title: Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.
abstract: The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.
authors.affiliation: From the Departments of Antibody Engineering.
authors.forename: Patrick
authors.initials: P
authors.lastname: Koenig
authors.affiliation: From the Departments of Antibody Engineering.
authors.forename: Chingwei V
authors.initials: CV
authors.lastname: Lee
authors.affiliation: From the Departments of Antibody Engineering.
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanowar
authors.affiliation: Structural Biology, Genentech Research and Early Development, South San Francisco, California 94080.
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.affiliation: Molecular Biology, and.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Stinson
authors.affiliation: Structural Biology, Genentech Research and Early Development, South San Francisco, California 94080.
authors.forename: Seth F
authors.initials: SF
authors.lastname: Harris
authors.affiliation: From the Departments of Antibody Engineering, gml@gene.com.
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D042702:Angiopoietin-2 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use; D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D015195:Drug Design; D005240:Feasibility Studies; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D008268:Macular Degeneration / Q000188:drug therapy / Q000276:immunology* / Q000378:metabolism; D008958:Models, Molecular; D058990:Molecular Targeted Therapy / Q000379:methods; D009154:Mutation; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism
chemical_list: D043925:Angiogenesis Inducing Agents; D042702:Angiopoietin-2; D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D042461:Vascular Endothelial Growth Factor A
keywords: angiopoietin2 (Ang2); antibody engineering; cooperativity; crystal structure; deep mutational scanning; deep sequencing; drug discovery; dual specificity; neovascular age-related macular degeneration; vascular endothelial growth factor (VEGF)