CONVERSION OF MURINE FABS ISOLATED FROM A COMBINATORIAL PHAGE DISPLAYLIBRARY TO FULL-LENGTH IMMUNOGLOBULINS

The use of combinatorial Ig libraries displayed on the surface of bact eriophage 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 a ntibodies were removed from the bacterial expression vector used for t he combinatorial library construction, and subcloned into individual m ammalian 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 u sed 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 th e culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versi ons of the mammalian expression vectors have been constructed for sing le step conversion of murine recombinant Fabs, recovered from combinat orial libraries, to IgG2a mAbs.