Affinity maturation of a V(H)H by mutational hotspot randomization.
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.
Yau
Kerrm Y F
KY
Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Dubuc
Ginette
G
Li
Shenghua
S
Hirama
Tomoko
T
Mackenzie
C Roger
CR
Jermutus
Lutz
L
Hall
J Christopher
JC
Tanha
Jamshid
J
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
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