Removal of amphipathic epitopes from genetically engineered antibodies: Production of modified immunoglobulins with reduced immunogenicity

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodi es often have decreased binding affinity. We described a new approach for r educing the immunogenicity of chimeric antibodies while maintaining the aff inity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipath ic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments c ontaining possible amphipathic epitopes were found in the heavy variable do main using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequ ence. No mutations were made in the murine light variable domain. Experimen ts in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in t he Kabat database revealed the presence of conserved patterns in the differ ent families of variable region sequences, suggesting that the proposed met hod may be of general applicability.