Culture medium for in-vitro amplification and activation of gamma delta T cells, culture method and application of culture medium

Abstract

The invention discloses a culture medium for in-vitro amplification and activation of gamma delta T cells, a culture method and application of the culture medium, and belongs to the technical field of in-vitro culture of immune cells. The culture medium comprises zoledronic acid, interleukin-2 (IL-2), wnt / beta-catenin signaling pathway activators (GSK-3alpha and GSK-3beta specific inhibitors), particularly 6-bromo indirubin-3 '-acetoxime (Bio-acetoxime), peripheral blood mononuclear cells (PBMC) can be used as a starting material, gamma delta T cells can be separated from the PBMC through amplification for 14 days, high-purity gamma delta T cells can be efficiently and repeatedly generated, and high-purity gamma delta T cells can be obtained. The yield of the gamma delta T cell can be obviously improved, the obtained gamma delta T cell has the functions of a T cell, an NK cell and an antigen presenting cell, and the anti-tumor efficiency is also obviously improved.

Description

Technical Field

[0001] The present application belongs to the technical field of in vitro culture of immune cells, and specifically relates to a culture medium, a culture method and an application thereof for in vitro expansion and activation of γδT cells. Background Art

[0002] Immunotherapy has become the fourth type of therapy for treating tumors after surgery, chemotherapy, and radiotherapy. T lymphocytes are mainly divided into two categories: αβT cells and γδT cells, of which γδT cells are considered to be potent cells that effectively resist infection and malignancy because they are strategically distributed in mucosal tissues, can specifically recognize target cells, and respond quickly after activation. Similar to αβT cells, γδT cells have the ability to trigger a strong cytotoxic response by releasing molecules such as TNF-α, IFN-γ, perforin, and granzymes after specific activation. Unlike αβ-TCR, the activation of γδ-TCR is usually independent of antigens presented on major histocompatibility complex I or II (MHC-I / II). In addition, γδT cells are fully mature when they leave the thymus, have a wide range of antigen recognition capabilities, and can respond quickly to antigens. Similar to αβT cells and natural killer (NK) cells, γδT cells express NKG2D receptors and can recognize stress-induced signals on tumors and infected cells. In addition, γδT cells also share other receptors with innate NK cells, such as FcγRIIa (CD16), DNAX accessory molecules (DNAM-1, CD226), and natural cytotoxicity receptors (NCRs) NKp46, NKp44, and NKp30 that promote antitumor cytotoxicity. According to the composition of the TCR chain, γδT cells can be divided into different subpopulations, of which Vδ1 and Vδ2 are the two main subpopulations found in humans. Both subpopulations can be found in peripheral blood, with Vδ2T cells being the main ones, while Vδ1T cells are mainly distributed in various tissues and mucosa, including skin, spleen, intestines, and lungs. The mechanism of action of γδT cells in antitumor immune response mainly includes direct cytolytic activity through NKG2D, FcγRIII, Fas / Fas ligand, DNAM-1 receptor-ligand interaction, and TNF-related apoptosis-inducing ligand (TRAIL) pathways, thereby releasing perforin, granzymes, IFN-γ, and TNF-α. In addition, γδT cells have the ability to activate other immune cells, such as promoting the activation of NK cells through co-stimulation, promoting the maturation of dendritic cells (DC), and supporting the initiation of humoral immune responses. The potential application of γδT cells in immunotherapy mainly stems from their unique MHC-independent activation characteristics, which makes their application not limited to the autologous environment. In addition, γδT cells may also target tumors that exhibit immune escape characteristics by downregulating MHC expression.

[0003] However, in peripheral blood, αβT cells account for about 95%, while the number of γδT cells is relatively small, accounting for only 0.5-5% of T cells. Especially in tumor tissues, the number of γδT cells is further reduced, which poses significant challenges to their application in basic research, preclinical research and in vitro research. Therefore, the method of expanding γδT cells is usually used to carry out related research.

[0004] Scientific studies using expanded γδT cells have mainly targeted specific subsets, and the isolation and expansion strategies used vary. The most common approach uses bisphosphonates, such as zoledronate, pamidronate, or bromohydron pyrophosphate (BrHPP), to activate specific Vδ2 T cells in total peripheral blood mononuclear cells (PBMCs). These compounds increase IPP levels by inhibiting the activity of analogous diphosphate synthase (FDPS), thereby promoting the expression of the BTN3A1 / BTN2A1 structure. Zoledronic acid-based expansion of Vδ2 T cells in total PBMCs can usually produce more than 90% pure Vδ2 T cells, but the yield varies greatly between different donors. In addition, other methods focus on the activation of TCR, such as stimulation with phytohemagglutinin (PHA) or anti-CD3 (OKT3), or in the presence of anti-CD28. In the literature, TCR-targeted activation methods are usually used to generate Vδ1 T cells in large quantities. Some less conventional methods have also been reported. For example, Siegers and colleagues found that pre-sorted Vδ2 T cells could be efficiently expanded by activation with the mitogen concanavalin A. Finally, K562 cells can be used as feeder cells and modified to express a variety of molecules, such as CD40L, CD70, CD80, CD86, CD83, CD137L, and the artificial membrane-bound cytokine IL-15. Typically, IL-2 is used to promote cell activation, growth, and proliferation, often in combination with other cytokines, such as IL-4, IL-7, IL-15, IL-18, and IL-21. During the isolation of γδ T cells, a depletion step of αβ T cells is often included, and additional depletion steps of NK cells or unwanted γδ subsets may be combined before or after the expansion period. The methods currently described have certain problems, mainly reflected in their limited applicability, often only targeting specific subsets, and may result in unpredictable or unrealistic purity levels of the final product.

[0005] Therefore, it is necessary to develop a practical protocol to specifically isolate Vδ2 T cells and to employ an expansion method that ensures cell purity and maintains their functional properties. Standardized isolation and expansion protocols will facilitate comparisons between studies and thus improve the understanding of γδ T cell differentiation, function, and their targets. Summary of the invention

[0006] 1. Purpose of the Invention

[0007] The purpose of the present invention is to provide a culture medium, a culture method and an application thereof for in vitro expansion and activation of γδT cells. The culture medium comprises zoledronic acid, interleukin-2 (IL-2), a wnt / β-catenin signaling pathway activator (GSK-3α and GSK-3β specific inhibitor), especially 6-bromoindirubin-3'-acetone oxime (Bio-acetoxime), and can use peripheral blood mononuclear cells (PBMC) as starting materials, and through a 14-day expansion, γδT cells, especially Vγ9Vδ2T cells, can be separated from PBMC, and high-purity γδT cells can be efficiently and repeatedly produced, and the yield of γδT cells can be significantly improved and the anti-tumor function of the cells can be improved.

[0008] 2. Technical solution

[0009] In order to achieve the above-mentioned invention object, the technical solution adopted in this application is as follows:

[0010] In a first aspect, the present application provides a culture medium for in vitro expansion and activation of γδT cells, the culture medium comprising: a basal culture medium, zoledronic acid, interleukin-2 (IL-2) and a wnt / β-catenin signaling pathway activator (GSK-3α and GSK-3β specific inhibitor).

[0011] Furthermore, the above-mentioned basic culture medium includes any one of RPMI-1640, DMEM, AIM-V, etc., but is not limited thereto; the culture medium needs to contain human AB serum (Human AB Serum, the optimal concentration is 5%).

[0012] Furthermore, the above-mentioned basal culture medium is RPMI-1640.

[0013] Furthermore, the concentration of the zoledronic acid is 4-6 μM.

[0014] Furthermore, the concentration of the zoledronic acid is 4, 5 or 6 μM. Furthermore, the concentration of the zoledronic acid is 5 μM.

[0015] Furthermore, the concentration of the interleukin-2 (IL-2) is 500 to 1500 U / ml.

[0016] Furthermore, the concentration of the interleukin-2 (IL-2) is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 U / ml. Still further, the concentration of the interleukin-2 (IL-2) is 1000 U / ml.

[0017] Furthermore, the above-mentioned wnt / β-catenin signaling pathway activator includes any one or more of Bio-acetoxime, CHIR-99021 (CT99021) HCl, CHIR-98014, AZD1080, LY2090314, etc., but is not limited thereto.

[0018] Furthermore, the above-mentioned wnt / β-catenin signaling pathway activator is Bio-acetoxime.

[0019] Furthermore, the concentration of the above-mentioned Bio-acetoxime is 0.5-4.0 μM.

[0020] Furthermore, the concentration of the above-mentioned Bio-acetoxime is 1.0-4.0 μM.

[0021] Furthermore, the concentration of the above-mentioned Bio-acetoxime is 1.0-3.0 μM.

[0022] Furthermore, the concentration of the above Bio-acetoxime is 1.0, 1.5, 2.0, 2.5 or 3.0.

[0023] Furthermore, the concentration of the above Bio-acetoxime is 1.5 μM.

[0024] In a second aspect, the present application provides a use of the above-mentioned culture medium for in vitro expansion and activation of γδT cells in in vitro expansion and activation of γδT cells.

[0025] Furthermore, the above applications include:

[0026] First, pre-culture is performed, and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells (but not αβT cells); the pre-culture medium includes a basal culture medium, zoledronic acid and interleukin-2;

[0027] Then, the cells are induced to culture using the culture medium for in vitro expansion and activation of γδT cells to induce expansion of γδT cells and obtain γδT cells.

[0028] Furthermore, the purity of the γδT cells is greater than 90%, and the expansion multiple is greater than 350 times.

[0029] Furthermore, the purity of the γδT cells is greater than 90%, and the expansion multiple is greater than 400 times.

[0030] Furthermore, the above-mentioned γδT cells mainly include Vγ9Vδ2T cells, and Vγ9Vδ2T cells account for more than 90% of the γδT cells.

[0031] Furthermore, in the above preculture medium, the concentration of zoledronic acid is 4-6 μM.

[0032] Furthermore, in the above preculture medium, the concentration of zoledronic acid is 4, 5 or 6 μM. Furthermore, further, in the above preculture medium, the concentration of zoledronic acid is 5 μM.

[0033] Furthermore, in the above preculture medium, the concentration of interleukin-2 is 50-100 U / ml.

[0034] Furthermore, in the above preculture medium, the concentration of interleukin-2 is 50, 60, 70, 80, 90 or 100 U / ml. Furthermore, in the above preculture medium, the concentration of interleukin-2 is 100 U / ml.

[0035] Furthermore, the pre-culture period is 1 to 5 days.

[0036] Furthermore, the pre-culture time is 1, 2, 3, 4 or 5 days. Furthermore, the pre-culture time is 5 days.

[0037] Furthermore, in the above pre-culture, the pre-culture medium is supplemented once in the middle of the culture.

[0038] Furthermore, the pre-culture period is 5 days, and half of the pre-culture medium is replenished once on the 3rd day.

[0039] Furthermore, the induction culture is carried out for 8 to 10 days. Furthermore, the pre-culture is carried out for 9 days.

[0040] Furthermore, in the above-mentioned induction culture, half of the above-mentioned culture medium for in vitro expansion and activation of γδT cells is supplemented every 2 to 3 days.

[0041] Furthermore, in the above application, the cell density during the culture process is 1.0×10 6 ~4.0×10 6 cells / ml.

[0042] Furthermore, in the above application, the culture conditions are 37° C. and 5% CO 2 , and the culture conditions can be controlled by an incubator.

[0043] Furthermore, in the above application, the culture container includes: any one of a 6-well plate, a 12-well plate, a 24-well plate, a shake flask, a T25 flask (T flask), a T75 flask (T flask), a T175 flask (T flask), a T225 flask (T flask), a disposable cell culture bag, etc., as well as any reaction container that can be easily adopted by a technician in this field.

[0044] In a third aspect, the present application provides a method for culturing γδT cells for in vitro expansion and activation, the method comprising:

[0045] First, pre-culture is performed, and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells (but not αβT cells); the pre-culture medium includes a basal culture medium, zoledronic acid and interleukin-2;

[0046] Then, the cells are induced to culture using the culture medium for in vitro expansion and activation of γδT cells to induce expansion of γδT cells and obtain γδT cells.

[0047] Furthermore, the purity of the γδT cells is greater than 90%, and the expansion multiple is greater than 350 times.

[0048] Furthermore, the purity of the γδT cells is greater than 90%, and the expansion multiple is greater than 400 times.

[0049] Furthermore, the above-mentioned γδT cells mainly include Vγ9Vδ2T cells, and Vγ9Vδ2T cells account for more than 90% of the γδT cells.

[0050] Furthermore, in the above preculture medium, the concentration of zoledronic acid is 4-6 μM.

[0051] Furthermore, in the above preculture medium, the concentration of zoledronic acid is 4, 5 or 6 μM. Furthermore, further, in the above preculture medium, the concentration of zoledronic acid is 5 μM.

[0052] Furthermore, in the above preculture medium, the concentration of interleukin-2 is 50-100 U / ml.

[0053] Furthermore, in the above preculture medium, the concentration of interleukin-2 is 50, 60, 70, 80, 90 or 100 U / ml. Furthermore, in the above preculture medium, the concentration of interleukin-2 is 100 U / ml.

[0054] Furthermore, the pre-culture period is 1 to 5 days.

[0055] Furthermore, the pre-culture time is 1, 2, 3, 4 or 5 days. Furthermore, the pre-culture time is 5 days.

[0056] Furthermore, in the above pre-culture, the pre-culture medium is supplemented once in the middle of the culture.

[0057] Furthermore, the pre-culture period is 5 days, and half of the pre-culture medium is replenished once on the 3rd day.

[0058] Furthermore, the induction culture is carried out for 8 to 10 days. Furthermore, the pre-culture is carried out for 9 days.

[0059] Furthermore, in the above-mentioned induction culture, half of the above-mentioned culture medium for in vitro expansion and activation of γδT cells is supplemented every 2 to 3 days.

[0060] Furthermore, in the above method, the cell density during the culture process is 1.0×10 6 ~4.0×10 6 cells / ml.

[0061] Furthermore, in the above method, the culture conditions are 37° C. and 5% CO 2 , and the culture conditions can be controlled by an incubator.

[0062] Furthermore, in the above method, the culture container includes: any one of a 6-well plate, a 12-well plate, a 24-well plate, a shake flask, a T25 flask (T flask), a T75 flask (T flask), a T175 flask (T flask), a T225 flask (T flask), a disposable cell culture bag, etc., and any reaction container that can be easily adopted by a person skilled in the art.

[0063] The present application also provides γδT cells cultured using the above-mentioned method for culture of γδT cells expanded and activated in vitro.

[0064] Furthermore, the above-mentioned γδT cells are mainly Vγ9Vδ2T, accounting for more than 90%.

[0065] Furthermore, the γδT cells mentioned above have the functions of T cells, NK cells and antigen presenting cells.

[0066] 3. Beneficial effects

[0067] Compared with the prior art, the present application has the following beneficial effects:

[0068] (1) The present application provides a culture medium, a culture method and its application for in vitro expansion and activation of γδT cells, which can use PBMC as raw materials to directly culture and expand γδT cells from peripheral blood. The purity of the obtained γδT cells and Vγ9Vδ2T cells exceeds 90%, the expansion multiple reaches more than 400 times, and the cell viability exceeds 95%. In addition, the obtained γδT cells have the functions of T cells, NK cells and antigen presenting cells, and also significantly improve the anti-tumor efficacy.

[0069] (2) Since the proportion of γδT cells in peripheral blood is relatively low, the culture medium, culture method and application thereof for in vitro expansion and activation of γδT cells provided in the present application provide a new idea for solving the problem of direct culture and expansion of γδT cells and further improving cell function. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1The results of comparative analysis of the purity of γδT cells on day 14 using peripheral blood mononuclear cells to culture γδT cells by using IL-2, IL-2 / zoledronic acid combination, IL-2 / zoledronic acid / TWS119 combination, and IL-2 / zoledronic acid / Bio-acetoxime combination.

[0071] Figure 2 The results of a comparative analysis of the purity of Vδ2Vγ9T cells on day 14 were shown when peripheral blood mononuclear cells were used to culture Vδ2Vγ9T cells by using IL-2, IL-2 / zoledronic acid combination, IL-2 / zoledronic acid / TWS119 combination, and IL-2 / zoledronic acid / Bio-acetoxime combination.

[0072] Figure 3 The results of comparative analysis of the proliferation rate of γδ T cells on day 14 are shown.

[0073] Figure 4 The results of comparative analysis of the viability of γδ T cells on day 14 are shown.

[0074] Figure 5 The results of comparative analysis of the expression of perforin and granzyme B markers in γδ T cells cultured on day 14 are shown.

[0075] Figure 6 The results of comparative analysis of the expression of CD69 and CXCR3 markers in γδ T cells cultured on day 14 are shown.

[0076] Figure 7 The results of comparative analysis of the expression of CD16, CD56 and NKG2D markers in γδ T cells cultured on day 14 are shown.

[0077] Figure 8 The figure shows the antigen phagocytosis function of γδT cells cultured on day 14, and the MFI analysis results of γδT cells positive for FITC-Survivin transfection.

[0078] Fig. 9 The results of comparative analysis of the expression of CD86 and CD40 markers in γδ T cells cultured on day 14 are shown.

[0079] Fig.10 The graph shows the killing of 786-0, MDA-MB-231, BCG-823, SKOV-3, H460 and HGC-27 tumor cells by γδ T cells. DETAILED DESCRIPTION

[0080] The present application is further described below in conjunction with specific embodiments.

[0081] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0082] 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 this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0083] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0084] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0085] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0086] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0087] As used herein, zoledronic acid is a bisphosphonate drug commonly used to treat osteoporosis, bone metastasis and hypercalcemia. It helps to increase bone density and reduce the risk of fractures by inhibiting the activity of osteoclasts and reducing bone resorption. Zoledronic acid is mainly used to treat the following conditions: osteoporosis (especially postmenopausal women), bone metastasis, hypercalcemia, and to prevent bone loss caused by treatment in certain cancer patients. The mechanism of zoledronic acid is mainly to inhibit the activity of osteoclasts and reduce bone resorption, thereby increasing bone density. It binds to hydroxyapatite in bone tissue, prevents the formation and function of osteoclasts, and promotes their apoptosis. This mechanism ultimately helps to maintain the strength and structure of bones. In clinical applications, zoledronic acid is administered by intravenous injection, usually once a year for the treatment of osteoporosis. For patients with bone metastasis, it can effectively reduce the risk of fractures and relieve pain. Renal function and electrolyte levels need to be monitored regularly to avoid potential side effects.

[0088] As used herein, Bio-acetoxime is a class of biologically active compounds that are usually derivatives of amine oxides and have potential biological activity. In drug synthesis, it may be used as an intermediate to prepare compounds with antibacterial, anticancer or anti-inflammatory properties. Its properties generally include good solubility and biocompatibility, but the specific properties may vary from derivative to derivative. The mechanism of Bio-acetoxime is generally related to its biological activity and chemical reactivity. Although the research on Bio-acetoxime is relatively limited, in general, it may work through the following mechanisms: (a) Enzyme inhibition: Bio-acetoxime may bind to certain enzymes and inhibit their activity, thereby affecting metabolic processes in organisms. This mechanism is particularly common in pesticides; (b) Cell signal interference: It may affect cell growth and differentiation by interfering with cell signal transduction pathways. For example, some Bio-acetoxime derivatives can regulate the Wnt signaling pathway; (c) Redox reaction: As a reactive compound, Bio-acetoxime may participate in redox reactions and affect the oxidative stress state in cells. The role of bio-acetoxime in cell expansion may be related to its properties as a bioactive compound. Although the specific mechanism may require more research to clarify, in general, similar compounds may affect cell expansion in the following ways: (a) Promoting cell proliferation: Some bio-acetoxime derivatives may promote cell proliferation by activating cell cycle-related genes or signaling pathways (such as the Wnt / β-catenin pathway); (b) Anti-apoptotic effects: They may inhibit apoptosis-related signals, thereby prolonging cell survival and indirectly promoting the increase in cell number; (c) Influencing the cell environment: Bio-acetoxime may create a microenvironment that is more conducive to cell expansion by regulating the extracellular matrix or affecting cell-to-cell interactions; (d) Regulating growth factors: Some bioactive compounds can upregulate the expression of growth factors, further promoting cell expansion.

[0089] As used herein, peripheral blood mononuclear cells (hPBMC or PBMC) refer to mononuclear cells in the blood circulation, including lymphocytes and monocytes, which are separated from peripheral blood by Ficoll-Hypaque gradient centrifugation. As an example, the isolation process of peripheral blood mononuclear cells in this application includes: 100 ml of blood collected from the donor is placed in a heparin tube by a medical professional; 25 ml of 1×DPBS (Dulbecco's Phosphate-Buffered Saline) and 25 ml of blood are respectively divided into four 50 ml conical tubes; 15 ml of Lymphoprep (AxisShield, UK) is carefully added vertically to a new 50 ml conical tube, trying to avoid contact with the tube wall; then, the diluted blood is slowly added to the top of the conical tube containing Lymphoprep solution along the tube wall; these conical tubes are placed in a centrifuge and centrifuged at 800×g for 20 minutes at room temperature; after centrifugation, Lymphoprep is collected in the four blood layers formed. The white brown-yellow cell layer between the p layer and the plasma layer was transferred to a new 50 ml conical tube (three or four) and centrifuged again at 500 × g for 10 min at room temperature; then, the cells were resuspended in 45 ml of 1× DPBS solution, divided into three or four 50 ml conical tubes, and finally collected in one 50 ml conical tube; the collected cells were centrifuged at 350 × g for 10 min at room temperature for a total of two centrifugations; medium was added to the stock solution of 1.0×10□ cells / ml to make the final volume reach 10 ml, and the cell concentration was 1.0×10 6 cells / ml; then perform two to three pipetting operations, and finally take 1.0 ml of cells as the culture sample on day 0, and the remaining cells are divided into freezing vials at 1.0×10□ cells per cell and stored in a liquid nitrogen tank (LN2 tank) to be thawed and used later as needed.

[0090] Example 1

[0091] This embodiment provides a culture medium for activating and expanding γδT cells in vitro.

[0092] The culture medium comprises: a basic culture medium, zoledronic acid, interleukin-2 (IL-2) and a wnt / β-catenin signaling pathway activator; wherein:

[0093] The basic culture medium includes any one of RPMI-1640, DMEM, AIM-V, etc., all of which must contain human AB serum (Human AB Serum, the optimal concentration is 5%);

[0094] The concentration of zoledronic acid was 4 to 6 μM;

[0095] The concentration of interleukin-2 (IL-2) is 500-1500 U / ml;

[0096] The wnt / β-catenin signaling pathway activators include any one or more of Bio-acetoxime, CHIR-99021 (CT99021) HCl, CHIR-98014, AZD1080, LY2090314, etc.

[0097] As one of the embodiments, the culture medium for in vitro expansion and activation of γδT cells is as follows: the basic culture medium is RPMI-1640 (containing human AB serum, 5%); the concentration of zoledronic acid is 5 μM; the concentration of interleukin-2 (IL-2) is 1000 U / ml; and the concentration of Bio-acetoxime is 1.5 μM.

[0098] Example 2

[0099] This embodiment provides a culture method for expanding and activating γδT cells in vitro.

[0100] The method includes:

[0101] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0102] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0103] There are four groups in total:

[0104] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0105] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0106] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0107] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0108] On day 14, the purity, expansion fold (proliferation rate) and cell viability of γδT cells were compared, where the purity, cell viability and proliferation rate of cells were evaluated using flow cytometry, CCK assay and direct counting, respectively.

[0109] Result analysis:

[0110] The results of γδT cell purity are as follows Figure 1 As shown, on the 14th day of culture, the purity of γδT cells in the IL-2 group was 5.4%, the purity of γδT cells in the IL-2 / zoledronic acid group was 64.9%, the purity of γδT cells in the IL-2 / zoledronic acid / TWS119 group was 79.2%, and the purity of γδT cells in the IL-2 / zoledronic acid / Bio-acetoxime group was 96.1%.

[0111] The purity of Vγ9Vδ2T cells was as follows Figure 2 As shown, on the 14th day of culture, the purity of γδT cells in the IL-2 group was 4.2%, the purity of Vγ9Vδ2T cells in the IL-2 / zoledronic acid group was 84.0%, the purity of Vγ9Vδ2T cells in the IL-2 / zoledronic acid / TWS119 group was 85.8%, and the purity of Vγ9Vδ2T cells in the IL-2 / zoledronic acid / Bio-acetoxime group was 96.4%.

[0112] The proliferation rate of γδT cells on day 14 was as follows Figure 3 As shown, on the 14th day of culture, the expansion fold of γδT cells in the IL-2 / zoledronic acid group was 120 times, the expansion fold of γδT cells in the IL-2 / zoledronic acid / TWS119 group was 256 times, and the expansion fold of γδT cells in the IL-2 / zoledronic acid / Bio-acetoxime group was 408 times.

[0113] The results of γδT cell viability on day 14 were as follows Figure 4 As shown, where:

[0114] A is the CCK assay results: the γδT cell viability in the IL-2 / zoledronic acid group was 1.5% compared with the IL-2 group, the γδT cell viability in the IL-2 / zoledronic acid / TWS119 group was 1.7% compared with the IL-2 group, and the γδT cell viability in the IL-2 / zoledronic acid / Bio-acetoxime group was 3.1% compared with the IL-2 group;

[0115] B is the result of direct counting method, the γδT cell viability in IL-2 / zoledronic acid group was 71.1%, the γδT cell viability in IL-2 / zoledronic acid / TWS119 group was 73.3%, and the γδT cell viability in IL-2 / zoledronic acid / Bio-acetoxime group was 92.0%.

[0116] Example 3

[0117] This example provides the expression of perforin and granzyme B in γδT cells obtained by using the culture medium and culture method for in vitro expansion and activation of γδT cells in the present application. The culture method refers to Example 2, and specifically includes:

[0118] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0119] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0120] There are four groups in total:

[0121] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0122] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0123] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0124] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0125] On day 14, the cells were analyzed by intracellular staining flow cytometry (Fixation / PermeabilizationSolution Kit with BD GolgiPlug TM ) to detect perforin and granzymes.

[0126] Result analysis:

[0127] The expression results of perforin and granzyme were as follows Figure 5 As shown, on the 14th day of culture, the expression of perforin was 44.8% in the IL-2 / zoledronic acid group, 53.1% in the IL-2 / zoledronic acid / TWS119 group, and 70.9% in the IL-2 / zoledronic acid / Bio-acetoxime group; the expression of granzyme B was 22.0% in the IL-2 / zoledronic acid group, 77.3% in the IL-2 / zoledronic acid / TWS119 group, and 80.8% in the IL-2 / zoledronic acid / Bio-acetoxime group.

[0128] Example 4

[0129] This example provides the expression of cell activation markers and chemokine receptors of γδT cells obtained by using the culture medium and culture method for in vitro expansion and activation of γδT cells in the present application. The culture method refers to Example 2, specifically comprising:

[0130] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0131] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0132] There are four groups in total:

[0133] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0134] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0135] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0136] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0137] On day 14, the expression of CD69 and CCR5 on T cells was detected by flow cytometry.

[0138] Result analysis:

[0139] The expression results of γδT cell activation markers and chemokine receptors are as follows Figure 6 As shown, on the 14th day of culture, the expression of CD69 in the IL-2 / zoledronic acid group was 77.4%, the expression of IL-2 / zoledronic acid / TWS119 group was 80.9%, and the expression of IL-2 / zoledronic acid / Bio-acetoxime group was 90.5%; the expression of CCR5 in the IL-2 / zoledronic acid group was 49.7%, the expression of IL-2 / zoledronic acid / TWS119 group was 72.1%, and the expression of IL-2 / zoledronic acid / Bio-acetoxime group was 83.4%.

[0140] Example 5

[0141] This example provides the expression of NK cell markers of γδT cells obtained by using the culture medium and culture method for in vitro expansion and activation of γδT cells in this application. The culture method refers to Example 2, specifically comprising:

[0142] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0143] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0144] There are four groups in total:

[0145] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0146] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0147] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0148] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0149] On day 14, the expression of CD16, CD56, and NKG2D on T cells was detected by flow cytometry.

[0150] Result analysis:

[0151] The expression results of NK cell markers of γδT cells are as follows Figure 7 As shown, on the 14th day of culture,

[0152] CD16: 67.0% in the IL-2 / zoledronic acid group, 83.8% in the IL-2 / zoledronic acid / TWS119 group, and 94.7% in the IL-2 / zoledronic acid / Bio-acetoxime group;

[0153] CD56: the expression of IL-2 / zoledronic acid group was 15.6%, the expression of IL-2 / zoledronic acid / TWS119 group was 37.8%, and the expression of IL-2 / zoledronic acid / Bio-acetoxime group was 95.7%;

[0154] NKG2D: The expression level in the IL-2 / zoledronic acid group was 73.3%, that in the IL-2 / zoledronic acid / TWS119 group was 83.0%, and that in the IL-2 / zoledronic acid / Bio-acetoxime group was 90.5%.

[0155] Example 6

[0156] This example provides a method for detecting the antigen presenting cell function of γδT cells obtained by using the culture medium and culture method for in vitro expansion and activation of γδT cells in this application. The culture method is referred to Example 2, and specifically includes:

[0157] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0158] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0159] There are four groups in total:

[0160] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0161] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0162] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0163] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0164] On day 14, the expression of CD16, CD56, and NKG2D on T cells was measured by flow cytometry. On day 14, γδ T cells were co-cultured with FITC-conjugated Survivin peptide for 4 hours, and their phagocytic efficiency was measured by flow cytometry. Flow cytometry was also used to detect the expression of CD86 and CD40.

[0165] Result analysis:

[0166] The results of γδT cell phagocytosis test are as follows Figure 8 As shown, on the 14th day of culture, the MFI of FITC-positive cells in the IL-2 / zoledronic acid group was 9431, the MFI of FITC-positive cells in the IL-2 / zoledronic acid / TWS119 group was 11632, and the MFI of FITC-positive cells in the IL-2 / zoledronic acid / Bio-acetoxime group was 15712.

[0167] The expression results of CD86 and CD40 on γδT cells are as follows Fig. 9 As shown, on the 14th day of culture,

[0168] CD86: the expression rate was 65.8% in the IL-2 / zoledronic acid group, 71.7% in the IL-2 / zoledronic acid / TWS119 group, and 78.6% in the IL-2 / zoledronic acid / Bio-acetoxime group;

[0169] CD40: The expression of IL-2 / zoledronic acid group was 12.3%, the expression of IL-2 / zoledronic acid / TWS119 group was 20.4%, and the expression of IL-2 / zoledronic acid / Bio-acetoxime group was 47.0%.

[0170] Example 7

[0171] This example provides a method for detecting the killing activity of γδT cells obtained by using the culture medium and culture method for in vitro expansion and activation of γδT cells in this application. The culture method is referred to Example 2, and specifically includes:

[0172] First, pre-culture is performed for 5 days (days 1 to 5), and the isolated peripheral blood mononuclear cells (PBMC) are cultured with a pre-culture medium to induce the activity of γδT cells; the pre-culture medium includes a basic culture medium RPMI-1640 (containing human AB serum, 5%), zoledronic acid (5 μM) and interleukin-2 (100 U / ml); the culture medium is supplemented once on day 3;

[0173] The induction culture was then carried out for 9 days (days 6 to 14), and the culture medium for in vitro expansion and activation of γδT cells described in Example 1 was used (1.0×10 6 The pre-cultured cells were adjusted to 1 ml and inoculated into each well of a 24-well plate) and the γδT cells were induced to expand at 37°C and 5% CO2 to obtain γδT cells; half of the medium for in vitro expansion and activation of γδT cells was supplemented every 2 to 3 days.

[0174] There are four groups in total:

[0175] Group 1 (control group): IL-2, including: IL-2 (1000U / ml);

[0176] The second group IL-2 / zoledronic acid combination, which included: IL-2 (1000 U / ml), zoledronic acid (5 μM);

[0177] Group 3: IL-2 / zoledronic acid / TWS119 combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), TWS119 (2 μM);

[0178] The fourth group: IL-2 / zoledronic acid / Bio-acetoxime combination, including: IL-2 (1000 U / ml), zoledronic acid (5 μM), Bio-acetoxime (1.5 μM).

[0179] On day 14, γδT cells were co-cultured with six tumor cell lines, as follows: 786-O (renal cell carcinoma), MDA-MB-231 (breast cancer cells), BCG-823 (gastric cancer cells), SKOV3 (ovarian adenocarcinoma), H460 (non-small cell lung cancer cells), HGC-27 (gastric cancer cells). Adherent tumor cells were used as target cells and γδT cells were used as effector cells for in vitro co-culture (effector cells: target cells ratio was 10:1), and cell killing function was detected by LDH assay.

[0180] Result analysis:

[0181] The results of the detection of γδT cell killing activity are as follows Fig.10 As shown,

[0182] 786-O cell line: The killing rate of IL-2 / zoledronic acid group was 67.1%, the killing rate of IL-2 / zoledronic acid / TWS119 group was 78.3%, and the killing rate of IL-2 / zoledronic acid / Bio-acetoxime group was 92.8%;

[0183] MDA-MB-231 cell line: The killing ratio of IL-2 / zoledronic acid group was 44.0%, the killing ratio of IL-2 / zoledronic acid / TWS119 group was 55.7%, and the killing ratio of IL-2 / zoledronic acid / Bio-acetoxime group was 65.4%;

[0184] BCG-823 cell line: The killing ratio of IL-2 / zoledronic acid group was 14.6%, the killing ratio of IL-2 / zoledronic acid / TWS119 group was 24.7%, and the killing ratio of IL-2 / zoledronic acid / Bio-acetoxime group was 37.3%;

[0185] SKOV3 cell line: The killing rate of IL-2 / zoledronic acid group was 33.1%, the killing rate of IL-2 / zoledronic acid / TWS119 group was 43.2%, and the killing rate of IL-2 / zoledronic acid / Bio-acetoxime group was 57.9%;

[0186] H460 cell line: The killing ratio of IL-2 / zoledronic acid group was 23.8%, the killing ratio of IL-2 / zoledronic acid / TWS119 group was 31.2%, and the killing ratio of IL-2 / zoledronic acid / Bio-acetoxime group was 47.1%;

[0187] HGC-27 cell line: The killing ratio of the IL-2 / zoledronic acid group was 20.6%, the killing ratio of the IL-2 / zoledronic acid / TWS119 group was 28.4%, and the killing ratio of the IL-2 / zoledronic acid / Bio-acetoxime group was 40.4%.

Claims

1. A culture medium for in vitro expansion and activation of γδT cells, characterized in that: The culture medium comprises: a basic culture medium, zoledronic acid, interleukin-2, and a wnt / β-catenin signaling pathway activator.

2. A culture medium for in vitro expansion and activation of γδT cells according to claim 1, characterized in that: The wnt / β-catenin signaling pathway activator includes any one or more of Bio-acetoxime, CHIR-99021 (CT99021) HCl, CHIR-98014, AZD1080, LY2090314, etc.

3. A culture medium for in vitro expansion and activation of γδT cells according to claim 2, characterized in that: The wnt / β-catenin signaling pathway activator is Bio-acetoxime.

4. A culture medium for in vitro expansion and activation of γδT cells according to claim 3, characterized in that: The concentration of zoledronic acid is 4 to 6 μM; and / or The concentration of interleukin-2 is 500-1500 U / ml; and / or The concentration of the Bio-acetoxime is 0.5-4.0 μM.

5. A culture medium for in vitro expansion and activation of γδT cells according to claim 4, characterized in that: The concentration of zoledronic acid is 5 μM; The concentration of interleukin-2 is 1000U / ml; The concentration of the Bio-acetoxime is 1.5 μM.

6. Use of a culture medium for in vitro expansion and activation of γδT cells according to any one of claims 1 to 5 in in vitro expansion and activation of γδT cells.

7. The use according to claim 6, characterized in that: The applications include: The method comprises the following steps: firstly performing pre-culture for 1 to 5 days, wherein the pre-culture comprises: using a pre-culture medium to culture the isolated peripheral blood mononuclear cells to induce the activity of γδT cells; the pre-culture medium comprises a basal culture medium, zoledronic acid and interleukin-2; the concentration of the zoledronic acid is 4 to 6 μM, and the concentration of the interleukin-2 is 50 to 100 U / ml; The induction culture is then carried out for 8 to 10 days. The induction culture comprises: using the culture medium for in vitro expansion and activation of γδT cells to carry out culture, induce the expansion of γδT cells, and obtain γδT cells.

8. A method for in vitro expansion and activation of γδT cells, characterized in that: The method comprises: The method comprises the following steps: firstly performing pre-culture for 1 to 5 days, wherein the pre-culture comprises: using a pre-culture medium to culture the isolated peripheral blood mononuclear cells to induce the activity of γδT cells; the pre-culture medium comprises a basal culture medium, zoledronic acid and interleukin-2; the concentration of the zoledronic acid is 4 to 6 μM, and the concentration of the interleukin-2 is 50 to 100 U / ml; The induction culture is then carried out for 8 to 10 days. The induction culture comprises: using the culture medium for in vitro expansion and activation of γδT cells to carry out culture, induce the expansion of γδT cells, and obtain γδT cells.

9. γδT cells obtained by culturing using the method for in vitro expansion and activation of γδT cells as claimed in claim 8.

10. The γδT cell according to claim 9, characterized in that Among the above-mentioned γδT cells, Vγ9Vδ2T accounts for more than 90%.

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