Engineered dendritic cell and use thereof

Abstract

The present application belongs to the technical field of biomedicine, and discloses a chimeric antigen receptor (CAR), an engineered dendritic cell (DC), and a use thereof. The CAR of the present application includes an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, and an intracellular domain. The extracellular domain includes a guide sequence and a single-chain antibody sequentially. The intracellular domain includes a Dectin-1 intracellular domain and an intracellular domain of FcR gamma. Chimeric antigen receptor-modified dendritic cells (CAR-DCs) prepared with the CAR of the present application can efficiently recognize a tumor antigen. The combined administration of the CAR-DC and radiotherapy for treating a solid tumor can effectively overcome the immunosuppression of the tumor microenvironment and improve the clinical treatment effect. Therefore, the present application provides an effective immunotherapy strategy for clinical tumor patients, and provides a new idea and method for tumor immunotherapy.

Claims

1. A chimeric antigen receptor (CAR), wherein the CAR has an amino acid sequence set forth in SEQ ID NO: 1.

2. A nucleic acid encoding the CAR according to claim 1, wherein the nucleic acid has a nucleotide sequence set forth in SEQ ID NO: 2.

3. An engineered dendritic cell (DC) comprising the CAR according to claim 1.

4. An engineered DC comprising a nucleic acid encoding the CAR according to claim 2.

5. The engineered DC according to claim 3, wherein the engineered DC is derived from at least one selected from the group consisting of a peripheral blood mononuclear cell, a hematopoietic stem cell, an induced pluripotent stem cell, and an embryonic stem cell.

6. A preparation method of an engineered DC, comprising the following steps: transforming DNA or mRNA encoding the CAR according to claim 1 into a DC, and allowing for expression.

7. The preparation method of an engineered DC according to claim 6, wherein the DNA or mRNA encoding the CAR is comprised in any one selected from the group consisting of an expression plasmid, a lentivirus, and a liposome.

8. A preparation comprising an engineered DC comprising the CAR according to claim 1.

9. A drug or preparation comprising the engineered DC according to claim 3, a pharmaceutically acceptable adjuvant, and/or a carrier or an excipient acceptable in a preparation process.

10. A method for treating a tumor, comprising: administering a population of engineered DCs modified with the CAR according to claim 1 to a tumor patient in combination with radiotherapy.

11. The method for treating a tumor according to claim 10, wherein the radiotherapy comprises external beam radiation therapy and/or intracavitary radiotherapy.

12. The method for treating a tumor according to claim 11, wherein the external beam radiation therapy comprises at least one selected from the group consisting of an X-knife, a gamma knife, and a linear accelerator; and the intracavitary radiotherapy comprises seed implantation.

13. The method for treating a tumor according to claim 10, wherein the tumor comprises any one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, melanoma, glioma, ovarian cancer, and prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the structure of CAR-DC;

(2) FIG. 2 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;

(3) FIG. 3 is a flow chart of an animal experiment in which colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(4) FIG. 4 shows tumor growth curves when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(5) FIG. 5 shows pictures of tumors (Day 9) when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(6) FIG. 6 shows tumor weights when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(7) FIG. 7 shows flow cytometry results of tumor single cells when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(8) FIG. 8 shows statistical charts of flow cytometry results of tumor single cells when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;

(9) FIG. 9 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;

(10) FIG. 10 is a flow chart of an animal experiment in which lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;

(11) FIG. 11 shows tumor growth curves when lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;

(12) FIG. 12 shows pictures of tumors (Day 7) when lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;

(13) FIG. 13 shows tumor weights when lung cancer LLC8 in mice is treated with a combination of CAR-DC and radiotherapy;

(14) FIG. 14 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;

(15) FIG. 15 is a flow chart of an animal experiment in which breast cancer 4T1 in mice is treated with a combination of CAR-DC and radiotherapy;

(16) FIG. 16 shows tumor growth curves when breast cancer 4T1 in mice is treated with a combination of CAR-DC and radiotherapy;

(17) FIG. 17 shows the expression of CAR on a surface of humanized mouse bone marrow-derived DCs that is detected by flow cytometry;

(18) FIG. 18 shows tumor growth curves when human breast cancer SKRBR3 is treated with a combination of CAR-DC and radiotherapy.

DETAILED DESCRIPTION

(19) To well explain the objective, technical solutions, and advantages of the present application, the present application will be further explained below with reference to specific examples. It should be understood by those skilled in the art that the specific examples described here are merely intended to explain the present application, rather than to limit the present application.

(20) In the examples, unless otherwise specified, the experimental methods used are conventional, and the materials and reagents used are commercially available.

(21) In the following examples, a method of plotting a tumor growth curve is as follows: A tumor is periodically measured for a length and a width by an electronic vernier caliper. A volume of the tumor is calculated based on the length and the width of the tumor according to the following calculation formula: (width{circumflex over ()}2*length)/2. The tumor growth curve is plotted with the Prism Graphad software.

Example 1: Engineered DC (CAR-DC)

(22) 1. Design of an Engineered DC (CAR-DC)

(23) Functional elements of CAR are shown in FIG. 1. The structure of the CAR includes an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain (an amino acid sequence of mouse Epha2 CAR is set forth in SEQ ID NO: 1).

(24) The extracellular domain includes a guide sequence (the amino acid sequence of a mouse Epha2 CAR guide sequence is set forth in SEQ ID NO: 3), a single-chain antibody (scFv) (the amino acid sequence of anti-mouse Epha2 VH is set forth in SEQ ID NO: 5, the amino acid sequence of anti-mouse Epha2 VL is set forth in SEQ ID NO: 7, and the amino acid sequence for a mouse Epha2 CAR linker is set forth in SEQ ID NO: 9). The hinge domain (the amino acid sequence of a mouse CD8 hinge domain is set forth in SEQ ID NO: 11) and the transmembrane domain (the amino acid sequence of a mouse CD8a transmembrane domain is set forth in SEQ ID NO: 13) are a hinge domain and a transmembrane domain of CD8a, respectively. The intracellular domain employs human or mouse Dectin-1 (the amino acid sequence of a mouse Dectin1 intracellular domain is set forth in SEQ ID NO: 15) and an intracellular domain of FcR gamma (the amino acid sequence of an intracellular domain of mouse FcR gamma is set forth in SEQ ID NO: 17). After being combined, the amino acid sequences of the elements were optimized by a codon optimization technology. A DNA sequence for mouse CAR-DC was cloned into a pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector.

(25) 2. In Vitro Transcription

(26) A gene for the CAR-DC was amplified by PCR. A PCR system (100 L system) was as follows: DNA (amplified with a codon-optimized base sequence set forth in SEQ ID NO: 2 as a template): 100 ng, 2Taq mix: 50 L, primer mix (5 M): 5 L, and H.sub.2O: to 100 L. Primer sequences were set forth in SEQ ID NOs: 19 and 20, respectively. Amplification conditions were as follows: 1. 94 C.: 5 min; 2. 94 C.: 20 s, 55 C.: 30 s, and 72 C.: 60 s, with 30 cycles; 3. 72 C.: 5 min; and 4. 4 C.: heat preservation.

(27) CAR-DC mRNA carrying a T7 promoter and a PolyA structure was produced through the transcription with a High Yield T7 RNA Synthesis Kit (HONGENE BIOTECH, Shanghai). A transcription system was as follows: template DNA (4 g), ATP (8 L), GTP (8 L), CTP (8 L), UTP (8 L), GAG (8 L), Reaction buffer (20 L), Enzyme (7.5 L), ultrapure water (to 100 L). The transcription system was incubated at 37 C. for 4 h, then 7.5 L of DNase was added, and a resulting mixture was incubated at 37 C. for 15 min. After a reaction was completed, mRNA was precipitated with lithium chloride. 150 L of ultrapure water and 150 L of a lithium chloride solution were added to a reaction system to precipitate RNA. A resulting mixed system was thoroughly mixed, refrigerated at 20 C. for 60 min, and centrifuged at 4 C. and a maximum rotational speed for 5 min to produce a supernatant and a precipitate. The supernatant was carefully removed. The precipitate was washed with 1 mL of about 70% ethanol, and then the 70% ethanol was carefully removed to produce a washed precipitate. The washed precipitate was air-dried and then resuspended with an appropriate amount of nuclease-free water.

(28) 3. Preparation of CAR-DCs

(29) Tibias and fibulas of hind legs of 6-8 week-old C57BL6 or Balbc mice were collected. The bone marrow was flushed out by a 1 ml syringe, properly ground into a single-cell suspension, and centrifuged at 1,500 rpm for 5 min, and a resulting supernatant was removed. Red blood cells were lysed with a 1ACK buffer. Centrifugation was conducted at 1,500 rpm for 5 min, and a resulting supernatant was removed. Cells were washed twice with phosphate buffered saline (PBS), counted, and inoculated in a 6-well plate at a density of 110.sup.6/mL. 100 ng/ml of GMCSF and IL4 were added to allow the differentiation of DCs. A medium was supplemented every 2 d to 3 d. After 8 d of differentiation, mouse DCs were harvested, counted, and electrotransformed with the above mRNA by a LONZA 4D system under the following conditions: 1E6-3E6/test, 100 L P3 solution, 5 g/test, and program CM150. The electrotransformed cells were inoculated in a 6-well plate and cultured for 24 h. Then an expression efficiency of CAR was measured with protein L. Resulting cells were used for downstream applications.

Example 2 Treatment of Colorectal Cancer with a Combination of CAR-DCs and Radiotherapy

(30) Tibias and fibulas of hind legs of 6-8 week-old C57BL6 mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 2. A transfection efficiency of CAR in DCs was 72.8%.

(31) Mouse colorectal cancer cells MC38 were transplanted into C57BL6 mice (6 weeks to 8 weeks). One week after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 1, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 310.sup.6 cells/mouse. On day 2, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 10 Gy. On day 4, CAR-DCs were infused through the tail vein for the second time at a dose of 310.sup.6 cells/mouse. The growth and survival of mouse tumors were continuously observed. A specific flow chart was shown in FIG. 3.

(32) At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected. Tumor tissues were digested with a collagenase to produce single cells. The single cells were labeled with CD45 (BD Pharmingen, 564279), CD3 (BD Pharmingen, 560591), CD8 (BD Pharmingen, 553031), CTLA4 (BD Pharmingen, 553720), and PDI (BD Pharmingen, 749422) antibodies, incubated for 30 min on ice in the dark, washed to remove non-specifically bound antibodies, resuspended with 400 L of PBS, and analyzed by flow cytometry for a cell subtype and cell status.

(33) A therapeutic effect of the combination of the CAR-DC and the radiotherapy for tumors was evaluated. It could be seen from tumor growth curves (FIG. 4), tumor sizes (FIG. 5), and tumor weights (FIG. 6) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the tumor growth (differences among groups were determined by t-test, **p<0.01 and ***p<0.001). In order to further analyze a phenotype of tumor-infiltrating T cells, single-cell suspensions were prepared from tumors, incubated with CD3, CD8, CTLA4, and PDI antibodies, and tested by flow cytometry for a cell subtype and phenotype (FIG. 7 and FIG. 8). Results showed that the combined use of the CAR-DC and the radiotherapy significantly increased the infiltration of T cells. In particular, the infiltration of effector T cells was significantly increased, and the infiltrating T cells exhibited lower exhausted phenotypes than T cells for animal tumors in other groups (differences among groups were determined by t-test, *p<0.05 and **p<0.01). It further explained a mechanism of the combination of the CAR-DC and the radiotherapy to achieve a prominent anti-tumor effect.

Example 3 Treatment of Lung Cancer with a Combination of CAR-DCs and Radiotherapy

(34) Tibias and fibulas of hind legs of 6-8 week-old Balbc mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 9. A transfection efficiency of CAR in DCs was 80.8%.

(35) Mouse lung cancer cells LLC were transplanted into C57BL6 mice (6 weeks to 8 weeks). 9 days after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 0, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 310.sup.6 cells/mouse. On day 1, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 5 Gy. On day 3, CAR-DCs were infused through the tail vein for the second time at a dose of 310.sup.6 cells/mouse. The growth and survival of mouse tumors were continuously observed.

(36) At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected.

(37) The prepared CAR-DCs were administered in combination with the radiotherapy to LLC tumor-bearing mice (a flow chart was shown in FIG. 10) to evaluate a therapeutic effect of the combination of the CAR-DC and the radiotherapy for the tumor. It could be seen from tumor growth curves (FIG. 11), tumor sizes (FIG. 12), and tumor weights (FIG. 13) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of lung cancer (differences among groups were determined by t-test, *p<0.05 and **p<0.01).

Example 4 Treatment of Breast Cancer with a Combination of CAR-DCs and Radiotherapy

(38) Tibias of hind legs of 6-8 week-old Balbc mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 14. A transfection efficiency of CAR in DCs was 8.86%.

(39) Mouse breast cancer cells 4T1 were transplanted into Balbc mice (6 weeks to 8 weeks). 9 days after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 0, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 310.sup.6 cells/mouse. On day 1, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 5 Gy. On day 3, CAR-DCs were infused through the tail vein for the second time at a dose of 310.sup.6 cells/mouse. The growth and survival of mouse tumors were continuously observed.

(40) At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected.

(41) The prepared CAR-DCs were administered in combination with the radiotherapy to LLC tumor-bearing mice (a flow chart was shown in FIG. 15) to evaluate a therapeutic effect of the combination of the CAR-DC and the radiotherapy for the tumor. It could be seen from tumor growth curves (FIG. 16) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of breast cancer.

Example 5 Treatment of Human Breast Cancer with a Combination of CAR-DCs and Radiotherapy

(42) Construction of humanized mice: Immunodeficient mice (purchased from Jiangsu GemPharmatech LLC., NCG, T001475) were irradiated at a sublethal dose. Then a human thymus tissue of about 1 mm.sup.3 was transplanted into a renal capsule of each immunodeficient mouse, and a wound was sutured after the surgery. The mice each were injected with CD34+ hematopoietic stem cells through the tail vein after wake. 10 weeks after the surgery, blood was collected and tested to determine the reconstruction of an immune system in mice. 2 to 3 drops of intravenous blood were collected from a hind leg of each mouse, added to an EDTA-PBS buffer, and centrifuged to settle cells. Red blood cells were completely lysed with a 1ACK lysis buffer until a transparent lysate solution was produced. The transparent lysate solution was centrifuged to produce a supernatant and a precipitate. The supernatant was removed. The precipitate was washed once with Dulbecco's phosphate-buffered saline (DPBS), then incubated with an anti-CAR antibody and an anti-CD11c antibody, then stained, washed once, resuspended in DPBS, and analyzed by flow cytometry to determine the successful reconstruction of humanized mice.

(43) Preparation of humanized CAR-DCs: Tibias and fibulas of hind legs of the humanized mice were collected. The bone marrow was flushed out by a 1 ml syringe, properly ground into a single-cell suspension, and centrifuged at 1,500 rpm for 5 min, and a resulting supernatant was removed. Red blood cells were lysed with a 1ACK buffer. Centrifugation was conducted at 1,500 rpm for 5 min, and a resulting supernatant was removed. Cells were washed twice with PBS, counted, and inoculated in a 6-well plate at a density of 110.sup.6/mL. 100 ng/ml of human GMCSF and human IL4 were added to allow the differentiation of DCs. A medium was supplemented every 2 d to 3 d. After 8 d of differentiation, DCs were harvested, counted, and transfected with a lentivirus encoding a human CAR-DC receptor (the amino acid sequence of the human Epha2 CAR protein was set forth in SEQ ID NO: 21). 48 h later, a CAR expression efficiency was detected with protein L. As shown in FIG. 17, a transfection efficiency of CAR in DCs was 45.3%.

(44) In vivo treatment: Human breast cancer cells SK-BR3 were transplanted into the humanized mice. One week after the tumor transplantation, the mice were randomly divided into the following five groups: a control group (PBS), a DC group, a CAR-DC group, a radiotherapy+DC group (IR+DC), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 2, mice in the radiotherapy+DC group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 1 Gy. On day 4, DCs and CAR-DCs each were infused through the tail vein at a dose of 310.sup.6 cells/mouse. The growth of mouse tumors were continuously observed. A length and width of a tumor were measured regularly with an electronic vernier caliper. A volume of the tumor was calculated based on the length and width of the tumor according to the following calculation formula: (width{circumflex over ()}2*length)/2. A tumor volume of each measurement was determined with a measurement on day 0 as a baseline. A baselined tumor growth curve (calculation formula: Vol.sub.Dayn/Vol.sub.Day0) was plotted with the Prism Graphad software. It could be seen from tumor growth curves (FIG. 18) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of breast cancer (differences among groups were determined by t-test, *p<0.05).

(45) The sequences involved in the present application include the following:

(46) TABLE-US-00001 AminoacidsequenceformouseEpha2CAR (SEQIDNO:1): MASPLTRFLSLNLLLLGESIILGSGEADIQMTQSPSSLSASVGDRVTIT CRASQYYSYYGVAWYQQKPGKAPKLLIYGASYLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQSFYPITFGQGTKVEIKGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNLSGGGVHWVRQAPGKGLEW VAGIYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC ARSSGGFDYWGQGTLVTVSSTTTKPVLRTPSPVHPTGTSQPQRPEDCRP RGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSRGVAIPR WPSSPAQSGKESGRPRKHIDQTSFDLQTYGDEDLNEIHSHYKMRLKIQV RKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ. NucleotidesequenceformouseEpha2CAR (SEQIDNO:2): ATGGCCAGCCCTCTGACCAGATTCCTGTCTCTGAACCTGCTCCTGCTGG GAGAGTCTATCATCCTGGGATCAGGAGAGGCTGACATCCAGATGACCCA GAGCCCTTCCTCACTGAGCGCTTCCGTGGGTGACAGAGTGACTATTACC TGCAGAGCCAGCCAGTACTACAGCTACTATGGAGTGGCCTGGTACCAGC AGAAGCCTGGCAAAGCTCCTAAGCTGCTGATCTATGGAGCTTCTTACCT GTACTCCGGGGTCCCATCTAGGTTCAGCGGCTCTAGGTCTGGCACCGAC TTCACTCTGACCATCTCCAGCCTGCAGCCAGAAGACTTCGCCACCTACT ACTGCCAGCAGAGTTTCTACCCCATCACCTTCGGACAGGGAACCAAGGT GGAAATCAAAGGCGGCGGCGGGAGCGGGGGCGGCGGCTCTGGAGGCGGC GGGTCCGAAGTCCAGCTGGTGGAGAGCGGCGGAGGTCTGGTGCAGCCAG GCGGCTCCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCAACCTGTCCGG GGGTGGAGTGCACTGGGTGAGGCAGGCTCCCGGCAAGGGACTGGAGTGG GTGGCTGGAATCTACTCCAGCTCCGGATACACATACTATGCCGACAGCG TGAAGGGCAGGTTTACCATCAGCGCCGACACCTCTAAAAACACCGCATA CCTGCAGATGAATAGCCTGCGAGCCGAGGATACAGCCGTGTATTACTGC GCCAGGAGCTCCGGCGGCTTTGATTACTGGGGGCAGGGCACTCTGGTGA CTGTGTCCTCTACAACAACTAAGCCTGTGCTGAGGACCCCTTCCCCTGT GCACCCAACCGGCACCAGCCAGCCCCAGCGACCTGAGGACTGCAGACCC CGGGGATCTGTGAAGGGCACCGGGCTGGATTTTGCATGTGACATTTATA TCTGGGCCCCTCTGGCCGGCATCTGCGTGGCCCTGCTGCTGTCTCTGAT CATTACCCTGATCTGCTATCATAGATCCAGAGGGGTGGCTATCCCCAGA TGGCCTAGCAGCCCAGCCCAGAGTGGAAAAGAGAGCGGCCGCCCTAGAA AGCACATCGACCAGACCTCTTTTGATCTGCAAACTTACGGTGACGAGGA TCTGAATGAGATCCACTCTCACTACAAGATGAGGCTGAAGATACAGGTG CGGAAGGCAGCCATCGCAAGCAGAGAGAAGGCCGACGCCGTGTACACAG GCCTGAACACAAGATCTCAGGAGACCTATGAGACCCTGAAGCATGAGAA GCCCCCCCAGTGA. Aminoacidsequencefortheguidesequenceof mouseEpha2CAR(SEQIDNO:3): MASPLTRFLSLNLLLLGESIILGSGEA. Nucleotidesequencefortheguidesequenceof mouseEpha2CAR(SEQIDNO:4): ATGGCCAGCCCTCTGACCAGATTCCTGTCTCTGAACCTGCTCCTGCTGG GAGAGTCTATCATCCTGGGATCAGGAGAGGCT. Aminoacidsequenceforanti-mouseEpha2VH (SEQIDNO:5): DIQMTQSPSSLSASVGDRVTITCRASQYYSYYGVAWYQQKPGKAPKLLI YGASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSFYPITF GQGTKVEIK. Nucleotidesequenceforanti-mouseEpha2VH (SEQIDNO:6): GACATCCAGATGACCCAGAGCCCTTCCTCACTGAGCGCTTCCGTGGGTG ACAGAGTGACTATTACCTGCAGAGCCAGCCAGTACTACAGCTACTATGG AGTGGCCTGGTACCAGCAGAAGCCTGGCAAAGCTCCTAAGCTGCTGATC TATGGAGCTTCTTACCTGTACTCCGGGGTCCCATCTAGGTTCAGCGGCT CTAGGTCTGGCACCGACTTCACTCTGACCATCTCCAGCCTGCAGCCAGA AGACTTCGCCACCTACTACTGCCAGCAGAGTTTCTACCCCATCACCTTC GGACAGGGAACCAAGGTGGAAATCAAA. Aminoacidsequenceforanti-mouseEpha2VL (SEQIDNO:7): EVQLVESGGGLVQPGGSLRLSCAASGFNLSGGGVHWVRQAPGKGLEWVA GIYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR SSGGFDYWGQGTLVTVSS. Nucleotidesequenceforanti-mouseEpha2VL (SEQIDNO:8): GAAGTCCAGCTGGTGGAGAGCGGCGGAGGTCTGGTGCAGCCAGGCGGCT CCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCAACCTGTCCGGGGGTGG AGTGCACTGGGTGAGGCAGGCTCCCGGCAAGGGACTGGAGTGGGTGGCT GGAATCTACTCCAGCTCCGGATACACATACTATGCCGACAGCGTGAAGG GCAGGTTTACCATCAGCGCCGACACCTCTAAAAACACCGCATACCTGCA GATGAATAGCCTGCGAGCCGAGGATACAGCCGTGTATTACTGCGCCAGG AGCTCCGGCGGCTTTGATTACTGGGGGCAGGGCACTCTGGTGACTGTGT CCTCT. AminoacidsequenceforthelinkerofmouseEpha2 CAR(SEQIDNO:9): GGGGSGGGGSGGGGS. NucleotidesequenceforthelinkerofmouseEpha2 CAR(SEQIDNO:10): GGCGGCGGCGGGAGCGGGGGCGGCGGCTCTGGAGGCGGCGGGTCC. AminoacidsequencefortheCD8ahingedomainof themouse(SEQIDNO:11): TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY. NucleotidesequencefortheCD8ahingedomainof themouse(SEQIDNO:12): ACAACAACTAAGCCTGTGCTGAGGACCCCTTCCCCTGTGCACCCAACCG GCACCAGCCAGCCCCAGCGACCTGAGGACTGCAGACCCCGGGGATCTGT GAAGGGCACCGGGCTGGATTTTGCATGTGACATTTAT. AminoacidsequencefortheCD8atransmembrane domainofthemouse(SEQIDNO:13): IWAPLAGICVALLLSLIITLICYHRSR. NucleotidesequencefortheCD8atransmembrane domainofthemouse(SEQIDNO:14): ATCTGGGCCCCTCTGGCCGGCATCTGCGTGGCCCTGCTGCTGTCTCTGA TCATTACCCTGATCTGCTATCATAGATCCAGA. AminoacidsequencefortheDectin1intracellular domainofthemouse(SEQIDNO:15): GVAIPRWPSSPAQSGKESGRPRKHIDQTSFDLQTYGDEDLNEIHSHYKM. NucleotidesequencefortheDectin1intracellular domainofthemouse(SEQIDNO:16): GGGGTGGCTATCCCCAGATGGCCTAGCAGCCCAGCCCAGAGTGGAAAAG AGAGCGGCCGCCCTAGAAAGCACATCGACCAGACCTCTTTTGATCTGCA AACTTACGGTGACGAGGATCTGAATGAGATCCACTCTCACTACAAGATG. Aminoacidsequencefortheintracellulardomain ofmouseFcRgamma(SEQIDNO:17): RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ. Nucleotidesequencefortheintracellulardomain ofmouseFcRgamma(SEQIDNO:18): AGGCTGAAGATACAGGTGCGGAAGGCAGCCATCGCAAGCAGAGAGAAGG CCGACGCCGTGTACACAGGCCTGAACACAAGATCTCAGGAGACCTATGA GACCCTGAAGCATGAGAAGCCCCCCCAG. ForwardstrandoftheprimerformouseCARIVT (invitrotranscription)(SEQIDNO:19): ATAATACGACTCACTATAGGGAGAGCCACCATGGCCAGCCCTCTGACCA G. ReversestrandoftheprimerformouseCARIVT (SEQIDNO:20): TTTTTTTTTTTTTCTGTCTTTTTATTGCCGTCACTGGGGGGGCTTCTCA T. AminoacidsequenceforhumanEpha2CAR (SEQIDNO:21): MALPVTALLLPLALLLHAARPQVQLLESGGGLVQPGGSLRLSCAASGFT FSSYTMSWVRQAPGQALEWMGTISSRGTYTYYPDSVKGRFTISRDNAKN SLYLQMNSLRAEDTAVYYCAREAIFTHWGRGTLVTVSSGGGGSGGGGSG GGGSDIQLTQSPSSLSASVGDRVTITCKASQDINNYHSWYQQKPGQAPR LLIYRANRLVDGVPDRESGSGYGTDFTLTINNIESEDAAYYFCLKYNVF PYTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRWPPSAACSGKESVVA IRTNSQSDFHLQTYGDEDLNELDPHYEMRLKIQVRKAAITSYEKSDGVY TGLSTRNQETYETLKHEKPPQ.

(47) Finally, it should be noted that the above examples are provided merely to describe the technical solutions of the present application, rather than to limit the protection scope of the present application. Although the present application is described in detail with reference to preferred examples, a person of ordinary skill in the art should understand that modifications or equivalent replacements may be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.