Blockade of Epidermal Growth Factor Receptor Function by Bivalent and Monovalent Fragments of 225 Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies1

We have previously described anti-epidermal growth factor (I t.r i re ceptor monoclonal antibodies i M MIMwhich can block binding of trans forming growth factor a (TGF-a) and EOF to receptors and inhibit activation of receptor protein l> rosine kinase. Studies with these M Mis 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-a 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')^ and monovalent 225 Fab' fragments. Both native 225 MAb and its frag ments could inhibit the binding of 'l-l (.I to EGF receptors. Scatchard analysis revealed that the Klt of 225 F(ab')2 is comparable to that of 225 MAb (I IIMI.whereas the A',,of 225 Fab' is 5 n\i. Both bivalent 225 MAb and 225 l iah i. and monovalent 225 Fab' were able to completely inhibit TGF-a-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on Ml I IOA nonmalignant human mammary cells. MUA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endog enous (autocrine) TGF-a in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-u in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-u upon MDA468 and A431 cell cultures. Monovalent 225 Fab' had weaker inhibi tory 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 I (¡ili11.and native 225 M Mi 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 I Mb11. 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')¿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 inhibi tor 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 partici pation of immune responses mediated by the Fc fragment of the antibody.