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Affinity maturation of a VHH by mutational hotspot randomization
- Source :
- Journal of Immunological Methods. 297:213-224
- Publication Year :
- 2005
- Publisher :
- Elsevier BV, 2005.
-
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.
- Subjects :
- Phage display
Amino Acid Motifs
Molecular Sequence Data
Immunology
Mutant
Antibody Affinity
Immunoglobulin Variable Region
Biology
Ribosome
Affinity maturation
Peptide Library
Animals
Immunology and Allergy
Amino Acid Sequence
Codon
Peptide library
Peptide sequence
Mammals
Genetics
Nucleic acid sequence
Single-Domain Antibodies
Complementarity Determining Regions
Molecular biology
Mutagenesis
Mutation
Ribosome display
Immunoglobulin Heavy Chains
Peptides
Ribosomes
Subjects
Details
- ISSN :
- 00221759
- Volume :
- 297
- Database :
- OpenAIRE
- Journal :
- Journal of Immunological Methods
- Accession number :
- edsair.doi.dedup.....3f8c329d46e50a3fafcab05e88fc1837