Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library

To generate antibodies to defined cell-surface antigens, we used a large ph age antibody fragment library to select on cell transfectants expressing on e of three chosen receptors. First, in vitro panning procedures and phage a ntibody screening ELISAs were developed using whole live cells stably expre ssing the antigen of interest. When these methodologies were applied to Chi nese hamster ovary (CHO) cells expressing one of the receptors for a neurop eptide, somatostatin, using either direct cell panning or a strategy of dep letion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panni ng on CHO-cells expressing the human membrane protein CD36, an extraordinar y high frequency of antigen specific phage antibodies was found. Fanning on myoblasts expressing the rat homologue of CD36 revealed a similar selectio n dominance for anti-(CD36). Binding of all selected 20 different anti-(CD3 6) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Simila r inhibition was found for seven anti-(rat) CD36 that cross-reacted with hu man CD36. Our results show that, although cells can be used as antigen carr iers to select and screen phage antibodies, the nature of the antigen targe t has a profound effect on the outcome of the selection.