title: Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display.
abstract: In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.
authors.affiliation: Global Drug Discovery, Global Biologics, Bayer HealthCare AG, Cologne, Germany. christian.votsmeier@bayer.com
authors.forename: Christian
authors.initials: C
authors.lastname: Votsmeier
authors.forename: Hanna
authors.initials: H
authors.lastname: Plittersdorf
authors.forename: Oliver
authors.initials: O
authors.lastname: Hesse
authors.forename: Andreas
authors.initials: A
authors.lastname: Scheidig
authors.forename: Michael
authors.initials: M
authors.lastname: Strerath
authors.forename: Uwe
authors.initials: U
authors.lastname: Gritzan
authors.forename: Klaus
authors.initials: K
authors.lastname: Pellengahr
authors.forename: Peter
authors.initials: P
authors.lastname: Scholz
authors.forename: Andrea
authors.initials: A
authors.lastname: Eicker
authors.forename: David
authors.initials: D
authors.lastname: Myszka
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Coco
authors.forename: Ulrich
authors.initials: U
authors.lastname: Haupts
mesh_terms: D000915:Antibody Affinity; D061505:Cell Surface Display Techniques; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology*; D057166:High-Throughput Screening Assays; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding
chemical_list: D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments