Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries. Issue 1 (5th January 2018)

• Record Type: Journal Article
• Authors: Kelly, Ryan L.; Le, Doris; Zhao, Jessie; Wittrup, K. Dane
• Is Part Of: Journal of molecular biology. Volume 430:Issue 1(2018)
• Page Start: 119
• Page End: 130
• Publication Date: 2018-01-05
• Subjects: nonspecificity -- monoclonal antibody -- synthetic scFv library -- complementarity-determining region -- cross-interaction; mAbs monoclonal antibodies -- CDRs complementarity-determining regions -- PSR polyspecificity reagent -- scFv single-chain variable fragment -- SCP or SMP soluble cytosolic or membrane preparations -- EMF enriched membrane fraction; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.11.008
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a "clean" antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties. Graphical abstract: Highlights: Screened two libraries for nonspecific antibodies using a high-throughput yeast display assay Identified motifs containing Trp, Val, Gly, and Arg as drivers of nonspecificity Created a new, Trp-free synthetic library based on natural frameworks that minimized these motifs. Isolated high-affinity binders to a wide panel of antigens.