Preparation method of nk cells and application thereof in treating tumors

Abstract

The application provides a preparation method of NK cells and application of the NK cells in tumor treatment, selects a tumor antigen CLEC12A specific to AML tumor cells as a target, screens and prepares an scFv region capable of specifically combining with the antigen, wherein an amino acid sequence of a heavy chain variable region is shown as SEQ ID NO:1, and an amino acid sequence of a light chain variable region is shown as SEQ ID NO:2; a second generation chimeric antigen receptor including the anti-CLEC12A scFv region is constructed, and an NK cell is used as an immune carrier; a CAR-NK cell is prepared and obtained, and the CAR-NK cell can effectively inhibit growth of tumor cells in vivo and in vitro; the CAR-NK cell can effectively stimulate an immune response in vivo, kill tumor cells, and the secretion level of the inflammatory factor is moderate, so that safety and effectiveness of treatment can be ensured.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology research and development, and specifically provides a method for preparing human collagen and its application in anti-aging. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. While existing first-line and consolidation chemotherapy is effective in 70% to 80% of patients, the 5-year survival rate is only 26%, highlighting the urgent need for new treatments (see Rotiroti MC, Arcangeli S., Casucci M., et al. Acute myeloidleukemia targeting by chimeric antigen receptor t cells: Bridging the gap from preclinical modeling to human studies. Hum. Gene Ther. 2017;28:231–241.). Genetically engineered patient-derived T cells to express chimeric antigen receptors (CARs), enabling malignant cell-targeting therapy independent of the major histocompatibility complex (MHC), has achieved widespread success in the treatment of B-cell leukemia. CD19-targeted CAR-T cells have been shown to achieve complete and durable remission in 70% to 90% of patients with acute lymphoblastic leukemia (ALL). Because AML has immunosuppressive properties, activating the immune system through genetically engineered T-cell therapy offers a promising cure option for patients (see Mussai F., DeSanto C., Abu-Dayyeh I., et al. Acute myeloid leukemia creates an arginase-dependent immunosuppressive microenvironment. Blood. 2013;122:749–758).

[0003] To apply the promising results of CAR-T cell therapy in ALL to AML, some research groups have developed CAR-T cells targeting CD33 and CD123, among others (see O'Hear C., Heiber JF, Schubert I., et al. Anti-cd33 chimeric antigen receptor targeting of acute myeloid leukemia. Haematologica. 2015;100:336–344; Wang QS, Wang Y., Lv HY, et al. Treatment of cd33-directed chimeric antigen receptor-modified t cells in one patient with relapsed and refractory acute myeloid leukemia. Mol. Ther. 2015;23:184–191). However, because CD33 and CD123 are also expressed on hematopoietic stem cells (HSCs), these CAR-T cells have the potential to cause prolonged bone marrow clearance, which contradicts the efficacy and durability of CAR-T cells.In fact, a recent fatal event occurred in a clinical trial of allogeneic CAR-T cells targeting CD123, with severe pancytopenia in an AML patient receiving anti-CD33 CAR-T cell therapy (see Bakker AB, van den Oudenrijn S., Bakker AQ, et al. C-type lectin-like molecule-1: A novel myeloid cellsurface marker associated with acute myeloid leukemia. Cancer Res. 2004;64:8443–8450), and hematopoietic impairment has also been reported in human hematopoietic xenotransplantation models (see Pizzitola I., Anjos-Afonso F., Rouault-Pierre K., et al. Chimeric antigen receptors against cd33 / cd123 antigens efficiently target primary acute myeloid leukemia cells in vivo. Leukemia. 2014;28:1596–1605). Furthermore, the high heterogeneity of AML necessitates the development of CAR-T cells to treat a broader patient population and reduce the risk of long-term adverse reactions.

[0004] In chimeric antigen receptor therapy, the selection of targets is crucial. For example, in CN119499368A, chimeric antigen receptors are constructed using CLL1, CD33, or CD123 as targets; in JP7621278B2, IL-1RAP is used as a target; and in CN118652336A, CD33 is used as a target. To rationally select AML targets and formulate a suitable targeting strategy for CAR-T therapy, Haubner et al. quantitatively analyzed the expression of surface target proteins in bone marrow and peripheral blood (PB) samples from AML patients and healthy donors. These patients belonged to different genotypes, including AML with relapsed gene abnormalities, TP53 mutations, and myelodysplastic anomalies. They found that CLEC12A distribution was more specific than the commonly used targets CD33 and CD123. CLEC12A levels were not detected in hematopoietic stem cells (HSCs), pluripotent progenitor cells (MPPs), and multi-lymphoid progenitor cells (MLPs), but were highly expressed in AML tumor cells (see Sascha H., Jorge MS, Sarah N., et al. Cooperative CAR targeting to selectively eliminate AML and minimize escape, Cancer Cell. 2023;41(11):1871-1891). Kenderian et al. found that CLEC12A is heterogeneously expressed in AML blast cells. CLEC12A was overexpressed in the bone marrow of AML patients who failed to achieve complete remission after induction chemotherapy, suggesting that it may be a marker of residual disease after chemotherapy. CLEC12A-positive blast cells screened from AML patients were more resistant to chemotherapy than CLEC12A-negative blast cells (see Kenderian S.S., Ruella M., Shestova O., et al. Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can Be Overcome By Targeting with Chimeric Antigen Receptor T Cells, Blood. 2016; 128 (22): 766).

[0005] In chimeric antigen receptor therapy, CAR-NK shows significant advantages over CAR-T in terms of safety, availability of donor sources, and production cost. CAR-NK almost never induces graft-versus-host disease (GvHD). NK cells recognize target cells through a "lack of self" mechanism, independent of TCR-HLA matching, and do not produce GvHD after reinfusion, thus allowing direct use of healthy donors or umbilical cord blood. Cytokine release syndrome (CRS) and neurotoxicity are significantly reduced. CAR-T activation releases large amounts of IL-6, IFN-γ, and TNF-α, leading to ≥ grade 3 CRS or neurotoxicity in 10–50% of patients; early CAR-NK trials (n=30+ cases) did not observe ≥ grade 2 CRS or neurotoxicity events. CAR-NK cells have a moderate duration of in vivo, reducing the risk of long-term bone marrow suppression. CAR-NK cells are generally cleared by the body in 2–4 weeks. Although this may limit the persistence of CAR-NK cells in hyperproliferative leukemia, it reduces late-stage toxicities such as long-term B-cell loss or bone marrow suppression, making it more suitable for sequential multiple infusion strategies (see Peng L., Sferruzza G., Yang L., et al. CAR-T and CAR-NK ascellular cancer immunotherapy for solid tumors, Cell Mol Immunol. 2024; 21(10):1089-1108).

[0006] To this end, we designed and optimized a CAR-NK cell targeting CLEC12A that can effectively recognize and kill AML cells, prevent tumor recurrence, and is both safe and effective. This may reduce production costs and improve the accessibility of immunotherapy. Summary of the Invention

[0007] To address the shortcomings of existing technologies, a first aspect of the present invention provides a chimeric antigen receptor (CAR), characterized in that the CAR comprises an scFv, a hinge region, a transmembrane region, a co-stimulatory factor domain, and an intracellular CD3ζ signaling segment; the scFv can specifically recognize the tumor antigen CLEC12A, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0008] Furthermore, the co-stimulatory factor domain is 4-1BB, having the amino acid sequence shown in SEQ ID NO.3.

[0009] Furthermore, the transmembrane region is a CD8 transmembrane region, having an amino acid sequence as shown in SEQ ID NO.4.

[0010] Furthermore, the hinge region is a CD8 hinge region, having an amino acid sequence as shown in SEQ ID NO.5.

[0011] Furthermore, the amino acid sequence of the CAR is shown in SEQ ID NO.6.

[0012] A second aspect of the invention provides a CAR-NK cell carrying the chimeric antigen receptor CAR.

[0013] A third aspect of the present invention provides a method for preparing the CAR-NK cells, comprising the following steps: preparing a viral vector capable of expressing the chimeric antigen receptor CAR; isolating mononuclear cells from peripheral blood, culturing and screening to obtain NK cells; expanding and culturing the NK cells, transducing the NK cells using the viral vector; passage culturing, collecting the cells, thereby obtaining the CAR-NK cells.

[0014] Furthermore, the viral vector is a lentiviral vector.

[0015] A fourth aspect of the present invention provides the use of the chimeric antigen receptor CAR or the CAR-NK cells described herein in the preparation of antitumor drugs.

[0016] Furthermore, the tumor is acute myeloid leukemia (AML).

[0017] Beneficial effects This invention provides a method for preparing NK cells and their application in tumor treatment, as detailed below: 1. The tumor antigen CLEC12A, which is specific to AML tumor cells, was selected as the target, and the scFv region that can specifically bind to the antigen was screened and prepared. 2. Construct a second-generation chimeric antigen receptor including the anti-CLEC12A scFv region, and use NK cells as the immune vector; 3. Prepare and obtain CAR-NK cells, which can effectively inhibit the growth of tumor cells in vivo and in vitro; 4. The CAR-NK cells can effectively stimulate the immune response in the body and kill tumor cells, and the secretion level of the inflammatory factors is moderate, which can ensure the safety and effectiveness of the treatment. Attached Figure Description

[0018] Figure 1 CAR-NK cells inhibit tumor cell proliferation; Figure 2 Lifespan of animal models; Figure 3 IL-2 expression level; Figure 4 IFN-γ expression level; Figure 5 TNF-α expression level. Detailed Implementation

[0019] Unless otherwise specified, 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.

[0020] Example 1: Preparation of CAR-NK cells 1.1 Structure of chimeric antigen receptor (CAR) This invention utilizes a chimeric antigen receptor based on the structural design of a second-generation CAR. The CAR includes an scFv, a hinge region, a transmembrane region, a co-stimulatory factor domain, and an intracellular CD3ζ signaling segment. The scFv specifically recognizes the tumor antigen CLEC12A, including a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The scFv was prepared by the inventors and exhibits high affinity for the target antigen, with a measured KD value of 5.89 nM.

[0021] The selection of components such as the hinge region, transmembrane region, co-stimulatory factor domain, and CD3ζ intracellular signaling segment is relatively flexible. In this embodiment, mature components commonly used in clinical trials were selected. The co-stimulatory factor domain is 4-1BB, with the amino acid sequence shown in SEQ ID NO.3; the transmembrane region is the CD8 transmembrane region, with the amino acid sequence shown in SEQ ID NO.4; and the hinge region is the CD8 hinge region, with the amino acid sequence shown in SEQ ID NO.5. After connecting these components, the amino acid sequence of the CAR is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.7.

[0022] 1.2 Preparation of Lentiviral Vectors The CAR gene fragment was introduced into the lentiviral plasmid vector pLTR-CMV-MCS, and the sequence was verified to be correct by enzyme digestion and sequencing. 293 T cells were revived and cultured, passaged to activate the cells, and then used when they reached the logarithmic growth phase. The plasmid vector, along with the packaging plasmids pCMV-VSVG and pC-MV-ΔR8.91, were co-transfected into 293 T cells. 24 h after transfection, the medium was replaced with fresh DMEM containing FBS and high glucose. 48 h later, the culture supernatant (virus stock solution) was collected, and fresh DMEM containing FBS and high glucose was added. The virus stock solution was collected again after 72 h. The combined virus stock solutions were concentrated to obtain a concentrated virus solution, and its titer was determined. The titer of the concentrated virus solution was 2.15 × 10⁻⁶. 8 TU / mL.

[0023] 1.3 NK cell preparation Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were obtained by separating the cells using Ficoll density gradient centrifugation after adding human lymphocyte separation medium. The PBMCs were seeded into culture flasks, and cultured in medium containing 450-550 U / ml IL-2 and 10-25 ng / ml IL-7. The flasks were then incubated at 37°C with 5% CO2. Cell growth was monitored daily, and the culture medium was changed as needed. Cells were harvested after 12-14 days of culture. After centrifugation and resuspending, the cell density was adjusted to 1×10⁶ cells / mL. 6 Cells / ml were added, and anti-CD3 and CD56 antibodies were added. CD10 cells were screened using flow cytometry. 3- CD 56+ NK cells.

[0024] 1.4 Preparation of CAR-NK cells Take 500 μL; the cell density is approximately 1 × 10⁻⁶. 6 NK cell suspension was seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Lentiviral vector was added at a multiplicity of infection (MOI) of 50, along with 10 μL each of HitransGA and HitransGP viral enhancement solutions to improve transfection efficiency. After incubation at 37°C with 5% CO2 for 72 h, cells were collected to obtain the CAR-NK cells.

[0025] Example 2: CAR-NK cells inhibit tumor cell growth Using human AML cell line kasumi-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) as target cells, the cells were seeded in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin, and cultured in an incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for subsequent experiments.

[0026] Adjust the density of Kasumi-1 cells and seed them into 96-well plates at 5 × 10⁶ cells per well. 4 Cells were added at effector-to-target ratios of 5:1, 1:1, and 1:5, and the culture volume was increased to 200 μL. Tumor-killing activity was assessed using the LDH assay (LDH kit purchased from Beyotime Biotechnology Co., Ltd.). 100 mL / L LDH dilution buffer was added, and the cells were cultured for another 24 h. After centrifugation at 1000 g for 5 min, 120 μL of the supernatant was mixed with 60 μL of working solution (a mixture of enzyme, lactate, and INT in equal proportions). The mixture was incubated at 37 °C for 30 min, and the absorbance (A value) was measured at 490 nm to calculate the cell lysis rate. Cell lysis rate % = (A... 实验孔 -A 自发释放孔 ) / (A LDH最大释放孔 - A 自发释放孔 ) × 100%.

[0027] like Figure 1 As shown, the CAR-NK cells provided in this invention can effectively kill AML cells and exhibit dose-dependent effects. The higher the effector-to-target ratio, the higher the tumor cell lysis rate. They have a strong specific lysis effect on AML cells, indicating that they can effectively recognize and kill tumor cells.

[0028] Example 3: CAR-NK cells inhibit tumor growth 3.1 Animal model construction and drug administration Eight-week-old NSG mice, half male and half female, were used to establish a mouse model by injecting kasumi-1 cells via the tail vein. Each mouse was injected with 5 × 10⁻⁶ cells. 5 Each cell was counted. Starting from the 3rd day after inoculation, the proportion of tumor cells in peripheral blood was periodically detected by flow cytometry. When the proportion reached or exceeded 0.1%, the model was considered to have been successfully established, and mice with successful modeling were selected for subsequent experiments.

[0029] Thirty mice that successfully developed the CAR-NK model were randomly divided into three groups of 10 mice each: the CAR-NK group, which received a tail vein injection of 2×10⁻⁶ mice. 6 The CAR-NK cells provided by this invention are prepared according to Example 1; NK group, 2×10⁶ cells were injected via tail vein. 6NK cells were prepared as described in Section 1.3 of Example 1; the model group received an equal volume of physiological saline via tail vein injection. Ten normal mice were used as the control group without any treatment.

[0030] 3.2 CAR-NK inhibits tumor growth The survival status of the experimental animals was observed and recorded daily, and survival curves were plotted based on the records. The results are as follows: Figure 2 As shown, immunotherapy can prolong the survival time of mouse models. Among them, the therapeutic effect of NK cells is significantly weaker than that of CAR-NK cells, indicating that NK cells modified in vitro have stronger anti-tumor capabilities.

[0031] 3.3 Peripheral blood count Two weeks after administration, blood samples were collected from mice using the orbital floor vein sampling method. After puncturing the orbital floor venous plexus with a capillary glass tube, 1 mL of blood was collected and placed in a heparin sodium anticoagulant tube. A fully automated blood cell analyzer was used for routine blood tests.

[0032] The results are shown in Table 1. Compared with the normal group, the model group showed a significant increase in white blood cell count and hemoglobin content, while a significant decrease in platelet count. After treatment, the blood count indicators of the experimental animals recovered to some extent. In the CAR-NK group, the white blood cell count decreased, the hemoglobin content also decreased significantly, and the platelet count recovered, which could significantly resist the blood count effects caused by tumor cells.

[0033] Table 1. Detection of peripheral blood counts in experimental animals 3.4 CAR-NK promotes the expression of inflammatory factors Two weeks after administration, peripheral blood was collected from mice, centrifuged at 1000g for 5 minutes, and tested using an ELISA kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The specific method was performed according to the kit instructions.

[0034] IL-2 is an important immunomodulatory factor in the body's immune regulation process. It can strongly induce the proliferation, survival, and expression of granzyme / perforin of tumor-specific CD8⁺ T cells (TST) and NK cells, directly killing tumor cells. However, high doses of IL-2 can stimulate endothelial cells to produce secondary cytokines (such as TNF-α and IL-1β), triggering vascular leakage syndrome and thus providing a pathway for tumor metastasis. Therefore, it has a double-edged sword effect. In this invention, as... Figure 3 As shown, the level of IL-2 in mice increased after tumor modeling, indicating that tumor modeling triggered an autoimmune response. The level of IL-2 tended to increase further after treatment with NK or CAR-NK cells, but it was generally within a controllable range and could effectively mediate the tumor apoptosis process.

[0035] IFN-γ is also an important anti-tumor factor that can directly inhibit proliferation or induce apoptosis, enhance immune recognition, activate innate immunity, and inhibit angiogenesis. It can suppress tumor growth through multiple immune regulatory mechanisms. In this invention, such as... Figure 4 As shown, CAR-NK cell therapy can significantly increase the secretion level of IFN-γ, thereby effectively inhibiting tumor growth.

[0036] TNF-α is a type II transmembrane glycoprotein that can activate immune cells such as macrophages, T cells, NK cells, neutrophils, and mast cells in vivo, thereby activating the NF-κB pathway and exerting anti-tumor effects. In this invention, such as... Figure 5 As shown, both NK cell injection and CAR-NK cell injection can effectively promote TNF-α secretion, but there seems to be no significant difference between the two. This may be related to the process of tumor development and progression, and further detection and research can be conducted at different time points.

[0037] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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. A chimeric antigen receptor CAR, characterized in that, The CAR includes scFv, hinge region, transmembrane region, costimulatory factor domain and CD3ζ intracellular signaling segment; the scFv can specifically recognize tumor antigen CLEC12A, including heavy chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The chimeric antigen receptor CAR according to claim 1, characterized in that, The co-stimulatory factor domain is 4-1BB and has the amino acid sequence shown in SEQ ID NO.

3.

3. The chimeric antigen receptor CAR according to claim 2, characterized in that, The transmembrane region is a CD8 transmembrane region, having the amino acid sequence shown in SEQ ID NO.

4.

4. The chimeric antigen receptor CAR according to claim 3, characterized in that, The hinge region is a CD8 hinge region, having an amino acid sequence as shown in SEQ ID NO.

5.

5. The chimeric antigen receptor CAR according to claim 4, characterized in that, The amino acid sequence of the CAR is shown in SEQ ID NO.

6.

6. A CAR-NK cell, said cell carrying the chimeric antigen receptor CAR as described in any one of claims 1-5.

7. A method for preparing CAR-NK cells as described in claim 6, characterized in that, The method includes the following steps: preparing a viral vector capable of expressing the chimeric antigen receptor CAR as described in any one of claims 1-5; isolating mononuclear cells from peripheral blood, culturing and screening to obtain NK cells; expanding and culturing the NK cells, transducing the NK cells using the viral vector; passage culturing, collecting the cells, and thus obtaining the CAR-NK cells.

8. The preparation method according to claim 7, characterized in that, The viral vector is a lentiviral vector.

9. The use of the chimeric antigen receptor CAR according to any one of claims 1-5, or the CAR-NK cell according to claim 6, in the preparation of an antitumor drug, wherein the tumor is acute myeloid leukemia (AML).

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