Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.
The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.
Ames
R S
RS
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
Tornetta
M A
MA
Deen
K
K
Jones
C S
CS
Swift
A M
AM
Ganguly
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D015936:Complement C5a / Q000276:immunology*; D015870:Gene Expression; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000276:immunology; D007158:Immunologic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D015936:Complement C5a