Chimeric Antigen Receptor and Method for Treating Cancers

Abstract

The present disclosure provides a chimeric antigen receptor and a combination of the chimeric antigen receptor and DAP10. The present disclosure provides an expression vector and a host cell for expressing the chimeric antigen receptor and said combination of the chimeric antigen receptor and DAP10. The present disclosure also provides use of the chimeric antigen receptor and the combination of the chimeric antigen receptor and DAP10 in the treatment of cancers or in the preparation of pharmaceutical compositions for treating cancers. The chimeric antigen receptor and the drugs provided in the present disclosure can effectively treat liver cancer, lung cancer and the like.

Claims

1. An isolated nucleic acid, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding DAP10 or a functional fragment thereof, wherein the CAR comprises: (a) an antigen binding domain comprising an NKG2D fragment corresponding to amino acid residues 82-216 of human NKG2D; (b) a transmembrane domain; and (c) an intracellular signaling domain.

2. The nucleic acid of claim 1, wherein the CAR comprises a nucleic acid sequence encoding a transmembrane domain of CD8 and/or CD28.

3. (canceled)

4. The nucleic acid of claim 1, wherein the intracellular signaling domain comprises CD28, 4-1BB and CD3ζ in an order from an N terminal to a C terminal thereof.

5. The nucleic acid of claim 1, wherein the CAR further comprises a hinge region between said antigen binding domain and said transmembrane domain.

6. The nucleic acid of claim 1, wherein the CAR further comprises a leader sequence upstream said NKG2D fragment.

7. (canceled)

8. The nucleic acid of claim 1, further comprising a nucleic acid sequence of IRES between the nucleotide sequence encoding said CAR and the nucleic acid sequence encoding said DAP10 or the functional fragment thereof.

9-13. (canceled)

14. A host cell, comprising the nucleic acid of claim 1.

15. The host cell of claim 14, wherein the host cell is a T cell.

16. The host cell of claim 14, wherein the host cell is a subject to be treated.

17-22. (canceled)

23. A method for treating or preventing cancer, using the nucleic acid of claim 1.

24. The nucleic acid of claim 1, wherein the nucleic acid is part of a recombinant expression vector expressing said nucleotide sequence encoding a chimeric antigen receptor (CAR) and the nucleic acid sequence encoding DAP10 or a functional fragment thereof.

25. The nucleic acid of claim 24, wherein the recombinant expression vector comprises a nucleic acid sequence encoding a leader sequence positioned upstream of said NKG2D fragment.

26. The nucleic acid of claim 25, wherein the recombinant expression vector further comprises a nucleic acid control element positioned upstream of the nucleic acid encoding said leader sequence.

27. The nucleic acid of claim 24, wherein the recombinant expression vector further comprises a promotor positioned upstream of the nucleotide sequence encoding the CAR.

28. The nucleic acid of claim 24, wherein the recombinant expression vector is a viral vector.

29. The method of claim 23, wherein said cancer is leukemia, lymphoma, multiple myeloma or a solid tumor.

30. The method of claim 29, wherein said leukemia is acute lymphocytic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, monocytic leukemia or hairy cell leukemia.

31. The method of claim 29, wherein said lymphoma is Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, or small lymphocytic lymphoma.

32. The method of claim 29, wherein said solid tumor is bladder cancer, urothelial cell carcinoma of urethra, ureter and renal pelvis, multiple myeloma, kidney cancer, breast cancer, colon cancer, head and neck cancer, lung cancer, prostate cancer, glioblastoma, osteosarcoma, liposarcoma, soft tissue sarcoma, ovarian cancer, melanoma, liver cancer, esophageal cancer, pancreatic cancer, or gastric cancer.

33. The method of claim 29, wherein said cancer is liver cancer or lung cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1 shows flow cytometry analysis result of detecting the expression of NKG2D ligand on Raji cell line.

[0078] FIG. 2 shows construction diagram of expression vectors. A: pCCL-DRCAR-IRES-DAP10 expression vector; B: pHAGE-DRCAR expression vector.

[0079] FIG. 3 shows the nucleotide sequence and description of the insert fragment of the expression vector pCCL-DRCAR-IRES-DAP10.

[0080] FIG. 4 shows the nucleotide sequence and description diagram of the insertion fragment of the expression vector pHAGE-DRCAR.

[0081] FIG. 5 shows flow cytometry analysis results of plasmid co-transfection 293T cells expressing DRCAR. The left is the control (only with the lentivirus packaging plasmid but without the recombinant plasmid). In the middle is the lentivirus with the pHAGE-DRCAR recombinant plasmid. On the right is the lentivirus with pCCL-DRCAR-IRES-DAP10 recombinant plasmid.

[0082] FIG. 6 shows titration of lentivirus expressing DRCAR. (A) is flow cytometry analysis results of lentiviruses expressing DRCAR in different amounts. (B) is the corresponding histogram. (C) Calculate the lentivirus titer formula and result based on the analysis data of the flow cytometer.

[0083] FIG. 7 shows the effect of DR-CAR-T cells in killing cancer cells. FIG. 7 (a)-FIG. 7 (u) show the DR-CAR-T cells killing effect on 21 kinds of cancer cell line expressing NKG2D ligand. FIG. 7(v) shows the DR-CAR-T cells killing effect on Raji cells that do not express NKG2D ligand. The percentage of cancer cells death was analyzed by flow cytometry.

[0084] FIG. 8 shows DR-CAR T cells inhibit tumors in human liver cancer transplantation animal models. FIG. 8 (a) is the experimental design diagram. FIG. 8(b) is a graph showing the survival curves of liver cancer transplanted mice in the experimental group injected with 4×10.sup.6 T cells or DR-CAR T cells. FIG. 8(c) is a graph showing the survival curves of liver cancer transplanted mice in the experimental group injected with 5×10.sup.6 T cells or DR-CAR T cells.

[0085] FIG. 9 shows the survival curves of animals injected with DR-CAR T cells in lung cancer transplanted mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] The inventions provided in the above section of the disclosure are further described with reference to the following examples, which are intended to illustrate the disclosure and not to limit the scope of the disclosure.

Example 1: Cell Production, Process, and Methods

[0087] Buffy coat was obtained from the Hong Kong Red Cross Blood Transfusion Service. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat by using Ficoll-Paque PLUS (GE Healthcare). T cells were isolated from PBMCs by using CD3/CD28 Dynabeads (Thermo). T cells isolated from PBMCs were cultured in initiate medium consisting of AIM-V medium (Thermo) supplemented with 5% human serum (Sigma), 2 mM L-glutamine (Thermo) and 50 U/ml IL-2 (Peprotech), or expansion medium consisting of AIM-V medium supplemented with 5% human serum, 2 mM L-glutamine and 300 U/ml IL-2.

[0088] All the cell lines mentioned below were obtained from ATCC, ECACC or Chinese Academy of Sciences Cell Bank.

[0089] 293T cells (ATCC #CRL-3216) were cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100 U/ml penicillin (Thermo) and 100 ug/ml streptomycin (Thermo).

[0090] Chronic myelogenous leukemia cell line-K562(ATCC #CCL-243), was cultured in IMEM medium (Thermo) supplemented with 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin.

[0091] Acute lymphocytic leukemia cell line MOLT-4 (ATCC #CRL-1582), non-Hodgkin's lymph tumor cell line KARPAS299 (ECACC #06072604), myeloma cell line-RPMI8226 (ATCC #CRM-CCL-155), NCI-H929 (ATCC #CRL-9608) and U266B1 (ATCC #TIB-196), acute T cells Leukemia cell line Jurkat (ATCC #TIB-152), gastric cancer cell line HGC27 (ECACC #94042256), lung cancer cell line NCI-H522 (ATCC #CRL-5810), breast cancer cell line MDA-MB-4355 (ATCC #HTB-129) and MDA-MB-231 (ATCC #HTB-26), bladder cancer cell line 5637 (ATCC #HTB-9), liver cancer cell line QGY7703, SMMC7721 and BEL7402, were cultured in RPMI1640 medium (Thermo) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin.

[0092] Cervical cancer cell line Hela (ATCC #CCL-2), neuroblastoma cell line SK-N-SH (ATCC #HTB-11) were cultured in MEM medium (Thermo) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin.

[0093] Lung cancer cell line A549 (ATCC #CCL-185) and prostate cancer cell line PC3 (ATCC #CRL-1435) were cultured in F12 medium (Thermo) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin.

[0094] Lung cancer cell line NCI-H1155 (ATCC #CRL-5818) and adenocarcinoma cell line NCI-H1355 (ATCC #CRL-5865) were cultured in serum-free ACL-4 medium (Thermo).

[0095] Construction of Retroviral Plasmids.

[0096] Lentivirus Packaging, Concentration and Purification

[0097] Lentivirus was produced by co-transfection of 3rd generation lentiviral plasmid-pMDLg/pRRE, pMD2.G, pRSV-Rev, and transfer plasmids in a ratio of 2:1:1:4 into 293T cells by calcium phosphate transfection method. The freshly collected or thawed supernatant containing lentivirus was put in a centrifuge at 300 g for 3 min to exclude cell debris in the supernatant. The supernatant was filtered by a 0.45-μm Minisart syringe filter attached to a 30-ml syringe (TERUMO). The supernatant was centrifuged at 20000 g, 4° C. for 90 minutes. After ultracentrifugation, the supernatant was removed. 1/10 of the initiate lentivirus volume of AIM-V medium was added into centrifuge tubes to re-suspend the pellets. The lentivirus suspension was mixed by pipetting. The concentrated lentivirus was aliquoted and stored in −80° C. freezer.

[0098] Titration of Lentivirus

[0099] 1×10.sup.5 Jurkat cells were seeded into each well of 12-well plate in 1 ml RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin. After overnight culture, different amounts (5 ul to 100 ul) of concentrated lentivirus were added into separate wells. The samples were duplicated or triplicated to increase accuracy. Polybrene (Sigma) was added to a final concentration of 6 ug/ml in each well. 24 hours later, cells were collected by centrifuge and re-suspended in 1 ml fresh RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin. Another 48 hours later, cells were harvested and the percentage of Jurkat cells expressing CAR was determined by flow cytometry. The titer of lentivirus was calculated by the following formula.

[00001]$\mathrm{Lentivirus}\mathrm{Titer}\left(\mathrm{TU}\text{/}\mathrm{ml}\right)=\frac{\mathrm{cell}\mathrm{number}\times \%\mathrm{of}\mathrm{reporter}\mathrm{positive}\mathrm{cells}\times \mathrm{dillution}\mathrm{factor}}{\mathrm{vector}\mathrm{volume}\left(\mathrm{ml}\right)\times 100}$

[0100] T Cells Isolation, Transduction and Cultivation

[0101] 1×10.sup.7 PBMC were isolated for CD3.sup.+ cells by using Dynabeads coated with antibodies to CD3 and CD28 at a bead-to-cell ratio of 3:1. The cell and bead mixture was incubated at room temperature for 1 hour on a shaker. The CD3.sup.+ cells enrichment was performed by magnet and re-suspended in initiation medium at 1×10.sup.6 cells/ml. 24 hours later, cells were collected by centrifugation (300×g, 3 minutes). The supernatant was discarded. 5×10.sup.8 TU lentivirus in 500 ul AIM-V medium was added into cells and centrifuged at 2000×g for 2 hours. Cells were re-suspended in the lentivirus culture and 1.5 ml initiation was added. The cells were put back into 6-well plates and placed into an incubator (37 C, 5% CO.sub.2). 24 hours later, Transduction was performed again. Another 24 hours later, cells were collected by centrifuge (300×g, 3 minutes) and re-suspended in 2 ml of expansion medium. The cells were put back into 6-wells plate and placed into incubator (37 degree, 5% CO.sub.2). 72 hours later, cells were transferred to a 100-cm dish and re-suspended in expansion media at concentration of 4×10.sup.5 cells/ml. Transduction rate can be determined by using Flow cytometer and cytotoxicity assay can be performed when T cells are sufficiently transduced.

[0102] Flow Cytometry Analysis

[0103] To detect CAR expression on the cell surface (both T cells and Jurkat cells), 1×10.sup.6 cells were re-suspended in 1 ml PBS buffer and stained with biotin goat anti-human IgG (H+L) (Jason Lab) followed by streptavidin-PE (eBioscience).

[0104] Cytotoxicity Assay

[0105] Target cells were collected by centrifuge and re-suspended in PBS at a concentration of 1×10.sup.6 cells/ml. 5 ml of the cells were stained with 2.5 ul of Oregon Green 488 (Thermo) for 20 minutes at 37 C. 20 ml of culture medium was added to absorb the excess dye. Target cells were re-suspended in culture medium at concentration of 4×10.sup.5 cells/ml.

[0106] T cells were collected by centrifuge and re-suspended with the medium required by the target cells at a concentration of 1.6×10.sup.7 cells/ml.

[0107] T cells and target cells were mixed in a ratio of 5, 10, 20 and 40 T-cells per target cell.

TABLE-US-00002 E:T ratio .sup.Note 40:1 20:1 10:1 5:1 0:1 target cell 500 μL 500 μL 500 μL  500 μL 500 μL T cell 500 μL 250 μL 125 μL 62.5 μL  0 μL Target cell  0 μL 250 μL 375 μL 437.5 μL  500 μL medium Note: E:T ratio = T cells and target cells ratio

[0108] Cells were incubated using an incubator for 5 hours or 8 hours accordingly. Cells were collected by centrifuge and re-suspended in 500 ul 7-AAD solution (1 ug/ml). Cells were incubated on ice for 30 minutes. Death rate was analyzed by flow cytometer.

Example 2: Expression of NKG2D Ligands in Various Cancer Cell Lines

[0109] Detect the expression of NKG2D ligand (human MICA/B and human ULBP1-ULBP6) on different cancer cell lines to determine whether CAR with NKG2D as the antigen domain can be used to kill these cell lines.

[0110] To detect human MICA/B, resuspend 1×10.sup.6 cells to be tested in 0.5 ml PBS buffer, and used monoclonal mouse anti-human MICA/B (R&D Cat #MAB13001) followed by biotin goat anti-mouse IgG (H+L) and then use streptavidin-APC staining.

[0111] In order to detect human ULBP2/5/6, resuspend 1×106 cells to be tested in 0.5 ml PBS buffer, and used monoclonal mouse anti-human ULBP2/5/6 (R&D Cat #MAB1298) followed by biotin Goat anti-mouse IgG (H+L) and then used streptavidin-APC staining.

[0112] To detect human ULBP1, 1×10.sup.6 cells to be tested were resuspended in 0.5 ml PBS buffer, and used monoclonal mouse anti-human ULBP1 (R&D Cat #MAB1380) followed by biotin goat anti-mouse IgG (H+L) and then used streptavidin-APC staining.

[0113] To detect human ULBP3, 1×10.sup.6 cells to be tested were resuspended in 0.5 ml PBS buffer, and used monoclonal mouse anti-human ULBP3 (R&D Cat #MAB1517) followed by biotin goat anti-mouse IgG (H+L) and then used streptavidin-APC staining.

[0114] In order to detect human ULBP4, resuspend 1×10.sup.6 cells to be tested in 0.5 ml PBS buffer, and use monoclonal mouse anti-human ULBP4 (R&D Cat #AF6285) followed by biotin bovine anti-goat IgG (H+L) and then used streptavidin-APC staining.

[0115] The following 21 cancer cell lines were tested for expression of NKG2D ligands (human MICA/B and human ULBP1-ULBP6):

[0116] Chronic myelogenous leukemia cell line K562 (ATCC #CCL-243), acute lymphocytic leukemia cell line MOLT-4 (ATCC #CRL-1582), non-Hodgkin lymphoma cell line KARPAS299 (ECACC #06072604), Myeloma cell line RPMI8226 (ATCC #CRM-CCL-155), NCI-H929 (ATCC #CRL-9608) and U266B1 (ATCC #TIB-196), acute T-cell leukemia cell line Jurkat (ATCC #TIB-152), gastric cancer cell line HGC27 (ECACC #94042256), lung cancer cell line NCI-H522 (ATCC #CRL-5810), breast cancer cell line MDA-MB-435S (ATCC #HTB-129) and MDA-MB-231 (ATCC #HTB-26), bladder cancer cell line 5637 (ATCC #HTB-9), liver cancer cell line QGY7703, SMMC7721 and BEL7402, cervical cancer cell line Hela (ATCC #CCL-2), neuroblast Tumor cell line SK-N-SH (ATCC #HTB-11), lung cancer cell line A549 (ATCC #CCL-185), prostate cancer cell line PC3 (ATCC #CRL-1435), lung cancer cell line NCI-H1155 (ATCC #CRL-5818) and adenocarcinoma cell line NCI-H1355 (ATCC #CRL-5865).

[0117] Experimental results proved that NKG2D ligands (human MICA/B or human ULBP1-ULBP6) are expressed in the 21 cancer cell lines mentioned above. The 21 cancer cell lines mentioned above were tested as target cells in the following CAR T cell killing experiment.

[0118] In addition, as shown in FIG. 1, Raji cells do not express NKG2D ligands (human MICA/B and human ULBP1-ULBP6). Raji cells were used as a negative control in CAR T cell killing experiments.

Example 3: Construction of Lentiviral Vector Expressing DRCAR

[0119] 1. Construction of pCCL-DRCAR-IRES-DAP10

[0120] pCCL-DRCAR-IRES-DAP10 has the structure shown in FIG. 2A. pCCL-DRCAR-IRES-DAP10 was constructed by the following method.

[0121] Synthesized a nucleic acid insert encoding DRCAR and DAP10, which has a nucleotide sequence as shown in FIG. 3. The insert includes, from 5′ to 3′, the fragments: 1. HpaI restriction site; 2. EF1α promoter; 3. Kozak sequence; 4. CD33 leader sequence; 5. aa82-216 fragment of NKG2D; 6. IgGH1 as the hinge region; 7. CD28 transmembrane domain; 8. CD28 intracellular signaling domain; 9. 4-1BB intracellular signaling domain; 10. CD3ζ intracellular signaling domain; 11. Ligation fragment; 12. IRES; 13. Ligation fragment; 14. DAP10; 15. Sal I restriction site.

[0122] The above insert fragment and plasmid Pax5 (Addgene, plasmid #35003) were double digested with restriction enzymes HpaI and Sal I. After the digested product was cut and recovered, it was ligated with T4 ligase overnight at 16° C. After ligation, transform the ligation product into competent E. coli, spread onto the plate, a single colony was selected and plasmid DNA was extracted for double enzyme digestion. The inserted sequence was underwent DNA sequencing. pCCL-DRCAR-IRES-DAP10 was then obtained.

[0123] 2. Construction of pHAGE-DRCAR

[0124] pHAGE-DRCAR has the structure shown in FIG. 2B. The pHAGE-DRCAR was constructed by the following method.

[0125] Synthesized a nucleic acid insert encoding DRCAR having a nucleotide sequence as shown in FIG. 4. The insert includes, from 5′ to 3′, the fragments: 1. HpaI restriction site; 2. EF1α promoter; 3. Kozak sequence; 4. CD33 leader sequence; 5. aa82-216 fragment of NKG2D; 6. IgG1H as the hinge region; 7. CD28 transmembrane domain; 8. CD28 intracellular signaling domain; 9. 4-1BB intracellular signaling domain; 10. CD3ζ intracellular signaling domain; 11. Sal I restriction site.

[0126] The inserted fragment and plasmid Pax5 were double-enzyme digested with restriction enzymes HpaI and Sal I. After the digested product was cut and recovered, it was ligated with the insert by using T4 ligase overnight at 16° C. After ligation, the ligated product was transformed into E. coli, spread onto the plate, and single colony was selected the next day for plasmid extraction. The plasmids underwent double enzyme digestion and sequencing. pHAGE-DRCAR was then obtained.

[0127] 3. Production of Lentiviral Vectors

[0128] Using PHAGE-DRCAR or pCCL-DRCAR-IRES-DAP10 as expression plasmids, and third-generation lentiviral plasmids pMDLg/pRRE, pMD2.G and pRSV-Rev were co-transfected to prepare corresponding lentiviral vectors.

[0129] Using method recited in Example 1, expression of DRCAR by the lentivirus were measured. FIG. 5 is a flow cytometric analysis result of 293T cells co-transfected with the plasmids expressing DRCAR. The results showed that, in comparison with pHAGE-DRCAR, the expression level of DRCAR by the lentivirus containing pCCL-DRCAR-IRES-DAP10 was significantly higher.

[0130] The titer of the lentivirus containing pCCL-DRCAR-IRES-DAP10 was calculated according to the method described in Example 1. FIG. 6 shows that the titer of pCCL-DRCAR-IRES-DAP10 packaged lentivirus is about 2×10.sup.7 TU/ml. Compared with pHAGE-DRCAR, the lentivirus containing pCCL-DRCAR-IRES-DAP10 gives a higher lentivirus titer.

Example 4: DR-CAR-T Cells Kill Cancer Cells In Vitro

[0131] According to the method described in Example 1, T cells were isolated from human PBMC. Then, the Lentivirus containing pCCL-DRCAR-IRES-DAP10 prepared in Example 3 was used to transduce T cells.

[0132] The cytotoxicity assay was performed according to the method described in Example 1, and the specific cytotoxic effects of T cells expressing DR-CAR on various tumor cells were examined. Among them, T cells expressing DR-CAR and untransduced T cells were used as effector cells, 21 different types of cancer cell lines were used as target cells.

[0133] The Raji cell line that does not express NKG2D ligand was used as a negative control.

[0134] The results are shown in FIG. 7(a)-FIG. 7(u). Under the same experimental conditions, compared with normal T cells, DR-CAR-T cells significantly kill NKG2D ligand expressing target tumor cells. The tumor cells include: Chronic myelogenous leukemia cell line-K562 (ATCC #CCL-243), acute lymphocytic leukemia cell line-MOLT-4 (ATCC #CRL-1582), non-Hodgkin lymphoma cell line-KARPAS299 (ECACC #06072604), Myeloma cell line-RPMI8226 (ATCC #CRM-CCL-155), NCI-H929 (ATCC #CRL-9608) and U266B1 (ATCC #TIB-196), acute T-cell leukemia cell line-Jurkat (ATCC #TIB-152)), gastric cancer cell line-HGC27 (ECACC #94042256), lung cancer cell line-NCI-H522 (ATCC #CRL-5810), breast cancer cell line-MDA-MB-435S (ATCC #HTB-129) and MDA-MB-231 (ATCC #HTB-26), bladder cancer cell line −5637 (ATCC #HTB-9), liver cancer cell line-QGY7703, SMMC7721 and BEL7402, cervical cancer cell line-Hela (ATCC #CCL-2), neuroblast Tumor cell line-SK-N-SH (ATCC #HTB-11), lung cancer cell line-A549 (ATCC #CCL-185), prostate cancer cell line-PC3 (ATCC #CRL-1435), lung cancer cell line-NCI-H1155 (ATCC #CRL-5818) and adenocarcinoma cell line-NCI-H1355 (ATCC #CRL-5865).

[0135] As shown in FIG. 7(v), the NKG2D ligand negative cell line, Raji, the cytotoxic effect of DR-CAR-T cells did not show any difference from normal T cells.

Example 5: DR-CAR T Cells Inhibit Tumors Growth in Human Liver Cancer Transplantation Animal Models

[0136] FIG. 8 (a) is the experimental design diagram.

[0137] A total of 20 experimental mice (NSG mice, 6-8 weeks old, provided by the Animal and Plant Car Facility of the Hong Kong University of Science and Technology) were divided into a DR-CAR T cell treatment group (12 randomly selected) and a T cells control group (8). All mice were intraperitoneal injected with liver cancer cells SMMC7721 (1×10.sup.6 cells) on day. Each group was injected with the same number of DR-CAR T cells (treatment group) or T cells (control group) in three times on the second, fifth and seventh weeks. Body weight and survival of mice was observed and recorded. The mice at the end of the experiment were characterized by death, or weight loss of 20% or more, sunken eyes or closed eyelids, unresponsive or outlier.

Experiment 1

[0138] In the second, fifth and seventh weeks of the experiment, 4×10.sup.6 DR-CAR T cells (treatment group) or T cells (control group) were injected three times

[0139] The experimental results are shown in FIG. 8(b).

Experiment 2

[0140] In the second, fifth and seventh weeks of the experiment, 5×10.sup.6 DRCAR T cells (treatment group) or T cells (control group) were injected three times

[0141] The experimental results are shown in FIG. 8(c).

[0142] From the experimental results, compared with the control group by using T cells that do not express DR-CAR, the DR-CAR T cells of the present disclosure can effectively protect the cancerous animals.

Example 6: DR-CAR T Cells Inhibit Tumors in Human Lung Cancer Transplantation Animal Models

[0143] The experimental arrangement is similar to Example 5, 24 experimental mice (NSG mice, 6-8 weeks old, provided by the Animal Feeding Room of the Department of Immunology of Peking University) which 16 mice are randomly divided into DR-CAR T cell therapy group (treatment group 1 and treatment group 2) and 8 mice are in control normal T cell group. All mice were injected with 1×10.sup.6 lung cancer cell line, A549, on the day 0, and each group was injected DRCAR T cells (treatment group) or T cells (control group) were injected three times at 2.sup.nd week (14th day), 4th week (28.sup.th day) and 6.sup.th week (42.sup.nd day). Body weight and survival of mice was observed and recorded. The mice at the end of the experiment were characterized by death, or weight loss of 20% or more, sunken eyes or closed eyelids, unresponsive or outlier.

[0144] Treatment group 1: In the second, fourth and sixth week of the experiment, 2.5×10.sup.6 DRCAR T cells were injected three times

[0145] Treatment group 2: In the second, fourth and sixth week of the experiment, 5×10.sup.6 DRCAR T cells were injected three times

[0146] Control group: 5×10.sup.6 T cells were injected three times in the second, fourth and sixth week of the experiment

[0147] The experimental results are shown in FIG. 9.

[0148] From the experimental results, compared with the control group by using T cells that do not express DR-CAR, the DR-CAR T cells of the present disclosure can effectively protect the cancerous animals.

[0149] The above experimental results show that the DRCAR and DRCAR-T cells with the NKG2D antigen receptor structure provided by the present disclosure can effectively recognize cancer cells with NKG2D ligands and activate tumor cell-specific anti-tumor cell immune responses and kill related tumor cells. The experiments results have also proved that the DRCAR and DRCAR-T cells provided by the present disclosure can kill various cancer cells in a broad spectrum of cancers, and have proven their effectiveness in inhibiting various cancers in animal.

[0150] Unless otherwise indicated, the practice of the present disclosure will employ common technologies of organic chemistry, polymer chemistry, biotechnology, and the like. It is apparently that in addition to the above description and examples than as specifically described, the present disclosure can also be achieved in other ways. Other aspects within the scope of the disclosure and improvement of the present disclosure will be apparent to the ordinary skilled in the art. According to the teachings of the present disclosure, many modifications and variations are possible, and therefore it is within the scope of the present disclosure.

[0151] Unless otherwise indicated herein, the temperature unit “degrees” refers to Celsius degrees, namely ° C.