Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.
The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.
Yang
Zhang-Min
ZM
Department of Biochemistry and Molecular Biology, Medical College, Shantou University, Shantou 515041, PR China. yzhangmin@yahoo.com
Li
En-Min
EM
Lai
Bao-Chang
BC
Wang
Yi-Li
YL
Si
Lu-Sheng
LS
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018122:B7-1 Antigen / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018122:B7-1 Antigen; D018106:CD28 Antigens; D017252:CD3 Complex; D011993:Recombinant Fusion Proteins