CAR-NK (chimeric antigen receptor-natural killer) cell and application thereof in preparation of medicine for treating tumors

Abstract

The invention belongs to the technical field of biology, and particularly relates to a CAR-NK cell and application of the CAR-NK cell in preparation of a medicine for treating tumors. 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 as shown in SEQ ID NO: 5. The HER2 CAR-NK cell disclosed by the invention has the effect of specifically cracking and killing HER2 positive tumors, can inhibit tumor growth, and has a very good application prospect in treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a CAR-NK cell and its application in the preparation of anti-tumor drugs. Background Art

[0002] Tumor, as a global health problem, seriously threatens human life 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 introduce a chimeric antigen receptor (CAR) that specifically recognizes tumor antigens into T cells through genetic engineering technology, enabling them to accurately recognize and kill tumor cells, showing significant therapeutic effects.

[0003] However, CAR-T cell therapy is not without limitations. First, the preparation process of CAR-T cells is complex and costly, restricting its wide application. Second, CAR-T cells may cause serious side effects during treatment, such as cytokine release syndrome (CRS) and neurotoxicity, threatening the lives of patients. In addition, the persistence of CAR-T cells is poor, which may lead to tumor recurrence. Moreover, for certain 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. As the natural killers of the immune system, NK cells have strong anti-tumor activity and fewer side effects. By introducing CAR into NK cells, CAR-NK cells can be constructed, which not only have the natural killing ability of NK cells but also the ability to specifically recognize tumor antigens by CAR. CAR-NK cells are not only relatively simple to prepare and have lower costs but also have fewer side effects, better persistence, and can effectively penetrate into tumor tissues, showing broader application prospects and more superior therapeutic potential. Therefore, the research and application of CAR-NK cells have become one of the important directions in the field of current tumor immunotherapy.

[0005] Breast cancer, as one of the most common malignant tumors in women globally, seriously threatens women's health and life. Although the treatment methods for breast cancer have been continuously improving in recent years, there are still some patients facing the risk of recurrence and metastasis. Especially those HER2-positive breast cancer patients, due to the overexpression of HER2 receptors on the surface of their tumor cells, the tumors are more invasive and drug-resistant. By introducing a chimeric antigen receptor (CAR) that specifically recognizes HER2 into NK cells through genetic engineering technology, the constructed HER2-targeted CAR-NK cells 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 cells targeting Her2 constructed in the present invention provide a new and potentially more effective treatment option for HER2-positive breast cancer patients, and are expected to improve the prognosis and quality of life of patients, becoming an important research direction in the field of breast cancer immunotherapy. Summary of the Invention

[0007] The present invention provides a chimeric antigen receptor that specifically binds to Her2. The chimeric antigen receptor includes a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory 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 as shown in SEQ ID NO: 5.

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

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

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

[0011] Preferably, the co-stimulatory domain consists of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain. Preferably, the amino acid sequence of the 4-1BB co-stimulatory domain is as shown in SEQ ID NO: 10.

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

[0013] Preferably, the intracellular signaling domain is the CD3ζ signaling domain, and its amino acid sequence is as shown in SEQ ID NO: 10.

[0014] In a preferred embodiment of the present invention, the present invention provides an expression vector, and the expression vector contains a nucleic acid molecule encoding the chimeric antigen receptor of the present invention.

[0015] In another preferred embodiment of the present invention, the present invention provides a CAR-NK cell, and the NK cell contains the expression vector of the present invention.

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

[0017] The present invention uses HER2 as a target for CAR-NK cell therapy 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 invention have the effect of specifically lysing and killing HER2-positive tumors, can inhibit tumor growth, and the effect is better than that of other CAR-NK cells targeting HER2 in the prior art. In addition, the present invention also finds that truncating the CD28 co-stimulatory domain can enhance the transfection rate, and has good application prospects in the treatment of breast cancer.

[0018] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Brief Description of the Drawings

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

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

[0021] Figure 3 is a bar chart of the killing rate of the CAR-NK cells prepared by the present invention against target cells.

[0022] Figure 4 is a tumor growth curve graph of the CAR-NK cells prepared by the present invention after being used in a mouse tumor model. Detailed Embodiments

[0023] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

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

[0025] To construct a CAR molecule targeting Her2, pUC57 was used as a vector, and the element sequence was: signal peptide - Her2ScFv - CD8 hinge region - CD28 transmembrane region - CD28 co-stimulatory region - 4-1BB co-stimulatory region - CD3ζ signal transduction region. After the Her2-CAR molecule was constructed, the positive clone transformants were sequenced, and the sequencing results were compared with the target gene. The comparison results showed a consistency of 100%, indicating that the overexpression of the Her2-CAR molecule was successfully constructed.

[0026] Among them, 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 Her2ScFv 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 light chain of Her2 ScFv are connected by a linker peptide, and its amino acid sequence 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 co-stimulatory region is shown in SEQ ID NO: 8:

[0039]

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

[0041]

[0042] The amino acid sequence of the 4-1BB co-stimulatory 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] The Her2-targeting CAR molecule 1 was constructed according to the above steps. Its structure is signal peptide-Her2ScFv-CD8 hinge region-CD28 transmembrane region-CD28 costimulatory region-4-1BB costimulatory region-CD3ζ signaling region, and its amino acid sequence is shown in SEQ ID NO: 12:

[0047]

[0048] According to the above steps, a Her2-targeting CAR molecule 2 was constructed. Its structure is signal peptide-Her2ScFv-CD8 hinge region-CD28 transmembrane region-CD28 costimulatory region (truncated)-4-1BB costimulatory region-CD3ζ signaling region, and its amino acid sequence is shown in 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 the coding genes of the Her2-CAR molecules 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. The digested products were recovered, and the vector fragments and the CAR gene were connected with T4 DNA ligase; the ligation product (pELNS-Her2-CAR) was transferred into competent cells for amplification, and the plasmid was extracted for enzyme digestion identification and sequencing identification.

[0053] (2) 24 hours before transfection, use about 8×10 6 Seed 293T cells into a 15 cm dish. Ensure that the cells are approximately 80% confluent and evenly distributed in the dish at the time of transfection.

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

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

[0056] (5) After 7 hours, discard the transfection medium and replace it with fresh DMEM complete medium. 48 hours after transfection, aspirate the culture medium containing viral particles, filter it with a 0.45 μm filter, and store it at 4°C.

[0057] Concentration of lentivirus:

[0058] Place the filtered virus stock solution in Beckman ultra-high-speed centrifuge tubes, ensuring strict balancing of each centrifuge tube with an error not exceeding ±0.001 g; the centrifugation conditions are: 4 °C, 70,000 g, 2 h; after centrifugation, discard the supernatant, and invert the centrifuge tubes on sterilized filter paper to absorb the remaining supernatant. Resuspend the virus pellet with serum-free and antibiotic-free DMEM medium, aliquot the resuspended virus concentrate, and store it in a -80 °C refrigerator.

[0059] Preparation of CAR-NK cells:

[0060] Adjust the density of NK-92 cells to 2 - 3×10 5 / mL, add the virus vector at a volume ratio (V / V) of virus vector: cell culture medium (RPMI 1640) = 1:6 - 10, and simultaneously add 8 μg / mL of polybrene. After 4 h, supplement an equal volume of fresh complete medium to adjust the cell density to 1×10 5 / mL and continue culturing. The next day, centrifuge all the cells, add fresh medium, and continue culturing. Replenish the liquid every 1 - 2 days to maintain the cell density at 2 - 3×10 5 / mL. After 72 h, perform CAR antibody staining, and simultaneously sort Her2-CAR NK-92 positive cells by flow cytometry and expand the culture.

[0061] Use flow cytometry to detect the transfection rate of CAR NK-92 cells. The results of flow cytometry for detecting the cell transfection rate are as Figure 2 shown. The transfection rate of Her2-CAR1 is 75.16%, and the transfection rate of Her2-CAR2 is 89.25%. It can be seen that truncation of the CD28 co-stimulatory domain can effectively improve the transfection rate of the Her2-CAR vector constructed in the present invention.

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

[0063] Seed CAR-NK cells (5×10 5 cells / well) and target cells SKBR-3 (1×10 5 cells / well) into 96-well plates, 100 μL / well, and divide them into three groups. The specific grouping is as follows: Experimental group A: Co-culture HER2-CAR1-NK cells with SKBR-3 cells; Experimental group B: Co-culture HER2-CAR2-NK cells with SKBR-3 cells; Control group: Co-culture NK cells with SKBR-3 cells.

[0064] Co-culture was carried out in an incubator with 5% CO2 at 37°C. After 24 hours, 20 μL of CCK-8 was added to each well. After continued incubation for 2 hours, the absorbance at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the OD value was read to calculate the cell killing rate. As Figure 3 shown, the truncation of CD28 co-stimulation did not affect its killing of cancer cells.

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

[0066] Male C57BL / 6J mice weighing 18 - 22 g were housed in an animal room (room temperature 23 ± 2°C, humidity 50% ± 10%). Breast cancer cells SKBR-3 in the logarithmic growth phase were collected and diluted to 2×10 5 cells / mL with phosphate-buffered saline (PBS) to obtain a SKBR-3 cell suspension. Under sterile conditions, 0.2 mL of the SKBR-3 cell suspension was inoculated into the left axilla of the mice. The appearance of a hard nodule the size of a soybean in the axilla was used as the standard for successful modeling, which was counted as day 0. The length and width of the tumor were measured with a vernier caliper, and the tumor volume was calculated (about 400 mm 3 3), and the tumor volume (mm3) = length × width 2 × 0.5.

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

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

[0069] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative labor. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the existing technology should be within the protection scope determined 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 signaling domain, wherein the extracellular antigen-binding domain targets Her2; the amino acid sequence of the HER2 antigen-binding domain is as shown in SEQ ID NO:

5.

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

1.

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

6.

4. The chimeric antigen receptor according to claim 1, wherein The transmembrane domain is a CD28 transmembrane domain, and its amino acid sequence is as shown in SEQ ID NO:

7.

5. The chimeric antigen receptor according to claim 1, wherein, The co-stimulatory domain consists 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 as shown in SEQ ID NO:

10.

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

9.

7. The chimeric antigen receptor according to claim 1, wherein The intracellular signaling domain is a CD3ζ signaling domain, and its amino acid sequence is as shown in SEQ ID NO:

10.

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

9. A CAR-NK cell, characterized in that, The NK cell contains the expression vector according to 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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