HUMAN-ENGINEERED MONOCLONAL-ANTIBODIES RETAIN FULL SPECIFIC BINDING-ACTIVITY BY PRESERVING NON-CDR COMPLEMENTARITY-MODULATING RESIDUES

Humanization of murine monoclonal antibodies for human therapy has com monly been achieved by complementarity-determining region (CDR) grafti ng, in which murine CDR loops are grafted onto human framework regions . Difficulties with that method have revealed the importance of certai n framework residues in determining both the 3-D structure of CDR loop s and the overall affinity of the molecule for its specific ligand. In the general model of structure-function relationships presented here, each amino acid position in the variable region is classified accordi ng to the benefit of achieving a more human-like antibody versus the r isk of decreasing or abolishing specific binding affinity. Substitutio ns of human residues at low-risk positions (exposed to solvent but not contributing to antigen binding or antibody structure) are likely to decrease inmunogenicity with little or no effect on binding affinity. Changes at high-risk positions (directly involved in antigen binding, CDR stabilization or internal packing) are avoided to preserve the bio logical activity of the antibody. Moderate-risk changes are made with caution. This model has been tested experimentally using H65, an anti- CDS murine monoclonal antibody, whose binding activity had been greatl y reduced by two previous attempts at humanizaton by conventional CDR grafting. The new 'human-engineered' H65 antibody containing 20 low-ri sk human consensus substitutions (expressed as either IgG or Fab) reta ins the full binding avidity of parental murine and chimeric H65 antib odies. A human-engineered antibody with an additional 14 moderate-risk substitutions has unexpectedly enhanced avidity (3- to 7-fold). This method is generally applicable to the design of other human-engineered antibodies with therapeutic potential.