title: A polar ring endows improved specificity to an antibody fragment.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois.
authors.forename: Zachary P
authors.initials: ZP
authors.lastname: Schaefer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois.
authors.forename: Lucas J
authors.initials: LJ
authors.lastname: Bailey
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D019943:Amino Acid Substitution; D000915:Antibody Affinity; D001224:Aspartic Acid; D001666:Binding Sites, Antibody; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D018360:Crystallography, X-Ray; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: C491641:ASF1A protein, human; D018797:Cell Cycle Proteins; D007140:Immunoglobulin Fab Fragments; D018832:Molecular Chaperones; D001224:Aspartic Acid
keywords: antibody engineering; phage display; protein engineering; protein solubility