Method for obtaining Vgamma9Vdelta2T cells with strong killing ability through serum-free specific amplification

Abstract

The invention discloses a method for obtaining Vgamma9Vdelta2T cells with strong killing ability through serum-free specific amplification, a serum-free culture system is used in the method, the scheme is simple, the operation is convenient and fast, the amplification efficiency and the killing effect on solid tumor cells can be greatly improved, and in addition, the method is suitable for large-scale production. The Vgamma9Vdelta2T cell prepared by adopting the method provided by the invention is high in positive rate and strong in killing ability for solid tumors, and has a good application prospect and important transformation significance in the technical field of tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to a method for obtaining Vγ9Vδ2T cells with strong killing ability through serum-free specific amplification. Background Art

[0002] The human immune system is composed of the innate immune system and the adaptive immune system, which help the body resist invasion by bacteria, viruses, etc. When innate immunity is not enough to eliminate pathogens, adaptive immunity will come into play. T cells are important members of adaptive immunity. They are produced in the bone marrow, migrate to the thymus to mature, and are responsible for the establishment and maintenance of immune responses, homeostasis, and memory in the body. T cells express a receptor (T cell receptor, abbreviated as TCR) on the surface of the membrane, which has the potential to recognize a variety of antigens from pathogens, tumors, and the environment, and maintain immune memory and self-tolerance. Depending on the type of peptide chain that makes up the TCR, T cells are mainly divided into αβT cells and γδT cells. αβT cells are the main subpopulation of CD3+ T cells in the immune repertoire, and mainly recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. In contrast, γδT cells utilize an alternative T cell receptor (TCR) structure composed of γ and δ chains, enabling them to recognize a wider range of antigens in an MHC-independent manner. This allows them to have "universal" and "off-the-shelf" applications superior to traditional T cells. γδT cells express the TCR γδ chain and the NK cell-activating receptor NKG2D, possessing characteristics of both T and NK cells, enabling them to directly recognize and kill tumor and infected cells. Furthermore, γδT cells can directly secrete perforins, granzymes, and cytokines, as well as activate death receptor / ligand binding and antibody-dependent cell-mediated cytotoxicity (ADCC). They can also indirectly regulate immune cells such as dendritic cells (DCs) and macrophages. Therefore, their relatively low cost, minimal side effects, and broad indications give γδT cells broad clinical potential in tumor immunotherapy.

[0003] Compared to currently approved CAR-T (αβT cell) cell therapies, γδT cells offer advantages such as low cost, minimal side effects, and a wide range of indications. However, naturally occurring γδT cells are relatively rare, accounting for only 0.5%-5% of human peripheral blood T cells. Of these, Vγ9Vδ2 subtype T cells account for 65%-90% of the γδT cell population in peripheral blood. In recent years, Vγ9Vδ2 subtype T cells have been used in experimental cancer therapies. These cells have been activated in vivo with various phosphonates and interleukin-2, or by using autologous or allogeneic Vδ2-enriched peripheral blood mononuclear cells (PBMCs), in combination with other adjuvant therapies, to treat various types of solid tumors. Their safety has been confirmed, and they have demonstrated efficacy against various cancer types. To further enhance efficacy and increase accessibility, it is crucial to easily obtain highly potent Vγ9Vδ2 subtype T cells. Currently, commonly used in vitro expansion methods utilize a combination of phosphonate small molecules and cytokines to co-stimulate the specific expansion of Vγ9Vδ2 subtype T cells. However, existing expansion protocols contain fetal bovine serum (which poses a safety risk), resulting in low expansion efficiency, a large number of added factors, long culture cycles, and cumbersome protocols. The expanded cells are also less able to kill solid tumor cells, limiting their application. Therefore, developing a serum-free, simple to operate, high-efficiency expansion method that can enhance the killing ability of the amplified Vγ9Vδ2 T cells is of great practical significance. Summary of the Invention

[0004] In view of this, in order to overcome the above-mentioned technical problems existing in the art, the purpose of the present invention is to provide a method for serum-free specific amplification to obtain Vγ9Vδ2T cells with strong killing ability.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides a method for obtaining Vγ9Vδ2T cells with strong killing ability through serum-free specific expansion.

[0007] Furthermore, the method comprises the following steps:

[0008] (1) PBMCs were inoculated into X-VIVO 15 serum-free medium and cultured with zoledronic acid;

[0009] (2) After culturing for 24 hours, interleukin-2 was added, and the culture was continued to expand and culture to obtain the Vγ9Vδ2 T cells with strong killing ability.

[0010] Furthermore, the seeding density of PBMC in step (1) is 0.1-10×10 6 cells / mL;

[0011] Optionally, the PBMC inoculation density in step (1) is 1×10 6 cells / mL;

[0012] Optionally, the final concentration of zoledronic acid in step (1) is 1-10 μM;

[0013] Optionally, the final concentration of zoledronic acid in step (1) is 5 μM;

[0014] Optionally, the culture conditions in step (1) are 37° C. and 5% CO 2 ;

[0015] Optionally, the PBMCs in step (1) are peripheral blood mononuclear cells extracted from human peripheral blood.

[0016] Furthermore, the final concentration of interleukin-2 in step (2) is 50-200 IU / mL;

[0017] Optionally, the final concentration of interleukin-2 in step (2) is 100 IU / mL;

[0018] Optionally, the expansion culture in step (2) is an expansion culture for 10-30 days;

[0019] Optionally, the expansion culture in step (2) is an expansion culture for 15 days.

[0020] Furthermore, the method also includes the following steps: on the third day of culture, expanding the culture system, and then changing the medium every two days and supplementing zoledronic acid and interleukin-2. After 10-30 days of expansion culture, the Vγ9Vδ2T cells with strong killing ability are obtained.

[0021] Furthermore, the expanded culture system is an expanded culture system at a ratio of 1:2-1:5;

[0022] Optionally, the expanded culture system is an expanded culture system at a ratio of 1:4;

[0023] Optionally, the cell density is adjusted to 200,000-800,000 cells / mL during the medium replacement;

[0024] Optionally, the cell density is adjusted to 500,000 cells / mL during the medium replacement;

[0025] Optionally, the final concentration of the supplemented zoledronic acid is 1-10 μM;

[0026] Optionally, the final concentration of the supplemented zoledronic acid is 5 μM;

[0027] Optionally, the final concentration of the supplemented interleukin-2 is 50-200 IU / mL;

[0028] Optionally, the final concentration of the supplemented interleukin-2 is 100 IU / mL;

[0029] Optionally, the expansion culture is an expansion culture for 15 days.

[0030] In the present invention, the Vγ9Vδ2T cells are the main subtype of γδT cells, and their T cell receptors (TCRs) are composed of Vγ9 and Vδ2 chains. They are mainly present in peripheral blood, accounting for more than 50% of peripheral blood γδT cells. This type of cell can recognize non-peptide antigens, such as phosphoantigens (derived from pathogens or human metabolites), does not require MHC molecule presentation, has the characteristics of rapid activation and broad-spectrum killing of tumor cells and pathogen-infected cells, and can secrete a variety of cytokines (such as IFN-γ, TNF-α) to regulate immune responses. Because it has both the rapid response of innate immunity and the specificity of adaptive immunity, it shows important research value in cancer immunotherapy, anti-infection and inflammatory diseases (such as rheumatoid arthritis and psoriasis), and is one of the hot cell types in the current field of immunotherapy.

[0031] In the present invention, the peripheral blood mononuclear cells (PBMCs) are the same as PBMCs, which refer to mononuclear cells isolated from peripheral blood and are commonly used in anti-cancer immunotherapy. PBMCs can be obtained from collected human blood using methods well known to those skilled in the art, such as the Ficoll-Hypaque density gradient method.

[0032] In some embodiments, the peripheral blood mononuclear cells can be obtained from a healthy individual, a subject at risk of a disease, or a patient. The peripheral blood mononuclear cells used herein do not necessarily need to be autologous, and allogeneic peripheral blood mononuclear cells can also be used.

[0033] The present invention has no particular limitation on the specific sources of the PBMC, zoledronic acid or interleukin-2, and those skilled in the art can obtain them through conventional routes.

[0034] A second aspect of the present invention provides an expansion medium for specifically expanding Vγ9Vδ2 T cells.

[0035] Furthermore, the expansion medium comprises X-VIVO 15 serum-free medium, zoledronic acid, and interleukin-2;

[0036] Optionally, the final concentration of zoledronic acid is 1-10 μM;

[0037] Optionally, the final concentration of zoledronic acid is 5 μM;

[0038] Optionally, the final concentration of interleukin-2 is 50-200 IU / mL;

[0039] Optionally, the final concentration of interleukin-2 is 100 IU / mL.

[0040] In some embodiments, PBMCs are inoculated into X-VIVO 15 serum-free medium in the expansion medium, and zoledronic acid in the expansion medium is added for culture; after 24 hours of culture, interleukin-2 in the expansion medium is added, and culture is continued to obtain the Vγ9Vδ2T cells with strong killing ability through expansion culture.

[0041] The third aspect of the present invention provides a Vγ9Vδ2T cell with strong killing ability obtained by expansion according to the method described in the first aspect of the present invention.

[0042] Furthermore, the CD3+TCRVγ9+ double-positive rate of the Vγ9Vδ2 T cells was ≥91.26%.

[0043] In a specific embodiment of the present invention, the present invention demonstrates through cell killing experiments that the positive rate of Vγ9Vδ2T cells prepared by amplification using the method described in the first aspect of the present invention is as high as 91.26%; its killing rate for the human ovarian cancer cell line SK-OV-3 is as high as 91.84%, the killing rate for the human liver cancer cell line HepG2 is as high as 95.1%, and the killing rate for the human prostate cancer cell line LNCaP is as high as 93.42%, which is significantly better than the current conventional Vγ9Vδ2T cell amplification method.

[0044] A fourth aspect of the present invention provides a pharmaceutical composition.

[0045] Furthermore, the pharmaceutical composition comprises Vγ9Vδ2T cells with strong killing ability obtained by amplification according to the method described in the first aspect of the present invention.

[0046] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, which are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used to help stabilize the formulation or to help improve the activity or its biological effectiveness as needed. The pharmaceutical composition thus formulated can be administered by any appropriate means known to those skilled in the art as needed.

[0047] Illustratively, representative examples of the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: one or more fillers, such as microcrystalline cellulose, lactose, sucrose, starch, modified starch, mannose, dextran, calcium carbonate, phosphate, sulfate, etc.; one or more binders, such as lactose, starch, modified starch, dextran, microcrystalline cellulose, sucrose, polyether polyols, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, methylcellulose, carboxymethyl cellulose, gelatin, polyvinyl pyrrolidone, magnesium aluminum silicate, etc.; one or more disintegrants, such as cross-linked polyvinyl pyrrolidone, cross-linked carboxymethyl starch, starch, microcrystalline cellulose, etc.

[0048] In addition, if necessary, the formulation of the pharmaceutical composition may further include surfactants and pigments, wherein the surfactants include but are not limited to solubilizers, emulsifiers, wetting agents, foaming agents, defoaming agents, disinfectants, and bactericides, and the pigments include but are not limited to natural pigments (such as caramel, chlorophyll, carotene, beet red, iron oxide, etc.) and artificial synthetic pigments.

[0049] In the present invention, the pharmaceutically acceptable refers to a substance (including but not limited to salts, carriers or diluents) that does not eliminate the biological activity or properties of the compound and is relatively non-toxic, that is, the substance can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any component of the composition into which it is incorporated.

[0050] A fifth aspect of the present invention provides a pharmaceutical preparation.

[0051] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the fourth aspect of the present invention.

[0052] In some embodiments, the pharmaceutical preparations of the present invention may have any dosage form selected from the group consisting of suspensions, solutions, sterile aqueous solutions, tablets, pills, powders, granules, capsules, emulsions, syrups, non-aqueous solutions, emulsions, lyophilized formulations, and suppositories. Furthermore, the formulations may be administered once or multiple times. Routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration.

[0053] In a specific embodiment, the pharmaceutical preparation provided by the present invention can be prepared into various dosage forms according to actual needs, and the clinical physician can determine the dosage that is beneficial to the patient based on factors such as the type, age, weight and general disease condition of the patient, and the method of administration.

[0054] A sixth aspect of the present invention provides any of the following applications:

[0055] (1) Use of the expansion medium described in the second aspect of the present invention for in vitro specific expansion of Vγ9Vδ2 T cells;

[0056] (2) Use of the Vγ9Vδ2 T cells with strong killing ability obtained by amplification according to the method of the first aspect of the present invention in the preparation of a drug for inhibiting, preventing or treating cancer, inflammatory diseases or infectious diseases;

[0057] Optionally, the cancer is ovarian cancer, liver cancer, prostate cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, multiple myeloma, kidney cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, testicular cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, melanoma, leukemia, lymphoma, osteosarcoma, neuroblastoma, meningioma or oral cancer;

[0058] Optionally, the inflammatory disease is rheumatoid arthritis, psoriasis, multiple sclerosis, alopecia areata, type I diabetes, Guillain-Barré syndrome, inflammatory bowel disease, scleroderma, polymyositis, vitiligo or systemic lupus erythematosus;

[0059] Optionally, the infectious disease is a viral infection, a bacterial infection, a parasitic infection or a fungal infection.

[0060] In the present invention, the cancer refers to a disease characterized by uncontrolled cell growth and is used in the broadest sense herein. Examples include, but are not limited to, ovarian cancer, liver cancer, prostate cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, multiple myeloma, kidney cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, testicular cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, melanoma, leukemia, lymphoma, osteosarcoma, neuroblastoma, meningioma or oral cancer, etc.

[0061] In the present invention, inflammatory diseases refer to a class of diseases characterized by an imbalance of local or systemic inflammatory responses in the body, caused by various causes (e.g., infection, autoimmunity, tissue damage, etc.). Examples include, but are not limited to, rheumatoid arthritis, psoriasis, multiple sclerosis, alopecia areata, type 1 diabetes, Guillain-Barré syndrome, inflammatory bowel disease, scleroderma, polymyositis, vitiligo, and systemic lupus erythematosus.

[0062] In the present invention, the infectious disease may be the result of any pathogenic agent, examples of which include but are not limited to viral infections such as AIDS, hepatitis B and C, bacterial infections, parasitic and fungal infections.

[0063] In addition, the present invention also provides a method for inhibiting, preventing or treating cancer, autoimmune diseases, inflammatory diseases or infectious diseases, which method comprises the following steps: administering a therapeutically or preventively effective amount of the Vγ9Vδ2T cells described in the third aspect of the present invention, the pharmaceutical composition described in the fourth aspect of the present invention, or the pharmaceutical preparation described in the fifth aspect of the present invention to a subject in need.

[0064] In some embodiments, the Vγ9Vδ2T cells described in the third aspect of the present invention, the pharmaceutical composition described in the fourth aspect of the present invention, or the pharmaceutical preparation described in the fifth aspect of the present invention can be administered for preventive and / or therapeutic treatment. In therapeutic applications, the Vγ9Vδ2T cells described in the third aspect of the present invention, the pharmaceutical composition described in the fourth aspect of the present invention, or the pharmaceutical preparation described in the fifth aspect of the present invention can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease or condition. The Vγ9Vδ2T cells described in the third aspect of the present invention, the pharmaceutical composition described in the fourth aspect of the present invention, or the pharmaceutical preparation described in the fifth aspect of the present invention can also be administered to reduce the likelihood of disease development, infection, or worsening.

[0065] In some embodiments, the effective amount of the Vγ9Vδ2T cells of the third aspect of the invention, the pharmaceutical composition of the fourth aspect of the invention, or the pharmaceutical preparation of the fifth aspect of the invention for therapeutic use may vary based on the severity and course of the disease or condition, previous treatment, the health status, weight and / or response to the drug of the subject and / or the judgment of the treating physician.

[0066] In some embodiments, the effective amount refers to the amount of a pharmaceutical composition or pharmaceutical preparation containing Vγ9Vδ2T cells as described above that is administered to a subject (e.g., a human patient already suffering from a disease, disorder, or condition), which is sufficient to cure or at least partially prevent or alleviate to some extent one or more symptoms of the disease, disorder, or condition being treated. The effectiveness of such a composition depends on multiple conditions, including but not limited to: the severity and progression of the disease, condition, or condition, previous therapy, the patient's health status and response to the drug, and the judgment of the attending physician. By way of example only, a therapeutically effective amount can be determined by routine experiments, including but not limited to dose-escalation clinical trials.

[0067] In some embodiments, the subject can be a human, a non-human primate, such as a chimpanzee and other apes and monkey species; a farm animal, such as a cow, horse, sheep, goat, pig; a domestic animal, such as a rabbit, dog, and cat; an experimental animal, including a rodent, such as a rat, mouse, and guinea pig, and the like. The subject can be of any age. The subject can be, for example, an elderly person, an adult, a teenager, a prepubescent, a child, a toddler, or an infant. In specific embodiments of the present invention, the subject is preferably a human.

[0068] The Vγ9Vδ2T cells of the third aspect of the present invention, the pharmaceutical composition of the fourth aspect of the present invention, or the pharmaceutical preparation of the fifth aspect of the present invention, and combinations thereof, can be administered in various regimens (e.g., time schedule, concentration, dosage, interval between treatments, and / or formulation). Prior to treatment with the Vγ9Vδ2T cells of the third aspect of the present invention, the pharmaceutical composition of the fourth aspect of the present invention, or the pharmaceutical preparation of the fifth aspect of the present invention, the subject may also be pretreated with, for example, chemotherapy, radiotherapy, or a combination of both.

[0069] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0070] The present invention provides a novel method for serum-free specific amplification to obtain Vγ9Vδ2T cells with strong killing ability. The method uses a serum-free culture system (using X-VIVO 15 medium as the basal culture medium). The protocol is simple and easy to operate. Only two molecules, zoledronic acid and interleukin-2, need to be added to greatly improve the amplification efficiency and the killing effect on solid tumor cells. In addition, the Vγ9Vδ2T cells prepared by the method provided by the present invention have a high positive rate and strong killing ability against solid tumors. It has good application prospects and important translational significance in the technical field of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Flow cytometry results of the proportion of Vγ9Vδ2T cells in T cell populations in peripheral blood mononuclear cells from different sources;

[0072] Figure 2 : Flow cytometry results of the proportion of Vγ9Vδ2T cells in the T cell population on day 10 after peripheral blood mononuclear cells from source 1 were expanded using different expansion schemes;

[0073] Figure 3 : Flow cytometry results of the proportion of Vγ9Vδ2T cells in the T cell population on day 10 after peripheral blood mononuclear cells from 2# were expanded using different expansion protocols;

[0074] Figure 4 : Figure 2 and Figure 3 The histogram of the data after aggregation;

[0075] Figure 5 : Flow cytometry results of Vγ9Vδ2T positive cells amplified on days 12, 15, 18, 21, 24, and 28 using the four optimized protocols;

[0076] Figure 6 : Figure 5Corresponding line chart;

[0077] Figure 7 : Detection results of the killing ability of specifically expanded Vγ9Vδ2T cells against human ovarian cancer cells SK-OV-3 at different effector-target ratios;

[0078] Figure 8 : Detection results of the killing ability of specifically amplified Vγ9Vδ2T cells against human liver cancer cells HepG2 at different effector-target ratios;

[0079] Figure 9 : Detection results of the killing ability of specifically amplified Vγ9Vδ2T cells against human prostate cancer cells LNCaP at different effector-target ratios. DETAILED DESCRIPTION

[0080] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0081] The reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0082] Example 1 Technical solution of the present invention

[0083] 1. Isolation of Peripheral Blood Mononuclear Cells (PBMC)

[0084] (1) Collect peripheral blood into a 50 mL centrifuge tube.

[0085] (2) Take 6 sterile 50mL centrifuge tubes, use a 25mL centrifuge tube to draw 15mL Ficoll reagent (purchased from Tianjin Haoyang Company) and add it directly to the bottom of the centrifuge tube slowly to prevent it from sticking to the wall. Adjust the speed of the electric pipette to 1.

[0086] (3) Slightly tilt the 50 mL centrifuge tube and carefully draw 15 mL of fresh umbilical cord blood into a 25 mL centrifuge tube and gently add it to the top layer of Ficoll without disturbing the Ficoll layer.

[0087] (4) Set the centrifuge parameters at room temperature to 460g, acceleration 9, deceleration 0, and centrifuge for 40 minutes.

[0088] (5) Carefully remove the upper serum layer with a rubber-tipped pipette and transfer the buffy coat layer 0.5 cm above the Ficoll to a new 50 mL centrifuge tube. Add 40 mL of DPBS (purchased from Cellmax) and centrifuge at 380 g for 10 minutes at room temperature.

[0089] (6) Discard the supernatant and resuspend each 50 mL centrifuge tube with 1 mL of DPBS, then combine. Rinse each centrifuge tube with 3 mL of DPBS and combine them into the previous tube. Repeat once.

[0090] (7) Centrifuge at 380 g for 10 minutes, then resuspend in 5 mL of DPBS and count.

[0091] (8) Cryopreservation or experimentation can be performed as needed. 9 million cells / 800 μL cryopreservation solution can be used for freezing.

[0092] 2. Flow cytometry

[0093] This example uses PBMCs from two different individual sources (PBMCs from 1# and PBMCs from 2#) as starting materials for flow cytometry. The PBMCs from 1# and 2# are both from Guangzhou Bairuikang Biotechnology Co., Ltd. The PBMCs used in the present invention are not specific, and PBMCs from other conventional sources or commercialized PBMCs can be amplified using the method provided by the present invention and similar technical effects can be achieved.

[0094] (1) Based on the cell counting results, the above cells were divided into 20,000 cells each.

[0095] (2) Centrifuge at 300g for 5 minutes at room temperature.

[0096] (3) Discard the supernatant, add 50 μL of DPBS containing 4% FBS, add 1 μL of antibody (all antibodies were purchased from Biolegend), and flick to mix. Incubate at room temperature in the dark for 15 minutes. In this experiment, 15 groups (the following 5 groups each correspond to 3 parallel groups) were incubated: negative control group, isotype control group (APC, FITC), APC-CD3 single staining group, FITC-Vγ9 single staining group, and APC-CD3 / FITC-Vγ9 double staining group.

[0097] (4) After the antibody incubation, add 1 mL of DPBS to the centrifuge tube and centrifuge at 300 g for 5 minutes.

[0098] (5) Resuspend the sample in 200 μL DPBS, filter into a 96-well plate, and use easyCyte TM 8HT was detected.

[0099] 3. Specific expansion of Vγ9Vδ2 T cells

[0100] (1) Two million peripheral blood mononuclear cells (PBMCs) isolated in the above steps were inoculated into the wells of a 12-well plate (corresponding to a density of 1×10 6 cells / mL), 2 mL of X-VIVO 15 medium (purchased from Lonza) was added, and on the same day, zoledronic acid (ZOL446; purchased from Selleck; hereinafter referred to as ZOL) was added to a final concentration of 5 μM. The cells were cultured in a 37°C, 5% CO2, saturated CO2 incubator (hereinafter referred to as incubator).

[0101] (2) After 24 hours, interleukin-2 (IL-2; purchased from Beijing Kelinen Company) was added at a final concentration of 100 IU / mL and the culture was continued.

[0102] (3) On the third day, the culture system was expanded at a ratio of 1:4.

[0103] (4) After that, fresh culture medium was replaced every two days, the cell density was adjusted to 500,000 cells per ml, and zoledronic acid and interleukin-2 were supplemented.

[0104] (5) The proportion of CD3 and TCRVγ9 double positive cells was detected at different time points.

[0105] (6) After 15 days, a killing experiment targeting different solid tumor cells was carried out.

[0106] 4. Killing experiments of Vγ9Vδ2T cells against solid tumor SK-OV-3, HepG2 and LNCaP cells

[0107] (1) One day in advance, target cells (SK-OV-3, Hep G2, and LNCaP cells) were plated in a 96-well plate at a density of 10,000 cells per well.

[0108] (2) The next day, effector cells (Vγ9Vδ2 T cells) were added. According to different effector-target ratios (0:1, 5:1, 10:1, and 20:1), a 1:1 mixture of target cell and effector cell culture medium was used as the resuspension medium to resuspend the Vγ9Vδ2 T cells. The supernatant in the 96-well plate was aspirated and 100 μL of target cells of the corresponding concentration was added.

[0109] (3) During plating, alamarBlue reagent (purchased from Thermo Fisher Scientific) was placed in a 15 mL centrifuge tube and centrifuged at the highest speed for 5 minutes; then 10 μL / well of alamarBlue reagent was added.

[0110] (4) After adding, tap the edges of the plate gently with your palm to mix, and place in an incubator for culture.

[0111] (5) Use an enzyme-labeled instrument to detect the absorbance of the samples at different time points and calculate the cell killing function.

[0112] Under this technical scheme, after 15 days of expansion, the Vγ9Vδ2T cell positivity rate was as high as 91.26%; its killing rate against the human ovarian cancer cell line SK-OV-3 was as high as 91.84%, its killing rate against the human liver cancer cell line HepG2 was as high as 95.1%, and its killing rate against the human prostate cancer cell line LNCaP was as high as 93.42%, which is significantly better than the current conventional Vγ9Vδ2T cell expansion method.

[0113] Example 2 Exploration of a Vγ9Vδ2 T cell-specific expansion scheme

[0114] 1. Experimental Materials

[0115] Peripheral blood mononuclear cells (PBMCs; purchased from Guangzhou Bairuikang Biotechnology Co., Ltd.), flow cytometry antibodies (all purchased from Biolegend), RPMI 1640 medium (purchased from Hyclone), X-VIVO 15 medium (purchased from Lonza), β-mercaptoethanol (purchased from Sigma), L-ascorbic acid (LAA, purchased from Sigma), fetal bovine serum (FBS, purchased from Cellmax), zoledronic acid (ZOL446, purchased from Selleck; hereinafter referred to as ZOL), pamidronate (PAM, purchased from Medlife; hereinafter referred to as PAM), interleukin-2 (IL-2, purchased from Beijing Kelinen), interleukin-15 (IL-15, purchased from PeproTech), phosphate-buffered saline (PBS), and 1,10-tetrahydrocannabinol (1,0-tetrahydrocannabinol) were used as the reagents. Saline, PBS; purchased from Cellmax Company), centrifuge (Xiangyi Company), carbon dioxide cell culture incubator (purchased from Esco Lifesciences Group Company), flow analyzer (purchased from Guava Company).

[0116] 2. Experimental methods

[0117] We initially set the commonly used concentrations of each reagent as the initial test concentrations, zoledronic acid (ZOL) concentrations of 5, 10, and 50 μM, pamidronate (PAM) concentration of 2.0 μg / mL, interleukin-2 (IL-2) concentrations of 100 IU / mL or 1000 IU / mL, and interleukin-15 (IL-15) concentrations of 100 IU / mL, and set up 8 different experimental schemes as shown in Table 1 below.

[0118] Table 1 Eight different specific expansion schemes of Vγ9Vδ2 T cells

[0119]

[0120] The initial number of cells was 2 million. After the second day, the medium was changed every two days. The proportion of CD3 and TCR Vγ9 double-positive cells was detected at different time points of amplification as an evaluation standard for the amplification effect.

[0121] The specific hallmark of Vγ9Vδ2 T cells is the co-expression of the TCR Vγ9 and TCR Vδ2 chains. The CD3 molecule, bound to the TCR to form a complex, is a characteristic hallmark of all T cells. Therefore, double positivity for CD3 and TCR Vγ9 by flow cytometry is a standard method for identifying Vγ9Vδ2 T cells in immunology.

[0122] 3. Experimental results

[0123] In this example, PBMCs from two different individuals were used as starting materials for flow cytometry detection. Figure 1 As shown by Figure 1 The results showed that in peripheral blood mononuclear cells, Vγ9Vδ2T cells accounted for 2.35% and 2.25% of the T cell population, respectively.

[0124] On this basis, we used these two samples to amplify the 8 schemes in Table 1. And performed flow cytometry detection on the 10th day. The test results are as follows Figure 2-4 As shown, according to Figure 2-4 The results show that the proportion of Vγ9Vδ2T amplified by Schemes 1 and 2 is the highest, reaching 40%-62% after 10 days of amplification. The proportion of Vγ9Vδ2T amplified by Schemes 4-1 and 4-2 is 33%-38%. The technical effect produced by Schemes 1 and 2 is a technical effect that is unexpected by those skilled in the art based on the existing technology.

[0125] Among them, the amplification efficiency of Scheme 1 and Scheme 2 is similar. Therefore, the amplification effect of adding 100 IU / mL IL-2 on Day 1 is similar to the amplification effect of adding 1000 IU / mL IL-2 on Day 0, and the former is considered in the design of subsequent schemes; the difference between Schemes 3-1, 3-2 and Schemes 4-1, 4-2 lies in the basal culture medium. The amplification effect of Schemes 4-1 and 4-2 is better than that of Schemes 3-1 and 3-2, which proves that X-VIVO 15 as a basal culture medium is more suitable for the amplification of Vγ9Vδ2T; the amplification efficiency of Scheme 4-1 and Scheme 4-2 is similar, while the amplification efficiency of Scheme 3-2 is slightly better than that of Scheme 3-1, which proves that the amplification effect of the combination of 100 IU / mL IL-2 and 100 IU / mL IL-15 is better than that of 1000 IU / mL IL-2 alone in the presence of fetal bovine serum, while in serum-free culture medium, the amplification effect of the combined factors is similar to that of the single factor. This technical effect is also a technical effect that is unexpected by those skilled in the art based on the existing technology. The amplification effect of scheme 5 was poor, suggesting that the concentration of pamidronate may need to be explored; the amplification effect of scheme 6 was poor, which may be related to the higher phosphonate concentration (50 μM).

[0126] We subsequently conducted a second round of method optimization for Protocol 1 and Protocol 4-1. The specific protocols are shown in Table 2. In the new Protocol 1 and Protocol 1-1, the basal culture medium RPMI1640 + serum in the first round was replaced with X-VIVO 15.

[0127] Table 2 Four optimized expansion schemes for Vγ9Vδ2 T cells

[0128]

[0129]

[0130] For the above four schemes, we performed flow cytometry tests on different culture days, and the test results are as follows: Figure 5-6 As shown, the above results indicate that Protocol 1 achieved the best expansion effect, with a positive rate of 91.26% after 15 days of expansion. Furthermore, Protocols 1 and 1-1 showed similar expansion effects, with phosphonate concentrations of 5 μM and 10 μM having no significant effect on the final percentage of amplified positive cells. Protocols 4-2 and 4-2-1 had significantly lower overall positive rates than Protocols 1 and 1-1, and took longer to reach peak positive rates. Therefore, among the above expansion protocols, Protocol 1 achieved the best expansion effect, with high amplification efficiency, simple operation, fewer culture medium components required, low cost, and short time. Next, we will perform a cell killing assay on cells expanded using Protocol 1.

[0131] Example 3 Detection of the cell killing function of specifically amplified Vγ9Vδ2T

[0132] 1. Experimental Materials

[0133] SK-OV-3 human ovarian cancer cells (purchased from Beina Chuanglian Company), HepG2 human liver cancer cells (purchased from Beina Chuanglian Company), LNCaP (purchased from the Cell Bank of the Chinese Academy of Sciences), alamarBlue cell viability detection reagent (purchased from Thermo Fisher Scientific), 96-well plates (purchased from Thermo Company), centrifuge (purchased from Xiangyi Company), carbon dioxide cell culture incubator (purchased from Esco Lifesciences Group), flow analyzer (purchased from Guava Company), and microplate reader (purchased from Thermo Fisher Scientific).

[0134] 2. Experimental methods

[0135] We used human ovarian cancer cell line SK-OV-3, human liver cancer cell line HepG2 and human prostate cancer cell line LNCaP as target cells, set four different effector-target ratios of 0:1, 5:1, 10:1 and 20:1, and used alamarBlue cell viability detection reagent to detect at different time points after incubation of effector cells and target cells, thereby obtaining the killing ability of effector cells (Vγ9Vδ2T cells specifically amplified using Scheme 1 described in Example 2) against different solid tumor cells.

[0136] 3. Experimental results

[0137] The experimental results are as follows Figure 7-9 As shown, the corresponding statistical results are shown in Tables 3-5 below. The above results show that after 15 days of amplification using the scheme 1 finally optimized in Example 2, the Vγ9Vδ2T cell positivity rate is as high as 91.26%; its killing rate against the human ovarian cancer cell line SK-OV-3 is as high as 91.84%, the killing rate against the human liver cancer cell line HepG2 is as high as 95.1%, and the killing rate against the human prostate cancer cell line LNCaP is as high as 93.42%.

[0138] Table 3 Figure 7 Statistics of killing efficiency of Vγ9Vδ2 T cells

[0139]

[0140] Table 4 Figure 8 Statistics of killing efficiency of Vγ9Vδ2 T cells

[0141]

[0142] Table 5 Figure 9 Statistics of killing efficiency of Vγ9Vδ2 T cells

[0143]

Claims

1. A method for obtaining Vγ9Vδ2 T cells with strong killing ability by serum-free specific amplification, characterized in that: The method comprises the following steps: (1) PBMCs were inoculated into X-VIVO 15 serum-free medium and cultured with zoledronic acid; (2) After culturing for 24 hours, interleukin-2 was added, and the culture was continued to expand and culture to obtain the Vγ9Vδ2 T cells with strong killing ability.

2. The method according to claim 1, characterized in that The seeding density of PBMC in step (1) is 0.1-10×10 6 cells / mL; Optionally, the PBMC inoculation density in step (1) is 1×10 6 cells / mL; Optionally, the final concentration of zoledronic acid in step (1) is 1-10 μM; Optionally, the final concentration of zoledronic acid in step (1) is 5 μM; Optionally, the culture conditions in step (1) are 37° C. and 5% CO 2 ; Optionally, the PBMCs in step (1) are peripheral blood mononuclear cells extracted from human peripheral blood.

3. The method according to claim 1, characterized in that The final concentration of interleukin-2 in step (2) is 50-200 IU / mL; Optionally, the final concentration of interleukin-2 in step (2) is 100 IU / mL; Optionally, the expansion culture in step (2) is an expansion culture for 10-30 days; Optionally, the expansion culture in step (2) is an expansion culture for 15 days.

4. The method according to claim 1, wherein The method also includes the following steps: on the third day of culture, expanding the culture system, then changing the medium every two days and supplementing zoledronic acid and interleukin-2, and after 10-30 days of amplification culture, obtaining the Vγ9Vδ2T cells with strong killing ability.

5. The method according to claim 4, characterized in that The expanded culture system is an expanded culture system at a ratio of 1:2-1:5; Optionally, the expanded culture system is an expanded culture system at a ratio of 1:4; Optionally, the cell density is adjusted to 200,000-800,000 cells / mL during the medium replacement; Optionally, the cell density is adjusted to 500,000 cells / mL during the medium replacement; Optionally, the final concentration of the supplemented zoledronic acid is 1-10 μM; Optionally, the final concentration of the supplemented zoledronic acid is 5 μM; Optionally, the final concentration of the supplemented interleukin-2 is 50-200 IU / mL; Optionally, the final concentration of the supplemented interleukin-2 is 100 IU / mL; Optionally, the expansion culture is an expansion culture for 15 days.

6. An expansion medium for specifically expanding Vγ9Vδ2 T cells, characterized in that The expansion medium comprises X-VIVO 15 serum-free medium, zoledronic acid, and interleukin-2; Optionally, the final concentration of zoledronic acid is 1-10 μM; Optionally, the final concentration of zoledronic acid is 5 μM; Optionally, the final concentration of interleukin-2 is 50-200 IU / mL; Optionally, the final concentration of interleukin-2 is 100 IU / mL.

7. A Vγ9Vδ2 T cell with strong killing ability obtained by amplification according to any one of claims 1 to 5, characterized in that: The CD3+TCRVγ9+ double-positive rate of the Vγ9Vδ2 T cells was ≥91.26%.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises Vγ9Vδ2 T cells with strong killing ability obtained by amplification according to the method of any one of claims 1 to 5.

9. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 8.

10. Any of the following applications: (1) Use of the expansion medium according to claim 6 for in vitro specific expansion of Vγ9Vδ2 T cells; (2) Use of the Vγ9Vδ2 T cells with strong killing ability amplified by the method of any one of claims 1 to 5 in the preparation of a drug for inhibiting, preventing or treating cancer, inflammatory diseases or infectious diseases; Optionally, the cancer is ovarian cancer, liver cancer, prostate cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, multiple myeloma, kidney cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, testicular cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, melanoma, leukemia, lymphoma, osteosarcoma, neuroblastoma, meningioma or oral cancer; Optionally, the inflammatory disease is rheumatoid arthritis, psoriasis, multiple sclerosis, alopecia areata, type I diabetes, Guillain-Barré syndrome, inflammatory bowel disease, scleroderma, polymyositis, vitiligo or systemic lupus erythematosus; Optionally, the infectious disease is a viral infection, a bacterial infection, a parasitic infection or a fungal infection.

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