Culture method of CAR-NK cells

Abstract

The invention discloses a culture method of CAR-NK cells, and relates to the technical field of biomedical engineering and cell therapy. According to the scheme, a mononuclear cell population containing NK cells is extracted from placenta tissue, a cell population rich in NK cells is obtained through adherent cell removal and affinity sorting, after in-vitro amplification culture is conducted, the cell population is in contact with a virus vector carrying a chimeric antigen receptor gene, and finally the NK cells expressing CAR are obtained. The method has the beneficial effects that the NK cell amplification efficiency, the virus transduction efficiency and the function maturity are improved through multi-dimensional regulation and control, the obtained CAR-NK cells are obviously superior to the CAR expression rate, the killing rate and the key factor secretion of the existing method, and the problems of unstable expression, weak function and the like are solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering and cell therapy technology, and in particular to a method for culturing CAR-NK cells. Background Art

[0002] Chimeric antigen receptor-modified natural killer (CAR-NK) cells have emerged as a significant branch of cellular immunotherapy in recent years, demonstrating a favorable safety profile and promising efficacy in the treatment of hematologic malignancies. Compared to CAR-T cells, CAR-NK cells offer advantages such as broad availability, the lack of HLA matching, and a lower risk of GVHD, making them suitable for the development of off-the-shelf, universal immune cell products. Currently, some CAR-NK cell technologies derived from umbilical cord blood, peripheral blood, and induced pluripotent stem cells (iPSCs) have entered clinical trials, exploring their potential in diseases such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma. The key to CAR-NK therapy lies in the efficient ex vivo expansion of primary NK cells and viral-mediated delivery of the chimeric antigen receptor gene to generate cell populations that express the CAR molecule and retain natural killer (NK) function. To this end, researchers have attempted to optimize various aspects, including cell culture systems, transduction techniques, virus types, and cell pretreatment procedures, to improve development efficiency and functional maturity. However, several key bottlenecks remain.

[0003] In the prior art, although a variety of in vitro culture strategies have been developed to expand primary NK cells, these strategies often have problems such as limited expansion multiples and unstable cell status. In particular, maintaining cell viability before and after transduction makes it difficult to meet the requirements of subsequent viral introduction and expression. In addition, the transduction efficiency of viral vectors in primary NK cells is usually significantly lower than that of T cells. The reasons include poor cell membrane permeability, weak endocytosis ability, low expression levels of target receptors, and insufficient cell metabolic support for viral expression. To this end, studies have introduced transduction enhancers, physical treatment methods, or adjustment of culture conditions for optimization. However, while these methods improve efficiency, they are often accompanied by problems such as decreased cell viability, functional loss, or enhanced stress response, making it difficult to balance the dual requirements of transduction efficiency and cell quality. In addition, the regulation of NK cell functional activation in existing expansion systems is relatively limited. It is often manifested that although the obtained CAR-NK cells express CAR molecules, they are insufficient in cytokine secretion levels and tumor cell killing ability, making it difficult to form an effective immune response. This problem is particularly evident in the expression of functional factors such as IFN-γ, Granzyme B, and Perforin, reflecting a regulatory disconnect between CAR structural expression and NK functional maturation. Therefore, simply increasing the expansion multiple or transduction rate cannot effectively construct CAR-NK cells with immune effector capacity, indicating that current technology still has significant shortcomings in achieving the simultaneous establishment of expression and function.

[0004] Therefore, a construction strategy that simultaneously addresses the following technical challenges is urgently needed: First, stable expansion of primary NK cells should be achieved, maintaining good cell viability and condition to support efficient viral vector delivery; second, the transduction efficiency of the CAR gene should be significantly improved to ensure that a sufficient proportion of cells express the functional CAR structure, laying the foundation for subsequent immune attacks; finally, the immune effector capacity of the transduced cells should be simultaneously enhanced, so that they not only express the CAR structure but also have strong targeted killing ability and stable secretion of key cytokines (such as IFN-γ, Granzyme B, and Perforin), thereby truly constructing a stable and functionally mature CAR-NK cell population. These technical challenges are interrelated and must be addressed in a coordinated manner. Existing technologies are difficult to address simultaneously, so a new multi-level optimization construction system is urgently needed to achieve breakthroughs. Summary of the Invention

[0005] The present invention provides a natural killer cell culture process for efficiently isolating, enriching, and amplifying natural killer cells from placental tissue and achieving efficient chimeric antigen receptor expression, so as to prepare CAR-NK cells with high purity, high activity and high transduction efficiency for subsequent tumor immunotherapy research and clinical application.

[0006] The present invention comprises the following steps: S1. Extracting mononuclear cell populations containing NK cells from placental tissue; S2. processing the mononuclear cell population to obtain a cell population rich in NK cells; S3, performing in vitro expansion and culture on the NK cell-rich cell population to obtain expanded NK cells; S4, contacting the amplified NK cells with a viral vector carrying a chimeric antigen receptor gene, so that the NK cells express the chimeric antigen receptor, and obtaining transduced NK cells; S5. Cultivate the transduced NK cells and detect the expression of the chimeric antigen receptor to obtain CAR-NK cells.

[0007] Preferably, in step S1, the placental tissue is perfused with physiological saline, followed by cascade centrifugation using an OptiPrep density gradient solution with a density of 1.073–1.081 g / mL to obtain a mononuclear cell population. This protocol effectively reduces contamination with blood and tissue debris, improves the purity and activity of mononuclear cells, and is suitable for the high-throughput processing of large volumes of placental tissue.

[0008] Preferably, in step S2, the mononuclear cell population is treated by inoculating the mononuclear cell population in serum-free medium, culturing the mononuclear cell population for 4–6 hours, removing adherent cells, collecting non-adherent cells, and separating the cells using an affinity chromatography column to obtain a cell population enriched in NK cells. The affinity column is preferably a negative-selective CD3 affinity adsorption column, which further excludes T cell components and retains the CD56-positive NK cell population, effectively improving the purity of NK cells.

[0009] Preferably, in step S3, when the NK cell-rich cell population is expanded and cultured, the culture medium used is X-VIVO15 culture medium, which further contains the following key components: IL-21, Thymosin-α1, plant-derived lipid nanodiscs, and N-acetyl-L-cysteine (NAC). The above components synergistically construct a culture environment that promotes proliferation and functional maturation: IL-21 continuously activates NK cell expansion and differentiation through the JAK / STAT3 pathway; Thymosin-α1 can enhance the responsiveness of NK cells to cytokines and improve their cytotoxic activity; Plant-derived lipid nanodiscs mimic physiological membrane structures, enhancing cell membrane signal transduction pathways and receptor stability; As an antioxidant protection factor, NAC effectively removes reactive oxygen free radicals accumulated during the culture process, improving cell viability and expansion stability.

[0010] Preferably, the concentration of IL-21 is 10-30 ng / mL on culture days 0-11 and 50 ng / mL on culture days 12-14, so as to achieve balanced regulation of early stable expansion and late functional maturation.

[0011] Preferably, the concentration of Thymosin-α1 is 0.1–2 μg / mL; the concentration of the plant-derived lipid nanodiscs is 0.5–5 mg / mL; and the concentration of N-acetyl-L-cysteine is 0.2–5 mM. These concentration ranges were determined through extensive experimental screening to ensure optimal combinations of cell proliferation rate, cytotoxic activity, and survival rate.

[0012] Preferably, the initial seeding density of the expansion culture in step S3 is 5×10 5 –2×10 6 cells / mL to achieve optimal cell contact, nutrient exchange, and spatial distribution, and to avoid apoptosis due to overseeding or delayed proliferation due to insufficient density.

[0013] Preferably, in step S4, before the expanded NK cells are contacted with the viral vector, the NK cells are first concentrated to 1×10 7cells / mL and treated with 5–20 μM Vectofusin-1 transduction-enhancing peptide for 30 minutes to enhance the cell membrane penetration ability and receptor expression synergy of AAV6-like viruses, thereby significantly improving viral transduction efficiency and chimeric antigen receptor (CAR) expression levels.

[0014] Furthermore, in step S5, the CAR molecule expression level of the transduced NK cells was detected by flow cytometry (FACS), and the functional activity of the CAR-positive cells was verified to finally obtain the CAR-NK cell population.

[0015] The technical solution provided by the present invention brings beneficial effects: The key ingredients used in this invention, such as IL-21, Thymosin-α1, plant-derived lipid nanodiscs, N-acetyl-L-cysteine and Vectofusin-1, regulate the CAR-NK cell construction process from multiple dimensions, including cytokine regulation, membrane structure optimization, antioxidant protection and viral transduction promotion. These components form a synergistic enhancement mechanism during the expansion and activation of NK cells, the introduction and expression maintenance of CAR genes, thereby effectively overcoming the technical difficulties such as low transduction rate and unstable CAR expression common in traditional methods.

[0016] Experimental results showed that under the complete combination conditions, the CAR conversion rate of NK cells was significantly higher than that of any single factor adjustment or deletion group. IL-21 promotes the continuous proliferation of NK cells by activating the STAT pathway, Thymosin-α1 enhances cellular immune response, lipid nanodiscs improve membrane fusion efficiency, NAC alleviates stress and maintains internal environment stability, and Vectofusin-1 promotes viral internalization efficiency. The above factors form a technical closed loop in the three links of viral entry, expression maintenance, and stable presentation of CAR, synergistically improving transduction efficiency and reflecting a nonlinear enhancement effect, verifying that this solution is not a simple superposition of the functions of each component.

[0017] In co-culture experiments with CD19-positive tumor cells, CAR-NK cells demonstrated a higher targeted killing rate, accompanied by high levels of secretion of key cytokines such as IFN-γ, Granzyme B, and Perforin, demonstrating mature immune effector capabilities. In contrast, removal of any of these key components resulted in a significant decrease in killing activity and immune factor secretion, further demonstrating the systemic effect of this optimized combination in functional activation.

[0018] This solution works synergistically on four levels: cell signal regulation, membrane fusion process optimization, internal environment protection, and transduction pathway enhancement. It achieves a CAR-NK cell construction strategy that takes into account both high expression and high functionality. It is an essential improvement over existing technologies, solves the bottleneck problems in multiple key links in CAR-NK construction, and is highly feasible and innovative. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The flow cytometry diagrams of the transduction efficiency of CAR-NK cells in the examples and comparative examples of the present invention are shown in FIG. 1(a). Example 3(b), Example 6(c), Example 7(d), Comparative Example 1(e), Comparative Example 2(f), Comparative Example 3(g), Comparative Example 4(h). DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the embodiments. Of course, the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] Example 1 The specific steps of this embodiment are as follows: S1: Mononuclear cell extraction Fresh placentas were minced into approximately 1 cm³ pieces. The pieces were placed in a 50-mL syringe and perfused with 30 mL of normal saline. The pieces were allowed to stand for 10 minutes to drain any residual blood. The pieces were then placed in RPMI-1640 enzymatic solution containing 2 mg / mL collagenase IV and 0.1 mg / mL DNase I and incubated at 37°C with shaking for 45 minutes. The cell suspension was collected and centrifuged, resuspended in PBS, and added to OptiPrep density gradient buffer at a density of 1.079 g / mL. The cells were then centrifuged at 800 × g for 20 minutes without brake. The mononuclear cell interface layer was collected, washed twice with PBS, and counted before subsequent processing.

[0022] S2: Enrichment of NK cell-rich populations The obtained mononuclear cell population was inoculated into serum-free X-VIVO15 basal medium at a cell density of 1×10 6 cells / mL, incubated at 37°C for 6 hours; non-adherent cells were collected; non-adherent cells were subjected to CD3 negative affinity chromatography to remove T cells; CD3 negative cells were collected as a population rich in NK cells; S3: In vitro expansion and culture of NK cells Resuspend the NK cell-enriched cell population in X-VIVO15 complete medium containing the following supplements: IL-21: The concentration was set at 20 ng / mL for the first 11 days and increased to 50 ng / mL on days 12–14; Thymosin-α1: added at a concentration of 1 μg / mL; Plant-derived lipid nanodiscs 2 mg / mL (Product Name: MSP1D1-His with DMPC Nanodisc Assembly Kit Supplier: Creative Biolabs, Product Number: MPN0081K Composition: membrane scaffold protein MSP1D1 + DMPC phospholipid Structure size: nanodisk diameter is approximately 9–10 nm); N-acetyl-L-cysteine (NAC): 2.5 mM; The initial seeding density was set at 1×10 6 cells / mL; cultured in a G-Rex 10 cell expansion container at 37°C and 5% CO2 for 14 days; the cell density was observed every 3 days, and the culture medium was supplemented appropriately to achieve a final density of 1×10 7 –1.5×10 7 cells / mL; on the 14th day, the cell viability was >95% as measured by the Trypan blue exclusion method, the total expansion fold was approximately 80 times, and the CD56⁺CD3⁻ NK cell purity was approximately 88%.

[0023] S4: CAR gene transduction The expanded NK cells were collected and centrifuged at 500 × g for 10 minutes, resuspended in serum-free X-VIVO15, and concentrated to 1 × 10 7 cells / mL; Vectofusin-1 transduction enhancer was added to a final concentration of 10 μM and incubated at 37°C for 30 minutes; CAR19 lentivirus was then added at an MOI of 50. Note: Anti-CD19 CAR lentivirus (CD19 ScFv-CD8-4-1BB-CD3ze; SIN vector), BPS Bioscience product number BPS-78601, was used to prepare CAR-NK cells. CAR positivity expression was detected after 72 hours.

[0024] Example 2 The culture method was the same as that in Example 1, except that the density of the OptiPrep density gradient solution was set to 1.073 g / mL.

[0025] Example 3 The same culture method as in Example 1 was followed, except that IL-21 was 10 ng / mL on days 0–11 and 50 ng / mL on days 12–14, Thymosin-α1 was 1.0 μg / mL, plant-derived lipid nanodiscs were 2.0 mg / mL, and N-acetyl-L-cysteine (NAC) was 2.5 mM.

[0026] Example 4 The same culture method as in Example 1 was followed, except that IL-21 was 30 ng / mL on days 0–11 and 50 ng / mL on days 12–14, Thymosin-α1 was 1.0 μg / mL, plant-derived lipid nanodiscs was 2.0 mg / mL, and N-acetyl-L-cysteine (NAC) was 2.5 mM.

[0027] Example 5 The same culture method as in Example 1 was followed, except that IL-21 was 20 ng / mL on days 0–11 and 50 ng / mL on days 12–14, Thymosin-α1 was 0.1 μg / mL, plant-derived lipid nanodiscs was 2.0 mg / mL, and N-acetyl-L-cysteine (NAC) was 2.5 mM.

[0028] Example 6 The same culture method as in Example 1 was followed, except that IL-21 was 20 ng / mL on days 0–11 and 50 ng / mL on days 12–14, Thymosin-α1 was 1.0 μg / mL, plant-derived lipid nanodiscs was 0.5 mg / mL, and N-acetyl-L-cysteine (NAC) was 2.5 mM.

[0029] Example 7 The same culture method as in Example 1 was followed, except that IL-21 was 20 ng / mL on days 0–11 and 50 ng / mL on days 12–14, Thymosin-α1 was 1.0 μg / mL, plant-derived lipid nanodiscs was 2.0 mg / mL, and N-acetyl-L-cysteine (NAC) was 0.2 mM.

[0030] Example 8 The same culture method as in Example 1 was used, except that the initial seeding density of NK cells in vitro expansion culture was 5×10 5 cells / mL.

[0031] Example 9 The same culture method as in Example 1 was used, except that the initial seeding density of NK cells in vitro expansion culture was 2×10 6 cells / mL.

[0032] Example 10 The same culture method as in Example 1 was followed, except that Vectofusin-1 transduction enhancer was added at a final concentration of 5 μM.

[0033] Example 11 The culture method was the same as in Example 1, except that Vectofusin-1 transduction enhancer was added to a final concentration of 20 μM.

[0034] Comparative Example 1 The culture method was the same as in Example 1, except that Thymosin-α1 was not added.

[0035] Comparative Example 2 The same culture method as in Example 1 was followed, except that plant-derived lipid nanodiscs were not added.

[0036] Comparative Example 3 The same culture method as in Example 1 was followed, except that N-acetyl-L-cysteine was not added.

[0037] Comparative Example 4 The same culture method as in Example 1 was followed, except that Thymosin-α1, plant-derived lipid nanodiscs, and N-acetyl-L-cysteine were not added.

[0038] Experimental test: 1. CAR conversion rate detection method Flow cytometry was used to detect the conversion rate of CAR-NK cells. The specific steps are as follows: Sample collection: NK cells were collected on day 5 after transduction, washed and resuspended in PBS buffer. The cell number was 1×10 6 cells; Antibody staining: Add anti-human F(ab')2 antibody (for detecting the IgG Fc segment in the CAR structure), anti-CD56 antibody, and anti-CD3 antibody, and incubate at 4°C in the dark for 30 minutes; Washing and resuspension: After staining, wash once with PBS and resuspend in flow cytometry buffer; Instrument detection: Flow cytometry was used to detect the proportion of CD56⁺CAR⁺ double-positive cells in CD56⁺ cells; Result determination: CAR conversion rate = number of CD56⁺CAR⁺ cells / total number of CD56⁺ cells × 100%.

[0039] 2. CAR-NK cells kill target cells The co-culture method was used to test the targeted killing ability of the CAR-NK cells of the present invention. CD22-positive tumor cells were selected as target cells, and untransduced NK cells were set as controls. Effector cells and target cells were co-cultured at different ratios of 1:1 for 4 hours. The apoptosis and death ratio of target cells were detected by flow cytometry, and the killing rate was calculated based on the positive staining results. The secretion levels of IFN-γ, Granzyme B, and Perforin in the cell supernatant were also detected to evaluate the functional activity of CAR-NK cells.

[0040] like Figure 1 As shown in Example 1, the optimal solution of the present invention, it shows a CAR conversion efficiency that is significantly better than all other groups, indicating that the combination of IL-21, Thymosin-α1, plant-derived lipid nanodiscs and NAC has a synergistic enhancement effect in promoting stable expansion and efficient transduction of NK cells. In contrast, in Examples 3, 6 and 7, the dosage of IL-21, lipid nanodiscs or NAC was reduced, and the conversion rate decreased, indicating that the dosage change of these key additives directly affects the permeability of the cell membrane, the maintenance of the antioxidant microenvironment and the responsiveness of the factor signal, thereby reducing the viral transduction efficiency and the expression level of the CAR structure. In addition, after removing one or more additive factors in each comparative group, the CAR conversion rate decreased significantly, especially the comparative group in which the three additives were completely removed, and its expression efficiency was the lowest, further verifying that the combination is not a simple additive effect, but relies on the systemic synergistic effect of multiple components in membrane stability, signal enhancement and cell metabolic protection.

[0041] Table 1 CAR-NK cell killing activity and functional factor detection results

[0042] As shown by the CAR-NK cell killing rate against CD22-positive tumor cells and the secretion of functional factors, Example 1 exhibited the best killing activity and immune factor release, fully demonstrating the synergistic effect of the complete optimized system (IL-21, Thymosin-α1, lipid nanodiscs, NAC, and Vectofusin-1) on CAR-NK cell function. In contrast, the remaining examples showed varying degrees of decrease in killing rate and factor secretion after adjusting the concentration of a key component (such as IL-21, lipid nanodiscs, or transduction enhancer), indicating that these components are dose-sensitive in the development of cellular effector functions, with a particularly strong effect on the expression of Perforin and Granzyme B. The functional data of the control group were significantly lower than those of any of the examples, especially in Control Example 4, which completely removed the synergistic factor, which nearly lost specific killing ability and immune factor release, further demonstrating the nonlinear synergistic enhancement mechanism between the key components of the inventive scheme. These results systematically verified that the present invention not only achieved a high conversion rate in the construction of CAR-NK cells, but also constructed immune effector cells with high functional activity, demonstrating the comprehensive advantages of this technical solution.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing CAR-NK cells, characterized in that: The following steps are involved: S1 extracts mononuclear cell populations containing NK cells from placental tissue; S2. processing the mononuclear cell population to obtain a cell population rich in NK cells; S3: performing in vitro expansion and culture on the NK cell-rich cell population to obtain expanded NK cells; S4: contacting the amplified NK cells with a viral vector carrying a chimeric antigen receptor gene, causing the NK cells to express the chimeric antigen receptor, thereby obtaining transduced NK cells; S5: culturing the transduced NK cells and detecting the expression of the chimeric antigen receptor to obtain CAR-NK cells.

2. The method for culturing CAR-NK cells according to claim 1, wherein: In step S1, the placental tissue is perfused with sterile physiological saline, and then cascade centrifugation is performed using an OptiPrep density gradient solution with a density of 1.073–1.081 g / mL to obtain a mononuclear cell population.

3. The method for culturing CAR-NK cells according to claim 1, wherein: The treatment of the mononuclear cell population in step S2 includes inoculating the mononuclear cell population in a serum-free culture medium, removing adherent cells after standing, collecting non-adherent cells and separating them through an affinity chromatography column to obtain a cell population rich in NK cells.

4. The method for culturing CAR-NK cells according to claim 1, wherein: When the NK cell-rich cell population is expanded and cultured in step S3, the culture medium used is X-VIVO15, and contains the following components: IL-21, Thymosin-α1, plant-derived lipid nanodiscs, and N-acetyl-L-cysteine.

5. The method for culturing CAR-NK cells according to claim 4, characterized in that: The concentration of IL-21 on culture days 0-11 was 10-30 ng / mL, and on culture days 12-14 was 50 ng / mL.

6. The method for culturing CAR-NK cells according to claim 4, characterized in that: Therefore, the concentration of Thymosin-α1 was 0.1–2 μg / mL.

7. The method for culturing CAR-NK cells according to claim 4, wherein: The concentration of the plant-derived lipid nanodiscs was 0.5–5 mg / mL.

8. The method for culturing CAR-NK cells according to claim 4, wherein: Therefore, the concentration of N-acetyl-L-cysteine was 0.2–5 mM.

9. The method for culturing CAR-NK cells according to claim 1, wherein: The initial seeding density of the expansion culture in step S3 was 5×10 5 –2×10 6 cells / mL.

10. The method for culturing CAR-NK cells according to claim 1, wherein: In step S4, before the expanded NK cells are contacted with the viral vector, the NK cells are first concentrated to 1×10 7 cells / mL and treated with 5–20 μM Vectofusin-1 for 30 min.

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