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 phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.
Hoogenboom
H R
HR
CESAME at the Department of Pathology, Maastricht University, The Netherlands. hho@lpat.azm.nl
Lutgerink
J T
JT
Pelsers
M M
MM
Rousch
M J
MJ
Coote
J
J
Van Neer
N
N
De Bruïne
A
A
Van Nieuwenhoven
F A
FA
Glatz
J F
JF
Arends
J W
JW
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000276:immunology; D018955:CD36 Antigens / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology*; D019151:Peptide Library; D051381:Rats; D017481:Receptors, Somatostatin / Q000276:immunology*
D000914:Antibodies, Viral; D018955:CD36 Antigens; D016056:Immunodominant Epitopes; D019151:Peptide Library; D017481:Receptors, Somatostatin