Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.
Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.
Evans
M J
MJ
Department of Molecular Development, Alexion Pharmaceuticals, Inc., New Haven, CT 06511, USA.
Hartman
S L
SL
Wolff
D W
DW
Rollins
S A
SA
Squinto
S P
SP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D003182:Complement C5 / Q000276:immunology*; D017931:DNA Primers; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013539:Simian virus 40 / Q000235:genetics
D000911:Antibodies, Monoclonal; D003182:Complement C5; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins