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
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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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