Construction and expression of humanized anti-EGFR antibody / 军事医学科学院院刊

ABSTRACT

Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.