Title: Fishing for High Affinity Antibody from Synthetic Antibody Phage Libraries Authors: Wei-Ching Liang*, Vivian Lee, Yvonne Chen, Frederic Fellouse, Sachdev Sidhu, and Germaine Fuh Institution: Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA. Abstract: Synthetic antibody phage libraries made with a single framework scaffold have been generated and characterized. This technology should be a useful complement to hybridoma technology in generating specific human antibodies against targets of interest, especially those targets which have proven difficult for conventional methods. We used humanized 4d5 as a scaffold and constructed phage-displayed libraries in Fab format. To generate library diversity, we chose to randomize surface exposed complementarity determining regions (CDR) residues that were also found to be highly diverse in the Kabat database of natural antibody sequences. Furthermore, we used site-directed mutagenesis with tailored degenerate codons to generate amino acid diversity that mimicked the natural immune repertoire at each CDR site. To our test target antigen, a two-step sorting strategy was developed such that, in step 1, potent binders could be isolated from naïve libraries by means of the solid-supported selection, and subsequently in step 2, those stronger affinity binders could be isolated from weaker ones by the progressive solution- binding method with adjusting target antigen concentration. Further, we developed a high throughput affinity scanning assay to isolate and verify the highest affinity Fabs against target. In this way, we identified Fab with subnanomolar affinity. By a quick epitope mapping assay using blocking receptor, we identify clones that are blocking antibodies. The high through process allow us to find functionally important antibodies with high affinity in a robust manner.
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