Blockade of Epidermal Growth Factor Receptor Function by Bivalent and Monovalent Fragments of 225 Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies

Abstract

We have previously described anti-epidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) which can block binding of transforming growth factor α (TGF-α) and EGF to receptors and inhibit activation of receptor protein tyrosine kinase. Studies with these MAbs involving cell cultures and nude mouse xenografts demonstrated their capacity to inhibit the growth of a variety of tumor cell lines, which express EGF receptors and TGF-α and appear to depend upon receptor activation for cell proliferation. To explore the mechanism(s) by which anti-EGF receptor 225 MAb inhibits cell proliferation, we have compared the activity of native 225 MAb with the response to bivalent 225 F(ab′)₂ and monovalent 225 Fab′ fragments. Both native 225 MAb and its fragments could inhibit the binding of ¹²⁵I-EGF to EGF receptors. Scatchard analysis revealed that the K_d of F(ab′)₂ is comparable to that of 225 MAb (1 nm), whereas the K_d of 225 Fab′ is 5 nm. Both bivalent 225 MAb and 225 F(ab′)₂ and monovalent 225 Fab′ were able to completely inhibit TGF-α-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on MCF10A nonmalignant human mammary cells, MDA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endogenous (autocrine) TGF-α in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-α in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-α upon MDA468 and A431 cell cultures. Monovalent 225 Fab′ had weaker inhibitiory effects upon the proliferation of these cell lines. To determine whether the in vivo antiproliferative activity of anti-EGF receptor MAb can occur without the participation of the Fc portion of MAb, the capacities of 225 F(ab′)₂ and native 225 MAb to inhibit growth of s.c. A431 cell xenografts were compared. Equimolar amounts of either 225 MAb or 225 F(ab′)₂ were administered at intervals equivalent to the half-lives of the molecules, to attempt to maintain comparable plasma levels. Both 225 MAb and 225 F(ab′)₂ inhibited A431 cell xenograft growth in a dose-dependent manner, with a more sustained response in the case of the intact antibody.

These experiments establish the capacity of the bivalent 225 F(ab′)₂ and monovalent 225 Fab′ fragments of an anti-EGF receptor antibody to mimic the properties of native 225 MAb in inhibiting ligand-induced tyrosine kinase activation, although the Fab′ fragment is a weaker inhibitor of proliferation compared with bivalent forms of antibody. They also provide strong evidence that inhibition of A431 cell growth in vivo can result from pharmacological blockade of EGF receptors, without participation of immune responses mediated by the Fc fragment of the antibody.