Method for culturing highly activated nk cell enriched lymphocytes

Abstract

The application discloses a culture method of high-activation-state NK cell enriched lymphocytes, which uses peripheral blood mononuclear cells as starting materials, applies stage stimulation to the PBMC and optimizes culture conditions, and realizes large-scale expansion of cells efficiently and repeatedly through three stages of an induction stage, a stimulation stage and an expansion stage, thereby significantly improving the purity of NK cells and related subgroups in the cell population and significantly improving the expansion efficiency. The cells prepared by the method are in a highly activated state and exhibit enhanced cytotoxicity and anti-tumor activity. The process is simple to operate, has strong adaptability and can be produced on a large scale, and is suitable for tumor immune cell treatment, immune regulation research and related biological medicine development and clinical transformation.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy technology, specifically relating to a method for culturing highly activated NK cell-enriched lymphocytes. Background Technology

[0002] Adoptive cell transfer (ACT) has become a significant strategy in the field of tumor immunotherapy in recent years. By activating, enhancing, and expanding effector immune cells in vitro, ACT can enhance the body's immune recognition and clearance of tumor cells, and has become a complementary therapeutic approach to immune checkpoint inhibitors and targeted therapy. T cells and natural killer (NK) cells are the two main effector cell types in ACT research and clinical applications. Among them, NK cells, because they can recognize and clear abnormal cells without prior antigen-specific sensitization, show unique advantages and broad development prospects in the immune intervention of solid tumors and hematological malignancies.

[0003] NK cells, as the main effector cells of innate immunity, participate in immune surveillance and exert antiviral and antitumor functions through multiple mechanisms. NK cells rely on a set of activating receptors (such as NKp46 and NKG2D) and co-stimulatory / regulatory molecules (such as 2B4 / CD244) to recognize stress-induced ligands or dysregulated MHC-I expression, and mediate direct cytotoxicity and immunomodulation by releasing perforin, granzymes, and producing cytokines such as IFN-γ. Extensive basic and preclinical research has systematically elucidated the recognition pathways, signal transduction, and effector mechanisms of NK cells. Meanwhile, multiple early clinical studies and phase I / II trials have demonstrated that the adoptive use of peripheral NK cells or engineered NK cells has a generally good safety profile and exhibits reproducible antitumor activity in several tumor types, proving the feasibility and potential of NK cells as a tool for tumor immunotherapy.

[0004] However, transforming NK cells into widely available clinical therapeutic products still faces key technological bottlenecks, particularly in obtaining sufficient quantities of functionally stable NK cells in vitro to meet clinical needs. The proportion of naturally occurring NK cells in peripheral blood is limited (5-10%), and their in vitro expansion capacity and effector function are significantly affected by individual differences, culture conditions, and process parameters. Existing processes still have shortcomings in terms of expansion fold, cell purity, consistency, and functional maintenance. These issues directly restrict the scalable production, quality control, and consistency of clinical efficacy of NK cell therapy.

[0005] Currently, there are various strategies for obtaining clinical-grade NK cells, which can be mainly described from three aspects. First, cell sources include peripheral blood mononuclear cells (PBMCs), cord blood, modified or differentiated stem / progenitor cells (such as iPSC / ESC-derived cells), and immortalized cell lines (such as NK-92), each with its own advantages and limitations (e.g., availability, differentiation maturity, degree of engineerability, and differences in in vivo persistence). Second, in vitro expansion and activation methods include combinations of cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, etc.), activating monoclonal antibodies, allogeneic (or genetically modified) feeder cells (such as modified K562 cells expressing membrane-bound IL-21 / 4-1BBL), and receptor / ligand co-stimulation strategies. Furthermore, cell sorting (positive / negative selection) is used in some processes to purify the initial NK population to improve consistency, but the sorting step usually increases process complexity, manpower and cost, and may reduce recovery rate. In autologous applications, if NK cell-rich products can be directly amplified from PBMCs without pre-sorting through culture strategies, the production process can be significantly simplified and the preparation cost reduced, which is particularly important for achieving scalable autologous NK cell formulations.

[0006] Therefore, developing a simple, reproducible, and scalable method for the in vitro expansion and activation of high-quality (e.g., highly activated) NK cells is a key step in advancing the clinical translation of NK cell immunotherapy. Summary of the Invention

[0007] 1. Purpose of the invention

[0008] The purpose of this invention is to provide a simple, reproducible, and scalable method for culturing highly activated NK cell-enriched lymphocytes. Using peripheral blood mononuclear cells (PBMCs) as the starting material, the method involves staged stimulation and optimized culture conditions, followed by three stages: induction, stimulation, and expansion. This process significantly enriches NK cells in vitro in a short period while maintaining their cytotoxicity and immunomodulatory activity, thereby improving cell yield, purity, and functional stability. It efficiently produces highly activated and pure NK cell-enriched lymphocytes, suitable for autologous or allogeneic applications in clinical and translational research, especially for the preparation of autologous NK cell therapy products, providing feasible immunotherapeutic cell products for clinical and translational research.

[0009] 2. Technical Solution

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a method for culturing highly activated NK cell-enriched lymphocytes, the method comprising the following steps:

[0012] S1, Obtain peripheral blood mononuclear cells (PBMCs).

[0013] S2, using peripheral blood mononuclear cells as starting material, was cultured, including:

[0014] S21, Induction Phase: PBMCs are seeded into antibody-coated culture dishes, and peripheral blood mononuclear cells are initially cultured using induction medium. The antibodies include anti-NKp46 antibody and anti-2B4 (CD244) antibody. The induction medium includes basal medium, autologous plasma, interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18). As a further explanation of the invention, the anti-NKp46 antibody and anti-2B4 (CD244) antibody are used to enhance the activation signal of NK cells through receptor cross-linking; autologous plasma is used to provide stable nutritional support; the combination of cytokines interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18) is used to promote NK cell proliferation and acquisition of a memory-like phenotype; simultaneous administration of specific antibodies and a combination of multiple cytokines can induce the initial activation and differentiation of NK cells.

[0015] S22, Stimulation Phase: After replacing the induction medium with a stimulation medium, the cells are cultured further. The stimulation medium includes: basal medium, autologous plasma, interleukin-2 (IL-2), interleukin-15 (IL-15), and a Wnt / β-catenin signaling pathway activator. As a further explanation of the invention, autologous plasma is used to provide stable nutritional support; the combination of interleukin-2 (IL-2) and interleukin-15 (IL-15) cytokines is used to maintain NK cell activation and expansion signals; the Wnt / β-catenin signaling pathway activator is used to optimize NK cell differentiation status and self-renewal capacity, and improve expansion efficiency and functional stability; the simultaneous administration of the cytokine combination and signaling pathway activator can enhance the functionalization and proliferative potential of NK cells.

[0016] S23, Amplification Phase: After replacing the stimulation medium with amplification medium, the cells are cultured again. The amplification medium includes: basal medium, autologous plasma, interleukin-2 (IL-2), and interleukin-15 (IL-15). As a further explanation of the present invention, autologous plasma is used to provide stable nutritional support; the combination of cytokines interleukin-2 (IL-2) and interleukin-15 (IL-15) is used to maintain continuous proliferation and effector function, thereby obtaining a large number of highly activated NK cells in a short period of time.

[0017] Through the combined effect of the three stages, the method of the present invention can significantly increase the enrichment ratio of NK cells in the PBMC population, improve the cytotoxicity and antitumor activity of the cells, simplify the process, reduce production costs, and take into account GMP operability.

[0018] Furthermore, the culture times for the induction, stimulation, and amplification phases are 18–24 h, 6–8 d, and 7–10 d, respectively. Even further, the culture times for the induction, stimulation, and amplification phases are 24 h, 6 d, and 7 d, respectively.

[0019] Furthermore, the plasma concentrations during the induction, stimulation, and amplification phases are 5%–10%, 5%–10%, and 5%–10%, respectively. Even further, the plasma concentrations during the induction, stimulation, and amplification phases are 10%, 10%, and 5%, respectively.

[0020] Furthermore, the method for preparing the antibody-coated culture dish includes:

[0021] Antibodies were dissolved in buffer solution to prepare a coating solution. The coating solution was added to a culture dish to ensure that it was in full contact with the inner surface of the culture dish. After full contact, the antibodies were adsorbed onto the inner surface of the culture dish, thus completing the coating process.

[0022] Furthermore, the above-mentioned buffer includes PBS or DPBS.

[0023] Furthermore, the coating was left to stand overnight at approximately 2–8°C to promote the binding of the antibody to the surface of the culture dish.

[0024] Furthermore, in the above coating solution, the concentration of anti-NKp46 antibody is 5~10 μg / mL; the concentration of anti-2B4 (CD244) antibody is 5~10 μg / mL. Even further, in the above coating solution, the concentration of anti-NKp46 antibody is 7.5 μg / mL; the concentration of anti-2B4 (CD244) antibody is 7.5 μg / mL.

[0025] Furthermore, the concentration of interleukin-12 (IL-12) in the above-mentioned induction medium is 10 ng / mL.

[0026] Furthermore, the concentration of interleukin-15 (IL-15) in the above-mentioned induction medium is 50 ng / mL.

[0027] Furthermore, the concentration of interleukin-18 (IL-18) in the above-mentioned induction medium is 100 ng / mL.

[0028] Furthermore, in the above-mentioned stimulation medium, the Wnt / β-catenin signaling pathway activators include GSK-3α and / or GSK-3β inhibitors.

[0029] Furthermore, the aforementioned GSK-3α and / or GSK-3β inhibitor is Bio-acetoxime, which has a specific inhibitory effect on GSK-3α and GSK-3β.

[0030] Furthermore, the concentration of interleukin-2 (IL-2) in the above-mentioned stimulation culture medium is 10 IU / mL.

[0031] Furthermore, the concentration of interleukin-15 (IL-15) in the above-mentioned stimulation culture medium is 50 ng / mL.

[0032] Furthermore, the concentration of the Wnt / β-catenin signaling pathway activator in the above-mentioned stimulation medium is 1.5~2.5 μM. Even further, the concentration of the Wnt / β-catenin signaling pathway activator in the above-mentioned stimulation medium is 2 μM.

[0033] Furthermore, the concentration of interleukin-2 (IL-2) in the above amplification medium is 10 IU / mL.

[0034] Furthermore, the concentration of interleukin-15 (IL-15) in the above amplification medium is 50 ng / mL.

[0035] Furthermore, the above-mentioned amplification stage utilizes the G-Rex suspension cell culture system.

[0036] Furthermore, the cell concentrations for the induction, stimulation, and expansion phases were (1~2)×10⁻⁶, respectively. 6 cells / mL, (0.8~1.5)×10 6 cells / mL, (0.11~0.22)×10 6 cells / mL.

[0037] Secondly, the present invention provides NK cell-enriched lymphocytes obtained by culturing using the above-described culture method; as a further explanation of the present invention, the lymphocytes contain highly activated NK cells and a high proportion of NKT cells.

[0038] Thirdly, the present invention provides the application of the above-mentioned NK cell-enriched lymphocytes in the preparation of tumor therapeutic drugs.

[0039] 3. Technical Effects

[0040] Compared with the prior art, the advantages of this invention are as follows:

[0041] (1) In achieving an efficient and scalable in vitro culture system, selecting appropriate starting materials and synergistically optimizing culture components and activation signals are key. The method for culturing highly activated NK cell-enriched lymphocytes provided by this invention uses peripheral blood mononuclear cells (PBMCs) as starting materials, which has the advantages of convenient source and clinical feasibility, and can induce selective expansion and functional enhancement of NK cells through exogenous signals in an optimized culture system. To improve the quality and consistency of cultured products, several factors need to be comprehensively considered and optimized. For example, using autologous donor plasma as a culture supplement can, to some extent, maintain the physiological environment of cells and reduce potential immune or batch-to-batch variations caused by allogeneic components. Specific monoclonal antibodies targeting NK cell activation receptors and co-stimulatory molecules (examples include anti-NKp46 and anti-2B4 / CD244) can be used to directly trigger or enhance NK cell recognition and effector programs. A well-optimized combination of cytokines (such as IL-2, IL-12, IL-15, and IL-18) can significantly promote NK cell proliferation, differentiation, and cytotoxic phenotypes. Furthermore, regulating intracellular signaling pathways (e.g., activating the Wnt / β-catenin pathway or inhibiting GSK-3α / β) can further contribute to the maintenance and enhancement of effector phenotypes. These elements can complement each other, and through rational design and phased coordination, provide theoretical and practical basis for an efficient and stable NK cell preparation system.

[0042] (2) The method for culturing highly activated NK cell-enriched lymphocytes provided by the present invention involves direct in vitro culture, activation, and expansion using PBMCs as the starting material to prepare NK cell-enriched lymphocyte products. The culture process is divided into three stages: induction, stimulation, and expansion. Throughout the entire culture process, donor autologous plasma is continuously used as a culture supplement. In the induction stage, monoclonal antibodies (anti-NKp46 and anti-2B4 / CD244) and a combination of cytokines (IL-12, IL-15, and IL-18) targeting NK activation and co-stimulatory molecules are used to induce the target phenotype. In the stimulation stage, IL-2 / IL-15 and Wnt / β-catenin pathway activators (specific inhibitors of GSK-3α / β) are introduced to enhance functional persistence while maintaining the cell effector phenotype. In the expansion stage, IL-2 / IL-15 is used as the main component for large-scale culture in a gas permeation or other amplification system (e.g., G-Rex). Evaluations have shown that the strategy provided by this invention has achieved high-purity NK cell-enriched lymphocyte products, significantly enhanced cell effector phenotypes and anti-tumor functions, good reproducibility, and process characteristics suitable for large-scale production, which is beneficial to the clinical application and industrialization of NK cells and their engineered formulations. Attached Figure Description

[0043] Figure 1 The comparative analysis of total cell count after 14 days of in vitro culture is shown. The comparison subjects are: the PBMC group at seed (day 0, baseline), the group without Bio-acetoxime and the group containing Bio-acetoxime cultured for 14 days using the described three-stage culture procedure. The three-stage culture procedure is briefly described as follows: induction phase (anti-NKp46 and anti-2B4 antibody + IL-12 / IL-15 / IL-18), stimulation phase (IL-2 / IL-15, comparing the groups without and with Bio-acetoxime), and expansion phase (IL-2 / IL-15 in the G-Rex system).

[0044] Figure 2 The results of NK cell purity assay by flow cytometry after 14 days of in vitro culture are shown. In A, the flow cytometry analysis of cells is shown; in B, CD3+ is shown. - CD16 + CD56 + A bar chart showing the percentage of cells, where C represents the mean fluorescence intensity (MFI) of CD56.

[0045] Figure 3 The results of NKT cell purity assay by flow cytometry after 14 days of in vitro culture are shown. In the figure: A is the flow cytometry analysis of cells, and B is the CD3+ chromatogram. + CD16 + CD56 + A bar chart showing the percentage of cells.

[0046] Figure 4 The results of NK cell activation marker expression detected by flow cytometry after 14 days of in vitro culture are shown, where: A is the expression ratio of NKG2D, B is the expression ratio of DNAM (DNAM-1), and C is the expression ratio of CD69.

[0047] Figure 5 The results of detecting IFN-γ expression levels in NK cells using ELISpot are shown. Cells were collected after 14 days of in vitro culture and then cultured for another 48 hours before detection.

[0048] Figure 6 The results of detecting granzyme B and perforin levels secreted by NK cells by ELISA are shown. Cells were collected after 14 days of in vitro culture and then cultured for another 48 hours before detection. A represents granzyme B and B represents perforin.

[0049] Figure 7 The results show the cytotoxic activity of NK cells against tumor cells as detected by LDH, where: A represents the cytotoxic activity against the HO-8910 cell line, and B represents the cytotoxic activity against the Colo205 cell line. Detailed Implementation

[0050] The present application will be further described below with reference to specific embodiments.

[0051] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0052] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0053] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0054] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0055] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0056] The materials, reagents, and equipment used in this invention are as follows:

[0057] Sample source: Human peripheral blood was obtained from healthy volunteers through routine venous blood collection using BDVacutainer® Heparin Tubes (BD 367874).

[0058] PBMC separation: Human lymphocyte separation medium (Shenzhen Dakwei Biotechnology Co., Ltd., 7111011) was used for separation. The method included: taking fresh anticoagulated whole blood as the starting sample, and diluting the anticoagulated whole blood with an equal volume of isotonic solution (phosphate-buffered saline (PBS) or physiological saline); then, adding a predetermined volume of separation medium to a centrifuge tube, and slowly spreading the diluted blood sample on top of the separation medium to maintain a clear and stable interface between the blood sample and the separation medium, wherein the volume ratio of the separation medium, anticoagulated and undiluted whole blood, and isotonic solution was 1:1:1; the above system was placed at room temperature and centrifuged using a horizontal rotor at 700 g to 800 g for 20 to 30 minutes. Centrifuge at a low speed (min) to minimize surface disturbance. After centrifugation, a layer of red blood cells, a separation fluid layer, and a plasma layer form sequentially from bottom to top. A thin, dense white membrane layer, enriched with mononuclear cells including lymphocytes and monocytes, forms between the plasma layer (the supernatant (plasma portion) obtained from separation is inactivated at 56°C for 30 minutes; the inactivated plasma is reserved as a supplement for culture medium and for NK cell expansion) and the separation fluid layer. This white membrane layer is enriched with mononuclear cells. Carefully aspirate the white membrane layer and transfer it to a new centrifuge tube. Then, dilute the cell suspension with isotonic solution (PBS, physiological saline, or culture medium) to a predetermined volume, gently mix, and centrifuge at approximately 250 g for 10 minutes at room temperature using a horizontal rotor. Discard the supernatant and repeat the washing 1-2 times as needed. Finally, resuspend the obtained cells in isotonic solution (PBS, physiological saline, or culture medium) to obtain a mononuclear cell suspension suitable for subsequent experiments or applications.

[0059] PBS: Phosphate Buffered Saline (1×), Cellmax, CBS101.05.

[0060] Culture medium: NK MACS® Medium, human (Miltenyi Biotec 130-114-429).

[0061] Inducing antibodies: anti-NKp46 antibody (BioTechne MAB1850); anti-2B4 (CD244) antibody (Invitrogen 16-5838-85).

[0062] Cytokines:

[0063] IL-2: Human interleukin-2 (rHuIL-2), Xiamen Tebo Biotechnology Co., Ltd., SJB03;

[0064] IL-12: Human interleukin-12 (rHuIL-12), Xiamen Tebo Biotechnology Co., Ltd., SJB12;

[0065] IL-15: Human interleukin-15 (rHuIL-15), Xiamen Tebo Biotechnology Co., Ltd., SJB08;

[0066] IL-18: Human interleukin-18 (rHuIL-18), Xiamen Tebo Biotechnology Co., Ltd., SJB06.

[0067] Wnt / β-catenin pathway activator: BIO-acetoxime (MedChemExpress HY-15356).

[0068] Antibody detection:

[0069] FITC anti-human CD3 (Biolegend 300440); Brilliant Violet 421 anti-humanCD56 (Biolegend 362552); APC anti-human CD56 (Biolegend 360706); PE anti-humanCD226 (DNAM-1) (Biolegend 338306); Brilliant Violet 605 anti-human CD314(NKG2D) (Biolegend 320832); Alexa Fluor 700 anti-human CD69 (Biolegend 310922).

[0070] Flow cytometer: DxFLEx (Beckman Coulter).

[0071] Microplate reader: Multiskan TM FC Microplate Photometer, Thermo Fisher Scientific, 51119080 (CB717732915).

[0072] Centrifuge: MULTIFUGE X1, Thermo Fisher Scientific.

[0073] Cell counter: Countess TM 3 Automated Cell Counter, Thermo Fisher Scientific, AMQAX2000.

[0074] G-Rex ® 100 Open System, Sterile Fluid Path (WILSONWOLF P / N 80500).

[0075] Test kit:

[0076] ELISpot: Human IFN-γ Pre-coated ELISpot strip plate (MABTECH 3420-3SPT);

[0077] Enzyme-linked immunosorbent assay kit: Human Granzyme B (MABTECH 3486-1H-20);

[0078] Enzyme-linked immunosorbent assay kit: Human Perforin (MABTECH 3465-1H-6);

[0079] LDH lactate dehydrogenase cytotoxicity assay kit, Beyotime Biotechnology Co., Ltd., C0016.

[0080] Example 1

[0081] This embodiment aims to provide a method for culturing highly activated NK cell-enriched lymphocytes by directly inducing, stimulating, and expanding peripheral blood mononuclear cells (PBMCs). This method achieves continuous activation and efficient expansion of NK cells through the combined use of specific monoclonal antibodies, cytokines, and Wnt / β-catenin signaling activators at different culture stages, thereby overcoming the technical bottlenecks of limited efficiency, unstable purity, and complex procedures in traditional in vitro NK cell expansion.

[0082] include:

[0083] S1, Obtain peripheral blood mononuclear cells (PBMCs).

[0084] S2, using peripheral blood mononuclear cells as starting material, was cultured, including:

[0085] S21, Induction Phase

[0086] (a) Antibody coating

[0087] Monoclonal antibodies against NKp46 and 2B4 (CD244) were dissolved in phosphate-buffered saline (PBS) to prepare a coating solution. The antibody concentration in the coating solution was approximately 5–10 μg / mL (preferably approximately 7.5 μg / mL in this example). The coating solution was added to a T25 culture flask (total volume 5–7 mL) to ensure that the inner surface of the flask was in full contact with the solution to achieve antibody adsorption.

[0088] The coating process can be performed under suitable low-temperature conditions, such as overnight incubation at approximately 2–8°C, to promote antibody binding to the surface of the culture dish. After coating, discard the remaining solution and wash 1–2 times with sterile PBS to remove unbound antibodies. The culture dishes treated in this way are then ready for use in subsequent cell seeding or culture processes.

[0089] (b) Induction culture

[0090] PBMCs were seeded into antibody-coated T25 culture flasks, and induction culture was performed using NK MACS. ® Medium was used as the basal medium, supplemented with 10% autologous plasma. IL-12 (10 ng / mL), IL-15 (50 ng / mL), and IL-18 (100 ng / mL) were added to the culture system to achieve inductive stimulation.

[0091] The cell density during the induction phase was 1.5 × 10⁻⁶ cells / year. 6 The cells / mL were cultured in a total volume of 10 mL; the induction culture lasted for about 24 hours; after induction, the cells were collected and washed to prepare for the stimulation phase.

[0092] S22, Stimulation Phase

[0093] After the induction phase, the recovered and washed cells were transferred into culture flasks for stimulation culture.

[0094] First, culture in a T75 culture flask for 3 days (total volume 20 mL), then transfer to a T175 culture flask and culture for another 3 days (total volume 30 mL).

[0095] The cell seeding density during the stimulation phase was 1.0 × 10⁶ cells / year. 6 cells / mL, the stimulation phase medium was also NK MACS ® Medium was used as the basal medium, supplemented with autologous plasma (10%), and IL-2 (10 IU / mL) and IL-15 (50 ng / mL) were used to maintain proliferation and activity. During the stimulation phase, Wnt / β-catenin signaling pathway activators were introduced, and Bio-acetoxime (2 μM), an inhibitor of GSK-3α and GSK-3β, was used as a time-dependent regulatory component to optimize cell proliferation potential and differentiation status.

[0096] S23, Amplification Phase

[0097] Following the stimulation phase, the cells were transferred to a large-volume gas-permeable amplification device for large-scale expansion. In this embodiment, G-Rex was used. ® Using a 100 Open System as the amplification vessel, the plating density was determined based on the total target cell count, with an initial cell density range of (0.11~0.22)×10⁻⁶. 6 The cells / mL culture volume was 400 mL.

[0098] The amplification medium is still NK MACS ® Medium was used as the basal medium, supplemented with 5% autologous plasma, and IL-2 (10 IU / mL) and IL-15 (50 ng / mL) were used as the main cytokines to support the amplification phase. The amplification phase lasted approximately 7 days.

[0099] After expansion, the cells were recovered and washed to obtain highly activated NK cell-enriched lymphocytes.

[0100] Example 2

[0101] This embodiment aims to provide an evaluation of cell expansion using the culture method described in Example 1.

[0102] To evaluate the effect of the NK cell-enriched lymphocyte culture method (system) described in this invention on overall cell expansion, two sets of experiments were set up:

[0103] Bio-acetoxime (–) group: No Wnt / β-catenin signaling activator added during the stimulation phase;

[0104] Bio-acetoxime (+) group: Wnt / β-catenin signaling activator (Bio-acetoxime) added during the stimulation phase;

[0105] The initial total cell count for both groups was 1.5 × 10⁻⁶. 7 .

[0106] The three stages of induction, stimulation, and expansion were as described in Example 1. During the induction stage, an anti-NKp46 and anti-2B4 coating system was used, supplemented with IL-12, IL-15, and IL-18 cytokines. During the stimulation stage, cells were cultured in T75 and T175 flasks with IL-2 and IL-15 for 6 days, with the experimental group receiving Bio-acetoxime during the stimulation phase. During the expansion stage, cells were transfected into G-Rex. ® The 100-cell culture system was cultured for another 7 days. Cells were collected and counted on day 14.

[0107] The results are as follows Figure 1 As shown:

[0108] Starting cell count (day 0): 1.5 × 10⁻⁶ 7 .

[0109] Bio-acetoxime (–) group: Total cell count on day 14 was 63.72 × 10⁻⁶. 7 The number of cells increased by 42.48 times compared to the initial number.

[0110] Bio-acetoxime (+) group: Total cell count on day 14 was 76.24 × 10⁻⁶. 7 The number of cells increased by 50.82 times compared to the initial number.

[0111] This embodiment demonstrates that the three-stage culture process itself can achieve significant cell expansion within 14 days (approximately 42.48× in the Bio-acetoxime (–) group), while the addition of the Wnt / β-catenin activator Bio-acetoxime during the stimulation phase further enhances the expansion efficiency (approximately 50.82× in the Bio-acetoxime (+) group). The two sets of results are significant: the scheme without Bio-acetoxime demonstrates the basic feasibility and stability of the process; the scheme containing Bio-acetoxime showcases a feasible improvement path to enhance yield through signaling pathway regulation.

[0112] Example 3

[0113] This embodiment aims to provide NK cells (CD3) obtained by the culture method of the present invention. - CD16 + CD56 + Evaluation of enrichment and phenotypic enhancement.

[0114] The effects of adding Wnt / β-catenin activator (Bio-acetoxime) during the stimulation phase on NK cell purity and CD56 expression intensity in expanded cells were compared. Referring to Example 2, CD3 expression was detected by flow cytometry on day 14 of culture. - CD16 + CD56 + The proportion of NK cells was compared with the mean fluorescence intensity (MFI) of CD56, and data from four donors were pooled to assess reproducibility.

[0115] The results are as follows Figure 2 As shown:

[0116] Figure 2 Middle A (Representative flow cytometry plot):

[0117] Bio-acetoxime (–) group: NK cell purity 64.71%; Bio-acetoxime (+) group: NK cell purity 69.50%.

[0118] Figure 2 Medium B (Summary of NK purity from four donors):

[0119] D0 (initial PBMC): 9.34%; Bio-acetoxime (–) group (day 14): 62.55%; Bio-acetoxime (+) group (day 14): 67.92%.

[0120] Figure 2 C (CD56 MFI comparison):

[0121] Bio-acetoxime (–) group: 3,153 (MFI); Bio-acetoxime (+) group: 5,261 (MFI).

[0122] This embodiment shows that after the three-stage culture process described above, the proportion of NK cells in the total cells and the CD56 expression intensity of the two groups (Bio-acetoxime(–) and Bio-acetoxime(+)) were significantly increased compared with the initial values. These results demonstrate that the culture process can stably enrich and promote the enhancement of NK cell phenotype, and that the proportion and CD56 expression intensity are even higher after the introduction of Bio-acetoxime.

[0123] Example 4

[0124] This embodiment aims to provide NK cell-enriched lymphocytes and NKT cells (CD3+) obtained by the culture method of the present invention. + CD16 + CD56 + ) Enrichment assessment.

[0125] Using the same three-stage culture procedure and group settings as in Example 2 (Bio-acetoxime (–) group and Bio-acetoxime (+) group), the culture products were analyzed by flow cytometry phenotyping on day 14, according to CD3. + CD16 + CD56 + A gating strategy was used to detect the proportion of NKT cells, and data from four donors were aggregated to assess consistency and reproducibility.

[0126] The results are as follows Figure 3 As shown:

[0127] Figure 3 Middle A (Representative flow cytometry plot):

[0128] Bio-acetoxime (–) group: NKT cell purity 4.32%; Bio-acetoxime (+) group: NKT cell purity 6.59%.

[0129] Figure 3 Medium B (Summary of NKT purity from four donors):

[0130] D0 (initial PBMC): 1.04%; Bio-acetoxime (–) group (day 14): 5.34%; Bio-acetoxime (+) group (day 14): 6.42%.

[0131] After the three-stage culture treatment, NKT (CD3) + CD16 + CD56 + The proportion of NK cells in the total cell count was significantly increased compared to the initial level. Both groups showed a trend of NKT enrichment, with a slight increase in the Bio-acetoxime(+) group compared to the control group. These results indicate that this culture system can not only enrich the conventional NK cell population but also increase the proportion of NKT cells to a certain extent.

[0132] Example 5

[0133] This embodiment aims to provide an analysis of the expression of NK cell activation markers obtained by the culture method of the present invention.

[0134] Using the same three-stage culture system as in Example 2, cells cultured on day 14 were subjected to CD3... - Gating, and on CD16 + CD56 + The positive rates of activation markers NKG2D (CD314), DNAM-1 (CD226), and CD69 were detected in the subgroups.

[0135] The results are as follows Figure 4 As shown:

[0136] Figure 4 Chinese A (NKG2D):

[0137] D0 (initial PBMC): 6.77%; Bio-acetoxime (–) group (day 14): 65.78%; Bio-acetoxime (+) group (day 14): 71.94%.

[0138] Figure 4 DNAM (DNAM):

[0139] D0 (initial PBMC): 8.04%; Bio-acetoxime (–) group (day 14): 67.17%; Bio-acetoxime (+) group (day 14): 67.50%.

[0140] Figure 4 C (CD69):

[0141] D0 (initial PBMC): 4.96%; Bio-acetoxime (–) group (day 14): 39.21%; Bio-acetoxime (+) group (day 14): 51.87%.

[0142] Compared with the initial sample, the three-stage culture significantly increased the expression ratios of NKG2D, DNAM-1, and CD69 in the expanded cells. In this test, the Bio-acetoxime(+) group showed a numerical increase in NKG2D and CD69, while DNAM-1 showed similar expression levels between the two groups. These results indicate that this culture system can enhance the activation phenotype of NK cells.

[0143] Example 6

[0144] This embodiment aims to provide a method for detecting the ability of cells to secrete IFN-γ obtained by the culture method of the present invention.

[0145] To evaluate the ability of expanded cells to secrete interferon-γ (IFN-γ) under conditions without exogenous cytokine stimulation, three groups were set up: PBMC control group (conventional culture for 14 days), Bio-acetoxime(-) group, and Bio-acetoxime(+) group. The Bio-acetoxime(-) group and the Bio-acetoxime(+) group were cultured for 14 days according to the three-stage culture system in Example 2.

[0146] After 14 days of culture, the cells were collected, washed, and then cultured for another 48 hours under cytokine-free conditions. The number of IFN-γ secretion spots was detected using ELISpot as an indicator of single-cell secretory activity.

[0147] After 48 hours of culture, the ability of cells to secrete IFN-γ was detected using ELISApot, and the results are as follows: Figure 5 As shown:

[0148] IFN-γ (number of spots):

[0149] PBMC (14-day culture control group): 20;

[0150] Bio-acetoxime (–) group: 551;

[0151] Bio-acetoxime (+) group: 701.

[0152] Compared with the control group of PBMCs that did not undergo the three-stage induction and expansion process, both the Bio-acetoxime(–) and Bio-acetoxime(+) groups showed significantly enhanced IFN-γ secretion capacity, demonstrating strong innate immune effector function. The Bio-acetoxime(+) group showed a further increase in the number of blots, suggesting that the addition of a Wnt / β-catenin pathway activator during the stimulation phase can enhance cellular secretory effector function to some extent. Overall, the results demonstrate that the three-stage culture system of this invention can effectively enhance the cytokine secretion activity of NK cell-enriched lymphocytes.

[0153] Example 7

[0154] This embodiment aims to provide an ELISA test for the ability of cells to secrete granzyme B and perforin obtained by the culture method of the present invention.

[0155] To evaluate the ability of expanded cells to secrete cytotoxic molecules, granzyme B and perforin, under conditions without exogenous cytokines, three experimental groups were set up: PBMC control group, Bio-acetoxime (–) group, and Bio-acetoxime (+) group. The Bio-acetoxime (–) group and the Bio-acetoxime (+) group were cultured for 14 days according to the three-stage culture system in Example 2.

[0156] After 14 days of culture, cells from each group were collected and washed, and then cultured for another 48 hours under conditions free of exogenous cytokines. The culture supernatant was then collected, and the secretion levels of granzyme B and perforin were detected by ELISA.

[0157] The results are as follows Figure 6 As shown:

[0158] Figure 6 In the middle group, A represents granzyme B (ng / mL): PBMC: 12.10; Bio-acetoxime (–) group: 1027.21; Bio-acetoxime (+) group: 1401.37.

[0159] Figure 6 In the middle group, B was perforin (ng / mL): PBMC: 3.71; Bio-acetoxime (–) group: 428.51; Bio-acetoxime (+) group: 632.61.

[0160] The experimental results showed that, compared with the basal culture PBMC control group, cells expanded through the three-stage process exhibited significantly enhanced granzyme B and perforin secretion capabilities even without cytokine stimulation, indicating that they acquired stronger cytotoxic effector functions. The Bio-acetoxime(+) group showed further increases in both indicators, suggesting that the Wnt / β-catenin pathway activator can further enhance the cytotoxic secretory potential of the expanded cells. These results further validate the significant advantages of the culture system of this invention in enhancing the effector function of NK cell-enriched lymphocytes.

[0161] Example 8

[0162] This embodiment aims to provide a method for detecting the tumor cell killing activity mediated by NK cells obtained by the culture method of the present invention.

[0163] To evaluate the direct killing effect of cells expanded using the three-stage culture system of this invention on tumor target cells, three groups were set up: a PBMC control group (not expanded according to the three-stage procedure of this invention, but cultured under basic conditions for 14 days), a Bio-acetoxime (–) group, and a Bio-acetoxime (+) group. The Bio-acetoxime (–) group and the Bio-acetoxime (+) group were cultured according to the three-stage culture system of Example 2. Cells from all three groups were collected on day 14 and used for subsequent killing experiments.

[0164] Two tumor cell lines were selected as target cells: the HO-8910 ovarian cancer cell line and the Colo205 colorectal cancer cell line. In the experiment, effector cells (E) and target cells (T) were co-cultured at an E:T ratio of 5:1 for 4 hours. Subsequently, the LDH release assay was used to assess the level of NK cell-mediated tumor cell lysis and the killing rate was calculated.

[0165] The results are as follows Figure 7 As shown:

[0166] Figure 7 In the figure, A represents the killing rate of the HO-8910 cell line: PBMC group: 5.09%; Bio-acetoxime (–) group: 50.06%; Bio-acetoxime (+) group: 66.43%.

[0167] Figure 7 B represents the killing rate against the Colo205 cell line: PBMC group: 4.71%; Bio-acetoxime (–) group: 34.81%; Bio-acetoxime (+) group: 46.79%.

[0168] The above results indicate that the cells obtained after expansion using the three-stage system of this invention all exhibited significantly enhanced killing ability, which was significantly better than the PBMC control group not cultured according to this system. Both the Bio-acetoxime(–) and Bio-acetoxime(+) groups were able to restore and maintain stable cytotoxic activity, while the Bio-acetoxime(+) group further enhanced the killing level under the experimental conditions, suggesting that Wnt / β-catenin signaling regulation has a potential promoting effector cell function enhancement, providing a valuable reference for subsequent system optimization.

Claims

1. A method for culturing high activated state NK cell enriched lymphocytes, characterized by, The method comprises the following steps: S1, obtaining peripheral blood mononuclear cells; S2, taking the peripheral blood mononuclear cells as starting material, and performing culture, comprising: S21, an induction stage: inoculating the peripheral blood mononuclear cells into a culture vessel coated with antibodies, and preliminarily culturing the peripheral blood mononuclear cells using an induction medium, the antibodies comprising: an anti-NKp46 antibody and an anti-2B4 antibody; the induction medium comprising: a basic culture medium, autologous plasma, the anti-NKp46 antibody, the anti-2B4 antibody, interleukin-12, interleukin-15, and interleukin-18; S22, a stimulation stage: continuing to culture after replacing the induction medium with a stimulation medium, the stimulation medium comprising: the basic culture medium, the autologous plasma, interleukin-2, interleukin-15, and a Wnt / β-catenin signaling pathway activator; S23, an expansion stage: continuing to culture after replacing the stimulation medium with an expansion medium, the expansion medium comprising: the basic culture medium, the autologous plasma, interleukin-2, and interleukin-15.

2. The culture method according to claim 1, characterized by, The culture time of the induction stage, the stimulation stage, and the expansion stage is respectively: 18-24 h, 6-8 d, and 7-10 d.

3. The culture method according to claim 1 or 2, characterized by, The antibody-coated culture vessel preparation method comprises: dissolving the antibodies in a buffer to prepare a coating solution; adding the coating solution into the culture vessel, so that the coating solution fully contacts the inner surface of the culture vessel; after full contact, the antibodies are adsorbed on the inner surface of the culture vessel, and the coating is completed.

4. The culturing method according to claim 3, wherein In the induction medium, the concentration of interleukin-12 is: 10 ng / mL; the concentration of interleukin-15 is: 50 ng / mL; the concentration of interleukin-18 is: 100 ng / mL.

5. The culturing method according to claim 4, wherein In the stimulation medium, the concentration of interleukin-2 is: 10 IU / mL; the concentration of interleukin-15 is: 50 ng / mL; the concentration of the Wnt / β-catenin signaling pathway activator is: 1.5-2.5 μM.

6. The culturing method according to claim 5, wherein The Wnt / β-catenin signaling pathway activator comprises a GSK-3α and / or GSK-3β inhibitor.

7. The culture method according to claim 5 or 6, characterized by, In the expansion medium, the concentration of interleukin-2 is: 10 IU / mL; the concentration of interleukin-15 is: 50 ng / mL.

8. The culture method according to claim 7, characterized by, The cell concentrations of the induction phase, stimulation phase and expansion phase are respectively: (1~2)×10 6 cells / mL, (0.8~1.5)×10 6 cells / mL, (0.11~0.22)×10 6 cells / mL.

9. NK cell-enriched lymphocytes obtained by the culture method according to any one of claims 1-8.

10. Use of the NK cell-enriched lymphocytes according to claim 9 in the preparation of a tumor treatment drug.

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