NK cell specific chimeric antigen receptor and application thereof

Abstract

The invention provides an NK cell specific chimeric antigen receptor and application thereof, and belongs to the technical field of immunotherapy. The invention provides a specific chimeric antigen receptor of an NK cell. The specific chimeric antigen receptor comprises an NKG2D transmembrane segment, a 2B4 intracellular segment structural domain and an FCER1G intracellular segment structural domain which are connected in sequence. The invention also discloses a coding gene of the specific chimeric antigen receptor, an expression vector containing the coding gene, a gene editing immune cell and application of the gene editing immune cell in preparation of medicines for preventing and / or treating tumors. According to the present invention, cell coupling formation, particle polarization, degranulation, in vitro killing experiments and cytokine secretion experiments prove that the specific chimeric antigen receptor has good functional performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and in particular relates to a NK cell-specific chimeric antigen receptor and an application thereof. Background Art

[0002] In recent years, chimeric antigen receptor T cells (CAR-T) have made great achievements in the treatment of hematological malignancies. However, CAR-T cells still face a series of challenges in clinical applications. For example, they show very low efficacy in the treatment of solid tumors; the immune escape mechanism of tumors through the loss of antigens causes off-target effects and drug resistance in CAR-T cell therapy. CAR-T cells require autologous adoptive cell transplantation, otherwise allogeneic T cells may cause graft-versus-host disease (GVHD); they are prone to adverse reactions such as cytokine storms and neurotoxicity, which endanger the patient's life. The latest studies have shown that CAR-NK cells may overcome the above-mentioned defects of CAR-T cells and show significant anti-tumor effects.

[0003] Unlike T cells, NK cells do not express a main recognition receptor, but express a series of inhibitory receptors and activating receptors. The state of NK cells depends on the balance of inhibitory and activating signals. Inhibitory receptors mainly include killer cell immunoglobulin-like receptors (KIRs) and NKG2A, which bind to MHC-I molecules to form immune tolerance and prevent NK cells from attacking their own healthy tissues. Activating receptors can be divided into three categories. The first type of receptors non-covalently bind to adapter molecules containing immunoreceptor tyrosine-based activation motifs (ITAMs), including CD16, NKp44, NKp46, NKp30, NKG2C and partially activated KIRs; the second type of receptors do not transmit activation signals through ITAMs, including the lymphocytic activation molecule (SLAM)-related receptor family represented by 2B4, NKG2D, and DNAM-1; the third type is adhesion molecules represented by LFA-1. Previous studies have shown that, for NK cells in a resting state, except for CD16, no single receptor can mediate NK cell degranulation to exert cytotoxic effects alone. For NK cells activated by IL-2, most activating receptors such as 2B4, NKG2D, and LFA-1 can mediate NK cell degranulation alone, and there is a synergistic effect between different activating receptors of NK cells.

[0004] Chimeric antigen receptors consist of three parts: an extracellular segment, a transmembrane segment, and an intracellular segment. The extracellular segment is usually a single-chain variable region fragment (scFv) of a monoclonal antibody that recognizes the target. The transmembrane segment connects the extracellular segment and the intracellular segment. The intracellular segment includes a co-stimulatory domain and a domain that transmits activation signals. CAR-NK refers to loading chimeric antigen receptors on NK cells. At present, the design of CAR-NK mainly follows the structure of CAR-T, that is, the second-generation CAR molecule that uses the intracellular segment of CD28 / 4-1BB as a co-stimulatory molecule and CD3ζ to transmit activation signals. Although its effectiveness has been verified in preclinical models and clinical trials, the activation mode and biological characteristics of NK cells are very different from those of T cells, and it is not suitable to directly use the CAR-T method to design and activate CAR-NK cells.

[0005] A deep understanding of the activation mode of immune cells is of great significance to the design of CAR molecules. For T cells, the T cell dual signal activation theory consisting of the first signal transmitted by the TCR-CD3 complex and the second signal transmitted by CD28 has established the core position of the CD28 / 4-1BB-CD3ζ second-generation CAR molecule in CAR-T. As mentioned above, for NK cells in a resting state, CD16 is the only receptor that can activate NK cells alone. Therefore, understanding the signal transduction mechanism of CD16 is crucial for designing NK cell-specific CAR molecules. CD16 recognizes the constant chain of immunoglobulin IgG and mediates ADCC (antibody-dependent cellular cytotoxicity) of NK cells. The intracellular segment of CD16 itself does not have a signal transduction function and needs to transmit activation signals through non-covalent pairing with ζζ or γγ homodimers or ζγ heterodimers. The γ subunit refers to the high-affinity immunoglobulin ε receptor subunit γ (FCER1γ), which is a component of the high-affinity immunoglobulin E receptor. Although co-transfection experiments have shown that either γ or ζ chain can support the expression of CD16, and ζζ, γγ and ζγ dimers can also be detected in the co-transfection system, under physiological conditions, CD16 prefers to pair with γγ homodimers, so compared with ζ chain, γ chain may be more suitable for CAR-NK. In fact, studies have shown that CAR-NK with γ chain as the intracellular segment has a stronger killing effect than ζ chain. In addition, there is a synergistic effect between different activation receptors of NK cells. For example, 2B4 can play a synergistic role by lowering the threshold of CD16-mediated degranulation. NKG2D and CD16 also have a synergistic effect on NK cell calcium ion influx and degranulation. 2B4 and NKG2D have a synergistic degranulation effect, but their performance in killing is average. Therefore, designing CAR molecules according to the activation mode of NK cells is expected to obtain more efficient CAR-NK. Summary of the invention

[0006] The present invention provides a NK cell-specific chimeric antigen receptor and application thereof. The specific chimeric antigen receptor is more suitable for NK cell activation and has a better ability to kill target cells.

[0007] The present invention provides a specific chimeric antigen receptor of NK cells, comprising a NKG2D transmembrane segment, a 2B4 intracellular segment domain and a FCER1G intracellular segment domain which are connected in sequence.

[0008] In a specific embodiment of the present invention, the amino acid sequence of the NKG2D transmembrane segment is shown as SEQ ID No.1; the amino acid sequence of the 2B4 intracellular segment domain is shown as SEQ ID No.2; and the amino acid sequence of the FCER1G intracellular segment domain is shown as SEQ ID No.3.

[0009] In a specific embodiment of the present invention, the amino acid sequence of the specific chimeric antigen receptor is shown in SEQ ID No.4.

[0010] The present invention provides a nucleic acid molecule, comprising a gene encoding the above-mentioned specific chimeric antigen receptor.

[0011] In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.5.

[0012] The present invention provides an expression vector carrying the above nucleic acid molecule.

[0013] The present invention provides a transgenic immune cell, comprising a cell expressing the above-mentioned specific chimeric antigen receptor, or carrying the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0014] The present invention also provides a method for constructing the above-mentioned transgenic immune cells, comprising the following steps: introducing the above-mentioned expression vector into immune cells to obtain the transgenic immune cells.

[0015] In a specific embodiment of the present invention, the immune cells include NK cells.

[0016] The present invention also provides the use of the above-mentioned specific chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, and the above-mentioned transgenic immune cell in the preparation of drugs for preventing and / or treating tumors.

[0017] Beneficial effects: The present invention provides a specific chimeric antigen receptor for NK cells, comprising a NKG2D transmembrane segment, a 2B4 intracellular segment domain and a FCER1G intracellular segment domain connected in sequence. Figure 1The NK cell-specific chimeric antigen receptor shown is: NKG2D (TM) -2B4-FCER1G, NKG2D is a type II transmembrane protein, to ensure the directionality of the chimeric antigen receptor, the amino acid sequence of the NKG2D transmembrane segment (TM) is inverted for connection, the 2B4 intracellular segment domain is used as a co-stimulatory sequence, and the FCER1G intracellular segment domain is used as a signal transduction sequence. In the embodiments of the present invention, cell coupling formation, particle polarization, degranulation, in vitro killing experiments and cytokine secretion experiments prove that the specific chimeric antigen receptor of the present invention has good functional performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a connection diagram of the NK cell-specific chimeric antigen receptor CAR4;

[0019] Figure 2 Design a schematic diagram for the CAR molecule and flow cytometry to verify the expression results of the CAR molecule;

[0020] Figure 3 The figure shows the results of B16 cells overexpressing B7H3 specifically promoting the expression of CAR-NK activation marker CD69;

[0021] Figure 4 The figure shows the result of B16 cells overexpressing B7H3 specifically coupling with CAR-NK cells;

[0022] Figure 5 This is the scattered distribution result of perforin in Mock or CAR-NK cells in the resting state;

[0023] Figure 6 This is a result diagram showing the specific promotion of CAR-NK particle polarization by B16 cells overexpressing B7H3. The left side of the figure shows only three representative groups of NK cell particle polarization result diagrams;

[0024] Figure 7 This is a result diagram showing that B16 cells overexpressing B7H3 specifically promote CAR-NK degranulation. The left side of the figure shows only three representative groups of NK cell flow cytometry results;

[0025] Figure 8 This is a graph showing that B16 cells overexpressing B7H3 can be specifically killed by CAR-NK;

[0026] Fig. 9 The results of NK and CAR-NK cells killing glioma stem cells are shown in Figure 2.

[0027] Fig.10 The figure shows the results of B16 cells overexpressing B7H3 promoting CAR-NK to secrete cytokines. DETAILED DESCRIPTION

[0028] The present invention provides a specific chimeric antigen receptor of NK cells, comprising a NKG2D transmembrane segment, a 2B4 intracellular segment domain and a FCER1G intracellular segment domain which are connected in sequence.

[0029] In a specific embodiment of the present invention, the amino acid sequence of the NKG2D transmembrane segment is as shown in SEQ ID No.1: AVMIIFRIGMAVAIFCCFFFP; the amino acid sequence of the 2B4 intracellular segment domain is as shown in SEQ ID No.2: WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQ SSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARL SRKELENFDVYS; the amino acid sequence of the intracellular segment domain of FCER1G is as shown in SEQ ID No.3: RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ.

[0030] In the present invention, NKG2D is a type II transmembrane protein. In order to ensure the directionality of the chimeric antigen receptor, the amino acid sequence of the NKG2D transmembrane segment (TM) is inverted for connection, the 2B4 intracellular segment domain is used as a co-stimulatory sequence, and the intracellular segment domain of FCER1G is used as a signal transduction sequence. Therefore, in a specific embodiment of the present invention, the amino acid sequence of the specific chimeric antigen receptor is as shown in SEQ ID No.4: AVMIIFRIGMAVAIFCCFFFPWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ.

[0031] The present invention provides a nucleic acid molecule, comprising a gene encoding the above-mentioned specific chimeric antigen receptor.

[0032] In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 5. The present invention can also perform codon optimization on the nucleic acid molecule, or perform mutation on one or more sites of the nucleic acid molecule, and obtain the amino acid sequence shown in SEQ ID No. 4 or a protein having the same function as the sequence shown in SEQ ID No. 4.

[0033] The present invention provides an expression vector carrying the above nucleic acid molecule.

[0034] The expression vector of the present invention can be a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector. In the embodiment, a plasmid vector is used as an example for illustration, and the sequence shown in SEQ ID No. 5 is inserted into the NheI / XhoI site of the plasmid vector pIRES-EGFP-puro (Addgene, #45567).

[0035] The present invention provides a transgenic immune cell, comprising a cell expressing the above-mentioned specific chimeric antigen receptor, or carrying the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0036] The immune cells of the transgenic immune cells of the present invention are NK cells.

[0037] The present invention also provides a method for constructing the above-mentioned transgenic immune cells, comprising the following steps: introducing the above-mentioned expression vector into immune cells to obtain the transgenic immune cells.

[0038] In a specific embodiment of the present invention, the immune cells include NK cells.

[0039] The present invention also provides the use of the above-mentioned specific chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, and the above-mentioned transgenic immune cell in the preparation of drugs for preventing and / or treating tumors.

[0040] The present invention designs a NK cell-specific CAR molecule combination based on the biological characteristics and activation mode of NK cells. Compared with traditional CAR molecules, the specific chimeric antigen receptor of the present invention is more suitable for NK cell activation and has a better ability to kill target cells. And based on the specific chimeric antigen receptor, CAR-NK targeting different targets can be constructed for cellular immunotherapy of solid tumors or lymphomas.

[0041] In order to further illustrate the present invention, a NK cell-specific chimeric antigen receptor and its application provided by the present invention are described in detail below in combination with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Sequence synthesis and vector construction: Gene sequence codon optimization was performed using GenScript codon optimization software (https: / / www.genscript.com.cn / tools / gensmart-codon-optimization), and the optimized gene sequence (SEQ ID No. 5) was sent to Anshengda for gene synthesis.

[0044] use MaxDNA Polymerase (Takara, Code No. R045Q) PCR was used to obtain the relevant target gene fragment, which was then double-digested with NheI / XhoI and connected to the plasmid vector pIRES-EGFP-puro (Addgene, #45567). After the CAR molecular screening plasmid was constructed, the plasmid was extracted using a midiprep kit (ZymoPURE II Plasmid Midiprep Kit Cat #D4201) for later use.

[0045] 2. CAR molecule electroporation: Take NK-92MI cells with good growth status, collect cells by centrifugation, resuspend cells with celetrix special electroporation solution 103buffer (partA: partB = 1:1), add CAR molecular screening plasmid to electroporate cells. Electroporation conditions: 100μL electroporation tube, 10M cells, 8μg CAR molecular screening plasmid, voltage cell line 430V, electroporation solution 103buffer, electroporator model Celetrix Cell Electroporator Model CTX-1500ALE+. The electroporation operation was carried out according to the celetrix electroporator manual. After electroporation, the cells were transferred to 1 well of a 6-well plate for culture, and 3ml NK-92MI cell special culture medium (Procell, CAT#CM-0533) was added to each well for culture.

[0046] 3. Screening of NK-92MI cells stably transfected with CAR molecules: After electroporation, the cells were transferred to 6-well plates, grown in a 37°C incubator for 3 days, and then cultured in separate wells. The cells in a single well of the 6-well plate were about 80% filled, and 2.0 μg / ml puromycin was added for screening. After two weeks, flow cytometry was used to identify the positive rate.

[0047] 4. In order to compare the effectiveness of the chimeric antigen receptor sequence specific to NK cells described in the present invention, a total of 9 CAR molecules and negative controls (Mock) of the same vector were designed. The extracellular segments were all single-chain variable fragments of monoclonal antibodies targeting B7H3 protein, and the intracellular segments used 2B4 or DAP10 or CD28 as costimulatory molecules, and FCER1G or CD3ζ chains to transmit activation signals. To facilitate subsequent functional experiments, each CAR molecule carried green fluorescent protein GFP. CAR-NK targeting B7H3 was constructed by an electroporation instrument (celetrix), and the double positivity of GFP and CAR molecules was detected by flow cytometry to prove the successful construction of CAR-NK ( Figure 2 ).

[0048] CAR1(SEQ ID No.6):AVMIIFRIGMAVAIFCCFFFPRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ;

[0049] CAR2 (SEQ ID No. 7):AVMIIFRIGMAVAIFCCFFFPRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

[0050] CAR3(SEQ ID No.8):AVMIIFRIGMAVAIFCCFFFPWRRKRKEKQSETSP KEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYS LIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS;

[0051] CAR4(SEQ ID No.4):AVMIIFRIGMAVAIFCCFFFPWRRKRKEKQSETS PKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLY SLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSR LKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ;

[0052] CAR5(SEQ ID No.9):AVMIIFRIGMAVAIFCCFFFPWRRKRKEKQSETSP KEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

[0053] CAR6(SEQ ID No.10):FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

[0054] CAR7 (SEQ ID No. 11):AVMIIFRIGMAVAIFCCFFFPLCARPRRSPAQEDG KVYINMPGRG;

[0055] CAR8(SEQ ID No.12):AVMIIFRIGMAVAIFCCFFFPLCARPRRSPAQEDG KVYINMPGRGRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPP Q;

[0056] CAR9 (SEQ ID No. 13):AVMIIFRIGMAVAIFCCFFFPLCARPRRSPAQEDG KVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR.

[0057] 5. The present invention constructs a B16 cell line overexpressing human B7H3 protein by lentiviral transfection, specifically: a stable cell line is constructed by lentiviral infection, the viral vector number is pHS-B-0085 (Beijing Hesheng Biotechnology Co., Ltd.), the element sequence is pLV-EF1a-hluc-P2A-Puro-CMV-MCS-3flag (Beijing Hesheng Biotechnology Co., Ltd.), and plasmid synthesis and virus packaging are completed by Beijing Hesheng Biotechnology Co., Ltd. The ratio of virus to cell number (MOI) during infection is 20, and after 72 hours of transfection, it is screened by 2.0 μg / ml puromycin, and the expression of the target protein is verified by flow cytometry for two weeks.

[0058] NK and CAR-NK cells themselves hardly express CD69, and B16 co-culture can hardly promote NK cells or CAR-NK to express CD69. However, B16 cells overexpressing human B7H3 protein can effectively promote CAR-NK to express the activation marker CD69 ( Figure 3 ), among which the CD69 of CAR4 increased most significantly.

[0059] 6.NK cells mainly release perforin and granzyme to kill target cells through steps such as cell coupling formation, particle polarization, and degranulation. Cell coupling formation refers to the formation of a stable connection between NK cells and ligands on the surface of target cells through surface-activated receptors. Cell coupling formation is the basis of particle polarization and degranulation. The most commonly used evaluation method for cell coupling formation is dual fluorescence flow cytometry detection. Mock and CAR-NK cells themselves carry green fluorescence, so the present invention uses live cell fluorescent dyes (AAT Bioquest, 22622, PE channel) to dye B16 cells overexpressing human B7H3 protein red, and after co-culture, the tissue fixative is fixed and flow-cytometry is detected. The red and green double positives represent cell coupling formation.

[0060] The representative results of three groups of NK cells are shown in Figure 4 As shown, Mock or CAR-NK cells rarely form cell coupling with B16 cells, while CAR-NK can efficiently form cell coupling with B16 cells expressing human B7H3 protein, among which CAR4 forms the most cell coupling with B16 cells expressing human B7H3 protein.

[0061] 7. Granule polarization refers to the aggregation of perforins and granzymes dispersed in the cytoplasm at the interface between NK cells and target cells after receiving activation signals. Granule polarization can mark the activation of NK cells and also prepare for efficient killing of target cells.

[0062] Mock or CAR-NK cells were directly fixed with tissue fixative, membrane permeabilized, and blocked, and then stained with fluorescent group-labeled perforin antibody (Biolegend, 308124). Under a confocal microscope, it can be clearly seen that the green cells are NK cells, and the perforin is red. The perforin in all NK cells is scattered ( Figure 5 ).

[0063] 8. After co-culture of Mock or CAR-NK with B16 cells overexpressing human B7H3 protein, the cells were fixed with tissue fixative, membrane permeabilized, blocked, and stained with fluorescent group-labeled perforin antibody (Biolegend, 308124). Under confocal microscopy, it can be clearly seen that the perforin of NK cells in the Mock group is still scattered after co-culture without particle polarization, while CAR-NK has different degrees of particle polarization, especially CAR4 ( Figure 6 ), 116 out of 129 NK cells underwent granule polarization.

[0064] 9. Degranulation refers to the process in which NK cells release perforin and granzyme in the cytoplasm after receiving activation signals. Degranulation more directly marks the activation of NK cells and is also the main means for NK cells to kill target cells. Currently, the most commonly used marker to evaluate degranulation is CD107a. After co-culture of Mock or CAR-NK with B16 cells overexpressing human B7H3 protein, they were fixed with tissue fixative and stained with CD107a antibody labeled with fluorescent group (Biolegend, 328620). Degranulation was observed by flow cytometry. The results showed that B16 cells could not promote the degranulation of Mock or CAR-NK cells, while B16 cells overexpressing human B7H3 protein could significantly increase the degranulation of CAR-NK, especially CAR4 ( Figure 7 ).

[0065] 10. In vitro killing experiment, B16 cells and B16 stable cells overexpressing B7H3 were co-cultured with Mock cells and CAR-NK cells respectively. Figure 8 As shown in the figure, neither the Mock group NK cells nor the CAR-NK cells can kill B16, while CAR-NK can efficiently kill B16 cells overexpressing human B7H3 protein, especially CAR4.

[0066] 11. To further verify the killing advantage of CAR4 in vitro, the present invention also conducted in vitro killing experiments in three brain glioma stem cell lines (BNI19-1-S, BNI 2-4-S, BNI 1-3-S). First, it was verified that all three brain glioma stem cells expressed the B7H3 target ( Fig. 9 ), after 4 hours of in vitro co-culture Fig. 9 As shown in the figure, although both Mock and CAR-NK cells can kill target cells, CAR4 has the best killing effect overall.

[0067] 12. In addition to directly killing target cells, cytokine secretion is also an important function of NK cells. After activation, NK cells can efficiently secrete cytokines to exert immunomodulatory effects. Next, the present invention explores the production of cytokines after co-culture of CAR-NK with B16 cells overexpressing human B7H3 protein.

[0068] The results are as follows Fig.10 As shown, both Mock group NK cells and CAR-NK cells could not secrete cytokines after co-culture with B16 cells, while CAR4 could efficiently secrete cytokines after co-culture with B16 cells overexpressing human B7H3 protein.

[0069] The cell coupling formation, particle polarization, degranulation, in vitro killing experiment and cytokine secretion experiment proved that the CAR4 (NKG2D (TM) -2B4-FCER1G) of the present invention has the best functional performance.

[0070] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A specific chimeric antigen receptor for NK cells, characterized in that: It includes the NKG2D transmembrane segment, the 2B4 intracellular segment domain and the FCER1G intracellular segment domain connected in sequence.

2. The specific chimeric antigen receptor according to claim 1, characterized in that: The amino acid sequence of the NKG2D transmembrane segment is shown in SEQ ID No.1; the amino acid sequence of the 2B4 intracellular segment domain is shown in SEQ ID No.2; and the amino acid sequence of the FCER1G intracellular segment domain is shown in SEQ ID No.

3.

3. The specific chimeric antigen receptor according to claim 1 or 2, characterized in that: The amino acid sequence of the specific chimeric antigen receptor is shown in SEQ ID No.

4.

4. A nucleic acid molecule, characterized in that It comprises a gene encoding the specific chimeric antigen receptor according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.

5.

6. An expression vector, characterized in that: Carrying the nucleic acid molecule according to claim 4 or 5.

7. A transgenic immune cell, characterized in that: The invention comprises expressing the specific chimeric antigen receptor according to any one of claims 1 to 3, or carrying the nucleic acid molecule according to claim 4 or 5 or the expression vector according to claim 6.

8. The method for constructing transgenic immune cells according to claim 7, characterized in that: The method comprises the following steps: introducing the expression vector according to claim 6 into immune cells to obtain the transgenic immune cells.

9. The construction method according to claim 8, characterized in that: The immune cells include NK cells.

10. Use of the specific chimeric antigen receptor according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the expression vector according to claim 6, or the transgenic immune cell according to claim 7 in the preparation of a drug for preventing and / or treating tumors.

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