CN114958770A

CN114958770A - Chimeric antigen receptor NK cell and preparation method thereof, cell medicine and application thereof

Info

Publication number: CN114958770A
Application number: CN202210678490.5A
Authority: CN
Inventors: 江文正; 郭丹丹
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-08-30

Abstract

The invention discloses a chimeric antigen receptor NK cell, a preparation method thereof and application of cell medicine, and relates to the technical field of CAR-NK cell treatment. The NK cells of the invention express a chimeric antigen receptor that targets a tumor associated antigen and in which the GPR116 gene is knocked out or its expression is inhibited. Compared with the original CAR-NK cell, the chimeric antigen receptor NK cell has stronger tumor cell killing capacity, and provides a new direction and thought for tumor treatment.

Description

Chimeric antigen receptor NK cell and preparation method thereof, cell medicine and application thereof

Technical Field

The invention relates to the technical field of CAR-NK cell therapy, and particularly relates to a chimeric antigen receptor NK cell, a preparation method thereof and a cell drug.

Background

Cancer is now the second leading cause of death worldwide, and about one sixth of all deaths worldwide are caused by cancer. Among all cancers, Pancreatic Cancer (PC) is called "king of cancer", and is extremely high in mortality and mortality, with a very low relative survival rate of less than 9% for 5 years. In recent years, with the development of medical technology, more and more treatment methods are applied to the treatment of pancreatic cancer, such as surgery, radiotherapy, chemotherapy, and immunotherapy. Cellular immunotherapy is one of the most advanced methods for treating cancer and infectious diseases at present, and particularly, Chimeric antigen receptor T cell therapy (CAR-T) brings new benefits to cancer patients. However, CAR-T cell therapy has certain problems, including Cytokine Release Syndrome (CRS), Graft Versus Host Disease (GVHD), and the production cycle of chimeric antigen receptor T cells (CAR-T). Furthermore, the high cost and expense of chimeric antigen receptor T cell (CAR-T) therapy also makes it burdensome for the average household. NK cells are considered to have the same potential to enhance the anti-tumor capacity of the NK cells through chimeric antigen receptor modification due to the advantages of the NK cells such as a special target cell recognition mechanism, a short culture period and wide tumor killing capacity.

The NKG2D (Natural killer group 2, member D) receptor belongs to the NKG2 family and is a type II transmembrane glycoprotein. The NKG2D receptor is an important activating receptor expressed on the surface of various immune cells, such as Natural killer (Natural killer NK) cells, NKT cells, gamma delta T cells, CD8 ⁺ T cells, and the like. When bound to a ligand, can initiate an immune response including an antibacterial immune response, immune surveillance, anti-tumor effects, and the like. Notably, NKG2D ligand is highly expressed in a variety of tumor cells, but is not generally expressed or is underexpressed in normal tissues or cells. We have therefore designed the NKG2D receptor into a CAR structure which, when bound to a ligand, activates NK cells and generates a series of anti-tumor responses.

The G protein-coupled receptors (GPCRs) are receptors with seven transmembrane structures and play important roles in various physiological reactions of organisms. The G-protein coupled receptor GPR116 belongs to a member of the family of adhesive G-protein coupled receptors, has a long N-terminal and an extracellular domain with C-like immunoglobulin repeats and SEA domains. It has been shown that the structural features of GPR116 are very similar to those of the LN-7TM receptor and may be involved in the regulation of the immune response as well as the LN-7TM receptor. The existing CAR-NK cells have the problem of poor effect of treating solid tumors and need to be solved.

Disclosure of Invention

The invention aims to provide a novel chimeric antigen receptor NK cell, a construction method of the chimeric antigen receptor NK cell and application of the chimeric antigen receptor NK cell in cell medicine. The effector CAR-NK cell of the chimeric antigen receptor NK cell can release stronger cell effector factors and has better tumor cell killing capacity.

The invention discloses a method for knocking down the expression of a GPR116 gene in CAR-NK cells by applying RNA interference for the first time, and aims to increase the proportion of effector CAR-NK cells and enhance the release of CAR-NK cell effector factors, thereby enhancing the killing capacity of the CAR-NK cells on tumor cells. The interference sequence of GPR116 is added into the structure of NKG2D-CAR to enhance the function of NKG 2D-CAR-NK.

The present invention provides a chimeric antigen receptor NK cell which expresses a chimeric antigen receptor targeting a tumor-associated antigen and whose GPR116 gene is knocked out or its expression is suppressed.

The invention innovatively provides that the expression of GPR116 is inhibited in CAR-NK cells for the first time, the anti-tumor capacity of the CAR-NK cells is obviously improved, and the invention provides a solid theoretical basis and a wide application prospect for applying the CAR-NK cells to treat tumors. The invention also provides that the inhibition or knockout of the expression of the GPR116 gene in CAR-NK cells can also achieve a remarkable good effect.

In the present invention, the expression of the GPR116 gene is inhibited by a combination of any one or more of the following molecules, including but not limited to: shRNA, antisense RNA, siRNA and antagomir; alternatively, the GPR116 gene of the above chimeric antigen receptor NK cell is knocked out by any one of methods including, but not limited to, the following: CRISPR/Cas9 technology, ZFN technology, and TALEN technology.

The present invention provides, based on the contents of the embodiments of the present invention, that the chimeric antigen receptor NK cell is modified by using techniques including but not limited to those common in the art, so as to inhibit the expression of GPR116 gene, or knock out GPR116 gene, and regardless of the technique, the chimeric antigen receptor NK cell obtained by any technique, as long as the GPR116 gene is inhibited from expressing or knocked out, is within the protection scope of the present invention.

In specific embodiments, the chimeric antigen receptor NK cells contain shRNA molecules by which expression of the GPR116 gene is inhibited.

In a specific embodiment, the target sequence of the shRNA molecule is shown as SEQ ID NO. 1. The embodiment of the invention shows that by utilizing the shRNA molecule to target a target sequence on the GPR116 gene as shown in SEQ ID No.1, the GPR116 gene shows a suppressed effect, and corresponding chimeric antigen receptor NK cells all have the expression of improving the anti-tumor capacity.

Preferably, the nucleotide sequence of the shRNA molecule is shown as SEQ ID NO. 13: GCAGUCGGAUUCGUCUAUUGU are provided.

In particular embodiments, the tumor-associated antigen includes, but is not limited to, a ligand selected from NKG 2D. In particular embodiments, the antigen binding domain of the chimeric antigen receptor is capable of specifically binding the tumor associated antigen.

It should be noted that, based on the disclosure of the present invention, those skilled in the art can select suitable tumor-associated antigens, and any selected tumor-associated antigen is within the scope of the present invention.

In a specific embodiment, when the tumor-associated antigen is a NKG2D ligand, the antigen-binding domain of the chimeric antigen receptor is a NKG2D protein, or a fragment with binding activity selected from the group consisting of NKG2D proteins.

In a specific embodiment, the tumor-associated antigen is a NKG2D ligand and the antigen-binding domain is an extracellular domain selected from the group consisting of NKG2D proteins.

In a specific embodiment, the amino acid sequence of the extracellular domain of the NKG2D protein is shown in SEQ ID No. 16.

In specific embodiments, the NKG2D ligand is selected from any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP 4, ULBP 5, and ULBP 6.

The immunological features of NKG2D and its ligands have been strongly correlated with tumors. The ligand for NKG2D includes six members: MICA, MICB, ULBP1-6, but most normal cells of the body generally do not express or express low-level ligands, and in some solid tumors, such as pancreatic cancer, prostate cancer, liver cancer cell, rectal cancer cell, gastric cancer cell, breast cancer cell and the like, NKG2D ligands are expressed to different degrees, and NKG2D receptor is designed into a CAR structure, and when the ligand is combined, NK cells are activated to generate a series of anti-tumor responses.

In particular embodiments, the chimeric antigen receptor further has a transmembrane domain and a costimulatory signaling region; the transmembrane domain is selected from: a transmembrane domain of one or more of CD8, CD28, CD33, CD37, CD8a, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154, CD19, CD45, CD4, and CD3 epsilon.

In a specific embodiment, the transmembrane domain is selected from the transmembrane domain of CD 8.

In particular embodiments, the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of: one or more of CD27, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79B, CD66d, CD2, CD4, CD5, CD28, CD30, CD40, CD134, CD137, ICOS, CD154, 4-1BB, OX40, CD7, LIGHT, NKG2C, and B7-H3.

In a specific embodiment, the co-stimulatory signaling region comprises the intracellular domain of 4-1BB and the intracellular domain of CD3 ζ.

The chimeric antigen receptor NK cells include but are not limited to NKG2D-CAR-NK cells.

The present invention also provides a method of making a chimeric antigen receptor NK cell as described in any one of the preceding claims, comprising the steps of: inhibiting expression of a GPR116 gene of the chimeric antigen receptor NK cell or knocking out a GPR116 gene of the chimeric antigen receptor NK cell.

It should be noted that, no matter what method is adopted to prepare the chimeric antigen receptor NK cell of the present invention, it is within the protection scope of the present invention.

The method can enhance the release of CAR-NK cell effector factors, and is beneficial to enhancing the killing capacity of CAR-NK cells to tumor cells.

The invention also provides a cell medicament which contains the chimeric antigen receptor NK cell as an active ingredient and pharmaceutically acceptable auxiliary materials.

The invention also provides an interference agent or inhibitor of GPR116 gene expression, which can interfere or inhibit GPR116 gene expression; the interference agent or inhibitor is any one or combination of shRNA, antisense RNA, siRNA and/or antagomir molecules.

The invention also provides the chimeric antigen receptor NK cell, the cell medicine, the application of the interference agent or the inhibitor in preparing a medicine for treating tumor diseases and the application in CAR-NK treatment.

In a specific embodiment, the cellular drug is used to treat a tumor.

In specific embodiments, the tumor is selected from a solid tumor or a non-solid tumor.

Based on the disclosure of the present invention, one skilled in the art can easily think that the chimeric antigen receptor NK cells of the present invention can be applied to the treatment of various tumors, not only solid tumors, but also non-solid tumors, and any tumors are within the scope of the present invention.

In a specific embodiment, the solid tumor is a tumor that specifically expresses NKG2D ligand.

In a specific embodiment, the solid tumor is selected from any one of pancreatic cancer, prostate cancer, liver cancer, rectal cancer, stomach cancer, and breast cancer.

According to the invention, the anti-tumor capability of the NKG2D-CAR-NK cell is improved by constructing the NKG2D-CAR-NK cell targeting the solid tumor and co-expressing an interference gene GPR116 capable of improving the anti-tumor capability of the CAR-NK cell; the NKG2D-CAR-NK cells target the treatment of pancreatic cancer; and/or, promoting NK92 cell killing ability by down-regulating GPR116 gene in NK92 cells; and/or, enhance the anti-tumor function of NKG2D-CAR-NK cells by interfering with GPR116 expression.

In the invention, the expression of GPR116 gene is knocked down in CAR-NK cells by an RNA interference method, and the expression of GPR116 gene is inhibited by any one or combination of shRNA, antisense RNA, siRNA and/or antagomir contained in the chimeric antigen receptor NK cells.

Chimeric antigen receptor T cell therapy, CAR-T therapy, has achieved good efficacy in the treatment of hematological neoplasms, and also faces significant challenges, mainly CRS, GVHD and manufacturing difficulties. Compared with CAR-T, CAR-NK has unique advantages, such as no host transplantation resistance, short production period, strong killing ability, low production cost, etc., and is gradually a new therapeutic method. According to the invention, through constructing the NKG2D-CAR-NK cell targeting the solid tumor and co-expressing the interference gene GPR116 capable of improving the anti-tumor capability of the CAR-NK cell, the result shows that the anti-tumor capability of the NKG2D-CAR-NK cell can be obviously and effectively improved after the interference of GPR116, and the researches provide a new direction and thought for treating the solid tumor.

The invention discloses that the GPR116 gene is down-regulated in NK92 cells for the first time, and the killing capability of NK92 cells can be effectively promoted. Meanwhile, the cell therapy of NKG2D-CAR-NK targeting pancreatic cancer cells is disclosed for the first time, and shRNA-GPR116 is also applied to CAR-NK therapy for the first time. In the research, in-vitro experiments prove that NKG2D-CAR-NK can effectively treat pancreatic cancer in a targeted manner, and meanwhile, the research proves that the anti-tumor function of NKG2D-CAR-NK cells can be effectively improved after GPR116 interference, so that a new direction is provided for the CAR-NK to be applied to solid tumor treatment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows that the expression of GPR116 receptor is down-regulated in NK92 cells, and the result shows that the NK92 cells promote the capability of NK92 cells to kill K562 after being interfered.

FIG. 2 is a graph showing the ability of GPR116 receptor deletion to promote NK cells to inhibit pancreatic cancer growth. The left graph shows the size of the tumor, and the right graph shows the weight of the tumor.

FIG. 3 is a schematic diagram of the structure of the partial expression elements of three plasmids, pLL3.7-NKG2D-CAR, pLL3.7-NC-NKG2D-CAR and pLL3.7-shRNA-NKG 2D-CAR.

FIG. 4 shows the results of double restriction with Nhe I and Xba I of the recombinant plasmids pLL3.7-NKG2D-CAR, pLL3.7-shRNA-A/B/C-NKG2D-CAR and pLL3.7-shRNA-NC-NKG 2D-CAR.

FIG. 5 shows the results of Q-PCR (left panel) and RT-PCR (right panel) identification of GPR116 gene in NK92 cells infected with lentivirus expressing shRNA-A/B/C-NKG2D-CAR and shRNA-NC-NKG 2D-CAR.

FIG. 6 is a result of efficiency of killing pancreatic cancer cells by shRNA-NC-NKG2D-CAR-NK92, shRNA-A-NKG2D-CAR-NK92 and NK92 cells infected with control virus. shRNA-A-NKG2D-CAR-NK92 cells kill more efficiently.

FIG. 7 is a fluorescent statistical graph of shRNA-NC-NKG2D-CAR-NK92, shRNA-A-NKG2D-CAR-NK92 and NK92 cell-killed pancreatic cancer infected with control virus.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Construction of pLL3.7-shRNA-GPR 116 interference vector

1RNAi target sequence design

The gene sequence number of Homo sapiens GPR116(ADGRF5)) is found in the NCBI website: 001098518.2. GPR116 gene RNAi target sequences were designed at the website (https:// rnaidesigner. thermofisher. com/rnainexpress/design. do) according to gene numbers, and the results are shown in Table 1.

TABLE 1 GPR116 Gene RNAi target sequences

Target sequence name	Starting position	Nucleotide sequence of RNAi target sequence	GC％	SEQ ID NO.
					Target sequence A	2673	GCAGTCGGATTCGTCTATTGT	52.1	SEQ ID NO.1

2 designing interference sequence according to target sequence:

based on the selected target sequence, the interference sequence is designed and determined by reference to the following principle: the 5 end is started with G, and the content of G + C is set to be 30-50%. According to the requirements of the pLL3.7 vector: (1) t is added at the 5' end of the sense strand to reconstruct T at the l position of the U6 promoter. (2) The Loop "TTCAAGAGAGA" is added after the interference target sequence. (3) The inverted complement and the termination signal "TTTTTT" are added. (4) An EcoR I cleavage site GAATTC is added at the 3' end to facilitate identification. (5) Then filling in Xho I enzyme cutting site to synthesize a pair of complementary fragments.

The sequence was scrambled and designed into NC (negative control) sequences, each of which is shown in Table 2 below.

TABLE 2 oligonucleotide sequences designed separately for target and negative control sequences

3 construction of targeting plasmid pLL3.7-shRNA-NKG2D-CAR

The sequence of the extracellular segment gene of the NKG2D protein was found by https:// www.ncbi.nlm.nih.gov/pubmed/and https:// www.uniprot.org/website and then the sequence of the CD8a signal peptide was added. The extracellular segment sequence of Sig-NKG2D obtained by RT-PCR amplification is shown as SEQ ID NO. 6:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGATGTTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG；

the amino acid sequence is as follows (SEQ ID NO. 16):

MALPVTALLLPLALLLHAARPMLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV。

the sigNKG2DEX sequence was ligated together with the secondary CAR sequence using overlap PCR. After sequencing and identification, the interference sequence of the inserted GPR116 is determined to be successfully constructed.

Example 2

Amplification and viral packaging of pLL3.7-NKG2D-CAR, pLL3.7-shRNA-NKG2D-CAR, and pLL3.7-shRNA-NC-NKG2D-CAR plasmids.

1 plasmid transfection

1) The plasmid, PEI and Opti-MEM medium was removed from the refrigerator and returned to room temperature;

2) uniformly mixing the plasmid/Opti-MEM and PEI/Opti-MEM culture mediums, and standing at room temperature for 5 min;

3) after standing, mixing the two solutions, vibrating the two solutions on a vortex oscillator until the two solutions are completely and uniformly mixed, and standing the solution at room temperature for 20 min;

4) slowly and dropwise adding 1ml of DNA/PEI mixture into a 293T culture dish, incubating in an incubator at 37 ℃, replacing a fresh culture medium after 6-8h, and continuously incubating in the incubator at 37 ℃.

2 Virus Collection and concentration

1) Collecting supernatants 48h and 72h after plasmid transfection;

2) centrifuging at 4 deg.C for 10min at 4000g, and filtering the supernatant with 0.45 μm filter;

3) transferring the filtered virus supernatant into an ultracentrifuge tube, centrifuging for 2h at 25000 r, subpackaging the virus liquid into 1.5ml EP tubes, and storing at-80 ℃.

3 viral titer assay

1) The density of the medium is 2X 10 in a 24-well culture plate ⁵ 293T cells per ml;

2) adding 0.1,0.5 and 1ul of virus concentrated solution into the 24-hole culture plate, continuously culturing for 16h, and then replacing a fresh culture medium;

3) after 48 hours, detecting the target gene expression of the infected cells in a flow mode;

4) the titer, titer 2 × 10, was calculated ⁵ X infection efficiency x 1000/dilution factor.

Example 3

pLL3.7-shRNA-NKG2D-CAR vector interference validation test

NK92 cells preserved in liquid nitrogen were removed, rapidly thawed and centrifuged at 800rpm/min for 5 minutes. The cell pellet was suspended with α -MEM in a complete medium and seeded in 24-well plates for culture. The recovered NK92 cells were divided into 3 groups: a virus infection-free negative control group, a LV-shRNA-NC-NKG2D-CAR group and a LV-shRNA-A-NKG2D-CAR lentivirus interference group, 2 wells in each group. The two experimental groups were each spiked with the corresponding volume of virus at MOI 10:1 and spiked with polybrene 10. mu.g/ml to facilitate infection. After 24h, the cells were collected, centrifuged at 1000g for 10min, the medium was discarded and fresh medium was added.

And collecting cells infected by lentivirus for 48h, extracting total RNA respectively, and detecting the expression of GPR116 gene by using Q-PCR and RT-PCR. As shown in fig. 5, the expression of GPR116 in the interfering group was significantly reduced compared to the control group, wherein the effect of shRNA-a-NKG2D-CAR group was more significant.

Example 4

Selection of target cells and CAR-NK killing function study

1, the pancreatic cancer cell line PANC28 is found to highly express NKG2D ligand in the early-stage research of a laboratory, so that PANC28 is selected as a target cell for the research of the subsequent experiments.

2 Mixed culture of target cells and effector cells

Respectively processing pancreatic cancer cell strain PANC28 according to 4 × 10 ⁴ The number of the wells is inoculated into a 96-well plate for culturing the ultra-low adsorption cells; inoculating shRNA-NC-NKG2D-CAR-NK92, shRNA-A-NKG2D-CAR-NK92 and NK92 cells infected with control viruses into target cells according to effective target ratios of 1:1, 2.5:1 and 5:1 respectively; the killing efficiency of the product is detected in a flow mode after 4 hours.

3 in vitro killing efficiency analysis

As shown in FIG. 6, both shRNA-NC-NKG2D-CAR-NK92 and shRNA-A-NKG2D-CAR-NK92 have a strong effect of killing pancreatic cancer, compared with NK 92. Compared with shRNA-NC-NKG2D-CAR-NK92, shRNA-A-NKG2D-CAR-NK92 has stronger killing efficiency, and the experiment shows that NKG2D can well target pancreatic cancer cell strains to play a role, and interference of GPR116 can enhance the killing efficiency of NKG2D-CAR-NK 92.

4 in vivo antitumor Effect analysis

As shown in FIG. 7, compared with NK92, both shRNA-NC-NKG2D-CAR-NK92 and shRNA-A-NKG2D-CAR-NK92 have better anti-tumor effect. And compared with shRNA-NC-NKG2D-CAR-NK92, shRNA-A-NKG2D-CAR-NK92 has stronger anti-tumor effect, and the experiment shows that NKG2D-CAR-NK92 has good anti-pancreatic cancer effect, and interference of GPR116 can enhance the anti-tumor effect of NKG2D-CAR-NK 92.

Comparative example

Different control interfering sequences were designed for a number of different target sequences and correlation experiments were performed. The experimental method was the same as the aforementioned method.

The target sequences of each proportion are shown in Table 3, the upstream and downstream segments of the interference sequences corresponding to the target sequences are shown in Table 4, wherein shRNA-A is the original sequence of the embodiment 1 of the invention.

TABLE 3 RNAi target sequences of the GPR116 gene

Examples	Name of target sequence	Starting position	Nucleotide sequence of RNAi target sequence	GC％	SEQ ID NO.
						Example 1	Target sequence A	2673	GCAGTCGGATTCGTCTATTGT	52.1	1
Comparative example C1	Target sequence B	1961	GCCCATCTATGAAGCTGAATC	52.39	7
						Comparative example C2	Target sequenceC	1175	GCAGTCAGGGTAATGTTAATT	47.62	8

TABLE 4 oligonucleotide sequences designed separately for target and negative control sequences

The RNA sequence of the shRNA targeting target sequence a is: GCAGUCGGAUUCGUCUAUUGU (SEQ ID NO. 13).

The RNA sequence of the shRNA targeting target sequence B is: GCCCAUCUAUGAAGCUGAAUC (SEQ ID NO. 14).

The RNA sequence of the shRNA targeting target sequence C is: GCAGUCAGGGUAAUGUUAAUU (SEQ ID NO. 15).

The same method as the above vector construction is used, after the interfering shRNA is designed, an interfering sequence DNA double strand is synthesized by a company and is connected to the pLL3.7 vector, the Nhe I and XbaI double enzyme digestion identification is carried out, and the result is shown in FIG. 4.

Lentivirus packaging was performed using the same method.

The results in FIG. 5 show that, from the expression of GPR116 mRNA, the expression level of GPR116 mRNA in sh-GPR116-A interfering group is lower than that in sh-GPR116-B and sh-GPR116-C interfering groups in a plurality of interfering RNAs, so that sh-GPR116-A fragment has the optimal interference effect. The results of the above experiments are unexpected to those skilled in the art.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met

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Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly

100 105 110

Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly

115 120 125

Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys

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Claims

1. A chimeric antigen receptor NK cell, wherein said chimeric antigen receptor NK cell is a chimeric antigen receptor NK cell whose expression is targeted to a tumor associated antigen and whose GPR116 gene is knocked out or its expression is suppressed.

2. The chimeric antigen receptor NK cell according to claim 1, characterized in that the expression of GPR116 gene is inhibited by any one or a combination of several of the following molecules: shRNA, antisense RNA, siRNA and/or antagomir; alternatively, the GPR116 gene of the chimeric antigen receptor NK cell is knocked out by any one of the following techniques: CRISPR/Cas9 technology, ZFN technology, and/or TALEN technology.

3. The chimeric antigen receptor NK cell according to claim 1, characterized in that it contains an shRNA molecule by which the expression of the GPR116 gene is inhibited.

4. The chimeric antigen receptor NK cell according to claim 3, characterized in that the target sequence of the shRNA molecule is shown in SEQ ID No. 1.

5. The chimeric antigen receptor NK cell according to claim 4, wherein the nucleotide sequence of the shRNA molecule is shown in SEQ ID No. 13.

6. The chimeric antigen receptor NK cell of claim 1, wherein said tumor-associated antigen is selected from the group consisting of NKG2D ligand; the antigen binding domain of the chimeric antigen receptor is capable of specifically binding the tumor associated antigen.

7. The chimeric antigen receptor NK cell according to claim 1, characterized in that, when the tumor-associated antigen is NKG2D ligand, the antigen binding domain of the chimeric antigen receptor is NKG2D protein or a fragment with binding activity selected from NKG2D protein.

8. The chimeric antigen receptor NK cell according to claim 6 or 7, characterized in that the NKG2D ligand is selected from any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP 4, ULBP 5 and ULBP 6.

9. The chimeric antigen receptor NK cell according to claim 7, characterized in that said tumor associated antigen is NKG2D ligand and said antigen binding domain is an extracellular segment selected from the NKG2D protein.

10. The chimeric antigen receptor NK cell according to claim 9, wherein the amino acid sequence of the extracellular domain of NKG2D protein is shown in SEQ ID No. 16.

11. The chimeric antigen receptor NK cell according to claim 6,

the chimeric antigen receptor further has a transmembrane domain and a costimulatory signaling region;

the transmembrane domain is selected from: a transmembrane domain of one or more of CD8, CD28, CD33, CD37, CD8a, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154, CD19, CD45, CD4, and CD3 epsilon.

12. The chimeric antigen receptor NK cell according to claim 11,

the transmembrane domain is selected from the transmembrane domain of CD 8; and/or the presence of a gas in the gas,

the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of: one or more of CD27, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79B, CD66d, CD2, CD4, CD5, CD28, CD30, CD40, CD134, CD137, ICOS, CD154, 4-1BB, OX40, CD7, LIGHT, NKG2C, and B7-H3; and/or the presence of a gas in the gas,

the costimulatory signaling region includes the intracellular domain of 4-1BB and the intracellular domain of CD3 ζ.

13. A method of producing the chimeric antigen receptor NK cell of any one of claims 1 to 12, comprising the steps of: inhibiting expression of a GPR116 gene of a chimeric antigen receptor NK cell or knocking out a GPR116 gene of said chimeric antigen receptor NK cell.

14. A cellular medicament, characterized in that it comprises as active ingredient the chimeric antigen receptor NK cells as defined in any one of claims 1 to 12 and pharmaceutically acceptable adjuvants.

15. An interfering or inhibiting agent for GPR116 gene expression, which interferes with or inhibits GPR116 gene expression; the interference agent or inhibitor is any one or combination of shRNA, antisense RNA, siRNA and/or antagomir molecules.

16. Use of a chimeric antigen receptor NK cell according to any one of claims 1 to 12, a cell medicament according to claim 14, or an interfering or inhibiting agent according to claim 15 for the preparation of a medicament for the treatment of a tumor disease, in CAR-NK therapy.

17. The use of claim 16, wherein the tumor is selected from a solid tumor or a non-solid tumor; and/or the solid tumor is a tumor specifically expressing the NKG2D ligand; and/or the solid tumor is selected from any one of pancreatic cancer, prostate cancer, liver cancer, rectal cancer, gastric cancer and breast cancer.

18. The use according to claim 16, wherein the anti-tumor capacity of NKG2D-CAR-NK cells is increased by constructing said solid tumor-targeted NKG2D-CAR-NK cells and co-expressing an interference gene GPR116 capable of increasing the anti-tumor capacity of the CAR-NK cells; the NKG2D-CAR-NK cells target the treatment of pancreatic cancer; and/or, by down-regulating the GPR116 gene in NK92 cells, promoting NK92 cell killing; and/or, enhance the anti-tumor function of NKG2D-CAR-NK cells by interfering with GPR116 expression; and/or, methods of RNA interference using the interferents or inhibitors knock down the expression of GPR116 gene in CAR-NK cells.