STEPWISE IN-VITRO AFFINITY MATURATION OF VITAXIN, AN ALPHA(V)BETA(3)-SPECIFIC HUMANIZED MAB

A protein engineering strategy based on efficient and focused mutagene sis implemented by codon-based mutagenesis was developed. Vitaxin, a h umanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alpha(v) beta(3) integrin complex, wa s used as a model system, Specifically, focused mutagenesis was used i n a stepwise fashion to rapidly improve the affinity of the antigen bi nding fragment by greater than 90-fold. In the complete absence of str uctural information about the Vitaxin-alpha(v) beta(3) interaction, ph age expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a q uantitative assay to identify variants with improved binding to alpha( v) beta(3). The Vitaxin variants in these libraries each contained a s ingle mutation, and all 20 amino acids were introduced at each complem entarity-determining region residue, resulting in the expression of 2, 336 unique clones. Multiple clones displaying 2- to 13-fold improved a ffinity were identified. Subsequent expression and screening of a libr ary of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These varian ts inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variant s with enhanced affinity were identified rapidly and required the synt hesis of only 2,592 unique variants. The use of such small focused lib raries obviates the need for phage affinity selection approaches typic ally used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.