Limited Diversity of Human scFv Fragments Isolated by Panning a Synthetic Phage-Display scFv Library with Cultured Human Melanoma Cells

Abstract

To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.

Evolution of phage display libraries for therapeutic antibody discovery

“…Antibody phage display was originally developed in three institutes: Institute of Cell and Tumor Biology at German Cancer Research Center (Germany), 39 MRC Laboratory of Molecular Biology (United Kingdom), 40 , 41 and Scripps Research Institute (United States). 42 , 43 Since then, various antibody fragment formats have been commonly used for phage display, such as single chain variable fragment (scFv), 44 , 45 antigen binding fragment (Fab), 24 , 46 single-domain antibody (VHH), 47 , 48 and bispecific antibody fragment. 49–51 Although display of full-length IgG on phage was also reported, 52 , 53 this platform was not widely used, likely due to unstable display, low display level, and bias on phage propagation, which resulted in limited functional diversity of the library.…”

Supplementary Methods, Figure Legends 1-3, Table Legend 1 from Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

“…o C with CSPG4 + cells (2 x 10 5 /50µL of RPMI 1640 medium) in a 96-well tissue culture plate (Falcon 3072, Becton Dickinson). Binding of biotinylated scFv antibodies and biotinylated mAb to target cells was measured by sequential incubation with SA-HRP and substrate as described (2).…”