title: Affinity maturation of a V(H)H by mutational hotspot randomization.
abstract: V(H)Hs from naive libraries have dissociation constants (K(D)s) in the low micromolar range and thus, for most antibody applications, their intrinsic affinities need to be improved significantly. Non-targeted in vitro affinity maturation approaches based on indiscriminate randomization of complementarity-determining region (CDR) residues or random mutagenesis of conventional antibody variable domains have been shown to improve the affinity of recombinant antibodies by 450- to over 6000-fold. A different, targeted approach based on selective randomization of CDR codons containing AGY/RGYW nucleotide mutational hotspots i.e., "hotspot codons", also promises to be very efficient for improving antibody affinities. Here we employed the latter approach for improving the affinity of PTH22, a parathyroid hormone (PTH)-derived peptide-specific V(H)H that was isolated from a naive llama phage display library. A PTH22 mutant ribosome display library was constructed by randomizing nine CDR2 and CDR3 hotspot codons. The affinity improvement of the lead binder was 30-fold, which seems somewhat low in view of the large number of randomized hotspot codons. Nucleotide sequence analyses of PTH22 and 23 naive V(H)Hs suggested that many AGY/RGYW mutational hotspots are not affinity mutational hotspots but play a role in V(H)H solubility, structure, and deletion/insertion events. Our results indicate that the mutagenesis approach described here is beneficial in terms of yielding moderate increases in affinity while fine-tuning physical properties of an antibody.
authors.affiliation: Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
authors.forename: Kerrm Y F
authors.initials: KY
authors.lastname: Yau
authors.forename: Ginette
authors.initials: G
authors.lastname: Dubuc
authors.forename: Shenghua
authors.initials: S
authors.lastname: Li
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hirama
authors.forename: C Roger
authors.initials: CR
authors.lastname: Mackenzie
authors.forename: Lutz
authors.initials: L
authors.lastname: Jermutus
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D003062:Codon / Q000235:genetics; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008322:Mammals; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology; D012270:Ribosomes / Q000502:physiology; D061905:Single-Domain Antibodies
chemical_list: D003062:Codon; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C501047:PTH22 protein, Lama glama; D019151:Peptide Library; D010455:Peptides; D061905:Single-Domain Antibodies