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) receptor monoclonal antibody (mAb) 225 can block receptor activation and inhibit proliferation of tumor cells bearing EGF receptors. To further explore the mechanism of mAb-mediated growth inhibition, we compared the capacities of bivalent 225 mAb and 225 F(ab')2, and monovalent 225 Fab' fragment to block ligand binding to EGF receptors, inhibit activation of receptor tyrosine kinase by exogenous and endogenous ligand, produce receptor dimerization, down-regulate receptors, and inhibit proliferation of cultured A431 squamous carcinoma cells. Unlike 225 mAb and 225 F(ab')2, 225 Fab' fragment was a poor inhibitor of A431 cell proliferation. The weak antiproliferative capacity of 225 Fab' was not due to depletion of active fragment from cultures. When cells were exposed to exogenous EGF, monovalent 225 Fab' remaining in conditioned culture medium could act as well as the bivalent forms of mAb to block binding and tyrosine kinase activation by exogenous EGF. However, unlike the bivalent forms, 225 Fab' fragment was unable to induce receptor dimerization and down-regulation, and it lacked the capacity to block autocrine activation of EGF receptors by endogenous ligand. These deficiencies 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 proliferation 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 nonmalignant cultured cell lines.
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York.
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D000911:Antibodies, Monoclonal; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D002455:Cell Division; D015536:Down-Regulation; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D066298:In Vitro Techniques; D011940:Receptor Aggregation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases