A polar ring endows improved specificity to an antibody fragment

作者：Schaefer Zachary P; Bailey Lucas J; Kossiakoff Anthony A^*

来源：Protein Science, 2016, 25(7): 1290-1298.

DOI：10.1002/pro.2888

• antibody engineering
• protein solubility
• phage display
• protein engineering

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 (similar to 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, Tyr(L93)Asp/His(L94)Asp/Thr(H100b) Asp, 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.

• 出版日期2016-7