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Embedded antigen acceptor and method for treating lung cancer through embedded antigen acceptor

A chimeric antigen receptor and antigen technology, applied in the fields of immunology and medicine, can solve problems such as damage to normal human cells

Pending Publication Date: 2019-10-18
DAREN BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But chemotherapy can not only kill tumor cells, but also damage normal human cells

Method used

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  • Embedded antigen acceptor and method for treating lung cancer through embedded antigen acceptor
  • Embedded antigen acceptor and method for treating lung cancer through embedded antigen acceptor
  • Embedded antigen acceptor and method for treating lung cancer through embedded antigen acceptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0105] Example 2 Expression of NKG2D ligands on various cancer cell lines

[0106] The expression of NKG2D ligands (human MICA / B and human ULBP1-ULBP6) was detected on different cancer cell lines to determine whether the CAR with NKG2D as the antigenic domain could be used to kill these cell lines.

[0107] To detect human MICA / B, 1×10 6 Each cell to be tested was resuspended in 0.5ml PBS buffer and treated with monoclonal mouse anti-human MICA / B (R&D Cat#MAB13001), followed by biotinylated goat anti-mouse IgG (H+L) and streptavidin Primer-APC staining.

[0108] To detect human ULBP2 / 5 / 6, mix 1×10 6 Each cell to be tested was resuspended in 0.5ml PBS buffer and treated with monoclonal mouse anti-human ULBP2 / 5 / 6 (R&D Cat#MAB1298), followed by biotinylated goat anti-mouse IgG(H+L) and streptavidin Avidin-APC staining.

[0109] To detect human ULBP1, 1×10 6 Each cell to be tested was resuspended in 0.5ml PBS buffer and treated with monoclonal mouse anti-human ULBP1 (R&D Cat#...

Embodiment 3

[0116] Example 3 Construction of a lentiviral vector expressing DRCAR

[0117] 1. Construction of pCCL-DRCAR-IRES-DAP10

[0118] pCCL-DRCAR-IRES-DAP10 has as figure 2 The structure shown in A. pCCL-DRCAR-IRES-DAP10 was constructed by the following method.

[0119] Synthetic nucleotide sequences such as image 3 Nucleic acid inserts encoding DRCAR and DAP10 are indicated. The insert fragment includes from the 5' end to the 3' end: 1. HpaI restriction site; 2. EF1α promoter; 3. Kozak; 4. CD33 leader sequence; 5. NKG2D a.a.82-216 fragment; IgG1H in the hinge region; 7. CD28 transmembrane domain; 8. CD28 intracellular signaling domain; 9.4-1BB intracellular signaling domain; 10. CD3ζ intracellular signaling domain; 11. connecting fragment; 12. IRES; 13. Junction fragment; 14. DAP10; 15. Sal I restriction site.

[0120] The above insert fragment and plasmid Pax5 (Addgene, plasmid #35003) were double-digested with restriction endonucleases HpaI and SalI, and the digested prod...

Embodiment 4

[0129] Example 4 DRCAR-T cells kill cancer cells in vitro

[0130] According to the method described in Example 1, T cells were extracted and obtained from human PBMC. Then, the lentivirus containing pCCL-DRCAR-IRES-DAP10 prepared in Example 3 was used to transfect T cells.

[0131] Cytotoxicity assays were performed according to the method described in Example 1 to examine the specific cytotoxic effects of DRCAR-expressing T cells on various tumor cells. Among them, T cells containing pCCL-DRCAR-IRES-DAP10 and T cells not transfected with lentivirus were used as effector cells, and four kinds of lung cancer cells were used as target cells.

[0132] A Raji cell line not expressing NKG2D ligand was used as a negative control.

[0133] The result is as Figure 7 (a)- Figure 7 As shown in (c), under the same experimental conditions, compared with normal T cells without DRCAR, T cells expressing DRCAR were able to induce significantly more target tumor cell death. The tumor ...

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Abstract

The invention relates to a new embedded antigen acceptor, and provides an expression vector for expressing the embedded antigen acceptor and a host cell. The invention further provides an applicationof the embedded antigen acceptor to treatment of cancer or preparation of medicines for treating cancer. The embedded antigen acceptor and the medicines, provided by the invention, can effectively treat lung cancer.

Description

[0001] This application claims the priority of the Chinese patent application filed on April 4, 2018 with application number 201810299324.8 and titled "NKG2D Chimeric Antigen Receptor and Its Method for Treating Cancer", the entire contents of which are incorporated herein by reference Applying. technical field [0002] The present invention relates to the fields of immunology and medicine. Specifically, the present invention provides novel chimeric antigen receptors. The present invention also provides the use of the new chimeric antigen receptor in treating cancer or preparing medicine for treating cancer. Background technique [0003] Lung cancer is one of the malignant tumors with the fastest increasing morbidity and mortality and the greatest threat to the health and life of the population. In the past 50 years, many countries have reported that the morbidity and mortality of lung cancer have increased significantly. The incidence and mortality of lung cancer in men r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C12N5/0636C12N15/86A61P35/00C07K2319/03C07K2319/33C12N2510/00C12N2740/15043C07K14/7056C07K14/4705A61K39/4611A61K2239/38A61K39/464429A61K2239/53A61K39/4631C12N5/10A61P35/02A61K31/7088C07K19/00C12N15/63A61K38/00C07K14/70517C07K14/70521C07K14/705C12N2740/16043C12N2840/203C07K16/2851A61K2039/844A61K2039/86C07K14/70578
Inventor 谢雍
Owner DAREN BIOTECH LTD
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