Construction, expression and functional analysis of anti-B7-H4- scFv-CH3 recombinant antibody.

The B7-H4 molecule, a unique negative regulator of T lymphocytes which is overexpressed on the surface of various tumor cells, is a particularly important target candidate for tumor therapy because it can be blocked with anti-B7-H4 antibodies to inhibit the B7-H4 signaling pathway. Our previous work established an anti-B7-H4 single-chain variable fragment (scFv) library, so we have now amplified the genes encoding anti-B7-H4-scFv and human IgG1 CH3 and ligated them by overlap extension PCR to obtain a recombinant gene. After sequencing, the gene was cloned into the expression vector pET43.1a and expression was induced in E. coli BL21 (DE3) by isopropyl-β-D-1-thiogalactopyranoside (IPTG). The protein was purified on a nickel-nitrilotriacetic acid (Ni-NTA) resin column and its antigen specificity and affinity were examined by ELISA and western blotting. We also established a Lewis lung cancer model in C57BL/6 mice to further identify the biological function of the scFv protein in vivo. The results showed that tumor volume, body weight and necrotic tissues in the control group were significantly greater than in the experimental group, indicating that selected scFvs had good biological activity and could inhibit tumor growth in tumor-bearing mice. Our work thus offers a new approach for the development of cancer-targeted therapy.