CN110903399B

CN110903399B - Chimeric antigen receptor, nucleic acid thereof, expression plasmid, cell, use and composition

Info

Publication number: CN110903399B
Application number: CN201811080479.9A
Authority: CN
Inventors: 周德阳; 邱绍智; 詹佳颖; 潘志明; 黄士维
Original assignee: Affiliated Hospital Of China Medical University In Taiwan
Current assignee: Affiliated Hospital Of China Medical University In Taiwan
Priority date: 2018-09-17
Filing date: 2018-09-17
Publication date: 2022-02-01
Anticipated expiration: 2038-09-17
Also published as: CN110903399A

Abstract

The invention discloses a chimeric antigen receptor, nucleic acid, an expression plasmid, cells, application and a composition thereof, wherein the chimeric antigen receptor is specifically combined with human leukocyte antigen G. The nucleic acid encodes the chimeric antigen receptor. The chimeric antigen receptor-expressing plastid can express the chimeric antigen receptor. The chimeric antigen receptor-expressing cell is obtained by transducing the chimeric antigen receptor into an immune cell. A pharmaceutical composition for treating cancer comprising a cell expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier. Thus, the chimeric antigen receptor-expressing cells can be used to induce tumor cell death in mammals.

Description

Chimeric antigen receptor, nucleic acid thereof, expression plasmid, cell, use and composition

Technical Field

The invention relates to a pharmaceutical product containing an antigen or an antibody, in particular to a chimeric antigen receptor, a nucleic acid for coding the chimeric antigen receptor, a chimeric antigen receptor expression plasmid, a cell for expressing the chimeric antigen receptor, a pharmaceutical composition for treating cancer and application of the cell for expressing the chimeric antigen receptor.

Background

Cancer, also known as malignant tumor, is an abnormal proliferation of cells, and these proliferating cells may invade other parts of the body, a disease caused by the malfunction of the mechanism controlling cell division and proliferation. There is a growing trend in the world population to develop cancer, which is one of ten causes of death among people in China and has been the leaderboard of ten causes of death for twenty-seven years.

Conventional methods of tumor therapy include surgical therapy, radiation therapy, chemotherapy, and targeted therapy. Tumor immunotherapy is another method for treating tumors besides the above-mentioned treatment methods, and is characterized by that it utilizes the tumor cells or tumor antigen substances to induce specific cellular immunity and humoral immune reaction of body to raise the anticancer capacity of body and prevent tumor growth, diffusion and recurrence so as to attain the goal of removing or controlling tumor.

There are three major approaches to tumor immunotherapy-tumor vaccines, cell therapy and immune checkpoint inhibitors, where the chimeric antigen receptor immunocyte technology is a very rapid cell therapy that has been developed in recent years. In the known technology, the chimeric antigen receptor immune cell is a chimeric protein formed by coupling the antigen binding part of an antibody capable of recognizing a certain tumor antigen and the intracellular part of a CD 3-delta chain or Fc epsilon RI gamma in vitro, and transfects the immune cell of a patient by a gene transduction method to express the chimeric antigen receptor. However, the efficacy of chimeric antigen receptors in solid tumors is greatly limited by the lack of unique tumor-associated antigens in the current cellular therapies of chimeric antigen receptor immune cells, the low efficiency of homing immune cells to the tumor site, and the inability to overcome the immunosuppressive microenvironment of solid tumors.

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor, an isolated nucleic acid, a chimeric antigen receptor expression plasmid, a cell expressing the chimeric antigen receptor, application thereof and a medical composition for treating cancer. The chimeric antigen receptor has excellent specific binding capacity to tumor cells. The nucleic acid encodes the chimeric antigen receptor. And said chimeric antigen receptor expressing plastid comprises said nucleic acid. The chimeric antigen receptor expressing cell comprises the chimeric antigen receptor expressing plastid, expresses the chimeric antigen receptor, can specifically target tumor cells, avoids off-target effect, and further effectively poisons the tumor cells, so that the chimeric antigen receptor expressing cell can be used for preparing medicaments for inducing the death of the tumor cells of mammals. The medicine composition for treating cancer comprises the chimeric antigen receptor-expressing cell, and can effectively poison tumor cells so as to treat cancer.

In one aspect of the present invention, there is provided a chimeric antigen receptor specifically binding to human leukocyte antigen G (HLA-G), the chimeric antigen receptor consisting of an anti-HLA-G antibody represented by seq id No. 1, an HLA-G receptor represented by seq id No. 2, a 2A peptide represented by seq id No. 10, and a co-stimulatory domain represented by seq id No. 3, which are sequentially arranged from N-terminus to C-terminus, the 2A peptide being in tandem with the HLA-G receptor and the co-stimulatory domain.

In another embodiment of one aspect of the invention, there is provided an isolated nucleic acid encoding the chimeric antigen receptor described in the preceding paragraph. The nucleic acid consists of an anti-HLA-G antibody fragment coded as shown in sequence identification number 11, an HLA-G receptor fragment coded as shown in sequence identification number 12, a 2A peptide sequence coded as shown in sequence identification number 15 and a costimulatory domain fragment coded as shown in sequence identification number 13 which are arranged in sequence, wherein the 2A peptide sequence is connected with the HLA-G receptor fragment and the costimulatory domain fragment in series.

In a further embodiment of one aspect of the present invention, there is provided a chimeric antigen receptor expressing plastid comprising a promoter as shown in seq id No. 16 and a nucleic acid as described in the preceding paragraph, arranged in sequence.

According to the chimeric antigen receptor-expressing plasmid, a suicide gene as shown in SEQ ID No. 14 can be further included, which is linked to the 3' end of the nucleic acid.

In yet another embodiment of one aspect of the invention there is provided a chimeric antigen receptor-expressing cell comprising an immune cell and a chimeric antigen receptor-expressing plastid as described in the preceding paragraph.

The chimeric antigen receptor-expressing cell according to the foregoing, wherein the immune cell may be a T cell or a natural killer cell. Preferably, the NK-92 cell line or primary NK-92 cell line can be the natural killer cell.

In another embodiment of one aspect of the invention, there is provided a pharmaceutical composition for treating cancer, comprising the chimeric antigen receptor-expressing cell of the preceding paragraph, and a pharmaceutically acceptable carrier.

The pharmaceutical composition for treating cancer according to the above may further comprise a chemotherapeutic agent. Preferably, the chemotherapeutic agent may be doxorubicin (doxorubicin), temozolomide (temozolomide), gemcitabine (gemcitabine) or carboplatin (carboplatin).

In another aspect of the present invention, there is provided a use of the chimeric antigen receptor-expressing cell as described in the previous paragraph for the preparation of a medicament for inducing death of mammalian tumor cells, which may be breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.

Compared with the prior art, the chimeric antigen receptor, the isolated nucleic acid, the chimeric antigen receptor expression plasmid, the chimeric antigen receptor expression cell, the application thereof and the pharmaceutical composition for treating cancer can be used for inducing the death of tumor cells of mammals.

The above summary of the present invention is intended to provide a simplified summary of the invention in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and is intended to neither identify key/critical elements of the embodiments nor delineate the scope of the invention.

Drawings

The above and other objects, features, and advantages of the present invention will become more apparent from the following description of the embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram showing the protein structure of an anti-HLA-G antibody of the present invention;

FIG. 2 is a schematic diagram showing the construction of the chimeric antigen receptor-expressing plastid of the present invention;

FIG. 3 is a graph showing the analysis of the expression level of the chimeric antigen receptor-expressing cell of example 1 of the present invention;

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H and FIG. 4I are graphs showing the results of the analysis of the tumor cell death induced by the chimeric antigen receptor-expressing cells according to example 1 of the present invention;

FIG. 5 is a graph showing an analysis of the expression level of a chimeric antigen receptor in a cell expressing the chimeric antigen receptor according to example 2 of the present invention;

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H and FIG. 6I are graphs showing the results of the analysis of the tumor cell death induced by the chimeric antigen receptor-expressing cells according to example 2 of the present invention;

FIG. 7 is a graph showing an analysis of the expression level of a chimeric antigen receptor in a cell expressing the chimeric antigen receptor according to example 3 of the present invention;

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H and FIG. 8I are graphs showing the results of the analysis of tumor cell death induced by the chimeric antigen receptor-expressing cells according to example 3 of the present invention;

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G and FIG. 9H are graphs showing immunofluorescence staining results for analyzing HLA-G expression of tumor cells after receiving chemotherapy;

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E are graphs showing the results of flow cytometry analysis of HLA-G expression levels after tumor cells have been subjected to chemotherapy; and

FIG. 11 is a schematic diagram showing the theoretical structure and mechanism of action of the chimeric antigen receptor of the present invention within the cytoplasmic membrane of the chimeric antigen receptor of the present invention.

Detailed Description

The present disclosure proposes a chimeric antigen receptor, a nucleic acid encoding the chimeric antigen receptor, a chimeric antigen receptor-expressing plastid comprising the nucleic acid, a chimeric antigen receptor-expressing cell comprising the chimeric antigen receptor-expressing plastid, uses thereof, and a pharmaceutical composition for treating cancer comprising the chimeric antigen receptor-expressing cell. Cell tests of tumor cells prove that the chimeric antigen receptor has excellent specific binding capacity to the tumor cells, and particularly specifically binds to human leukocyte antigen G expressed on cell membranes of the tumor cells, so that the chimeric antigen receptor expressing the chimeric antigen receptor can target the tumor cells specifically to avoid off-target effect, and further effectively poison the tumor cells. The pharmaceutical composition for treating cancer of the present invention comprises the chimeric antigen receptor-expressing cell of the present invention and may further comprise a chemotherapeutic agent, which is effective in poisoning tumor cells and thus treating cancer.

As used herein, "human leukocyte antigen G (HLA-G)" is encoded by HLA-G gene, and is atypical Major Histocompatibility Complex (MHC), and has a heavy chain of about 45 kDa. HLA-G is expressed on placental cells of fetal origin and is active in the down-regulation of immune responses, the main role of which is to inhibit cytotoxic immune cell function.

The present invention is further illustrated by the following specific test examples, which are included to facilitate one of ordinary skill in the art to which the invention pertains and which are not to be construed as limiting the scope of the invention but as illustrating how the materials and methods of the present invention may be practiced, without undue interpretation.

Test examples

One, the chimeric antigen receptor of the invention, the isolated nucleic acid and the chimeric antigen receptor-expressing plasmid

The chimeric antigen receptor specifically binds to human leukocyte antigen G, and comprises an anti-HLA-G antibody shown in sequence identification number 1, an HLA-G receptor shown in sequence identification number 2 and a co-stimulatory domain shown in sequence identification number 3, which are arranged in sequence from the N-terminal to the C-terminal. Preferably, the C-terminal of the co-stimulatory domain may be further linked to a suicide protein as shown in seq id No. 4, and further comprising a 2A peptide as shown in seq id No. 10 in tandem with the HLA-G receptor and the co-stimulatory domain. Specifically, the anti-HLA-G antibody represented by SEQ ID No. 1 comprises a Heavy Chain (HC) immunoglobulin variable domain sequence and a Light Chain (LC) immunoglobulin variable domain sequence. Wherein the heavy chain immunoglobulin variable domain sequence is CDRH1 shown in sequence identification number 5, CDRH2 shown in sequence identification number 6 and CDRH3 shown in sequence identification number 7. The light chain immunoglobulin variable domain sequence is CDRL2 as shown in seq id No. 8 and CDRL3 as shown in seq id No. 9. FIG. 1 is a schematic diagram showing the protein structure of the anti-HLA-G antibody of the present invention, wherein the dotted circle region represents the variable domain of the anti-HLA-G antibody of the present invention. The HLA-G receptor shown in SEQ ID NO. 2 is a killer cell immunoglobulin-like receptor 2DS4(Killer cell immunoglobulin-like receptor 2DS4, KIR2DS 4). The co-stimulatory domain shown in SEQ ID NO. 3 is DNAX activating protein 12(DNAX activating protein 12, DAP 12). The suicide protein shown in the sequence identification number 4 is iCas9 protein.

The isolated nucleic acids of the invention are those that encode a chimeric antigen receptor of the invention. The nucleic acid comprises an anti-HLA-G antibody-encoding fragment shown in SEQ ID No. 11, an HLA-G receptor-encoding fragment shown in SEQ ID No. 12, and a co-stimulatory domain-encoding fragment shown in SEQ ID No. 13, which are sequentially arranged. Preferably, the 3' end of the co-stimulatory domain fragment can be further connected with a suicide gene as shown in SEQ ID No. 14, and a 2A peptide coding sequence as shown in SEQ ID No. 15 is further connected in series with the HLA-G receptor coding fragment and the co-stimulatory domain coding fragment. The anti-HLA-G antibody fragment encoded by SEQ ID NO. 11 encodes the anti-HLA-G antibody represented by SEQ ID NO. 1, the HLA-G receptor fragment encoded by SEQ ID NO. 12 encodes the HLA-G receptor represented by SEQ ID NO. 2, the co-stimulatory domain fragment encoded by SEQ ID NO. 13 encodes the co-stimulatory domain represented by SEQ ID NO. 3, the suicide gene represented by SEQ ID NO. 14 encodes the suicide protein represented by SEQ ID NO. 4, and the 2A peptide sequence encoded by SEQ ID NO. 15 encodes the 2A peptide represented by SEQ ID NO. 10.

FIG. 2 is a schematic diagram showing the construction of the chimeric antigen receptor-expressing plasmid of the present invention. In detail, in one embodiment shown in this experimental example, the chimeric antigen receptor-expressing plasmid is a Lenti-EF1a-CAR-100517-S1A plasmid, and its insert (insert) fragment comprises a promoter, a fragment encoding an anti-HLA-G antibody, a fragment encoding an HLA-G receptor, and a fragment encoding a co-stimulatory domain, which are arranged in this order. The promoter is EF-1alpha promoter shown in sequence identification number 16, the sequence for coding the anti-HLA-G antibody fragment is shown in sequence identification number 11, the sequence for coding the HLA-G receptor fragment is shown in sequence identification number 12, and the sequence for coding the co-stimulatory domain fragment is shown in sequence identification number 13. In addition, the insert contains the coded information peptide segment shown in the sequence identification number 17, the suicide gene shown in the sequence identification number 14 and the coded 2A peptide sequence shown in the sequence identification number 15. The coding information peptide segment is connected to the 5 'end of the coding anti-HLA-G antibody segment, the suicide gene is connected to the 3' end of the costimulatory domain segment, and the coding 2A peptide sequence is connected in series with the coding HLA-G receptor segment and the coding costimulatory domain segment. The resulting intercalator fragment was then constructed on Creative Biolabs vector (Creative Biolabs, NY, USA) to obtain Lenti-EF1a-CAR-100517-S1A plasmid. The vector used is a lentivirus (lentivirus) vector system, so that the constructed chimeric antigen receptor expression plasmid can be transfected into an expression cell to produce lentivirus, and the lentivirus can be used for transducing the chimeric antigen receptor into an immune cell.

Second, the chimeric antigen receptor-expressing cell of the present invention, its use, and a pharmaceutical composition for treating cancer

The chimeric antigen receptor-expressing cell of the present invention is obtained by transducing the chimeric antigen receptor of the present invention into an immune cell using a lentivirus. Preferably, the immune cell may be a T cell or a natural killer cell. More preferably, the NK-92 cell line or the primary NK cell can be the natural killer cell. In detail, the constructed lentivirus plasmid of Lenti-EF1a-CAR-100517-S1A was transfected into 293T cell line using lipofectamine 3000(Invitrogen) to prepare lentivirus carrying the chimeric antigen receptor of the present invention, and the primary T cells were cultured for 3 days with the supernatant of the prepared lentivirus and Opti-MEM (Invitrogen) containing 8. mu.g/ml polybrene (Sigma-Aldrich) to transduce the chimeric antigen receptor of the present invention into the primary T cells. And Opti-MEM (Invitrogen) containing the supernatant of the prepared lentivirus and 50. mu.g/ml Protamine (Sigma-Aldrich) was cultured for 7 days to transduce the chimeric antigen receptor of the present invention into the primary natural killer cell or NK-92 cell line, thereby obtaining the chimeric antigen receptor-expressing cell of the present invention. The obtained chimeric antigen receptor-expressing cell has the effect of inducing the death of mammalian tumor cells, and can be used for preparing medicaments for inducing the death of mammalian tumor cells. Preferably, the tumor cell can be a breast cancer cell, a glioblastoma multiforme cell, a pancreatic cancer cell, or an ovarian cancer cell.

The pharmaceutical composition for treating cancer of the present invention comprises the chimeric antigen receptor-expressing cell of the present invention and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition for treating cancer may further comprise a chemotherapeutic agent. More preferably, the chemotherapeutic agent may be doxorubicin (doxorubicin), temozolomide (temozolomide), gemcitabine (gemcitabine) or carboplatin (carboplatin).

The following test examples will demonstrate that the chimeric antigen receptor-expressing cells of the present invention and the pharmaceutical compositions for cancer therapy comprising the chimeric antigen receptor-expressing cells of the present invention have a good effect of inducing death of tumor cells of various mammals. The tumor cells used in the test were human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG-05MG (hereinafter referred to as DBTRG), human pancreatic cancer cell line AspC1 and human ovarian cancer cell line SKOV 3. The tumor cell lines used were all purchased from the American Type Culture Collection (ATCC). Human breast cancer cell line MDA-MB-231 cell line is a triple negative breast cancer cell line, namely hormone receptors (ER, PR) and HER-2 receptors are negative, and is cultured in RPMI culture solution containing 10% Fetal Bovine Serum (FBS). The human malignant brain tumor cell strain DBTRG is cultured in DMEM culture solution containing 10% fetal calf serum. The human pancreatic cancer cell line AspC1 was cultured in RPMI medium containing 10% fetal bovine serum. Human ovarian cancer cell line SKOV3 was cultured in McCoy's 5A medium containing 10% fetal bovine serum.

2.1. Example 1

In this test example, the chimeric antigen receptor of the present invention was transduced into NK-92 cell line to obtain the chimeric antigen receptor-expressing cell of example 1 of the present invention, and the expression level of the chimeric antigen receptor of the obtained chimeric antigen receptor-expressing cell of example 1 was analyzed by flow cytometry. FIG. 3 is a graph showing the analysis of the expression level of the chimeric antigen receptor-expressing cell of example 1, FIG. 3 is a graph showing the analysis of the expression level of the chimeric antigen receptor of the parent NK-92 cell line, to which the chimeric antigen receptor of the present invention is not transduced, and FIG. 1 is a graph showing the analysis of the expression level of the chimeric antigen receptor-expressing cell of example 1 on days 3 and 7 after transduction, respectively. As can be seen from the data of FIG. 3, the Mean Fluorescence Intensity (MFI) of the parental NK-92 cell line was only 9.98%, whereas the mean fluorescence intensity of the cells expressing the chimeric antigen receptor of example 1 at day 3 and day 7 after transduction could reach 20.11% and 65.07%, respectively, showing that the cells expressing the chimeric antigen receptor of example 1 could indeed stably express the chimeric antigen receptor of the present invention.

The chimeric antigen receptor-expressing cells of example 1 of the present invention and the pharmaceutical composition for treating cancer comprising the chimeric antigen receptor-expressing cells of example 1 of the present invention were further tested for their effects of inducing death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells.

Firstly, respectively using human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AspC1 and human ovarian cancer cell line SKOV3 at a ratio of 1 × 10⁵cells/wells were plated in 12-well plates and incubated for an additional 48 hours before testing. Each tumor cell was divided into 6 groups in the experiment, including untreated control group, test group 1 for treatment of chemotherapeutic drug, and treatment parentTest group 2 which substitutes for the NK-92 cell line, test group 3 which treated the parent NK-92 cell line and the chemotherapeutic agent, test group 4 which treated the chimeric antigen receptor-expressing cells of example 1, and test group 5 which treated the chimeric antigen receptor-expressing cells of example 1 and the chemotherapeutic agent. Wherein in the group of human breast cancer cell line MDA-MB-231, the used chemotherapeutic drug is adriamycin (200 nM); in the group of human malignant brain tumor cell lines DBTRG, the chemotherapeutic drug used is temozolomide (80 mug/ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapeutic agent used was gemcitabine (20 μ M); in the group of human ovarian cancer cell line SKOV3, the chemotherapeutic agent used was carboplatin (20 μ M). In the test group 4 and the test group 5, the number of cells expressing the chimeric antigen receptor of example 1 was 1X 10⁵cells, the number of cells of the parental NK-92 cell line treated in Experimental group 2 and Experimental group 3 was also 1X 10⁵cells. Then, each group of treated cells are subjected to cell staining by Annexin V-FITC and a nucleic acid stain Propidium Iodide (PI), the apoptosis and death conditions of the cells are detected by a flow cytometer, and the sum of the percentages of the cells stained with Annexin V-FITC and/or PI (namely the percentages of the cells in the first quadrant, the second quadrant and the fourth quadrant in the double variable flow cytometer scattergram) is calculated to obtain the cytotoxic effect. After each group was subjected to independent triplicate experiments, the cytotoxic effect was counted.

Referring to FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E and FIG. 4F, there are shown the results of the analysis of the tumor cell death induced by the chimeric antigen receptor-expressing cells of example 1 of the present invention. Wherein FIG. 4A is a graph showing the results of the analysis of the human breast cancer cell line MDA-MB-231 death induced by the chimeric antigen receptor-expressing cells of example 1, FIG. 4B is a statistical chart of FIG. 4A after the three-repeat test, FIG. 4C is a chart of the analysis results of the human malignant brain tumor cell line DBTRG death induced by the chimeric antigen receptor-expressing cells of example 1, FIG. 4D is a statistical chart of FIG. 4C after the three-repeat test, FIG. 4E is a chart of the result of the analysis of the human pancreatic cancer cell line AsPC1 death induced by the chimeric antigen receptor-expressing cells of example 1, FIG. 4F is a statistical chart of FIG. 4E after the three-fold experiment, FIG. 4G is a chart of the results of the analysis of the human ovarian cancer cell line SKOV3 death induced by the chimeric antigen receptor-expressing cells of example 1, fig. 4H is a statistical chart of fig. 4G after the triple trial, and fig. 4I is a statistical chart of fig. 4A, 4C, 4E and 4G after the triple trial. In the figure, P represents the parent NK-92 cell line, H represents the chimeric antigen receptor-expressing cell of example 1, H represents doxorubicin, T represents temozolomide, G represents gemcitabine, and C represents carboplatin.

As shown by the results of FIGS. 4A and 4B, only about 10% of the deaths of the human breast cancer cell line MDA-MB-231 were observed in the untreated control group, while the mortality of the human breast cancer cell line MDA-MB-231 was increased but there was no statistical difference between the doxorubicin-treated test group 1 and the parental NK-92 cell line-treated test group 2. In test group 3, which treated the parental NK-92 cell line and adriamycin, the human breast cancer cell line MDA-MB-231 mortality was increased to 40%, which was statistically different (p <0.05) from that of test group 2. The mortality rate of human breast cancer cell line MDA-MB-231 induced by the test group 4 expressing chimeric antigen receptor cells of example 1 was about 60%, which is statistically different from that of the test group 2 (p < 0.001). In addition, the experimental group 5 treated with the chimeric antigen receptor-expressing cells of example 1 and doxorubicin induced mortality of the human breast cancer cell line MDA-MB-231 more than about 80%, and had a statistically significant difference (p <0.05) compared to the experimental group 4 and a statistically significant difference (p <0.01) compared to the experimental group 3.

As shown in FIGS. 4C and 4D, only 10% of the human malignant brain cell lines DBTRG were found dead in the untreated control group, while the mortality rate of the human malignant brain cell lines DBTRG was increased but there was no statistical difference between the test group 1 treated with temozolomide and the test group 2 treated with the parent NK-92 cell line. And in the test group 3 for treating the parent NK-92 cell strain and the temozolomide, the death rate of the human malignant brain tumor cell strain DBTRG can be improved to 40 percent, and compared with the test group 2, the human malignant brain tumor cell strain DBTRG has statistical significance (p is less than 0.05). On the other hand, the test group 4 treated with the chimeric antigen receptor-expressing cells of example 1 induced a mortality rate of over 60% in the human malignant brain cell line DBTRG, which was statistically different from that of the test group 2 (p < 0.001). In addition, the experimental group 5 treated with the chimeric antigen receptor cells and temozolomide of example 1 induced more than 80% of human malignant brain cell lines DBTRG, which was statistically significant (p <0.05) compared to experimental group 4 and statistically significant (p <0.001) compared to experimental group 3.

As shown in FIGS. 4E and 4F, only 10% of the human pancreatic cancer cell line AsPC1 died in the untreated control group, whereas the mortality rate of the human pancreatic cancer cell line AsPC1 was increased in gemcitabine-treated test group 1 and parental NK-92 cell line-treated test group 2, but there was no statistically significant difference. Whereas, in test group 3, which treated the parental NK-92 cell line and gemcitabine, the human pancreatic cancer cell line AspC1 mortality could be increased to about 30%, but there was still no statistically significant difference. On the other hand, the human pancreatic cancer cell line AsPC1 mortality induced by the test group 4 treated with the chimeric antigen receptor-expressing cells of example 1 was close to 40%, which was statistically different from that of the test group 2 (p < 0.01). In addition, the test group 5 treated with the chimeric antigen receptor cells and gemcitabine of example 1 induced a mortality of human pancreatic cancer cell line AsPC1 of about 60%, which was statistically significant (p <0.001) compared to the test group 4 and statistically significant (p <0.001) compared to the test group 3.

The results in FIGS. 4G and 4H show that only 10% of the human ovarian cancer cell line SKOV3 died in the untreated control group, whereas the mortality rate of the human ovarian cancer cell line SKOV3 was increased but not statistically different between test group 1 treated with carboplatin and test group 2 treated with the parental NK-92 cell line. Whereas, in test group 3, which treated the parental NK-92 cell line and carboplatin, the mortality of the human ovarian cancer cell line SKOV3 could be increased to about 30%, with a statistically significant difference (p <0.05) compared to test group 2. In contrast, the mortality rate of human ovarian cancer cell line SKOV3 induced by test group 4 treated with the chimeric antigen receptor-expressing cells of example 1 was close to 40%, which was statistically different (p <0.01) from that of test group 2. In addition, the test group 5 treated with the chimeric antigen receptor cells and carboplatin of example 1 induced a mortality rate of the human ovarian cancer cell line SKOV3 of about 60%, which was statistically significant (p <0.05) compared to test group 4 and statistically significant (p <0.01) compared to test group 3.

The results shown in FIG. 4I show that the chimeric antigen receptor-expressing cells of example 1 have excellent poisoning effect on human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AsPC1 and human ovarian cancer cell line SKOV3, and that the chimeric antigen receptor-expressing cells of the present invention can be used for preparing drugs for inducing the death of tumor cells in mammals. Preferably, the tumor cell may be a breast cancer cell, a glioblastoma multiforme cell, a pancreatic cancer cell, or an ovarian cancer cell. In addition, the expression chimeric antigen receptor cell and the chemotherapeutic drug in example 1 were treated simultaneously, and the poisoning effect on human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AsPC1 and human ovarian cancer cell line SKOV3 was more significant, which shows that the pharmaceutical composition for treating cancer of the present invention can effectively poison tumor cells and further treat cancer. Preferably, the pharmaceutical composition for treating cancer of the present invention may further comprise a chemotherapeutic drug, which may include, but is not limited to, doxorubicin, temozolomide, gemcitabine and carboplatin.

2.2. Example 2

In this test example, the chimeric antigen receptor of the present invention was transduced into primary natural killer cells to obtain the chimeric antigen receptor-expressing cells of example 2 of the present invention, and the expression level of the chimeric antigen receptor of the obtained chimeric antigen receptor-expressing cells of example 2 was analyzed by flow cytometry. FIG. 5 is a diagram showing the analysis of the expression level of the chimeric antigen receptor-expressing cell of example 2 of the present invention. FIG. 5 is a graph showing the analysis of the expression level of the chimeric antigen receptor in the parent primary NK cell not transduced with the chimeric antigen receptor of the present invention, and the analysis of the expression level of the chimeric antigen receptor in the chimeric antigen receptor-expressing cell of example 2 on days 3 and 7 after transduction, respectively. As can be seen from the data of fig. 5, the mean fluorescence intensity of the parental primary natural killer cells was 22.09%, whereas the mean fluorescence intensity of the chimeric antigen receptor-expressing cells of example 2 at 3 and 7 days after transduction could reach 29.02% and 50.21%, respectively, showing that the chimeric antigen receptor-expressing cells of example 2 could indeed stably express the chimeric antigen receptor of the present invention.

The chimeric antigen receptor-expressing cells of example 2 of the present invention and the pharmaceutical composition for treating cancer comprising the chimeric antigen receptor-expressing cells of example 2 of the present invention were further tested for their effects of inducing death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells.

Firstly, respectively using human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AspC1 and human ovarian cancer cell line SKOV3 at a ratio of 1 × 10⁵cells/wells were plated in 12-well plates and incubated for an additional 48 hours before testing. Each tumor cell was experimentally divided into 6 groups, untreated control group, test group 1 for treatment of chemotherapeutic drugs, test group 2 for treatment of parent primary NK cells, test group 3 for treatment of parent primary NK cells and chemotherapeutic drugs, test group 4 for treatment of chimeric antigen receptor-expressing cells of example 2, and test group 5 for treatment of chimeric antigen receptor-expressing cells of example 2 and chemotherapeutic drugs. Wherein in the group of human breast cancer cell line MDA-MB-231, the used chemotherapeutic drug is adriamycin (200 nM); in the group of human malignant brain tumor cell lines DBTRG, the chemotherapeutic drug used is temozolomide (80 mug/ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapeutic agent used was gemcitabine (20 μ M); in the group of human ovarian cancer cell line SKOV3, the chemotherapeutic agent used was carboplatin (20 μ M). In the test group 4 and the test group 5, the number of cells expressing the chimeric antigen receptor of example 2 was 1X 10⁵cells, the number of cells of the parental primary natural killer cells treated in test group 2 and test group 3 was also 1X 10⁵cells. Then, the treated cells of each group are subjected to cell staining by Annexin V-FITC and PI, and the sum of the percentage of cells stained with Annexin V-FITC and/or PI is calculated to obtain the cytotoxic effect. After each group was subjected to independent triplicate experiments, the cytotoxic effect was counted.

Referring to FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H and FIG. 6I, there are graphs showing the results of the analysis of the tumor cell death induced by the chimeric antigen receptor-expressing cells of example 2 according to the present invention. FIG. 6A is a graph showing the analysis results of the chimeric antigen receptor-expressing cells of example 2 inducing death of human breast cancer cell line MDA-MB-231, and FIG. 6B is a statistical graph of FIG. 6A after three replicates. FIG. 6C is a graph showing the analysis results of the induction of DBTRG death, which is a human malignant brain tumor cell line, by the chimeric antigen receptor-expressing cells of example 2, and FIG. 6D is a statistical graph of FIG. 6C after the three-repeat test. FIG. 6E is a graph showing the results of analysis of human pancreatic cancer cell line AsPC1 death induced by the chimeric antigen receptor-expressing cells of example 2, and FIG. 6F is a statistical graph of FIG. 6E after three replicates. FIG. 6G is a graph showing the results of the analysis of example 2 in which the chimeric antigen receptor-expressing cells induced death of human ovarian cancer cell line SKOV3, and FIG. 6H is a statistical chart of FIG. 6G after the three-repeat test. Fig. 6H is a statistical chart of fig. 6G, and fig. 6I is a statistical chart of fig. 6A, 6C, 6E, and 6G after three repeated experiments. In the figure, P represents parental primary natural killer cells, H represents the chimeric antigen receptor-expressing cells of example 2, H represents doxorubicin, T represents temozolomide, G represents gemcitabine, and C represents carboplatin.

As shown in FIGS. 6A and 6B, only about 10% of the deaths of the human breast cancer cell line MDA-MB-231 were observed in the untreated control group, while the mortality rate of the human breast cancer cell line MDA-MB-231 was increased but there was no statistical difference between the doxorubicin-treated test group 1 and the parental naive killer cell-treated test group 2. In test group 3, which treated parental primary natural killer cells and doxorubicin, the human breast cancer cell line MDA-MB-231 mortality was increased to about 30%, which was statistically significant (p <0.05) compared to test group 2. On the other hand, the test group 4 treated with the chimeric antigen receptor-expressing cells of example 2 induced a mortality rate of more than 50% in human breast cancer cell line MDA-MB-231, which was statistically different from that of the test group 2 (p < 0.01). In addition, the experimental group 5 treated with the chimeric antigen receptor-expressing cells of example 2 and doxorubicin induced mortality of the human breast cancer cell line MDA-MB-231 more than about 80%, and had a statistically significant difference (p <0.05) compared to the experimental group 4 and a statistically significant difference (p <0.01) compared to the experimental group 3.

As shown in fig. 6C and 6D, only 10% of the human malignant brain cell lines DBTRG died were observed in the untreated control group, while the human malignant brain cell lines DBTRG died in temozolomide-treated test group 1 and parental primary natural killer cell-treated test group 2, although the mortality rate of the human malignant brain cell lines DBTRG increased, they did not have a statistically significant difference. And in the test group 3 for treating parent primary natural killer cells and temozolomide, the death rate of the human malignant brain tumor cell strain DBTRG can be improved to be close to 30 percent. The test group 4 treated with the chimeric antigen receptor-expressing cells of example 2 induced a mortality rate of the human malignant brain tumor cell line DBTRG of more than 20%, which was statistically different from that of the test group 2 (p < 0.05). In addition, the test group 5 treated in example 2 expressing chimeric antigen receptor cells and temozolomide induced mortality of human malignant brain cell line DBTRG more close to 60%, and had a statistically significant difference (p <0.01) compared to test group 4 and a statistically significant difference (p <0.05) compared to test group 3.

As shown in fig. 6E and 6F, only 10% of the human pancreatic cancer cell line AsPC1 died was observed in the untreated control group, whereas gemcitabine-treated test group 1 and parental primary killer cell-treated test group 2 showed no statistically significant difference in the mortality rate of human pancreatic cancer cell line AsPC 1. In test group 3, which treated the parental primary natural killer cells and gemcitabine, the human pancreatic cancer cell line AsPC1 was found to have an increased mortality rate of about 30%, which was statistically significant (p <0.05) compared to test group 2. On the other hand, the test group 4 treated with the chimeric antigen receptor-expressing cells of example 2 induced a mortality rate of about 20% for the human pancreatic cancer cell line AsPC1, which was statistically different from that of the test group 2 (p < 0.01). In addition, the test group 5 treated with the chimeric antigen receptor cells and gemcitabine of example 2 induced a mortality of human pancreatic cancer cell line AsPC1 of about 50%, which was statistically significant (p <0.01) compared to the test group 4 and statistically significant (p <0.05) compared to the test group 3.

The results in fig. 6G and 6H show that only 10% of the human ovarian cancer cell line SKOV3 died were seen in the untreated control group, whereas test group 1 treated with carboplatin showed no statistically significant difference in the mortality rate of the human ovarian cancer cell line SKOV 3. The cytotoxic effect of test group 2 treated with parental primary natural killer cells was comparable to the control group. In test group 3, which treated parental primary natural killer cells and carboplatin, the human ovarian cancer cell line SKOV3 mortality rate could exceed 20%, with statistically significant differences (p <0.05) compared to test group 2. In contrast, the mortality rate of human ovarian cancer cell line SKOV3 induced by test group 4 treated with the chimeric antigen receptor cells of example 2 was about 20%, which was statistically different (p <0.05) from that of test group 2. In addition, the test group 5 treated with the chimeric antigen receptor cells and carboplatin of example 2 induced a mortality rate of human ovarian cancer cell line SKOV3 that was more close to 50%, statistically significant difference (p <0.01) compared to test group 4 and statistically significant difference (p <0.05) compared to test group 3.

The results in FIG. 6I show that the chimeric antigen receptor-expressing cells of example 2 have excellent cytotoxic activity against breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells, or ovarian cancer cells, and thus the chimeric antigen receptor-expressing cells of the present invention can be used to prepare drugs for inducing tumor cell death in mammals. In addition, the concurrent treatment of the chimeric antigen receptor-expressing cells of example 2 and the chemotherapeutic agent showed more significant toxic effects on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, indicating that the pharmaceutical composition for treating cancer of the present invention, preferably containing the chemotherapeutic agent, can effectively poison tumor cells and thus treat cancer.

2.3. Example 3

In this test example, the chimeric antigen receptor of the present invention was transduced into primary T cells to obtain the chimeric antigen receptor-expressing cells of example 3 of the present invention, and the obtained chimeric antigen receptor-expressing cells of example 3 were analyzed for the expression level of the chimeric antigen receptor by flow cytometry. FIG. 7 is a graph showing the analysis of the expression level of the chimeric antigen receptor of the cells expressing the chimeric antigen receptor of example 3 of the present invention, FIG. 7 is a graph showing the analysis of the expression level of the chimeric antigen receptor of primary T cells not transduced with the chimeric antigen receptor of the present invention, and FIG. 3 is a graph showing the analysis of the expression level of the chimeric antigen receptor of the cells expressing the chimeric antigen receptor of example 3 on days 3 and 7 after transduction, respectively. As can be seen from the data in fig. 7, the mean fluorescence intensity of the primary T cells was only 9.36%, whereas the mean fluorescence intensity of the chimeric antigen receptor-expressing cells of example 3 at day 3 and day 7 after transduction could reach 34.1% and 88.64%, respectively, showing that the chimeric antigen receptor-expressing cells of example 3 could indeed stably express the chimeric antigen receptor of the present invention.

The chimeric antigen receptor-expressing cells of example 3 of the present invention and the pharmaceutical composition for treating cancer comprising the chimeric antigen receptor-expressing cells of example 3 of the present invention were further tested for their effects of inducing death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells.

Firstly, respectively using human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AspC1 and human ovarian cancer cell line SKOV3 at a ratio of 1 × 10⁵cells/wells were plated in 12-well plates and incubated for an additional 48 hours before testing. Each tumor cell was experimentally divided into 6 groups, untreated control group, test group 1 for treatment of chemotherapeutic drugs, test group 2 for treatment of primary T cells, test group 3 for treatment of primary T cells and chemotherapeutic drugs, test group 4 for treatment of chimeric antigen receptor cells of example 3, and test group 5 for treatment of chimeric antigen receptor cells of example 3 and chemotherapeutic drugs. Wherein in the group of human breast cancer cell line MDA-MB-231, the used chemotherapeutic drug is adriamycin (200 nM); in the group of human malignant brain tumor cell lines DBTRG, the chemotherapeutic drug used is temozolomide (80 mug/ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapeutic agent used was gemcitabine (20 μ M); in the group of human ovarian cancer cell line SKOV3, the chemotherapeutic agent used was carboplatin (20 μ M). In test group 4 and test group 5, the expression of example 3 was treatedThe number of cells of the chimeric antigen receptor cell was 1X 10⁵cells, the number of primary T cells treated in Experimental group 2 and Experimental group 3 was also 1X 10⁵cells. Then, the treated cells are subjected to cell staining by Annexin V-FITC and PI, the apoptosis and death conditions of the cells are detected by a flow cytometer, and the sum of the percentage of the cells stained with Annexin V-FITC and/or PI is calculated to obtain the cytotoxic effect. After each group was subjected to independent triplicate experiments, the cytotoxic effect was counted.

Please refer to fig. 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H and 8I, which are graphs showing the results of the analysis of tumor cell death induced by the chimeric antigen receptor-expressing cells of example 3 according to the present invention. Wherein FIG. 8A is a graph showing the analysis results of the chimeric antigen receptor-expressing cells of example 3 inducing death of human breast cancer cell line MDA-MB-231, and FIG. 8B is a statistical graph of FIG. 8A after three replicates. FIG. 8C is a graph showing the analysis results of the induction of DBTRG death, which is a human malignant brain tumor cell line, by the chimeric antigen receptor-expressing cells of example 3, and FIG. 8D is a statistical graph of FIG. 8C after the three-repeat test. FIG. 8E is a graph showing the results of analysis of human pancreatic cancer cell line AsPC1 death induced by the chimeric antigen receptor-expressing cells of example 3, and FIG. 8F is a statistical graph of FIG. 8E after three replicates. FIG. 8G is a graph showing the results of analysis of the death of human ovarian cancer cell line SKOV3 induced by the chimeric antigen receptor-expressing cells of example 3, and FIG. 8H is a statistical graph of FIG. 8G. Fig. 8H is a statistical chart of fig. 8G, and fig. 8I is a statistical chart of fig. 8A, 8C, 8E, and 8G after three repeated experiments. In the figure, P represents parental primary T cells, H represents the chimeric antigen receptor-expressing cell of example 3, H represents doxorubicin, T represents temozolomide, G represents gemcitabine, and C represents carboplatin.

As shown in FIGS. 8A and 8B, only about 10% of the deaths of the human breast cancer cell lines MDA-MB-231 were observed in the untreated control group, while the mortality of the human breast cancer cell lines MDA-MB-231 was increased but not statistically significant between the doxorubicin-treated test group 1 and the parental primary T-cell-treated test group 2. In test group 3, which treated parental primary T cells and doxorubicin, the human breast cancer cell line MDA-MB-231 mortality was increased to about 20%, which was statistically significant compared to test group 2 (p < 0.01). On the other hand, the test group 4 treated with the chimeric antigen receptor-expressing cells of example 3 induced a mortality rate of more than 30% in human breast cancer cell line MDA-MB-231, which was statistically different from that of the test group 2 (p < 0.001). In addition, the experimental group 5 treated with the chimeric antigen receptor-expressing cells and doxorubicin of example 3 induced a mortality rate of human breast cancer cell line MDA-MB-231 of approximately 50%, which was statistically significant (p <0.05) compared to experimental group 4 and statistically significant (p <0.001) compared to experimental group 3.

As shown in fig. 8C and 8D, only 20% of the human malignant brain cell lines DBTRG died were observed in the untreated control group, while the human malignant brain cell lines DBTRG died with temozolomide in test group 1 and the parent primary T-cell in test group 2, although the death rate was increased, they did not have a statistically significant difference. In the test group 3 for treating parent primary T cells and temozolomide, the human malignant brain tumor cell strain DBTRG mortality can be improved to 30%, but the difference has no statistical significance. On the other hand, the test group 4 treated with the chimeric antigen receptor-expressing cells of example 3 induced a mortality rate of the human malignant brain tumor cell line DBTRG of more than 50%, which was statistically different from that of the test group 2 (p < 0.001). In addition, the test group 5 treated with the chimeric antigen receptor cells and temozolomide of example 3 induced a mortality rate of the human malignant brain cell line DBTRG that was closer to 80%, statistically significant difference (p <0.05) compared to test group 4, and statistically significant difference (p <0.001) compared to test group 3.

The results in fig. 8E and 8F show that only 20% of the human pancreatic cancer cell line AsPC1 died in the untreated control group, whereas gemcitabine-treated test group 1 had an increased mortality rate of human pancreatic cancer cell line AsPC1, but had no statistically significant difference. The cytotoxic effect of test group 2 treated with parental primary T cells was comparable to that of the control group. Whereas, in test group 3, which treated parental primary T cells and gemcitabine, the human pancreatic cancer cell line AsPC1 mortality was increased to over 30%, with a statistically significant difference (p <0.05) compared to test group 2. In contrast, the mortality rate of human pancreatic cancer cell line AsPC1 induced by the treatment group 4 expressing chimeric antigen receptor cells of example 3 was increased to more than 50%, which was statistically significant (p <0.001) compared to the treatment group 2. In addition, the test group 5 treated with the chimeric antigen receptor cells and gemcitabine of example 3 induced a mortality of human pancreatic cancer cell line AsPC1 of about 60%, which was statistically significant (p <0.01) compared to the test group 4 and statistically significant (p <0.01) compared to the test group 3.

The results in fig. 8G and 8H show that only 10% of the human ovarian cancer cell line SKOV3 died were seen in the untreated control group, whereas test group 1 treated with carboplatin and test group 2 treated with parental primary T cells showed no statistically significant difference in the mortality rate of the human ovarian cancer cell line SKOV 3. In the test group 3 for treating parent primary natural killer cells and carboplatin, the mortality rate of the human ovarian cancer cell line SKOV3 can reach about 30%, and compared with the test group 2, the human ovarian cancer cell line SKOV3 has statistical significance (p is less than 0.05). The mortality rate of human ovarian cancer cell line SKOV3 induced by test group 4 treated with the chimeric antigen receptor-expressing cells of example 3 was close to 60%, which was statistically significant different (p <0.001) from that of test group 2. In addition, the test group 5 treated with the chimeric antigen receptor cells and carboplatin of example 3 induced a mortality rate of SKOV3 of more than 60%, which was statistically significant (p <0.05) compared to test group 4 and statistically significant (p <0.001) compared to test group 3.

The results in FIG. 8I show that the chimeric antigen receptor-expressing cells of example 3 have excellent cytotoxic activity against breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells, or ovarian cancer cells, and thus the chimeric antigen receptor-expressing cells of the present invention can be used to prepare drugs for inducing tumor cell death in mammals. In addition, the combination of the chimeric antigen receptor-expressing cells of example 3 and the chemotherapeutic agent shows a more significant poisoning effect on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, indicating that the pharmaceutical composition for treating cancer of the present invention may preferably contain a chemotherapeutic agent, which can effectively poison tumor cells and thus treat cancer.

2.4. Treatment with chemotherapeutic agents increases the expression of human leukocyte antigen G on the cell membrane of tumor cells

The experimental example further discusses the reason why the group simultaneously treating the chimeric antigen receptor-expressing cell of the present invention and the chemotherapeutic agent has more excellent effect of poisoning tumor cells.

Firstly, respectively using human breast cancer cell line MDA-MB-231, human malignant brain tumor cell line DBTRG, human pancreatic cancer cell line AspC1 and human ovarian cancer cell line SKOV3 at 2 x 10⁵cells/wells were seeded in 6-well plates and cultured until every other day for testing after cell attachment. Each tumor cell was experimentally divided into 2 groups, an untreated control group and a test group treated with chemotherapeutic drugs for 48 hours. Wherein in the group of human breast cancer cell line MDA-MB-231, the used chemotherapeutic drug is adriamycin (200 nM); in the group of human malignant brain tumor cell lines DBTRG, the chemotherapeutic drug used is temozolomide (80 mug/ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapeutic agent used was gemcitabine (20 μ M); in the group of human ovarian cancer cell line SKOV3, the chemotherapeutic agent used was carboplatin (20 μ M). And detecting the expression of human leukocyte antigen G on the cell surface of the tumor cells by using an immunofluorescence staining method and a flow cytometer for each group of treated tumor cells.

Please refer to fig. 9A, 9B, 9C, 9D, 9E, 9F, 9G and 9H, which are graphs showing the results of immunofluorescence staining for analyzing the HLA-G expression of tumor cells after receiving chemotherapy. Fig. 9A is a graph of immunofluorescent staining results of human breast cancer cell lines MDA-MB-231 in the control group, fig. 9B is a graph of immunofluorescent staining results of human breast cancer cell lines MDA-MB-231 treated with doxorubicin, fig. 9C is a graph of immunofluorescent staining results of human malignant brain tumor cell lines DBTRG in the control group, fig. 9D is a graph of immunofluorescent staining results of human malignant brain tumor cell lines DBTRG treated with temozolomide, fig. 9E is a graph of immunofluorescent staining results of human pancreatic cancer cell lines AsPC1 in the control group, fig. 9F is a graph of immunofluorescent staining results of human pancreatic cancer cell lines AsPC1 treated with gemcitabine, fig. 9G is a graph of immunofluorescent staining results of human ovarian cancer cell lines SKOV3 in the control group, and fig. 9H is a graph of immunofluorescent staining results of human ovarian cancer cell lines SKOV3 treated with carboplatin. In addition, in order to make the immunofluorescent staining results of fig. 9A to 9H clear, the appendix 1 is the color drawing of fig. 9A, the appendix 2 is the color drawing of fig. 9B, the appendix 3 is the color drawing of fig. 9C, the appendix 4 is the color drawing of fig. 9D, the appendix 5 is the color drawing of fig. 9E, the appendix 6 is the color drawing of fig. 9F, the appendix 7 is the color drawing of fig. 9G, and the appendix 8 is the color drawing of fig. 9H. Wherein Pan-cadherin is an epithelial-mesenchymal cell transformation-associated protein which can represent the position of a cell membrane; and the location of the nuclei is indicated by the staining of DAPI (blue fluorescence).

FIG. 9A and FIG. 9B show that doxorubicin exposure increased the expression of human leukocyte antigen G on the cell membrane of human breast cancer cell line MDA-MB-231. The results in FIG. 9C and FIG. 9D show that temozolomide treatment increases the expression of human leukocyte antigen G on the cell membrane of DBTRG, a human malignant brain tumor cell line. The results in fig. 9E and 9F show that gemcitabine treatment increases the expression of human leukocyte antigen G on the cell membrane of human pancreatic cancer cell line AsPC 1. The results in FIG. 9G and FIG. 9H show that carboplatin treatment increased the expression of human leukocyte antigen G on the cell membrane of human ovarian cancer cell line SKOV 3.

Referring to fig. 10A, 10B, 10C, 10D and 10E, the results of flow cytometry analysis of HLA-G expression after tumor cells received chemotherapy are shown. Wherein fig. 10A is a flow cytometry analysis result graph of human breast cancer cell line MDA-MB-231, fig. 10B is a flow cytometry analysis result graph of human malignant brain tumor cell line DBTRG, fig. 10C is a flow cytometry analysis result graph of human pancreatic cancer cell line AsPC1, fig. 10D is a flow cytometry analysis result graph of human ovarian cancer cell line SKOV3, and fig. 10E is a statistical graph of fig. 10A to 10D.

The results of fig. 10A to 10E show that the mean fluorescence intensity of the human breast cancer cell line MDA-MB-231 of the control group was only 12.27%, whereas the mean fluorescence intensity of the human breast cancer cell line MDA-MB-231 of the test group increased to 64.45%, which was statistically significant different (p < 0.001). The mean fluorescence intensity of the human malignant brain tumor cell line DBTRG in the control group is only 14.01 percent, while the mean fluorescence intensity of the human malignant brain tumor cell line DBTRG in the test group is 22.33 percent, which has statistically significant difference (p < 0.001). The mean fluorescence intensity of the human pancreatic cancer cell line AsPC1 in the control group was only 13.18%, while the mean fluorescence intensity of the human pancreatic cancer cell line AsPC1 in the test group was increased to 41.44%, which was statistically significant different (p < 0.01). The mean fluorescence intensity of the human ovarian cancer cell line SKOV3 in the control group was only 14.69%, while the mean fluorescence intensity of the human ovarian cancer cell line SKOV3 in the test group was 38.58%, which had statistically significant differences (p < 0.01).

The results in fig. 9A to fig. 10E show that the treatment of chemotherapeutic drugs can increase the expression of human leukocyte antigen G on the cell membrane of tumor cells, and the chimeric antigen receptor expressed by the chimeric antigen receptor-expressing cells of the present invention can specifically bind to human leukocyte antigen G, so that the treatment of the chimeric antigen receptor-expressing cells of the present invention after the treatment of chemotherapeutic drugs, or the simultaneous treatment of the chimeric antigen receptor-expressing cells of the present invention and chemotherapeutic drugs can have more excellent effect of poisoning tumor cells.

Referring to FIG. 11, a schematic diagram of the theoretical structure and mechanism of action of the chimeric antigen receptor of the present invention in the cytoplasmic membrane of the chimeric antigen receptor of the present invention is shown. The chimeric antigen receptor-expressing cell of the invention is a genetically engineered natural killer cell or T cell expressing the chimeric antigen receptor of the invention, and the chimeric antigen receptor system of the invention is a tumor-targeting receptor complex consisting of an anti-HLA-G antibody (scFv), an HLA-G receptor (KIR) and a costimulatory domain (DAP12), and preferably, can further comprise a suicide protein-iCas 9. The chimeric antigen receptor expressing cell can specifically identify human leukocyte antigen G on a tumor cell membrane, and further has a poisoning effect on tumor cells. Preferably, when the tumor cell is treated with a chemotherapeutic drug, the expression of human leukocyte antigen G on the plasma membrane of the tumor cell can be up-regulated, so that the chimeric antigen receptor-expressing cell of the present invention is combined with the human leukocyte antigen G specifically recognized on the surface of the tumor cell to trigger signal transduction, generate a signal cascade to lead the activation and proliferation of the chimeric antigen receptor-expressing cell of the present invention, further induce the exocytosis of the lytic granule and kill the target tumor cell.

In summary, the chimeric antigen receptor of the present invention has excellent ability of specifically binding to tumor cells, especially human leukocyte antigen G expressed on the cell membrane of tumor cells, so that the chimeric antigen receptor of the present invention can be expressed to target tumor cells specifically to the chimeric antigen receptor cells, thereby avoiding off-target effect, and further effectively poisoning tumor cells. The pharmaceutical composition for treating cancer of the present invention comprises the chimeric antigen receptor-expressing cell of the present invention, and can effectively poison tumor cells and further treat cancer. The pharmaceutical composition for treating cancer further comprises chemotherapeutic drugs, which can further induce the cell membrane of tumor cells to express a large amount of human leukocyte antigen G, thereby enhancing the poisoning effect of the cells expressing chimeric antigen receptors on the tumor cells, so as to have more excellent tumor cell poisoning effect.

Therefore, it is intended that the present invention not be limited to the particular embodiments disclosed, but that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> China university Hospital

<120> chimeric antigen receptor, nucleic acid, expression plasmid, cell, use and composition thereof

<160>　17

<210>　1

<211>　246

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> anti-HLA-G antibody

<400>　1

Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Phe Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met

65 70 75 80

Leu Ser Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Val Arg Gly Gly Tyr Trp Ser Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Ile Thr Gln Thr Thr Pro Ser

130 135 140

Val Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu

165 170 175

Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Ser Arg Met Ser Ser

180 185 190

Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val

210 215 220

Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys

245

<210>　2

<211> 75

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HLA-G receptor

<400>　2

Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His

1 5 10 15

Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu

20 25 30

Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val

35 40 45

Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser

50 55 60

Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

65 70 75

<210>　3

<211>　113

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Co-stimulatory domain fragment

<400>　3

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys

<210>　4

<211>　439

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　iCas9

<400>　4

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly

100 105 110

Ser Thr Asn Arg Gln Ala Ala Lys Leu Ser Lys Pro Thr Leu Glu Asn

115 120 125

Leu Thr Pro Val Val Leu Arg Pro Glu Ile Arg Lys Pro Glu Val Leu

130 135 140

Arg Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Phe Gly

145 150 155 160

Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr

165 170 175

Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val

180 185 190

Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile

195 200 205

Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val

210 215 220

Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu

225 230 235 240

Glu Leu Ala Gln Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val

245 250 255

Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala

260 265 270

Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn

275 280 285

Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu

290 295 300

Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu

305 310 315 320

Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu

325 330 335

Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu

340 345 350

Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr

355 360 365

Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser

370 375 380

Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser

385 390 395 400

Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val

405 410 415

Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys

420 425 430

Lys Leu Phe Phe Lys Thr Ser

435

<210>　5

<211>　8

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　CDRH1

<400>　5

Gly Phe Thr Phe Asn Thr Tyr Ala

1 5

<210>　6

<211>　10

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　CDRH2

<400>　6

Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr

1 5 10

<210>　7

<211>　10

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　CDRH3

<400>　7

Val Arg Gly Gly Tyr Trp Ser Phe Asp Val

1 5 10

<210>　8

<211>　11

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　CDRL2

<400>　8

Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr

1 5 10

<210>　9

<211>　9

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　CDRL3

<400>　9

Met Gln His Leu Glu Tyr Pro Tyr Thr

1 5

<210>　10

<211>　22

<212>　PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 2A peptide

<400>　10

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210>　11

<211>　738

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding anti-HLA-G antibody fragment

<400> 11

gaggttcagc tgcaagagtc tggcggagga ctggtgcagc ctaagggaag cctgaagctg 60

agctgtgccg ccttcggctt caccttcaac acctacgcca tgcactgggt ccgacaggcc 120

cctggaaaag gccttgaatg ggtcgcccgg atcagaagca agagcaacaa ttacgccacc 180

tactacgccg acagcgtgaa ggacagattc accatcagcc gggacgacag ccagagcatg 240

ctgagcctgc agatgaacaa cctgaaaacc gaggacaccg ccatctacta ctgcgtcaga 300

ggcggctact ggtccttcga tgtttgggga gccggcacca ccgtgacagt ttctagcgga 360

ggcggtggat ctggcggcgg aggaagtggt ggcggaggtt ctgatatcgt gatcacccag 420

accacaccta gcgtgccagt gacacctggc gagagcgtgt ccatcagctg cagaagcagc 480

aagagcctgc tgcacagcaa cggcaatacc tacctgtact ggttcctgca gaggcccgga 540

cagtctcctc agctgctgat ctccagaatg agcagcctgg ctagcggcgt gcccgataga 600

ttttctggca gcggctctgg caccgccttc acactgagaa tcagcagagt ggaagccgag 660

gacgtgggcg tgtactactg tatgcagcac ctggaatacc cctacacctt cggcggaggc 720

accaagctgg aaatcaag 738

<210>　12

<211>　225

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding HLA-G receptor fragment

<400>　12

tcacccactg aaccaagctc caaaaccggt aaccccagac acctgcatgt tctgattggg 60

acctcagtgg tcaaaatccc tttcaccatc ctcctcttct ttctccttca tcgctggtgc 120

tccaacaaaa aaaatgctgc tgtaatggac caagagcctg cagggaacag aacagtgaac 180

agcgaggatt ctgatgaaca agaccatcag gaggtgtcat acgca 225

<210>　13

<211>　339

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding Co-stimulatory Domain fragments

<400>　13

atggggggac ttgaaccctg cagcaggctc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggc ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaa 339

<210>　14

<211>　1317

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>　iCas9

<400>　14

atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60

cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt ggattcctcc 120

cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180

gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240

tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300

gatgtggagc ttctaaaact ggaatctgga ggaggttcta ctaacaggca agcagcaaag 360

ttgtcgaagc caaccctaga aaaccttacc ccagtggtgc tcagaccaga gattcgcaaa 420

ccagaggttc tcagaccgga aacacccaga ccagtggaca ttggttctgg aggatttggt 480

gatgtcggtg ctcttgagag tttgagggga aatgcagatt tggcttacat cctgagcatg 540

gagccctgtg gccactgcct cattatcaac aatgtgaact tctgccgtga gtccgggctc 600

cgcacccgca ctggctccaa catcgactgt gagaagttgc ggcgtcgctt ctcctcgctg 660

catttcatgg tggaggtgaa gggcgacctg actgccaaga aaatggtgct ggctttgctg 720

gagctggcgc agcaggacca cggtgctctg gactgctgcg tggtggtcat tctctctcac 780

ggctgtcagg ccagccacct gcagttccca ggggctgtct acggcacaga tggatgccct 840

gtgtcggtcg agaagattgt gaacatcttc aatgggacca gctgccccag cctgggaggg 900

aagcccaagc tctttttcat ccaggcctgt ggtggggagc agaaagacca tgggtttgag 960

gtggcctcca cttcccctga agacgagtcc cctggcagta accccgagcc agatgccacc 1020

ccgttccagg aaggtttgag gaccttcgac cagctggacg ccatatctag tttgcccaca 1080

cccagtgaca tctttgtgtc ctactctact ttcccaggtt ttgtttcctg gagggacccc 1140

aagagtggct cctggtacgt tgagaccctg gacgacatct ttgagcagtg ggctcactct 1200

gaagacctgc agtccctcct gcttagggtc gctaatgctg tttcggtgaa agggatttat 1260

aaacagatgc ctggttgctt taatttcctc cggaaaaaac ttttctttaa aacatca 1317

<210>　15

<211>　66

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 2A peptide

<400>　15

ggatctggcg ccaccaactt cagcctgctg aagcaggcag gcgacgtgga agagaaccct 60

ggccct 66

<210>　16

<211>　1335

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> promoter

<400>　16

gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60

ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120

gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180

gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240

agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300

ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360

acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420

ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480

attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540

ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600

cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660

aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720

caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780

tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840

gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900

ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960

cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020

ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080

tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140

tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200

gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260

gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320

tttcaggtgt cgtga 1335

<210>　17

<211>　63

<212>　DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding informative peptide fragments

<400>　17

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

Claims

1. A chimeric antigen receptor specifically binding to human leukocyte antigen G (HLA-G), comprising an anti-HLA-G antibody represented by SEQ ID NO 1, an HLA-G receptor represented by SEQ ID NO 2, a 2A peptide represented by SEQ ID NO 10, and a co-stimulatory domain represented by SEQ ID NO 3, arranged in this order from the N-terminus to the C-terminus, wherein the 2A peptide is in tandem with the HLA-G receptor and the co-stimulatory domain.

2. An isolated nucleic acid encoding the chimeric antigen receptor of claim 1, wherein the nucleic acid comprises an anti-HLA-G antibody-encoding fragment shown in SEQ ID NO. 11, an HLA-G receptor-encoding fragment shown in SEQ ID NO. 12, a 2A peptide-encoding sequence shown in SEQ ID NO. 15, and a costimulatory domain-encoding fragment shown in SEQ ID NO. 13, arranged in sequence, wherein the 2A peptide-encoding sequence is concatenated with the HLA-G receptor-encoding fragment and the costimulatory domain-encoding fragment.

3. A chimeric antigen receptor-expressing plastid, comprising:

the promoter as shown in SEQ ID No. 16 and the nucleic acid of claim 2, arranged sequentially.

4. The chimeric antigen receptor-expressing plastid of claim 3, further comprising a suicide gene as set forth in SEQ ID No. 14 linked to the 3' end of said nucleic acid.

5. A cell expressing a chimeric antigen receptor, comprising:

an immune cell; and

the chimeric antigen receptor-expressing plastid of claim 3 or 4.

6. The chimeric antigen receptor-expressing cell of claim 5, wherein the immune cell is a T cell.

7. The chimeric antigen receptor-expressing cell of claim 5, wherein the immune cell is a natural killer cell.

8. The chimeric antigen receptor-expressing cell of claim 7, wherein the natural killer cell is an NK-92 cell line or a primary natural killer cell.

9. A pharmaceutical composition for treating cancer, comprising:

the chimeric antigen receptor-expressing cell of claim 5; and

a pharmaceutically acceptable carrier.

10. The pharmaceutical composition for treating cancer according to claim 9, further comprising a chemotherapeutic agent.

11. The pharmaceutical composition for treating cancer according to claim 10, wherein the chemotherapeutic agent is doxorubicin, temozolomide, gemcitabine or carboplatin.

12. Use of the chimeric antigen receptor-expressing cell of claim 5, for the preparation of a medicament for inducing death of mammalian tumor cells, wherein the tumor cells are breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells, or ovarian cancer cells.