title: A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.
abstract: There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.
authors.affiliation: a Institute for Organic Chemistry and Biochemistry ; Technische Universität Darmstadt ; Darmstadt , Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schröter
authors.forename: Ralf
authors.initials: R
authors.lastname: Günther
authors.forename: Laura
authors.initials: L
authors.lastname: Rhiel
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.forename: Lars
authors.initials: L
authors.lastname: Toleikis
authors.forename: Achim
authors.initials: A
authors.lastname: Doerner
authors.forename: Janine
authors.initials: J
authors.lastname: Becker
authors.forename: Andreas
authors.initials: A
authors.lastname: Schönemann
authors.forename: Daichi
authors.initials: D
authors.lastname: Nasu
authors.forename: Berend
authors.initials: B
authors.lastname: Neuteboom
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
authors.forename: Björn
authors.initials: B
authors.lastname: Hock
mesh_terms: D000068879:Adalimumab; D019943:Amino Acid Substitution; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D006639:Histidine; D000068879:Adalimumab
keywords: Fab display; Yeast surface display; adalimumab; antibody engineering; pH-dependent antigen binding; pH-switch; protein engineering