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 cel ls 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 i n the H chain second CDR. Here a mutational analysis was used to ident ify 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 s timulating 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 1 4- and 3-fold, respectively. The Ag binding efficiencies of anti-CD3 v ariants correlate directly with their potencies in stimulating the pro liferative activity of both resting human PBMC and IL-2-activated huma n T lymphocytes. Humanized variant v9 is equipotent to the murine pare nt Ab in stimulating ATL activity. PBMC activated by variants v1 and v 9 IgG in a short term culture are equally cytotoxic against human brea st carcinoma cells. Thus, high efficiency Ag binding by anti-CD3 varia nts is important for stimulating efficient T cell proliferation, but n ot cytotoxicity, in vitro.