A car-nk cell and application thereof in preparation of a drug for treating tumors

Abstract

The application belongs to the technical field of biology and particularly relates to a CAR-NK cell and application thereof in preparation of a tumor treatment drug. The CAR comprises a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and an intracellular signal transduction domain, wherein the extracellular antigen binding domain targets Her2; the amino acid sequence of the HER2 antigen binding domain is shown as SEQ ID NO: 5. The HER2 CAR-NK cell of the application has the effect of specifically lysing and killing HER2 positive tumors and can inhibit tumor growth, and has a good application prospect in the treatment of breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a CAR-NK cell and application thereof in preparation of a tumor treatment drug. BACKGROUND

[0002] Tumor is a global health problem that seriously threatens human life and safety. With the in-depth medical research, the emergence of immunotherapy, especially CAR-T cell therapy, has brought revolutionary progress to tumor treatment. CAR-T cells, through genetic engineering technology, introduce a chimeric antigen receptor (CAR) that specifically recognizes tumor antigens into T cells, enabling them to accurately recognize and kill tumor cells, and showing significant therapeutic effect.

[0003] However, CAR-T cell therapy is not without limitations. First, the preparation process of CAR-T cells is complex and costly, limiting its widespread application. Second, CAR-T cells may cause severe side effects during treatment, such as cytokine release syndrome (CRS) and neurotoxicity, threatening patient life. In addition, the persistence of CAR-T cells is poor, which may lead to tumor recurrence. Also, for some types of tumors, CAR-T cells may not be able to effectively penetrate, affecting the treatment effect.

[0004] To overcome these limitations of CAR-T cells, researchers have begun to explore the use of natural killer (NK) cells to construct CAR-NK cells. NK cells, as natural killers of the immune system, have strong anti-tumor activity and fewer side effects. By introducing CAR into NK cells, CAR-NK cells can be constructed that not only have the natural killing ability of NK cells, but also have the ability to specifically recognize tumor antigens. CAR-NK cells not only have relatively simple preparation and lower cost, but also have fewer side effects, better persistence, and can effectively penetrate into tumor tissues, showing more extensive application prospects and superior therapeutic potential. Therefore, the research and application of CAR-NK cells have become one of the important directions in the field of tumor immunotherapy.

[0005] Breast cancer, as one of the most common malignant tumors in women worldwide, seriously threatens women's health and life. Although the treatment of breast cancer has made progress in recent years, some patients still face the risk of recurrence and metastasis, especially those with HER2-positive breast cancer, whose tumor cells overexpress HER2 receptors on the surface, making the tumor more invasive and drug-resistant. By introducing a chimeric antigen receptor (CAR) that specifically recognizes HER2 into NK cells through genetic engineering technology, CAR-NK cells targeting Her2 can not only retain the natural killing activity of NK cells, but also enhance their targeting and killing ability against HER2-positive breast cancer cells.

[0006] The CAR-NK cell targeting Her2 provides a new and potentially more effective treatment option for HER2-positive breast cancer patients, and is expected to improve the prognosis and quality of life of patients, and become an important research direction in the field of breast cancer immunotherapy. SUMMARY

[0007] The application provides a chimeric antigen receptor specifically binding to Her2, comprising a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and an intracellular signaling domain, wherein the extracellular antigen binding domain targets Her2; the amino acid sequence of the HER2 antigen binding domain is shown as SEQ ID NO: 5.

[0008] Preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 1.

[0009] Preferably, the hinge region is a CD8 hinge region, and the amino acid sequence thereof is shown as SEQ ID NO: 6.

[0010] Preferably, the transmembrane domain is a CD28 transmembrane domain, and the amino acid sequence thereof is shown as SEQ ID NO: 7.

[0011] Preferably, the costimulatory domain is composed of a CD28 costimulatory domain and a 4-1BB costimulatory domain, and preferably, the amino acid sequence of the 4-1BB costimulatory domain is shown as SEQ ID NO: 10.

[0012] Preferably, the amino acid sequence of the CD28 costimulatory domain is shown as SEQ ID NO: 8 or SEQ ID NO: 9.

[0013] Preferably, the intracellular signaling domain is a CD3 zeta signaling domain, and the amino acid sequence thereof is shown as SEQ ID NO: 10.

[0014] In a preferred embodiment of the application, the application provides an expression vector containing a nucleic acid molecule encoding the chimeric antigen receptor of the application.

[0015] In another preferred embodiment of the application, the application provides a CAR-NK cell containing the expression vector of the application.

[0016] In still another preferred embodiment of the application, the application provides the use of the chimeric antigen receptor of the application and / or the CAR-NK cell of the application in the preparation of a medicament for treating breast cancer.

[0017] The present application takes HER2 as a target point of CAR-NK cell treatment of solid tumors, constructs a specific CAR molecule targeting HER2, and prepares CAR-NK cells (HER2 CAR-NK). The HER2 CAR-NK cells of the present application have the effect of specifically lysing and killing HER2 positive tumors, can inhibit tumor growth, and the effect is better than other HER2 targeted CAR-NK cells in the prior art. In addition, it is found that truncation of the CD28 co-stimulatory domain can enhance the transfection rate, and has good application prospect in the treatment of breast cancer.

[0018] The concept, specific structure and technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a structural schematic diagram of the CAR prepared by the present application.

[0020] Figure 2 is a column chart of the transfection rate of the CAR lentiviral vector transfected NK cells prepared by the present application.

[0021] Figure 3 is a column chart of the target cell killing rate of the CAR-NK cells prepared by the present application.

[0022] Figure 4 is a tumor growth curve diagram of the CAR-NK cells prepared by the present application after being used for a mouse tumor model. DETAILED DESCRIPTION

[0023] The present application will be described in detail below in combination with the drawings and specific examples, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0024] Example 1 Construction of Chimeric Antigen Receptor Targeting Her2

[0025] To construct the CAR molecule targeting Her2, pUC57 is used as the vector, and the element order is: signal peptide-Her2 ScFv-CD8 hinge region-CD28 transmembrane region-CD28 co-stimulatory region-4-1BB co-stimulatory region-CD3ζ signaling region. After the Her2-CAR molecule is constructed, the positive clone transformant is sequenced, and the sequencing result is compared with the target gene. The comparison result shows that the consistency is 100%, indicating that the overexpression Her2-CAR molecule is successfully constructed.

[0026] The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1: MALPVTALLLPLALLLHAARP. The amino acid sequence of the heavy chain of Her2 ScFv is shown in SEQ ID NO: 2:

[0027]

[0028] The amino acid sequence of the light chain of Her2 ScFv is shown in SEQ ID NO: 3:

[0029]

[0030] The heavy chain and the light chain of Her2 ScFv are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 4:

[0031]

[0032] The amino acid sequence of Her2 ScFv is shown in SEQ ID NO: 5:

[0033]

[0034] The amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 6:

[0035]

[0036] The amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 7:

[0037]

[0038] The amino acid sequence of the CD28 costimulatory region is shown in SEQ ID NO: 8:

[0039]

[0040] The amino acid sequence of the CD28 costimulatory region is shown in SEQ ID NO: 9:

[0041]

[0042] The amino acid sequence of the 4-1BB costimulatory region is shown in SEQ ID NO: 10:

[0043]

[0044] The amino acid sequence of the CD3ζ signaling region is shown in SEQ ID NO: 11:

[0045]

[0046] CAR molecule 1 targeting Her2 was constructed according to the above steps, which has the structure of signal peptide-Her2 ScFv-CD8 hinge region-CD28 transmembrane region-CD28 costimulatory region-4-1BB costimulatory region-CD3 zeta signaling region, and its amino acid sequence is shown as SEQ ID NO: 12:

[0047]

[0048] CAR molecule 2 targeting Her2 was constructed according to the above steps, which has the structure of signal peptide-Her2 ScFv-CD8 hinge region-CD28 transmembrane region-CD28 costimulatory region (truncated)-4-1BB costimulatory region-CD3 zeta signaling region, and its amino acid sequence is shown as SEQ ID NO: 13:

[0049]

[0050] Example 2 Preparation of CAR-NK cells

[0051] Preparation of lentiviral expression vector:

[0052] (1) The two pUC57 vectors containing Her2-CAR molecule coding genes constructed in Example 1 were double digested with Nhe I and Xho I, and the lentiviral vector plasmid pELNS was double digested with Nhe I and Xho I, then the digested products were recovered, and the vector fragments and CAR genes were ligated with T4 DNA ligase; the ligation product (pELNS-Her2-CAR) was transformed into competent cells for amplification, and the plasmid was extracted for enzyme digestion and sequencing identification.

[0053] (2) 24 hours before transfection, about 8x10 6 The 293T cells were inoculated into a 15 cm culture dish. Ensure that the cells are about 80% confluent and evenly distributed in the culture dish at the time of transfection.

[0054] (3) 60 min before transfection, replace the DMEM culture medium (10% FBS, 1% HEPES, without double antibiotics).

[0055] (4) Add 4.5 mL of 0.1 mol / L CaCl2 solution to a 50 mL centrifuge tube, add pELNS-Her2-CAR, and transfect, then place the cells in a 37°C, 5% CO2 incubator for culture.

[0056] (5) After 7 hours, discard the transfection culture medium and replace it with fresh DMEM complete medium. After 48 hours of transfection, the culture medium containing virus particles was aspirated, filtered with a 0.45 μm filter, and stored at 4°C.

[0057] Concentration of lentivirus:

[0058] The filtered virus stock solution was placed in a Beckman ultracentrifuge tube, and each centrifuge tube was strictly balanced, with an error of not more than ±0.001 g; the centrifugation conditions were: 4°C, 70000 g, 2 h; after centrifugation, the supernatant was discarded, and the centrifuge tube was inverted on a sterile filter paper to absorb the remaining supernatant. The virus precipitate was resuspended in serum-free and antibiotic-free DMEM medium, and the resuspended virus concentrate was aliquoted and stored in a -80°C refrigerator.

[0059] Preparation of CAR-NK cells:

[0060] The density of NK-92 cells was adjusted to 2-3×10 5 / mL, and the virus vector was added at a ratio of virus vector: cell culture medium (RPMI 1640) = 1:6-10 (V / V), and polybrene was added at 8 μg / mL. After 4 h, the cell density was adjusted to 1×10 5 / mL by adding an equal amount of fresh complete medium, and the cells were cultured. The next day, all the cells were centrifuged, fresh medium was added, and the cells were cultured. The medium was replaced every 1-2 days to maintain the cell density at 2-3×10 5 / mL. After 72 h, CAR antibody staining was performed, and Her2-CAR NK-92 positive cells were sorted by flow cytometry and expanded in culture.

[0061] The transfection rate of CAR NK-92 cells was detected by flow cytometry, and the results of flow cytometry detection of cell transfection rate are shown in Figure 2 Table 1. The transfection rate of Her2-CAR1 was 75.16%, and the transfection rate of Her2-CAR2 was 89.25%. It can be seen that the truncation of the CD28 costimulatory domain can effectively improve the transfection rate of the Her2-CAR vector constructed in the application.

[0062] Example 3: In vitro killing experiment of CAR-NK cells

[0063] CAR-NK cells (5×10 5 cells / well) were inoculated with target cells SKBR-3 (1×10 5 cells / well) in a 96-well plate, 100 μL / well, and divided into three groups, as follows: experimental group A: HER2-CAR1-NK cells were co-cultured with SKBR-3 cells; experimental group B: HER2-CAR2-NK cells were co-cultured with SKBR-3 cells; control group: NK cells were co-cultured with SKBR-3 cells.

[0064] The cells were co-cultured in a 5% CO2, 37°C incubator. After 24 hours, 20 mL of CCK-8 was added to each well, and incubation continued for another 2 hours. Cells were then measured at 450 nm using a microplate reader to read the OD value and calculate the cell killing rate. Figure 3 As shown, truncating CD28 co-stimulation did not affect its killing effect on cancer cells.

[0065] Example 4: Inhibitory effect of CAR-NK cells on tumors in vivo

[0066] 18-22g male C57BL / 6J mice were housed in the animal room (room temperature 23±2℃, humidity 50%±10%). SKBR-3 breast cancer cells in the logarithmic growth phase were collected and diluted to 2×10⁻⁶ cells / mL with phosphate-buffered saline (PBS). 5 SKBR-3 cell suspension was obtained by inoculating 0.2 mL of the suspension into the left axilla of mice under aseptic conditions. The appearance of a firm nodule the size of a soybean in the axilla was considered a successful modeling criterion and marked as day 0. The length and width of the tumor were measured using calipers, and the tumor volume (approximately 400 mm²) was calculated. 3 Tumor volume (mm3) = length × width 2 ×0.5.

[0067] C57BL / 6J breast cancer model mice were randomly divided into 3 groups of 5 mice each, as follows: Experimental group A: injected intravenously with 2×10 6 HER2-CAR1-NK cells / animal; Experimental group B: 2×10 HER2-CAR1-NK cells injected via tail vein. 6 HER2-CAR2-NK cells / animal; Control group: 2×10 HER2-CAR2-NK cells injected via tail vein. 6 NK cells / animal; injected via tail vein on days 0 and 7, and tumor volume was measured on days 0, 7, 14, 21, 28 and 35, and tumor growth curves were plotted using the tumor volume.

[0068] like Figure 4 As shown, tumors in experimental groups A and B grew slowly, indicating that the CAR-NK cells in the experimental groups had a significant inhibitory effect on tumors.

[0069] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A chimeric antigen receptor that specifically binds to Her2, the chimeric antigen receptor comprising a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain targets Her2; the amino acid sequence of the HER2 antigen-binding domain is shown in SEQ ID NO:

5.

2. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO:

1.

3. The chimeric antigen receptor according to claim 1, characterized in that, The hinge region is a CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO:

6.

4. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequence of the transmembrane domain is shown in SEQ ID NO:

7.

5. The chimeric antigen receptor according to claim 1, characterized in that, The co-stimulatory domain is composed of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:

10.

6. The chimeric antigen receptor according to claim 5, characterized in that, The amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID NO: 8 or SEQ ID NO:

9.

7. The chimeric antigen receptor according to claim 1, characterized in that, The intracellular signal transduction domain is the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO:

11.

8. An expression carrier, characterized in that, The expression vector contains a nucleic acid molecule encoding the chimeric antigen receptor as described in any one of claims 1-7.

9. A CAR-NK cell, characterized in that, The NK cells contain the expression vector as described in claim 8.

10. Use of the chimeric antigen receptor according to any one of claims 1-7 and / or the CAR-NK cell according to claim 9 in the preparation of a medicament for treating breast cancer.

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