Gamma delta T cell preparation as well as preparation method and application thereof

Abstract

The invention provides a gamma delta T cell preparation as well as a preparation method and application thereof, and relates to the technical field of biology. The composition provided by the invention comprises coenzyme Q10, vitamin C, human serum albumin, trehalose, sodium pyruvate and amifostine. By using the composition and the related preparation provided by the invention, the killing ability of gamma delta T cells can be effectively improved, the functions of the gamma delta T cells can be maintained, and tumor growth can be effectively inhibited.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a kind of γδT cell preparation and its preparation method and application. BACKGROUND

[0002] Gamma delta T cells (gamma delta T cells) are innate lymphocytes not limited by major histocompatibility complex (MHC). This cell can secrete a lot of substances, which can effectively kill cancer cells. Gamma delta T cells are one of the main components of human intraepithelial lymphocytes and mucosal epithelial lymphocytes (IEL).

[0003] Gamma delta T cells are composed of gamma chain and delta chain. Due to its unique advantages and practicability of realizing quantitative proliferation, there are a large number of clinical studies to explore the potential of gamma delta T cells in tumor treatment. A number of clinical studies have confirmed the safety and effectiveness of gamma delta T cells in the field of tumor immunotherapy; a large number of studies have confirmed that gamma delta T cells show good anti-tumor effect in tumors of the digestive system, urinary system, blood system, reproductive system, respiratory system and other systems, and cell immunotherapy based on gamma delta T cells is a powerful supplement to tumor immunotherapy, but further improvement of its clinical efficacy still faces many challenges and needs to be improved and optimized in many aspects.

[0004] The prior art Lv J, Liu Z, Ren X, Song S, Zhang Y, Wang Y. γδT cells, a key subset of T cell for cancer immunotherapy. Front Immunol. mentioned that a key area for future research is to enhance the persistence of gamma delta T cells in the tumor microenvironment (TME). Achieving sustained activity of gamma delta T cells in TME is crucial for long-term treatment success, especially in overcoming tumor recurrence and immune escape. Strategies to optimize the transport, homing and infiltration of gamma delta T cells to solid tumors are crucial for improving clinical outcomes. In addition, maintaining target antigen expression on tumor cells is key to preventing immune escape and ensuring sustained response. SUMMARY

[0005] To solve the above problems, the present application provides a kind of gamma delta T cell preparation and its preparation method and application.

[0006] In one aspect, the present application provides a kind of gamma delta T cell preparation, comprising: coenzyme Q10, vitamin C, human blood albumin, trehalose, sodium pyruvate, amifostine.

[0007] Specifically, the γδ T cell preparation comprises: 10-20 μM coenzyme Q10, 40-60 μM vitamin C, 0.1-0.2 mg / mL human blood albumin, 50-70 mM trehalose, 5-8 mM sodium pyruvate and 10-20 mg / mL amifostine.

[0008] Further specifically, the γδ T cell preparation comprises: 15-20 μM coenzyme Q10, 40-50 μM vitamin C, 0.15-0.2 mg / mL human blood albumin, 60-70 mM trehalose, 5-6 mM sodium pyruvate and 15-20 mg / mL amifostine.

[0009] Further specifically, the γδ T cell preparation comprises: 10-15 μM coenzyme Q10, 50-60 μM vitamin C, 0.1-0.15 mg / mL human blood albumin, 50-60 mM trehalose, 6-8 mM sodium pyruvate and 10-15 mg / mL amifostine.

[0010] Further specifically, the γδ T cell preparation can be stored stably at -80℃ for at least 6 months, and / or the cell preparation can be stored stably at -196℃ for at least one year.

[0011] Further preferably, the γδ T cell preparation is suitable for injection by intravenous drip injection.

[0012] In another aspect, the present application provides a method for preparing the above-mentioned γδ T cell preparation, comprising the following steps: (1) preparing γδ T cells; (2) taking water for injection, adding coenzyme Q10, vitamin C, human blood albumin, trehalose, sodium pyruvate and amifostine, and then adding an electrolyte solution to be isotonic with blood plasma.

[0013] Specifically, the final concentration of the γδ T cells is 1×10 6 -1×10 8 mg / mL.

[0014] Specifically, the components of the electrolyte solution include, but are not limited to, any one or more of sodium chloride, sodium gluconate, sodium acetate, potassium chloride or magnesium chloride.

[0015] Preferably, the electrolyte solution is an aqueous NaCl solution.

[0016] In yet another aspect, the present application provides the use of the above-mentioned γδ T cell preparation in the preparation of a product for treating a tumor disease.

[0017] Specifically, the tumor diseases include acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, multiple myeloma, liver cancer, kidney cancer, prostate cancer or lung cancer.

[0018] Compared with the prior art, the present application has the following advantages: 1. The gamma delta T cell preparation provided by the present application has strong killing ability to cancer cells, and also has good tumor inhibition rate in mouse in-vivo experiments.

[0019] 2. The composition provided by the present application is safe and reliable, and each component used in the present application can be used to prepare an injection solution, wherein coenzyme Q 10 The injection solution and vitamin C injection solution are described in the 2025 edition of Chinese Pharmacopoeia (Volume 2), human blood albumin and trehalose are both common injection solution components in the art, the safety of pyruvate sodium injection solution is described in the related literature "Application of pyruvate sodium in preparation of drugs for relieving high-chlorine acidosis", and the safety of amifostine injection solution is described in the related literature "Protective effect of domestic amifostine (WR-2721) on cisplatin-induced nephrotoxicity"; the safety of the injection solution is also detected in the experimental process, and no adverse reactions are found in healthy mice after injection of the gamma delta T cell preparation provided by the present application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the marker identification result in experimental example 1.

[0021] Figure 2 It is the killing effect of gamma delta T cells on THP-1 cells in experimental example 2.

[0022] Figure 3 It is the killing effect of gamma delta T cells on A549 cells in experimental example 2.

[0023] Figure 4 It is the killing effect of gamma delta T cells on Hep2G cells in experimental example 2.

[0024] Figure 5 It is the killing effect of gamma delta T cells on SW1353 cells in experimental example 2.

[0025] Figure 6 It is the secretion amount of cytokine IFN-gamma in experimental example 3.

[0026] Figure 7 It is the secretion amount of cytokine TNF-alpha in experimental example 3.

[0027] Figure 8 It is the mouse experiment result in experimental example 4.

[0028] Figure 9For comparison of the cell activity change trends of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 in Experimental Example 5.

[0029] Figure 10 For comparison of the cell activity change trends of Example 2, Comparative Example 4, Comparative Example 5 and Comparative Example 6 in Experimental Example 5.

[0030] Figure 11 For comparison of the cell activity change trends of Example 3, Comparative Example 7, Comparative Example 8 and Comparative Example 9 in Experimental Example 5.

[0031] Figure 12 For comparison of the cell activity change trends of Example 1, Comparative Example 10 and Comparative Example 11 in Experimental Example 5. DETAILED DESCRIPTION

[0032] The application will be further described in conjunction with specific examples, which are not intended to limit the application, but merely to illustrate the application. The experimental methods used in the following examples are not specifically described, and the experimental methods not specifically described in the examples are generally performed under conventional conditions. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0033] Table 1 Reagents

[0034] Example 1 Preparation of γδ T cells The human γδ T cell expansion kit (Sedo, item number CT-004) was used according to the instructions.

[0035] Examples 1-3 In Examples 1-3, the amounts of the components in each group of cell preparations are shown in Table 2.

[0036] Table 2 Grouping of Examples 1-3

[0037] Preparation method: The amounts of the components in 100 mL of injection solution were calculated according to Table 2, 85 mL of water for injection was taken, and the components were added according to the calculation results. The osmotic pressure was adjusted to 300 Osm / kg by adding NaCl, and the volume was adjusted to 100 mL by adding water for injection.

[0038] Comparative Examples 1-9 In Comparative Examples 1-9, the amounts of the components in each group of cell preparations are shown in Table 3, and the preparation method is the same as that of Examples 1-3.

[0039] Table 3 Grouping of Comparative Examples 1-9 Comparative Examples 1-9

[0040] Comparative Example 10 The difference from Example 1 is that the density of γδT cells is 1 x 10 5 cells / mL.

[0041] Comparative Example 11 The difference from Example 1 is that the density of γδT cells is 1 x 10 9 cells / mL.

[0042] Experimental Example 1 Marker identification After 10 days of culture, the expression of CD107a, a marker related to the killing activity of γδT cells, was detected by flow cytometry, and the results are shown in Table 4 and Figure 1 .

[0043] Table 4 CD107a expression

[0044] Note: Compared with Example 1, *** represents p<0.001.

[0045] As can be seen from Table 4 and Figure 1 , the cell preparations prepared by each group of the present application can effectively stimulate the expression of CD107a by γδT cells, and the expression of CD107a in the cell preparations prepared by each group of the comparative examples is significantly decreased.

[0046] As can be seen from Examples 1-3 and Comparative Examples 1-9, there is a synergistic effect between the components of the preparation adjuvant, and the overall components have a significant effect on the expression of the killing activity related markers of γδT cell preparations.

[0047] As can be seen from Example 1 and Comparative Examples 10-11, too low or too high a cell density significantly affects the expression of killing activity related markers of γδT cell preparations. Too low a density can not provide the best intercellular interaction and activation signal, while only when the cells are fully activated, the synthesis and degranulation capacity of their cytotoxic granules will reach the best state. Too high a density can induce cell exhaustion phenotype, and the most typical feature of exhausted T cells is the loss of cytotoxic function. Therefore, these exhausted γδT cells cannot effectively mobilize CD107a to the cell surface, and the positive rate of detected CD107a will be significantly reduced.

[0048] Experimental Example 2 In vitro killing activity experiment of cells The cell preparations prepared in Examples 1-3 or Comparative Examples 1-11 were stored at -196°C, and after 48h and 240h of storage, the killing activity of γδT cells on THP-1 cells was detected.

[0049] THP-1 cells were added to centrifuge tubes, serum-free RPMI-1640 medium was added, and the final cell density was adjusted to 2×10 5 cells / ml, and the cells were labeled with CFSE.

[0050] The γδT cell preparation was centrifuged at 1600 rpm for 10 min, the supernatant was discarded, the cells were resuspended in RPMI-1640 medium containing 10% FBS, the final cell density was adjusted to 2×10 6 cells / ml, and was named as Example 1-3 group and Comparative Example 1-11 group, respectively.

[0051] THP-1 cells and γδT cells were mixed in equal volume, 3 parallel groups were set for each group, and after mixing, they were placed in a 37℃, 5% CO2 cell incubator for 4 h, stained with propidium iodide (PI), and the dead cells (CFSE and PI double positive, cells as dead target cells) were detected by flow cytometry.

[0052] The killing ability of γδT cells on lung cancer cell line (A549), liver cancer cells (HepG2) and human chondrosarcoma cells (SW1353) was detected according to the above steps.

[0053] The experimental results are shown in Table 5 and Figures 2-5 .

[0054] Table 5 Killing activity experiment results

[0055] According to the comparison and analysis of the experimental data in Table 5 and Figures 2-5 It can be clearly seen that the γδT cell preparation prepared in the examples of the present application exhibits significantly better killing performance than the comparative examples. Specifically, the killing rate of Example 1-3 group on various tumor cells (THP-1, A549, HepG2, SW1353) is 90.4%-98.5% when stored for 48h, which is much higher than that of Comparative Example 1-11 group (not more than 88.3%); after storage for 240h, the example still maintains a killing rate of 88.4%-94.6%, while the highest of the comparative example is only 77.2%.

[0056] According to Example 1-3 and Comparative Example 1-9, it can be seen that the preparation adjuvant has a significant effect on the preservation and freeze-thaw recovery of the γδT cell preparation, so that the γδT cell preparation can still maintain high cell killing activity after long-term preservation and freeze-thaw recovery.

[0057] According to Example 1 and Comparative Examples 10-11, low cell density has little effect on the preservation, cryopreservation and recovery of the γδ T cell preparation, but as mentioned above, too low density can not provide the best cell-cell interaction and activation signal. If the cells themselves are not sufficiently activated, even if co-cultured with target cells, the up-regulation of CD107a can be weak, resulting in a significant decrease in the killing ability of the cell preparation. Too high cell density significantly affects the preservation, cryopreservation and recovery of the γδ T cell preparation, and high-density cryopreservation can lead to a sharp decrease in the survival rate after recovery (due to uneven penetration of cryoprotective agents, ice crystal damage, necrosis of cell clumps in the center, etc.). Although the E:T Ratio of all groups was adjusted to be consistent at the time of detection, this ratio was calculated based on the "number of viable cells after recovery". Toxic substances produced by a large number of dead cells and debris can further inhibit the function of surviving cells. And before cryopreservation, cells can have gone into exhaustion (low expression of CD107a) due to high density. This exhausted state can be preserved or even exacerbated after recovery. The cells that survive after recovery are likely to be "exhausted" cells or resting cells that are no longer strong in function.

[0058] This fully demonstrates that the preparation of the present application can significantly improve the killing ability of γδ T cells on tumor cells, and can still maintain high activity after a longer period of preservation and cryopreservation and recovery, proving that the γδ T cell preparation prepared in the examples of the present application has excellent preservation stability. This property has important practical application value for cell therapy products that need to be stored and transported for a long time.

[0059] Experimental Example 3 The cell culture solution of each group of THP-1 in Experimental Example 2 was collected, and the cytokine secretion was detected by ELISA kit. The experimental results are shown in Table 6 and Figures 6-7 .

[0060] Table 6

[0061] Note: Compared with Example 1, *** represents p<0.001.

[0062] Table 6 and Figures 6-7The experimental data fully show that the γδ T cell preparation prepared by the embodiment of the present application can effectively enhance the ability of γδ T cells to produce a variety of key cytokines. The levels of IFN-γ (480.7-495.6 pg / μL) and TNF-α (68.3-71.3 pg / μL) secreted by the γδ T cells in groups 1-3 of the examples are significantly higher than those in groups 1-11 of the comparative examples (IFN-γ is 368.3-426.0 pg / μL, and TNF-α is 40.3-60.1 pg / μL). These cytokines play an important role in the process of immune regulation and anti-tumor, in which IFN-γ can activate macrophages and enhance their killing ability, and TNF-α can directly induce tumor cell apoptosis. The experimental data fully show that the γδ T cell preparation prepared by the present application can effectively improve the cytokine synthesis and secretion function of γδ T cells, thereby enhancing their immune regulation and anti-tumor activity, and providing an important experimental basis for clinical application.

[0063] Experimental Example 4 Mouse Experiment 1. Tumorigenicity experiment: 40 one-day-old C57BL / 6J mice were randomly divided into 4 groups, namely a positive control group, an example 1 group, an example 2 group, and an example 3 group. The cell preparations of each group were inoculated subcutaneously in mice, and whether progressive nodules formed at the inoculation site was observed for 18 weeks. The experimental results were as follows: in the positive control group, all mice produced nodules, while no nodules were produced in the example 1-3 groups, indicating that the cell preparation prepared by the present application has no tumorigenicity.

[0064] 2. Tumor inhibition rate experiment: 18-22 g C57BL / 6J mice were inoculated with S180 solid tumors under aseptic conditions according to the transplanted tumor research method, and randomly divided into groups, with 10 mice in each group, including a negative control group, a positive drug group (cyclophosphamide 0.03 g / kg), an example 1 group, an example 2 group, an example 3 group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, a comparative example 4 group, a comparative example 5 group, a comparative example 6 group, a comparative example 7 group, a comparative example 8 group, a comparative example 9 group, a comparative example 10 group, and a comparative example 11 group. Each group was intravenously injected with an equal volume of normal saline and the corresponding prescription drugs, with a drug volume of 20 mL / kg, and continuous administration for 7 days. The tumor-bearing mice were sacrificed and weighed on the 2nd day after the last administration, and the tumor mass was separated and weighed. The negative control group was used as a reference for the other groups, and the tumor inhibition rate was calculated. The results are shown in Table 7 and Figure 8 .

[0065]

[0066] Table 7

[0067] Note: compared with example 1, *** represents p<0.001.

[0068] Table 7 and Figure 8 The experimental data in Table 7 and Table 8 fully show that the tumor inhibition rates (66.3%-69.8%) of the groups of Examples 1-3 are close to that of the positive control group (69.6%), and are significantly higher than those of the groups of Comparative Examples 1-11 (49.6-63.2%), indicating that the preparation of the application can effectively inhibit tumor growth in vivo. In the cell preparation provided by the application, each adjuvant is indispensable and the density of γδ T cells needs to be strictly controlled. If the total number of cells is small, the total secretion amount of various effector cytokines (such as IFN-γ and TNF-α) will decrease, and if the density is too high, it may lead to functional exhaustion, and the cytotoxic function of γδ T cells in the exhausted state will be severely impaired, and their ability to recognize and kill tumor cells will be significantly reduced, so that even if the number of cells is large, the overall tumor inhibition effect will be worse.

[0069] Example 5 Long-term storage stability experiment of γδ T cell preparation The stability of the preparations prepared in each embodiment and the comparative examples of the application was detected when stored at-196℃ for a long time.

[0070] (1) Cell activity checking method: After mixing the culture solution with an equal amount of trypan blue dye (0.2%), the mixture was added to a cell counting plate, and a cell counter was used to count the total number of cells and the number of living cells, and the proportion of living cells was calculated. If the proportion of living cells is more than 90%, this index is considered to be qualified.

[0071] (2) Cell property checking method: The cell surface markers CD3 (+) and TCR-γδ (+) were detected.

[0072] (3) Sterility test: The long-term stored preparation was detected according to the 1101 sterility test method recorded in the 4th part of the 2025 edition of the Chinese Pharmacopoeia, using culture method for detection, and the result was negative, then this index was considered to be qualified.

[0073] (4) Endotoxin test: The long-term stored preparation was detected according to the 1143 bacterial endotoxin test method recorded in the 4th part of the 2025 edition of the Chinese Pharmacopoeia, using endotoxin gel limit method, endotoxin <0.5 EU / mL, then this index was considered to be qualified.

[0074] (5) Mycoplasma test: The long-term stored preparation was detected according to the 3301 mycoplasma test method recorded in the 4th part of the 2025 edition of the Chinese Pharmacopoeia, using PCR method for detection, and the result was negative, then this index was considered to be qualified.

[0075] (6) pH test: The long-term stored preparation was detected according to the 0631 pH value test method recorded in the 4th part of the 2025 edition of the Chinese Pharmacopoeia, using a pH meter for detection, and the range was 6.8-7.4, then this index was considered to be qualified.

[0076] (7) Osmolarity test: The preparations stored for a long time are tested according to the O632 osmolarity determination method recorded in Part 4 of the 2025 edition of the Chinese Pharmacopoeia. The freezing point depression method is used for testing. If the range is 275-325 mOsmol / kg, the indicator is considered qualified.

[0077] All indicators of the preparations met the requirements after long-term storage. The test methods and results are shown in Table 8.

[0078] Table 8. Long-term stability test of γδT cell preparations

[0079] Cell marker identification: All groups met the cell characteristics requirements of CD3 (+) and TCR-γδ (+) within 12 months.

[0080] Sterility testing, endotoxin testing, and mycoplasma testing: No related contaminants were detected in any of the groups within 12 months, meeting the requirements for sterility, endotoxin-free, and mycoplasma-free, indicating that the formulation of the present invention can guarantee good safety.

[0081] pH and osmotic pressure tests: The pH value of all groups was within the range of 6.8-7.4 and the osmotic pressure was within the range of 275-325 mOsmol / kg within 12 months, which met the prescribed standards, indicating that the physicochemical properties of the preparation were stable.

[0082] Visible foreign matter: No visible foreign matter was found in any of the groups within 12 months, indicating that the appearance quality of the formulation was stable.

[0083] Cell viability assay results as follows Figures 9-12 As shown.

[0084] The synergistic effect of formulation excipients is a key factor in ensuring the long-term survival of γδT cells. Figures 9-11 The importance of this can be clearly seen by comparing the examples with the comparative examples: In Examples 1-3, the percentage of live cells decreased very little during the 12-month storage period and remained at a high level. The complete excipient system constructed a "protective barrier" that can effectively resist cell damage during long-term storage.

[0085] In Comparative Examples 1-9, the proportion of live cells decreased significantly faster during storage than in the Examples. This was because the synergistic effect of the excipients was disrupted, ultimately leading to a significant reduction in the number of live cells during long-term storage.

[0086] Final cell concentration (1×10) 6 -1×10 8 Strict control of cell / mL is another core factor ensuring the long-term viability of γδT cells. Figure 12The difference between the examples and the comparative examples can be seen: the cell concentration of examples 1-3 is in the range of 1x10 6 -1x10 8 The proportion of viable cells within 12 months not only decreases slowly, but also the cell function (such as killing activity, cytokine secretion) can be maintained (combined with the results of experimental examples 2-4).

[0087] The key reason is that at this density, cells can not only form effective interaction (such as signal transmission activation) between each other, but also will not cause "resource competition" due to excessive crowding. Cells can uniformly obtain nutrients and protection provided by auxiliary materials, avoiding problems such as local hypoxia, accumulation of metabolic waste, and thus maintaining long-term activity.

[0088] Comparative example 10 (density is too low, 1x10 5 Low density leads to insufficient signal transmission between cells, making it difficult to maintain an activated state, and a small amount of cells are more susceptible to environmental fluctuations during preservation, resulting in poor activity stability.

[0089] Comparative example 11 (density is too high, 1x10 9 The proportion of viable cells decreases rapidly, and high density can cause serious "resource competition", leading to deterioration of the local microenvironment; in addition, too high a density can induce cells to enter a "depleted state", even if they survive, their tumor-killing and cytokine-secreting functions will also decrease significantly (combined with the results of experimental examples 2-4), losing clinical application value.

[0090] In summary, the composition and preparation and method for the preparation of γδT cell products provided by the present application can significantly enhance the killing ability of γδT cells, promote cytokine secretion, improve the in vivo tumor suppression effect, and have excellent long-term preservation stability and high safety, wherein the synergistic effect of coenzyme Q10 and vitamin C and the strict control of the density of γδT cells are the key to achieving these advantages, providing an efficient, stable and safe cell preparation and related preparation method for tumor treatment.

[0091] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A preparation of γδ T cells, characterized in that, comprising: Coenzyme Q10, Vitamin C, human blood albumin, trehalose, sodium pyruvate and amifostine.

2. The preparation of gd T cells according to claim 1, characterized in that, comprising: 10-20 μΜ Coenzyme Q10, 40-60 μΜ Vitamin C, 0.1-0.2 mg / mL human blood albumin, 50-70 mM trehalose, 5-8 mM sodium pyruvate and 10-20 mg / mL amifostine.

3. The preparation of gd T cells according to claim 2, characterized in that, comprising: 15-20 μΜ Coenzyme Q10, 40-50 μΜ Vitamin C, 0.15-0.2 mg / mL human blood albumin, 60-70 mM trehalose, 5-6 mM sodium pyruvate and 15-20 mg / mL amifostine.

4. The preparation of γδ T cells according to claim 2, characterized in that, comprising: 10-15 μΜ Coenzyme Q10, 50-60 μΜ Vitamin C, 0.1-0.15 mg / mL human blood albumin, 50-60 mM trehalose, 6-8 mM sodium pyruvate and 10-15 mg / mL amifostine.

5. The preparation of γδ T cells according to any one of claims 1 to 4, characterized in that, The γδ T cell preparation can be stored stably at -80°C for at least 6 months, and / or the cell preparation can be stored stably at -196°C for at least one year.

6. The preparation of γδ T cells according to any one of claims 1 to 4, characterized in that, The injection method is selected from intravenous drip injection.

7. A method for the preparation of a preparation of γδ T cells according to any one of claims 1 to 4, characterized in that, comprising the following steps: (1) preparing γδ T cells; (2) taking water for injection, adding Coenzyme Q10, Vitamin C, human blood albumin, trehalose, sodium pyruvate and amifostine, and then adding an electrolyte solution to be isotonic with blood plasma, wherein the electrolyte solution comprises at least one of sodium chloride, sodium gluconate, sodium acetate, potassium chloride or magnesium chloride.

8. The preparation method according to claim 7, characterized in that, The final concentration of the γδ T cells is 1 x 10 6 -1 x 10 8 6 cells / mL.

9. Use of the γδ T cell preparation of any one of claims 1-4 in the preparation of a product for treating a tumor disease.

10. Use according to claim 9, characterized in that, The tumor disease includes acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, multiple myeloma, liver cancer, kidney cancer, prostate cancer or lung cancer. The tumor disease includes acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, multiple myeloma, liver cancer, kidney cancer, prostate cancer or lung cancer.

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