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Logo of bmcbiotBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Biotechnology
BMC Biotechnol. 2012; 12: 23.
Published online May 7, 2012. doi:  10.1186/1472-6750-12-23
PMCID: PMC3425314
Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library
Yonghai Li,2 Donald L Siegel,3 Nathalie Scholler,4 and David E Kaplancorresponding author1,2
1Medicine and Research Services, Philadelphia VA Medical Center, Philadelphia, PA, 19104, USA
2Division of Gastroenterology, Department of Medicine, University of Pennsylvania, 600 CRB, 415 Curie Blvd, Philadelphia, PA, 19104, USA
3Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
4Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Women’s Health (CRRWH), Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, 19104, USA
corresponding authorCorresponding author.
Yonghai Li: yonghai/at/; Donald L Siegel: siegeld/at/; Nathalie Scholler: naths/at/; David E Kaplan: dakaplan/at/
Received November 21, 2011; Accepted May 7, 2012.
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.
Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3550-558 and a truncated GPC3368-548 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 EC50 ranging from 5.0-110.9nM were identified.
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.
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