A COMPARISON OF 2 MURINE MONOCLONAL-ANTIBODIES HUMANIZED BY CDR-GRAFTING AND VARIABLE DOMAIN RESURFACING

The variable domain resurfacing and CDR-grafting approaches to antibod y humanization mere compared directly on the two murine monoclonal ant ibodies 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 consi sts of transferring the CDRs from a rodent antibody onto the Fv framew ork of a human antibody, Computer-aided molecular modelling was used t o design the initial CDR-grafted and resurfaced versions of these two antibodies, The initial versions of resurfaced N901 and resurfaced ant i-B4 maintained the full binding affinity of the original murine paren t antibodies and further refinements to these versions described herei n generated five new resurfaced antibodies that contain fewer murine r esidues at surface positions, four of which also have the full parenta l binding affinity, A mutational study of three surface positions with in 5 Angstrom of the CDRs of resurfaced anti-B4 revealed a remarkable ability of the resurfaced antibodies to maintain binding affinity desp ite dramatic changes of charges near their antigen recognition surface s, suggesting that the resurfacing approach can be used with a high de gree of confidence to design humanized antibodies that maintain the fu ll parental binding affinity, By comparison CDR-grafted anti-B4 antibo dies with parental affinity were produced only after seventeen version s were attempted using two different strategies for selecting the huma n acceptor frameworks, For both the CDR-grafted anti-B4 and N901 antib odies, full restoration of antigen binding affinity was achieved when the most identical human acceptor frameworks were selected, The CDR-gr afted anti-B4 antibodies that maintained high affinity binding for CD1 9 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 antibo dies with reduced antigenic potential relative to their corresponding CDR-grafted versions.