title: Robust in vitro affinity maturation strategy based on interface-focused high-throughput mutational scanning.
abstract: Development of protein therapeutics or biosensors often requires in vitro affinity maturation. Here we report a robust affinity engineering strategy using a custom designed library. The strategy consists of two steps beginning with identification of beneficial single amino acid substitutions then combination. A high quality combinatorial library specifically customized to a given binding-interface can be rapidly designed by high-throughput mutational scanning of single substitution libraries. When applied to the optimization of a model antibody Fab fragment, the strategy created a diverse panel of high affinity variants. The most potent variant achieved 2110-fold affinity improvement to an equilibrium dissociation constant (Kd) of 3.45 pM with only 7 amino acid substitutions. The method should facilitate affinity engineering of a wide variety of protein-protein interactions due to its context-dependent library design strategy.
authors.affiliation: Advanced Medical Research Department, Mitsubishi Tanabe Pharma Corporation, 3-16-89 Kashima, Osaka 532-8505, Japan. Fujino.Yasuhiro@mb.mt-pharma.co.jp
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Fujino
authors.forename: Risako
authors.initials: R
authors.lastname: Fujita
authors.forename: Kouichi
authors.initials: K
authors.lastname: Wada
authors.forename: Kotomi
authors.initials: K
authors.lastname: Fujishige
authors.forename: Takashi
authors.initials: T
authors.lastname: Kanamori
authors.forename: Lindsey
authors.initials: L
authors.lastname: Hunt
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
mesh_terms: D019943:Amino Acid Substitution; D020650:Combinatorial Chemistry Techniques; D022801:Complementarity Determining Regions / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D011485:Protein Binding / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology*; D054852:Small Molecule Libraries
chemical_list: D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D018124:Receptors, Tumor Necrosis Factor; D054852:Small Molecule Libraries