A method for preparing NK cells and its application in treating cancer

Abstract

The present invention provides a method for preparing NK cells and their application in treating cancer. First, a monoclonal antibody targeting PD-L1 is screened and obtained, which can bind to the target antigen with high affinity. Second, the activation mode of NK cells is optimized, and cytokines such as IL-15, IL-12 and IL-18 are used to progressively activate NK cells, thereby improving the activation level of NK cells. Third, the combination of NK cells and antibodies targeting PD-L1 can effectively prevent tumor immune escape and enhance anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a method for preparing NK cells and their application in treating cancer. Background Art

[0002] Malignant tumors are serious diseases that severely impact human health. While the immune system can inhibit the growth and reproduction of tumor cells to a certain extent, tumor cells can exploit immune escape mechanisms to suppress the anti-tumor immune response of immune cells in the tumor microenvironment, thereby promoting tumor development and progression. Therefore, the emerging field of tumor immunotherapy aims to alter the suppressive tumor microenvironment, restore immune system activity, and ultimately eliminate tumor cells.

[0003] NK cells play a vital role in controlling the occurrence and development of tumors. NK cells are cytotoxic lymphocytes in the innate immune system that play a direct killing role. Their anti-tumor effect does not require antigen sensitization and is independent of MHC-I molecules. Compared with CD8+ T cells, their recognition mechanism is more flexible and has the ability to rapidly kill tumor cells. They are currently the most promising tumor-killing cells besides T cells. This non-specific recognition mechanism and efficient anti-tumor activity may supplement the deficiencies of anti-tumor T cell therapy (see Terrén I, Orrantia A, Vitallé J, et al. NK cell metabolism and tumor microenvironment. Front Immunol. (2019) 10: 2278). Based on this, NK cells have become an important research subject in tumor treatment, and immunotherapy strategies to enhance NK cell anti-tumor responses have developed rapidly, including adoptive NK cell therapy, cytokine therapy, antibody-based NK cell therapy, and so on.

[0004] Adoptive NK cell therapy involves the infusion of autologous or allogeneic NK cells that have been expanded or genetically modified in vitro into tumor patients to increase the number and anti-tumor activity of NK cells in the patient's body. The NK cells used in this therapy come from a variety of sources, including peripheral blood, umbilical cord blood, NK cells differentiated from induced pluripotent stem cells (iPSCs), and NK cell lines cultured in vitro (see Laskowski TJ, A, Rezvani K. Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer. (2022) 22:557–75). Early adoptive NK cell therapy directly infused NK cells, but this approach lacked targeted therapy and clinical efficacy. With the development of chimeric antigen receptor (CAR) technology, researchers have developed CAR-NK cells, which recognize tumor antigens through the single-chain variable region of the CAR structure, enhancing the specificity of NK cells. CAR-NK cells can simultaneously use CAR-dependent and CAR-independent mechanisms to identify and kill tumor cells. They can effectively kill tumor cells even when MHC-I is downregulated, lost, or tumor antigens are mutated. This dual mechanism helps overcome CAR-T cell therapy resistance caused by antigen loss and reduces the risk of recurrence caused by loss of specific tumor antigens targeted by CAR. Compared with CAR-T cells, CAR-NK cells are more stable and have fewer side effects (see Wang W, Jiang J, Wu C. CAR-NK for tumor immunotherapy: Clinical transformation and future prospects. Cancer Lett. (2020) 472: 175–80). However, CAR-NK cell therapy also has some challenges and obstacles, such as the difficulty in obtaining a large number of highly pure NK cells, because the number of NK cells from a single donor is insufficient for treatment, and the time required to culture NK cells (see Chu J, Gao F, Yan M, et al. Natural killer cells: apromising immunotherapy for cancer. J Transl Med. (2022) 20: 240).

[0005] Cytokine therapy is the use of cytokines to promote the mobilization of endogenous NK cells, thereby regulating anti-tumor immune responses. It has been observed that cytokines such as IL-2, IL-12, IL-15, IL-18, and TGF-β can regulate NK cell-mediated anti-tumor immune responses. IL-2 can activate the cytotoxicity of NK cells and is currently widely used as a cytokine in clinical cancer treatment. However, the excessive induction of Tregs, activation-induced cell death of NK cells, and serious side effects of IL-2 on vascular leakage syndrome limit the application of IL-2. In clinical treatment, the dosage of IL-2 needs to be precisely controlled (see Mitra S, Leonard WJ. Biology of IL-2 and its therapeutic modulation: Mechanisms and strategies. J Leukoc Biol. 2018; 103(4): 643–655). Therefore, researchers have gradually shifted their research focus to cytokines other than IL-2, such as IL-12, IL-15, IL-18, and TGF-β inhibitors. However, most of these studies are still in the research stage and have not yet been widely used in clinical practice.

[0006] Antibody-based NK cell therapy combines antibodies with NK cells to enhance the tumor immunity effect of NK cells. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism for NK cells to specifically target and kill tumor cells. Utilizing NK cell-mediated ADCC to specifically eliminate tumor cells is an important strategy for NK cell-based tumor immunotherapy (see Decaup E, Rossi C, Gravelle P, et al. A tridimensional model for NK cell-mediated ADCC of follicular lymphoma. Front Immunol. (2019) 10: 1943). For example, rituximab is used to treat follicular lymphoma, trastuzumab is used to treat human epidermal growth factor receptor 2 (HER2)-positive breast cancer, cetuximab and avelumab are used to treat non-small cell lung cancer, and daratumumab and all-trans retinoic acid are used to treat multiple myeloma. These monoclonal antibodies enhance the NK cell's anti-tumor activity by enhancing the ADCC effect, thereby achieving good therapeutic effects. These results highlight the great potential of enhancing NK cell-mediated ADCC to kill tumor cells in tumor immunotherapy.

[0007] Immune checkpoints can hinder NK cell function by recognizing and binding to specific ligands on tumor cells, leading to NK cell exhaustion and promoting tumor immune escape (see Gemelli M, Noonan DM, Carlini V, et al. Overcoming resistance to checkpoint inhibitors: natural killer cells in non-small cell lung cancer. Front Oncol. (2022) 12: 886440). The NK cell immune checkpoints that have been discovered include NKG2A, PD-1, TIGIT, TIM-3, KIR, LIR, CD96, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), B7-H3 (CD276), LAG-3, Siglec-7 / 9, SIRPα, CD200R and CD47, etc. Based on this discovery, a variety of monoclonal antibodies targeting different immune checkpoints are continuously being developed for clinical tumor treatment. They have been shown to be safe and effective both in vivo and in vitro.

[0008] Although NK cells can effectively kill tumor cells, there is still room for improvement in terms of efficient activation and improved anti-tumor efficacy. Therefore, the present invention provides a method for preparing NK cells and their application in treating cancer. NK cells are activated by a combination of multiple cytokines and are combined with NK cells and antibodies targeting PD-L1 to enhance anti-tumor activity. Summary of the Invention

[0009] A first aspect of the present invention provides an antibody targeting PD-L1, comprising a heavy chain and a light chain, wherein the heavy chain comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, and the light chain comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7.

[0011] Furthermore, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8.

[0012] PD-1 is an important immunosuppressive molecule expressed in CD4+ and CD8+ T cells, NK cells, NKT cells, B cells, and other innate lymphocytes. Upregulation of PD-1 expression has been observed in peripheral blood and tumor-infiltrating NK cells in patients with various cancers, leading to a weakening of NK cell responses. Blocking the PD-1 / PD-L1 interaction can alleviate NK cell inhibition and thus enhance its anti-tumor immune function (see Hsu J, Hodgins JJ, Marathe M, et al. Contribution of NK cells to immunotherapy mediated by PD-1 / PD-L1 blockade. J Clin Invest. (2018) 128:4654–68). Researchers have attempted to use inhibitors targeting PD-1 / PD-L1 to enhance NK cell activity. For example, avelumab promotes breast cancer cell killing by inducing NK cells to produce cytokines (Juliá EP, Amante A, Pampena MB, et al. An igG1 anti-PD-L1 immune checkpoint inhibitor, triggers NK cell-mediated cytotoxicity and cytokine production against triple-negative breast cancer cells. Front Immunol. (2018) 9: 2140). Currently, a variety of antibodies targeting PD-1 or PD-L1 have been developed in the prior art, such as CN115925943A, which discloses an anti-PD-L1 antibody and its use, CN109053891A, which discloses an anti-PD-L1 antibody and its preparation method and use, and CN110590955A, which discloses a bispecific antibody that targets PD-L1 and CD3, respectively.

[0013] In the present invention, hybridoma technology was used to screen and prepare a novel monoclonal antibody targeting PD-L1. The antibody has a novel heavy chain and light chain structure, can effectively bind to the target antigen, block the PD-1 / PD-L1 signaling pathway, inhibit the immune escape of tumor cells, and help enhance the anti-tumor activity of NK cells.

[0014] The second aspect of the present invention provides a therapeutic composition comprising the antibody and NK cells.

[0015] Furthermore, the method for preparing NK cells includes: extracting mononuclear cells, inoculating them into a culture medium containing 10% FBS, and sequentially adding IL-15, IL-12 and IL-18 for culturing.

[0016] Furthermore, the NK cell preparation method also includes: first adding IL-15 with a final concentration of 10 ng / mL, culturing for 3 days, then adding IL-12 with a final concentration of 20 ng / mL, culturing for 7 days, adding IL-18 with a final concentration of 50 ng / mL, and collecting the cells after culturing for 14 days.

[0017] The present invention optimizes the activation mode of NK cells, and gradually activates immune cells by increasing the concentration of stimulating factors in sequence. Compared with the traditional activation mode using IL-2 cytokines, not only the activation degree of NK cells is greatly improved, but also the CD3 - CD56 + The cell ratio limit is increased, and the potential risks caused by high-dose use of IL-2 can be effectively avoided.

[0018] The third aspect of the present invention provides the use of the therapeutic composition in the preparation of anti-tumor drugs.

[0019] Furthermore, the tumor includes one or more of lung cancer, gastric cancer, colorectal cancer, melanoma, glioma, pancreatic cancer, leukemia, myeloma, and lymphoma.

[0020] Furthermore, the tumor is a glioma.

[0021] Beneficial effects

[0022] The present invention provides a method for preparing NK cells and its application in treating cancer, as follows

[0023] Beneficial effects:

[0024] 1. We screened and obtained a monoclonal antibody targeting PD-L1 that binds to the target antigen with high affinity;

[0025] 2. Optimized the activation mode of NK cells and improved the activation level of NK cells;

[0026] 3. Combining NK cells with antibodies targeting PD-L1 can effectively prevent tumor immune escape and enhance anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : NK cell purity;

[0028] Figure 2 : Tumor cell killing rate;

[0029] Figure 3 : Changes in tumor volume in animal models;

[0030] Figure 4 : TNF-α expression level in animal model serum;

[0031] Figure 5 : IL-6 expression levels in serum of animal models. DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0033] Example 1 Preparation and activation of NK cells

[0034] 1.1 Extraction of mononuclear cells

[0035] Peripheral blood was collected from healthy volunteers and placed in a sodium heparin blood collection tube. The blood was slowly added along the tube wall into a centrifuge tube containing mononuclear cell separation medium (purchased from Tianjin Haoyang Biotechnology Co., Ltd.). The tube was centrifuged at 800 g for 25 min at room temperature. The middle buffy coat cells were carefully aspirated with a pipette into a new centrifuge tube and washed three times with sterile PBS.

[0036] 1.2 NK cell activation

[0037] This invention provides a novel NK cell activation method that uses a "cocktail" activation procedure, i.e., multiple cytokines are used in a programmed activation process. To compare the activation effect of this method, a traditional method was used as a control.

[0038] The traditional activation method includes: inoculating NK cells in 1640 culture medium, adding 200 IU / mL IL-2 and 10% FBS to the culture medium, culturing for 7 days, increasing the IL-2 concentration to 1000 IU / mL, and collecting the cells after 14 days of culture.

[0039] The "cocktail" activation method provided in the present invention is to inoculate NK cells in 1640 culture medium, add 10% FBS, first add IL-15 (purchased from PeproTech) at a final concentration of 10 ng / mL, and then add IL-12 (purchased from Sigma) at a final concentration of 20 ng / mL after culturing for 3 days. After culturing for 7 days, add IL-18 (purchased from R&D Systems) at a final concentration of 50 ng / mL, and collect the cells after culturing for 14 days.

[0040] Flow cytometry was used to detect the purity of NK cells. Cells were collected by centrifugation and resuspended in PBS. 1 μL of anti-CD3-FITC antibody and anti-CD56-PE antibody were added respectively. The cells were incubated in the dark at room temperature for 30 min. CD3 was detected by flow cytometry. - CD56 + Cell content was analyzed using FlowJo V10 software. Figure 1As shown, CD3 - CD56 + The cell content is as high as 88%, which is significantly higher than the traditional activation method, indicating that the use of multiple cytokine gradient activation methods can effectively activate NK cells.

[0041] 1.3NK cells inhibit tumor cell growth in vitro

[0042] Conventional NK cells and improved NK cells obtained in Section 1.2 were used as effector cells, U251 glioma cells were used as target cells, and PBS was used as a control. The effector and target cells were seeded into 96-well plates at a 10:1 effector-target ratio and incubated at 37°C, 5% CO₂ for 12 hours. Then, 20 μl of MTT (5 mg / ml) was added to each well and incubated for another 4 hours. Then, 100 μl of DMSO was added to each well and mixed by pipetting. The optical density (A) at a wavelength of 570 nm was measured using a microplate reader to calculate the killing rate.

[0043] The results are as follows Figure 2 As shown, although NK cells prepared by traditional methods can inhibit the growth of tumor cells, their anti-tumor activity is limited. The improved NK cells provided by the present invention can greatly increase the tumor cell killing rate, indicating that it can effectively activate NK cells and promote their anti-tumor effect.

[0044] Example 3 Preparation of monoclonal antibodies targeting PD-L1

[0045] In this invention, hybridoma technology is used to screen and prepare monoclonal antibodies targeting PD-L1. The PD-L1 extracellular domain gene (GenBank: AY254342.1) was selected and transiently expressed in CHO cells. The immunogen was purified by Protein A affinity chromatography and its purity was determined by polyacrylamide gel electrophoresis to obtain the target immunogen.

[0046] The immunogen was emulsified with Freund's adjuvant and injected subcutaneously into Balb / c mice at multiple sites every two weeks, at a dose of 100 μg per mouse, for a total of four immunizations. Five days after the final immunization, blood was collected from the tail vein, and serum titers were determined by ELISA. Mice with high titers were selected for pulse immunization. Mouse spleens were isolated and electrofused with Sp2 / 0-Ag14 cells in a 2:1 ratio, using 75V AC for 30 seconds, 1500V DC for 40 μs, and then AC for 5 seconds. After fusion, positive clones were screened, and hybridoma RNA was extracted to analyze the antibody gene sequence. Chimeric antibody plasmids were prepared, and the plasmids were expressed in cells using polyethyleneimine. The antibodies were purified by protein A affinity chromatography. The half-maximal effect concentration (EC50) of the antibody and the immunogen was determined by ELISA. The EC50 of the antibody reached 8.53 ng / mL, indicating high affinity for the target immunogen.

[0047] After bioinformatics identification, the amino acid sequences of the heavy chain variable region HCDR1-3 of the antibody are shown as SEQ ID NOs: 1-3, respectively, the amino acid sequences of the light chain variable region LCDR1-3 are shown as SEQ ID NOs: 4-6, respectively, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8.

[0048] Example 4 NK cells and monoclonal antibodies targeting PD-L1 inhibit tumor cell growth in vivo

[0049] 4.1 Animal model preparation and drug administration

[0050] Balb / c nude mice were used to digest U251 cells in the logarithmic growth phase and reconstituted into 1×10 7 100 / mL solution, and then 0.2mL of cell suspension was injected into the right anterior armpit of the mouse. After 10-14 days, the formation of tumor could be felt, indicating that the model was successful. 30 nude mice with successful model were randomly divided into 3 groups: control group, NK cell group and NK+antibody group, with 10 mice in each group. Among them, 3×10 6 Improved NK cells were injected 4 times in total; NK+antibody group was injected 3×10 6 The improved NK cells and 100 mg / kg of the PD-L1 targeting antibody were administered for 4 times in total; the control group was injected with an equal amount of normal saline every week for 4 times in total.

[0051] 4.2 Tumor volume observation

[0052] Tumor volume was observed weekly. The length and width of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the formula: volume = 0.52 × length × width 2 The results are as follows Figure 3 As shown, NK cells can effectively inhibit tumor growth in vivo, and their growth rate is significantly slower than that of the control group; when NK cells are used in combination with antibodies targeting PD-L1, the tumor volume is further reduced, indicating that immune checkpoint inhibitors can effectively prevent tumor immune escape, thereby exerting a greater anti-tumor effect.

[0053] 4.3 Serum cytokine detection

[0054] After the last administration, blood was collected from the eyeballs of nude mice, kept at 4°C for 1 hour, and centrifuged at 3000 rpm for 15 minutes to collect serum. The ELISA kit (purchased from Shanghai Fanke Industrial Co., Ltd.) was used according to the instructions to detect the levels of cytokines TNF-α and IL-6 in the serum.

[0055] The results are as follows Figure 4 , Figure 5 As shown, NK cell therapy can promote the expression of cytokines TNF-α and IL-6, and the addition of antibodies targeting PD-L1 further enhances cytokine secretion, especially the expression of IL-6. This shows that the NK cells and antibodies provided by the present invention can effectively activate immune pathways in the body and exert a synergistic anti-tumor effect.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody targeting PD-L1, characterized in that The antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, and the light chain comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively.

2. The antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

8.

3. A therapeutic composition, characterized in that The therapeutic composition comprises the antibody according to any one of claims 1-2 and NK cells, and the preparation method of the NK cells comprises: extracting mononuclear cells, inoculating them in a culture medium containing 10% FBS, first adding IL-15 with a final concentration of 10 ng / mL, culturing for 3 days, then adding IL-12 with a final concentration of 20 ng / mL, culturing for 7 days, adding IL-18 with a final concentration of 50 ng / mL, and collecting the cells after 14 days of culture.

4. Use of the therapeutic composition according to claim 3 in the preparation of an anti-tumor drug, wherein the tumor comprises one or more of lung cancer, gastric cancer, colorectal cancer, melanoma, glioma, and pancreatic cancer.

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