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

Abstract

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 antigenspecific 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.

Inhibiting HLA–B27 homodimer–driven immune cell inflammation in spondylarthritis

“…Diversity of the selected HLA-binding phages was determined by colony polymerase chain reaction (PCR) (Red-Taq Readymix; Sigma) and fingerprinting (Bst NI digestion) and confirmed by DNA sequencing with heavy and light chain-specific primers. Fab were expressed, purified, and converted into chimeric IgG molecules exhibiting mouse Fc, using a glutamine synthetase gene expression system (Lonza) as previously described (25). Purity and molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

“…22,23 Phagebound V H -V L constructs, selected by successive rounds of enrichment, successfully identified the tumor-associated antigen C1q-R; it is immaterial whether a particular V H -V L combination exists in vivo or is the result of an in vitro construct. Previous studies have reported that nonspecific phage binding interfered with specific selection in both nitrocellulose-bound antigens 17 and immobilized lambda phage cDNA expression libraries hybridized to a phagebound anti-CD30 scfv. 43 Serial dilutions of phage-to-target were needed to calibrate binding specificity.…”

“…15,16 Combinatorial Ig libraries have most often been used to identify antibodies to either known, purified proteins or proteins specifically engineered to be overexpressed by cells. [15][16][17][18] Identifying previously unrecognized antigens on the surface of whole tumor cells has remained somewhat more difficult for several reasons. Ongoing cell culture and in vitro manipulation may adversely affect preservation of folded 3-D epitopic sites on surface antigens.…”

Application of colony lift assay in the medullary thyroid carcinoma screening of single-chain variable fragment antibody phage library

“…This method is applicable to cell-surface specific antibody screening, [11] but some reports suggest that the screening results method may be unpredictable. [12] (2) Liquid antigen screening, [13] soluble resistance factors combined with phage antibody, original and combined with the support of antibodies in the packaging phage. This method can better retain the natural conformation of the antigen.…”