A polar ring endows improved specificity to an antibody fragment.

TitleA polar ring endows improved specificity to an antibody fragment.
Publication TypeJournal Article
Year of Publication2016
AuthorsSchaefer, Zachary P., Bailey Lucas J., and Kossiakoff Anthony A.
JournalProtein Sci
Volume25
Issue7
Pagination1290-8
Date Published2016 07
ISSN1469-896X
KeywordsAmino Acid Substitution, Antibody Affinity, Aspartic Acid, Binding Sites, Antibody, Cell Cycle Proteins, Cell Surface Display Techniques, Crystallography, X-Ray, Humans, Immunoglobulin Fab Fragments, Models, Molecular, Protein Conformation, Protein Engineering
Abstract

<p>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, 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.</p>

DOI10.1002/pro.2888
Alternate JournalProtein Sci.
PubMed ID27334407
PubMed Central IDPMC4918417
Grant ListR01 GM117372 / GM / NIGMS NIH HHS / United States
U54 GM087519 / GM / NIGMS NIH HHS / United States
U01 GM094588 / GM / NIGMS NIH HHS / United States
U54 HG006436 / HG / NHGRI NIH HHS / United States

University of Toronto  UCSF  The University of Chicago  QB3  Chicago Biomedical Consortium