A method for preparing CCR2+ gamma delta T cells with anti-tumor activity and migration ability
By culturing peripheral blood PBMCs from healthy individuals with the addition of factors such as IL-15, metformin, glucocorticoids, and nicotinamide in stages, Vδ2T cells expressing high levels of CCR2 were prepared. This solved the preparation problem in existing technologies, significantly improved their expansion rate and migration ability, and enhanced their killing activity against specific tumor cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING WEICHUANG BOJING BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-13
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies make it difficult to efficiently prepare Vδ2T cells that highly express CCR2, which limits their efficacy in tumor treatment. Furthermore, gene-edited CAR-T cells have significant side effects and are costly.
By adding factors such as IL-15, metformin, glucocorticoids and nicotinamide in stages, peripheral blood PBMCs from healthy individuals were cultured to prepare Vδ2T cells that highly express CCR2, with a culture period of 16 days.
It achieved high expression of CCR2 in Vδ2T cells, significantly improved their expansion and migration ability, and enhanced their killing activity against tumor cells such as breast cancer, ovarian cancer, and lung cancer.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing CCR2+γδT cells with anti-tumor activity and migration ability. Background Technology
[0002] γδT cells recognize a broad range of target ligands, but are limited by major histocompatibility complex (MHC) classes, thus triggering a rapid and early response. In equal numbers, γδT cells outperform conventional αβT cells in controlling tumor growth, indicating that γδT cells possess a superior ability to inhibit tumor growth and target and kill tumor cells.
[0003] γδT cells mainly include two types: Vδ1T and Vδ2T. Vδ2T cells are more abundant in peripheral blood. High-purity Vδ2T cells can be prepared by isolating peripheral blood mononuclear cells (PBMCs) from healthy donors and using stimulants such as zoledronic acid and IL-2. However, natural Vδ2T cells do not express or express low levels of CCR2, making it difficult to obtain CCR2+ Vδ2T cells with strong antitumor activity using conventional methods. This limits their effectiveness in cancer treatment.
[0004] CCR2 ligands include CCL2, and tumors with high CCL2 expression include breast cancer, ovarian cancer, lung cancer, and colorectal cancer. CCL2 ligands can recruit immune cells that highly express CCR2, and these recruited immune cells can exert a killing effect, thereby inhibiting tumor progression. Currently, adoptive immunotherapy typically uses gene editing to overexpress CCR2 in T cells; however, gene-edited CAR-T cells have significant side effects and are very expensive to produce, greatly limiting their clinical application.
[0005] Therefore, it is crucial to develop a method for preparing Vδ2T cells with high CCR2 expression without gene editing and through factor induction. Summary of the Invention
[0006] In view of this, the present invention provides a method for preparing CCR2+γδT cells, with a total culture period of 16 days. PBMCs are extracted from the peripheral blood of healthy individuals and cultured together with substances such as IL-15, metformin, glucocorticoids, and nicotinamide. This method overcomes the problems existing in the prior art and provides a method for preparing CCR2+γδT cells with stronger anti-tumor activity and migration ability.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a method for preparing CCR2+γδT cells with anti-tumor activity and migration ability, comprising the following steps: Step (1): At D0, PBMCs were extracted from peripheral blood of healthy individuals and inoculated into complete culture medium at a concentration of 1×10⁻⁶ cells / mL. 6 / mL~2×10 6 / mL, then add zoledronic acid, IL-15, and metformin, and place in a T25 culture flask for culture. Define day 0 of the initial culture as D0. Step (2): On day 2, count the cells cultured in step (1), replenish the complete culture medium according to the counting results, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, transfer cells from T25 culture flask to T75 culture flask for culture; Step (3): On day 4, count the cells cultured in step (2), replenish the complete culture medium according to the counting results, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, then add glucocorticoids and nicotinamide for culture, and transfer the cells from the T75 culture flask to the T150 culture flask for culture; Step (4): On day 6, collect the cells cultured in step (3), rinse with physiological saline, resuspend the cells in complete culture medium, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, inoculated into T300 culture flasks for culture; Step (5): From D8 to D16, replenish the complete culture medium every two days to maintain the cell concentration at 1×10⁻⁶. 6 / mL~2×10 6 / mL, and harvest the cells at D16 to obtain CCR2+Vδ2T cells with anti-tumor activity and migration ability; The complete culture medium in steps (1) to (5) consists of KBM581 medium, SR, Glutamax and IL2.
[0008] As a further aspect of the present invention: the concentration of zoledronic acid in step (1) is 5 μM.
[0009] As a further aspect of the present invention: the concentration of IL-15 in step (1) is 5-30 ng / ml.
[0010] As a further aspect of the present invention: the concentration of glucocorticoids in step (3) is 0.05-0.3 μM.
[0011] As a further aspect of the present invention, the concentration of nicotinamide in step (3) is 1-10 μM.
[0012] As a further aspect of the present invention: the culture conditions in steps (1) to (5) are all 37°C and 5% CO2.
[0013] As a further aspect of the present invention: the volume ratio of KBM581 culture medium:SR:Glutamax is 100:3:1; the final concentration of IL2 is 200U / ml.
[0014] The present invention relates to CCR2+γδT cells prepared by the above preparation method, wherein the cells are Vδ2T cell subtypes.
[0015] The present invention provides CCR2+γδT cells prepared by the above-described method. These CCR2+γδT cells can be used in the preparation of anti-tumor drugs, wherein the tumor is one or more of breast cancer, ovarian cancer, lung cancer, and colorectal cancer with high CCL2 expression.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves high CCR2 expression in Vδ2T cells by adding inducing factors such as IL-15, metformin, glucocorticoids, and nicotinamide in stages, resulting in a Vδ2T cell expansion rate several times higher than that of conventional methods.
[0017] The Vδ2T cells of this invention exhibit stronger migration ability than those obtained using conventional methods (Transwell assay).
[0018] The Vδ2T cells of this invention exhibit significantly higher killing activity against breast cancer, ovarian cancer, lung cancer, and colon cancer cells than conventional methods, by several times.
[0019] The Vδ2T cells of this invention can highly express chemokine receptors such as CCR2. Detailed Implementation
[0020] This invention discloses a method for preparing CCR2+γδT cells with anti-tumor activity and migration ability. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications that are obvious to those skilled in the art are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0021] Peripheral blood mononuclear cells (PBMCs) are cells in peripheral blood that possess a single nucleus, including lymphocytes, monocytes, dendritic cells (DCs), and a small number of other cells. PBMCs can be obtained from peripheral blood of healthy human or animal donors through steps such as Ficoll density gradient centrifugation and magnetic bead sorting. They mainly consist of lymphocytes and monocytes, and PBMCs are an important source for the in vitro preparation of γδT cells.
[0022] Ficoll method and white film layer: Ficoll is a density gradient centrifugation medium with a specific density of approximately 1.077 g / mL, widely used for the separation of highly active mononuclear cells from peripheral blood, bone marrow, and umbilical cord blood. The Ficoll method utilizes the differences in volume and specific gravity of various cellular components in human blood, separating cells of different densities through gradient centrifugation. Plasma and platelets, due to their lower density, remain in the upper layer of the separation medium after centrifugation; erythrocytes and granulocytes, with higher density, remain at the bottom; because PBMCs constitute a small proportion of blood and have a slightly higher density than the separation medium, they appear as a thin white film floating on top of the separation medium, below the plasma layer, and are called the "white film layer."
[0023] Unless otherwise specified, the reagents and consumables involved in this invention are all commercially available conventional products. KBM581 culture medium, SR, and Glutamax were purchased from a reputable biological reagent company. Zoledronic acid (MCE, HY-13777), IL-2 (Bepsys, GMP-L02H14), IL-15 (Nearshore Protein, C016), metformin (MCE, HY-B0627), glucocorticoids (MCE, HY-N0583), and nicotinamide (MCE, HY-B0150) are all analytical grade reagents. T25, T75, T150, and T300 culture flasks are conventional cell culture consumables. Example 1
[0024] A method for preparing CCR2+γδT cells with antitumor activity and migration ability: (1) Preparation of complete culture medium: Take 1000ml of KBM581 basal culture medium, add 30ml of SR to it with a pipette, then add 10ml of Glutamax, and finally add IL2 to make the final concentration reach 200U / ml. Mix thoroughly and store at 4℃ for later use.
[0025] (2) D0: Peripheral blood was collected from healthy individuals, and PBMCs were isolated. The white membrane layer of the PBMCs was washed twice with physiological saline, and the cells were resuspended in 5 ml of the KBM581 complete culture medium prepared above. 1 ml of the resuspended cells were used for cell counting and flow cytometry. Based on the counting results, the cells were divided into 1×10⁻⁶ cells. 6 / m PBMCs were inoculated into T25 culture flasks, 10ml of complete culture medium was added, along with 5μM zoledronic acid, 20ng / ml IL-15 and 10-50μM metformin. The T25 culture flasks were then incubated statically in a 37℃, 5% CO2 incubator.
[0026] (3) When cultured to day 2, add 20 ml of complete culture medium to adjust the cell concentration to 1 × 10⁻⁶. 6 / mL~2×10 6 / mL, cells were transferred from T25 culture flask to T75 culture flask, and the T75 culture flask was incubated statically in a 37℃ 5% CO2 incubator.
[0027] (4) At day 4, count the cells in the T75 culture flask, add 60 ml of complete culture medium, and simultaneously add 0.05-0.3 μM glucocorticoids and 1-10 μM nicotinamide to adjust the cell concentration to 1 × 10⁻⁶ cells / year. 6 / mL~2×10 6 / mL, and then all cells were transferred to T150 culture flasks and incubated statically in a 37°C, 5% CO2 incubator.
[0028] (5) At day 6, collect all cells from the T150 culture flask, rinse twice with physiological saline, discard the rinsing waste liquid, resuspend the cells in 300 ml of complete culture medium, and adjust the cell concentration to 1 × 10⁻⁶. 6 / mL~2×10 6 / mL, and inoculated into two T300 culture flasks, and incubated statically in a 37℃, 5% CO2 incubator.
[0029] (6) During D8-D16: Count the cells in the T300 culture flasks every two days, and replenish the culture medium each time at a ratio of 1:2 to 2:1 (original culture medium volume: replenished culture medium volume) to maintain the cell concentration at 1×10⁻⁶. 6 / mL~2×10 6 / mL, and cultured in a 37℃, 5% CO2 incubator. When cultured to D16, all cultured cells in the T300 culture flask were harvested to obtain the CCR2+γδT cells of this invention.
[0030] Comparative Example 1 Conventional methods for preparing CCR2+γδT cells: Prepare the complete culture medium according to the method in step 1 of Example 1.
[0031] D0: The PBMC extraction and processing method is the same as in step 2 of the embodiment, but the PBMCs are processed in a 1×102 ratio. 6 Inoculate the culture medium at a concentration of / ml into a T25 culture flask, add 10ml of complete culture medium and 5μM zoledronic acid, without adding any other inducing factors, and incubate statically at 37℃ in a 5% CO2 incubator.
[0032] At day 2, after cell counting, add 20 ml of complete culture medium to adjust the concentration to 1 × 10⁻⁶. 6 / mL~2×10 6 / mL, and continue to incubate in T25 culture flasks at 37℃ in a 5% CO2 incubator.
[0033] At day 4, after cell counting, add 60 ml of complete culture medium to adjust the concentration to 1 × 10⁻⁶. 6 / mL~2×10 6 / mL, transfer cells to T75 culture flasks and incubate at 37℃ in a 5% CO2 incubator.
[0034] At day 6, cells were collected, washed with physiological saline, resuspended in 300 ml of complete culture medium, and the concentration was adjusted to 1 × 10⁻⁶. 6 / mL~2×10 6 / mL, transferred to two T150 culture flasks for incubation.
[0035] During D8-D16, cell counts were performed every two days, and complete culture medium was added at a ratio of 1:2 to 2:1 (original culture medium volume: supplemental culture medium volume) to maintain a concentration of 1×10⁻⁶. 6 / mL~2×10 6 / mL, transferred to T300 culture flasks according to cell growth, and harvested at D16 to obtain γδT cells prepared by conventional methods.
[0036] Performance check: Cells harvested from Example 1 and Comparative Example 1 D16 were subjected to expansion fold detection, CCR2 expression level flow cytometry detection, Transwell migration assay, and tumor cell killing assay, respectively. The results are shown in Table 1.
[0037] Table 1: Performance indicators of cells harvested from D16 in Example 1 and Comparative Example 1 The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing CCR2+γδT cells with antitumor activity and migration ability, characterized in that, Includes the following steps: Step (1): At D0, PBMCs were extracted from peripheral blood of healthy individuals and inoculated into complete culture medium at a concentration of 1×10⁻⁶ cells / mL. 6 / mL~2×10 6 / mL, then add zoledronic acid, IL-15, and metformin, and place in a T25 culture flask for culture. Define day 0 of the initial culture as D0. Step (2): On day 2, count the cells cultured in step (1), replenish the complete culture medium according to the counting results, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, transfer cells from T25 culture flask to T75 culture flask for culture; Step (3): On day 4, count the cells cultured in step (2), replenish the complete culture medium according to the counting results, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, then add glucocorticoids and nicotinamide for culture, and transfer the cells from the T75 culture flask to the T150 culture flask for culture; Step (4): On day 6, collect the cells cultured in step (3), rinse with physiological saline, resuspend the cells in complete culture medium, and adjust the cell concentration to 1×10⁻⁶. 6 / mL~2×10 6 / mL, inoculated into T300 culture flasks for culture; Step (5): From D8 to D16, replenish the complete culture medium every two days to maintain the cell concentration at 1×10⁻⁶. 6 / mL~2×10 6 / mL, and harvest the cells at D16 to obtain CCR2+γδT cells with anti-tumor activity and migration ability; The complete culture medium in steps (1) to (5) consists of KBM581 medium, SR, Glutamax and IL2.
2. The preparation method according to claim 1, characterized in that, The concentration of zoledronic acid in step (1) is 5 μM.
3. The preparation method according to claim 1, characterized in that, The concentration of IL-15 in step (1) is 5-30 ng / ml.
4. The preparation method according to claim 1, characterized in that, The metformin concentration in step (1) is 10-50 μM.
5. The preparation method according to claim 1, characterized in that, In step (3), the concentration of glucocorticoids is 0.05-0.3 μM.
6. The preparation method according to claim 1, characterized in that, The concentration of nicotinamide in step (3) is 1-10 μM.
7. The preparation method according to claim 1, characterized in that, The culture conditions in steps (1) to (5) are all 37°C and 5% CO2.
8. The preparation method according to claim 1, characterized in that, The KBM581 medium:SR:Glutamax volume ratio is 100:3:1; the final concentration of IL2 is 200 U / ml.
9. CCR2+γδT cells prepared by any one of the preparation methods according to claims 1-8, characterized in that, The cells in question are Vδ2T cell subtypes.
10. The application of CCR2+γδT cells according to claim 9 in the preparation of antitumor drugs, characterized in that, The tumor is one or more of the following: breast cancer, ovarian cancer, lung cancer, and colorectal cancer, which highly express CCL2.