Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody.
We have examined the plasticity of the antigen-combining site of a high-affinity antibody. In phage-displayed Fab libraries, selected CDR positions and one FR position of the humanized anti-Her2 antibody hu4D5 were substituted with all 20 amino acids. Antigen-binding selections were used to enrich for high-affinity variants, and a large number of sequences were obtained prior to convergence of the selected pool to a small set of clones. As expected, sequence variability of the antigen-binding site is overall diminished compared to known IgG sequences; however, certain positions retain much higher variability than others. The sequence variability map of the hu4D5 binding site is compared with a map derived from previous alanine-scanning of the antibody. Affinities of soluble Fab fragments for antigen confirm that multiple variants were selected with high affinity for antigen, including one variant with a single point mutation that was about threefold improved in affinity compared to the parental hu4D5. Interestingly, this mutation is one of the most radical in terms of changing side-chain chemistry (Trp for Asp) and occurs at the most plastic site as calculated by the Wu-Kabat variability coefficient. Thus variability mapping yields information about the antibody-antigen interaction that is useful and complementary to that obtained by alanine scanning mutagenesis.
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Resi B
RB
Gerstner
Paul
P
Carter
Henry B
HB
Lowman
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000276:immunology* / Q000627:therapeutic use; D001666:Binding Sites, Antibody; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D008958:Models, Molecular; D009154:Mutation; D009369:Neoplasms / Q000628:therapy; D019151:Peptide Library; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D000068878:Trastuzumab
D000409:Alanine; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab