Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

摘要: A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against conformational epitope αvβ3 integrin complex, used as model system. Specifically, in stepwise fashion to rapidly improve affinity antigen binding fragment greater than 90-fold. In complete absence structural information about Vitaxin-αvβ3 interaction, phage-expressed libraries for all six Ig heavy light chain complementarity-determining regions were expressed screened quantitative assay identify variants with improved αvβ3. The Vitaxin these each contained single mutation, 20 amino acids introduced at region residue, resulting expression 2,336 unique clones. Multiple clones displaying 2- 13-fold identified. Subsequent screening library 256 combinatorial optimal mutations identified from primary resulted identification multiple 50-fold enhanced affinity. These inhibited ligand receptor more potently demonstrated inhibition cell adhesion competition assays. Because limited approach, required synthesis only 2,592 variants. use such small obviates need phage selection approaches typically used, permitting functional assays proteins mammalian culture.