Designing human consensus antibodies with minimal positional templates.

A humanized antibody retains from the original murine antibody the variable region amino acid residues that are required for antigen binding. These generally include the grafted complementarity determining regions, as well as a few key framework residues. Although the remainder of the framework sequences are imported from a human antibody, they nevertheless differ at a few positions from the human consensus sequences. These atypical residues, which arose by somatic mutation during the affinity maturation of the chosen human antibody, could elicit an immune response in some of the patients receiving the humanized antibody. Thus, ideally one should, instead, choose human consensus frameworks for humanizing murine antibodies. Because there is a different consensus sequence for each of the subclasses of variable light and heavy chains, a method is needed to choose the most appropriate one. We are developing a minimal positional template for such a purpose. A minimal positional template indicates which positions in the variable region frameworks are absolutely required for maintaining the integrity of the binding domains. Therefore, to choose a human framework for humanization, one screens the available human consensus sequences for those that are most similar to the original murine sequence, specifically at the positions indicated by the template. In the subsequent humanization protocol, one then retains all of the murine residues found in the positions indicated in the template while humanizing the residues found at all other positions. A conservative positional template has been applied to the humanizations of the antibreast epithelial mucin antibodies BrE-3 and KC4-G3 without loss of binding affinity. Now we are using progressive cycles of computer modeling and laboratory testing to develop a minimal template. The first of such cycles produced template B, which has been used successfully in the humanization of the antibreast epithelial antigen BA46 antibody Mc3. This prompted us to design template C, which further approaches the desired minimal template. Future constructs will test the validity of this template as well as the validity of this novel humanization approach.