Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation.
A previously identified humanized anti-CD3 Ab variant, v9, binds T cells with > 100-fold higher efficiency than the original variant, v1, and almost as efficiently as a chimeric molecule containing corresponding murine variable domains. Variants v1 and v9 differ at six positions in the H chain second CDR. Here a mutational analysis was used to identify which of these six replacements are primarily responsible for the difference in binding efficiency. These anti-CD3 variants were used to probe the relationship between Ag binding efficiency and potency in stimulating T cell proliferation. The human to mouse mutations T57S and V63F increase the binding efficiency of variant v1 for T cells by 8- and 12-fold, respectively, and together in variant M18 enhance binding by 26-fold to within 4-fold of variant v9. A framework mutation, 169L, was identified that enhances the binding of variants v1 and M18 by 14- and 3-fold, respectively. The Ag binding efficiencies of anti-CD3 variants correlate directly with their potencies in stimulating the proliferative activity of both resting human PBMC and IL-2-activated human T lymphocytes. Humanized variant v9 is equipotent to the murine parent Ab in stimulating ATL activity. PBMC activated by variants v1 and v9 IgG in a short term culture are equally cytotoxic against human breast carcinoma cells. Thus, high efficiency Ag binding by anti-CD3 variants is important for stimulating efficient T cell proliferation, but not cytotoxicity, in vitro.
Zhu
Z
Z
Department of Cell Genetics, Genentech, Inc., South San Francisco, CA 94080, USA.
Carter
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P
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001667:Binding, Competitive; D017252:CD3 Complex / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D013601:T-Lymphocytes / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D017252:CD3 Complex; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains