Comparative expression of different antibody formats in mammalian cells and Pichia pastoris.
Generation of recombinant antibody fragments has been advanced by phage display technology but their broad use in biochemical or analytical applications is often hindered by their univalence. For enhancement of functional affinity and overall applicability, the fusion of scFvs (single-chain variable fragments) to IgG constant domains has become an attractive approach. In order to evaluate characteristics and expression behaviour of different IgG-analogous antibody formats, we fused an scFv to different portions of the heavy chain constant region of human IgG1. Two types of antibodies, an scFv-C(H)2-3 antibody and an scFv-C(H)1-3 antibody, a new intermediate with retained C(H)1 domain, were generated with or without an affinity tag for purification. Additionally, the scFv was reconverted into the heterotetrameric IgG molecule. To allow a reliable comparison of expression behaviours of different antibodies, we established vector systems that allow isogenetic and efficient expression of the recombinant antibodies based on site-specific recombination. Upon recombinant expression in mammalian cells and the methylotrophic yeast Pichia pastoris, disulfide-linked and glycosylated oligomers were obtained. Establishment of isogenetic cell lines revealed that the presence of the C(H)1 domain is not critical for secretion efficiency. Reactivity of the different constructs with antigen and Fc receptors was verified by ELISA, surface plasmon resonance approaches, as well as FACS analysis of HEK-293 cells (human embryonic kidney cells) stably transfected with human FcgammaRI (high-affinity IgG receptor) (CD64). In summary, the results obtained provide evidence for comparable behaviour of the different antibody formats and the vectors for isogenetic expression will contribute to a broader application of phage display-derived antibodies.
Braren
Ingke
I
Division of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany.
Greunke
Kerstin
K
Umland
Oliver
O
Deckers
Susanne
S
Bredehorst
Reinhard
R
Spillner
Edzard
E
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000502:physiology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013045:Species Specificity
D000911:Antibodies, Monoclonal