title: Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library.
abstract: BACKGROUND
  Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application.
  
  
  RESULTS
  Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3(550-558) and a truncated GPC3(368-548) fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC(50) ranging from 5.0-110.9 nM were identified.
  
  
  CONCLUSIONS
  Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.
authors.affiliation: Medicine and Research Services, Philadelphia VA Medical Center, PA 19104, USA.
authors.forename: Yonghai
authors.initials: Y
authors.lastname: Li
authors.forename: Donald L
authors.initials: DL
authors.lastname: Siegel
authors.forename: Nathalie
authors.initials: N
authors.lastname: Scholler
authors.forename: David E
authors.initials: DE
authors.lastname: Kaplan
mesh_terms: D000937:Antigen-Antibody Reactions; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D053673:Glypicans / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D053673:Glypicans; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D005982:Glutathione Transferase