Cytokine combination, culture medium, tumor-specific T cell population and application thereof

Abstract

The present invention relates to the field of cells, and in particular to a cytokine combination, a culture medium, a tumor-specific T cell group and its application. The present invention first activates the T cells by solidifying CD3 monoclonal antibodies, and the T cells secrete some cytokines. At the same time, under the action of exogenous cytokines, the monocytes in the peripheral blood lymphocytes mature into dendritic cells; unactivated peripheral blood lymphocytes are added again, and under the action of dendritic cells and cytokines, the T cells are activated and proliferated; then the activation and proliferation of T cells are further promoted, thereby obtaining a tumor-specific T cell group, which can directly kill the tumor and inhibit its growth.

Description

Technical Field

[0001] The present invention relates to the field of cells, and in particular to a cytokine combination, a culture medium, a tumor-specific T cell population and applications thereof. Background Art

[0002] Lung cancer is one of the most common and lethal cancers. It is highly malignant, progresses rapidly, has a high recurrence rate after surgery, and has poor treatment effects. Non-small cell lung cancer (NSCLC) is a common form of lung cancer. About 2 million people are diagnosed with lung cancer each year, resulting in 20,000 deaths. Non-small cell lung cancer accounts for 80-85% of lung cancer cases. Patients with advanced NSCLC can currently receive a variety of treatments, including traditional radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy. At present, antibody-drug conjugates (ADC), immune checkpoint inhibitors, and cell therapy products (TIL, CAR-T, DC, CIK, DC-CIK, NKT, γδT) are also used in lung cancer research. Cell therapy for lung cancer has established its anti-tumor effect in basic research and clinical trials. Further enhancing the specificity, recognition function, and anti-tumor ability of immune cells through genetic engineering modification, bispecific antibodies, and immune checkpoint molecular intervention is an important means of immunotherapy in the future. Although several cell therapy products for certain types of cancer have been approved for marketing worldwide, cell therapy products specifically for lung cancer have not yet been approved for marketing. Lung cancer has the highest incidence rate among all cancers, so there is an urgent need for cell therapy products that can target lung cancer and be used in the treatment of lung cancer. Summary of the invention

[0003] In view of this, the present invention provides a cytokine combination, culture medium, tumor-specific T cell group and its application. The tumor-specific T cell group provided by the present invention is a non-genetically modified autologous T cell group. It is a mixed T lymphocyte obtained by in vitro activation and amplification culture of mononuclear cells (PBMC) in the patient's own peripheral blood by CD3 antibody combined with cytokines, and then frozen and stored by adding a therapeutic cell cryoprotectant. After being re-infused into the patient's body, it can supplement the patient's body with specific T lymphocytes with natural anti-tumor ability, improve the body's immunity, and enhance the monitoring and killing of tumor cells by T cells.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a cytokine combination, the cytokine combination comprising a primary activation cytokine and an amplification cytokine:

[0006] The primary activated cytokines include:

[0007] rhIL-2: 400~600 IU / mL;

[0008] rhIL-4: 40-60 IU / mL; and

[0009] G-CSF: 3500~4500 IU / mL;

[0010] The amplification cytokines include:

[0011] rhIL-2: 400~600 IU / mL.

[0012] In some specific embodiments of the present invention, the primary activation cytokine comprises:

[0013] rhIL-2: 400 IU / mL;

[0014] rhIL-4: 40 IU / mL; and

[0015] G-CSF: 3500 IU / mL;

[0016] The amplification cytokines include:

[0017] rhIL-2: 400 IU / mL; or

[0018] The primary activated cytokines include:

[0019] rhIL-2: 600 IU / mL;

[0020] rhIL-4: 60 IU / mL; and

[0021] G-CSF: 4500 IU / mL;

[0022] The amplification cytokines include:

[0023] rhIL-2: 600 IU / mL.

[0024] In a second aspect, the present invention further provides the use of the cytokine combination in any of the following:

[0025] (I) preparing tumor-specific T cell populations;

[0026] (II), preparing a culture medium for tumor-specific T cell populations; and / or

[0027] (III) Preparation of anti-lung cancer drugs.

[0028] In a third aspect, the present invention also provides a culture medium for preparing a tumor-specific T cell population, wherein the culture medium comprises the cytokine combination and a complete culture medium;

[0029] The complete culture medium includes a basal culture medium and 0.5% to 10% autologous plasma; preferably, the amount of autologous plasma added includes 1% to 3%;

[0030] The basal culture medium includes a serum-free culture medium.

[0031] In a fourth aspect, the present invention further provides the use of the culture medium in any of the following:

[0032] (I), preparing tumor-specific T cell populations; and / or

[0033] (II) Preparation of anti-lung cancer drugs.

[0034] In a fifth aspect, the present invention further provides a tumor-specific T cell population, the T cell population comprising:

[0035] CD3+ / CD45+: 85%~95%;

[0036] CD3+CD8+CD4- / CD3+: 30%~65%;

[0037] CD3-(CD16+or CD56+) / CD45+: 1%~10%;

[0038] CD3-CD19+ / CD45+: 1%~5%; and / or

[0039] CD3+CD8+Granzyme B+ / CD3+CD8+: 70%~90%; or

[0040] The T cell population includes:

[0041] CD3+ / CD45+: 94.07±3.04%~97.83±1.50%;

[0042] CD3+CD8+CD4- / CD3+: 57.13±9.00%~65.28±0.35%;

[0043] CD3-(CD16+or CD56+) / CD45+: 1.45±0.31%~2.72±0.32%;

[0044] CD3-CD19+ / CD45+: 0.27±0.12%~3.00±1.55%.

[0045] In some specific embodiments of the present invention, the T cell population comprises:

[0046] CD3+ / CD45+: 94.07±3.04%, 97.83±1.50%, 97.63±1.39%, 96.27±1.46%, 96.92±2.68%, 96.57±0.57%, 94.13±3.88%, or 96.90±1.04%;

[0047] CD3+CD8+CD4- / CD3+: 57.98±9.66%, 62.56±2.84%, 62.91±4.83%, 57.13±9.00%, 61.87±4.54%, 65.28±0.35%, 63.76±0.53% or 67.26±0.53%;

[0048] CD3-(CD16+or CD56+) / CD45+: 2.62±1.58%, 1.45±0.31%, 1.53±0.42%, 2.32±0.36%, 1.48±0.57%, 2.72±0.32%, 2.19±0.58% or 2.06±0.56%; or

[0049] CD3-CD19+ / CD45+: 3.00±1.55%, 0.33±0.06%, 0.27±0.12%, 1.27±1.16%, 0.35±0.09%, 0.73±0.40%, 0.70±0.46% or 0.57±0.32%.

[0050] In some specific embodiments of the present invention, the T cell population comprises:

[0051] CD3+ / CD45+: 94.07±3.04%;

[0052] CD3+CD8+CD4- / CD3+: 57.98±9.66%;

[0053] CD3-(CD16+or CD56+) / CD45+: 2.62±1.58%;

[0054] CD3-CD19+ / CD45+: 3.00±1.55%; or

[0055] The T cell population includes:

[0056] CD3+ / CD45+: 97.83±1.50%;

[0057] CD3+CD8+CD4- / CD3+: 62.56±2.84%;

[0058] CD3-(CD16+or CD56+) / CD45+: 1.45±0.31%;

[0059] CD3-CD19+ / CD45+: 0.33±0.06%; or

[0060] The T cell population includes:

[0061] CD3+ / CD45+: 97.63±1.39%;

[0062] CD3+CD8+CD4- / CD3+: 62.91±4.83%;

[0063] CD3-(CD16+or CD56+) / CD45+: 1.53±0.42%;

[0064] CD3-CD19+ / CD45+: 0.27±0.12%; or

[0065] The T cell population includes:

[0066] CD3+ / CD45+: 96.27±1.46%;

[0067] CD3+CD8+CD4- / CD3+: 57.13±9.00%;

[0068] CD3-(CD16+or CD56+) / CD45+: 2.32±0.36%;

[0069] CD3-CD19+ / CD45+: 1.27±1.16%; or

[0070] The T cell population includes:

[0071] CD3+ / CD45+: 96.92±2.68%;

[0072] CD3+CD8+CD4- / CD3+: 61.87±4.54%;

[0073] CD3-(CD16+or CD56+) / CD45+: 1.48±0.57%;

[0074] CD3-CD19+ / CD45+: 0.35±0.09%; or

[0075] The T cell population includes:

[0076] CD3+ / CD45+: 96.57±0.57%;

[0077] CD3+CD8+CD4- / CD3+: 65.28±0.35%;

[0078] CD3-(CD16+or CD56+) / CD45+: 2.72±0.32%;

[0079] CD3-CD19+ / CD45+: 0.73±0.40%; or

[0080] The T cell population includes:

[0081] CD3+ / CD45+: 94.13±3.88%;

[0082] CD3+CD8+CD4- / CD3+: 63.76±0.53%;

[0083] CD3-(CD16+or CD56+) / CD45+: 2.19±0.58%;

[0084] CD3-CD19+ / CD45+: 0.70±0.46%; or

[0085] The T cell population includes:

[0086] CD3+ / CD45+: 96.90±1.04%;

[0087] CD3+CD8+CD4- / CD3+: 67.26±0.53%;

[0088] CD3-(CD16+or CD56+) / CD45+: 2.06±0.56%;

[0089] CD3-CD19+ / CD45+: 0.57±0.32%.

[0090] In some specific embodiments of the present invention, the preparation method comprises the following steps:

[0091] Step 1, separation of peripheral PBMC-enriched blood: diluent is diluted at a ratio of 1:4 to obtain diluted PBMC;

[0092] Step 2, PBMC pre-culture: taking the diluted PBMC and culturing them with basal culture medium and CD3 monoclonal antibody to obtain pre-cultured PBMC, taking another diluted PBMC and refrigerating and preserving them to obtain refrigerated PBMC;

[0093] Step 3, primary activation culture: adding the primary activation cytokine in the cytokine combination to the pre-cultured PBMC, and continuing the culture to obtain a primary activation culture sample;

[0094] Step 4, secondary activation culture: adding the primary activation culture sample to the refrigerated PBMC, and continuing to culture to obtain an activated culture;

[0095] Step 5, expansion culture of tumor-specific T cell population: using the complete medium in the culture medium and the expansion cytokine in the cytokine combination to expand and culture the activated culture for 96 hours;

[0096] The basal culture medium includes a serum-free culture medium.

[0097] In some specific embodiments of the present invention, the CD3 monoclonal antibody comprises OKT3;

[0098] The CD3 monoclonal antibody concentration ranges from 1 to 16 μg / mL.

[0099] In some specific embodiments of the present invention, the CD3 monoclonal antibody concentration is 2-8 μg / mL.

[0100] In a sixth aspect, the present invention further provides a method for preparing a tumor-specific T cell population, the preparation method comprising the following steps:

[0101] Step 1, separation of peripheral PBMC-enriched blood: diluent is diluted at a ratio of 1:4 to obtain diluted PBMC;

[0102] Step 2, PBMC pre-culture: taking the diluted PBMC and culturing them with the basal medium and CD3 monoclonal antibody to obtain pre-cultured PBMC, and taking another diluted PBMC and refrigerating and preserving them to obtain refrigerated PBMC;

[0103] Step 3, primary activation culture: adding the primary activation cytokine in the cytokine combination to the pre-cultured PBMC, and continuing the culture to obtain a primary activation culture sample;

[0104] Step 4, secondary activation culture: adding the primary activation culture sample to the refrigerated PBMC, and continuing to culture to obtain an activated culture;

[0105] Step 5, expansion culture of tumor-specific T cell population: using the complete culture medium in the culture medium and the expansion cytokine in the cytokine combination to expand and culture the activated culture for 96 hours.

[0106] In some specific embodiments of the present invention, the CD3 monoclonal antibody comprises OKT3;

[0107] The CD3 monoclonal antibody concentration ranges from 1 to 16 μg / mL.

[0108] In some specific embodiments of the present invention, the CD3 monoclonal antibody concentration is 2-8 μg / mL.

[0109] In a seventh aspect, the present invention also provides a T cell population obtained by the preparation method.

[0110] In an eighth aspect, the present invention also provides the use of the T cell population in the preparation of anti-lung cancer drugs.

[0111] In some specific embodiments of the present invention, the lung cancer comprises non-small cell lung cancer.

[0112] In a ninth aspect, the present invention also provides an anti-lung cancer drug, comprising the T cell population.

[0113] The present invention first activates T cells through solidified CD3 monoclonal antibodies, and T cells auto-secrete some cytokines. At the same time, under the action of exogenous cytokines, monocytes in peripheral blood lymphocytes mature into dendritic cells. Unactivated peripheral blood lymphocytes are added again, and under the action of dendritic cells and cytokines, T cells are activated and proliferate. Subsequently, the activation and proliferation of T cells are further promoted, thereby obtaining a tumor-specific T cell population, which can directly kill the tumor and inhibit its growth.

[0114] The beneficial effects of the tumor-specific T cell population provided by the present invention are as follows:

[0115] 1) Tumor specificity: It has specific recognition of tumor antigens and can be reactivated by tumor antigens after being infused back into the body, thus killing tumor cells quickly and directly;

[0116] 2) Highly efficient in vivo expansion: Overcoming the immunosuppression of the tumor microenvironment, having the ability to self-renew and replicate, being able to efficiently expand in vitro, having a long survival time in vivo, and having long-term anti-tumor effects;

[0117] 3) Anti-tumor persistence: Overcoming the genetic and epigenetic variability of tumors, recognizing tumor antigens and transforming them into new tumor-specific T cells, it produces a large amount of anti-tumor factors such as Granzyme B+, IFN-γ, IL-2, or kills tumors through apoptosis pathways, and continuously exerts anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0119] Figure 1 The PBMC harvest volume / 1mL peripheral PBMC-enriched blood is shown under different dilution ratios;

[0120] Figure 2 Shows the expansion fold of tumor-specific T cell population under different dilution ratios;

[0121] Figure 3 The cell phenotype proportions (%) of tumor-specific T cell populations cultured under different dilution ratios are shown;

[0122] Figure 4 The expansion times of tumor-specific T cell populations under different cytokine concentration ratios are shown;

[0123] Figure 5 The viability of tumor-specific T cell populations cultured with different complete culture medium formulas is shown;

[0124] Figure 6 The expansion times of tumor-specific T cell populations cultured with different complete culture medium formulas are shown;

[0125] Figure 7 The phenotype proportions of tumor-specific T cell populations cultured with different complete culture medium formulas (%) are shown;

[0126] Figure 8 Shows the expansion fold of tumor-specific T cell population under different expansion time conditions;

[0127] Fig. 9 The comparison of the killing activity of tumor-specific T cell populations cultured at different expansion times with the same effector target ratio is shown;

[0128] Fig.10 The cell viability was shown at different autologous plasma addition amounts;

[0129] Fig.11 Shows the expansion times of different autologous plasma addition amounts;

[0130] Fig.12 The phenotype of cells was shown with different amounts of autologous plasma added;

[0131] Fig.13 The statistics of cytokine secretion content of tumor-specific T cell populations in each batch are shown;

[0132] Fig.14 The data of the in vitro killing of tumor-specific T cell population by injection (RTCA method) changes with the co-culture time; a is the normalized cell index graph; b is the killing activity graph;

[0133] Fig.15 The results of in vitro killing by injection of tumor-specific T cell population (RTCA method) and co-culture for 72 hours are shown; a is the normalized cell index graph; b is the killing activity graph;

[0134] Fig.16The figure shows the comparison of fluorescence intensity values ​​(RLU) of each effector-target ratio in the in vitro killing test of the injection of tumor-specific T cell population after sorting and purification (3 batches combined);

[0135] Fig.17 The comparison of killing activity (%) of each effector-target ratio in the in vitro killing test of the injection of tumor-specific T cell population after sorting and purification (3 batches combined);

[0136] Fig.18 The figure shows the comparison of fluorescence intensity values ​​(RLU) of each effector-target ratio in the in vitro killing blocking test (3 batches combined) of tumor-specific T cell population injection liquid;

[0137] Fig.19 The comparison of killing activity (%) of each effector-target ratio in the in vitro killing blocking test of tumor-specific T cell population injection (3 batches combined);

[0138] Fig. 20 The fluorescence intensity values ​​of each effector-target ratio (RLU) of the tumor-specific T cell population injection in vitro killing test after different antigen stimulation are shown;

[0139] Fig.21 The comparison of killing activity (%) of each effector-target ratio in the in vitro killing test of tumor-specific T cell population injection after different antigen stimulation is shown;

[0140] Fig. 22 Shows the ELISPOT-IFN-γ positive spot statistics; among them, represents p<0.05, represents p<0.01, represents p < 0.001;

[0141] Fig.23 ELISPOT-Granzyme B positive spot statistics are shown; represents p<0.05, represents p<0.01, represents p < 0.001;

[0142] Fig.24 Shows the ELISPOT-IFN-γ plate layout and positive spot diagram;

[0143] Fig.25 Shows the ELISPOT-Granzyme B well plate layout and positive spot diagram;

[0144] Fig.26 The weight change curve of each group of animals during the test period is shown; the ordinate is the weight and the abscissa is the date;

[0145] Fig. 27 The curves showing the changes in tumor volume during the experimental period of each group of animals are shown; the ordinate is the tumor volume, and the abscissa is the date;

[0146] Fig.28 The RTV change curves of each group of animals during the test are shown; the ordinate is RTV and the abscissa is date;

[0147] Fig.29 The curves of T / C (%) changes during the animal experiment in each group are shown; the ordinate is T / C and the abscissa is date;

[0148] Fig.30 The IRTV (%) variation curves of each group of animals during the experimental period are shown; the ordinate is IRTV and the abscissa is date;

[0149] Fig.31 The graphs show the tumor weight of each group of animals during the experimental period; the ordinate is the tumor weight, and the abscissa is the group;

[0150] Fig.32 The tumor pictures of each animal at the end of the experiment are shown;

[0151] Fig.33 The curve showing the change of CD45+ cell ratio during the experiment;

[0152] Fig.34 The curve showing the change of CD45+CD3+ cell ratio during the experiment;

[0153] Fig.35 The curve showing the change of cytokine IFN-γ content during the experiment;

[0154] Fig.36 The curve showing the change of cytokine IL-6 content during the experiment;

[0155] Fig.37 The curve showing the change of cytokine IL-8 content during the experiment;

[0156] Fig.38 The curve showing the change of cytokine IL-10 content during the experiment;

[0157] Fig.39 The curve showing the change of cytokine IL-2 content during the experiment;

[0158] Fig.40 The curve showing the change of cytokine IL-4 content during the experiment;

[0159] Fig.41 The curve showing the change of cytokine IL-17 content during the experiment;

[0160] Fig.42 The curve showing the change of cytokine TNF-α content during the experiment;

[0161] Fig.43The figure shows the changes in tumor volume during the experiment; a is the change in tumor volume of male mice; b is the change in tumor volume of female mice;

[0162] Fig.44 The tumor metastasis during the experiment was shown. DETAILED DESCRIPTION

[0163] The present invention discloses a cytokine combination, a culture medium, a tumor-specific T cell population and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0164] Explanation of relevant terms:

[0165] PBMC: Peripheral blood mononuclear cells, refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells and a small number of other cells.

[0166] NSCLC: Non-small cell lung cancer, is any type of epithelial cell lung cancer except small cell lung cancer; non-small cell lung cancer includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, its cancer cells grow and divide more slowly, and spread and metastasize relatively late.

[0167] rhIL-2: Recombinant human interleukin-2, is a genetically recombinant product, a non-glycosylated protein with the same biological activity as natural IL-2.

[0168] rhIL-4: recombinant human interleukin-4, is a cytokine recombinantly expressed in vitro through biotechnology and has the same biological activity as IL-4 naturally produced in the human body.

[0169] IL-2: Interleukin-2, mainly produced by activated CD4+T cells and CD8+T cells.

[0170] IL-4: Interleukin-4 is a cytokine in the chemokine family and is a cytokine secreted by type II helper T cells (Th2 cells).

[0171] IL-6: Interleukin-6 is a glycoprotein produced by a variety of cells with a molecular weight of approximately 26kDa.

[0172] IL-8: Interleukin-8 is a cytokine in the chemokine family and is expressed in a variety of tumor cells.

[0173] IL-10: Interleukin-10 is a multi-cell-derived, multifunctional cytokine.

[0174] IL-17: Interleukin-17 is an inflammatory cytokine mainly produced by activated T cells.

[0175] TNF-α: Tumor necrosis factor-α, mainly secreted by macrophages, monocytes, neutrophils, CD4+T cells, and NK cells.

[0176] G-CSF: granulocyte colony stimulating factor.

[0177] IFN-γ: short for gamma-interferon, mainly produced by activated T cells and NK cells.

[0178] Granzyme B: Granzyme B is a neutral serine protease with a molecular weight of 29 kDa. It is specifically present in the granules of cytotoxic T lymphocytes (CTL) and natural killer cells (NK) and is released during the degradation of effector cells.

[0179] HCC827: human non-small cell lung cancer cell line.

[0180] HCC827-Luc-GFP: A human non-small cell lung cancer cell line that stably expresses firefly luciferase and green fluorescent protein.

[0181] APC: Antigen presenting cell, refers to a type of immune cell that can take up, process antigens, and present the processed antigens to T cells.

[0182] DC: Dendritic cell, is the most powerful antigen-presenting cell, named because it extends many dendritic or pseudopodia-like processes when mature.

[0183] TCR: T cell receptor, responsible for recognizing specific antigen peptides presented by MHC on the surface of APC, forming a TCR-antigen peptide-MHC complex. TCR is a heterodimer composed of two different peptide chains: about 95% of T cells are αβT cells, whose TCR is composed of α and β chains. MHC molecules are mainly divided into two categories, namely MHC-I molecules and MHC-II molecules.

[0184] HLA-A: Human leukocyte antigen-A.

[0185] HLA-B: Human leukocyte antigen-B.

[0186] HLA-C: human leukocyte antigen-C.

[0187] HLA-DR: human leukocyte antigen-DR.

[0188] HLA-DP: Human leukocyte antigen-DP.

[0189] HLA-DQ: human leukocyte antigen-DQ.

[0190] RTCA: Real-time label-free cell analysis technology. This technology uses a special process to integrate a microelectrode sensor array at the bottom of each cell growth well in the cell culture plate to construct a cell impedance detection sensor system that can track cell morphology, proliferation and differentiation changes in real time, dynamically and quantitatively. When cells growing on the surface of the microelectrode cause changes in the impedance of the adherent electrode interface, this change is correlated with the real-time state change of the cell. Through real-time detection of impedance values, biological information related to physiological functions such as cell growth, extension, morphological changes, infiltration and migration, death and adhesion can be obtained.

[0191] EasySep® is a novel and highly efficient immunomagnetic cell sorting technology that combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system. This technology can be used to separate and purify the main cellular components of tumor-specific T cell population injections.

[0192] RLU (Relative light unit): Relative light unit, used to compare the luminous intensity of different samples. The larger the RLU value, the higher the luminous intensity.

[0193] Cell proliferation detection: It is a basic experimental method to evaluate cell health, genetic toxicity and the effect of anti-tumor drugs. The CellTrace™ Far Red Cell Proliferation Kit uses dye dilution to track multiple generations of cells through flow cytometry analysis. Its red excitation wavelength is 630 nm and the emission wavelength is 661 nm. It can visualize six or more generations of proliferating cells before the cell's inherent autofluorescence masks the labeled fluorescent signal. It is suitable for labeling cells in vitro and in vivo.

[0194] CDX: cell derived xenograft, a human tumor cell line xenograft, refers to a tumor model established by inoculating human tumor cells into immunodeficient mice after in vitro subculture. The common inoculation sites are subcutaneous, in situ or intravenous. Because of its clear tumor growth dynamics, easy genetic manipulation, high tumor formation rate, low price, and ability to generate consistent and reproducible data, it is widely used in cancer research and anticancer drug development, and is the preferred model for in vivo drug screening.

[0195] PDX: A human-derived tumor xenograft model, is a new tumor research model established by directly inoculating patient tumor tissue into immunodeficient mice. This model retains the main histological and genetic characteristics of its donor tumor and can remain stable in subsequent passages. It is a preclinical research model that can maintain high consistency with clinical samples and has high clinical predictive value. It can be used in preclinical drug evaluation, biomarker identification and personalized precision medicine research.

[0196] ELISPOT: Solid-phase enzyme-linked immunosorbent spot technique uses coated antibodies to capture cytokines secreted by cells in culture and presents them in the form of enzyme-linked spot color development.

[0197] RTV: relative tumor volume, refers to the tumor volume at the end of an experimental cycle.

[0198] IRTV: relative tumor inhibition rate, used to evaluate the inhibitory effect of test drugs on tumor growth in vivo (i.e. animal experiments).

[0199] The cytokine combination, culture medium, tumor-specific T cell population and raw materials and reagents used in their application provided by the present invention can all be purchased from the market.

[0200] The present invention will be further described below in conjunction with embodiments:

[0201] Example 1 Preparation of tumor-specific T cell population and parameter optimization

[0202] 1. Preparation of tumor-specific T cell populations

[0203] 1.1. Reagent and solution preparation

[0204] 1.1.1 Preparation of coating solution and coating of culture flasks

[0205] 1.1.1.1 Sterilize the sodium hydroxide volumetric analysis standard solution in an autoclave at 121°C for 30 min. Use the sterilized sodium hydroxide volumetric analysis standard solution to adjust the pH of Dulbecco's phosphate buffered saline (DPBS) to 7.5-10.5.

[0206] 1.1.1.2 Add 500 μg / tube of Recombinant anti-Human CD3 mAb (OKT3) to 50-1000 mL of DPBS with adjusted pH. The concentration of OKT3 (Shanghai Jinan) antibody is 1-16 μg / mL (preferably 2-8 μg / mL).

[0207] 1.1.1.3 Culture flask coating: Add 14±2 mL of coating solution to a 75 cm2 cell culture flask and store it horizontally at room temperature for 4-12 h.

[0208] 1.1.2 Preparation of culture medium

[0209] 1.1.2.1 Serum-free culture medium: CTS™ AIM-V™ Medium (volume: 1 L / bottle) (Thermo).

[0210] 1.1.2.2 Complete culture medium 1:

[0211] rhIL-2: 10-2000 IU / mL (Beijing Shuanglu), rhIL-4: 10-800 IU / mL (Shanghai Jinan), G-CSF: 1000-10000 IU / mL (Qilu Pharmaceutical) (preferably rhIL-2: 400-600 IU / mL, rhIL-4: 40-60 IU / mL, G-CSF: 3500-4500 IU / mL), prepared in serum-free medium.

[0212] 1.1.2.3 Complete culture medium 2:

[0213] rhIL-2: 10-2000 IU / mL (preferably rhIL-2: 400-600 IU / mL) in serum-free medium.

[0214] 1.1.3 Preparation of cleaning solution

[0215] Human albumin (Octefama) was added to DPBS at 1.8% (g / mL) to prepare a washing solution.

[0216] 1.2. Peripheral PBMC-enriched blood collection

[0217] Single blood collection is performed by a qualified institution or hospital, and the peripheral blood mononuclear cells (PBMC) collected are no less than 0.5×10 9 The samples were transported to the laboratory at 2-8℃ cold chain within 24 hours.

[0218] 1.3. PBMC Isolation

[0219] 1.3.1 Compound electrolyte injection (Sichuan Kelun) was used to dilute and mix peripheral PBMC-enriched blood at a ratio of 4:1 (v / v) to prepare a cell suspension. The cell suspension was dispensed into 50 mL conical centrifuge tubes (each centrifuge tube was pre-filled with 13 mL lymphocyte separation solution) at 25 mL / tube and centrifuged at 810±20×g for 20 min. After centrifugation, the PBMC cell layer was aspirated into a 50 mL conical centrifuge tube and centrifuged at 750±20×g for 12 min.

[0220] 1.3.2 Discard the supernatant, take 50-200 mL CTS™ AIM-V™ Medium to mix the cells, and then take samples for counting. Centrifuge at 210±20×g for 20 min, discard the supernatant, take 50-200 mL CTS™ AIM-V™ Medium to mix the cells, divide evenly into 2 centrifuge tubes, and centrifuge at 610±20×g for 10 min.

[0221] 1.4. PBMC pre-culture

[0222] 1.4.1 Washing of coated bottles: discard the coating solution in the bottle, add 30 mL of DPBS solution per bottle to N culture bottles, rinse gently, discard the washing solution, and repeat the washing twice.

[0223] 1.4.2 Take the cells after centrifugation and discard the supernatant. Take one of the cells and add an appropriate amount of CTS™ AIM-V™ Medium to mix the cells. The PBMC seeding density is 1.0-5.0×10 6 / mL (optimum is 2.0×10 6 / mL) and dispense the cell suspension into the washed coated bottles (about 20-40 mL per bottle), and culture in a 38.5℃, 6.5% CO2 constant temperature incubator for 3 h. Adjust the concentration of another cell and place it in a 2-8℃ refrigerator for storage.

[0224] 1.5. One-time activation culture

[0225] 1.5.1 Take out the pre-cultured sample, calculate the required volume of added factors according to the volume of culture medium in the culture bottle, make it into complete culture medium 1 (the final concentration of cytokines in the culture system is rhIL-2: 10-2000 IU / mL, rhIL-4: 10-800 IU / mL, G-CSF: 1000-10000 IU / mL), and continue to culture for 3 hours.

[0226] 1.6. Secondary activation culture

[0227] 1.6.1 Take out the cell suspension from the refrigerator and centrifuge it at 610±20×g for 10 min (for a small number of platelets) or 210±20×g for 20 min (for a large number of platelets).

[0228] 1.6.2 Take out the centrifuged sample, discard the supernatant, and mix the cell pellet with 1 mL of CTS™ AIM-V™ Medium per bottle. Take out the activated culture sample once, add the cell suspension evenly to the culture bottle, and culture it in a constant temperature incubator at 38.5℃ and 6.5% CO2 for 18-24 hours.

[0229] 1.7. Expansion and culture of tumor-specific T cell populations

[0230] 1.7.1 Collect the activated culture into a 250 ml conical bottom centrifuge tube and centrifuge at 610±20×g for 10 min. Discard the supernatant and take complete culture medium 2 to mix the cells and then take a sample for counting.

[0231] 1.7.2 Inoculate at 1000-1500 mL / bag, add autologous plasma at 0.5-10% (preferably 1%-3%) of the inoculation volume, take complete culture medium 2 and adjust the cell suspension to a density of (1.0-5.0)×10 5 The cells were inoculated with 100 viable cells / mL into a cell culture bag and placed in a constant temperature incubator at 38.5°C and 6.5% CO2 for 90-102 h.

[0232] 1.8. Collection and washing of tumor-specific T cell populations

[0233] 1.8.1 Collect the cell suspension in the culture bag into a 250 mL conical centrifuge tube and centrifuge at 610±20×g for 10 min. Discard the supernatant, add the cleaning solution, mix well and count the cells.

[0234] 1.8.2 Centrifuge at 610±20×g for 10 min. Discard the supernatant, add cleaning solution again for washing, and centrifuge under the same conditions.

[0235] 1.9. Finished product mixing, packaging and testing

[0236] 1.9.1 Discard the supernatant and adjust the concentration by (1.0-3.0)×10 7The cryopreservation density was 100 viable cells / mL, and commercially available cryopreservation protective agent for treatment (manufacturer: Nanjing Sansheng; trade name: Gennuo) was added to mix the cells to make a finished product of tumor-specific T cell population injection. The surface leukocyte differentiation antigens and intracellular factor secretion of the finished product cells were detected by flow cytometry, CD3+ / CD45+: 85%-95%, CD3+CD8+CD4- / CD3+: 30%-65%, CD3-(CD16+or CD56+) / CD45+: 1%-10%, CD3-CD19+ / CD45+: 1%-5%, CD3+CD8+Granzyme B+ / CD3+CD8+: 70%-90%.

[0237] 1.10. Program cooling

[0238] The injection solution was divided into 250 mL cell cryopreservation bags at 50 mL / bag, sealed and labeled, and placed in a customized aluminum cryopreservation box. The aluminum cryopreservation box containing the sample was transferred to the program cooling instrument for program cooling (the ambient temperature of the operating room corresponding to the program cooling instrument was set to 18°C ​​in advance, the computer was started to set the relevant parameters of the program cooling instrument, the cryopreservation box containing the sample was transferred to the program cooling instrument, the temperature probe was connected to the corresponding position, the program cooling instrument was started, and the program was set according to the program cooling instrument. The specific operation steps are shown in the table below). After the program cooling is completed, the sample is transferred to liquid nitrogen for storage.

[0239] Table 1

[0240]

[0241] Note: ℃ / m represents the temperature change per minute, S represents the sample temperature, and C represents the chamber temperature.

[0242] Parameter Optimization

[0243] The parameters during the culture of tumor-specific T cell populations were optimized, including the dilution ratio of peripheral PBMC-enriched blood, the concentration of cytokines used in the culture system, the formula of complete culture medium, the expansion culture time, the amount of autologous plasma added, the secretion of IL-2 after cells were cultured with different concentrations of CD3 coating fluid, and the secretion of cytokines by tumor-specific T cell populations.

[0244] 2. Optimization of parameters for preparation of tumor-specific T cell populations

[0245] 2.1. Peripheral PBMC Enrichment Blood Dilution Ratio

[0246] The tumor-specific T cell population was cultured according to "1. Preparation method of tumor-specific T cell population" under Example 1, except that the dilution ratio of peripheral PBMC-enriched blood before PBMC separation was different:

[0247] Group 1: peripheral PBMC-enriched blood: diluent = 1:1;

[0248] Group 2: peripheral PBMC-enriched blood: diluent = 1:2;

[0249] Group 3: peripheral PBMC-enriched blood: diluent = 1:4;

[0250] Group 4: peripheral PBMC-enriched blood: diluent = 1:6.

[0251] After dilution using the above four dilution ratios, PBMCs were centrifuged and separated respectively. Samples were taken and counted, and the PBMC yield / 1mL peripheral PBMC-enriched blood under different dilution ratios was calculated ( Figure 1 , Table 2), and then the tumor-specific T cell populations of each group were prepared according to the method of Example 1, and the viability and proliferation times of the tumor-specific T cell populations cultured in each group were compared ( Figure 2 ), purity (Table 3, Figure 3 ).

[0252] Table 2 PBMC harvest volume / 1mL peripheral PBMC-enriched blood under different dilution ratios

[0253]

[0254] Table 3 Viability and phenotype proportion of tumor-specific T cell populations isolated under different dilution ratios

[0255]

[0256] The results in Tables 2 and 3 show that the PBMC yield in Group 3 was the highest, at 75.36×10 6 There was a significant difference between group 1 and group 3 (p<0.05); there was no statistically significant difference in the proliferation times and purity of the tumor-specific T cell population cultured among the four groups of samples. The optimal dilution ratio of PBMC-enriched blood was determined to be 1:4 for peripheral PBMC-enriched blood: diluent.

[0257] 2.2. Cytokine concentrations used in the culture system

[0258] The tumor-specific T cell population was cultured according to "1. Preparation method of tumor-specific T cell population" under Example 1, except that the concentration of cytokines in the complete culture medium during the primary activation culture and the expansion culture was different:

[0259] Group 1: CAPRI cell culture medium (Publication No.: CN 105524883 B, invention name: cell culture medium formula in patent "CAPRI cells and preparation method thereof")

[0260] Group 2: primary activation cytokine concentrations: rhIL-2: 10 IU / mL, rhIL-4: 10 IU / mL, G-CSF: 1000 IU / mL; amplification cytokine concentrations: rhIL-2: 10 IU / mL;

[0261] Group 3: primary activation cytokine concentrations: rhIL-2: 2000 IU / mL, rhIL-4: 800 IU / mL, G-CSF: 10000 IU / mL; amplification cytokine concentrations: rhIL-2: 2000 IU / mL;

[0262] Group 4: primary activation cytokine concentrations: rhIL-2: 400 IU / mL, rhIL-4: 40 IU / mL, G-CSF: 3500 IU / mL; expansion cytokine concentrations: rhIL-2: 400 IU / mL;

[0263] Group 5: primary activation cytokine concentrations: rhIL-2: 500 IU / mL, rhIL-4: 50 IU / mL, G-CSF: 4000 IU / mL; expansion cytokine concentrations: rhIL-2: 500 IU / mL;

[0264] Group 6: Primary activation cytokine concentrations: rhIL-2: 600 IU / mL, rhIL-4: 60 IU / mL, G-CSF: 4500 IU / mL; amplification cytokine concentration: rhIL-2: 600 IU / mL.

[0265] After the pre-culture, different concentrations of cytokines were added to the pre-cultured cell solution according to the preset combination, and the tumor-specific T cell population was cultured and harvested according to the preparation method of Example 1, and the cell expansion times of each group were compared ( Figure 4 ), cell purity (Table 4). At the same time, the harvested tumor-specific T cell population was co-cultured with HCC827 to detect its killing activity (Table 5).

[0266] Table 4 Expansion folds and cell phenotypes of tumor-specific T cell populations under different cytokine concentrations

[0267]

[0268] Table 5 Cytotoxic activity of tumor-specific T cell populations under different cytokine concentrations (%)

[0269]

[0270] The results in Tables 4 and 5 show that the expansion multiple of tumor-specific T cell population was the highest in Group 4, followed by Group 5 and Group 6; and there was a significant difference in the expansion multiple between Group 4 and other groups (p<0.05), and there was no statistically significant difference in the expansion multiple of tumor-specific T cell population between the other groups (p>0.05), and the lowest was Group 1 (CAPRI control). The cell phenotypes CD3+ / CD45+ and CD3+CD8+CD4- / CD3 accounted for a higher proportion in Group 4, Group 5, and Group 6; the two cell phenotypes accounted for the lowest proportion in Group 1 (CAPRI control). The CD3-(CD16+orCD56+) / CD45+ and CD3-CD19+ / CD45+ accounted for a lower proportion in Group 4, Group 5, and Group 6; the highest proportion was in Group 1 (CAPRI control). The cytotoxic activity was higher in Group 4, Group 5, and Group 6; the lowest cytotoxic activity was in Group 1 (CAPRI control).

[0271] Based on the above results, it was confirmed that the primary activation cytokine concentrations: rhIL-2: 400-600 IU / mL, rhIL-4: 40-60 IU / mL, G-CSF: 3500-4500 IU / mL, and the expansion cytokine concentrations: rhIL-2: 400-600 IU / mL are the cytokine concentrations used in the optimal culture system for activation and culture of tumor-specific T cell populations.

[0272] 2.3. Complete medium formulation

[0273] Tumor-specific T cell populations were cultured according to "1. Preparation of Tumor-Specific T Cell Populations" under Example 1, with the only difference being the formula of the complete culture medium used for expansion (the basal culture medium for each group was CTS™ AIM-V™ Medium).

[0274] Group 1: basal medium;

[0275] Group 2: basal medium + 1% autologous plasma;

[0276] Group 3: basal medium + human serum albumin (1:10);

[0277] Group 4: basal medium + human serum albumin (1:20);

[0278] Group 5: basal medium + human serum albumin (1:50);

[0279] Group 6: basal medium + CTS™ Immune Cell SR (1:20);

[0280] Group 7: basal medium + CTS™ Immune Cell SR (1:50);

[0281] By detecting the viability, proliferation ability, and cell phenotype of PBMC and tumor-specific T cell populations (Table 6, Figure 5 , Figure 6 , Figure 7 ) and other projects to determine the optimal complete culture medium formula for the culture of tumor-specific T cell populations.

[0282] Table 6 The viability, expansion times and cell phenotype of tumor-specific T cell populations cultured with different complete medium formulas

[0283]

[0284] The results in Table 6 show that the cell viability of Group 2 was maintained above 95%, while that of the other groups was below 90%. Among the cell populations in Group 2, Tc cells (CD3+CD8+CD4- / CD3+) accounted for the highest proportion, while B cells (CD3-CD19+ / CD45+ / %) accounted for the lowest proportion. The culture medium of Group 2, i.e., basal culture medium + 1% autologous plasma, was most conducive to the culture of tumor-specific T cell populations.

[0285] 2.4. Expansion culture time

[0286] According to the method for preparing tumor-specific T cell populations in Example 1, after the activation culture is completed, the expansion culture is carried out according to the preset culture time, and finally the tumor-specific T cell population is harvested. The only difference is the expansion culture time. At the same time, the harvested tumor-specific T cell population is co-cultured with HCC827 and its killing activity is detected to compare the expansion multiples (Table 7, Figure 8 ), purity, in vitro killing (Table 8), etc.

[0287] Table 7 Tumor-specific T cell population viability and expansion times at different expansion times

[0288]

[0289] Table 8 The same effector target killing activity of tumor-specific T cell populations cultured at different expansion times

[0290]

[0291] The results in Tables 7 and 8 show that as the expansion culture time increases, the proliferation times of the tumor-specific T cell population increase, and the cell viability does not show a significant decrease. The one-way analysis of variance (LSD method) was used to evaluate the expansion times of the tumor-specific T cell population among the groups. The results showed that the differences in the expansion times of the tumor-specific T cell population between the 72h group and the 120h group, and between the 96h group and the 120h group were statistically significant (p < 0.01). At the effector-target ratio of 20:1 and 10:1, there was no difference in the killing activity of the tumor-specific T cell population in the 96h group and the 120h group compared with the killing activity of the tumor-specific T cell population in the 72h group ( Fig. 9 ), which was not statistically significant (p > 0.05), indicating that the extension of the expansion culture time did not affect the killing effect. The killing activity of the effector-target ratio (5:1) group was selected to calculate the total killing rate, and the total killing rate of tumor-specific T cell populations at different expansion times was calculated using the expansion multiple × killing activity (Table 9). After analysis, it was found that the 96 h group had the highest total killing rate, so the expansion culture time was preferably 96 h.

[0292] Table 9 Total killing rate at different amplification times

[0293]

[0294] 2.5. Different amounts of autologous plasma added

[0295] The tumor-specific T cell population was cultured according to the method of "1. Preparation of tumor-specific T cell population" under Example 1, except for the amount of autologous plasma added during the expansion culture.

[0296] Group 1: no addition of autologous plasma;

[0297] Group 2: 1% autologous plasma added;

[0298] Group 3: Add 2% autologous plasma;

[0299] Group 4: 3% autologous plasma added;

[0300] By detecting the activity rate, proliferation ability, and cell phenotype of tumor-specific T cell population (Table 10, Table 11, Fig.10 , Fig.11 , Fig.12 ) and other items to determine the optimal amount of autologous plasma to be added during the cultivation of tumor-specific T cell populations.

[0301] Table 10 The viability and expansion times of tumor-specific T cell populations at different autologous plasma addition amounts

[0302]

[0303] Table 11 Phenotypes of tumor-specific T cell populations at different autologous plasma addition amounts

[0304]

[0305] The results in Table 10 show that the cell viability of the group with added autologous plasma (Group 2, Group 3, Group 4) is significantly higher than that of the group without added autologous plasma (Group 1), and the cell viability gradually increases with the increase in the amount of autologous plasma added. The expansion multiples were the highest in Group 3 and the lowest in Group 1. The results in Table 11 show that the proportion of CD3+ / CD45+ phenotype in the group with added autologous plasma (Group 2, Group 3, Group 4) is higher than that in the group without added autologous plasma (Group 1); the proportion of CD3-CD19+ / CD45+ (Group 2, Group 3, Group 4) is lower than that in the group without added autologous plasma (Group 1). This indicates that autologous plasma should be added during the culture process, and the optimal amount of autologous plasma added is 1%-3%.

[0306] 2.6. IL-2 secretion after cells were cultured with different concentrations of CD3 coating medium

[0307] Six CD3 coating concentrations (0, 1, 2, 4, 8, 16 μg / mL) were set in the experiment, and the amount of IL-2 secreted by Jurkat cells stimulated by the six groups of CD3 concentrations was counted. The results are shown in Table 12.

[0308] Table 12 IL-2 secretion after cells were cultured with different concentrations of CD3 coating solution

[0309]

[0310] As shown in Table 12, the secretion of IL-2 was the highest when the CD3 coating concentration was 4 μg / mL, which was 482.21 ± 39.57. The next highest was the CD3 coating concentration groups of 8 μg / mL and 2 μg / mL, with the IL-2 contents of the two groups being 464.63 ± 32.05 and 444.91 ± 14.21, respectively. When the coating concentration was lower than 4 μg / mL, the amount of IL-2 secreted by Jurkat cells continued to increase with the increase of CD3 coating concentration. However, when the coating concentration was higher than 4 μg / mL, the amount of IL-2 secreted by Jurkat cells continued to decrease with the increase of CD3 coating concentration.

[0311] In summary, a CD3 coating concentration of 1-16 μg / mL is more conducive to stimulating the secretion of IL-2, but the most preferred CD3 coating concentration is 2-8 μg / mL.

[0312] 2.7. Cytokine secretion by tumor-specific T cell populations

[0313] In order to confirm the secretion range of intracellular factors of tumor-specific T cell populations, six batches of tumor-specific T cell populations were prepared using "1. Preparation method of tumor-specific T cell populations" under Example 1, and the secretion of IFN-γ and Granzyme B of each batch of samples was compared (Table 13, Fig.13 ):

[0314] Table 13 Statistics of cytokine secretion content of tumor-specific T cell populations in each batch

[0315]

[0316] According to the data of 6 batches of repeated preparations, the average content of Granzyme B secreted by the harvested tumor-specific T cell population was 78.70 ± 4.79%, with the highest being 87.48% and the lowest being 73.26%, both within the range of 70-90%; the average content of IFN-γ secreted was 25.56 ± 14.26, with the highest being 12.65% and the lowest being 48.16%, both within the range of 10-50%.

[0317] Example 2 Verification of the function of tumor-specific T cell population in killing lung cancer cells in vitro

[0318] 1. RTCA method verifies the ability of tumor-specific T cell population to kill lung cancer cells in vitro

[0319] The RTCA method was used to verify the killing of lung cancer cells by tumor-specific T cell populations. The tumor-specific T cell population injection prepared according to Example 1 was used to co-culture the injection with HCC827 cells to observe the proliferation and growth of HCC827 cells on the xCELLigence RTCA Instrument system after adding the tumor-specific T cell population injection, and to detect the killing effect of the tumor-specific T cell population injection on lung cancer cells.

[0320] HCC827 cells were cultured overnight to allow them to fully adhere to the wall. Tumor-specific T cell population injection was added to the HCC827 cell culture wells in proportion. The proliferation and growth process of HCC827 cells after adding tumor-specific T cell population injection was monitored, and the impedance signal of target cells was measured. This experiment was divided into 6 groups, each group was plated with 0.5×10 4 HCC827 cells, and the number of tumor-specific T cell populations added to each group is as follows:

[0321] Group 1: Add 20×10 4 Tumor-specific T cell population injection cells, that is, E:T is 40:1

[0322] Group 2: Add 10×10 4Tumor-specific T cell population injection cells, that is, E:T is 20:1

[0323] Group 3: Add 5×10 4 Tumor-specific T cell population injection cells, that is, E:T is 10:1

[0324] Group 4: Add 2.5 × 10 4 Tumor-specific T cell population injection cells, that is, E:T is 5:1

[0325] Group 5: Add 0.5×10 4 Tumor-specific T cell population injection cells, that is, E:T is 1:1

[0326] Group 6: Only the same volume of culture medium was added without adding tumor-specific T cell population, i.e., the control group.

[0327] Table 14 Fig.14 , Fig.15 The results showed that after 72 hours of co-culture, the Normalized Cell Index of the co-culture test groups with various effector-target ratios (40:1, 20:1, 10:1, and 5:1) of the tumor-specific T cell population injection solution was significantly decreased compared with that of the target cell control group, which was statistically significant (p <0.001); the killing activity (%) of the co-culture test groups with various effector-target ratios (40:1, 20:1, 10:1, and 5:1) of the tumor-specific T cell population injection solution was also significantly different from that of the target cell control group, which was statistically significant (p <0.001); and when the effector-target ratio was 40:1 and 20:1, the killing activity reached more than 90%.

[0328] The results showed that the effector-target ratios of tumor-specific T cell population injection had significant killing activity against HCC827 cells, with a clear action time-effect curve. After 72 hours of co-culture, the effector-target ratios of tumor-specific T cell population injection (40:1, 20:1, 10:1, 5:1, 1:1) showed a clear dose-effect curve.

[0329] Table 14 Results of in vitro killing by injection of tumor-specific T cell population (RTCA method) and co-culture for 72 hours

[0330]

[0331] Note: Sample size is 9, One-way ANOVA followed by LSD, P<0.05, P<0.01, P<0.001 vs. target cell control group.

[0332] 2. Study on the in vitro killing of lung cancer cells by injection of sorted and purified tumor-specific T cell population (luciferase method)

[0333] In the present invention, HCC827-Luc-GFP, a lung cancer cell transfected, is oxidized to oxyluciferin in the cell under the catalysis of firefly luciferase. During the oxidation of luciferin, biological fluorescence with a wavelength of about 560 nm is emitted, which can be measured by a chemiluminometer or a microplate reader with a chemiluminescence detection module to detect surviving lung cancer cells. The present invention uses 3 batches of test samples for 3 tests, and the tumor cells are cultured overnight to fully adhere to the wall. The main cell components of the tumor-specific T cell group injection are sorted and purified by immunomagnetic bead cell sorting technology. Different cells after sorting are inoculated in vitro with different effect-target ratios, and the RLU detection of surviving target cells is performed after 64 hours to analyze the killing of tumor cells. There were 5 groups in this study: group 1 (NK cell co-culture test group sorted and purified from tumor-specific T cell population injection), group 2 (CD8+T cell Tc cell co-culture test group sorted and purified from tumor-specific T cell population injection), group 3 (CD4+T cell Th cell co-culture test group sorted and purified from tumor-specific T cell population injection), group 4 (tumor-specific T cell population injection co-culture test group), group 5 (target cell plating 1×10 5 The non-small cell lung cancer line HCC827 carrying the stable Luc gene and the target cell control group without effector cells). Groups 1-4 were further divided into 3 groups according to the amount of effector cells added: (1) E:T 15:1 group (effector cells: tumor cells), (2) E:T 10:1 group (effector cells: tumor cells), and (3) E:T 7.5:1 group (effector cells: tumor cells). There were 13 groups in total.

[0334] Table 15 Fig.16 , Fig.17The results showed that in the in vitro killing test of the injection of the sorted and purified tumor-specific T cell population, at each effector-target ratio (15:1, 10:1, 7.5:1), the killing effect of the NK cell co-culture test group was significantly lower than that of the unsorted test group and was negative, which was statistically significant (p < 0.001); the killing effect of the CD4+T cell co-culture test group decreased with the decrease of the effector-target ratio. When the effector-target ratio was 10:1 and 5:1, it was significantly lower than the killing effect of the unsorted test group, which was statistically significant (P < 0.001); at each effector-target ratio (15:1, 10:1, 7.5:1), the killing effect of the CD8+T cell test group was not statistically significant compared with the unsorted test group (p > 0.05), but its killing activity was slightly lower than that of the unsorted cell group, indicating that the killing effect was played by the entire T cell population (mainly CD8+ and CD4+).

[0335] In the tumor-specific T cell group injection, after immunomagnetic bead sorting, the killing effect of tumor-specific T cell group cells with different surface specificities in the tumor-specific T cell group injection on the non-small cell lung cancer line HCC827 is from high to low: CD8+T cells>CD4+T cells>CD56+ cells. It can be inferred that the effective component of the tumor-specific T cell group injection that has a killing effect on lung cancer cells is the T cell group.

[0336] Table 15 Fluorescence intensity values ​​(RLU) and cytotoxicity (%) of each effector-target ratio in vitro killing test of injection liquid of tumor-specific T cell population after sorting and purification

[0337]

[0338] Note: Sample size is 18, One-way ANOVA followed by LSD, #P<0.001 vs. target cell control group, P<0.05, P<0.01, P<0.001 vs. unsorted group.

[0339] 3. Study on in vitro killing blocking test of tumor-specific T cell population injection

[0340] Three batches of test products were used for three experiments, using Anti-human HLA-DR, DP, and DQ Antibodies (16 μg / 1.0×10 5 cell) and Anti-human HLA-A, B, C Antibody (18μg / 1.0×10 5The non-small cell lung cancer line HCC827 carrying the stably transfected Luc gene was treated with the cell culture medium and cultured overnight to allow it to fully adhere to the wall. The tumor-specific T cell population was injected with Anti-human TCR a / β Antibody (32μg / 1.0×10 6 After treatment with T cells, the cells were added to the tumor cell culture wells in proportion. After 64 hours, the luciferase method was used to detect the RLU (Relative light unit) of the surviving target cells to analyze the killing of tumor cells, evaluate the blocking effect of the corresponding treatment, and explore the anti-tumor killing mechanism of tumor-specific T cell group injection. The experiment was divided into 4 groups:

[0341] Group 1: Tumor cells co-cultured with HLA-II blocking treatment

[0342] Group 2: Tumor cells co-cultured with HLA-I blocking treatment

[0343] Group 3: Co-culture test group in which tumor cells and tumor-specific T cell population injections were not subjected to blocking treatment

[0344] Group 4: Tumor-specific T cell population injection and TCR-α, β blocking treatment co-culture test group

[0345] 1.5×10 target cells were plated in each group 4 The HCC827 non-small cell lung cancer line carrying a stable Luc gene was further divided into 4 groups according to the amount of effector cells added: (1) E:T 20:1 group (effector cells: tumor cells), (2) E:T 15:1 group (effector cells: tumor cells), (3) E:T 10:1 group (effector cells: tumor cells), and (4) E:T 0:1 group (effector cells: tumor cells, i.e., tumor cell control group). There are 16 groups in total.

[0346] Table 16 Fig.18 , Fig.19 The results showed that in the in vitro killing blocking test of tumor-specific T cell group injection, compared with the unblocked control group, tumor cells treated with HLA-A, B, C and HLA-DR, DP, DQ blocking treatment, and tumor-specific T cell group injection with TCR a / β blocking treatment can significantly increase the fluorescence intensity (p <0.05); reduce the killing of lung cancer cells by tumor-specific T cell group injection (p <0.05), and when the effect-target ratio (15:1, 10:1), the killing activity of target cells treated with HLA-A, B, C blocking treatment was the most significantly reduced (p <0.001). After blocking HLA-I, HLA-II and TCR molecules, the T cell MHC-TCR recognition process in target cells and tumor-specific T cell groups was blocked.

[0347] Tumor-specific T cell population injection can kill lung cancer cells by binding and interacting with MHC-I, MHC-II and TCR-α / β.

[0348] Table 16 Fluorescence intensity values ​​(RLU) and killing activity (%) of each effector-target ratio in vitro killing blocking test of tumor-specific T cell population injection

[0349]

[0350] Note: Sample size is 15, Independent-Samples T Test, P<0.05, P<0.01, P<0.001 vs. untreated group.

[0351] 4. Study on DC cell maturation detection and antigen stimulation effect in the preparation of tumor-specific T cell population injection

[0352] The tumor-specific T cell population injection was prepared using the cryopreserved and resuscitated samples of volunteer PBMC. During the preparation process, DC cell maturity was detected at five time points, including PBMC inoculation, secondary activation, expansion culture, 48h expansion culture, and cryopreservation, to confirm the DC cell maturation in the preparation process of the tumor-specific T cell population injection. Since the monocytes of tumor patients have been exposed to tumor cell antigens in vivo, they can mature into DCs carrying tumor antigens in the culture system and stimulate and activate tumor-specific T lymphocytes. In this experiment, tumor cell lysates prepared from tumor cell lines homologous to target cells and plasma from patients with non-small cell lung cancer were selected to simulate the tumor antigens of patients. Antigen stimulation was added during the expansion culture in the preparation process, and the cell killing effect of the prepared tumor-specific T cell population injection was detected by luciferase method to evaluate the difference in the culture effect of tumor antigens on the tumor-specific T cell population injection. In order to verify the feasibility of the experimental plan and the reliability of the experimental results, the experiment was repeated using the same experimental conditions and two different PBMCs.

[0353] This experiment was divided into 3 major groups: Group 1 (group in which non-patient plasma was added during amplification, i.e., group without tumor antigens), Group 2 (group in which patient plasma was added during amplification, i.e., group with non-homologous tumor antigens), and Group 3 (group in which non-patient plasma and tumor cell line tumor lysate were added during amplification, i.e., group with homologous tumor antigens). When conducting killing experiments, each group was further divided into 3 subgroups according to the effector-target ratio: (1) E:T 20:1 group (effector cells: tumor cells), (2) E:T 10:1 group (effector cells: tumor cells), and (3) E:T 5:1 group (effector cells: tumor cells). In addition, an E:T 0:1 group (target cell control group) was added, for a total of 10 subgroups, with 1.0×10 target cells in each group. 4 A non-small cell lung cancer line HCC827 carrying a stably transfected Luc gene was prepared. Tumor cells were cultured overnight to allow them to fully adhere to the wall. Tumor-specific T cell population injection was added to the tumor cell culture wells according to the effect-target ratio. After 72 hours, the luciferase method was used to detect the RLU (Relative light unit) of surviving target cells to analyze the killing of tumor cells.

[0354] Table 17, Table 18, Fig. 20 , Fig.21 The results showed that in the in vitro killing test of the first and second batches of tumor-specific T cell population injections after stimulation with different antigens, when the effector-target ratio was 20:1, the tumor-specific T cell population injection containing non-homologous tumor antigens, the tumor-specific T cell population injection containing homologous tumor antigens, and the tumor-specific T cell population injection without tumor antigens all showed strong killing activity, and there was no significant difference in killing activity among the groups, which was not statistically significant (p>0.05); when the effector-target ratio was 10:1, the tumor-specific T cell population injection containing non-homologous tumor antigens The killing activity of the tumor-specific T cell group injection containing homologous tumor antigen culture was significantly increased compared with the tumor-specific T cell group injection without tumor antigen culture, which was statistically significant (p<0.01); when the effect-target ratio was 5:1, the tumor-specific T cell group injection co-cultured with homologous tumor antigen cultured group showed significant killing activity, and compared with the killing activity of the tumor-specific T cell group injection co-cultured group without tumor antigen cultured, the killing activity was significantly increased, which was statistically significant (p<0.001). From the results of the in vitro killing test of tumor-specific T cell group injection after stimulation with two batches of different antigens, it can be seen that the killing activity is from high to low: tumor-specific T cell group injection containing homologous tumor antigen cultured>tumor-specific T cell group injection containing non-homologous tumor antigen cultured>tumor-specific T cell group injection without tumor antigen plasma cultured.

[0355] The above results show that the tumor-specific T cell population injection can effectively deliver tumor antigens. In the killing test, the killing effect of the tumor-specific T cell population injection containing homologous tumor antigens was higher than that of the tumor-specific T cell population injection containing non-homologous tumor antigens. The reason may be that the tumor lysate added during the culture process comes from the HCC827 cell line that is homologous to the target cells of the killing test.

[0356] Table 17 Fluorescence intensity values ​​of each effector-target ratio (RLU) of the tumor-specific T cell population injection in vitro killing test after different antigen stimulation

[0357]

[0358] Note: Sample size is 6, One-way ANOVA followed by LSD and Games-Howell, ##P<0.01 ###P<0.001 vs. target cell control group, P<0.05, P<0.01, P<0.001 vs. group without tumor antigen.

[0359] Table 18 Killing activity of each effective target in the in vitro killing test of tumor-specific T cell population injection after different antigen stimulation (%)

[0360]

[0361] Note: Sample size is 6, One-way ANOVA followed by LSD and Games-Howell, P<0.05, P<0.01, P<0.001 vs. group without tumor antigen.

[0362] 5. Study on the in vitro T cell reactivity of tumor-specific T cell population injection on primary human non-small cell lung cancer

[0363] The secretion of IFN-γ by autologous tumor cells and the in vitro T cell reactivity of tumor-specific T cell population injection were evaluated by ELISPOT detection and Granzyme B was used to evaluate the in vitro T cell reactivity of tumor-specific T cell population injection. In the present invention, fresh lung adenocarcinoma tumors removed by surgical operation are inoculated subcutaneously on the back of immunodeficient mice for preparing PDX (Patient derived Xenograft) models, and anticoagulated peripheral whole blood is obtained to prepare autologous cell-derived test sample tumor-specific T cell group injection solution. After tissue processing and cell sorting of the PDX model tumor, the tumor-specific T cell group injection solution and the sorted tumor cells are simultaneously added to IFN-γ and Granzyme B ELISPOT plates at effector-target ratios of 16:1, 8:1, 4:1, 2:1, and 1:1. The number of spots of interferon-γ (IFN-γ) and granzyme B (Granzyme B) secreted by the tumor-specific T cell group injection solution stimulated by the autologous PDX model P0 generation tumor cells is detected by enzyme-linked immunospot (ELISPOT) to evaluate the in vitro T cell reactivity of the tumor-specific T cell group injection solution.

[0364] A total of 4 experimental groups were set up, namely, tumor-specific T cell population injection only control, tumor cell only control, culture medium control, and positive stimulation control. Three replicate wells were set for each condition and cultured in a 37°C, 5% CO2 incubator for 40-44 hours. The plates were washed and developed according to the instructions of the ELISPOT kit. After the ELISPOT plate was dried, the number of positive spots was read using an enzyme-linked immunosorbent assay analyzer.

[0365] Table 19, Table 20, Fig. 22 , Fig.23 , Fig.24 , Fig.25 The results showed that the release of IFN-γ and Granzyme B increased significantly after the test cell tumor-specific T cell population was co-cultured with autologous tumor cells. At the effector-target ratio of 16:1, 8:1, 4:1, 2:1 and 1:1, the number of co-cultured IFN-γ spots was significantly higher than that of the tumor-specific T cell population only (p<0.05). And as the effector-target ratio decreased, the difference between the two groups became more obvious. Compared with the number of spots of the tumor-specific T cell population only, the multiple of the number of co-cultured IFN-γ spots was between 1.62 and 12.67. At the effector-target ratio of 4:1 and 2:1, the number of co-cultured Granzyme B spots was significantly higher than that of the tumor-specific T cell population only (p<0.05). At the effector-target ratio of 8:1, 4:1, 2:1 and 1:1, compared with the number of spots of the tumor-specific T cell population only, the multiple of the number of co-cultured Granzyme B spots was between 1.66 and 2.03.

[0366] Tumor-specific T cell population injection can release IFN-γ and Granzyme B under the stimulation of autologous tumors, and has a significant response to autologous tumor cells.

[0367] Table 19 Summary of ELISPOT (IFN-γ) positive spot counts (pcs / well)

[0368]

[0369] Note: The multiple is the average number of co-culture spots divided by the average number of tumor-specific T cell population-only spots.

[0370] Table 20 Summary of ELISPOT (Granzyme B) positive spot numbers (pcs / well)

[0371]

[0372] Note: The multiple is the average number of co-culture spots divided by the average number of tumor-specific T cell population-only spots.

[0373] Example 3 In vivo study of tumor-specific T cell population killing tumors

[0374] 1. Pharmacodynamics study of tumor-specific T cell population injection on non-small cell lung cancer (HCC827) tumor-bearing mouse model

[0375] All animals that passed the health examination were inoculated subcutaneously in the right axilla with 0.57×10 7 When the tumor volume grew to an appropriate size, 40 animals with tumor volumes ranging from 50.45 to 126.96 mm3 were randomly divided into 4 groups, with 10 animals in each group, namely, vehicle control group, low-dose tumor-specific T cell population injection group (0.5×10 7 cells / each), medium-dose group (1×10 7 cells / each), high-dose group (2×10 7 cells / animal / time), 0.2 mL / animal of the test or control solution of the corresponding dose was injected into the tail vein on D1, D3, and D5. The first day of administration was designated as D1, and the administration was performed 3 times in total. The tumor volume (mm) of all animals in Groups 1 to 4 was measured twice a week after tumor inoculation. 3) and body weight, and the changes in tumor volume and relative tumor volume of animals in each group during the experiment were recorded. Blood was collected from orbital veins of all animals in Groups 1 to 4 on D7, D14, D21, and D27. After centrifugation at 3000 rpm for 10 min, the upper plasma was used to detect the levels of human cytokines IL-2, IL-4, IL-6, IL-8, IL -10, IL-17, TNF-α, and IFN-γ (using CBA (CytometricBeadArray, sample multiple protein quantification technology) kit to detect human cytokines), and the lower blood cells were resuspended in PBS and used for CD45+, CD45+CD3+ cell ratio lymphocyte subset detection (flow cytometer detection). At the end of the experiment, the tumor tissue was removed, photographed, weighed, fixed with neutral formalin, and sent to the client. The relative tumor proliferation rate (T / C, %) and relative tumor inhibition rate (IRTV, %) were calculated and statistically analyzed.

[0376] Table 21-Table 29, Figure 26-Figure 42 The results showed that in the toxicity test of repeated intravenous injection of tumor-specific T cell population injection into NOG tumor-bearing mice (subcutaneously transplanted with human non-small cell lung cancer HCC827) for 1 week and 4 weeks of recovery, the results of histopathological examination after euthanasia of D37 animals showed that metastatic tumor cells (human non-small cell lung cancer HCC827) were infiltrated in the lungs of the tumor-bearing control group animals, while no tumor cell infiltration was observed in any organs of the test sample group, indicating that the test sample has the effect of preventing tumor metastasis.

[0377] Under the conditions of this experiment, the tumor-specific T cell population injection was 0.5×10 7 cells / each~2.0×10 7 cells / mouse / time, and a total of 3 administrations could significantly inhibit the growth and metastasis of tumors in non-small cell lung cancer (HCC827) tumor-bearing mice.

[0378] Table 21 Body weight of non-small cell lung cancer (HCC827) tumor-bearing mouse model (g)

[0379]

[0380] Note: Compared with the vehicle control group, indicates P < 0.01, Indicates P<0.001.

[0381] Table 22 Tumor volume (mm) of non-small cell lung cancer (HCC827) tumor-bearing mouse model 3 )

[0382]

[0383] Note: Compared with the vehicle control group, indicates P < 0.05, indicates P < 0.01, Indicates P < 0.001.

[0384] Table 23 Relative tumor volume of non-small cell lung cancer (HCC827) tumor-bearing mouse model

[0385]

[0386] Note: Compared with the vehicle control group, indicates P < 0.01, Indicates P<0.001.

[0387] Table 24 Relative tumor proliferation rate (T / C, %) of non-small cell lung cancer (HCC827) tumor-bearing mouse model

[0388]

[0389] Table 25 Relative tumor inhibition rate (IRTV, %) of non-small cell lung cancer (HCC827) tumor-bearing mouse model

[0390]

[0391] Table 26 Tumor weight of non-small cell lung cancer (HCC827) bearing mice (g)

[0392]

[0393] Note: Compared with the vehicle control group, Indicates P<0.001.

[0394] Table 27 CD45+ cell subset ratio (%)

[0395]

[0396] Note: Compared with the vehicle control group, indicates P < 0.05, Indicates P < 0.001. Compared with the low-dose group, ++ indicates P < 0.01, and +++ indicates P < 0.001. Compared with the medium-dose group, %%% indicates P < 0.001.

[0397] Table 28 CD45+CD3+ cell subset ratio (%)

[0398]

[0399] Note: Compared with the vehicle control group, indicates P < 0.05, Indicates P < 0.001. Compared with the low-dose group, ++ indicates P < 0.01, and +++ indicates P < 0.001. Compared with the medium-dose group, %%% indicates P < 0.001.

[0400] Table 29 Cytokine statistics (pg / mL)

[0401]

[0402] Note: Compared with the vehicle control group, indicates P < 0.05, indicates P < 0.01, Indicates P < 0.001. Compared with the low-dose group, + indicates P < 0.05, ++ indicates P < 0.01, and +++ indicates P < 0.001. Compared with the medium-dose group, % indicates P < 0.05.

[0403] 2. Verification of the in vivo effect of tumor-specific T cell populations on tumor killing

[0404] 196 NOG mice (98 / sex) were randomly divided into 4 groups according to the animal temporary number and the tumor volume measured before administration. Group 1 used non-tumor-bearing mice (26 / sex / group), and groups 2 to 4 used tumor-bearing mice (human non-small cell lung cancer HCC827, subcutaneously transplanted with human non-small cell lung cancer HCC827 5 days before administration), including 20 / sex / group in group 2, and 26 / sex / group in groups 3 and 4. Group 1 animals were given a solvent control (therapeutic cell cryoprotectant (Genno)) as a non-tumor-bearing control group, group 2 was given a solvent control (therapeutic cell cryoprotectant (Genno)) as a tumor-bearing control group, and group 3 was given 1×10 7 cells / cells of tumor-specific T cell population injection as the low-dose group, and the fourth group was given 3×10 7 The high-dose group was treated with a tumor-specific T cell population injection of cells / animal. The animals were intravenously administered with drugs on D1, D3 and D5. The first 13 animals / sex / group in groups 1, 3 and 4 and the first 10 animals / sex / group in group 2 were used for dissection on D9, and the remaining animals were used for dissection on D37. During the experiment, the animals were subjected to clinical observation, body weight, food intake, tumor volume measurement, blood cell count, blood biochemistry, immune cell phenotype, cytokine, tissue distribution detection, gross dissection, organ weighing and histopathological examination.

[0405] Table 30 Fig.43 , Fig.44The results showed that compared with the same-sex tumor-bearing control group of the same period, the tumor volume of male and female animals in the low- and high-dose groups of the test article began to shrink from D21, and continued until the end of the observation period D35. Among them, the tumor volume of male animals in the high-dose group of the test article at D35 was reduced by 88.1%, and the tumor volume of female animals was reduced by 90.7%. The tumor volume of male animals in the low-dose group of the test article at D35 was reduced by 84.0%, and the tumor volume of female animals was reduced by 92.5%, which is considered to be related to the anti-tumor effect of the test article. Metastatic tumor cells (human non-small cell lung cancer HCC827) infiltration can be seen in the lungs of animals in the tumor-bearing control group; no tumor cell infiltration was found in any organ of the low- and high-dose groups of the test article, which is considered to be related to the anti-tumor effect of the test article. Compared with the tumor-bearing control group, no tumor cell metastasis was found in the animals in the test article group, indicating that the test article has a certain inhibitory effect on tumor cells.

[0406] Tumor-specific T cell population injection at 0.5×10 7 cells / each~2.0×10 7 cells / mouse / time, and a total of 3 administrations within the dosage range can significantly inhibit the growth and metastasis of tumors in non-small cell lung cancer (HCC827) tumor-bearing mice, and show a certain tumoricidal effect.

[0407] Table 30 Tumor volume changes (mm 3 )

[0408]

[0409] Note:" ” indicates P ≤ 0.05 compared with the tumor-bearing control group; “↓” indicates a decrease. The data in brackets are shown as the percentage difference from the tumor-bearing control value: (mean value-tumor-bearing control value) / tumor-bearing control value × 100%.

[0410] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cytokine combination, characterized in that The cytokine combination consists of a primary activation cytokine and an amplification cytokine: The primary activated cytokine is composed of the following components: rhIL-2: 400~600 IU / mL; rhIL-4: 40-60 IU / mL; and G-CSF: 3500-4500 IU / mL; The amplification cytokine is composed of the following components: rhIL-2: 400~600 IU / mL.

2. The cytokine combination according to claim 1, characterized in that The primary activated cytokine is composed of the following components: rhIL-2: 400 IU / mL; rhIL-4: 40 IU / mL; and G-CSF: 3500 IU / mL; The amplification cytokine is composed of the following components: rhIL-2: 400 IU / mL; or The primary activated cytokine is composed of the following components: rhIL-2: 600 IU / mL; rhIL-4: 60 IU / mL; and G-CSF: 4500 IU / mL; The amplification cytokine is composed of the following components: rhIL-2: 600 IU / mL.

3. Use of the cytokine combination according to claim 1 or 2 in any of the following: (I) preparing tumor-specific T cell populations; (II) Preparation of culture medium for tumor-specific T cell population.

4. A culture medium for preparing a tumor-specific T cell population, characterized in that: The culture medium consists of the cytokine combination as claimed in claim 1 or 2 and a complete culture medium; The complete culture medium is a basal culture medium and 1% to 3% autologous plasma; The basic culture medium is a serum-free culture medium.

5. Use of the culture medium as claimed in claim 4 in preparing a tumor-specific T cell population.

6. A method for preparing a tumor-specific T cell population, characterized in that: Its preparation method is: Step 1, separation of peripheral PBMC-enriched blood: diluent is diluted at a ratio of 1:4 to obtain diluted PBMC; Step 2, PBMC pre-culture: taking the diluted PBMC and culturing them with basal culture medium and CD3 monoclonal antibody to obtain pre-cultured PBMC, taking another diluted PBMC and refrigerating and preserving them to obtain refrigerated PBMC; Step 3, primary activation culture: adding the primary activation cytokine in the cytokine combination as claimed in claim 1 or 2 to the pre-cultured PBMC, and continuing to culture to obtain a primary activation culture sample; Step 4, secondary activation culture: adding the primary activation culture sample to the refrigerated PBMC, and continuing to culture to obtain an activated culture; Step 5, expansion culture of tumor-specific T cell population: using the complete culture medium in the culture medium as claimed in claim 4 and the expansion cytokine in the cytokine combination as claimed in claim 1 or 2 to expand and culture the activated culture for 96 hours; The basic culture medium is a serum-free culture medium.

7. The preparation method according to claim 6, characterized in that: The CD3 monoclonal antibody is OKT3; The CD3 monoclonal antibody concentration is 1-16 μg / mL.

8. The preparation method according to claim 7, characterized in that: The CD3 monoclonal antibody concentration is 2-8 μg / mL.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The tumor-specific T cell population is: CD3+ / CD45+: 85%~95%; CD3+CD8+CD4- / CD3+: 30%~65%; CD3-(CD16+or CD56+) / CD45+: 1%~10%; CD3-CD19+ / CD45+: 1%~5%; and / or CD3+CD8+Granzyme B+ / CD3+CD8+: 70%~90%; or The T cell population is: CD3+ / CD45+: 94.07±3.04%~97.83±1.50%; CD3+CD8+CD4- / CD3+: 57.13±9.00%~65.28±0.35%; CD3-(CD16+or CD56+) / CD45+: 1.45±0.31%~2.72±0.32%; CD3-CD19+ / CD45+: 0.27±0.12%~3.00±1.55%.

10. The preparation method according to claim 9, characterized in that: The tumor-specific T cell population is: CD3+ / CD45+: 94.07±3.04%, 97.83±1.50%, 97.63±1.39%, 96.27±1.46%, 96.92±2.68%, 96.57±0.57%, 94.13±3.88%, or 96.90±1.04%; CD3+CD8+CD4- / CD3+: 57.98±9.66%, 62.56±2.84%, 62.91±4.83%, 57.13±9.00%, 61.87±4.54%, 65.28±0.35%, 63.76±0.53% or 67.26±0.53%; CD3-(CD16+or CD56+) / CD45+: 2.62±1.58%, 1.45±0.31%, 1.53±0.42%, 2.32±0.36%, 1.48±0.57%, 2.72±0.32%, 2.19±0.58% or 2.06±0.56%; or CD3-CD19+ / CD45+: 3.00±1.55%, 0.33±0.06%, 0.27±0.12%, 1.27±1.16%, 0.35±0.09%, 0.73±0.40%, 0.70±0.46% or 0.57±0.32%.

11. The preparation method according to claim 10, characterized in that: The tumor-specific T cell population is: CD3+ / CD45+: 94.07±3.04%; CD3+CD8+CD4- / CD3+: 57.98±9.66%; CD3-(CD16+or CD56+) / CD45+: 2.62±1.58%; CD3-CD19+ / CD45+: 3.00±1.55%; or The T cell population is: CD3+ / CD45+: 97.83±1.50%; CD3+CD8+CD4- / CD3+: 62.56±2.84%; CD3-(CD16+or CD56+) / CD45+: 1.45±0.31%; CD3-CD19+ / CD45+: 0.33±0.06%; or The T cell population is: CD3+ / CD45+: 97.63±1.39%; CD3+CD8+CD4- / CD3+: 62.91±4.83%; CD3-(CD16+or CD56+) / CD45+: 1.53±0.42%; CD3-CD19+ / CD45+: 0.27±0.12%; or The T cell population is: CD3+ / CD45+: 96.27±1.46%; CD3+CD8+CD4- / CD3+: 57.13±9.00%; CD3-(CD16+or CD56+) / CD45+: 2.32±0.36%; CD3-CD19+ / CD45+: 1.27±1.16%; or The T cell population is: CD3+ / CD45+: 96.92±2.68%; CD3+CD8+CD4- / CD3+: 61.87±4.54%; CD3-(CD16+or CD56+) / CD45+: 1.48±0.57%; CD3-CD19+ / CD45+: 0.35±0.09%; or The T cell population is: CD3+ / CD45+: 96.57±0.57%; CD3+CD8+CD4- / CD3+: 65.28±0.35%; CD3-(CD16+or CD56+) / CD45+: 2.72±0.32%; CD3-CD19+ / CD45+: 0.73±0.40%; or The T cell population is: CD3+ / CD45+: 94.13±3.88%; CD3+CD8+CD4- / CD3+: 63.76±0.53%; CD3-(CD16+or CD56+) / CD45+: 2.19±0.58%; CD3-CD19+ / CD45+: 0.70±0.46%; or The T cell population is: CD3+ / CD45+: 96.90±1.04%; CD3+CD8+CD4- / CD3+: 67.26±0.53%; CD3-(CD16+or CD56+) / CD45+: 2.06±0.56%; CD3-CD19+ / CD45+: 0.57±0.32%.

12. Use of the T cell population prepared by the preparation method according to any one of claims 6 to 11 in the preparation of an anti-non-small cell lung cancer drug.

13. An anti-non-small cell lung cancer drug, characterized in that: The method comprises a T cell population prepared by the preparation method according to any one of claims 6 to 11.

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