Materials and methods for improving gd t cell expansion and uses of gd t cells

Abstract

The application discloses materials and methods for improving gamma delta T cell expansion and uses of gamma delta T cells, and relates to the technical field of biotechnology. The application provides a culture system for expanding gamma delta T cells in vitro, wherein the culture system is a serum-free culture medium containing L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin 2, human interleukin 7, interleukin 15, vitamin C and zoledronic acid. The human gamma delta T cells cultured by using the culture system provided by the application have the advantages of fast cell growth speed, large quantity, high purity, strong killing power to tumor cells and stable activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to materials and methods for improving the expansion of γδT cells and the uses of γδT cells. Background Technology

[0002] Tumors are caused by the body's insufficient immune function to overcome various pathogenic factors, and their occurrence and development are closely related to the host's immune status. Currently, surgery, radiotherapy, chemotherapy, and targeted therapy have become the main means of treating tumors in clinical practice, and have achieved certain therapeutic effects. However, the recurrence rate and mortality rate of tumors remain high, mainly due to tumor cells' ability to escape or develop drug resistance. Since its inception in the 1960s and 1970s, tumor cell immunotherapy has been one of the most promising treatment methods in the field of tumor immunotherapy. From early LAK and CIK cells to TILs cells and CAR-T cells, they have all played important roles in clinical tumor immunotherapy. However, LAK / CIK cells have limited their further clinical application due to the emergence of negative regulatory cells such as Treg cells during in vitro expansion and culture; TILs have been greatly limited in their clinical application due to difficulties in obtaining them; CAR-T cells have achieved significant therapeutic effects in the treatment of hematological malignancies, but their application in solid tumors has not yet made a breakthrough, and off-target effects and cytokine storms in CAR-T cell therapy have become obstacles to its clinical application. Therefore, researchers have been working hard to explore better treatment strategies and to apply other cell sources such as NK cells, NKT cells, and γδT cells to tumor immunotherapy.

[0003] As a non-classical T cell group, γδT cells, unlike classical T cells which express αβTCR, express only γδTCR. They play multiple roles, including "the first line of defense in the immune system," "a bridge between innate and adaptive immunity," and "immunomodulation." γδT cells can recognize tumor-specific antigens not only through TCR but also through their innate immune receptors such as NKG2D, DNAM-1, NCR, and TLR, thereby exerting their anti-tumor effects. They possess strong tumor-killing capabilities without genetic modification. The mechanisms by which γδT cells directly kill tumor cells include: ① secreting perforin / granzyme and TNF-α; ② expressing cytotoxic molecules such as TRAIL and FasL; and ③ CD16-mediated ADCC effects. γδT cells can recognize tumor cells through a non-classical MHC-dependent pathway, avoiding attack on normal tissues, making the clinical use of allogeneic γδT cells in cancer patients possible. In addition, γδT cells express various chemokine receptors such as CXCR3, CCR1, CCR5, and CCR8, exhibiting strong tissue chemotaxis capabilities. They can rapidly reach tumor tissues and activate to produce various effector molecules. Activated γδT cells can also interact with antigen-presenting cells and other immune cells, promoting the body's innate and adaptive anti-tumor immune responses. For example, activated γδT cells secrete large amounts of TNF-α and IFN-γ, thereby promoting the maturation of dendritic cells (DCs) and the activation of macrophages; activated γδT cells directly perform antigen-presenting cell functions, activating NKT cells and αβT cells; and activated γδT cells activate the anti-tumor effect of NK cells by upregulating the expression of the co-stimulatory molecule CD137L. Due to their unique immunological characteristics, γδT cells have become a highly promising and important cell source for adoptive cell immunotherapy of tumors, and γδT cell-based tumor immunotherapy has received increasing attention.

[0004] Numerous clinical trials have demonstrated that autologous γδT cells exhibit good safety and therapeutic efficacy in the antitumor immunotherapy of various solid tumors and hematological malignancies. However, activation-induced γδT cell anergy and reduced cell numbers remain unresolved issues. Furthermore, obtaining a sufficient number of γδT cells from patients with advanced cancer for in vitro expansion is challenging. Compared to patients, allogeneic immune cells derived from healthy individuals possess specific advantages, and the therapeutic application of allogeneic γδT cells has attracted attention; however, current clinical research on allogeneic γδT cells is still in its early stages both domestically and internationally. Therefore, expanding a sufficient number of γδT cells in vitro and using them for the clinical treatment of malignant tumors has significant scientific and practical value. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a culture system for in vitro expansion of γδT cells, wherein the culture medium is a serum-free medium containing one or a combination of L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C, and zoledronic acid. Using the culture system provided by this invention, γδT cells can be activated and expanded in vitro with rapid cell growth, high expansion rate, high purity, strong killing effect on tumor cells, and stable activity.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a culture system for in vitro expansion of γδT cells, wherein the culture system is a serum-free culture medium containing one or a combination of L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C and zoledronic acid.

[0007] In one embodiment, the culture system is a serum-free culture medium containing L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C, and zoledronic acid.

[0008] In one embodiment, the concentration of L-glutamine in the culture system is 0.20 g / L-0.40 g / L; and / or the concentration of ribonucleoside is 5.0 mg / L-10.0 mg / L; and / or the concentration of deoxyribonucleoside is 5.0 mg / L-10.0 mg / L; and / or the concentration of recombinant human albumin is 1.0 g / L-3.5 g / L; and / or the concentration of human interleukin-2 is 1 g / L. 0.0×10⁵ IU / L-5.0×10⁵ IU / L; and / or the concentration of human interleukin-7 is 5.0 μg / L-30.0 μg / L; and / or the concentration of interleukin-15 is 5.0 μg / L-40.0 μg / L; and / or the concentration of vitamin C is 5.0 mg / L-15.0 mg / L; and / or the concentration of zoledronic acid is 0.15 mg / L-2.0 mg / L.

[0009] In one embodiment, the concentration of L-glutamine in the culture system is 0.30 g / L; and / or the concentration of ribonucleoside is 7.5 mg / L; and / or the concentration of deoxyribonucleoside is 7.5 mg / L; and / or the concentration of recombinant human albumin is 2.0 g / L; and / or the concentration of human interleukin-2 is 2.0 × 10⁻⁶ g / L. 5 IU / L; and / or the concentration of human interleukin-7 is 10.0 μg / L; and / or the concentration of interleukin-15 is 10.0 μg / L; and / or the concentration of vitamin C is 7.5 mg / L; and / or the concentration of zoledronic acid is 0.45 mg / L.

[0010] In one embodiment, the concentration of L-glutamine in the culture system is 0.30 g / L; the concentration of ribonucleoside is 7.5 mg / L; the concentration of deoxyribonucleoside is 7.5 mg / L; the concentration of recombinant human albumin is 2.0 g / L; and the concentration of human interleukin-2 is 2.0 × 10⁻⁶ g / L. 5 IU / L; the concentration of human interleukin-7 was 10.0 μg / L; the concentration of interleukin-15 was 10.0 μg / L; the concentration of vitamin C was 7.5 mg / L; and the concentration of zoledronic acid was 0.45 mg / L.

[0011] In one embodiment, the ribonucleoside includes one or a combination of adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, and uracil ribonucleoside.

[0012] In one embodiment, the ribonucleosides include adenine ribonucleosides, guanine ribonucleosides, cytosine ribonucleosides, and uracil ribonucleosides.

[0013] In one embodiment, the mass ratio of adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, and uracil ribonucleoside is 1:1:1:1.

[0014] In one embodiment, the deoxyribonucleoside includes one or a combination of adenine deoxyribonucleoside, guanine deoxyribonucleoside, cytosine deoxyribonucleoside, and thymine deoxyribonucleoside.

[0015] In one embodiment, the deoxyribonucleoside includes adenine deoxyribonucleoside, guanine deoxyribonucleoside, cytosine deoxyribonucleoside, and thymine deoxyribonucleoside.

[0016] In one embodiment, the mass ratio of adenine deoxyribonucleoside, guanine deoxyribonucleoside, cytosine deoxyribonucleoside, and thymine deoxyribonucleoside is 1:1:1:1.

[0017] In another aspect, the present invention also provides a method for activating and expanding γδT cells in vitro, the method comprising: a) Introduce the cell population containing T cells into the culture system described above; b) Culture the cell population in vitro in the culture system to activate and expand γδT cells.

[0018] In one embodiment, the method further includes obtaining the cell population from a subject. In one embodiment, the subject is healthy. In one embodiment, the subject is unhealthy.

[0019] In one embodiment, the cell population is a mammalian cell population. In one embodiment, the mammalian cells are human cells. In one embodiment, the human cells are engineered cells. In one embodiment, the human cells are unengineered cells.

[0020] In one embodiment, the cell population is a peripheral blood mononuclear cell (PBMC) population. In one embodiment, the PBMC is freshly obtained PBMC. In one embodiment, the PBMC is frozen PBMC.

[0021] In one embodiment, the cell population is derived from human tissue. In one embodiment, the human tissue is fresh. In one embodiment, the human tissue is frozen.

[0022] In one embodiment, the cell population is cultured in the culture system for 0-24 days.

[0023] In one embodiment, the cell population is cultured in the culture system for 16-18 days.

[0024] In one embodiment, the culture temperature is 36°C-37°C.

[0025] In one embodiment, the culture temperature is 37°C.

[0026] In one embodiment, the CO2 volume percentage content in the culture air is 4.0%-8.0%.

[0027] In one embodiment, the CO2 volume percentage content in the culture air is 5.0%.

[0028] In one embodiment, the humidity of the air used for cultivation is saturated humidity.

[0029] In one embodiment, the cell concentration in the culture is (1.0-3.5) × 10⁻⁶. 6 per ml.

[0030] In one embodiment, the cell concentration in the culture is (1.5-2.5) × 10⁻¹⁰ on days 0-13. 6 Cells / ml. In one embodiment, the cell concentration in the culture was (2.0-3.5) × 10⁻⁶ cells / ml on days 14-18. 6 per ml.

[0031] In one embodiment, the method increases the percentage of γδT cells in the cell population to 10-100%. In another embodiment, the method increases the percentage of γδT cells in the cell population to 90-95%.

[0032] In one embodiment, the method further includes enriching in vitro expanded γδT cells from the cell population.

[0033] In another aspect, the present invention also provides an isolated population of γδT cells, which is generated by the method described above.

[0034] In another aspect, the present invention also provides isolated cell populations, wherein the percentage of γδT cells in the isolated cell populations is 10-100%.

[0035] In one embodiment, the percentage of γδT cells in the isolated cell population is 90-100%.

[0036] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) Therapeutic effective amount of the above-mentioned γδT cell population; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0037] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0038] In another aspect, the present invention also provides the use of the above-described isolated cell populations, pharmaceutical compositions, or pharmaceutical preparations in the preparation of medicaments for the prevention and / or treatment of diseases.

[0039] In one embodiment, the disease is cancer.

[0040] In one embodiment, the cancer includes blood cancer or solid tumor cancer.

[0041] In one embodiment, the solid tumor cancer is lung cancer or breast cancer.

[0042] In one embodiment, the lung cancer is squamous cell carcinoma of the lung, such as squamous cell carcinoma of the lung induced by SK-MES-1 cells, or adenocarcinoma of the lung induced by A549 cells.

[0043] The breast cancer mentioned includes breast cancer induced by SK-BR-3 cells.

[0044] (III) Beneficial Effects This invention provides a culture system for in vitro expansion of γδT cells. Compared with existing technologies, it has the following advantages: 1. The culture system provided by this invention enables the in vitro expansion of γδT cells, resulting in rapid cell growth, high fold expansion, high purity, strong killing effect on tumor cells, and stable activity. After 14-18 days of culture, the γδT cell expansion can reach more than 1500-fold, with a total cell count of up to 1.5 × 10⁻⁶ cells. 10 More than one, including CD3 +γδTCR + Cells account for more than 90% of the composition, and there is no contamination from bacteria, fungi, mycoplasma, or viral agents. 2. Using killing experiments, the results showed that γδT cells cultured for 14-18 days had high killing activity against SK-MES-1 cells, A549 cells and SK-BR-3 cells.

[0045] (iv) Terms and Definitions As used in this article, the term "allogeneic" refers to grafts derived from different individuals of the same species.

[0046] As used herein, the terms "isolation" or "isolating" refer to a process that increases the percentage of a substance in a composition. For example, isolating a type of cell from a cell population means producing a cell population in which the percentage of that type of cell increases compared to the percentage of that type of cell in the original cell population. Therefore, when used in the context of a type of cell, the term "isolated" does not mean that the isolated cell population contains 100% of that type of cell, but rather that the percentage of that type of cell in the cell population increases after the isolation process.

[0047] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0048] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0049] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0050] As used herein, the terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammal, such as a non-primate or a primate (e.g., a human). In a particular embodiment, the subject is a human. In one embodiment, the subject is a mammal diagnosed with a disease or disorder, such as a human. In another embodiment, the subject is a mammal at risk of developing a disease or disorder, such as a human.

[0051] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0052] As used in this article, the term "prevention" (preventing and prevention) refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 This is a graph showing the change in the total number of γδT cells over time during the culture process.

[0055] Figure 2 This is a graph showing the results of the γδT cell purity test.

[0056] Figure 3 This is a graph showing the detection results of γδT cell killing activity. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0060] In the following examples, the AIM V culture medium can specifically be a product of Thermo Fisher Scientific (China) Co., Ltd. Ribonucleotides and deoxyribonucleosides can both be products of BioBASic. L-glutamine can be a product of Sigma-Aldrich. Recombinant human albumin can specifically be a product of Hualan Biological Technology Co., Ltd. Human interleukin-2 can be recombinant human interleukin-2, specifically a product of Shanghai Huaxin Biotechnology Co., Ltd. Human interleukin-7 can be recombinant human interleukin-7, specifically a product of Peprotech. Human interleukin-15 can be recombinant human interleukin-15, specifically a product of Peprotech. Vitamin C can specifically be a product of Zhejiang Ruixin Pharmaceutical Co., Ltd. Zoledronic acid can be a product of Sichuan Hairong Pharmaceutical Co., Ltd.

[0061] Example 1 Expansion of human γδT cells: I. Isolation of peripheral blood mononuclear cells (PBMCs): 1. Peripheral blood sample: 50ml of peripheral blood was taken from healthy individuals, anticoagulated with heparin, and informed consent was obtained from the blood donors.

[0062] 2. Separation steps: (1) Centrifuge peripheral blood samples at 800g and 25℃ for 10 minutes, and collect the supernatant (plasma) into a 50ml sterile centrifuge tube and store at -80℃. Resuspend the precipitate with sterile PBS solution and dilute the peripheral blood cells to 60ml to obtain a peripheral blood cell suspension; add 20ml of human lymphocyte separation medium to two 50ml sterile centrifuge tubes respectively, and then gently add 30ml of peripheral blood cell suspension to the upper layer of human lymphocyte separation medium, and then centrifuge at 800g and 25℃ for 20 minutes.

[0063] (2) Take the mononuclear cells from the middle boundary layer, wash them three times with PBS solution, centrifuge at 1500 rpm for 10 minutes at 25°C, discard the supernatant, resuspend the mononuclear cells in 1.0 ml of AIM V medium, count them, and place them at 4°C for later use.

[0064] II. In vitro expansion culture using culture medium specifically designed for in vitro expansion of γδT cells: 1. Preparation of culture medium for in vitro expansion of γδT cells L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C, and zoledronic acid were added to AIM V medium to obtain a culture medium for in vitro expansion of γδT cells.

[0065] In this embodiment, five culture media for in vitro expansion of γδT cells were prepared, namely γδT cell culture medium A, γδT cell culture medium B, γδT cell culture medium C, γδT cell culture medium D, and γδT cell culture medium E. The concentrations of L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C, and zoledronic acid in γδT cell culture medium A, γδT cell culture medium B, γδT cell culture medium C, γδT cell culture medium D, and γδT cell culture medium E are shown in Table 1.

[0066] Table 1. Preparation of γδT cell culture medium 2. Expansion and culture of γδT cells The γδT cells were expanded using the five different γδT cell culture media mentioned above, and the specific steps are as follows: (1) Dilute the obtained PBMC cells with 30ml-50ml of γδT cell culture medium to make the cell concentration (1.0-1.5)×10⁻⁶. 6 Cells / ml were transferred to culture flasks and incubated at saturated humidity, 37°C, and 5.0% (v / v) CO2.

[0067] (2) On days 3-4 of culture, add γδT cell culture medium according to the cell density to maintain the cell density at (1.5-2.5)×10⁻⁶. 6 Cells / ml, continue incubation at saturated humidity, 37℃, and 5.0% (volume percentage) CO2.

[0068] (3) On days 6-8 of culture, carefully observe the cell growth status, resuspend the γδT cells in γδT cell culture medium to 300 ml, and transfer the cells into GT-T610 culture bags for culture. During the culture process, the cell density is controlled at (1.5-2.5) × 10⁻⁶. 6 The samples were cultured at 1 ml per 1 ml volume in an incubator with saturated humidity, 37°C, and 5.0% CO2. Simultaneously, 1 ml of the sample was collected in a 15 ml centrifuge tube for bacterial and fungal testing.

[0069] On day 6 of culture, the number of cells cultured using γδT cell culture medium D and γδT cell culture medium E did not increase significantly, and the cell growth status was poor.

[0070] Trypan blue staining showed that the number of cells stained with trypan blue was greater than 50%, indicating that γδT cell culture medium D and γδT cell culture medium E were not suitable for the expansion culture of γδT cells. In subsequent implementation, only experiments and tests were conducted on three culture medium systems: γδT cell culture medium A, γδT cell culture medium B, and γδT cell culture medium C.

[0071] (4) Carefully observe the cell growth status on days 9-11 of culture. Based on the cell growth status, add γδT cell culture medium to the cell culture bag. During the culture process, control the cell density at (1.5-2.5) × 10⁻⁶. 6 Cells / ml were placed in an incubator with saturated humidity, 37°C, and 5.0% CO2 for further incubation.

[0072] (5) On days 11-12 of culture, carefully observe the cell growth status. Based on the cell growth status, transfer half of the cells to another GT-T610 culture bag. Add γδT cell culture medium to both cell culture bags. During the culture process, control the cell density at (1.5-2.5) × 10⁻⁶. 6 The samples were cultured at a concentration of 1 / ml and placed in an incubator with saturated humidity, 37°C, and 5.0% CO2 for further incubation. Simultaneously, 1 ml of the culture was sampled and placed in a 15 ml centrifuge tube for bacterial and fungal testing.

[0073] (6) Carefully observe the cell growth status on days 14-16 of culture. Based on the cell growth status, add γδT cell culture medium to both cell culture bags. During the culture process, control the cell density at (2.0-3.5) × 10⁻⁶. 6Cells / ml were placed in an incubator with saturated humidity, 37°C, and 5.0% CO2 for further incubation.

[0074] (7) On the 17th-18th day of culture, collect γδT cells according to the cell growth status, transfer the cells in the culture bag to a 250ml sterile centrifuge cup, centrifuge at 800g for 10min.

[0075] (8) Discard the supernatant, resuspend the cell pellet in the centrifuge cup with 100ml of sterile saline for injection, wash twice, centrifuge at 800g for 10min.

[0076] (9) Discard the supernatant, resuspend the cell pellet with sterile saline for injection, and transfer it to a 250ml sterile centrifuge cup to obtain γδT cells. Place the cells on ice for later use. At the same time, sample 1ml into a 15ml centrifuge tube for bacterial and fungal testing.

[0077] Example 2 Biological characteristics and functional assays of γδT cells: I. Proliferation rate of γδT cells: Samples were taken at different time points during the culture process to detect the growth status of γδT cells, and the cells were counted using trypan blue staining. The number of cells stained with trypan blue was less than 10%, indicating that the γδT cells were in good growth status.

[0078] During this period, the growth rate of γδT cells was recorded, and cell growth curves were constructed (e.g., Figure 1 The results showed that when γδT cells were expanded using the above method, the cells grew rapidly, and the number of cells obtained could meet the needs of clinical infusion (Table 2).

[0079] Table 2. Average total number of γδT cells during culture II. Purity of γδT cells: Take 1×10 γδT cells obtained in Example 1 6Each cell was placed in a centrifuge tube and resuspended in 100 μl of 1×PBS solution to form a single-cell suspension. 2 μl of mouse IgG (BD) solution (0.1 μg / μl) was added to each tube, and the cells were blocked at room temperature for 30 min. BD fluorescently labeled antibodies (FITC-conjugated anti-γδTCR (clone B1), percp-cy5.5-conjugated anti-CD3e (clone UCHT1)) were added to the corresponding sample tubes, and the cells were labeled at 4°C in the dark for 30 min. 1.0 ml of 1×PBS solution was added to each tube, the cells were mixed, and the cells were centrifuged at 3000 rpm for 5 min at 4°C, discarding the supernatant. The washing was repeated twice. The labeled cells were resuspended in 300 μl of 1×PBS solution, and the data were analyzed using a BD LSR Fortessa flow cytometer and FlowJo V10 software.

[0080] The results are as follows Figure 2 As shown, in γδT cells cultured in media A, B, and C, CD3... + γδTCR + T cells (CD3) + γδTCR + γδT cells accounted for 94.5%, 93.8%, and 94.7% of the total cells, respectively, and the purity of γδT cells was over 90%.

[0081] III. Killing activity of γδT cells against SK-MES-1 cells, A549 cells, and SK-BR-3 cells: The cytotoxic activity of the γδT cells prepared above against SK-MES-1 cells, A549 cells, and SK-BR-3 cells was detected using the following experimental methods: Target cells, namely SK-MES-1 cells (ATCC, catalog number HTB-58), A549 cells (ATCC, catalog number CCL-185), and SK-BR-3 cells (ATCC, catalog number HTB-30), were labeled with a red fluorescent dye (CellTrace Far Red, Invitrogen). The red fluorescent dye and target cells were incubated at 37°C in the dark for 20 minutes, with shaking every 5–10 minutes to mix the cells. The cells were then washed three times with 1640 medium containing 10% fetal bovine serum (FBS) at 3000 rpm for 10 minutes at 25°C. The cell concentration was adjusted to 1 × 10⁻⁶ cells using 1640 medium containing 10% FBS. 5 Quantity / ml, for later use.

[0082] 4-hour killing assay: Labeled target cells were added to sterile FACs tubes, 100 μl per tube. The concentration of effector cells (γδT cells) was adjusted according to the effector-to-target cell ratio (10:1), and γδT cell suspension was added to each tube at a volume of 100 μl / well. Control target cell tubes did not contain effector cells, but only 100 μl of 1640 medium containing 10% fetal bovine serum. The mixture was gently mixed and incubated at 37°C, 5.0% CO2 for 4 hours. Flow cytometry was used to detect the fluorescence intensity of the target cells, distinguishing between fluorescently positive and fluorescently negative target cells. Calculation of killing activity: expressed as a percentage of cytotoxicity: Cytotoxicity (%) = [(Number of target cells in control tube - Number of target cells in experimental tube) / Number of target cells in control tube] × 100%.

[0083] The results are as follows Figure 3 As shown in Table 3.

[0084] Table 3. Cytotoxic activity of γδT cells (%) The results showed that γδT cells cultured in γδT cell culture medium A, γδT cell culture medium B, and γδT cell culture medium C all exhibited strong killing activity against SK-MES-1 cells, A549 cells, and SK-BR-3 cells.

[0085] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture system for in vitro expansion of γδT cells, characterized in that, The culture system is a serum-free culture medium containing L-glutamine, ribonucleoside, deoxyribonucleoside, recombinant human albumin, human interleukin-2, human interleukin-7, interleukin-15, vitamin C, and zoledronic acid.

2. The culture system according to claim 1, characterized in that, The concentration of L-glutamine in the culture system was 0.20 g / L to 0.40 g / L; The concentration of ribonucleoside and / or ribonucleoside is 5.0 mg / L-10.0 mg / L; The concentration of and / or deoxyribonucleosides is 5.0 mg / L-10.0 mg / L; The concentration of recombinant human albumin is 1.0 g / L to 3.5 g / L; And / or the concentration of human interleukin-2 is 1.0 × 10⁻⁶. 5 IU / L - 5.0×10 5 IU / L; The concentration of human interleukin-7 is 5.0 μg / L to 30.0 μg / L; And / or the concentration of interleukin-15 is 5.0 μg / L-40.0 μg / L; And / or the concentration of vitamin C is 5.0 mg / L-15.0 mg / L; The concentration of zoledronic acid is 0.15 mg / L to 2.0 mg / L.

3. The culture system according to claim 2, characterized in that, The ribonucleosides include one or a combination of adenosine ribonucleosides, guanine ribonucleosides, cytosine ribonucleosides, and uracil ribonucleosides.

4. The culture system according to claim 3, characterized in that, The mass ratio of adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, and uracil ribonucleoside is 1:1:1:

1.

5. The culture system according to claim 2, characterized in that, The deoxyribonucleosides include one or a combination of adenine deoxyribonucleoside, guanine deoxyribonucleoside, cytosine deoxyribonucleoside, and thymine deoxyribonucleoside.

6. The culture system according to claim 5, characterized in that, The mass ratio of adenine deoxyribonucleoside, guanine deoxyribonucleoside, cytosine deoxyribonucleoside, and thymine deoxyribonucleoside is 1:1:1:

1.

7. A method for activating and expanding γδT cells in vitro, characterized in that, The method includes: a) Contacting a cell population containing T cells with the culture system according to any one of claims 1-6; b) Culture the cell population in vitro in the culture system to activate and expand γδT cells.

8. The method according to claim 7, characterized in that, The culture temperature is 36℃-37℃; And / or, the CO2 volume percentage content in the air during cultivation is 4.0%-8.0%; And / or, the humidity of the air used for cultivation is saturated humidity; And / or, the cell concentration in the culture system is (1.0-3.5) × 10⁻⁶. 6 per ml.

9. An isolated population of γδT cells, said isolated population of γδT cells being generated by the method according to claim 7 or 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (1) A therapeutically effective amount of the γδT cell population as described in claim 9; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

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