ANTIBODY-INDUCED EPIDERMAL GROWTH-FACTOR RECEPTOR DIMERIZATION MEDIATES INHIBITION OF AUTOCRINE PROLIFERATION OF A431 SQUAMOUS CARCINOMA-CELLS

We previously reported that anti-epidermal growth factor (EGF) recepto r monoclonal antibody (mAb) 225 can block receptor activation and inhi bit proliferation of tumor cells bearing EGF receptors. To further exp lore the mechanism of mAb-mediated growth inhibition, we compared the capacities of bivalent 225 mAb and 225 F(ab')(2), and monovalent 225 F ab' fragment to block ligand binding to EGF receptors, inhibit activat ion of receptor tyrosine kinase by exogenous and endogenous ligand, pr oduce receptor dimerization, down-regulate receptors, and inhibit prol iferation of cultured A431 squamous carcinoma cells. Unlike 225 mAb an d 225 F(ab')(2), 225 Fab' fragment was a poor inhibitor of A431 cell p roliferation. The weak antiproliferative capacity of 225 Fab' was not due to depletion of active fragment from cultures. When cells were exp osed to exogenous EGF, monovalent 225 Fab' remaining in conditioned cu lture medium could act as well as the bivalent forms of mAb to block b inding and tyrosine kinase activation by exogenous EGF. However unlike the bivalent forms, 225 Fab' fragment was unable to induce receptor d imerization and down-regulation, and it lacked the capacity to block a utocrine activation of EGF receptors by endogenous ligand. These defic iencies were corrected by addition of rabbit anti-mouse IgG antibody, which also enabled 225 Fab' fragment to inhibit cell proliferation. We conclude that in A431 cells, inhibition of autocrine-stimulated proli feration by anti-EGF receptor mAbs requires antibody bivalency, which provides the capacity to produce EGF receptor dimerization accompanied by receptor down-regulation. These properties may explain the greater efficacy of bivalent mAb and F(ab')(2), compared with monovalent Fab' fragment, in inhibiting proliferation of a variety of malignant and n onmalignant cultured cell lines.