Affinity maturation of a VHH by mutational hotspot randomization

摘要: 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 vitro affinity maturation approaches based on indiscriminate randomization of complementarity-determining region (CDR) residues or random mutagenesis conventional variable domains been shown improve recombinant antibodies by 450- over 6000-fold. A different, targeted approach selective CDR codons containing AGY/RGYW nucleotide mutational hotspots i.e., "hotspot codons", also promises very efficient improving affinities. Here we employed latter PTH22, a parathyroid hormone (PTH)-derived peptide-specific V(H)H that was isolated llama phage display library. PTH22 mutant ribosome library constructed randomizing nine CDR2 CDR3 hotspot codons. The improvement lead binder 30-fold, which seems somewhat view large number randomized Nucleotide sequence analyses 23 suggested many are not but play role solubility, structure, deletion/insertion events. Our results indicate described here is beneficial terms yielding moderate increases while fine-tuning physical properties an antibody.