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Archive > Volume 23 Issue 5 > 2016,23(5):613-619. DOI:10.3872/j.issn.1007-385X.2016.05.005 Prev Next
Prompt Report
Killing effect of NK cells modified with pCD19-CAR on B-cell lymphoma
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Abstract:
Objective:To screen and prepare theraputic monoclonal antibody CD19 using hybridoma technique in mice, and to explore killing effect of NK cells modified with pCD19-CAR which made by CD19 scFv sequence on B-cell lymphoma cells. Methods:CD19 monoclonal antibody was prepared in mice immunized with conjugated polypeptide, then sequence of the obtained antibody was obtained by gene sequencing method. The antibody sequence was analyzed and pCD19-CAR fragments were constructed via gene synthesis and molecular cloning PCR-based gene synthesis techniques. Then the pCD19-CAR fragments were cloned into lentiviral vectors, and transfected into NK-92MI cells after packaging preparation. and cloned into lenti-virus using molecular cloning method; and then the positive hybridoma cells were analyzed for scFv sequences. One of the scFv sequence was used to construct CAR (pCD19-CAR).Finally, the killing rates of various pCD19-CAR-NK-92MI cells on B-cell lymphoma cells were examined using Flow cytometry assay. Results: (1) CD19 monoclonal antibody pCD19 with high specificity was successfully screened out. (2) The measurement of the antibody showed that the positive rates of Ramos and Raji cells were 84.3% and 85.6% respectively, which were similar to commercial CD19 antibody. (3) The killing efficacy of pCD19-CAR-NK-92MI cells, of which modification rate was 28.72%, on CD+Ramos cells and CD+Raji cells was apparently higher than those of non-modified NK-92MI cells (\[47.0±1.7\]% vs \[24.7±6.2\]% and \[51.8±7.9\]% vs \[27.6±9.3\]%, all P<0.05). Specific killing effects of unmodified and modified NK-92 cells on CD19- Jurkat cells were not almost found (\[16.1±0.7\]% vs \[17.7±2.9\]%, P>0.05). Conclusion：pCD19-CAR was successfully constructed ; pCD19-CAR-NK-92MI cells could specifically recognize CD19 antigen and kill CD19+ B-cell lymphoma cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China（No.31471283）
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