Construction, Expression And Targeting Studies Of Mouse ScFv₂₅, Humanized ScFv₂₅ And Their Fusion To Mutant TNF-α Against Hepatocellular Carcinoma

In the last years, our group had successfully constructed and highly expressed the mouse scFv₂₅ and humanized scFv₂₅ against hepatocellular carcinoma. After linking them to human natural TNF-α, the prokaryotic, eukaryotic expression and targeting therapy were studied early or late. Those experiments all had obtained certain therapeutic efficacy. In order to avoid the serious side-etfect of human natural TNF-α, the mutant TNF-α with relative high biologic activity and low toxicity was used so that a new m/hscFv₂₅-mTNF-α anti-HCC bifunctional antibody with high effect, low toxicity was constructed. After induction and purification, we carried on the targeting research to HCC SMMC-7721 cells and HCC xenografts in nude mice. In the hope to get anti-HCC bifunctional antibody, which could be used to HCC targeting therapy in the future. 1. The construction and expression of m/hscFv₂₅-niTNF-αTwo relevant sites of enzymatic digestion were added to the mTNF-α by PCR. The mTNF-α was linked to the 3' end of m/hscFv₂₅ in pGEX4T-l vector. The prokaryotic expression vector pGEX4T-lm/hscFv₂₅-mTNF-a was constructed successfully. After induction and expression by IPTG, the expression of two kinds of fusion protein is 15% and 12% of total bacteria proteins respectively. The anti-HCC bifunctional antibodies...