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Volume 271, Number 37, Issue of September 13, 1996 pp. 22611-22618
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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A Combinatorial Library Strategy for the Rapid Humanization of Anticarcinoma BR96 Fab

(Received for publication, April 19, 1996, and in revised form, June 25, 1996)

Mae Joanne Rosok , Dale E. Yelton , Linda J. Harris , Jürgen Bajorath , Karl-Erik Hellström , Ingegerd Hellström , Gina A. Cruz ^¶ , Karin Kristensson ^¶ , Huey Lin ^¶ , William D. Huse ^¶ and Scott M. Glaser ^¶

From  Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121 and ^¶ Ixsys, Inc., San Diego, California 92121

We have used a combinatorial mutagenesis strategy to humanize BR96, a monoclonal antibody that binds to the Lewis Y class of tumor antigens. This approach allows simultaneous assessment of hundreds of humanized variable regions to identify the molecules that best preserve affinity, thus overcoming the major drawback of current humanization procedures, the requirement to construct and analyze each humanized antibody separately. Murine residues of BR96 were mutated to human if they were solvent-exposed residues that did not participate in the formation of the antigen binding site and were not at the interface of the light and heavy chain. At positions that might be involved in binding to antigen, the choice between the murine and human residue was more difficult. Murine and human alternatives were incorporated into a combinatorial library at positions representing buried residues that might affect the structural integrity of the antigen binding site. By encoding this library of humanized BR96 Fabs in an M13 phage vector, we rapidly identified several candidates with nearly identical antigen binding, within 2-fold, of the chimeric Fab. Additional mutagenesis directed at sites suggested in the literature as potentially important for antigen binding in a similar anti-Lewis Y antibody yielded no further improvements.

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