Improved Yield and Stablity of L49-sFv-beta-Lactamase,a Single-Chain Antibody Fusion Protein for Anticancer Prodrug Activaton,by Protein Engineering

摘要

The L49 single-chain Fv fused to beta-lactamease (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen.However,large-scale production of L49-sFv-bL from refolded E.coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein.Sequence analysis of theL49-sFv framework regions revealed three residues in the framework regions at positions L2,H82B,and H91,which are not conserved for their position,occurring in <1% ofsequences in Fv sequence dataases.one further unusual residue,found in <3% of variable sequences,was observed at position H39.Each unusual residue was mutated to a conserved residue for its position and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein.In an attempt to further imporve yield,the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resultin gin a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein.The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations.Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts,respectively.These reslts show that a rational protein-engineering approch improved the yield,stability,and refolding characteristics of L49-sFv-bL while maintanining binding affinity and therapeutic efficacy.