(CANCER RESEARCH 53. 4322-432«. September 15. I9V3|
Blockade of Epidermal Growth Factor Receptor Function by Bivalent and
Monovalent Fragments of 225 Anti-Epidermal Growth Factor Receptor
Monoclonal Antibodies1
Zhen Fan, Hideo Masui, Ian Altas, and John Mendelsohn2
Laboratory of Receptor Biology, Memorial Slaan-Kellering
Cancer Center ¡2.E, H. M., I. A., J. M./, and Cornell University Medical Colicué\J. M./. New York, New York 10021
ABSTRACT
tors (2). One of these, the EGF-1receptor, appears to he an appropriate
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
target for antireceptor therapy. Cell transfection experiments have
demonstrated that the EGF receptor can function as a ligand-dependent oncoprotein and play a causal role in cell transformation (3-5).
Many human epithelial tumors have been found to express high num
bers of EGF receptors (6-15), and receptor levels are associated with
a poor clinical prognosis in cancers involving the bladder (10, 13),
breast (10, 11), and lung (14, 15).
We have produced and characterized two MAbs against the human
EGF receptor, 225 IgGl and 528 IgG2a (16, 17). These MAbs bind to
the EGF receptor with affinity similar to those of the natural ligands,
EGF and TGF-a, compete with these ligands for EGF receptor bind
ing, and prevent ligand-induced activation of EGF receptor tyrosine
phosphorylation (16-19). The antibodies also can inhibit proliferation
of a variety of cultured tumor cell lines which express EGF receptors
and TGF-a, including vulva (16-18), breast (20, 21), colon (22, 23),
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.
INTRODUCTION
The rationale for attempting to block the function of receptors for
growth factors as an approach to cancer therapy is compelling. Nearly
all cells require growth factors for proliferation in culture (1). A large
number of cellular oncogenes code for growth factors or their recep-
Reccived -V24/93; accepted 7/12/93.
The costs »Ipublication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by NIH Grants CA420M) and CA37641.
2 To whom requests for reprints should be addressed, at Box 15ft. Memorial SloanKctlcring Cancer Center. 1275 York Avenue. New York. NY 10021.
lung (24). renal (25), and prostate cells (26). MAb 455. which binds
to EGF receptors without inhibiting the binding of ligand, has no
effect on proliferation (16, 27). The antagonistic effects of the antireceptor MAbs upon cell growth have been postulated to result from
a blockade of receptor activation by TGF-a/EGF, either provided in
the culture medium or produced and released by the cells in an
autocrine fashion (28-30). Experiments with these MAbs provide
strong evidence for an EGF receptor-mediated autocrine growth-pro
moting pathway in many types of tumor cells.
Additional experiments have demonstrated the capacity of MAbs
225 IgGl and 528 lgG2a to prevent the growth of human tumor
xenografts in nude mice. Inhibition was seen against xenografts of cell
lines which appeared to depend upon autocrine EGF receptor stimu
lation for growth, based on the observation of MAb-mediated inhibi
tion of cells in culture. We and others have reported successful treat
ment of xenografted tumor cell lines from vulva, breast, lung, and
colon (23, 24, 28, 29, 31-33).
As a result of these experimental observations, we are undertaking
clinical studies to test the capacity of anti-EGF receptor MAb to act
therapeutically in patients with malignancies expressing high levels of
EGF receptor. The initial Phase 1 clinical trial demonstrated the safety
of administering 225 MAb in doses adequate to image tumors and to
maintain blood levels >30 HM(>30 X K,, of both ligand and antibody) for more than 3 days (34).
It is important to further explore the mechanism(s) by which antiEGF receptor MAbs inhibit cell proliferation. Two issues are of spe
cial interest. First is bivalency of antibody required to block receptor
activation by TGF-a/EGF, or can this be mediated by the monovalent
Fab' fragment, which lacks the capacity to cross-link two EGF recep
tors? This question is important when considering the possibility of
constructing low molecular weight peptides that may duplicate MAbmediated inhibition of receptor activation. A second question relates to
the mechanism of inhibiting tumor growth in the in vivo xenograft
'The
abbreviations
used are: KGK epidermal growth factor; TGF-u. transforming
growth factor u: MAb. monoclonal antibody: SUS. sodium dodecyl sulfate: FBS. phos
phate-buffered saline: DME. Dulbccco's modified liagle's medium: BSA. bovine serum
albumin: t-'BS, fetal bovine serum.
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BLOCKADE
OF EOF RECEPTOR
BY BIVALENT
model system. Does antitumor activity require the presence of the Fc
portion of the MAh. which has the capacity to activate complement
and to attract macrophages, natural killer cells, and lymphocytes that
bear Fc receptors? Demonstration of in vivo antitumor effect by
F(ab')j fragment of antibody would support the conclusion that inhi
bition of growth can be achieved by pharmacological blockade of
EOF receptors, without participation of immune and inflammatory
mechanisms.
As an approach to addressing these issues, we have examined the
activity of antireceptor 225 MAb and its bivalent and monovalent
fragments. Four different measures of MAb effects upon the activities
of EOF receptor were assayed: (a) inhibition of EOF binding; (b)
inhibition of receptor autophosphorylation by activated tyrosine pro
tein kinase: (c) inhibition of cell proliferation in culture; and (</)
inhibition of xenograft growth. For these studies, three cell lines were
investigated. MDA468 breast adcnocarcinoma cells and A431 vulvar
squamous carcinoma cells express high levels of EOF receptors and
produce TGF-a, and we have observed a complex bidirectional re
sponse to activation by additional exogenous ligand (17-19, 21).
Nonmalignant MCF10A mammary cells respond to TGF-a and they
also produce the ligand, but in quantities inadequate to stimulate
optimal proliferation (35, 36). The results of these studies strongly
support the hypothesis that EOF receptor blockade by antireceptor
antibody may provide a new form of anticancer therapy.
MATERIALS
Materials.
AND
MONOVALENT
MAh
buffer (50 ITIMTris-HCI-15 /AM¡odoaceticacid-300 JXMphenylmethylsulfonylfluoride) and disrupted in a 40-ml Dounce homogenizer. The cell homogenale
was centrifuged at 2000 rpm for 10 min at 4°Cto remove the nuclear pellet.
The supernatant was centrifuged at 15,(XM)rpm for 20 min at 4°C,and the
pelleted material was resuspended in PBS, pH 7.4. The homogenate was (hen
aliquoted in %-well microtiter plates with glass fiber filters in the well bottoms
(V&P Enterprise, San Diego. CA) at 0.6 /xg/well and incubated in DME plus
0.2% BSA al room temperature for 1 h. For the competition assays, I2SI-EGF
(5 nM) with indicated concentrations of 225 MAb, 225 F(ab')2, or 225 Fab'
were added to the wells. For the binding assays, indicated concentrations of
125I-225 MAb, 125I-225 F(ab')2, or 125I-225 Fab' were added to the wells. Both
assays were conducted
in the presence or absence of a 100-fold excess of
unlabeled material. After incubation for 2 h. the plates were washed 5 times
with PBS containing 0.2% BSA and 0.3% gelatin. The radioactivity hound to
filters was measured with a gamma counter. The binding data were analyzed by
the method of Scatchard (41). We have demonstrated that the binding charac
teristics of EGF receptors from Dounce homogenates of A431 cells, deter
mined with this method, are comparable to those obtained with assays of intact
cells.-4
Cell Culture. Immortalized, nonmalignant human mammary epithelial cell
line MCF10A(35,36)wasgrown
¡nDME:Ham's F-12 (1:1, v/v) supplemented
with 5% horse serum, 0.5 fig/ml hydrocortisone, 10 ftg/ml insulin. 100 ng/ml
cholera enterotoxin. 20 ng/ml TGF-a or EGF, and 1.5 mg/ml amphotericin.
Human breast adenocarcinoma cell line MDA468 (21 ) was maintained in DME
medium supplemented with 10% FBS and 2 mm glutamine. Human vulvar
squamous carcinoma cell line A431 (17-19) was cultured in DME-Ham's F-12
(1:1, v/v) containing 10% FBS.
Cell Growth Assay. MCF10A, MDA468. and A431 cells were seeded onto
12-well plates in their appropriate cell culture media described above. On the
AND METHODS
Anti-EGF receptor 225 MAb has been described previously (16,
17). Pepsin was purchased from Worthington BiomédicalCorp. (Freehold, NJ).
Anti-phosphotvrosine
antibody PY69 was obtained from IC'N Biochemical.
Inc. (Cleveland. OH). Anti-EGF receptor carboxyl-terminal peptide antiserum
RK-2 was u gift from Dr. J. Schlessinger (New York University Medical
Center). Anti-EGF receptor MAb EGFR1 against a carbohydrate component of
the extracellular portion of EGF receptors was kindly provided by Professor A.
L. Harris (Oxford University) (37). EGF was from Collaborative Research
(Watham, MA). TGF-a was from Intergen Company (Purchase, NY). Na'25I
and I25l-labeled protein A were from DuPont-New England Nuclear Research
(Boston. MA). All of the other chemicals were from Sigma (St. Louis. MO).
BALB/c nude mice used in this study were bred and maintained at the animal
facility at Memorial Sloan-Kettering Cancer Center, in accord with institu
tional standards.
Preparation and Characterization of Bivalent and Monovalent Frag
ments of 225 MAb. The F(ab')2 fragment of 225 MAb was obtained by
pepsin digestion (38, 39). Briefly, concentrated 225 MAb (10 mg/ml) in 0.1 M
sodium acetate buffer (pH 3.7) was digested with 10 fig/ml of pepsin for 75
min at 37°C.We aimed at 80 to 90% cleavage to avoid potential damage to the
immunoreactivity of the antibody fragments. Digestion was terminated by
increasing the pH to 8.0 with 3 M Tris-HCl buffer and was followed by
extensive overnight dialysis against 25 mw Tris-HCl (pH 7.6) containing 25
mm NaCI. Pepsin and small peptides generated by further digestion of MAb
fragments were removed by gel filtration on a Sephadex G-75 column (Pharmacia-LKB Biotechnology. Inc.). Undigested 225 MAb was separated from
225 F(ab'), on a Bio-Rad DEAE-gel column (Bio-Rad Laboratories); 225
F(ab')2 can be eluted with the same buffer (pH 7.6), while 225 MAb requires
a 10-fold higher NaC'l concentration (250 HIM)to be eluted. The monovalent
225 Fab' was made by reducing the bivalent 225 F(ab')2 (2 mg/ml) with 10 mvi
i.-cysteine in Tris-HCl buffer (pH 7.6) containing 25 HIMNaCI for l h in a 37°C
water bath and then alkylating with 40 IHMiodoacetamide for 30 min at room
temperature. This was followed by extensive dialysis against the same buffer
and gel filtration on Sephadex G-75 to remove any remaining unreduced
F(ab')2. The purity of the fragments was analyzed by 10% SDS-polyacrylamide gel electrophoresis. The antibody and fragments were sterilized by
filtration and stored in aliquots at 4°C.
next day, MCF10A cells were switched to the same medium, lacking exog
enous TGF-a or EGF; MDA468 and A431 cells were switched to medium
containing 0.5% dialyzcd FBS. The cells then were incubated for 5 days with
indicated concentrations of TGF-a. 225 MAb. or 225 Fab' as described in Figs.
4 and 5. The medium and added agents were replenished after 3 days. Cell
proliferation was assayed by trypsinizing and counting with a Coulter Counter
after 5 days of incubation.
Western Blot Analysis. Cells were treated with TGF-a plus or minus
antibody or fragments as described in the text and figure legend, lysed with
sample buffer (2% SDS-0.5% 2-mercaptoethanol-10%
glycerol in 62.5 mw
Tris-HCl. pH 6.8). boiled for 5 min. and analyzed by 7% SDS-polyacrylamide
gel electrophoresis. The proteins on the gels were then transferred to nitrocel
lulose and reacted with either anti-phosphotyrosine monoclonal antibody PY69
or anti-EGF receptor polyclonal antiserum RK-2 (42). Sample protein concen
trations were equalized before loading onto the gels.
Blood Clearance of 225 MAb and I i;ib'i. in Nude Mice. BALB/c nude
mice (8 to 12 weeks of age) were used in the present studies. I25l-labeled 225
MAb (1.6 x IO7 cpm/100 pig 225 MAb/mouse) or 225 F(ab')2 (3.9 X 10fi
cpm/67 fig 225 F(ab'):/mouse) was injected i.p. into nude mice, and 10 fil of
blood were taken from the tail veins at intervals and mixed with 1 ml of 0.1%
BSA solution. Trichloroacetic acid was added to a final concentration of 10%
and acid-precipitable radioactivity was counted with a gamma counter (31).
A431 Cell Xenografts in
trypsinized and washed with
cells (5 X K)1'cells/mouse)
Treatment consisted of 225
Nude Mice. A431 cells from stock cultures were
scrum free medium by centrifugation. The tumor
were injected s.e. into the backs of nude mice.
MAh administered twice weekly, or 225 F(ab')2
administered twice daily, injected ¡.p.into mice in PBS. The tumor size was
measured twice weekly by a vernier caliper and calculated by the formula
TT
-ab-
where a is length, /> is width, and a a b. Both earlier and concurrent studies
have demonstrated that treatment with an irrelevant MAb does not inhibit the
growth of A431 cell xenografts (31. 43).
EGF Receptor Binding and Competition Binding Assay. EGF. 225
MAb, 225 F(ab')2, and 225 Fab' were labeled with Na'25I by the chloramine-T
4 Unpublished observations.
method (40). A431 cells were scraped from culture dishes in homogenizing
4323
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BLOCKADE
OF EOF RECEPTOR
BY BIVALENT
AND
MONOVALENT
MAb
RESULTS
Preparation and Characterization of Bivalent and Monovalent
Fragments of 225 MAb. The digestion of 225 MAb with pepsin and
(he purification of antibody fragments were described in the experi
mental procedures. Fig. 1 shows that there was no detectable crosscontamination in the preparations of 225 MAb, 225 F(ab')2, and 225
Fab' after the various purification steps. This is important for the
interpretation of the subsequent experiments. Lanes I, 2, and 3 are
from a nonreducing gel, and Lanes 4, 5, and 6 are from a reducing gel
containing 2-mercaptoethanol. Under nonreducing conditions, 225
MAb (Lane 1), 225 F(ab')2 (Lane 2), and 225 Fab' (Lane 3) show
their expected migration positions. Under reducing conditions, 225
MAb (Lane 4) is resolved into characteristic heavy and light chains,
while 225 F(ab')2 (Lane 5) and 225 Fab' (Lane 6) display identical
0
24
5
10
15
20
25
225 MAb, F(ab')2 or Fab' (nM)
6 8 10 12 14 16
'25l-225MAb(nM)
D.
14
modified heavy chains and unmodified light chains.
225 F(ab')2 and 225 Fab', in addition to the native 225 MAb, were
able to compete with the binding of 125I-EGF to EOF receptors on
12
A431 cell homogenates (Fig. 2A). To further assess the capacities of
225 F(ab')2 and 225 Fab' to bind to EOF receptors, we performed
10
binding assays with labeled antibody and fragments. Scatchard analy
ses revealed that the binding of 225 F(ab')2 is comparable to 225
MAb, with Kj values of 1 nw (Fig. 2, B and C). The K,, of 225 Fab'
was 5 nw, indicating that the affinity of monovalent 225 Fab' is 5-fold
?
8
Z
E
6
O
4
weaker than the bivalent forms of the antibody (Fig. 2D). Under
identical experimental conditions, Scatchard analysis of I25I-EGF
binding to A431 cell homogenates showed a biphasic curve, with a
small number of higher affinity binding sites in addition to the more
prevalent site with K,¡similar to that of 225 MAb (data not shown).
Modulation of EGF Receptor Tyrosine Phosphorylation by Bi
valent and Monovalent Forms of Anti-EGF Receptor MAb. After
determining the activities of bivalent and monovalent forms of 225
MAb in blocking EGF receptor binding, we explored their capacities
to modulate TGF-a-induced activation of EGF receptor protein tyrosine kinase. The targets were MCF10A, MDA468, and A431 cells.
Fig. 3 shows the capacities of high concentrations of bivalent 225
MAb and 225 F(ab')2, and of monovalent 225 Fab', to inhibit TGFa-induced EGF receptor tyrosine kinase activation as assayed by
autophosphorylation of EGF receptors on tyrosine residues. The ad
dition of TGF-a into cultures of these three cell lines for 30 min
markedly stimulated tyrosine phosphorylation of the EGF receptor.
Concurrent treatment with TGF-a plus 225 MAb, 225 F(ab')2, or 225
O 0.4 0.8 1.2 1.6
Bound "'I-Fati (nM)
2
4 6 8 10 12 14 16
125l-F(ab')2 (nM)
6
8
10 12 14 16
125l-Fab'(nM)
homogenates were incubated for 2 h. Binding assay: indicated concentrations of (B)
'25I-225 MAb, (C) 125I-225 F(ab')2, and (D) l25I-225 Fab' were added to the wells and
the cell homogenates were incubated for 2 h. In both assays, after the filters were washed,
the associated radioactivity was measured in a gamma counter. Each point represents the
mean of duplicate determinations after subtracting the background, obtained by adding
100-fold excess unlabeled 225 MAb or its fragment. Insets, Scalchard analyses of the
binding data. B, bound; F, free.
Fab' could completely block the phosphorylation of EGF receptors on
tyrosine residues. This inhibition by 225 MAb and fragments was
concentration dependent (data not shown). 225 MAb, 225 F(ab')2, or
225 Fab' were unable to directly activate the receptor tyrosine kinase.
In these and subsequent experiments, the concentration of monovalent
•¿
—¿
Reduced gel
-200-
- 97 •¿
- 69
Fig. 1. SDS-polyacrylamide gel clectrophoresis
analysis of 225 MAb and its F(ab')i and Fab' frag
ments. 225 MAb and its fragments were separated
by 10% SDS-polyacrylamide gel electrophoresis
under nonreducing conditions (Lanes 1-3) and un
der reducing conditions with 2-mercaptoethanol
(Lanes 4-6). Lañen1 and 4. 225 MAh; Lanes 2 and
5, 225 F(ab')2; Lanes 3 and 6, 225 Fab'. kDa,
4
Fig. 2. EGF receptor binding and competition binding studies. Aliquots of A431 cell
homogenates (see "Materials and Methods") were plated in 96-well microtiler piales with
filters in the well bottoms. Competition assay (A ): I25I-EGF (5 nM) was added to the wells
with indicated concentrations of 225 MAb, 225 F(ab')2, and 225 Fab' and the cell
Non-reducedgel
"~
~~" kDa
225
MAb
F(ab')2
2
H chain
0» - 46 •¿
Fab
molecular weight in thousands.
H'chain
- 30 •¿
L chain
- 21 Lane: 1
2
3
456
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BLOCKADE
A.
OF EOF RECEPTOR BY BIVALENT
MCF10A
cell lines, reflecting the capacity of bivalent or monovalent antibody
to modulate responses to endogenous TGF-a and to block regulation
of cell growth by exogenous TGF-a.
MCF10A cells express endogenous TGF-a, but at levels that are
sufficient for only limited growth (Fig. 4/4). Addition of exogenous
TGF-a or EOF to cultures of these cells stimulates optimal growth. In
the absence of added exogenous TGF-a, the addition of bivalent or
monovalent 225 antibody produced identical and complete inhibition
of cell growth, suggesting comparable capacity to inhibit an autocrine
TGF-a-mediated stimulatory pathway (Fig.44 ). Addition of TGF-a to
the cultures resulted in a dose-dependent 4-fold increase in prolifera
tion. This was inhibited by bivalent 225 MAb, as evidenced by a shift
of the growth curve to the right. Treatment of cultures with mono
valent 225 Fab' produced a shift of the growth curve, but to a reduced
RK-2
B. MDA468
PY69
extent.
The results of experiments with MDA468 and A431 cell cultures
are complex. These cells produce endogenous TGF-a which stimu
lates proliferation. We have reported that growth is modestly further
stimulated by addition of low concentrations of exogenous ligand, but
proliferation is inhibited in a dose-dependent manner by higher con
centrations of ligand (17-19, 21).
Bivalent 225 MAb (30 nM),and to a lesser extent 225 Fab'(150 nM),
RK-2
A431
partially inhibited proliferation of MDA468 cells in the absence of
added exogenous TGF-a, suggesting interference with an autocrinestimulatory pathway (Fig. 40). Bivalent 225 MAb prevented the
stimulation of MDA468 cells by low doses of added exogenous
TGF-a; it also shifted to the right the growth curve showing concen
tration-dependent inhibition of MDA468 cells mediated by higher
doses of TGF-a (Fig. 4ß).Monovalent 225 Fab' had modest impact
PY69
RK-2
Lanes:
MAb
Cell Proliferation. Experiments were carried out to determine the
effects of 225 MAb and its F(ab')2 and Fab' fragments upon the three
PY69
C.
AND MONOVALENT
Activity of Bivalent 225 Antibody and Monovalent 225 Fab' on
on the response to added TGF-a (Fig. 4ß).
Bivalent 225 MAb (30 nM), but not monovalent 225 Fab' (150 nw),
1 2345678
Fig. 3. Inhibition of TGF-a-induced tyrosine phosphorylation of EOF receptor by
bivalent and monovalent forms of 225 MAb in MCF10A, MDA468, and A431 cell lines.
The cells were incubated for 30 min at 37°Cwith no addition (Lane 1 ), 10 nw TGF-a
(Lane 2), 200 nM 225 MAb (Lane.?), the mixture of 10 nM TGF-a and 200 niu 225 MAb
(Lane 4), 200 nM 225 F(ab'), (Lane 5), the mixture of 10 nM TGF-a and 200 nM 225
F(ab')2 (Lane 6), KXX)nu 225 Fab' (Lane 7), and the mixture of 10 nMTGF-a and KXM)
nM 225 Fab' (Lane N). The cells were then lysed with sample buffer and resolved by 7%
SDS-polyacrylamide gel clectrophorcsis. The proteins were transferred to nitrocellulose
and immunoblotted with either anti-phosphotyrosine antibody PY69 or anti-EGF receptor
polyclonal antiserum RK-2.
225 Fab' was 5-fold higher than that of bivalent 225 MAb or 225
F(ab')2, because of the 5-fold weaker binding affinity of the mono
valent fragment.
caused approximately 50% inhibition of A431 cell proliferation in the
absence of exogenous TGF-a, suggesting blockade of an autocrinestimulatory pathway mediated by endogenous TGF-a (Fig. 4C). The
bivalent antibody also blocked the capacity of low doses of added
exogenous TGF-a to stimulate proliferation. The addition of higher,
nanomolar concentrations of exogenous TGF-a to A431 cell cultures
caused a concentration-dependent
inhibition of growth, as reported
previously (18). The concurrent addition of either 225 MAb or 225
Fab' attenuated the TGF-a-induced inhibitory effect on cell growth,
but again, the monovalent fragment had weaker effect.
To further explore whether antibody bivalency is required for
modulating the growth regulation of A431 cells by TGF-a, this ex-
B. MDA468
A. MCF10A
50 r
Fig. 4. Inhibition of TGF-a-induced effects on
cell proliferation by bivalent and monovalent 225
MAb in MCF10A. MDA468, and A431 cell lines.
MCF10A (in 5% horse serum) and MDA468 and
A431 cells (in 0.5% dialyzed FBS) were grown
with concentrations of exogenous TGF-a varying
from 0 to 100 nM (•).Parallel cultures were incu
bated with 30 nM 225 MAb (A) or 150 nM225 Fab'
—¿30
(D). Cells were trypsinized and assayed by Coulter
Counter after 5 days of culture. The results are
expressed as the means of triplicates: bars, SE. The
initial cell counts were 2.0 X IO4, 1.8 X IO4, and
2.1 X IO4 in the respective cultures.
O
10r
40
20
0)
10
O
0.1
10
100
O 0.01
TGF-a (nM)
4325
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BLOCKADE
OF EOF RECEPTOR
BY BIVALENT
AND
MONOVALENT
MAti
périmentwas repeated using higher concentrations of monovalent 225
Fab'. The results in Fig. 5 demonstrate that 225 Fab' was able to
modestly effect growth stimulation by endogenous TGF-a and growth
inhibition by exogenous TGF-a.
When cultures were incubated with bivalent 225 F(ab')2 fragment,
the results in each case were comparable to those obtained with intact
225 MAb (data not shown). These experiments demonstrate that bi
valent forms of 225 antibody inhibited both the stimulatory and in
hibitory effects of TGF-a upon EGF receptor function. Monovalent
225 Fab' had a definite, but weaker, capacity to modulate growth
24
regulation dependent upon activation of EGF receptors by endogenous
or exogenous TGF-a, even though the concentration of the 225 Fab'
was adjusted to 5-fold greater than bivalent antibody.
Control experiments were performed with anti-EGF receptor MAb
EGFR1, which binds to EGF receptors but does not alter the binding
or activity of EGF (37). EGFR1 MAb had no effect upon proliferation
in cultures of the three cell lines, in the absence or presence of added
exogenous TGF-a (data not shown).
Inhibition of Human Tumor Xenografts by Bivalent 225 F(ab')?
Fragment. We previously demonstrated that treatment of nude mice
with 225 MAb can prevent the formation of tumor xenografts (23,
31-33, 43). The antitumor activity of antibody is concentration de
pendent. To determine whether this effect can be achieved by phar
macological EGF receptor blockade in vivo, without the participation
of Fc fragment-mediated activation of immune or inflammatory re
sponses, we compared native 225 MAb and bivalent 225 F(ab')2 for
their capacities to inhibit A431 cell xenograft growth. Because of the
differences in the molecular weights of these molecules and the an
ticipated differences in clearance by the reticuloendothelial system
and excretion by the kidneys (44), we initially determined the serum
clearance rates in vivo of both native 225 MAb and 225 F(ab')2.
Equimolar concentrations of 12SI-labeled 225 MAb or 225 F(ab')2
were injected i.p. into nude mice. Blood samples were taken from the
tail vein at intervals for radioactivity determinations. Fig. 6 shows the
serum clearance rates following i.p. injection of a single dose of native
225 MAb or 225 F(ab')2. 225 MAb had a serum half-life of approxi
mately 70 h. while 225 F(ab')2 had a serum half life of approximately
10 h.
Experiments were then performed to compare the antitumor activi
ties of 225 MAb and 225 F(ab')2. BALB/c mice were given s.c.
injections of 5 X 10<>A431cells/mouse and treated with i.p. injections
of either 225 MAb or 225 F(ab')2, beginning on the day of xenograft
inoculation. Our previous pharmacological studies demonstrated that
a treatment schedule of 1 mg i.p. twice weekly could produce stable
48
72
96
120 144
168
Hours
Fig. 6. Scrum clearance of 225 MAb and 225 F(ab'); in nude mice. Equimolar con
centrations of either '-5I-225 MAb(l(X)/ig/mouse)(tl)or
L25I-225F(ab')2(67/xg/mousc)
(•)were injected i.p. into ñnude mice. Tail vein blood samples were collected and
trichloroacetic acid-precipitable radioactivity was counted with a gamma counter. I'oinls,
means; bars, SE.
blood levels of native antireceptor MAb that would be capable of
saturating xenograft EGF receptors (>30 X K,,) (31). Considering the
observed differences in serum half-lives, 225 F(ab')2 was adminis
tered twice daily instead of the twice weekly injection schedule for
225 MAb. The doses of 225 MAb and 225 F(ab')2 were adjusted to
attempt to obtain equimolar concentrations of these two molecules
(225 MAb, M, 180,000; 225 F(ab')2, M, 120,000). Fig. 7 shows the
inhibitory effects upon A431 cell xenograft growth of 225 MAb and
225 F(ab')2 in high doses (1.5 mg 225 MAb and 1.0 mg 225 F(ab')2)
and in low doses (0.5 mg 225 MAb and 0.33 mg 225 F(ab')2). Animals
were treated for a period of 15 days and followed for 26 days. The
results clearly demonstrate the capacity of 225 F(ab')2 to produce a
concentration-dependent inhibition of A431 cell xenografts, indicating
that pharmacological blockade of EGF receptors can mediate inhibi
tion of A431 cell growth in vivo. This response pattern was observed
in three separate experiments. With this schedule of antibody and
fragment administration, the 225 F(ab')2 fragment appears to produce
less complete growth inhibition than the 225 MAb, suggesting the
possible involvement of additional immune mechanisms in the antitumor activity of complete antibody.
DISCUSSION
These experiments provide information concerning the mechanism
by which 225 MAb against the EGF receptor mediates its effects upon
receptor function. The capacity of an antibody to bind to the EGF
B. 225F(ab')2
A. 225MAb
50r
9
£
033 mg
20 -
Jt 10 0
75
250
750
225Fab'(nM)
2500
Days
Days
Fig. 7. Inhibition of A431 cell xenografts by 225 MAb and 225 F(ab'),. BALB/c nude
mice were given s.c. injections of 5 X IO6 A431 cells/mouse. Groups of K-l> mice were
Fig. 5. Effccls of 225 Fab' on A43I cell growth in the presence or absence of exog
enous TGF-tt. The cells were grown in 0.5% dialyzcd FBS with increasing concentrations
of monovalent 225 Fab' (H). Parallel cultures were incubated with the identical concen
trations of 225 Fah' plus 3 nMTGF-a (•).Cells were trypsinized and assayed by Coulter
Counter after 5 days of culture. The results are expressed as the means of triplicates; bars,
SE. The initial counts were 2.1 X IO4 cells.
treated beginning on the day of xenograft inoculation with i.p. injection of equimolar
concentrations of cither (A ) 225 MAb (0.5 and 1.5 mg) or (B ) 225 F(ab' )2 (0.33 and 1.0
mg). The 7-fold difference in the serum half-lives of the two molecules was accounted for
by treating the 225 F(ab')2 group twice daily, and the 225 MAb group twice weekly
(arrows at lops). I'oinls, means: bars, SE.
4326
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BLOCKADE
OF KOF RF.Å’PTOR
BY B1VALKNT AND
receptor and immunoprecipitate it does not necessarily confer prop
erties of functional blockade of receptor activity. For example, EGFR1
MAb does not block binding of EOF (37). This is also the case for
MAb 455 from our laboratory (16, 17). These MAbs have served as
controls in our current and earlier studies with MAbs that do block
EOF binding (16, 17, 27). Furthermore, two studies with unique IgM
MAbs against the EOF receptor reported agonistic effects, with the
activity of the antibody mimicking the stimulatory properties of EOF
upon receptor tyrosine phosphorylation and cell proliferation (45, 46).
In addition to antibodies 225 IgGl and 528 lgG2a, produced by our
laboratory, a number of other anti-EGF receptor MAbs have been
shown to block the binding of EOF. These include MAb 425 (47, 48),
MAb 108 (49), and MAb 2E9, which appears to block only low
affinity binding sites (50).
Does receptor blockade by MAb require antibody bivalency, which
provides the potential for linking together two different EOF receptor
molecules on the cell surface? Our results show that while bivalent
225 MAb and 225 F(ab')2 were capable of preventing EOF binding
MAh
the growth of KB cells in nude mice, while the complete MAb showed
antitumor activity (49). The investigators suggested that the lack of
antitumor activity by the 108 Fab' fragment might be due to the
inability of 108 Fab' to induce receptor aggregation (49). However,
the experiment may not have been a valid test of the efficacy of Fab'
compared with native 108 MAb, because of the short plasma half-life
which would be expected for this molecule (44). Because the reduced
binding affinity and short half-life of 225 Fab' suggest that extremely
large amounts of the fragment would have to be continuously infused,
in order to obtain stable plasma concentrations which could saturate
EGF receptors, we have not pursued this question further in the
present studies.
The results of present experiments support our postulation that EGF
receptor blockade may be useful in antitumor therapy. The demon
stration of i«vivo activity of bivalent 225 F(ab'), fragment establishes
that 225 MAb can act as a pharmacological EGF receptor blocking
agent. However, the evidence that monovalent 225 Fab' fragments
completely block ligand-mediated activation of receptor protein tyro
sine kinase but weakly inhibit growth stimulated by TGF-a suggests
and inhibiting ligand-induced tyrosine kinase activation and cell pro
liferation, the monovalent 225 Fab' fragment also had the capacity to
that the monovalent fragment of 225 antibody regulates some, but not
all, EGF receptor activities. These observations raise the interesting
possibility that optimal regulation of proliferation by 225 antibody
may involve activities beyond blockade of receptor tyrosine kinase,
which require antibody bivalency. Planned future trials of anti-EGF
receptor MAb treatment, using a murine/human chimeric antibody in
repeated doses, will determine the clinical efficacy of this therapeutic
approach.
block binding and inhibit kinase activation but had weaker effects on
cell proliferation. The binding affinity of 225 Fab' was 5-fold weaker
than the affinities of either of the bivalent forms of the antibody, but
this small difference cannot fully explain its weaker effect upon pro
liferation. The possibility must be considered that inhibition of pro
liferation by anti-EGF receptor MAbs may involve other pathways, in
addition to blocking EGF receptor tyrosine kinase activation.
All of the bivalent and monovalent forms of 225 antibody had
monophasic Scatchard plots, with a single Kt¡in the low nanomolar
range. This is consistent with our previous findings with native 225
MAb (16, 17). In contrast, EGF binding curves displayed a second,
minority population of receptors with higher affinity, as has been
observed in many laboratories (51). The capacity of 225 MAb and its
fragments to completely block tyrosine kinase activation is consistent
with the concept that the higher affinity state may be due to oligomerization of a single class of receptors, which individually display
lower affinity for ligand (51).
In previous studies we have demonstrated that treatment of nude
mice with 225 or 528 MAb against the EGF receptor can prevent
growth of xenografts of A431 vulvar squamous carcinoma cells (31,
32, 43), MDA 468 breast adenocarcinoma cells (29), and DiFi colon
adenocarcinoma cells (23). Inhibition by MAb is dose dependent, and
delay of therapy with MAb until xenografts have become well estab
lished results in varying responses, from elimination of tumors in the
case of DiFi cells to a modest reduction in proliferation rate in the case
of well established A431 cell xenografts (23, 31).5
MONOVALF.NT
ACKNOWLEDGMENTS
The present study of nude mouse xenografts treated with 225
F(ab')2 clearly demonstrates that inhibition of A431 tumor growth can
be achieved in vivo by anti-EGF receptor antibody without the par
ticipation of immune or inflammatory mechanisms mediated by the Fc
portion of the MAb. However, the finding that a less complete inhi
bition of tumor growth was observed with 225 F(ab')2 suggests the
possibility that dual activities, involving both pharmacological recep
tor blockade and immune mechanisms, may be contributing to the
antitumor activity observed with intact 225 MAb.
These observations do not address the question of whether the
monovalent 225 Fab' fragment could mediate in vivo antitumor ac
tivity. The data from the cell culture experiments suggest that receptor
blockade by 225 Fab' fragment inhibits proliferation less effectively
than bivalent antibody forms. In in vivo studies with anti-EGF recep
tor MAb 108, injections of 108 Fab' fragment were unable to inhibit
5 Z. Fan el ai, manuscript in preparation.
We thank Dr. Dan Shochat for helpful udvicc on preparation of antibody
fragments.
REFERENCES
1. Barnes. D., and Sato, G. Serum-free cell eulture: u unifying approach. Cell, 22:
649-655, 1980.
2. Weinberg, R. A. (ed.). Oncogcnes and the Molecular Origin of Cancer. C'old Spring
Harbor. NY: Cold Spring Harbor Laboratory, 1989.
3. Ricdel. H.. Massoglia, S.. Schlessinger. J.. and Ullrich. A. Ligand activation oÃoverexpressed epidermal growth factor receptors transforms N1H 3T3 mouse fihroblasls. Proc. Nati. Acad. Sci. USA, 85: 1477-1481. 1988.
4. Di Fiore. P. P., Pierce, J. H., Fleming. T. P., Hazan. R.. Ullrich. A., King, C. R.,
Schlessinger, J.. and Aaronson. S. A. Overexpression of the human EGF receptor
confers an EGF-dependenl transformed phenotype to NIH 3T3 cells. Cell. 51: 10631070, 1987.
5. Velu. T. J.. Bcguinot, L., Vass. W. C. Willingham, M. C., Merlino, G. T., Pastan, !..
and Lowy, D. R. Epidermal growth factor-dependent transformation by a human EGF
receptor proto-oncogene. Science (Washington DC), 2.M: 1408-1410, 1987.
6. Ozanne. B.. Richards, C. S., Hendler, F., Burns, D., and Gusterson, B. Over-expres
sion of the EGF receptor is a hallmark of squamous cell carcinomas. J. Pathol.. 149:
9-14, 1986.
7. Sobol, R. E., Astarita. R. W., Hofeditz, C., Masui, H., Fairshter, R.. Royston, I., and
Mendelsohn. J. Epidermal growth factor receptor expression in human lung carcino
mas defined by a monoclonal antibody. J. Nati. Cancer Inst.. 7V; 403—107, 1987.
8. Véale,D.. Kerr, N., Gibson, G. H., and Harris. A. L. Characterization of epidermal
growth factor receptor in primary human non-small cell lung cancer. Cancer Res., 49:
1313-1317. 1989.
9. Libermann, T. A., Razón,N., Bartal, A. D., Yarden, Y, Schlessinger, J.. and Soreq. H.
Expression of epidermal growth factor receptors in human hrain tumors. Cancer Res..
44: 753-760, 1984.
10. Harris, A. L., Nicholson, S., Sainsbury, J. R. C., Neal. D., Smith, K.. Farndon, J. R.,
and Wright, C. Epidermal growth factor receptors: a marker of early relapse in breast
cancer and tumor state progression in bladder cancer: interactions with neu. Cancer
Cells (Cold Spring Harbor). 7: 353-357. 1989.
11. Sainsbury, J. R. C., Malcolm, A. J., Appleton, D. R., Farndon, J. R., and Harris, A. L.
Presence of epidermal growth factor receptor as an indicator of poor prognosis in
patients with breast cancer. J. Clin. Pathol., J«: 1225-1228, 1985.
12. Eishruch. A.. Blick. M.. Lee. J. S.. Sacks, P. G., and Gutterman. J. Analysis of the
epidermal growth factor receptor gene in fresh human head and neck tumors. Cancer
Res., 47: 3603-3605. 1987.
13. Neal, D. E., Bennett, M. K., Hall, R. R., Marsh, C., Abel, P. D., Sainsbury, J. R. C..
and Harris. A. L. Epidermal growth factor receptors in human bladder cancer: com
parison of invasive and superficial tumors. Lancet. /: 366-368, 1985.
14. Hendler, F., Shum-Siu. A., Nanu, L., Yuan, D.. and Ozanne. B. Increased EGF
4327
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
HIWKADi:
15.
16.
17.
18.
OF t:OI
RECEPTOR
BY BIVALENT
receptors and the absence of an alveolar differentiation marker predici a poor survival
in lung cancer. Proc. Am. Soc. Clin. Oncol., 8: 223. 1989.
Vealc. D.. Ashcroft. T.. Marsh. C, Gibson. G. J.. and Harris, A. L. Epidermal growth
factor receptors in non-small cell lung cancer. Br. J. Cancer. 55: 513-516, 1987.
Sato, J. D., Kawamoto, T., Le. A. D., Mendelsohn. J.. Polikoff. J.. and Sato. G. H.
Biological effect in vitro of monoclonal antibodies to human epidermal growth factor
receptors. Mol. Biol. Med.. /: 511-529, 1983.
Kawamoto, T., Sato, J. D., Le, A.. Polikoff. J., Sato, G. H., and Mendelsohn. J. Growth
stimulation of A431 cells by epidermal growth factor: identification of high affinity
receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc.
Nail. Acad. Sci. USA, SO: 1337-1341, 1983.
Kawamoto. T., Mendelsohn. J.. Le. A.. Sato. G. H., Lazar, C. S., and Gill, G. N.
Relation of epidermal growth factor receptor concentration to growth of human
epidermoid carcinoma A431 cells. J. Biol. Chcm., 259.- 7761-7766, 1984.
19. Gill. G. N.. Kawamoto, T., Cochet, C.. Le. A.. Salo, J. D.. Masui. H.. MacLeod, C. L..
and Mendelsohn. J. Monoclonal anti-epidermal growth factor receptor antibodies
which are inhibitors of epidermal growth factor binding and antagonists of epidermal
growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem.. 259: 7755776(1, 1984.
20. Arteaga, C. L.. Coronado, E., and Osborne. C. K. Blockade of the epidermal growth
factor receptor inhibits transforming growth factor a-induced but not estrogen-induced growth of hormone-dependent human breast cancer. Mol. EndocrÃ-nol., 2:
1064-1(169. 1988.
21. Ennis, B. W.. Valverius. E. M.. Lippman. M. E.. Bello!. F.. Kris. R., Schlcssinger. J..
Masui. H.. Goldenberg. A.. Mendelsohn. J.. and Dickson. R. B. Monoclonal anti-EGF
receptor antibodies inhibit the growth of malignant and non-malignant human mammary epithelial cells. Mol. Endocrino!.,.?: 1830-1838, 1989.
22. Karnes, W. E., Jr.. Walsh, J. 11., Wu, S. V, Kim, R. S.. Martin, M. G., Wong, H. C.,
Mendelsohn. J.. Gazdar. A. F., and Cullitta, F. Autocrine stimulation of EGF receptors
by TGF-a regulates autonomous proliferation of human colon cancer cells. Gastroenlerology, 102: 474-485. 1992.
23. Masui. II.. Boman. B., Hyman. J., Castro, L.. and Mendelsohn. J. Treatment with
anti-EGF receptor monoclonal antibody causes regression of DiFi human colorectal
carcinoma xenografts. Proc. Am. Assoc. Cancer Res., 32: 394. 19l>|.
24. Reiss, M.. Slash, E. B., Vellucci, V. F., and Zhou. Z-I. Activation of the autocrine
transforming growth factor «pathway in human squamous carcinoma cells. Cancer
Res., 51: 6254-6262. 1992.
25. Alias. I.. Mendelsohn. J.. Baselga. J.. Fair. W. R., Masui, H.. and Kumar. R. Growth
regulation of human renal carcinoma cells: role of transforming growth factor-a.
Cancer Res.. 52: 3335-3339, 1992.
26. Hofcr, D. R.. Sherwood, E. R., Bromberg, W. D., Mendelsohn, J.. Lee. C.. and
Ko/lowski, J. M. Autonomous growth of androgen-independent
human prostatic
carcinoma cells: role of transforming growth factor a. Cancer Res., 51: 278(1-2785,
1991.
27. Markowitz. S. D., Molkcntin. K.. Gcrbic. C., Jackson, J., Stellato. T.. and Willson. J.
K. V. Growth stimulation by coexpression of transforming growth factor-a and
epidermal growth factor-receptor in normal and adenomatous human colon epithe
lium. J. Clin. Invest., 86: 356-362, 1990.
28. Mendelsohn. J.. Masui. H.. and Goldenherg. A. Anti-epidermal growth factor receptor
monoclonal antibodies may inhibit A431 tumor cell proliferation by blocking on
aulocrine pathway. Trans. Õssoc. Am. Phys., 100: 173-178, 1987.
29. Mendelsohn. J. Potential clinical applications of anti-EGF receptor monoclonal an(¡bodies.Cancer Cells (Cold Spring Harbor). 7: 359-362, 1989.
30. Van de Vijver. M., Kumar. R.. and Mendelsohn. J. Ligand-induced activation of A431
cell epidermal growth factor receptors occurs primarily hy an autocrine pathway that
acts upon receptors on (he surface rather than intracellularly. J. Biol. Chem.. 266:
7503-7508. 1991.
31. Masui. H.. Kawamoto. T., Sato, J. D.. Wolf. B., Salo, G. H., and Mendelsohn, J.
Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth
factor receptor monoclonal antibodies. Cancer Res.. 44: 1002-1007. 1984.
32. Masui. H., Morayama. T.. and Mendelsohn. J. Mechanism of antitumor activity of
mice for anti-epidermal growth factor receptor monoclonal antibodies with different
isotypes. Cancer Res.. 46: 5592-5598. 1986.
AND
MONOVALENT
MAh
33. Goldenberg. A., Masui. H.. Divgi. C.. Kamrath. H.. Pcntlow. K.. and Mendelsohn. J.
Imaging of human lumor xenografts with an indium-Ill
labelled anti-epidermal
growth factor receptor monoclonal antibody. J. Nati. Cancer Inst-, HI: 1616-1625,
1989.
34. Divgi. C. R., Welt, C., Kris, M., Real, F. X., Yen. S. D. J., Gralla. R.. Merchant. B..
Schwcighart, S.. Unger. M.. Larson. S. M.. and Mendelsohn. J. Phase I and imaging
trial of indium-Ill labeled anti-epidermal growth factor receptor monoclonal anti
body 225 in patients with squamous cell lung carcinoma. J. Nati. Cancer Inst.. 83:
97-104, 1991.
35. Ciardiello. F.. McGeady. M. L.. Kim, N.. Basólo. F., llynes. N.. Langton, B. C.,
Yoko/aki. H.. Sacki. T.. Elliot, J. W.. Masui. H.. Mendelsohn. J., Soule. H.. Russo. J.,
and Salomon, D. S. Transforming growth factor-a expression is enhanced in human
mammary epithelial cells transformed by an activated c-Ha-n/.v proto-oncogene hut
not by the c-neu proto-oncogene, and overexpression of the transforming growth
factor-a complementary DNA leads to transformation. Cell Growth Differ.. /: 407420. 1990.
36. Soule. H. D., Maloney, T. M., Woltnan, S. R., Pelerson, W. D., Jr., Brenz, R.,
McGrath, C. M.. Russo. J.. Pauley, R. J.. Jones, R. F. and Brooks. S. C. Isolation and
characterization of a spontaneously immortalized human breast epithelial cell line.
MCF-IO. Cancer Res.. 50: 6075-6086. 1990.
37. Walerfield. M. D., Mayes, E. L. V.. Stroobanl. P.. Benne!. P. L. P.. Young, S.,
Goodfellow, P. N., Banting, G. S.. and Ozanne. B. A monoclonal antibody to the
human epidermal growth factor receptor. J. Cell. Biochem.. 20: 149-161. 1982.
38. Parham, P. On the fragmentation of moni>clonal IgCil. lgG2a and lgCi2b form
BALB/c mice. J. Immunol.. 131: 2895-2902. 1983.
39. Demignot. S.. Garnctt. M. C., and Baldwin, R. W. Mouse lgG2b monoclonal anlibody
fragmentation: preparation and purification of Fab. Fc and Fab/c fragments. J. Im
munol. Methods, /2/: 209-217. 1989.
40. Hunter. W. M.. and Greenwood, F. C. Preparation of iodine-131 labelled human
growth hormone of high specific activity. Nature (Lond.). 194: 495—496. 1962.
41. Scatchard. G. The attractions of proteins for small molecules and ions. Ann. NY Acad.
Sci., SI: 660-672. 1949.
42. Kris, R. M., Lax. I.. Gullick, W., Waterficld. M. D.. Ullrich. A.. Fridkin. M., and
Schlessinger, J. Antibodies againsl a synthetic peptide as a probe for the kinase
activity of the avian EGF receptor and v-erb B protein. Cell. 40: 619-625, 1985.
43. Baselga. J.. Norton. L.. Masui. H.. Pandiella, A.. Copian, K.. Cordon-Cardo. C..
Miller, W.. and Mendelsohn. J. Anti-tumor effects of doxorubicin in combination with
anti-epidermal growth factor receptor monoclonal antibodies. J. Nati. Cancer Insl.,
«5:1327-1333, 1993.
44. Covell, D. G., Barbel, J.. Hollon. O. D.. Black, C. D. V.. Parker. R. J.. and Weinslein.
J. N. Pharmacokmetics of monoclonal immunoglobulin Gl. F(ab')^, and Fab' in mice.
Cancer Res.. 46: 3969-3978. 1986.
45. Schreiber. A. B., Libermann. T. A.. Lax. I.. Yarden, Y., and Schlessinger. J. Biological
role of epidermal growth factor-receptor clustering. Investigation with monoclonal
anti-receptor antibodies. J. Biol. Chem., 25«:846-853, 1983.
46. Fernandez-Pol, J. A. Epidermal growth factor receptor of A431 cells. J. Biol. Chem.,
26(1: 5003-5011, 1985.
47. Rodeck. U., Hcrlyn. M.. Herlyn. D.. Molthoff. C.. Atkinson, B., Varello, M.,
Steplewski, Z.. and Koprowski. H. Tumor growth modulation hy a monoclonal
antibody to the epidermal growth factor receptor: immunologically mediated and
effector cell-independent effects. Cancer Res., 47: 3692-3696. 1987.
48. Rodeck. U.. Williams. N.. Murthy. U.. and Hcrlyn. M. Monoclonal antibody 425
inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived
EGFATGF-a. J. Cell. Biochem.. 44: 69-79. 1990.
49. Aboud-Pirak, E.. Hurwitz. E.. Pirak. M. E.. Bcllot, F. Schlessinger. J.. and Seht. M.
Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in
vitro and in nude mice. J. Nati. Cancer Inst.. 80: 1605-1611. 1988.
50. Defizc. H. K., Boonstra, J.. Mcisenhcldcr. J.. Kruijer. W.. Tertoolcn, L. Ci. J.. Tilly. B.
C.. Hunter. T.. van Bergen en Henegouwen. P. M. P.. Moolcnaar. W. H.. and de Laal.
S. W. Signal transduction by epidermal growth factor occurs through the subclass of
high affinity receptors. J. Cell Biol.. 709: 2495-2507, 1989.
51. Ullrich. A., and Schlessinger. J. Signal transduction hy receptors with tyrosine kinase
activity. Cell. 61: 2(13-212. 1990.
4328
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Blockade of Epidermal Growth Factor Receptor Function by
Bivalent and Monovalent Fragments of 225 Anti-Epidermal
Growth Factor Receptor Monoclonal Antibodies
Zhen Fan, Hideo Masui, Ian Altas, et al.
Cancer Res 1993;53:4322-4328.
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