BLOCKADE OF EPIDERMAL GROWTH-FACTOR RECEPTOR FUNCTION BY BIVALENT ANDMONOVALENT FRAGMENTS OF 225 ANTIEPIDERMAL GROWTH-FACTOR RECEPTOR MONOCLONAL-ANTIBODIES
Z. Fan et al., BLOCKADE OF EPIDERMAL GROWTH-FACTOR RECEPTOR FUNCTION BY BIVALENT ANDMONOVALENT FRAGMENTS OF 225 ANTIEPIDERMAL GROWTH-FACTOR RECEPTOR MONOCLONAL-ANTIBODIES, Cancer research, 53(18), 1993, pp. 4322-4328
We have previously described anti-epidermal growth factor (EGF) recept
or monoclonal antibodies (MAbs) which can block binding of transformin
g growth factor alpha (TGF-alpha) and EGF to receptors and inhibit act
ivation of receptor protein tyrosine kinase. Studies with these MAbs i
nvolving cell cultures and nude mouse xenografts demonstrated their ca
pacity to inhibit the growth of a variety of tumor cell lines, which e
xpress EGF receptors and TGF-alpha and appear to depend upon receptor
activation for cell proliferation. To explore the mechanism(s) by whic
h anti-EGF receptor 225 MAb inhibits cell proliferation, we have compa
red 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 I-125-EGF to EGF receptors. Sca
tchard analysis revealed that the K(d) of 225 F(ab')2 is comparable to
that of 225 MAb (1 nM), whereas the K(d) of 225 Fab' is 5 nm. Both bi
valent 225 MAb and 225 F(ab')2 and monovalent 225 Fab' were able to co
mpletely inhibit TGF-alpha-induced EGF receptor tyrosine kinase activa
tion, as assayed by autophosphorylation of tyrosine residues of EGF re
ceptors on MCF10A nonmalignant human mammary cells, MDA468 human breas
t adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells
. The bivalent forms of MAb could inhibit proliferation stimulated by
endogenous (autocrine) TGF-alpha in cultures of these three cell lines
. They also blocked growth stimulation by added exogenous TGF-alpha in
cultures of MCF10A cells and the growth-inhibitory effect of exogenou
s TGF-alpha upon MDA468 and A431 cell cultures. Monovalent 225 Fab' ha
d weaker inhibitory 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 g
rowth of s.c. A431 cell xenografts were compared. Equimolar amounts of
either 225 MAb or 225 F(ab')2 were administered at intervals equivale
nt to the half-lives of the molecules, to attempt to maintain comparab
le plasma levels. Both 225 MAb and 225 F(ab')2 inhibited A431 cell xen
ograft growth in a dose-dependent manner, with a more sustained respon
se in the case of the intact antibody. These experiments establish the
capacity of the bivalent 225 F(ab')2 and monovalent 225 Fab' fragment
s of an anti-EGF receptor antibody to mimic the properties of native 2
25 MAb in inhibiting ligand-induced tyrosine kinase activation, althou
gh the Fab' fragment is a weaker inhibitor of proliferation compared w
ith bivalent forms of antibody. They also provide strong evidence that
inhibition of A431 cell growth in vivo can result from pharmacologica
l blockade of EGF receptors, without participation of immune responses
mediated by the Fc fragment of the antibody.