CN112707967B

CN112707967B - HLA-G specific chimeric antigen receptor, nucleic acid, expression plasmid, cell, use and composition thereof

Info

Publication number: CN112707967B
Application number: CN201911017626.2A
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: 2019-10-24
Filing date: 2019-10-24
Publication date: 2024-12-13
Anticipated expiration: 2039-10-24
Also published as: CN112707967A

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

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

Technical Field

The invention relates to a medical product containing an antigen or an antibody, in particular to a chimeric antigen receptor, nucleic acid for encoding the chimeric antigen receptor, chimeric antigen receptor expression plastid, a pharmaceutical composition for expressing chimeric antigen receptor cells and treating cancers and application of the chimeric antigen receptor expressing cells.

Background

Cancer is also known as malignancy, is abnormal proliferation of cells, and these proliferated cells may invade other parts of the body, a disease caused by the malfunction of the mechanism of controlling cell division proliferation. There is an increasing trend in the world population suffering from cancer, which is one of the ten major causes of death in the state and has been the top of the ten major causes of death for twenty-seven years in succession.

Conventional methods of tumor treatment include surgical treatment, radiation treatment, chemotherapy, targeted therapy, and the like. Tumor immunotherapy is another method for treating tumors, except the treatment method, by activating the autoimmune system of a patient, inducing specific cellular immunity and humoral immunity of an organism by tumor cells or tumor antigen substances, enhancing the anticancer capability of the organism, and preventing the growth, diffusion and recurrence of tumors so as to achieve the aim of eliminating or controlling the tumors.

Tumor immunotherapy is mainly three major directions-tumor vaccine, cytotherapy and immune checkpoint inhibitor, wherein chimeric antigen receptor immune cell technology is a cytotherapy that has been developed very rapidly in recent years. In the prior art, chimeric antigen receptor immune cells are obtained by coupling an antigen binding portion of an antibody capable of recognizing a tumor antigen with an intracellular portion of a CD 3-delta chain or an Fc epsilon RI gamma in vitro to form a chimeric protein, and transfecting immune cells of a patient by a gene transduction method to express the chimeric antigen receptor, and the chimeric antigen receptor immune cells have remarkable curative effects on the treatment of acute leukemia and non-Hodgkin lymphoma, and are considered as one of the most promising tumor treatment modes. However, the cell therapy of the immune cells of the chimeric antigen receptor is greatly limited in the efficacy of the chimeric antigen receptor in the solid tumor due to the lack of unique tumor-related antigens, the low efficiency of homing the immune cells to the tumor site and the inability to overcome the immunosuppressive microenvironment of the solid tumor.

Disclosure of Invention

The invention aims at providing 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 an application of the HLA-G specific chimeric antigen receptor expression cell. HLA-G specific chimeric antigen receptors have excellent ability to specifically bind tumor cells. The nucleic acid encodes an HLA-G specific chimeric antigen receptor. While HLA-G specific chimeric antigen receptor-expressing plastids comprise nucleic acids. The HLA-G specific chimeric antigen receptor expressing cells comprise HLA-G specific chimeric antigen receptor expressing plastids which express the HLA-G specific chimeric antigen receptor, can specifically target tumor cells, avoid off-target effect, and further effectively poison the tumor cells, and can be used for preparing medicaments for inducing death of the tumor cells of mammals. And the pharmaceutical composition for treating cancer comprises cells expressing HLA-G specific chimeric antigen receptor, which can effectively poison tumor cells and thus treat cancer.

An embodiment according to an object of the present invention is to provide an HLA-G specific chimeric antigen receptor comprising an antigen recognition domain, a transmembrane domain, an IL2 receptor beta chain information transfer domain and a CD3 zeta information transfer domain. The antigen recognition domain comprises a monoclonal antibody fragment specific for human leukocyte antigen G (human leukocyte antigen G, HLA-G), and the amino acid sequence of the antigen recognition domain is shown as sequence recognition number 1. The amino acid sequence of the transmembrane domain is shown in SEQ ID NO. 2. The amino acid sequence of the IL2 receptor beta chain information transfer domain is shown as sequence identification number 3. The amino acid sequence of the CD3 zeta information transfer domain is shown as sequence identification number 4.

According to the aforementioned HLA-G specific chimeric antigen receptor, a signal peptide chain as shown in SEQ ID No. 5 may be further included, which is linked to the N-terminus of the antigen recognition domain.

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

Another embodiment of an object of the present invention is to provide an isolated nucleic acid encoding an HLA-G specific chimeric antigen receptor of the preceding paragraph. The nucleic acid comprises, in order, a coding antigen recognition domain fragment as shown in SEQ ID NO. 12, a coding transmembrane domain fragment as shown in SEQ ID NO. 13, a coding IL2 receptor beta chain information transfer domain fragment as shown in SEQ ID NO. 14, and a coding CD3 zeta information transfer domain fragment as shown in SEQ ID NO. 15.

According to the aforementioned isolated nucleic acid, the coding signal peptide chain fragment shown in SEQ ID No. 16 may be further included, which is linked to the 5' end of the coding antigen recognition domain fragment.

According to the aforementioned isolated nucleic acid, it may further comprise a sequence encoding a CD8 hinge region, wherein the sequence encoding a CD8 hinge region is linked to the fragment encoding the antigen recognition domain and the fragment encoding the transmembrane domain.

Yet another embodiment of the present invention provides an HLA-G specific chimeric antigen receptor-expressing plasmid comprising a promoter as shown in SEQ ID No. 18 and a nucleic acid as in the preceding paragraph in sequence.

Yet another embodiment of an object of the present invention is to provide an HLA-G specific chimeric antigen receptor-expressing cell comprising an immune cell and an HLA-G specific chimeric antigen receptor-expressing plastid as in the preceding paragraph.

According to the aforementioned HLA-G specific chimeric antigen receptor expressing cells, 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.

Yet another embodiment of an object of the present invention is to provide a pharmaceutical composition for treating cancer, comprising the HLA-G specific chimeric antigen receptor-expressing cells of the preceding paragraph, and a pharmaceutically acceptable carrier.

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

Another object of the present invention is to provide a use of the HLA-G specific chimeric antigen receptor expressing cell as in the preceding paragraph for the preparation of a medicament for inducing death of tumor cells of a mammal, which can be breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.

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

Drawings

In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the following description is made with reference to the accompanying drawings in which:

FIG. 1 is a schematic diagram showing the protein structure of the antigen recognition domain of the present invention;

FIG. 2 is a schematic diagram showing construction of HLA-G specific chimeric antigen receptor-expressing plastids according to the present invention;

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G and 3H are graphs showing the results of an assay for HLA-G specific chimeric antigen receptor-expressing cells of example 1 of the present invention to induce tumor cell death;

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H are graphs showing the results of an assay for HLA-G specific chimeric antigen receptor-expressing cells of example 2 of the present invention to induce tumor cell death, and

FIG. 5 is a statistical chart of results of flow cytometry analysis of HLA-G expression levels after tumor cells received chemotherapy.

Detailed Description

The present disclosure proposes 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-expressing plasmid comprising the nucleic acid, an HLA-G specific chimeric antigen receptor-expressing cell comprising the HLA-G specific chimeric antigen receptor-expressing plasmid, uses thereof, and a pharmaceutical composition for treating cancer comprising the HLA-G specific chimeric antigen receptor-expressing cell. In the specification, the HLA-G specific chimeric antigen receptor has excellent specific binding capability to tumor cells, especially to human leukocyte antigen G (human leukocyte antigen G, HLA-G) expressed on the cell membrane of the tumor cells, so that the HLA-G specific chimeric antigen receptor expressing the HLA-G specific chimeric antigen receptor can specifically target tumor cells, avoid off-target effect and further effectively poison the tumor cells, and can be used for preparing medicines for inducing death of the tumor cells of mammals. The pharmaceutical composition for treating cancer of the present invention comprises the HLA-G specific chimeric antigen receptor cell of the present invention, and may further comprise a chemotherapeutic agent, which is effective for killing tumor cells and thus treating cancer.

As used herein, the term "human leukocyte antigen G (human leukocyte antigen G, HLA-G)" is defined by the HLA-G gene, and is an atypical first class major histocompatibility complex (major histocompatibility complex, MHC), with a heavy chain of about 45kDa. HLA-G is expressed on fetal-derived placental cells and is active in the down regulation of immune responses, which primarily function to suppress cytotoxic immune cell functions.

The present invention is further illustrated by the following specific examples, which are presented to facilitate a person skilled in the art to which the invention pertains and to make a complete use and practice of the invention without undue interpretation, and are not to be construed as limiting the scope of the invention, but as illustrating how the materials and methods of the invention may be practiced.

Test example

1. HLA-G specific chimeric antigen receptor, isolated nucleic acid and HLA-G specific chimeric antigen receptor-expressing plastid of the present invention

The HLA-G specific chimeric antigen receptor of the present invention comprises an antigen recognition domain as shown in sequence recognition number 1, a transmembrane domain as shown in sequence recognition number 2, an IL2 receptor beta chain information transfer domain as shown in sequence recognition number 3, and a CD3 zeta information transfer domain as shown in sequence recognition number 4. The antigen recognition domain comprises a monoclonal antibody fragment specific for human leukocyte antigen G (human leukocyte antigen G, HLA-G). Preferably, it may further be linked to a signal peptide chain as shown in SEQ ID No. 5 at the N-terminus of the antigen recognition domain, and further comprises a CD8 hinge region as shown in SEQ ID No. 11 linking the antigen recognition domain and the transmembrane domain. Specifically, the antigen recognition domain shown in sequence recognition number 1 includes 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 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 light chain immunoglobulin variable domain sequence is CDRL1 as shown in sequence identification number 9, CDRL2 as shown in sequence identification number 10, and CDRL2 as shown in RMS. Referring to FIG. 1, a schematic protein structure of the antigen recognition domain of the present invention is shown, wherein the dotted circular region represents the variable domain in the antigen recognition domain of the present invention.

The isolated nucleic acids of the invention are those encoding HLA-G specific chimeric antigen receptors of the invention. The nucleic acid comprises, in order, a coding antigen recognition domain fragment as shown in SEQ ID NO. 12, a coding transmembrane domain fragment as shown in SEQ ID NO. 13, a coding IL2 receptor beta chain information transfer domain fragment as shown in SEQ ID NO. 14, and a coding CD3 zeta information transfer domain fragment as shown in SEQ ID NO. 15. Preferably, it may further comprise a coding signal peptide chain fragment as shown in SEQ ID NO. 16 attached to the 5' end of the coding antigen recognition domain fragment, and may further comprise a coding CD8 hinge region sequence as shown in SEQ ID NO. 17 attached to the coding antigen recognition domain fragment and the coding transmembrane domain fragment. The encoded antigen recognition domain fragment shown as sequence recognition number 12 is the antigen recognition domain shown as sequence recognition number 1, the encoded transmembrane domain fragment shown as sequence recognition number 13 is the transmembrane domain shown as sequence recognition number 2, the encoded IL2 receptor beta chain information transfer domain fragment shown as sequence recognition number 14 is the IL2 receptor beta chain information transfer domain shown as sequence recognition number 3, the encoded CD3 zeta information transfer domain fragment shown as sequence recognition number 15 is the CD3 zeta information transfer domain shown as sequence recognition number 4, the encoded signal peptide chain fragment shown as sequence recognition number 16 is the signal peptide chain shown as sequence recognition number 5, and the encoded CD8 hinge region shown as sequence recognition number 17 is the CD8 hinge region shown as sequence recognition number 11.

Referring to FIG. 2, a schematic diagram of construction of HLA-G specific chimeric antigen receptor expression plasmids of the present invention is shown. In particular, in one embodiment shown in this test example, the HLA-G specific chimeric antigen receptor-expressing plastid is a Lenti-EF1a-H-28-IL2RB-Z plastid, and the insert (insert) fragment comprises, in order, a promoter, a coding antigen-recognition domain fragment, a coding transmembrane domain fragment, a coding IL2 receptor beta chain information-transmitting domain fragment, and a coding CD3 zeta information-transmitting domain fragment. The promoter is EF-1alpha promoter shown as sequence identification number 18, the sequence for encoding antigen identification domain fragment is shown as sequence identification number 12, the sequence for encoding transmembrane domain fragment is shown as sequence identification number 13, the sequence for encoding IL2 receptor beta chain information transmission domain fragment is shown as sequence identification number 14, and the sequence for encoding CD3 zeta information transmission domain fragment is shown as sequence identification number 15. In addition, the insert also contains a coding signal peptide chain fragment shown as sequence identification number 16 and a coding CD8 hinge region sequence shown as sequence identification number 17. The coded signal peptide chain fragment is connected with the 5' end of the coded antigen recognition domain fragment, and the coded CD8 hinge region sequence is connected with the coded antigen recognition domain fragment and the coded transmembrane domain fragment. The insert was then constructed on a Creative Biolabs vector (Creative Biolabs, NY, USA) to give Lenti-EF1a-H-28-IL2RB-Z plastids. The vector used is a lentivirus (lentivirus) vector system, so that the constructed HLA-G specific chimeric antigen receptor expression plasmid can be transfected into a expressing cell to produce a lentivirus, which can be subsequently used to transduce the HLA-G specific chimeric antigen receptor into an immune cell.

2. HLA-G specific chimeric antigen receptor-expressing cells of the invention, uses thereof, and pharmaceutical compositions for the treatment of cancer

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 an immune cell using a lentivirus. Preferably, the immune cells may be T cells or natural killer cells. More preferably, the natural killer cells may be NK-92 cell lines or primary natural killer cells. Specifically, constructed Lenti-EF1a-H-28-IL2RB-Z plastids were transfected into 293T cell lines using lipofectamine 3000 (Invitrogen) to prepare lentiviruses with HLA-G specific chimeric antigen receptors of the present invention, and then Opti-MEM (Invitrogen) containing the prepared lentiviruses supernatant and 8. Mu.g/ml polybrene (Sigma-Aldrich) were cultured for 3 days to transduce HLA-G specific chimeric antigen receptors of the present invention into primary T cells. And culturing the primary natural killer cells with Opti-MEM (Invitrogen) containing the supernatant of the prepared lentivirus and 50. Mu.g/ml protamine (Protamine sulfate, sigma-Aldrich) for 7 days to transduce the chimeric antigen receptor of the present invention into the primary natural killer cells or NK-92 cell lines, thereby obtaining the HLA-G specific chimeric antigen receptor-expressing cells of the present invention. The obtained HLA-G specific chimeric antigen receptor expressing cells have the effect of inducing the death of tumor cells of mammals, and can be used for preparing medicines for inducing the death of tumor cells of mammals. Preferably, the tumor cells may be breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells, or ovarian cancer cells.

The pharmaceutical composition for treating cancer of the present invention comprises the HLA-G specific chimeric antigen receptor-expressing cells 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 agent may be doxorubicin (doxorubicin), temozolomide, gemcitabine (gemcitabine), or carboplatin (carboplatin).

The following test examples will demonstrate, as data, that the HLA-G specific chimeric antigen receptor-expressing cells of the present invention and the pharmaceutical composition for treating cancer comprising the HLA-G specific chimeric antigen receptor-expressing cells of the present invention have good effect of inducing death in various mammalian tumor cells. 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 abbreviated as DBTRG), human pancreatic cancer cell line AsPC1 and human ovarian cancer cell line SKOV3, respectively. The tumor cell lines used were all purchased from the American type culture Collection (AMERICAN TYPE Culture Collection, ATCC). The human breast cancer cell line MDA-MB-231 cell line was a triple negative breast cancer cell line, i.e., hormone receptor (ER, PR) and HER-2 receptor were negative, which was cultured in RPMI medium containing 10% Fetal Bovine Serum (FBS). Human malignant brain tumor cell line DBTRG was cultured in DMEM medium containing 10% fetal bovine serum. 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.

2.1. Example 1

In this test example, the HLA-G specific chimeric antigen receptor-expressing cells of example 1 of the present invention (hereinafter referred to simply as example 1) were obtained by transduction of the HLA-G specific chimeric antigen receptor of the present invention into primary natural killer cells. And further testing 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.

The human breast cancer cell line MDA-MB-231, the human malignant brain tumor cell line DBTRG, the human pancreatic cancer cell line AsPC1 and the human ovarian cancer cell line SKOV3 were each grown in 12-well plates at a density of 1X 10 ⁵ cells/well for 48 hours, and then subjected to experiments. Each tumor cell on the test was divided into 6 groups, untreated control group, test group 1 treated with chemotherapeutic agent, test group 2 treated with parent primary natural killer cell, test group 3 treated with parent primary natural killer cell and chemotherapeutic agent, test group 4 treated with example 1, and test group 5 treated with example 1 and chemotherapeutic agent, respectively. Wherein the chemotherapeutic agent used is doxorubicin (200 nM) in the group of human breast cancer cell line MDA-MB-231, temozolomide (80 μg/ml) in the group of human malignant brain tumor cell line DBTRG, gemcitabine (20 μM) in the group of human pancreatic cancer cell line AsPC1, and carboplatin (20 μM) in the group of human ovarian cancer cell line SKOV 3. In test groups 4 and 5, the number of cells of example 1 treated was 1X 10 ⁵ cells, and the number of cells of the parent primary natural killer cells treated in test groups 2 and 3 was also 1X 10 ⁵ cells. The treated cells of each group were then stained with Annexin V-FITC and PI, and the sum of the percentages of cells stained with Annexin V-FITC and/or PI was calculated to give the cytotoxic effect. After independent triplicate experiments were performed for each group, the cytotoxic results were counted.

Fig. 3A, 3B, 3C, 3D, 3E, 3F, 3G and 3H are graphs of the results of the assays of example 1 for inducing tumor cell death. Wherein FIG. 3A is a graph showing the results of the analysis of the death of the human breast cancer cell line MDA-MB-231 induced in example 1, and FIG. 3B is a statistical graph of the human breast cancer cell line after the three-fold test of FIG. 3A. Fig. 3C is a graph showing the results of the analysis of example 1 for inducing death of human malignant brain tumor cell line DBTRG, and fig. 3D is a statistical graph of the three-fold test of fig. 3C. FIG. 3E is a graph showing the results of the analysis of the death of the human pancreatic cancer cell line AsPC1 induced in example 1, and FIG. 3F is a statistical graph after the three-fold test of FIG. 3E. FIG. 3G is a graph showing the results of the analysis of the death of the human ovarian cancer cell line SKOV3 induced in example 1, and FIG. 3H is a statistical graph of the human ovarian cancer cell line after the three-fold test of FIG. 3G. P in fig. 3B, 3D, 3F and 3H represents the parent primary natural killer cells.

The results from FIGS. 3A and 3B show that in the untreated control group, only about 5% of the human breast cancer cell line MDA-MB-231 died, while there was no statistically significant difference in mortality, although there was an increase in the mortality of the human breast cancer cell line MDA-MB-231, in the test group 1 treated with doxorubicin, the test group 2 treated with the parent primary natural killer cell, and the test group 3 treated with the parent primary natural killer cell and doxorubicin. Whereas treatment of test group 4 of example 1 induced a mortality of approximately 70% for human breast cancer cell line MDA-MB-231, there was a statistically significant difference (p < 0.001) compared to test group 2. In addition, treatment of test group 5 of examples 1 and doxorubicin resulted in a higher mortality of more than 80% of the human breast cancer cell line MDA-MB-231, with a statistically significant difference (p < 0.05) compared to test group 4 and a statistically significant difference (p < 0.001) compared to test group 3.

From the results of fig. 3C and 3D, it is shown that in the untreated control group, only less than 5% of the human malignant brain tumor cell lines DBTRG die, whereas the test group 1 treated with temozolomide, the test group 2 treated with the parent primary natural killer cells, and the test group 3 treated with the parent primary natural killer cells and temozolomide, although there was an increase in mortality of the human malignant brain tumor cell lines DBTRG, there was no statistically significant difference. Whereas the mortality of human malignant brain tumor cell line DBTRG induced by treatment of test group 4 of example 1 may be over 60%, there is a statistically significant difference (p < 0.001) compared to test group 2. In addition, mortality rates of human malignant brain tumor cell lines DBTRG induced by treatment of test group 5 of example 1 and temozolomide were more nearly 90%, with statistically significant differences (p < 0.01) compared to test group 4, and also statistically significant differences (p < 0.001) compared to test group 3.

From the results of fig. 3E and 3F, it was shown that in the untreated control group, only less than 5% of human pancreatic cancer cell line AsPC1 died, whereas the gemcitabine-treated test group 1, the parental primary natural killer cell-treated test group 2, and the parental primary natural killer cell-treated test group 3, although the human pancreatic cancer cell line AsPC1 died at an increased rate, had no statistically significant difference. Whereas the mortality of human pancreatic cancer cell line AsPC1 induced by treatment of test group 4 of example 1 was about 50%, there was a statistically significant difference (p < 0.01) compared to test group 2. In addition, treatment of test group 5 of example 1 and gemcitabine resulted in a mortality of approximately 70% of human pancreatic cancer cell line AsPC1, a statistically significant difference (p < 0.05) compared to test group 4, and a statistically significant difference (p < 0.001) compared to test group 3.

From the results of fig. 3G and 3H, it was shown that in the untreated control group, only less than 5% of the human ovarian cancer cell line SKOV3 died, whereas the carboplatin treated test group 1 and the parent primary natural killer cell treated test group 2 had increased mortality but no statistically significant differences. Test group 3, which treated the parental primary natural killer cells and carboplatin, had a mortality rate of nearly 40% for the human ovarian cancer cell line SKOV3, but had no statistically significant differences. Whereas the mortality of the human ovarian cancer cell line SKOV3 induced by treatment of test group 4 of example 1 was approximately 50%, there was a statistically significant difference (p < 0.05) compared to test group 2. In addition, mortality of human ovarian cancer cell line SKOV3 induced by treatment of test group 5 of example 1 and carboplatin was more nearly 70%, with statistically significant differences (p < 0.05) compared to test group 4, and also statistically significant differences (p < 0.01) compared to test group 3.

As shown in the results of fig. 3A to 3H, the treatment example 1 has excellent poisoning effect on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, and is useful for preparing a medicament for inducing tumor cell death in mammals by using the HLA-G specific chimeric antigen receptor expressing cells of the present invention. In addition, the simultaneous treatment of example 1 and the chemotherapeutic agent, which is more pronounced against breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, shows that the pharmaceutical composition for treating cancer of the present invention, preferably, may contain the chemotherapeutic agent, can effectively poison tumor cells and thus treat cancer.

2.2. Example 2

In this test example, the HLA-G specific chimeric antigen receptor-expressing cells of example 2 of the present invention (hereinafter referred to simply as example 2) were obtained by transducing the HLA-G specific chimeric antigen receptor of the present invention into primary T cells. And further testing the effect of example 2 and the pharmaceutical composition for treating cancer comprising example 2 on inducing death of breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells and ovarian cancer cells.

The human breast cancer cell line MDA-MB-231, the human malignant brain tumor cell line DBTRG, the human pancreatic cancer cell line AsPC1 and the human ovarian cancer cell line SKOV3 were each grown in 12-well plates at a density of 1X 10 ⁵ cells/well for 48 hours, and then subjected to experiments. Each tumor cell on the test was divided into 6 groups, untreated control group, test group 1 treated with chemotherapeutic agent, test group 2 treated with parent primary T cell, test group 3 treated with parent primary T cell and chemotherapeutic agent, test group 4 treated with example 2, and test group 5 treated with example 2 and chemotherapeutic agent, respectively. Wherein the chemotherapeutic agent used is doxorubicin (200 nM) in the group of human breast cancer cell line MDA-MB-231, temozolomide (80 μg/ml) in the group of human malignant brain tumor cell line DBTRG, gemcitabine (20 μM) in the group of human pancreatic cancer cell line AsPC1, and carboplatin (20 μM) in the group of human ovarian cancer cell line SKOV 3. In test groups 4 and 5, the number of cells of example 2 treated was 1X 10 ⁵ cells, and the number of cells of primary T cells treated in test groups 2 and 3 was also 1X 10 ⁵ cells. And then, staining the treated cells with Annexin V-FITC and PI, detecting the apoptosis and death conditions of the cells by using a flow cytometer, and calculating the sum of the percentages of the cells stained with Annexin V-FITC and/or PI to obtain the cytotoxic effect. After independent triplicate experiments were performed for each group, the cytotoxic results were counted.

Referring to fig. 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H, the results of the analysis of the induction of tumor cell death in example 2 are shown. Wherein FIG. 4A is a graph showing the results of the analysis of the death of example 2 induced human breast cancer cell line MDA-MB-231 and FIG. 4B is a statistical graph of FIG. 4A after a three-repeat test. Fig. 4C is a graph showing the results of the analysis of example 2 for inducing death of human malignant brain tumor cell line DBTRG, and fig. 4D is a statistical graph of the three-fold test of fig. 4C. Fig. 4E is a graph showing the results of the analysis of the death of example 2 induced human pancreatic cancer cell line AsPC1, and fig. 4F is a statistical graph after the triple-repeat test of fig. 4E. FIG. 4G is a graph showing the results of the analysis of the death of the human ovarian cancer cell line SKOV3 induced in example 2, and FIG. 4H is a statistical chart of FIG. 4G. P in FIGS. 4B, 4D, 4F and 4H represents a parent primary T cell.

The results from FIGS. 4A and 4B show that in the untreated control group, only about 10% of the human breast cancer cell line MDA-MB-231 died, while there was no statistically significant difference in mortality, though there was an increase in the mortality of the human breast cancer cell line MDA-MB-231, 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. Whereas the mortality of the human breast cancer cell line MDA-MB-231 induced by treatment of test group 4 of example 2 was nearly 70%, there was a statistically significant difference (p < 0.01) compared to test group 2. In addition, mortality of the human breast cancer cell line MDA-MB-231 induced by treatment of test group 5 of example 2 and doxorubicin was more nearly 80%, with statistically significant differences (p < 0.05) compared to test group 4, and also statistically significant differences (p < 0.01) compared to test group 3.

From the results of fig. 4C and 4D, it is shown that in the untreated control group, only less than 10% of human malignant brain tumor cell lines DBTRG die, whereas in the test group 1 treated with temozolomide and the test group 2 treated with parental primary T cells, there was an increase in the mortality of human malignant brain tumor cell lines DBTRG, but no statistically significant difference. Whereas in test group 3, where parental primary T cells and temozolomide were treated, mortality of human malignant brain tumor cell line DBTRG could be increased to nearly 40%, but there was still no statistically significant difference. Whereas mortality of human malignant brain tumor cell line DBTRG induced by treatment of test group 4 of example 2 may be over 70%, there is a statistically significant difference (p < 0.001) compared to test group 2. In addition, mortality of human malignant brain tumor cell line DBTRG induced by treatment of test group 5 of example 2 and temozolomide was more nearly 80%, with statistically significant differences (p < 0.05) compared to test group 4, and also statistically significant differences (p < 0.001) compared to test group 3.

From the results of fig. 4E and 4F, it was shown that in the untreated control group, only about 5% of human pancreatic cancer cell line AsPC1 died, whereas the test group 1 treated with gemcitabine and the test group 2 treated with parental primary T cells had increased mortality but no statistically significant difference. Whereas in test group 3, where parental primary T cells and gemcitabine were treated, the mortality of human pancreatic cancer cell line AsPC1 could be increased to nearly 40%. Whereas the mortality of human pancreatic cancer cell line AsPC1 induced by treatment of test group 4 of example 2 could be increased to more than 30%, with statistically significant differences (p < 0.01) compared to test group 2. Furthermore, the mortality of human pancreatic cancer cell line AsPC1 induced by treatment example 2 and gemcitabine, test group 5, was more than 50%, with a statistically significant difference (p < 0.01) compared to test group 4, and also with a statistically significant difference (p < 0.05) compared to test group 3.

From the results of FIGS. 4G and 4H, it was shown that in the untreated control group, only less than 5% of the human ovarian cancer cell line SKOV3 was seen to die, while there were no statistically significant differences in mortality of the human ovarian cancer cell line SKOV3, though there were increases in the test group 1 treated with carboplatin, the test group 2 treated with the parent primary T cells, and the test group 3 treated with the parent primary T cells and carboplatin. Whereas the mortality of human ovarian cancer cell line SKOV3 induced by treatment of test group 4 of example 2 was nearly 80%, with statistically significant differences (p < 0.001) compared to test group 2. In addition, mortality of human ovarian cancer cell line SKOV3 induced by treatment of test group 5 of example 2 and carboplatin was more than 80%, with statistically significant differences (p < 0.05) compared to test group 4, and also statistically significant differences (p < 0.001) compared to test group 3.

As shown in the results of fig. 4A to 4H, the treatment example 2 has excellent poisoning effect on breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, and is useful for preparing a medicament for inducing tumor cell death in mammals by using the HLA-G specific chimeric antigen receptor expressing cells of the present invention. In addition, the simultaneous treatment of example 2 and the chemotherapeutic agent, which is more pronounced against breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells, shows that the pharmaceutical composition for treating cancer of the present invention, preferably, may contain the chemotherapeutic agent, can effectively poison tumor cells and thus treat cancer.

2.3. Treatment with chemotherapeutic agents increases the expression of human leukocyte antigen G on tumor cell membranes

The present test example further examined the reason why the group of the HLA-G-specific chimeric antigen receptor-expressing cells and the chemotherapeutic agent of the present invention treated simultaneously had a more excellent effect of poisoning tumor cells.

The human breast cancer cell line MDA-MB-231, the human malignant brain tumor cell line DBTRG, the human pancreatic cancer cell line AsPC1 and the human ovarian cancer cell line SKOV3 are planted on 6-hole plates at the density of 2×10 ⁵ cells/well, and then are cultured until the cells are attached every other day, and then the experiments are carried out. Each tumor cell was experimentally divided into 2 groups, untreated control and chemotherapy groups treated with chemotherapeutic drugs for 48 hours. Wherein the chemotherapeutic agent used is doxorubicin (200 nM) in the group of human breast cancer cell line MDA-MB-231, temozolomide (80 μg/ml) in the group of human malignant brain tumor cell line DBTRG, gemcitabine (20 μM) in the group of human pancreatic cancer cell line AsPC1, and carboplatin (20 μM) in the group of human ovarian cancer cell line SKOV 3. And detecting the HLA-G expression of the tumor cells on the cell surface of each group of treated tumor cells by using a flow cytometer.

Referring to FIG. 5, a statistical chart of results of flow cytometry analysis for analyzing HLA-G expression levels of tumor cells after chemotherapy is shown. In FIG. 5, treatment with doxorubicin increased HLA-G expression on the membrane of human breast cancer cell line MDA-MB-231 (p < 0.001), treatment with temozolomide increased HLA-G expression on the membrane of human malignant brain tumor cell line DBTRG (p < 0.001), treatment with gemcitabine increased HLA-G expression on the membrane of human pancreatic cancer cell line AsPC1 (p < 0.001), and treatment with carboplatin increased HLA-G expression on the membrane of human ovarian cancer cell line SKOV3 (p < 0.001) compared to the control and chemotherapy groups of the same tumor cells. As shown in the results of FIG. 5, treatment of the chemotherapeutic agent can increase the expression of HLA-G on the cell membrane of tumor cells, while 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 by treating the HLA-G specific chimeric antigen receptor-expressing cells of the present invention after the chemotherapeutic agent has been treated, or by treating both the HLA-G specific chimeric antigen receptor-expressing cells of the present invention and the chemotherapeutic agent, can have a more excellent effect of poisoning tumor cells.

In summary, the HLA-G specific chimeric antigen receptor of the present invention has excellent specific binding capability to tumor cells, particularly to HLA-G specific binding expressed on the cell membrane of tumor cells, so that the HLA-G specific chimeric antigen receptor of the present invention can target tumor cells expressing the specificity of HLA-G specific chimeric antigen receptor cells, avoid off-target effect, and further effectively poison tumor cells, and can be used for preparing drugs for inducing death of tumor cells of mammals. The pharmaceutical composition for treating cancer of the present invention, comprising the HLA-G specific chimeric antigen receptor expressing cell of the present invention, can effectively poison tumor cells to treat cancer. And a pharmaceutical composition for treating cancer further comprising a chemotherapeutic agent, which can further induce the cell membrane of tumor cells to express HLA-G in a large amount, thereby enhancing the poisoning effect of cells expressing HLA-G specific chimeric antigen receptor on tumor cells, thereby having more excellent tumor cell poisoning effect.

The present invention has been described in terms of embodiments, but it is not limited thereto, and various modifications and variations can be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims.

Sequence listing

<110> Taiwan university of Chinese medicine attached Hospital

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

<160> 18

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<210> 1

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<212> PRT

<213> Artificial sequence (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 (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 (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 (ARTIFICIAL SEQUENCE)

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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 (ARTIFICIAL SEQUENCE)

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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 (ARTIFICIAL SEQUENCE)

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Gly Phe Thr Phe Asn Thr Tyr Ala

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr

1 5 10

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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Val Arg Gly Gly Tyr Trp Ser Phe Asp Val

1 5 10

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

Met Gln His Leu Glu Tyr Pro Tyr Thr

1 5

<210> 11

<211> 45

<212> PRT

<213> Artificial sequence (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 (ARTIFICIAL SEQUENCE)

<400> 12

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> 13

<211> 204

<212> DNA

<213> Artificial sequence (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 (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 (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 (ARTIFICIAL SEQUENCE)

<400> 16

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 17

<211> 135

<212> DNA

<213> Artificial sequence (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 (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

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

Claims

1. An HLA-G specific chimeric antigen receptor, comprising:

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, further comprising 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, further comprising 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, wherein 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 of claim 4, further comprising 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, further comprising 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, comprising:

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, comprising:

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, wherein the immune cell is a T cell.

10. The cell expressing HLA-G specific chimeric antigen receptor according to claim 8, wherein the immune cell is a natural killer cell.

11. The cell expressing HLA-G specific chimeric antigen receptor according to claim 10, wherein the natural killer cell is a NK-92 cell line or a primary natural killer cell.

12. A pharmaceutical composition for treating cancer, comprising:

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, further comprising a chemotherapeutic drug.

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 the cell expressing HLA-G specific chimeric antigen receptor as claimed in claim 8, characterized in that it is used to prepare a drug for inducing death of tumor cells in mammals, wherein the tumor cells are breast cancer cells, glioblastoma multiforme cells, pancreatic cancer cells or ovarian cancer cells.