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

J Immunol Methods. 2001 May 1;251(1-2):123-35. doi: 10.1016/s0022-1759(00)00290-8.

Abstract

Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results 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 fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody-Dependent Cell Cytotoxicity
  • Base Sequence
  • DNA Primers / genetics
  • Genetic Vectors
  • Humans
  • Immunoglobulin Fc Fragments / biosynthesis
  • Immunoglobulin Fc Fragments / genetics*
  • Immunoglobulin Fc Fragments / isolation & purification
  • Immunoglobulin Fragments / biosynthesis
  • Immunoglobulin Fragments / genetics*
  • Immunoglobulin Fragments / isolation & purification
  • Immunoglobulin G / biosynthesis
  • Immunoglobulin G / genetics
  • Immunoglobulin G / isolation & purification
  • Male
  • Mice
  • Mice, SCID
  • Molecular Sequence Data
  • Peptide Library
  • Pichia / genetics*
  • Plasmids / genetics
  • Protein Engineering
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification

Substances

  • DNA Primers
  • Immunoglobulin Fc Fragments
  • Immunoglobulin Fragments
  • Immunoglobulin G
  • Peptide Library
  • Recombinant Fusion Proteins
  • immunoglobulin Fv