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ABSTRACT: The variable domain resurfacing and CDR-grafting approaches to antibody humanization were compared directly on the two murine monoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19). Resurfacing replaces the set of surface residues of a rodent variable region with a human set of surface residues. The method of CDR-grafting conceptually consists of transferring the CDRs from a rodent antibody onto the Fv framework of a human antibody. Computer-aided molecular modeling was used to design the initial CDR-grafted and resurfaced versions of these two antibodies. The initial versions of resurfaced N901 and resurfaced anti-B4 maintained the full binding affinity of the original murine parent antibodies and further refinements to these versions described herein generated five new resurfaced antibodies that contain fewer murine residues at surface positions, four of which also have the full parental binding affinity. A mutational study of three surface positions within 5 Å of the CDRs of resurfaced anti-B4 revealed a remarkable ability of the resurfaced antibodies to maintain binding affinity despite dramatic changes of charges near their antigen recognition surfaces, suggesting that the resurfacing approach can be used with a high degree of confidence to design humanized antibodies that maintain the full parental binding affinity. By comparison CDR-grafted anti-B4 antibodies with parental affinity were produced only after seventeen versions were attempted using two different strategies for selecting the human acceptor frameworks. For both the CDR-grafted anti-B4 and N901 antibodies, full restoration of antigen binding affinity was achieved when the most identical human acceptor frameworks were selected. The CDR-grafted anti-B4 antibodies that maintained high affinity binding for CD19 had more murine residues at surface positions than any of the three versions of the resurfaced anti-B4 antibody. This observation suggests that the resurfacing approach can be used to produce humanized antibodies with reduced antigenic potential relative to their corresponding CDR-grafted versions.