HLA-G specific chimeric antigen receptor, nucleic acid, expression plasmid, cell, use and composition thereof

Abstract

The present invention discloses an HLA-G specific chimeric antigen receptor, its nucleic acid, expression plasmid, cell, use and composition, HLA-G specific chimeric antigen receptor, isolated nucleic acid, HLA-G specific chimeric antigen receptor expression plasmid, HLA-G specific chimeric antigen receptor cell, pharmaceutical composition for treating cancer and use of HLA-G specific chimeric antigen receptor cell. HLA-G specific chimeric antigen receptor binds specifically to human leukocyte antigen G. Cells expressing HLA-G specific chimeric antigen receptor are obtained by transducing HLA-G specific chimeric antigen receptor into immune cells. A pharmaceutical composition for treating cancer comprises cells expressing HLA-G specific chimeric antigen receptor and a pharmaceutically acceptable carrier. Thereby, cells expressing HLA-G specific chimeric antigen receptor can be used to induce tumor cell death in mammals.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical product containing an antigen or an antibody, in particular to a chimeric antigen receptor, a nucleic acid encoding a chimeric antigen receptor, a chimeric antigen receptor expression plasmid, a chimeric antigen receptor expressing cell, a pharmaceutical composition for treating cancer, and a use of a chimeric antigen receptor expressing cell. Background Art

[0002] Cancer, also known as malignant tumor, is an abnormal proliferation of cells that may invade other parts of the body. It is a disease caused by the abnormal control of cell division and proliferation. The number of people suffering from cancer in the world is increasing. Cancer is one of the top ten causes of death in China and has been ranked first for 27 consecutive years.

[0003] Conventional tumor treatment methods include surgery, radiotherapy, chemotherapy and targeted therapy. Tumor immunotherapy is another method of treating tumors besides the above treatment methods. It activates the patient's own immune system and uses tumor cells or tumor antigens to induce the body's specific cellular and humoral immune responses, enhance the body's anti-cancer ability, prevent tumor growth, spread and recurrence, and achieve the purpose of eliminating or controlling tumors.

[0004] There are three main directions in tumor immunotherapy - tumor vaccines, cell therapy and immune checkpoint inhibitors, among which chimeric antigen receptor immune cell technology is a cell therapy that has developed very rapidly in recent years. In the known technology, chimeric antigen receptor immune cells are a chimeric protein that couples the antigen binding part of an antibody that can recognize a certain tumor antigen with the intracellular part of the CD3-δ chain or FcεRIγ in vitro, and transfects the patient's immune cells through gene transduction to express chimeric antigen receptors. It has significant therapeutic effects in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered to be one of the most promising tumor treatment methods. However, the current cell therapy of chimeric antigen receptor immune cells is greatly limited in the efficacy of chimeric antigen receptors in solid tumors due to the lack of unique tumor-associated antigens, the low efficiency of immune cells homing to the tumor site, and the inability to overcome the immunosuppressive microenvironment of solid tumors. Summary of the invention

[0005] The purpose of the present invention is to provide an HLA-G specific chimeric antigen receptor, an isolated nucleic acid, an HLA-G specific chimeric antigen receptor expression plasmid, an HLA-G specific chimeric antigen receptor expression cell, a pharmaceutical composition for treating cancer, and the use of an HLA-G specific chimeric antigen receptor expression cell. The HLA-G specific chimeric antigen receptor has excellent specific binding ability to tumor cells. The nucleic acid encodes the HLA-G specific chimeric antigen receptor. The HLA-G specific chimeric antigen receptor expression plasmid contains the nucleic acid. The HLA-G specific chimeric antigen receptor expression cell contains the HLA-G specific chimeric antigen receptor expression plasmid, which expresses the HLA-G specific chimeric antigen receptor, can specifically target tumor cells, avoid off-target effects, and effectively kill tumor cells, so that it can be used to prepare a drug that induces tumor cell death in mammals. The pharmaceutical composition for treating cancer contains the HLA-G specific chimeric antigen receptor expression cell, which can effectively kill tumor cells and treat cancer.

[0006] According to one embodiment of one purpose of the present invention, there is provided an HLA-G specific chimeric antigen receptor, comprising an antigen recognition domain, a transmembrane domain, an IL2 receptor β chain signaling domain, and a CD3ζ signaling domain. The aforementioned antigen recognition domain comprises a monoclonal antibody fragment specific to human leukocyte antigen G (human leukocyte antigen G, HLA-G), and the amino acid sequence of the antigen recognition domain is shown in sequence identification number 1. The amino acid sequence of the aforementioned transmembrane domain is shown in sequence identification number 2. The amino acid sequence of the aforementioned IL2 receptor β chain signaling domain is shown in sequence identification number 3. The amino acid sequence of the aforementioned CD3ζ signaling domain is shown in sequence identification number 4.

[0007] According to the aforementioned HLA-G specific chimeric antigen receptor, it may further comprise a signal peptide chain as shown in sequence identification number 5, which is connected to the N-terminus of the antigen recognition domain.

[0008] According to the aforementioned HLA-G specific chimeric antigen receptor, it may further comprise a CD8 hinge region, wherein the aforementioned CD8 hinge region connects the antigen recognition domain and the transmembrane domain.

[0009] Another embodiment of one object of the present invention is to provide an isolated nucleic acid encoding the HLA-G specific chimeric antigen receptor as described in the preceding paragraph. The nucleic acid comprises a fragment encoding an antigen recognition domain as shown in sequence identification number 12, a fragment encoding a transmembrane domain as shown in sequence identification number 13, a fragment encoding an IL2 receptor β chain signaling domain as shown in sequence identification number 14, and a fragment encoding a CD3ζ signaling domain as shown in sequence identification number 15, which are arranged in sequence.

[0010] The isolated nucleic acid described above may further comprise a signal peptide chain fragment as shown in sequence identification number 16, which is connected to the 5' end of the fragment encoding the antigen recognition domain.

[0011] The isolated nucleic acid described above may further comprise a sequence encoding a CD8 hinge region, wherein the sequence encoding a CD8 hinge region is connected to a fragment encoding an antigen recognition domain and a fragment encoding a transmembrane domain.

[0012] Another embodiment of one object of the present invention is to provide an HLA-G specific chimeric antigen receptor expression plasmid, wherein the chimeric antigen receptor expression plasmid comprises a promoter as shown in sequence identification number 18 and a nucleic acid as described in the preceding paragraph.

[0013] Another embodiment of one object of the present invention is to provide a cell expressing an HLA-G specific chimeric antigen receptor, comprising an immune cell and an HLA-G specific chimeric antigen receptor expression plasmid as described in the preceding paragraph.

[0014] According to the aforementioned cells expressing HLA-G specific chimeric antigen receptor, the immune cells may be T cells or natural killer cells. Preferably, the natural killer cells may be NK-92 cell lines or primary natural killer cells.

[0015] Another embodiment of an object of the present invention is to provide a pharmaceutical composition for treating cancer, comprising a cell expressing HLA-G specific chimeric antigen receptor as described in the preceding paragraph, and a pharmaceutically acceptable carrier.

[0016] The pharmaceutical composition for treating cancer described above may further comprise a chemotherapeutic drug. Preferably, the chemotherapeutic drug may be doxorubicin, temozolomide, gemcitabine or carboplatin.

[0017] Another object of the present invention is to provide a use of a cell expressing an HLA-G specific chimeric antigen receptor as described in the preceding paragraph for preparing a drug for inducing death of tumor cells in mammals, wherein the tumor cells may be breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.

[0018] The above summary of the invention is intended to provide a simplified summary of the invention so that readers can have a basic understanding of the invention. This summary of the invention is not a complete overview of the invention and is not intended to point out important / critical elements of the embodiments of the invention or to define the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the following description is made with reference to and in conjunction with the accompanying drawings:

[0020] Figure 1 A schematic diagram of the protein structure of the antigen recognition domain of the present invention is depicted;

[0021] Figure 2 A schematic diagram showing the construction of the HLA-G specific chimeric antigen receptor expression plasmid of the present invention is shown;

[0022] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F , Figure 3G and Figure 3H This is a diagram showing the analysis results of tumor cell death induced by cells expressing HLA-G specific chimeric antigen receptor according to Example 1 of the present invention;

[0023] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F , Figure 4G and Figure 4H This is a diagram showing the analysis results of the HLA-G-specific chimeric antigen receptor-expressing cells inducing tumor cell death according to Example 2 of the present invention; and

[0024] Figure 5 This is a statistical graph showing the results of flow cytometry analysis of HLA-G expression in tumor cells after chemotherapy. DETAILED DESCRIPTION

[0025] The disclosure of this specification provides an HLA-G specific chimeric antigen receptor, a nucleic acid encoding the HLA-G specific chimeric antigen receptor, an HLA-G specific chimeric antigen receptor expression plasmid containing the nucleic acid, an HLA-G specific chimeric antigen receptor expressing cell containing the HLA-G specific chimeric antigen receptor expression plasmid, uses thereof, and a pharmaceutical composition for treating cancer containing the HLA-G specific chimeric antigen receptor expressing cell. In the specification, a cell experiment of tumor cells is used to prove that the HLA-G specific chimeric antigen receptor of the present invention has excellent specific binding ability to tumor cells, especially specific binding to human leukocyte antigen G (HLA-G) expressed on the cell membrane of tumor cells, so that the HLA-G specific chimeric antigen receptor expressing cell expressing the HLA-G specific chimeric antigen receptor of the present invention can specifically target tumor cells, avoid off-target effects, and then effectively kill tumor cells, so that it can be used to prepare a drug for inducing tumor cell death in mammals. The pharmaceutical composition for treating cancer of the present invention comprises the cell expressing HLA-G specific chimeric antigen receptor of the present invention, and may further comprise a chemotherapeutic drug, which can effectively kill tumor cells and thus treat cancer.

[0026] The "human leukocyte antigen G (HLA-G)" mentioned in this specification is encoded by the HLA-G gene, which is an atypical class I major histocompatibility complex (MHC) with a heavy chain of approximately 45 kDa. HLA-G is expressed on placental cells of fetal origin and is very active in the negative regulation of immune responses. Its main function is to inhibit the function of cytotoxic immune cells.

[0027] The present invention is further illustrated by the following specific test examples, which are used to facilitate those skilled in the art to which the present invention belongs, so that the present invention can be fully utilized and practiced without excessive interpretation. These test examples should not be regarded as limiting the scope of the present invention, but are used to illustrate how to implement the materials and methods of the present invention.

[0028] [Test example]

[0029] 1. HLA-G specific chimeric antigen receptor, isolated nucleic acid and HLA-G specific chimeric antigen receptor expression plasmid of the present invention

[0030] The HLA-G specific chimeric antigen receptor of the present invention comprises an antigen recognition domain as shown in sequence identification number 1, a transmembrane domain as shown in sequence identification number 2, an IL2 receptor β chain signaling domain as shown in sequence identification number 3, and a CD3ζ signaling domain as shown in sequence identification number 4. The antigen recognition domain comprises a monoclonal antibody fragment specific to human leukocyte antigen G (human leukocyte antigen G, HLA-G). Preferably, the N-terminus of the antigen recognition domain may also be connected to a signal peptide chain as shown in sequence identification number 5, and further comprises a CD8 hinge region as shown in sequence identification number 11 to connect the antigen recognition domain and the transmembrane domain. In detail, the antigen recognition domain shown in sequence identification number 1 comprises a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. The heavy chain immunoglobulin variable domain sequence is CDRH1 as shown in sequence identification number 6, CDRH2 as shown in sequence identification number 7, and CDRH3 as shown in sequence identification number 8. The sequence of the light chain immunoglobulin variable domain is CDRL1 as shown in sequence identification number 9, CDRL2 with the sequence RMS, and CDRL3 as shown in sequence identification number 10. Figure 1 , a schematic diagram of the protein structure of the antigen recognition domain of the present invention is drawn, wherein the dotted ring region represents the variable domain in the antigen recognition domain of the present invention.

[0031] The isolated nucleic acid of the present invention encodes the HLA-G specific chimeric antigen receptor of the present invention. The nucleic acid comprises a sequence-arranged fragment encoding an antigen recognition domain as shown in sequence identification number 12, a sequence-arranged fragment encoding a transmembrane domain as shown in sequence identification number 13, a sequence-arranged fragment encoding an IL2 receptor β chain signaling domain as shown in sequence identification number 14, and a sequence-arranged fragment encoding a CD3ζ signaling domain as shown in sequence identification number 15. Preferably, the 5' end of the fragment encoding the antigen recognition domain may be further connected to a sequence-arranged fragment encoding a signal peptide chain as shown in sequence identification number 16, and may further comprise a sequence-arranged fragment encoding a CD8 hinge region as shown in sequence identification number 17 to connect the fragment encoding the antigen recognition domain and the fragment encoding the transmembrane domain. The antigen recognition domain encoding fragment shown in sequence identification number 12 encodes the antigen recognition domain shown in sequence identification number 1, the transmembrane domain encoding fragment shown in sequence identification number 13 encodes the transmembrane domain shown in sequence identification number 2, the IL2 receptor β chain signaling domain encoding fragment shown in sequence identification number 14 encodes the IL2 receptor β chain signaling domain shown in sequence identification number 3, the CD3ζ signaling domain encoding fragment shown in sequence identification number 15 encodes the CD3ζ signaling domain shown in sequence identification number 4, the signal peptide chain encoding fragment shown in sequence identification number 16 encodes the signal peptide chain shown in sequence identification number 5, and the CD8 hinge region encoding fragment shown in sequence identification number 17 encodes the CD8 hinge region shown in sequence identification number 11.

[0032] Please refer to Figure 2, depicting a schematic diagram of the construction of the HLA-G specific chimeric antigen receptor expression plasmid of the present invention. In detail, in one embodiment shown in this test example, the HLA-G specific chimeric antigen receptor expression plasmid is a Lenti-EF1a-H-28-IL2RB-Z plasmid, and its insert fragment includes a promoter, a fragment encoding an antigen recognition domain, a fragment encoding a transmembrane domain, a fragment encoding an IL2 receptor β chain signaling domain, and a fragment encoding a CD3ζ signaling domain. The promoter is the EF-1alpha promoter as shown in sequence identification number 18, the sequence encoding the antigen recognition domain fragment is as shown in sequence identification number 12, the sequence encoding the transmembrane domain fragment is as shown in sequence identification number 13, the sequence encoding the IL2 receptor β chain signaling domain fragment is as shown in sequence identification number 14, and the sequence encoding the CD3ζ signaling domain fragment is as shown in sequence identification number 15. In addition, the insert also includes a coding signal peptide chain fragment as shown in sequence identification number 16 and a coding CD8 hinge region sequence as shown in sequence identification number 17. The coding signal peptide chain fragment is connected to the 5' end of the coding antigen recognition domain fragment, and the coding CD8 hinge region sequence connects the coding antigen recognition domain fragment and the coding transmembrane domain fragment. The insert fragment is then constructed on a Creative Biolabs vector (Creative Biolabs, NY, USA) to obtain a Lenti-EF1a-H-28-IL2RB-Z plasmid. The vector used is a lentivirus vector system, so the constructed HLA-G specific chimeric antigen receptor expression plasmid can be transfected into the expression cell to produce a lentivirus, and the lentivirus can be used to transduce the HLA-G specific chimeric antigen receptor into immune cells.

[0033] 2. HLA-G-specific chimeric antigen receptor expressing cells of the present invention, their uses, and pharmaceutical compositions for treating cancer

[0034] The HLA-G specific chimeric antigen receptor expressing cell of the present invention is obtained by transducing the HLA-G specific chimeric antigen receptor of the present invention into immune cells using lentivirus. Preferably, the immune cell may be a T cell or a natural killer cell. More preferably, the natural killer cell may be an NK-92 cell line or a primary natural killer cell. In detail, the constructed Lenti-EF1a-H-28-IL2RB-Z plasmid is first transfected into a 293T cell line using lipofectamine 3000 (Invitrogen) to prepare a lentivirus with the HLA-G specific chimeric antigen receptor of the present invention, and then the primary T cells are cultured for 3 days with Opti-MEM (Invitrogen) containing the supernatant of the prepared lentivirus and 8 μg / ml of polybrene (Sigma-Aldrich) to transduce the HLA-G specific chimeric antigen receptor of the present invention into the primary T cells. And the primary natural killer cells are cultured for 7 days in Opti-MEM (Invitrogen) containing the supernatant of the prepared lentivirus and 50 μg / ml protamine (Protamine sulfate, Sigma-Aldrich) to transduce the chimeric antigen receptor of the present invention into the primary natural killer cells or NK-92 cell lines, and the HLA-G specific chimeric antigen receptor expressing cells of the present invention can be obtained. The obtained HLA-G specific chimeric antigen receptor expressing cells have the effect of inducing tumor cell death in mammals, and can be used to prepare drugs for inducing tumor cell death in mammals. Preferably, the tumor cells can be breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.

[0035] The pharmaceutical composition for treating cancer of the present invention comprises the cell expressing HLA-G specific chimeric antigen receptor of the present invention and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition for treating cancer may further comprise a chemotherapeutic drug. More preferably, the chemotherapeutic drug may be doxorubicin, temozolomide, gemcitabine or carboplatin.

[0036] The following test examples will use data to prove that the HLA-G specific chimeric antigen receptor expressing cells of the present invention and the pharmaceutical composition for treating cancer containing the HLA-G specific 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 experiment are 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 SKOV3. The tumor cell lines used are all purchased from the American Type Culture Collection (ATCC). The human breast cancer cell line MDA-MB-231 cell line is a triple-negative breast cancer cell line, that is, hormone receptors (ER, PR) and HER-2 receptors are negative, and it is cultured in RPMI culture medium containing 10% fetal bovine serum (FBS). The human malignant brain tumor cell line DBTRG is cultured in DMEM culture medium containing 10% fetal bovine serum. The human pancreatic cancer cell line AsPC1 was cultured in RPMI medium containing 10% fetal bovine serum. The human ovarian cancer cell line SKOV3 was cultured in McCoy's 5A medium containing 10% fetal bovine serum.

[0037] 2.1. Example 1

[0038] In this test example, the HLA-G specific chimeric antigen receptor of the present invention is transduced into primary natural killer cells to obtain the HLA-G specific chimeric antigen receptor expressing cells of Example 1 of the present invention (hereinafter referred to as Example 1). The effect of Example 1 and the pharmaceutical composition for treating cancer comprising Example 1 on inducing death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells was further tested.

[0039] First, 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 were respectively cultured at 1×10 5The cells were seeded in a 12-well plate at a density of 10 cells / well and then cultured for 48 hours before the test. In the test, each tumor cell was divided into 6 groups, namely, an untreated control group, a test group 1 treated with chemotherapy drugs, a test group 2 treated with parental primary natural killer cells, a test group 3 treated with parental primary natural killer cells and chemotherapy drugs, a test group 4 treated with Example 1, and a test group 5 treated with Example 1 and chemotherapy drugs. In the group of human breast cancer cell line MDA-MB-231, the chemotherapy drug used was doxorubicin (200nM); in the group of human malignant brain tumor cell line DBTRG, the chemotherapy drug used was temozolomide (80μg / ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapy drug used was gemcitabine (20μM); in the group of human ovarian cancer cell line SKOV3, the chemotherapy drug used was carboplatin (20μM). In test groups 4 and 5, the number of cells treated with Example 1 was 1×10 5 cells. The number of parental primary natural killer cells treated in Experimental Groups 2 and 3 was also 1×10 5 The treated cells in each group were then stained with Annexin V-FITC and PI, and the total percentage of cells stained with Annexin V-FITC and / or PI was calculated to obtain the cytotoxic effect. Each group was tested independently in triplicate, and the cytotoxic results were statistically analyzed.

[0040] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F , Figure 3G and Figure 3H , is a diagram showing the analysis results of inducing tumor cell death in Example 1. Figure 3A This is a diagram showing the analysis results of inducing death of human breast cancer cell line MDA-MB-231 in Example 1. Figure 3B for Figure 3A Statistical graph after three repeated experiments. Figure 3C This is the analysis result of inducing death of human malignant brain tumor cell line DBTRG in Example 1. Figure 3D for Figure 3C Statistical graph after three repeated experiments. Figure 3E This is a diagram showing the analysis results of inducing death of human pancreatic cancer cell line AsPC1 in Example 1. Figure 3F for Figure 3E Statistical graph after three repeated experiments. Figure 3G This is the analysis result of inducing death of human ovarian cancer cell line SKOV3 in Example 1. Figure 3H for Figure 3G Statistical graph after three repeated experiments. Figure 3B , Figure 3D , Figure 3F and Figure 3H The P in represents parental primary natural killer cells.

[0041] Depend on Figure 3A and Figure 3B The results showed that in the untreated control group, only about 5% of the human breast cancer cell line MDA-MB-231 died, while the mortality rate of the human breast cancer cell line MDA-MB-231 increased in the test group 1 treated with doxorubicin, the test group 2 treated with parental primary natural killer cells, and the test group 3 treated with parental primary natural killer cells and doxorubicin, but there was no statistically significant difference. The mortality rate of the human breast cancer cell line MDA-MB-231 induced by the test group 4 treated with Example 1 was about 70%, which was statistically significant compared with the test group 2 (p<0.001). In addition, the mortality rate of the human breast cancer cell line MDA-MB-231 induced by the test group 5 treated with Example 1 and doxorubicin was as high as more than 80%, which was statistically significant compared with the test group 4 (p<0.05) and the test group 3 (p<0.001).

[0042] Depend on Figure 3C and Figure 3D The results showed that in the untreated control group, less than 5% of the human malignant brain tumor cell line DBTRG died, while in the test group 1 treated with temozolomide, the test group 2 treated with parental primary natural killer cells, and the test group 3 treated with parental primary natural killer cells and temozolomide, although the mortality rate of the human malignant brain tumor cell line DBTRG increased, there was no statistically significant difference. The mortality rate of the human malignant brain tumor cell line DBTRG induced by the test group 4 treated with Example 1 can exceed 60%, which is statistically significant compared with the test group 2 (p<0.001). In addition, the mortality rate of the human malignant brain tumor cell line DBTRG induced by the test group 5 treated with Example 1 and temozolomide can be close to 90%, which is statistically significant compared with the test group 4 (p<0.01) and the test group 3 (p<0.001).

[0043] Depend on Figure 3E and Figure 3FThe results showed that in the untreated control group, less than 5% of the human pancreatic cancer cell line AsPC1 died, while in the experimental group 1 treated with gemcitabine, the experimental group 2 treated with parental primary natural killer cells, and the experimental group 3 treated with parental primary natural killer cells and gemcitabine, although the mortality rate of the human pancreatic cancer cell line AsPC1 increased, there was no statistically significant difference. The mortality rate of the human pancreatic cancer cell line AsPC1 induced by the experimental group 4 treated with Example 1 was about 50%, which was statistically significant compared with the experimental group 2 (p<0.01). In addition, the mortality rate of the human pancreatic cancer cell line AsPC1 induced by the experimental group 5 treated with Example 1 and gemcitabine reached about 70%, which was statistically significant compared with the experimental group 4 (p<0.05), and also statistically significant compared with the experimental group 3 (p<0.001).

[0044] Depend on Figure 3G and Figure 3H The results showed that in the untreated control group, less than 5% of the human ovarian cancer cell line SKOV3 died, while in the test group 1 treated with carboplatin and the test group 2 treated with parental primary natural killer cells, although the mortality rate of the human ovarian cancer cell line SKOV3 increased, there was no statistically significant difference. In the test group 3 treated with parental primary natural killer cells and carboplatin, the mortality rate of the human ovarian cancer cell line SKOV3 reached nearly 40%, but there was still no statistically significant difference. The mortality rate of the human ovarian cancer cell line SKOV3 induced by the test group 4 treated with Example 1 was about 50%, which was statistically significant compared with the test group 2 (p<0.05). In addition, the mortality rate of the human ovarian cancer cell line SKOV3 induced by the test group 5 treated with Example 1 and carboplatin reached nearly 70%, which was statistically significant compared with the test group 4 (p<0.05) and the test group 3 (p<0.01).

[0045] Depend on FIG. 3A to FIG. 3H The results show that the treatment example 1 has excellent cytotoxic effects on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, and the cells expressing the HLA-G specific chimeric antigen receptor of the present invention can be used to prepare drugs for inducing tumor cell death in mammals. In addition, the simultaneous treatment of example 1 and chemotherapeutic drugs has a more significant cytotoxic 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 preferably includes chemotherapeutic drugs, which can effectively kill tumor cells and thus treat cancer.

[0046] 2.2. Example 2

[0047] In this test example, the HLA-G specific chimeric antigen receptor of the present invention is transduced into primary T cells to obtain the HLA-G specific chimeric antigen receptor expressing cells of Example 2 of the present invention (hereinafter referred to as Example 2). The effect of Example 2 and the pharmaceutical composition for treating cancer comprising Example 2 on inducing the death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells was further tested.

[0048] First, 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 were respectively cultured at 1×10 5 The cells were seeded in a 12-well plate at a density of 10 cells / well and then cultured for 48 hours before the test. In the test, each tumor cell was divided into 6 groups, namely, an untreated control group, a test group 1 treated with chemotherapy drugs, a test group 2 treated with parental primary T cells, a test group 3 treated with parental primary T cells and chemotherapy drugs, a test group 4 treated with Example 2, and a test group 5 treated with Example 2 and chemotherapy drugs. In the group of human breast cancer cell line MDA-MB-231, the chemotherapy drug used was doxorubicin (200nM); in the group of human malignant brain tumor cell line DBTRG, the chemotherapy drug used was temozolomide (80μg / ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapy drug used was gemcitabine (20μM); in the group of human ovarian cancer cell line SKOV3, the chemotherapy drug used was carboplatin (20μM). In test groups 4 and 5, the number of cells treated with Example 2 was 1×10 5 cells. The number of primary T cells treated in Experimental Groups 2 and 3 was also 1×10 5 The treated cells in each group were then stained with Annexin V-FITC and PI, and the cell apoptosis and death were detected by flow cytometry, and the total percentage of cells stained with Annexin V-FITC and / or PI was calculated to obtain the cytotoxic effect. Each group was tested independently in triplicate, and the cytotoxic results were statistically analyzed.

[0049] Please refer to Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F , Figure 4G and Figure 4H , is the analysis result diagram of tumor cell death induced by Example 2. Figure 4A This is the analysis result of Example 2 inducing the death of human breast cancer cell line MDA-MB-231. Figure 4B for Figure 4A Statistical graph after three repeated experiments. Figure 4C This is the analysis result of inducing death of human malignant brain tumor cell line DBTRG in Example 2. Figure 4D for Figure 4C Statistical graph after three repeated experiments. Figure 4E This is a diagram showing the analysis results of inducing death of human pancreatic cancer cell line AsPC1 in Example 2. Figure 4F for Figure 4E Statistical graph after three repeated experiments. Figure 4G This is the analysis result of inducing death of human ovarian cancer cell line SKOV3 in Example 2. Figure 4H for Figure 4G Statistical chart of . Figure 4B , Figure 4D , Figure 4F and Figure 4H The P in the figure indicates parental primary T cells.

[0050] Depend on Figure 4A and Figure 4B The results showed that in the untreated control group, only about 10% of the human breast cancer cell line MDA-MB-231 died, while the mortality rate of the human breast cancer cell line MDA-MB-231 increased in the test group 1 treated with doxorubicin, the test group 2 treated with parental primary T cells, and the test group 3 treated with parental primary T cells and doxorubicin, but there was no statistically significant difference. The mortality rate of the human breast cancer cell line MDA-MB-231 induced by the test group 4 treated with Example 2 was close to 70%, which was statistically significant compared with the test group 2 (p<0.01). In addition, the mortality rate of the human breast cancer cell line MDA-MB-231 induced by the test group 5 treated with Example 2 and doxorubicin was close to 80%, which was statistically significant compared with the test group 4 (p<0.05) and the test group 3 (p<0.01).

[0051] Depend on Figure 4C and Figure 4DThe results showed that in the untreated control group, less than 10% of the human malignant brain tumor cell line DBTRG died, while in the test group 1 treated with temozolomide and the test group 2 treated with parental primary T cells, although the mortality rate of the human malignant brain tumor cell line DBTRG increased, there was no statistically significant difference. In the test group 3 treated with parental primary T cells and temozolomide, the mortality rate of the human malignant brain tumor cell line DBTRG can be increased to nearly 40%, but there is still no statistically significant difference. The mortality rate of the human malignant brain tumor cell line DBTRG induced by the test group 4 treated with Example 2 can exceed 70%, which is statistically significant compared with the test group 2 (p<0.001). In addition, the mortality rate of the human malignant brain tumor cell line DBTRG induced by the test group 5 treated with Example 2 and temozolomide can be close to 80%, which is statistically significant compared with the test group 4 (p<0.05), and also statistically significant compared with the test group 3 (p<0.001).

[0052] Depend on Figure 4E and Figure 4F The results showed that in the untreated control group, only about 5% of the human pancreatic cancer cell line AsPC1 died, while in the experimental group 1 treated with gemcitabine and the experimental group 2 treated with parental primary T cells, although the mortality rate of the human pancreatic cancer cell line AsPC1 increased, it was not statistically significant. In the experimental group 3 treated with parental primary T cells and gemcitabine, the mortality rate of the human pancreatic cancer cell line AsPC1 can be increased to nearly 40%. The mortality rate of the human pancreatic cancer cell line AsPC1 induced by the experimental group 4 treated with Example 2 can be increased to more than 30%, which is statistically significant compared with the experimental group 2 (p<0.01). In addition, the mortality rate of the human pancreatic cancer cell line AsPC1 induced by the experimental group 5 treated with Example 2 and gemcitabine is more than 50%, which is statistically significant compared with the experimental group 4 (p<0.01), and also statistically significant compared with the experimental group 3 (p<0.05).

[0053] Depend on Figure 4G and Figure 4HThe results showed that in the untreated control group, less than 5% of the human ovarian cancer cell line SKOV3 died, while the mortality rate of the human ovarian cancer cell line SKOV3 in the test group 1 treated with carboplatin, the test group 2 treated with parental primary T cells, and the test group 3 treated with parental primary T cells and carboplatin increased, but there was no statistically significant difference. The mortality rate of the human ovarian cancer cell line SKOV3 induced by the test group 4 treated with Example 2 reached nearly 80%, which was statistically significant compared with the test group 2 (p<0.001). In addition, the mortality rate of the human ovarian cancer cell line SKOV3 induced by the test group 5 treated with Example 2 and carboplatin reached more than 80%, which was statistically significant compared with the test group 4 (p<0.05) and the test group 3 (p<0.001).

[0054] Depend on 4A to 4H The results show that the treatment example 2 has excellent cytotoxic effects on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, and the cells expressing the HLA-G specific chimeric antigen receptor of the present invention can be used to prepare drugs for inducing tumor cell death in mammals. In addition, the simultaneous treatment of example 2 and chemotherapeutic drugs has a more significant cytotoxic 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 preferably includes chemotherapeutic drugs, which can effectively kill tumor cells and thus treat cancer.

[0055] 2.3. Treatment with chemotherapy drugs increases the expression of human leukocyte antigen G on the cell membrane of tumor cells

[0056] This test example further explores the reason why the group treated simultaneously with the HLA-G-specific chimeric antigen receptor cells of the present invention and the chemotherapeutic drugs has a more excellent tumor cell-killing effect.

[0057] First, 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 were cultured at 2×10 5The cells were seeded in a 6-well plate at a density of cells / well and then cultured the next day for the cells to attach before the test. In the experiment, each tumor cell was divided into two groups, namely an untreated control group and a chemotherapy group treated with chemotherapy drugs for 48 hours. In the group of human breast cancer cell line MDA-MB-231, the chemotherapy drug used was doxorubicin (200nM); in the group of human malignant brain tumor cell line DBTRG, the chemotherapy drug used was temozolomide (80μg / ml); in the group of human pancreatic cancer cell line AsPC1, the chemotherapy drug used was gemcitabine (20μM); in the group of human ovarian cancer cell line SKOV3, the chemotherapy drug used was carboplatin (20μM). The treated tumor cells in each group were then used to detect the expression of HLA-G on the cell surface of the tumor cells by flow cytometry.

[0058] Please refer to Figure 5 , is a statistical graph showing the results of flow cytometry analysis of HLA-G expression in tumor cells after chemotherapy. Figure 5 Compared with the control group and chemotherapy group of the same tumor cells, treatment with doxorubicin can increase the expression of HLA-G on the cell membrane of human breast cancer cell line MDA-MB-231 (p<0.001), treatment with temozolomide can increase the expression of HLA-G on the cell membrane of human malignant brain tumor cell line DBTRG (p<0.001), treatment with gemcitabine can increase the expression of HLA-G on the cell membrane of human pancreatic cancer cell line AsPC1 (p<0.001), and treatment with carboplatin can increase the expression of HLA-G on the cell membrane of human ovarian cancer cell line SKOV3 (p<0.001). Figure 5 The results show that treatment with chemotherapy drugs can increase the expression of HLA-G on the cell membrane of tumor cells, and the HLA-G specific chimeric antigen receptor expressed by the HLA-G specific chimeric antigen receptor expressing cells of the present invention can specifically bind to HLA-G, so that treating the HLA-G specific chimeric antigen receptor expressing cells of the present invention after treating with chemotherapy drugs, or treating the HLA-G specific chimeric antigen receptor expressing cells of the present invention and chemotherapy drugs at the same time, can have a better effect of killing tumor cells.

[0059] In summary, the HLA-G specific chimeric antigen receptor of the present invention has excellent specific binding ability to tumor cells, especially specific binding to HLA-G expressed on the cell membrane of tumor cells, so that the HLA-G specific chimeric antigen receptor expressing cells expressing the HLA-G specific chimeric antigen receptor of the present invention can specifically target tumor cells, avoid off-target effects, and effectively kill tumor cells, so that it can be used to prepare drugs that induce tumor cell death in mammals. The pharmaceutical composition for treating cancer of the present invention, comprising the cell expressing the HLA-G specific chimeric antigen receptor of the present invention, can effectively kill tumor cells and thus treat cancer. The pharmaceutical composition for treating cancer further comprising a chemotherapeutic drug can further induce the cell membrane of tumor cells to express HLA-G in large quantities, thereby enhancing the cytotoxic effect of the cell expressing the HLA-G specific chimeric antigen receptor on tumor cells, and thus has a more excellent tumor cell cytotoxic effect.

[0060] Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Any technician in the field can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims. Sequence Listing <110> China Medical University Hospital, Taiwan <120> HLA-G specific chimeric antigen receptor, nucleic acid, expression plasmid, cell, use and composition thereof <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 246 <212> PRT <213> Artificial Sequence <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> 68 <212> PRT <213> Artificial Sequence <400> 2 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser 65 <210> 3 <211> 94 <212> PRT <213> Artificial Sequence <400> 3 Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn 1 5 10 15 Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly 20 25 30 Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45 Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50 55 60 Arg Asp Lys Val Thr Gln Leu Leu Pro Leu Asn Thr Asp Ala Tyr Leu 65 70 75 80 Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 85 90 <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <400> 4 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Tyr Arg His Gln Ala Leu Pro Pro 100 105 110 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <400> 5 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <400> 6 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <400> 7 Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Val Arg Gly Gly Tyr Trp Ser Phe Asp Val 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <400> 9 Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Met Gln His Leu Glu Tyr Pro Tyr Thr 1 5 <210> 11 <211> 45 <212> PRT <213> Artificial Sequence <400> 11 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 12 <211> 738 <212> DNA <213> Artificial Sequence <400> 12 60. gaggttcagc tgcaagagtc tggcggagga ctggtgcagc ctaaggaag cctgaagctg agctgtgccg ccttcggctt caccttcaac acctacgcca tgcactgggt ccgacaggcc cctggaaaag gccttgaatg ggtcgcccgg atcagaagca agagcaacaa ttacgccacc tactacgccg acagcgtgaa ggacagattc accatcagcc gggacgacag ccagagcatg ctgagcctgc agatgaacaa cctgaaaacc gaggacaccg ccatctacta ctgcgtcaga 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> 13 <211> 204 <212> DNA <213> Artificial Sequence <400> 13 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120 aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180 cgcgacttcg cagcctatcg ctcc 204 <210> 14 <211> 282 <212> DNA <213> Artificial Sequence <400> 14 aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc 60 tcgaagttct tttcccagct gagctcagag catggaggag acgtccagaa gtggctctct 120 tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta 180 gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg 240 tccctccaag aactccaggg tcaggaccca actcacttgg tg 282 <210> 15 <211> 339 <212> DNA <213> Artificial Sequence <400> 15 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300 acctacgacg cctatcgcca ccaggccctg cccccttaa 339 <210> 16 <211> 63 <212> DNA <213> Artificial Sequence <400> 16 atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60 ccc 63 <210> 17 <211> 135 <212> DNA <213> Artificial Sequence <400> 17 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 18 <211> 1335 <212> DNA <213> Artificial Sequence <400> 18 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 agaaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300 ccgagggtgg gggagaaccg tatataagtg footgtcgc 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

Claims

1. An HLA-G specific chimeric antigen receptor, characterized in that Include: An antigen recognition domain, wherein the antigen recognition domain comprises a monoclonal antibody fragment specific to human leukocyte antigen G (HLA-G), and the amino acid sequence of the antigen recognition domain is shown in sequence identification number 1; A transmembrane domain, wherein the amino acid sequence of the transmembrane domain is shown in sequence identification number 2; An IL2 receptor β chain signaling domain, wherein the amino acid sequence of the IL2 receptor β chain signaling domain is as shown in sequence identification number 3; and CD3ζ signal transduction domain, wherein the amino acid sequence of the CD3ζ signal transduction domain is shown in sequence identification number 4.

2. The HLA-G specific chimeric antigen receptor according to claim 1, characterized in that It also comprises a signal peptide chain as shown in sequence identification number 5, which is connected to the N-terminus of the antigen recognition domain.

3. The HLA-G specific chimeric antigen receptor according to claim 1, characterized in that It also comprises a CD8 hinge region, wherein the CD8 hinge region connects the antigen recognition domain and the transmembrane domain.

4. An isolated nucleic acid, characterized in that It encodes the HLA-G specific chimeric antigen receptor according to claim 1, and the nucleic acid comprises: Arranged in sequence are the fragment encoding the antigen recognition domain as shown in sequence identification number 12, the fragment encoding the transmembrane domain as shown in sequence identification number 13, the fragment encoding the IL2 receptor β chain signal transduction domain as shown in sequence identification number 14, and the fragment encoding the CD3ζ signal transduction domain as shown in sequence identification number 15.

5. The nucleic acid according to claim 4, characterized in that It also comprises a signal peptide chain fragment as shown in sequence identification number 16, which is connected to the 5' end of the fragment encoding the antigen recognition domain.

6. The nucleic acid according to claim 4, characterized in that It also comprises a sequence encoding a CD8 hinge region, wherein the sequence encoding the CD8 hinge region connects the fragment encoding the antigen recognition domain and the fragment encoding the transmembrane domain.

7. An HLA-G specific chimeric antigen receptor expression plasmid, characterized in that: Include: The promoter as shown in sequence identification number 18 and the nucleic acid as claimed in claim 4 are arranged in sequence.

8. A cell expressing an HLA-G specific chimeric antigen receptor, characterized in that: Include: immune cells; and The HLA-G specific chimeric antigen receptor expression plasmid according to claim 7.

9. The cell expressing HLA-G specific chimeric antigen receptor according to claim 8, characterized in that: The immune cells are T cells.

10. The cell expressing HLA-G specific chimeric antigen receptor according to claim 8, characterized in that: The immune cells are natural killer cells.

11. The cell expressing HLA-G specific chimeric antigen receptor according to claim 10, characterized in that: The natural killer cells are NK-92 cell lines or primary natural killer cells.

12. A pharmaceutical composition for treating cancer, characterized in that: Include: The cell expressing HLA-G specific chimeric antigen receptor according to claim 8; and Pharmaceutically acceptable carrier.

13. The pharmaceutical composition for treating cancer according to claim 12, wherein It also contains chemotherapy drugs.

14. The pharmaceutical composition for treating cancer according to claim 13, wherein The chemotherapy drug is doxorubicin, temozolomide, gemcitabine or carboplatin.

15. A use of a cell expressing an HLA-G specific chimeric antigen receptor as claimed in claim 8, characterized in that: The invention is used for preparing a drug for inducing death of tumor cells of mammals, wherein the tumor cells are breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.

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