A polar ring endows improved specificity to an antibody fragment

Engineering monovalent Fab fragments into bivalent formats like IgGs or F(ab’)2 can lead to aggregation presumably because of nonspecific off‐target interactions that induce aggregation. In an effort to further understand the molecular determinants of nonspecific interactions for engineered antibodies and natively folded proteins in general, we focused on a synthetic Fab with low nanomolar affinity to histone chaperone Anti‐silencing factor 1 (Asf1) that demonstrates off‐target binding through low solubility (∼5 mg/mL) in the multivalent F(ab') 2 state. Here, we generated phage display‐based shotgun scanning libraries to introduce aspartate as a negative design element into the antibody paratope. The antibody‐combining site was amenable to aspartate substitution at numerous positions within the antigen binding loops and one variant, TyrL93Asp/HisL94Asp/ThrH100bAsp, possessed high solubility (>100 mg/ml). Furthermore, the mutations decreased nonspecific interactions measured by column interaction chromatography and ELISA in the multivalent antibody format while maintaining high affinity to the antigen. Structural determination of the antibody‐antigen complex revealed that the aspartate‐permissive residues formed a polar ring around the structural and functional paratope, recapitulating the canonical feature of naturally occurring protein‐protein interactions. This observation may inform future strategies for the design and engineering of molecular recognition.

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