 / αCD28 in 10 mL of RPMI1640 complete medium for 14 days (37°C, 5% CO2, medium was changed every two days) to expand T cells (cell viability 75%) and obtain T cells 3.

[0346] (6) Allogeneic cell systems for the prevention of cancer metastasis

[0347] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. One day before adoptive transfer of cells into mice, recipient mice were intraperitoneally injected with 100 mg / kg of cyclophosphamide to eliminate immune cells in the recipient mice. On day 0, mice were intravenously injected with 100 μL of 500,000 antigen-specific T cells. At the same time, each mouse was intravenously injected with 0.5×10 5 The mice were killed on the 14th day, and the number of melanoma foci in the lungs of the mice was observed and recorded.

[0348] (7) Experimental results

[0349] like Figure 3 As shown, the PBS control group mice had many cancer foci, while the mice treated with T cells had fewer cancer foci. Compared with the control group, the mice treated with cancer cell-specific T cells obtained by sorting and amplification had significantly fewer cancer foci. Moreover, antigen-specific T cells cultured under hypoxic conditions had better effects.

[0350] This embodiment uses hypoxic conditions to culture antigen-specific T cells. In practical applications, other pressure conditions such as radiation, electromagnetic fields, high temperatures, hypoxia, low pH, ultrasound, terahertz electromagnetic waves, microwaves, electron beams, high CO2, oxidation, reduction, etc. can also be used to culture or expand antigen-specific T cells.

[0351] Example 4: Antigen-specific T cells amplified by X-ray irradiation pressure culture for the treatment of lung cancer

[0352] (1) Preparation of antigen components

[0353] The cultured LLC lung cancer cell line was collected and lysed with 8M urea aqueous solution, and the whole cell components after lysis were dissolved to obtain the antigen components for preparing micron particles.

[0354] (2) Preparation of micron particles

[0355] In this embodiment, micron particles 1 are prepared by double emulsion method. The molecular weight of the micron particle preparation material PLGA used is 38KDa-54KDa, and the immune adjuvant used is CpG2395. The preparation method is as described above. In the preparation process, the antigen component and adjuvant are first loaded inside the micron particles by double emulsion method, and then 100mg of micron particles are centrifuged at 8000g for 15 minutes, and then 10mL of ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h. The average particle size of the micron particles is about 1.1μm, and each 1mg of PLGA micron particles is loaded with about 540μg of protein or polypeptide components, and the loaded CpG2395 is 0.1mg.

[0356] (3) Isolation of cancer cell-specific T cells

[0357] Each C57BL / 6 mouse was subcutaneously inoculated with 2.0 × 10 6 LLC cells were injected intraperitoneally with 100 μg of αPD-1 antibody on days 8, 10, and 12. The mice were killed on day 20, and their spleens were collected to prepare single-cell suspensions of mouse spleen cells. 80 million spleen cells were mixed with 20 μg of microparticles 1 and incubated in 10 mL of RPMI complete medium (containing 5% FSBS) for 24 hours, and then CD3 + CD39 + T cells are tumor antigen-specific T cells activated by cancer antigens (cell viability 75%).

[0358] (4) Cultivation and expansion of tumor antigen-specific T cells

[0359] The 1 million tumor antigen-specific T cells obtained by the above sorting were cultured with IL-2 (20 ng / mL) and αCD3 / αCD28 antibody (20 ng / mL) in 10 mL of DMEM high-glucose complete medium (37°C, 5% CO2) under X-ray irradiation conditions (6 Gy) for 1.5 hours (T cell viability after culture was 20%), and then cultured with IL-2 (20 ng / mL) and αCD3 / αCD28 antibody (200 ng / mL) in 10 mL of DMEM high-glucose complete medium (37°C, 5% CO2) without X-ray conditions for 6 days (the medium was changed every two days) to expand antigen-specific T cells to obtain T cells 1;

[0360] The 1 million tumor antigen-specific T cells obtained by the above sorting were directly cultured with IL-2 (20 ng / mL) and αCD3 / αCD28 antibody (200 ng / mL) in 10 mL of DMEM high-glucose complete medium (37° C., 5% CO2) under X-ray-free conditions for 6 days (the medium was changed every two days) to expand the antigen-specific T cells and obtain T cells 2.

[0361] (5) Antigen-specific T cells expanded for cancer prevention

[0362] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, each mouse was subcutaneously injected with 2.0×10 6 LLC cells. On day 5, recipient mice were intraperitoneally injected with 100 mg / kg of cyclophosphamide to eliminate immune cells in the recipient mice. On days 6 and 16, mice were intravenously injected with 1 million tumor antigen-specific T cells (5000 U IL-2 was injected intravenously every two days starting from day 6). The methods for monitoring mouse tumor volume and survival were the same as above.

[0363] (6) Experimental results

[0364] like Figure 4 As shown, the tumor growth rate of mice in the PBS control group was very fast and the survival period was very short, while the tumor growth rate of mice treated with T cells was significantly slowed down and the survival period was significantly prolonged. Moreover, the effect of stimulating cultured tumor antigen-specific T cells under X-ray conditions was even better.

[0365] Example 5: Use of tumor antigen-specific T cells cultured under ultraviolet irradiation stress to treat colorectal cancer

[0366] (1) Preparation of antigen components

[0367] The cultured MC38 colorectal cancer cells were collected, and 8M urea PBS solution was used to lyse the cancer cells and dissolve the whole cell components of the cancer cells, which are the antigen components for preparing nanoparticles.

[0368] (2) Preparation of Nanoparticles

[0369] In the present embodiment, nanoparticles are prepared by double emulsion method. The molecular weight of the material PLGA for preparing nanoparticle 1 is 12KDa-15KDa, and the immunoadjuvants used are Poly(I:C) and CpG1018. The preparation method is as described above, first adopting double emulsion method to load antigen components and adjuvant inside nanoparticles, then centrifuging 100mg nanoparticles at 15000g for 20 minutes, and resuspending and freeze-drying for 48h after using 10mL ultrapure water containing 4% trehalose. The average particle size of the nanoparticle 1 is about 280nm, and each 1mg PLGA nanoparticle is loaded with about 100μg protein or polypeptide components, and loaded with 0.05mg of CpG1018 and Poly(I:C).

[0370] The nanoparticles 2 are prepared using the same materials and methods, and only carry antigen components without adjuvants. The particle size is about 280 nm, and each 1 mg of PLGA nanoparticles 2 carries about 100 μg of protein or polypeptide components.

[0371] (3) Isolation of tumor antigen-specific T cells

[0372] Each C57BL / 6 mouse was subcutaneously inoculated with 1.0 × 10 6 MC38 cells. Mice were killed on day 20, and their spleens were collected to prepare single cell suspensions of mouse spleen cells. 50 million spleen cells were incubated with 50 mg of nanoparticle 1 or 50 mg of nanoparticle 2 in 2 mL of RPMI complete medium for 96 hours (37.0°C, 5.0% CO2), and then CD3 + CD39 + T cells (cell viability 70%) are tumor antigen-specific T cells.

[0373] (4) Cultivation and expansion of tumor antigen-specific T cells

[0374] The 500,000 tumor antigen-specific T cells obtained by the above sorting were irradiated with 300nm ultraviolet light (50μW / cm 2) and IL-2 (20 ng / mL) and αCD3 / αCD28 antibodies were co-incubated in 10 mL DMEM high glucose complete medium (37.0°C, 5.0% CO2) for 7 days (the medium was changed every two days) to expand antigen-specific T cells (cell viability 60%). The tumor antigen-specific T cells activated by nanoparticles 1 were expanded under stress conditions to obtain T cells 1 (T cells 1); the tumor antigen-specific T cells activated by nanoparticles 2 were expanded under stress conditions to obtain T cells 2 (T cells 2).

[0375] Alternatively, the 500,000 tumor antigen-specific T cells obtained by the above sorting (obtained after activation and sorting by nanoparticle 2) were directly incubated with IL-2 (20 ng / mL) and αCD3 / αCD28 (1000 ng / mL) antibodies in 10 mL DMEM high-glucose complete medium (37.0°C, 5.0% CO2) for 7 days (the medium was changed every two days) to amplify the sorted cancer cell-specific T cells (cell viability 80%) to obtain T cells 3 (T cells 3).

[0376] (5) Antigen-specific T cells for cancer treatment

[0377] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 2×10 6 100 μL of 100,000 tumor antigen-specific T cells were injected into mice on days 4, 7, 10, 15, and 20. The methods for monitoring tumor growth and survival of mice were the same as above.

[0378] (6) Experimental results

[0379] like Figure 5 As shown, the tumor growth rate of mice in the PBS control group was very fast. The antigen-specific T cells obtained by activation, sorting and amplification of nanoparticles 1 and 2 could effectively delay the tumor growth rate and increase the survival time of mice.

[0380] Example 6: Low pH (acidic conditions) pressure culture of tumor antigen-specific T cells for the treatment of breast cancer

[0381] (1) Preparation of antigen components

[0382] The cultured 4T1 breast cancer cells were inactivated and denatured by ultraviolet light and high temperature heating at 400 g, and then the 4T1 cells were lysed with an appropriate amount of 8M urea and the lysate was dissolved to prepare the antigen component of the particles.

[0383] (2) Preparation of micron particles

[0384] In this embodiment, the preparation of micron particles adopts the double emulsion method. The molecular weight of the PLGA skeleton material of micron particle 1 is 30KDa-36KDa, and the immune adjuvant used is Poly ICLC. During the preparation, the internal loaded antigen component and adjuvant are prepared by the double emulsion method, and then 100mg of micron particles are centrifuged at 8000g for 10 minutes, resuspended with 10mL of ultrapure water containing 4% trehalose, and dried for 48h before use. The average particle size of the micron particle system is about 2.5μm, and each 1mg of PLGA micron particle is loaded with about 310μg of protein or polypeptide components.

[0385] (3) Isolation of tumor antigen-specific T cells

[0386] Female BALB / c mice aged 6-8 weeks were selected and inoculated subcutaneously on the back of the mice with 4.5×10 4 4T1 cells were injected subcutaneously on days 8, 10, and 12. Mice were killed on day 14, peripheral blood was collected, and PBMCs were isolated. 500,000 PBMCs were incubated with 400 μg of microparticles in 2 mL of RPMI1640 complete medium for 48 hours. CD3 + CD137 + T cells (cell viability 85%) are tumor antigen-specific T cells.

[0387] (4) Cultivation and expansion of tumor antigen-specific T cells

[0388] The tumor antigen-specific T cells obtained by the above sorting were cultured in DMEM medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody) at 37.0°C and 5.0% CO2 for 7 days under acidic conditions of pH=5.5 to expand the antigen-specific T cells (cell viability 55%). The acidic conditions were prepared by adding lactic acid to the culture medium. The T cells cultured and expanded under the acidic conditions were T cells 1.

[0389] The tumor antigen-specific T cells obtained by the above sorting were cultured in DMEM medium with pH = 7.2-7.4 (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2 for 7 days to expand the antigen-specific T cells (cell viability 85%). The T cells expanded under pH 7.2-7.4 conditions were T cells 2.

[0390] (5) T cells expanded by stress conditions for cancer treatment

[0391] Female BALB / c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. At the same time, each mouse was subcutaneously injected with 4.5×10 5 4T1 cells. One day before adoptive transfer of cells into mice, recipient mice were intraperitoneally injected with 100 mg / kg of cyclophosphamide to eliminate immune cells in the recipient mice. On the 6th and 16th days, mice were intravenously injected with 1 million amplified T cells (5000 U of IL-2 was intravenously injected every two days from the 6th day). The methods for monitoring mouse tumor growth and survival were the same as above.

[0392] (6) Experimental results

[0393] like Figure 6 As shown in the figure, compared with the control group (PBS), the tumor growth rate in the T cell treatment group was significantly slowed down and the survival of mice was significantly prolonged. Moreover, T cells cultured under acidic stress conditions were more effective in inhibiting tumor growth and delaying survival.

[0394] Example 7 Tumor antigen T cells cultured under reducing conditions (glutathione, GSH) are used to prevent cancer metastasis

[0395] (1) Antigenic components

[0396] This example uses 4 new antigens of B16F10 melanoma mice as antigen components: B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), TRP2:180-188 (SVYDFFVWL).

[0397] (2) Preparation of Nanoparticles

[0398] In the present embodiment, nanoparticle 1 adopts solvent evaporation method to prepare, and the molecular weight of nanoparticle preparation material PLGA is 24KDa-38KDa, and the immunoadjuvant adopted is CpG7909 and Poly (I: C). Preparation method is as mentioned above, first adopts multiple emulsion method to load antigen component and adjuvant in nanoparticle internal and then 100mg nanoparticle is centrifuged at 16000g for 20 minutes, and uses 10mL4% trehalose aqueous solution to resuspend and freeze-dry after 48h for standby use. This nanoparticle 1 mean particle diameter is about 270nm, and every 1mg PLGA nanoparticle 1 is about loaded with 200μg antigen component, and each 20μg of load CpG7909 and Poly (I: C).

[0399] (3) Isolation of tumor antigen-specific T cells

[0400] Female C57BL / 6 mice aged 6-8 weeks were selected and 0.5×10 5 B16F10 cells. Mice were killed on day 14, peripheral blood was collected and PBMCs were isolated. 700,000 PBMCs were incubated with 4 mg of nanoparticles in 1 mL of RPMI1640 complete medium for 48 hours. Flow cytometry was then used to sort the CD3 + 137 + CD134 + T cells are tumor antigen-specific T cells (cell viability 80%).

[0401] (4) Cultivation and expansion of tumor antigen-specific T cells

[0402] The 100,000 tumor antigen-specific T cells obtained by the above sorting were cultured in 1 mL of RPMI1640 complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody) containing 40 μmol / L glutathione (GSH) at 37.0°C and 5.0% CO2 for 14 days to expand the T cells (cell viability 60%), which were T cells 1.

[0403] The 100,000 tumor antigen-specific T cells obtained by the above sorting were cultured in 1 mL of normal RPMI1640 complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2 for 14 days to expand the T cells (cell viability 80%) to T cells 2.

[0404] (5) Antigen-specific T cells for the prevention of cancer metastasis

[0405] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. One day before adoptive cell transfer, the recipient mice were intraperitoneally injected with 100 mg / kg of cyclophosphamide to eliminate immune cells in the recipient mice. On day 0, 2 million tumor antigen-specific T cells (T cells 1 or T cells 2) were intravenously injected into the mice. At the same time, each mouse was intravenously inoculated with 0.5×10 5 The mice were killed on the 14th day, and the number of melanoma foci in the lungs of the mice was observed and recorded.

[0406] (6) Experimental results

[0407] like Figure 7 As shown, tumor antigen-specific T cells can effectively prevent cancer metastasis. Moreover, T cells cultured under high GSH stress conditions prevent the metastasis of cancer lesions.

[0408] Example 8: T cells cultured under high CO2 concentration pressure conditions are used to treat pancreatic cancer

[0409] (1) Preparation of antigen components

[0410] The cultured Pan02 pancreatic cancer cells were collected, and the cancer cells were lysed with 8M urea aqueous solution, and then the cancer cell lysate components were dissolved, which were the antigen components.

[0411] (2) Preparation of Nanoparticles

[0412] In this embodiment, nanoparticles are prepared by multiple emulsion method. The PLGA molecular weight of the nanoparticles used in nanoparticle 1 is 7KDa-17KDa, and Poly (I: C) and CpG7909 are used as adjuvants. The preparation method is as described above, firstly, the antigen component and adjuvant are loaded inside the nanoparticles, and then 100mg of nanoparticles are centrifuged at 12000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h. The average particle size of nanoparticle 1 is about 360nm, and each 1mg of PLGA nanoparticle 1 is loaded with about 650μg of protein or polypeptide components, and 0.06mg of each of Poly (I: C) and CpG7909 is loaded.

[0413] (3) Isolation of tumor antigen-specific T cells

[0414] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1×10 6 Pan02 pancreatic cancer cells were cultured in 400 mice. 200 μg of αPD-1 antibody was injected subcutaneously on days 8, 10, 12, 14, and 16. Mice were sacrificed on day 18, and spleen cell single cell suspensions were collected. 50 million spleen cells were incubated with 500 μg of nanoparticles for 16 hours. CD3 + CD137 + T cells (cell viability 75%) are antigen-specific T cells.

[0415] (4) Cultivation and expansion of tumor antigen-specific T cells

[0416] One million antigen-specific T cells were cultured and expanded in 1 mL of RPMI1640 complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody) at 37.0°C and 7.0% CO2 for 14 days (the medium was changed every two days). The T cells obtained by culture and expansion were T cells 1.

[0417] One million antigen-specific T cells were cultured and expanded in 1 mL of RPMI1640 complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody) at 37.0°C and 5.0% CO2 for 14 days (the medium was changed every two days). Among them, the T cells cultured and expanded under high CO2 (7.0%) conditions were T cells 2.

[0418] (5) Antigen-specific T cells for the treatment of pancreatic cancer

[0419] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. At the same time, each mouse was subcutaneously injected with 1×10 6 Pan02 pancreatic cancer cells. Two million tumor antigen-specific T cells were intravenously injected into the mice on days 8 and 18 (10,000 U of IL-2 was intravenously injected every two days starting from day 8). The tumor volume of mice was monitored and recorded as above.

[0420] (6) Experimental results

[0421] like Figure 8 As shown, tumor antigen-specific T cells can effectively treat pancreatic cancer, and antigen-specific T cells cultured under high CO2 stress conditions are more effective in inhibiting tumor growth and delaying survival.

[0422] Example 9 Antigen-specific T cells cultured and expanded under ultrasonic pressure conditions are used to prevent esophageal cancer

[0423] (1) Preparation of antigen components

[0424] After collecting the cultured AKR cancer cells, 8M urea aqueous solution was used to lyse the cancer cells and dissolve the lysate, which is the antigen component.

[0425] (2) Preparation of Nanoparticles

[0426] In the present embodiment, nanoparticle 1 is prepared by multiple emulsion method. The nanoparticle preparation materials adopted are PLGA and mannose-modified PLGA. When preparing nanoparticles with target heads, the mass ratio of the two is 4:1 when used together, and the molecular weight is 7KDa-17KDa. The preparation method is as mentioned above, first the antigen component is loaded inside the nanoparticle, then 100mg nanoparticles are centrifuged at 12000g for 25 minutes, and 10mL ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h for standby use. The average particle size of the nanoparticles is about 220nm, and each 1mg PLGA nanoparticle is about loaded with 80μg protein or polypeptide components.

[0427] (3) Preparation and activation of dendritic cells (DCs)

[0428] This example uses bone marrow-derived dendritic cells (BMDC) as antigen presenting cells. The preparation method is the same as above. Mouse BMDCs are plated in a cell culture plate, 10 mL of RPMI 1640 (10% FBS) medium is added to every 10 million BMDC cells, and then 20 mg of nanoparticles are added and incubated with BMDCs for 48 h (37.0°C, volume fraction 5.0% CO2), and then BMDCs are collected and centrifuged at 300g for 5 minutes, washed twice with phosphate buffered saline (PBS), and then resuspended in PBS for use.

[0429] (4) Preparation of tumor antigen-specific T cells

[0430] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1.0×10 6 On day 16, the mice were killed and spleen cells were collected. CD3 + T cells. The 5 million CD3 + T cells were co-incubated with 10 million BMDCs activated in step (3) in 4 mL DMEM high-glucose complete medium for 18 hours. CD3 + CD39 + T cells (cell viability 75%) are tumor antigen-specific T cells.

[0431] (5) Cultivation and expansion of tumor antigen-specific T cells

[0432] The 200,000 antigen-specific T cells obtained from the above sorting were divided into 2×10 5 Hz ultrasound stimulation (0.3W / cm 2) in 1 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody) at 37.0°C and 5.0% CO2 for 3 days to expand antigen-specific T cells (cell viability 50%), and then at 0.5×10 5 Hz ultrasonic stimulation in 1 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibody), 37.0°C, 5.0% CO2, and cultured for another 3 days to expand antigen-specific T cells (cell viability 45%) to obtain T cells 1 (Tcells1);

[0433] The 200,000 antigen-specific T cells obtained by the above sorting were cultured in 1 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 500 ng / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2 for 6 days to expand the antigen-specific T cells (cell viability 85%) and obtain T cells 2.

[0434] (6) Cancer cell-specific T cells for cancer prevention

[0435] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare esophageal cancer tumor-bearing mice. One day before adoptive transfer of cells into mice, 100 mg / kg of cyclophosphamide was intraperitoneally injected into the recipient mice to eliminate immune cells in the recipient mice. Then, 1 million tumor antigen-specific T cells were intravenously injected into the recipient mice. The next day, 1.0×10 6 The methods for monitoring the growth rate of mouse tumors and the survival of mice were the same as above.

[0436] (7) Experimental results

[0437] As shown in Figure 9, the tumor growth rate of mice treated with T cells was significantly slower than that of the PBS control group. Moreover, antigen-specific T cells cultured and expanded under ultrasonic stress conditions had better effects.

[0438] Example 10 Tumor antigen-specific T cells cultured under hypoxia and acidic stress conditions are used to treat liver cancer

[0439] (1) Preparation of antigen components

[0440] After collecting the cultured Hepa 1-6 liver cancer cells, 8M urea was used to lyse and dissolve the whole cell components of the cancer cells, which were the antigen components.

[0441] (2) Preparation of Nanoparticles

[0442] In this embodiment, the nanoparticles are prepared by solvent evaporation method, and the molecular weight of the PLGA material for preparing the nanoparticles is 24KDa-38KDa. The preparation method is as described above, firstly, the antigen component and adjuvant are loaded inside the nanoparticles, then 100mg of the nanoparticles are centrifuged at 10000g for 25 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48h before use. The particle size of the nanoparticles is about 270nm; each 1mg of PLGA nanoparticles is loaded with 100μg of protein or polypeptide components.

[0443] (3) Isolation of tumor antigen-specific T cells

[0444] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously on the back of each mouse with 1.0×10 6 The mice were killed on day 18 and their spleen cells were collected. 80 million spleen cells and 50 mg nanoparticles were co-incubated in 50 mL of DMEM complete medium for 72 hours. CD8 + CD134 + T cells (cell viability 80%) are tumor antigen-specific T cells.

[0445] (4) Cultivation and expansion of tumor antigen-specific T cells

[0446] Five million of the above tumor antigen-specific T cells were cultured in 5 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) under hypoxic (oxygen volume fraction of 5.0%) and acidic (pH = 5.0, prepared by adding carbonic acid to the culture medium) conditions at 37.0°C and 5.0% CO2 for 7 days to expand the sorted cancer cell-specific T cells (cell viability 30%), thereby obtaining T cells 1;

[0447] Five million of the above tumor antigen-specific T cells were cultured in 5 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) under hypoxic conditions (oxygen volume fraction of 5.0%) at 37.0°C and 5.0% CO2 volume fraction for 7 days to expand the sorted cancer cell-specific T cells (cell viability 30%) to obtain T cells 2 (Tcells2);

[0448] Five million of the above tumor antigen-specific T cells were cultured in 5 mL DMEM complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) at 37.0° C. and 5.0% CO2 for 7 days under acidic conditions (pH=5.0, prepared by adding carbonic acid to the culture medium) to expand the sorted cancer cell-specific T cells (cell viability 30%), and obtain T cells 3;

[0449] Five million of the above tumor antigen-specific T cells were cultured in 5 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibodies) under normal conditions at 37.0°C and 5.0% CO2 for 7 days to expand the sorted cancer cell-specific T cells (cell viability 30%) and obtain T cells 4.

[0450] (5) Cancer cell-specific T cells for cancer prevention

[0451] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare liver cancer-bearing mice. On day 0, each mouse was subcutaneously injected with 1.0×10 6 Hepa1-6 liver cancer cells were implanted in the recipient mice. On the 5th day, 100 mg / kg of cyclophosphamide was intraperitoneally injected into the recipient mice to eliminate the immune cells in the recipient mice. On the 6th and 13th days, 2 million tumor antigen-specific T cells were intravenously injected into the mice (and 5000 U IL-2 was injected every two days starting from the 6th day). Tumor growth and mouse survival were recorded in the same way as above.

[0452] (6) Experimental results

[0453] like Fig.10 As shown, the tumor growth rate of mice treated with T cells was significantly slower than that of the control group. Moreover, the antigen-specific T cells expanded by culturing under hypoxic and acidic conditions had a better effect.

[0454] Example 11: Tumor antigen-specific T cells cultured and amplified under terahertz electromagnetic wave pressure conditions for the treatment of melanoma

[0455] (1) Preparation of antigen components

[0456] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to about 1000mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were lysed with 8M urea aqueous solution and the lysate components were dissolved to obtain the antigen components for preparing nanoparticles.

[0457] (2) Preparation of Nanoparticles

[0458] In this embodiment, the nanoparticles are prepared by the double emulsion method. The molecular weight of the PLGA material for preparing the nanoparticles is 7KDa-17KDa. The preparation method is as described above, firstly the antigen group is loaded inside the nanoparticles, then 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. The average particle size of the nanoparticles is about 250nm, and each 1mg of PLGA nanoparticles is loaded with about 200μg of the protein or polypeptide component in the antigen component.

[0459] (3) Isolation of tumor-specific T cells

[0460] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously on the back of the mice with 1.5×10 5 B16F10, and then injected 200μg of αPD-L1 antibody into the mice intraperitoneally on days 8, 10, 12, 14, and 16. The mice were killed on day 20 and the spleen cells were collected. 80 million spleen cells were co-incubated with nanoparticles in 2mL RPMI1640 complete medium for 48 hours. Then, CD8 + CD137 + T cells (cell viability 80%) are tumor antigen-specific T cells.

[0461] (4) Cultivation and expansion of tumor antigen-specific T cells

[0462] Five million tumor antigen-specific T cells were exposed to terahertz electromagnetic waves (300 μm wavelength, 0.3 W / cm 2 ) Under stress conditions, the cells were cultured in 20 mL of RPMI1640 complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) at 37.0°C and 5.0% CO2 for 7 days (the medium was changed every two days) to expand T cells and obtain T cells 1;

[0463] Five million tumor antigen-specific T cells were cultured directly in 20 mL of RPMI1640 complete medium (37.0°C, 5.0% CO2, containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) for 7 days (the medium was changed every two days) to expand T cells and obtain T cells 2.

[0464] (4) Using expanded cancer cell-specific T cells to treat cancer

[0465] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells. Cyclophosphamide (100 mg / kg) was injected intraperitoneally to the recipient mice on the 5th day to eliminate the immune cells in the recipient mice. Two million tumor antigen-specific T cells were injected intravenously on the 6th and 20th days after melanoma inoculation (and 5000U IL-2 was injected every two days starting from the 6th day). The group that only used cyclophosphamide to ablate the myeloablation and the group that only gave the same dose of IL-2 served as the control group. In the experiment, the tumor volume and survival monitoring methods of the mice were the same as above.

[0466] (5) Experimental results

[0467] like Fig.11 As shown, the cancer cell-specific T cells sorted and amplified under terahertz electromagnetic wave pressure conditions in the present invention have a good therapeutic effect on melanoma.

[0468] Example 12 Antigen-specific T cells cultured under microwave pressure conditions are used for the treatment of breast cancer

[0469] (1) Preparation of antigen components

[0470] EO771 mouse breast cancer cells were lysed with 8M urea aqueous solution and the lysate components were dissolved to prepare the antigen components of micron particles.

[0471] (2) Preparation of micron particles

[0472] In this embodiment, the preparation of micron particles 1 adopts the double emulsion method. The molecular weight of the micron particle 1 skeleton material PLA is 30-40KDa. During the preparation, the antigen component is first loaded inside, and then 100mg of the micron particles are centrifuged at 8000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The average particle size of the micron particles is about 3.0μm, and each 1mg of PLGA micron particles is loaded with about 600μg of protein or polypeptide components.

[0473] (3) Sorting of tumor antigen-specific T cells

[0474] Female C57BL / 6 mice aged 6-8 weeks were selected and injected subcutaneously with 1.0×10 6 EO771 mouse breast cancer cells. The mice were sacrificed on day 18 and the mouse spleen cells were collected. 80 million spleen cells and microparticles 1 (50 mg) were co-incubated in 5 mL RPMI1640 complete medium for 24 hours, and then CD3 + CD137+ T cells (cell viability 80%) are tumor antigen-specific T cells.

[0475] (4) Cultivation of tumor antigen-specific T cells

[0476] Two million tumor antigen-specific T cells were cultured in 10 mL of DMEM complete medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) at 37.0°C, volume fraction 5.0% CO2 for 10 minutes (cell viability 10%) under microwave stress conditions (wavelength of 1 cm), and then cultured in DMEM complete medium (37.0°C, 5.0% CO2) for 4 days to expand T cells (cell viability 80%), and the resulting cells were T cells 1 (T cells 1).

[0477] Two million tumor antigen-specific T cells were directly cultured in DMEM complete medium (37°C, 5% CO2, containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibodies) for 4 days to expand T cells (cell viability 90%), and the resulting cells were T cells 2 (T cells 2).

[0478] (5) Antigen-specific T cells for cancer prevention

[0479] Female BALB / c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. One day before adoptive transfer of cells into mice, 100 mg / kg of cyclophosphamide was intraperitoneally injected into the recipient mice to eliminate immune cells in the recipient mice. On day 0, 1 million T cells 1 (T cells 1) or T cells 2 (T cells 2) were subcutaneously injected into the mice. At the same time, 1.0×10 6 EO771 cells, mouse tumor volume and survival monitoring methods are the same as above.

[0480] (6) Experimental results

[0481] like Fig.12 As shown, compared with the control group, the tumor growth rate in the T cell treatment group was significantly slowed down and the survival period was significantly prolonged. Moreover, the effect of T cell 1 was significantly better than that of T cell 2.

[0482] Example 13 Antigen-specific T cells cultured and expanded under high temperature, acidic and oxidative stress conditions efficiently kill lung cancer cells

[0483] In this example, mixed cell lysates of various human lung cancer cell lines were loaded onto nanoparticles, and then the nanoparticles were used to sort and amplify tumor antigen-specific T cells in the peripheral immune organs of lung cancer patients. After culturing and amplifying the T cells under stress conditions, they were used to kill autologous cancer cells.

[0484] (1) Preparation of antigen components

[0485] Human lung cancer cell lines A549 cells, H1975 cells, H1650 cells, H1299 cells, PC9 cells, H358 cells, H226 cells, H520 cells, H460 cells and SK-MES-1 cells were cultured respectively.

[0486] The above 8 types of cells were collected separately and mixed at a cell number ratio of 1:1. The mixed cancer cells were then lysed with a 6M guanidine sulfate aqueous solution and the lysate was completely dissolved to prepare the antigen component of the nanoparticles.

[0487] (2) Preparation of Nanoparticles Loaded with Whole Cell Components

[0488] In the present embodiment, nanoparticle 1 is prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA is 20KDa-40KDa, and the adjuvant is poly(I:C), CpG7909 and CpG2395. The preparation method is as described above, firstly, the antigen component and adjuvant are loaded inside the nanoparticle, and then 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and lyophilized for 48h after resuspending with 10mL of ultrapure water containing 4% trehalose. The average particle size of the nanoparticles is about 280nm, and each 1mg of PLGA nanoparticles is loaded with about 800μg of protein or polypeptide components, and each of poly(I:C), CpG7909 and CpG2395 is loaded with 0.05mg.

[0489] (3) T cell sorting and expansion

[0490] The tumor tissue of non-small cell lung cancer patient A shrank after immunotherapy. 10mL of peripheral blood was extracted from non-small cell lung cancer patient A two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMC) were separated from 10mL of peripheral blood of non-small cell lung cancer patients using gradient centrifugation. Nanoparticles (10mg) and PBMC (10 million) were co-incubated in 10mL of AIM V serum-free medium for 48 hours (37.0℃, 5.0% CO2). CD3 was then sorted from the cells using flow cytometry. + CD137 + T cells are tumor antigen-specific T cells.

[0491] (4) Cultivation and expansion of tumor antigen-specific T cells

[0492] 100,000 tumor antigen-specific T cells were cultured in 10 mL of LONZAX-VIVO 15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibodies) under high temperature (38.5°C), acidic (pH = 4.0) and oxidative (10 nM hypochlorous acid) conditions at 37.0°C, with a volume fraction of 5.0% CO2, for 12 days (the medium was changed every two days) to expand the T cells (cell viability 5%) and obtain T cells 1.

[0493] 100,000 tumor antigen-specific T cells were cultured at high temperature (38.5°C) in 10 mL of LONZAX-VIVO 15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) at 37.0°C and 5.0% CO2 for 12 days (the medium was changed every two days) to expand the T cells (cell viability 20%) and obtain T cells 2;

[0494] Alternatively, 100,000 tumor antigen-specific T cells were cultured in 10 mL of LONZAX-VIVO15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) under acidic conditions (pH = 4.0) at 37.0°C and 5.0% CO2 for 12 days (the medium was changed every two days) to expand the T cells (cell viability 25%) and obtain T cells 3;

[0495] 100,000 tumor antigen-specific T cells were cultured in 10 mL of LONZA X-VIVO15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibody) under oxidative conditions (10 nM hypochlorous acid) at 37.0°C and 5.0% CO2 for 12 days (medium was changed every two days) to expand T cells (cell viability 10%) and obtain T cells 4;

[0496] Under conventional conditions, 100,000 tumor antigen-specific T cells were cultured in 10 mL of LONZA X-VIVO 15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2 for 12 days (the medium was changed every two days) to expand the T cells (cell viability 85%) and obtain T cells 5.

[0497] (4) Efficacy of expanded T cells in killing cancer cells

[0498] Select 6-8 week old severely immunodeficient NSG mice and inoculate each nude mouse subcutaneously with 2.0×10 6 Patient A's lung cancer cells were obtained by amplifying cancer cells from the patient. On the 6th and 14th days after tumor inoculation, mice were intravenously injected with 5 million different tumor-specific T cells (T cells 1, T cells 2, T cells 3, T cells 4, T cells 5) or 100 μL PBS. The tumor growth rate and survival of the mice were monitored.

[0499] (5) Experimental results

[0500] like Fig.13 As shown, tumor antigen-specific T cells sorted and expanded in vitro can effectively kill cancer cells in patients. Moreover, tumor antigen-specific T cells expanded under various stress conditions have better effects.

[0501] Example 14: CAR-T cells expanded by culture under high temperature and low oxygen pressure conditions for the treatment of gastric cancer

[0502] (1) Cultivation and expansion of CAR-T cells

[0503] 500,000 mouse CAR-T cells recognizing the antigen Claudin18.2 were cultured and expanded in 10 mL LONZAX-VIVO 15 medium under high temperature (39.0°C) and low oxygen (5.0% oxygen content by volume) stress conditions for 7 days (5.0% CO2 by volume) to obtain T cells 1.

[0504] 500,000 CAR-T cells with the recognition antigen Claudin18.2 were cultured and expanded in 10 mL LONZAX-VIVO 15 medium under high temperature (39.0°C) stress conditions for 7 days (volume fraction 5.0% CO2) to obtain T cells 2 (Tcells2);

[0505] Alternatively, 500,000 CAR-T cells with the recognition antigen Claudin18.2 were cultured and expanded in 10 mL LONZA X-VIVO 15 medium under hypoxic (5.0% oxygen content by volume) stress conditions for 7 days (37.0° C., 5.0% CO2 by volume) to obtain T cells 3;

[0506] Under conventional conditions, 500,000 CAR-T cells recognizing the antigen Claudin18.2 were cultured and expanded in 10 mL LONZAX-VIVO 15 medium for 7 days (37.0° C., volume fraction 5.0% CO2) to obtain T cells 4.

[0507] (2) CAR-T cells expanded by stress culture are used to treat gastric cancer

[0508] On day 0, 1.0×106 MFC mouse gastric cancer cells were subcutaneously inoculated on the back of each mouse. On the 6th, 12th, and 19th days after tumor inoculation, 5 million T cells (T cell 1, or T cell 2, or T cell 3, and 10,000 U IL-2 were injected every two days from day 6) or 100 μL PBS were injected intravenously into the mice. The tumor growth rate and survival of the mice were monitored.

[0509] (3) Experimental results

[0510] like Fig.14 As shown, the mice treated with antigen-specific T cells cultured and expanded under high temperature and low oxygen stress conditions had significantly slower tumor growth and longer survival. Moreover, T cell 1 was more effective than T cell 2 and T cell 3.

[0511] Example 15 Antigen-specific T cells activated by high temperature and acidic stress conditions efficiently kill lung cancer cells

[0512] In this example, mixed cell lysates of various human lung cancer cell lines were loaded onto nanoparticles, and then the nanoparticles were used to sort and amplify tumor antigen-specific T cells in the peripheral immune organs of lung cancer patients. After culturing and amplifying the T cells under stress conditions, they were used to kill autologous cancer cells.

[0513] (1) Preparation of antigen components

[0514] Human lung cancer cell lines A549 cells, H1975 cells, H1650 cells, H1299 cells, PC9 cells, H358 cells, H226 cells, H520 cells, H460 cells and SK-MES-1 cells were cultured respectively.

[0515] The above 8 types of cells were collected separately and mixed at a cell number ratio of 1:1. The mixed cancer cells were then lysed with a 6M guanidine sulfate aqueous solution and the lysate was completely dissolved to prepare the antigen component of the nanoparticles.

[0516] (2) Preparation of Nanoparticles Loaded with Whole Cell Components

[0517] In the present embodiment, nanoparticle 1 is prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA is 20KDa-40KDa, and the adjuvant is poly(I:C), CpG7909 and CpG2395. The preparation method is as described above, firstly, the antigen component and adjuvant are loaded inside the nanoparticle, and then 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and lyophilized for 48h after resuspending with 10mL of ultrapure water containing 4% trehalose. The average particle size of the nanoparticles is about 280nm, and each 1mg of PLGA nanoparticles is loaded with about 800μg of protein or polypeptide components, and each of poly(I:C), CpG7909 and CpG2395 is loaded with 0.05mg.

[0518] (3) T cell activation under stress conditions

[0519] The tumor tissue of non-small cell lung cancer patient A shrank after immunotherapy. 10 mL of peripheral blood of non-small cell lung cancer patient A was extracted two weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were separated from 10 mL of peripheral blood of non-small cell lung cancer patient A using gradient centrifugation.

[0520] Nanoparticles (10 mg) were co-incubated with PBMC (10 million) in 10 mL of AIM V serum-free medium under high temperature (38.5°C) and acidic (pH = 6.8) pressure conditions for 48 h (volume fraction 5.0% CO2). CD3 + HLA-DR + T cells are tumor antigen-specific T cells 1 (T cells 1, cell viability 15%).

[0521] Nanoparticles (10 mg) were co-incubated with PBMC (10 million) in 10 mL of AIM V serum-free medium under high temperature (38.5°C) pressure conditions for 48 h (volume fraction 5.0% CO2). CD3 + HLA-DR + T cells, namely tumor antigen-specific T cells 2 (T cells 2).

[0522] Nanoparticles (10 mg) were co-incubated with PBMC (10 million) under acidic (pH = 6.8) stress conditions in 10 mL of AIM V serum-free medium for 48 h (37.0°C, 5.0% CO2). CD3 + HLA-DR +T cells, namely, tumor antigen-specific T cells 3 (T cells 3, cell viability 40%).

[0523] Nanoparticles (10 mg) were co-incubated with PBMC (10 million) in 10 mL of AIM V serum-free medium under conventional conditions for 48 h (37.0°C, 5.0% CO2). CD3 + HLA-DR + T cells, namely, tumor antigen-specific T cells 4 (T cells 4, cell viability 80%).

[0524] (4) Cultivation and expansion of tumor antigen-specific T cells

[0525] The sorted tumor antigen-specific T cells (T cells 1, T cells 2, T cells 3, T cells 4) were then cultured and expanded in 10 mL of LONZA X-VIVO 15 medium (containing 20 ng / mL IL-2 and 5 μg / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2 for 8 days (the medium was changed every two days).

[0526] (4) Efficacy of expanded T cells in killing cancer cells

[0527] Select 6-8 week old severely immunodeficient NSG mice and inoculate each nude mouse subcutaneously with 2.0×10 6 Patient A's lung cancer cells were obtained by amplifying cancer cells from the patient. On the 6th and 16th days after tumor inoculation, mice were intravenously injected with 5 million different tumor-specific T cells (T cells 1, T cells 2, T cells 3, T cells 4) or 100 μL PBS. The tumor growth rate and survival of the mice were monitored.

[0528] (5) Experimental results

[0529] like Fig.15 As shown, tumor antigen-specific T cells activated, sorted and expanded in vitro can effectively kill the patient's own cancer cells. Moreover, the effect of culturing and expanding tumor antigen-specific T cells (T cells 1) under various stress conditions is even better.

[0530] Example 16 T cells activated and cultured under high temperature and oxidative stress conditions are used to treat pancreatic cancer

[0531] (1) Preparation of antigen components

[0532] The cultured Pan02 pancreatic cancer cells were collected, and the cancer cells were lysed with 8M urea aqueous solution, and then the cancer cell lysate components were dissolved, which were the antigen components.

[0533] (2) Preparation of Nanoparticles

[0534] In this embodiment, nanoparticles are prepared by multiple emulsion method. The PLGA molecular weight of the nanoparticles used in nanoparticle 1 is 7KDa-17KDa, and CpG7909 is used as an adjuvant. The preparation method is as described above, firstly, the antigen component and adjuvant are loaded inside the nanoparticles, then 100mg of nanoparticles are centrifuged at 15000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h. The average particle size of nanoparticle 1 is about 260nm, and each 1mg of PLGA nanoparticle 1 is loaded with about 250μg of protein or polypeptide components and about 0.05mg of CpG7909.

[0535] (3) Isolation of tumor antigen-specific T cells

[0536] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1×10 6 Pan02 pancreatic cancer cells were placed in the 400-well plate. 200 μg of αPD-1 antibody was subcutaneously injected on days 8, 10, 12, 14, and 16. Mice were sacrificed on day 18, and spleen cell single cell suspensions were collected.

[0537] 50 million spleen cells were incubated with 5 mg of nanoparticles under hot (39.0°C) and oxidative (100 nM hydrogen peroxide) conditions for 36 h. CD3 + CD137 + T cells (cell viability 10%) are antigen-specific T cells, namely T cells 1.

[0538] 50 million spleen cells were incubated with 5 mg of nanoparticles at high temperature (39.0°C) for 36 hours. CD3 + CD137 + T cells (cell viability 30%), that is, antigen-specific T cells, T cells 2 (Tcells2);

[0539] Alternatively, 50 million spleen cells were incubated with 5 mg of nanoparticles under oxidative conditions (100 nM hydrogen peroxide) for 36 hours. CD3 + CD137 + T cells (cell viability 20%), namely antigen-specific T cells, are T cells 3 (T cells 3);

[0540] Alternatively, 50 million spleen cells were incubated with 5 mg of nanoparticles under normal conditions (37.0°C) for 36 hours. CD3 + CD137 + T cells (cell viability 70%), namely antigen-specific T cells, are T cells 4 (Tcells 4).

[0541] (4) Cultivation and expansion of tumor antigen-specific T cells

[0542] Ten million antigen-specific T cells (T cells 1, T cells 2, T cells 3, T cells 4) were cultured and expanded under high temperature (39.0°C) and oxidative (100 nM hydrogen peroxide) conditions in 50 mL of RPMI1640 complete medium (containing 20 ng / mL IL-2 and 1000 ng / mL αCD3 / αCD28 antibodies) at 37.0°C and 5.0% CO2.

[0543] (5) Antigen-specific T cells for the treatment of pancreatic cancer

[0544] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. At the same time, each mouse was subcutaneously injected with 1×10 6 Pan02 pancreatic cancer cells. On days 8 and 18, mice were intravenously injected with 500,000 tumor antigen-specific T cells (10,000 U of IL-2 were intravenously injected every two days starting from day 8). The tumor volume of mice was monitored and recorded as above.

[0545] (6) Experimental results

[0546] like Fig.16 As shown, tumor antigen-specific T cells can effectively treat pancreatic cancer, and the antigen-specific T cells activated, sorted, cultured and amplified under the pressure conditions of the present invention are more effective.

[0547] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A method for preparing antigen-specific T cells, characterized in that: The method comprises activating and amplifying immune cells separated from peripheral blood, tumor tissue or peripheral immune organs to obtain the antigen-specific T cells; or, amplifying and culturing the immune cells obtained by gene editing technology to obtain the antigen-specific T cells; Among them, the activation culture and / or expansion culture step is to culture the separated cells or gene-edited cells under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction.

2. The preparation method according to claim 1, characterized in that: Controlling the high temperature to be 37.5°C to 42.0°C; And / or, controlling the low temperature to be 4.0°C to 32.0°C; And / or, controlling the hypoxia to have an oxygen volume content of 3.0% to 16.0%; And / or, controlling the low pH to a pH of 4.0 to 6.8; And / or, controlling the high pH to be pH 7.6-8.0; And / or, controlling the high CO2 to have a CO2 volume content of 5.1% to 15.0%; And / or, the condition for controlling the ultrasound is that the ultrasound frequency is greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 ; and / or, controlling the electromagnetic field dose to be 20 μSv-1000 mSv; and / or, controlling the electron beam dose to be 0.2 to 20 Gy; And / or, the irradiation includes irradiation with ultraviolet rays, infrared rays, X-rays, α rays, β rays or γ rays; preferably, the intensity of the ultraviolet rays is controlled to be 5 to 200 μW / cm 2 , with a wavelength of 100 to 400 nm; preferably, the dose of the X-ray is 0.2 to 20 Gy; and / or, controlling the terahertz wavelength of the terahertz electromagnetic wave to be 30 to 3000 μm; And / or, controlling the wavelength of the microwave to be 1 mm to 1 m; And / or, the oxidation is activation culture and / or expansion culture in the presence of an oxidant; And / or, the reduction is activation culture and / or expansion culture in the presence of a reducing agent.

3. The preparation method according to claim 2, characterized in that: The oxidant is selected from one or more of peroxide, hypochlorous acid, sulfate, dichromate, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, KIO3, KBrO3, ClO3-, ClO4-, Na2O2, K2O2, MgO2, CaO2, BaO2, NO3-, MnO4-, F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3; preferably peroxide and / or hypochlorous acid; Preferably, the peroxide is selected from hydrogen peroxide, peracetic acid and sodium peroxide; Preferably, the concentration of the oxidant is controlled to be 0.0001-1000 μM.

4. The preparation method according to claim 2, characterized in that: The reducing agent is selected from a sulfur-containing reducing agent, a ferrous salt, and a copper-containing reducing agent; Preferably, the sulfur-containing reducing agent is selected from one or more of glutathione, thiourea dioxide, sodium sulfide (Na2S), thiourea, thioacetic acid, TCEP, DTT, and mercaptoethanol; preferably glutathione; Preferably, the concentration of the reducing agent is controlled to be 0.0001-1000 μM.

5. The preparation method according to claim 1, characterized in that: The antigen-specific T cells are one or more of tumor antigen-specific T cells, bacterial antigen-specific T cells, viral antigen-specific T cells or self-antigen-specific T cells; Preferably, the tumor antigen-specific T cells include monoclonal tumor antigen-specific T cells and polyclonal tumor antigen-specific T cells; Preferably, the gene-edited cells include one or more of CAR-T, TCR-T, and TILs; Preferably, the activation culture time is 0.05 hours to 168 hours, preferably 4 hours to 96 hours; Preferably, the amplification culture time is 0.05 hours to 42 days, preferably 1 hour to 30 days.

6. The preparation method according to claims 1 to 5, characterized in that: The antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises: (1) activating and culturing the isolated immune cells, and then sorting the activated and cultured cells to obtain positive cells; (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells; Wherein, the activation culture is carried out under one or more conditions selected from the group consisting of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction; Preferably, the step of activating the culture is selected from any one or more of the following: (1-1) co-culturing the separated immune cells and the particles loaded with the antigen components at 37.5° C. to 42.0° C. and pH 4.0 to 6.8; (1-2) co-culturing the isolated immune cells and the particles loaded with the antigen components under the conditions of pH 4.0 to 6.8 and an oxidant concentration of 0.0001 to 1000 μM; (1-3) The separated immune cells and the particles loaded with the antigen components are co-cultured at 37.5°C to 42.0°C and in an oxidant concentration of 0.0001 to 1000 μM.

7. The preparation method according to claims 1 to 5, characterized in that: The antigen-specific T cells are polyclonal tumor antigen-specific T cells, and the method comprises: (1) activating and culturing the isolated immune cells, and sorting the activated and cultured cells to obtain positive cells; (2) amplifying and culturing the positive cells, and sorting to obtain the antigen-specific T cells; Wherein, the amplification culture is carried out under one or more conditions of high temperature, low temperature, low oxygen, low pH, high pH, ​​ultrasound, radiation, electromagnetic field, terahertz electromagnetic wave, microwave, electron beam, high CO2, oxidation and reduction; The step of activating the culture is selected from any one or more of the following: (1-4) co-culturing the isolated immune cells, particles loaded with antigen components, and antigen presenting cells; (1-5) co-culturing the isolated immune cells and particles loaded with antigen components; (1-6) The particles loaded with antigen components are first contacted with antigen presenting cells, and the antigen presenting cells after contact are then co-cultured with immune cells.

8. The preparation method according to claim 7, characterized in that: The step of amplification and cultivation is specifically selected from one or more of the following: (2-1) culturing the positive cells at 37.5°C to 42.0°C or 4.0°C to 32.0°C for 1 hour to 42 days, preferably 1 hour to 21 days; (2-2) culturing the positive cells in the presence of an oxidant or a reducing agent for 1 hour to 42 days, preferably 1 day to 21 days; (2-3) culturing the positive cells at pH 4.0 to 6.8 or pH 7.6 to 8.0 for 1 hour to 42 days, preferably 1 day to 21 days; (2-4) culturing the positive cells under ultraviolet light, infrared light, X-ray, α-ray, β-ray or γ-ray irradiation for 0.05 hours to 42 days; preferably 1 hour to 21 days; preferably, controlling the intensity of the ultraviolet light to be 5 to 200 μW / cm 2 , with a wavelength of 100 to 400 nm; preferably, the X-ray dose is 0.2 to 20 Gy, and the irradiation time is 0.05 hours to 24 hours; (2-5) culturing the positive cells under conditions of an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days; (2-6) culturing the positive cells under conditions of a CO2 volume content of 5.1% to 15.0% for 1 hour to 42 days, preferably 1 day to 21 days; (2-7) Make positive cells at an ultrasonic frequency greater than 2×10 4 Hz, ultrasonic intensity greater than 0.003W / cm 2 Under the conditions, culture for 1 hour to 42 days, preferably 1 day to 21 days; (2-8) culturing the positive cells under the condition of a terahertz wavelength of 30 to 3000 μm for 1 hour to 42 days, preferably 1 day to 21 days; (2-9) culturing the positive cells under microwave conditions with a wavelength of 1 mm to 1 m or a frequency of 300 MHz to 300 GHz for 5 min to 600 min; (2-10) culturing the positive cells under conditions of pH 4.0 to 6.8 and oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days; (2-11) culturing the positive cells at 37.5°C to 42.0°C, pH 4.0 to 6.8, and in the presence of an oxidant at a concentration of 0.0001 to 1000 μM for 1 hour to 42 days, preferably 1 day to 21 days; (2-12) The positive cells are cultured at 37.5°C to 42.0°C and an oxygen volume content of 3.0% to 16.0% for 1 hour to 42 days, preferably 1 day to 21 days.

9. The preparation method according to claim 1, characterized in that: The expansion culture is carried out in the presence of interleukin, antibody and / or granulocyte-macrophage colony stimulating factor; Preferably, the interleukin is selected from one or more of IL-2, IL-7, IL-5, IL-12, IL-15, IL-17, and IL-21, preferably one or more of IL-2, IL-15, and IL-7; Preferably, the antibody is selected from anti-CD3 antibody and / or anti-CD28 antibody.

10. The preparation method according to claim 6 or 7, characterized in that: The antigen component is selected from one or more of the following: (a) Whole cell lysate fraction of cancer cells / tumor tissues; (b) A portion of the whole cell fraction consisting of the protein and peptide components of the whole cell lysate fraction of cancer cells / tumor tissues; (c) The protein and peptide components of the whole cell fraction of cancer cells / tumor tissues plus the mRNA component; (d) Tumor specific antigen (TSA); (e) Tumor associated antigen (TAA); Preferably, the concentration of the particles loaded with antigen components is 2.5 ng / mL to 50 mg / mL; Preferably, the particles are nanoparticles and / or microparticles; Preferably, the concentration of the antigen presenting cells is 1 to 50 million / mL; Preferably, the antigen presenting cells are selected from one or more of B cells, DC cells and macrophages.

11. An antigen-specific T cell obtained by the preparation method according to any one of claims 1 to 10, Preferred site, specific antigen specific T cell selection own CD3 + CD69 + , CD3 + CD8 + CD69 + , CD3 + CD4 + CD69+, CD3 + CD137 + , CD3 + CD4 + CD137 + , CD3 + CD8 + CD137 + , CD3 + CD25 + , CD3 + CD8 + CD25 + , CD3 + CD4 + CD25 + , CD3 + CD134 + , CD3 + CD8 + CD134 + , CD3 + CD4 + CD134 + , CD3 + IL-2R + , CD3 + CD8 + IL-2R + , CD3 + CD4 + IL-2R + , CD3 + HLA-DR + , CD3 + CD8 + HLA-DR + , CD3 + CD4 + HLA-DR + , CD3 + FAS L + , CD3 + CD8 + FAS L + , CD3 + CD4 + FAS L + , CD3 + OX40 + , CD3 + CD8 + OX40 + 、CD3 + CD4 + OX40 + 、CD3 + TCF-1 + 、CD3 + CD8 + TCF-1 + 、CD3 + CD4 + TCF-1 + 、CD3 + PD-1 + 、CD3 + CD8 + PD-1 + 、CD3 + CD4 + PD-1 + 、CD3 + CD39 + 、CD3 + CD8 + CD39 + 、CD3 + CD4 + CD39 + 、CD3 + CD38 + 、CD3 + CD8 + CD38 + 、CD3 + CD4 + CD38 + 、CD3 + CD28 + 、CD3 + CD8 + CD28 + 、CD3 + CD4 + CD28 + 、CD3 + CD71 + 、CD3 + CD8 + CD71 + 、CD3 + CD4 + CD71 + 、CD3 + CD44 + 、CD3 + CD8 + CD44 + 、CD3 + CD4 + CD44 + 、CD3 + CXCR3 + 、CD3 + CD8 + CXCR3 + 、CD3 + CD4 + CXCR3 + 、CD3 + CXCR1 + 、CD3 + CD8 + CXCR1 + 、CD3 + CD4 + CXCR1 + 、CD3 + ICAM-1 + 、CD3 + CD8 + ICAM-1 + 、CD3 + CD4 + ICAM-1 + 、CD3 + CD70 + 、CD3 + CD8 + CD70 + 、CD3 + CD4 + CD70 + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD62L + 、CD3 + CD8 + CD62L + 、CD3 + CD4 + CD62L + 、CD3 + CD154 + 、CD3 + CD8 + CD154 + 、CD3 + CD4 + CD154 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + ICOS + 、CD3 + CD8 + ICOS + 、CD3 + CD4 + ICOS + 、CD3 + CD27 + 、CD3 + CD8 + CD27 + 、CD3 + CD4 + CD27 + 、CD3 + CD107A + 、CD3 + CD8 + CD107A + and CD3 + CD4 + CD107A + Any one or more of the above T cells.

12. Use of the antigen-specific T cells obtained by the preparation method according to any one of claims 1 to 10 or the antigen-specific T cells according to claim 11 in the preparation of drugs for treating or preventing diseases, wherein the diseases include cancer, autoimmune diseases, diseases caused by bacterial infections, and diseases caused by viral infections; Preferably, the tumor is selected from solid tumors and / or hematological tumors; Preferably, the tumor includes lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant hematologic tumor, brain tumor, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, One or more of the following: thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, metastatic cancer, circulating tumors.

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