Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library

Abstract

BackgroundGlypican-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.ResultsUsing 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.ConclusionsUsing 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.

Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

“…In conclusion, we, along with other investigators [39] , [75] have shown that affinity reagents raised against peptide fragments of membrane proteins are robust binders of the native receptor expressed both heterologously and in animal tissues. We have also shown that the K d values of these binders can be enhanced substantially by dimerization, which enables them to be useful tools in varied pharmacological and biochemical applications.…”

“…Traditionally, using membrane proteins as targets for selections has been problematic, in part due to the difficulty in expressing and purifying native protein that retains its structure and function [36] , [37] , [38] . However, scFvs against membrane proteins can be generated to peptide fragments of these proteins, thereby overcoming the need for generating purified protein targets [39] .…”

<p>Preparation and Characterization of Anti-GPC3 Nanobody Against Hepatocellular Carcinoma</p>

“…Most studies on improved antibody applicability for immunotherapy have focused on the modification of conventional antibody molecules, such as single-chain antibodies, Fab antibodies, and Fv single domain antibodies. 33,34 However, these antibodies have many shortcomings such as cumbersome preparation processes, poor water solubility, and easy aggregation. Therefore, it is necessary to obtain more advantageous GPC3 antibodies to improve diagnostic sensitivity, targeting and metabolism.…”

The Dual Role of the Liver in Nanomedicine as an Actor in the Elimination of Nanostructures or a Therapeutic Target

“…Recently, a novel scFv able to bind CD24, a target overexpressed by hepatocellular carcinoma (HCC), has been developed, showing a higher accumulation in the hepatocellular carcinoma xenograft mouse model and proving its potential as a therapeutic and diagnostic agent [128]. Another promising target expressed by malignant hepatocytes called Glypican-3 (GPC3) has already been exploited to isolate different scFvs with future potential applications [129,130].…”