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 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.
Powers
D B
DB
Departments of Anesthesia and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94110, USA.
Amersdorfer
P
P
Poul
M
M
Nielsen
U B
UB
Shalaby
M R
MR
Adams
G P
GP
Weiner
L M
LM
Marks
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008297:Male; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D019151:Peptide Library; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv