Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

Abstract

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. Coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K d = 4.8×10 -9 M and K d =5.1×10 -9 M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Preventive Strategies in Epithelial Ovarian Cancer

“…In addition to these efforts, we have generated a cost-and time-effective method for generating site-specific in vivo biotinylated recombinant antibodies secreted by yeast (Biobodies 5,95 ). Biobodies have been generated against HE4 95 and mesothelin 96 .…”

“…Biobodies have been generated against HE4 95 and mesothelin 96 . We have also demonstrated that this technology can be used reliably in a highly-sensitive bead-based ELISA assay for screening large populations of women for ovarian cancer 5,67 and for serum biomarker discovery 6 .…”

Use of Yeast-SecretedIn vivoBiotinylated Recombinant Antibodies (Biobodies) in Bead-Based ELISA

“…The Bb dissociation K s , as measured by surface plasmon resonance sensor, were of K d = 4.8 Â 10 -9 mol/L and K d = 5.1 Â 10 -9 mol/L before and after purification, respectively (8). We also developed and validated anti-mesothelin Bbs of high affinity that could detect both membrane-bound and soluble forms of mesothelin (9).…”

“…8). Bbs are recombinant antibodies secreted by diploid yeast as HIS-tagged, in vivo biotinylated proteins.…”

“…This makes supplementary steps of chemical biotinylation unnecessary, thereby reducing preparation time relative to hybridoma supernatant or ascites purification. In addition and importantly, in vivo biotinylation preserves the recognition function of recombinant antibodies through a targeted biotinylation (8).…”