Antibody humanization often requires the replacement of key residues in the framework regions with corresponding residues from the parent non-human antibody. These changes are in addition to grafting of the antigen-binding loops. Although guided by molecular modeling, assessment of which framework changes are beneficial to antigen binding usually requires the analysis of many different antibody mutants. Here we describe a phage display method for optimizing the framework of humanized antibodies by random mutagenesis of important framework residues. We have applied this method to humanization of the anti-vascular endothelial growth factor murine monoclonal antibody A4.6.1. Affinity panning of a library of humanized A4.6.1 antibody mutants led to the selection of one variant with greater than 125-fold enhanced affinity for antigen relative to the initial humanized antibody with no framework changes. A single additional mutation gave a further 6-fold improvement in binding. The affinity of this variant, 9.3 nM, was only 6-fold weaker than that of a murine/human chimera of A4.6.1. This method provides a general means of rapidly selecting framework mutations that improve the binding of humanized antibodies to their cognate antigens and may prove an attractive alternative to current methods of framework optimization based on cycles of site-directed mutagenesis. Monoclonal antibodies (mAbs)1 have enormous potential as therapeutic agents; however, most mAbs are derived from murine or other non-human sources, which severely limits their clinical efficacy. In addition to the immunogenicity of rodent mAbs when administered to humans (1-3), further limitations arise from weak recruitment of effector function (4, 5) and rapid clearance from serum (6, 7). As a means of circumventing these deficiences, the antigen-binding properties of murine mAbs can be conferred to human antibodies through a process known as antibody "humanization" (4,8). A humanized antibody contains the amino acid sequences from the six complementarity-determining regions (CDRs) of the parent murine mAb, which are grafted onto a human antibody framework. For this reason, humanization of non-human antibodies is commonly referred to as CDR grafting. The low content of nonhuman sequence in humanized antibodies (ϳ5%) has proven effective in both reducing the immunogenicity and prolonging the serum half-life in humans (7,9).Unfortunately, simple grafting of CDR sequences often yields humanized antibodies that bind antigen much more weakly than the parent murine mAb (4, 10 -16), and decreases in affinity of up to several hundredfold have been reported (13)(14)(15). To restore high affinity, the antibody must be further engineered to fine tune the structure of the antigen-binding loops. This is achieved by replacing key residues in the framework regions of the antibody variable domains with the matching sequence from the parent murine antibody. These framework residues are usually involved in supporting the conformation of the CDR loops (17), although some framew...