Expression of single-chain Fv-Fc fusions in Pichia pastoris

Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is u seful to restore Fc mediated antibody functions such as avidity, effector f unctions and a prolonged serum half-life. We have constructed vectors for t he convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia p astoris. The scFv-Fc fusion protein is secreted and recovered from the cult ure medium as a disulfide-linked, glycosylated homodimer. The increased siz e of the dimer (similar to 106 kDa vs. similar to 25 kDa for a scFv) result s in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fu sion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral bl ood mononuclear cells as effecters. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, e liminating the need for proteolytically sensitive epitope and/or affinity t ags on the scFv. (C) 2001 Elsevier Science B.V. All rights reserved.