Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries

J Mol Biol. 2018 Jan 5;430(1):119-130. doi: 10.1016/j.jmb.2017.11.008. Epub 2017 Nov 26.


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.

Keywords: complementarity-determining region; cross-interaction; monoclonal antibody; nonspecificity; synthetic scFv library.

MeSH terms

  • Amino Acid Motifs / genetics*
  • Amino Acid Sequence
  • Amino Acids / genetics
  • Animals
  • Antibody Affinity / genetics
  • Cell Line
  • Complementarity Determining Regions / genetics
  • HEK293 Cells
  • Humans
  • Peptide Library
  • Sf9 Cells
  • Single-Chain Antibodies / genetics*
  • Tryptophan / genetics
  • Yeasts / genetics


  • Amino Acids
  • Complementarity Determining Regions
  • Peptide Library
  • Single-Chain Antibodies
  • Tryptophan