A gamma delta t cell preparation, method of making and use thereof

Abstract

The application provides a kind of gamma delta T cell preparation and its preparation method and application, it is related to the field of biotechnology.The composition provided by the application includes: coenzyme Q10, vitamin C, human blood albumin, trehalose, sodium pyruvate and amifostine.Using the composition and related preparation provided by the application can effectively improve the killing ability of gamma delta T cells and maintain the function of gamma delta T cells, and can effectively inhibit tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a γδT cell preparation, its preparation method, and its application. Background Technology

[0002] Gamma delta T cells are innate lymphocytes that are not restricted by the major histocompatibility complex (MHC). These cells can secrete many substances that effectively kill cancer cells. Gamma delta T cells are one of the main components of human intraepithelial lymphocytes and mucosal intraepithelial lymphocytes (IELs).

[0003] γδT cells are composed of γ and δ chains. Due to their unique advantages and the practicality of achieving quantitative proliferation, numerous clinical studies are currently exploring the potential of γδT cells in tumor treatment. Multiple clinical studies have confirmed the safety and efficacy of γδT cells in the field of tumor immunotherapy. Numerous studies have demonstrated that γδT cells have shown good anti-tumor effects in tumors of the digestive, urinary, hematologic, reproductive, and respiratory systems. Cell immunotherapy based on γδT cells is a powerful complement to tumor immunotherapy, but further improvement in its clinical efficacy still faces many challenges and requires improvement and optimization in multiple aspects.

[0004] The existing research, Lv J, Liu Z, Ren X, Song S, Zhang Y, Wang Y. γδT cells, a key subset of T cells for cancer immunotherapy. Front Immunol., mentions that a key area for future research is enhancing the persistence of γδT cells in the tumor microenvironment (TME). Achieving sustained γδT cell activity in the TME is crucial for long-term therapeutic success, particularly in overcoming tumor recurrence and immune escape. Optimizing strategies for the transport, homing, and invasion of γδT cells into solid tumors is essential for improving clinical outcomes. Furthermore, maintaining the expression of target antigens on tumor cells is key to preventing immune escape and ensuring a durable response. Summary of the Invention

[0005] To address the above problems, this invention provides a γδT cell preparation, its preparation method, and its application.

[0006] On one hand, the present invention provides a γδT cell preparation comprising: coenzyme Q10, vitamin C, human serum albumin, trehalose, sodium pyruvate, and amifostine.

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

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

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

[0010] More specifically, the γδT cell preparation can be stably stored at -80°C for at least 6 months, and / or the cell preparation can be stably stored at -196°C for at least 1 year.

[0011] More preferably, the injection method suitable for the γδT cell preparation is selected from: intravenous drip injection.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned γδT cell preparation, comprising the following steps:

[0013] (1) Preparation of γδT cells;

[0014] (2) Take water for injection, add coenzyme Q10, vitamin C, human serum albumin, trehalose, sodium pyruvate and amifostine, and then add electrolyte solution until it is isotonic with plasma.

[0015] Specifically, the final concentration of the γδT cells was 1×10⁻⁶. 6 -1×10 8 per mL.

[0016] 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.

[0017] Preferably, the electrolyte solution is an aqueous NaCl solution.

[0018] In another aspect, the present invention provides the application of the above-mentioned γδT cell preparation in the preparation of products for treating tumor diseases.

[0019] 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.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The γδT cell preparation provided by this invention has a strong killing ability against cancer cells and also has a good tumor inhibition rate in mouse in vivo experiments.

[0022] 2. The composition provided by this invention is safe and reliable. All components used in this invention can be used to prepare injection solutions, including coenzyme Q. 10 Information regarding the injection and vitamin C injection is recorded in Part II of the 2025 edition of the Chinese Pharmacopoeia. Human serum albumin and trehalose are commonly used components of injections in this field. The relevant literature on sodium pyruvate injection, "Application of sodium pyruvate in the preparation of drugs to relieve hyperchloremic acidosis," records the safety of pyruvate injection. The relevant literature on amifostine injection, "Protective effect of domestic amifostine (WR-2721) against cisplatin-induced nephrotoxicity," records the safety of amifostine injection. The safety of the injections was also tested during the experiment. No adverse reactions were observed in healthy mice after injection of the γδT cell preparation provided by this invention. Attached Figure Description

[0023] Figure 1 The results of marker identification in Experiment Example 1.

[0024] Figure 2 The killing effect of γδT cells on THP-1 cells in Experiment Example 2.

[0025] Figure 3 The killing effect of γδT cells on A549 cells in Experiment Example 2.

[0026] Figure 4 The killing effect of γδT cells on Hep2G cells in Experiment Example 2.

[0027] Figure 5 The killing effect of γδT cells on SW1353 cells in Experiment Example 2.

[0028] Figure 6 The amount of IFN-γ secreted in Experiment Example 3.

[0029] Figure 7 The amount of TNF-α secreted in Experiment Example 3.

[0030] Figure 8 The results are from the mouse experiment in Experiment Example 4.

[0031] Figure 9 This is a comparison of the trends in cell activity changes in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 in Experiment 5.

[0032] Figure 10 This is a comparison of the trends in cell activity changes in Example 2, Comparative Example 4, Comparative Example 5, and Comparative Example 6 in Experiment 5.

[0033] Figure 11 This is a comparison of the cell activity change trends of Example 3, Comparative Example 7, Comparative Example 8 and Comparative Example 9 in Experiment 5.

[0034] Figure 12 This is a comparison of the trends in cell activity changes in Example 1, Comparative Example 10, and Comparative Example 11 in Experiment 5. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0036] Table 1 Reagents

[0037]

[0038] Basic Example 1: Preparation of γδT cells

[0039] Human γδT cell expansion kit (Sydopeptide, catalog number CT-004) was used for culturing according to the instructions.

[0040] Examples 1-3

[0041] In Examples 1-3, the dosage of each component in each group of cell preparations is shown in Table 2.

[0042] Table 2 Grouping of Examples 1-3

[0043]

[0044] Preparation method:

[0045] Calculate the amount of each substance in 100mL of injection solution according to Table 2. Take 85mL of water for injection, add each component according to the calculation results, add NaCl to adjust the osmotic pressure to 300 Osm / kg, and then add water for injection to make up to 100mL.

[0046] Comparative Examples 1-9

[0047] In Comparative Examples 1-9, the dosage of each component in each group of cell preparations is shown in Table 3, and the preparation method is the same as in Examples 1-3.

[0048] Table 3 Grouping of Comparative Examples 1-9

[0049]

[0050] Comparative Example 10

[0051] The difference from Example 1 is that the γδT cell density is 1×10⁻⁶. 5 per mL.

[0052] Comparative Example 11

[0053] The difference from Example 1 is that the γδT cell density is 1×10⁻⁶. 9 per mL.

[0054] Experimental Example 1: Marker Identification

[0055] After 10 days of culture, the expression of CD107α, a marker related to γδT cell cytotoxicity, was detected by flow cytometry. The results are shown in Table 4. Figure 1 As shown.

[0056] Table 4. CD107α expression status

[0057]

[0058] Note: Compared with Example 1, *** represents p < 0.001.

[0059] From Table 4 and Figure 1 It can be seen that the cell preparations prepared in each group of the embodiments of the present invention can effectively stimulate γδT cells to express CD107α, and the expression of CD107α in the cell preparations prepared in each group of the comparative example is significantly reduced.

[0060] According to Examples 1-3 and Comparative Examples 1-9, there is a synergistic effect among the components of the formulation excipients, and the overall components have a significant impact on the expression of markers related to the killing activity of γδT cell preparations.

[0061] As demonstrated in Example 1 and Comparative Examples 10-11, both excessively low and high cell densities significantly affect the expression of cytotoxic activity markers in γδT cell preparations. Low densities may fail to provide optimal cell-cell interactions and activation signals, while the synthesis and degranulation of cytotoxic granules only reach their optimal state after cells are fully activated. Conversely, excessively high densities may induce a cell exhaustion phenotype; the most typical characteristic of exhausted T cells is the loss of cytotoxic function. Therefore, these exhausted γδT cells cannot effectively mobilize CD107α to the cell surface, resulting in a significantly reduced CD107α positivity rate.

[0062] Experiment Example 2: In vitro cell killing activity experiment

[0063] The cell preparations prepared in Examples 1-3 or Comparative Examples 1-11 were stored at -196°C and revived after 48h and 240h, and the killing activity of γδT cells against THP-1 cells was detected.

[0064] Add THP-1 cells to centrifuge tubes, add serum-free RPMI-1640 medium, and adjust the final cell density to 2 × 10⁶ cells / mL. 5 cells / ml, cells labeled with CFSE.

[0065] Centrifuge the γδT cell preparation at 1600 rpm for 10 min, discard the supernatant, resuspend the cells in RPMI-1640 medium containing 10% FBS, and adjust the final cell density to 2 × 10⁻⁶ cells / mL. 6 The cells / ml were named Examples 1-3 and Comparative Examples 1-11, respectively.

[0066] THP-1 cells and γδT cells were mixed in equal volumes, with three replicates per group. After thorough mixing, the mixture was incubated at 37°C.

[0067] Cells were incubated in a 5% CO2 cell culture incubator for 4 h, stained with propidium iodide (PI), and flow cytometry was used to detect dead cells (CFSE and PI double positive, indicating that the cells were dead target cells).

[0068] The killing ability of γδT cells against lung cancer cell line (A549), liver cancer cell line (HepG2), and human chondroma cell line (SW1353) was tested according to the above steps.

[0069] The experimental results are shown in Table 5 and Figures 2-5 .

[0070] Table 5 Results of lethality experiments

[0071]

[0072] According to Table 5 and Figures 2-5The comparative analysis of experimental data clearly shows that the γδT cell preparations prepared in the embodiments of this invention exhibit significantly superior killing performance compared to the comparative examples. Specifically, the killing rates of the Examples 1-3 groups against various tumor cells (THP-1, A549, HepG2, SW1353) after 48 hours of storage were 90.4%-98.5%, far exceeding those of the comparative examples 1-11 (which did not exceed 88.3%). After 240 hours of storage, the Examples still maintained a killing rate of 88.4%-94.6%, while the comparative examples only achieved a maximum of 77.2%.

[0073] According to Examples 1-3 and Comparative Examples 1-9, the formulation excipients have a significant impact on the preservation and cryopreservation and thawing of γδT cell preparations, enabling γδT cell preparations to maintain high cell-killing activity even after long-term preservation and cryopreservation and thawing.

[0074] As shown in Example 1 and Comparative Examples 10-11, low cell density has a relatively small impact on the preservation and cryopreservation recovery ability of γδT cell preparations. However, as mentioned above, excessively low density may not provide optimal cell-cell interactions and activation signals. If the cells themselves are not sufficiently activated, even when co-cultured with target cells, the upregulation of CD107α may be very weak, leading to a significant decrease in the cytotoxicity of the cell preparation. Excessively high cell density significantly affects the preservation, cryopreservation recovery, and cytotoxicity of γδT cell preparations. High-density cryopreservation leads to a sharp decline in recovery survival rate (due to uneven penetration of cryoprotectant, ice crystal damage, and necrosis at the center of cell clumps). Although the E:T Ratio of all groups was adjusted to be consistent during the test, this ratio was calculated based on the number of viable cells after recovery. The toxic substances produced by a large number of dead cells and debris may further inhibit the function of surviving cells. Moreover, before cryopreservation, the cells may have already become exhausted due to excessive density (low expression of CD107a). This state of exhaustion may be retained or even aggravated after recovery. The cells that survive resuscitation are likely to be "exhausted" or resting cells that are no longer functioning well.

[0075] This fully demonstrates that the formulation of the present invention can significantly improve the killing ability of γδT cells against tumor cells, and can maintain high activity even after long-term storage and cryopreservation and thawing. This proves that the γδT cell formulation prepared in the embodiments of the present invention has excellent storage stability. This characteristic has important practical application value for cell therapy products that require long-term storage and transportation.

[0076] Experimental Example 3

[0077] The cell culture medium of THP-1 cells from each group in Experiment 2 was collected, and the cytokine secretion was detected using an ELISA kit. The experimental results are shown in Table 6 and 7. Figures 6-7 .

[0078] Table 6

[0079]

[0080] Note: Compared with Example 1, *** represents p < 0.001.

[0081] Table 6 and Figures 6-7 The experimental data fully demonstrate that the γδT cell preparation prepared in the embodiments of this invention can effectively enhance the ability of γδT 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 γδT cells in Examples 1-3 were significantly higher than those in Comparative Examples 1-11 (IFN-γ: 368.3-426.0 pg / μL, TNF-α: 40.3-60.1 pg / μL). These cytokines play important roles in immune regulation and anti-tumor processes. IFN-γ can activate macrophages and enhance their killing ability, while TNF-α can directly induce tumor cell apoptosis. The experimental data fully demonstrate that the γδT cell preparation prepared in this invention can effectively improve the cytokine synthesis and secretion function of γδT cells, thereby enhancing their immune regulation and anti-tumor activity, providing important experimental evidence for clinical application.

[0082] Experiment Example 4: Mouse Experiment

[0083] 1. Tumorigenicity test: Forty 1-day-old C57BL / 6J mice were randomly divided into 4 groups: positive control group, Example 1 group, Example 2 group, and Example 3 group. The cell preparation of each group was inoculated subcutaneously into the mice, and it was observed whether progressive nodule formation occurred at the inoculation site after 18 weeks. The results showed that nodules were generated in all mice in the positive control group, while no nodules were generated in Example 1-3 groups, indicating that the cell preparation prepared in this invention is non-tumorigenic.

[0084] 2. Tumor Inhibition Rate Experiment: 18-22g C57BL / 6J mice were inoculated with S180 solid tumors under aseptic conditions according to the transplanted tumor research method. Mice were randomly divided into groups of 10 each, including a negative control group, a positive drug group (cyclophosphamide 0.03g / kg), Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 7 group, Comparative Example 8 group, Comparative Example 9 group, Comparative Example 10 group, and Comparative Example 11 group. Each group received an equal volume of physiological saline and the corresponding prescribed drug intravenously at a volume of 20mL / kg for 7 consecutive days. On the second day after the last administration, the tumor-bearing mice were sacrificed and weighed, and the tumor fragments were dissected and weighed. The negative control group served as a baseline reference for other groups. The tumor inhibition rate was calculated, and the results are shown in Table 7. Figure 8 .

[0085]

[0086] Table 7

[0087]

[0088] Note: Compared with Example 1, *** represents p < 0.001.

[0089] Table 7 and Figure 8 The experimental data clearly show that the tumor inhibition rate of groups 1-3 (66.3%-69.8%) is close to that of the positive control group (69.6%), and significantly higher than that of groups 1-11 (49.6-63.2%), indicating that the formulation of the present invention can effectively inhibit tumor growth in vivo. In the cell formulation provided by the present invention, each adjuvant is indispensable and the density of γδT cells needs to be strictly controlled. If the total number of cells is low, the total secretion of various effector cytokines (such as IFN-γ, TNF-α) will decrease. If the density is too high, it may lead to functional exhaustion. The cytotoxic function of γδT cells in an exhausted state will be severely impaired, and their ability to recognize and kill tumor cells will be significantly reduced. Even if the number of cells is high, the overall tumor inhibition effect will be worse.

[0090] Experiment Example 5: Long-term stability experiment of γδT cell preparation

[0091] The stability of the formulations prepared in the various embodiments and comparative examples of the present invention was tested when stored at -196°C for a long period of time.

[0092] (1) Cell viability test method: Mix the culture medium with an equal amount of trypan blue dye (0.2%), add it to the cell counting chamber, use a cell counter to count the total number of cells and the number of live cells, and calculate the percentage of live cells. If the percentage of live cells exceeds 90%, the indicator is considered qualified.

[0093] (2) Cell characteristic examination method: Detect cell surface markers, CD3 (+) and TCR-γδ (+).

[0094] (3) Sterility test: The long-term stored preparations are tested according to the 1101 sterility test method recorded in Part 4 of the 2025 edition of the Chinese Pharmacopoeia. The culture method is used for testing. If the result is negative, the indicator is considered qualified.

[0095] (4) Endotoxin test: The preparations stored for a long time were tested according to the bacterial endotoxin test method 1143 recorded in Part 4 of the 2025 edition of the Chinese Pharmacopoeia. The endotoxin gel limit method was used. If the endotoxin < 0.5 EU / mL, the indicator was considered qualified.

[0096] (5) Mycoplasma test: The long-term stored preparations were tested according to the Mycoplasma test method 3301 recorded in Part 4 of the 2025 edition of the Chinese Pharmacopoeia. The test was performed using PCR. If the result was negative, the indicator was considered qualified.

[0097] (6) pH test: The pH test method recorded in Part 4 of the 2025 edition of the Chinese Pharmacopoeia is used to test the long-term stored preparations. The pH test is performed using a pH meter. If the pH is within the range of 6.8-7.4, the indicator is considered qualified.

[0098] (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.

[0099] All indicators of the preparations met the requirements after long-term storage. The test methods and results are shown in Table 8.

[0100] Table 8. Long-term stability test of γδT cell preparations

[0101]

[0102] Cell marker identification: All groups met the cell characteristics requirements of CD3 (+) and TCR-γδ (+) within 12 months.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Cell viability assay results as follows Figure 9-12 As shown.

[0107] The synergistic effect of formulation excipients is a key factor in ensuring the long-term survival of γδT cells. Figure 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.

[0108] 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.

[0109] 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 12 The differences between the Chinese examples and the comparative examples show that the cell concentrations in Examples 1-3 were all around 1×10⁻⁶. 6 -1×10 8 Within the range of cells / mL, the percentage of live cells not only decreased slowly over 12 months, but cell functions (such as cytotoxic activity and cytokine secretion) were also maintained (based on the results of Experiments 2-4).

[0110] The key reason is that at this density, cells can form effective interactions (such as signal transduction activation) without experiencing "resource competition" due to overcrowding. Cells can uniformly obtain the nutrients and protection provided by the excipients, avoiding problems such as local hypoxia and accumulation of metabolic waste, thereby maintaining long-term activity.

[0111] Comparative Example 10 (density too low, 1×10) 5 Low cell density (cells / mL) leads to insufficient intercellular signal transduction, making it difficult to maintain an activated state. Furthermore, a small number of cells are more susceptible to environmental fluctuations during preservation, resulting in poor activity stability.

[0112] Comparative Example 11 (Density too high, 1×10) 9 (cells / mL): The percentage of live cells decreases rapidly, and high density can trigger severe "resource competition" and lead to the deterioration of the local microenvironment. In addition, excessively high density can also induce cells to enter a "depletion state". Even if they survive, their ability to kill tumors and secrete cytokines will be significantly reduced (based on the results of Experiment 2-4), thus losing their clinical application value.

[0113] In summary, the compositions, formulations, and methods for preparing γδT cell products provided by this invention can significantly enhance the killing ability of γδT cells, promote cytokine secretion, and improve the in vivo tumor-suppressing effect. They also have excellent long-term storage stability and high safety. The synergistic effect of coenzyme Q10 and vitamin C, as well as the strict control of γδT cell density, are the key to achieving these advantages. This provides a highly efficient, stable, and safe cell preparation and related preparation method for tumor treatment.

[0114] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A γδT cell preparation, characterized in that, It consists of 15 μM coenzyme Q10, 50 μM vitamin C, 0.15 mg / mL human serum albumin, 60 mM trehalose, 6 mM sodium pyruvate, 15 mg / mL amifostine, and γδT cells, with a final concentration of 1 × 10⁻⁶ γδT cells. 6 -1×10 8 per mL.

2. The method for preparing the γδT cell preparation according to claim 1, characterized in that, Includes the following steps: (1) Preparation of γδT cells; (2) Take water for injection, add coenzyme Q10, vitamin C, human serum albumin, trehalose, sodium pyruvate and amifostine, and then add electrolyte solution until it is isotonic with plasma. The electrolyte solution includes at least one of sodium chloride, sodium gluconate, sodium acetate, potassium chloride or magnesium chloride. The final concentration of the γδT cells in the γδT cell preparation was 1×10⁻⁶. 6 -1×10 8 per mL.

3. The use of the γδT cell preparation according to claim 1 in the preparation of a drug for treating tumors.

4. The application according to claim 3, characterized in that, The tumors mentioned are: acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, B-cell lymphoma, multiple myeloma, liver cancer, kidney cancer, prostate cancer, or lung cancer.

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