1045
Bioscience Reports 3, 1045-1052 (1983)
Printed in Great Britain
Monoclonal antibody r e a c t i v e with t h e human
e p i d e r m a l - g r o w t h - f a c t o r r e c e p t o r recognizes
t h e b l o o d - g r o u p - A antigen
H. C. GOOII~ 3. SCHLESSINGER2, I. LAX2~ Y. YARDEN2~
T. A. LIBERMANN 2, and T. FEIZIt
IApplied Immunochemistry Research Group,
Division of Communicable Diseases, Clinical Research Centre,
Watford Road, Harrow, Middlesex HA1 3UJ, U.K.; and
2Department of Chemical Immunology,
The Weizmann Institute of Science, Rehovot, 76100, Israel
(Received 12 September 1983)
The h y b r i d o m a antibody TLS, which precipitates the
EGF receptor from the human epidermoid carcinoma
cell line A#31, has been shown to recognize the
blood-group-A carbohydrate structure.
This conclusion
has been reached from studies of (a) the binding of
the antibody to glycoproteins and haemagglutination of
erythrocytes with known blood-group-antigen activities
and (b) the inhibition of binding of the antibody to a
r a d i o l a b e l l e d b l o o d - g r o u p - A - a c t i v e g l y c o p r o t e i n by
structurally defined oligosaccharides.
An i n c i d e n t a l though important development resulting from the
widespread use of the hybridoma technique (1) in cell biology has
been the generation of antibodies with specificities for carbohydrate
s e q u e n c e s in g l y c o c o n j u g a t e s .
Thus t h e r e is a growing list of
h y b r i d o m a - d e r i v e d a n t i b o d i e s originally raised a g a i n s t antigenic
'markers' that distinguish embryonic cells from those of adults, tumour
cells from their normal counterparts, or differentiated cells from one
another, which upon detailed investigation are found to be directed
against carbohydrate structures of glycoproteins and glycolipids (see
refs 2-4 for example).
These hybridoma-derived antibodies together
with c e r t a i n natural-monoclonal autoantibodies against the developmentally regulated carbohydrate antigens I and i (5,6) are proving to
be useful reagents :[or the structural characterization of the glycoc o n j u g a t e s of intact cells (7,8) and of isolated glycoproteins and
glycolipids (9-12).
S e v e r a l h y b r i d o m a a n t i b o d i e s r e a c t i v e with the receptor for
epidermal growth factor (EGF) have been produced (13-18). One of
these was reported recently (19) to show specificity for the bloodgroup-H (Type l) carbohydrate structure.
We report here that the
mouse hybridoma antibody TLS, which was raised (15) against the
epidermoid carcinoma cell line A431 and precipitates the EGF receptor
from these ceI!s , recognizes the blood-group-A trisaccharide structure.
01983
The Biochemical Society
10146
Materials
GOOI
ET
AL.
and Methods
Hybridoma antibody
The mouse hybridoma antibody TL5 was isolated from ascites fluid
by p r e c i p i t a t i o n with 35% a m m o n i u m s u l p h a t e followed by gel
chromatography on an Ultrogel AcA-3t~ Column, LKB (15).
Immunological procedures
Immunostaining of glycoproteins on nitrocellulose paper (Bio-Rad
Trans-Blot TM) was carried out as follows: l IJl of a l-mg/ml solution
of each glycoprotein was applied to the nitrocellulose sheets, air dried~
and incubated in 0.5 M NaC% 20 mM sodium phosphate buffer pH 7.5
containing 2% bovine serum albumin (solution A) at 37~ for 1 h.
The sheets were incubated at 4~ for z~ h in a humidified chamber
with TL5 antibody, l0 mg per ml of solution A, or normal mouse
immunoglobulins as a control.
The sheets were rinsed in solution A,
washed twice in solution A containing 1% Triton-X-100 and once in
s o l u t i o n A (30 min each)~ and incubated at 4~
for 16 h with
tz~I-labelled rabbit immunoglobulins to mouse immunoglobulins.
The
rabbit immunoglobulins (Dako~ Copenhagen) were radioiodinated by the
chloramine-T method (20); specific activity was 5 laCi/pg of protein
and the antibodies were used at 2 x l06 c.p.m./ml of solution A. The
nitrocellulose sheets were washed as described above in solution A
containing I% Triton-X-100 with a final rinse in solution A and air
dried.
A u t o r a d i o g r a p h y was p e r f o r m e d using X-ray film from
Ceaverken AB, Strangras, Sweden.
Haemagglutination of erythrocytes of blood groups O, A9 and B was
carried out essentially as described previously (21).
D o u b l e - a n t i b o d y r a d i o i m m u n o a s s a y was performed as described
p r e v i o u s l y (22~23) using r a d i o i o d i n a t e d glycoprotein enriched for
b l o o d - g r o u p - A and -I activities from sheep gastric mucosae (22).
Inhibition of binding assays was carried out using TL5 antibody at 6
IJg/ml.
B a t c h w i s e a b s o r p t i o n of TL5 a n t i b o d y was carried out using
a d s o r b e n t s (Synsorb C h e m b i o m e d L t d . , Alberta, Canada) ol five
oligosaccharides (Table 1) coupled via spacer arms to silica (2t~);
these were kindly provided by Dr. Winifred M. Watkins and contained:
A-disaccharide, 0.8 IJmol/g (lot MBI-152); A-trisaccharide, 0.7 pmol/g
( l o t DB2-559); B-trisaccharide, 0.7 9mol/g (lot MBI-96); Forssman
disaccharide (lot AEI-238); and GalNAcCd-3GIcNAc (lot AB3-i8); the
saccharide content of the latter two adsorbants was not assayed. To
g m g of adsorbent was added 80 IJl of TL5 antibody (6 ~g/ml); after
overnight absorption the supernatant was tested for binding to the
r a d i o i o d i n a t e d blood-group-A-active glycoprotein by double-antibody
radioimmunoassay.
Glycoproteins
The following glycoproteins were used as reference blood-group
substances.
Ovarian c y s t glycoproteins: MSS, with blood-group-A
a c t i v i t y (25); Beach~ blood-group-B-active (26); 3S, blood-groupHLeb-active (27); N-l, blood-group-Lea-I-active (25); these were gifts
MONOCLONAL ANTIBODY TO EGF RECEPTOR
1047
of Dr. E. A. Kabat, Columbia Medical Center, New York. A bloodgroup precursor glycoprotein F1 (28) with Ii-activity, but lacking ABH
activity, was a gift from Dr. Winifred M. Watkins. A preparation of
sheep gastric mucins with blood-group-A and -I activities has been
described previously ( 2 2 ) .
01igosaccharides
N-acetyigalactosamine was from Sigma (Poote, U.K.); the bloodgroup-A-active disaccharide designated A-disaccharide (Fig. i), isolated
from partial acid hydrolysis products of an ovarian-cyst glycoprotein
with blood-group-A activity (29) was gift from Dr. W. M. Watkins,
Clinical Research Centre, Harrow, U.K.; the A-trisaccharide and the
A-pentasaccharide (sample GS) isolated from human urine (30) were
gifts of Dr. G. Strecker, Universit~ des Sciences et Techniques, Lille,
France; the A-tetra-, penta- (sample HS), hexa-, and heptasaccharides
isolated from faeces of breast-fed children (31) were gifts from Drs.
H.. Sabharwal and A. Lundblad, University Hospital, Lund, Sweden.
Results
In the nitrocellulose-immunostaining experiments using the reference
glycoproteins with known activities for blood groups A, B~ H, Le a ,
Le b, I, and i~ the TL5 antibody was found to react strongly with the
two blood-group-A-active glycoproteins derived from an ovarian cyst
and from sheep gastric mucins (results not shown).
There was no
i m m u n o s t a i n i n g os the precursor glycoprotein nor of the reference
o v a r i a n - c y s t g l y c o p r o t e i n s with blood groups B, HLe b, and Le a
activities.
Like p r e v i o u s l y d e s c r i b e d m o n o c l o n a l a n t i - A a n t i b o d i e s (see
references 52 and 33, for example) the TL5 antibody was found to
agglutinate human erythrocytes of blood group A (minimum agglutinating concentration 2.5 pg/ml) but not those of blood groups O and B.
The a n t i g e n i c d e t e r m i n a n t r e c o g n i z e d by TL5 a n t i b o d y was
i n v e s t i g a t e d by r a d i o i m m u n o a s s a y s in which structurally defined
oligosaccharides were used as inhibitors of the binding of the antibody
to the r a d i o i o d i n a t e d b l o o d - g r o u p - A - a c t i v e glycoprotein (Fig. i ) .
N-acetylgalactosamine inhibited the binding of TL5 antibody at a high
c o n c e n t r a t i o n (2600 nmol was required for 50% inhibition).
The
blood-group-A disaccharide was one thousand times more active (2.6
nmol for 50% inhibition), while the blood-group-A trisaccharide was
even more a c t i v e (0.6 nmol).
The antibody does not distinguish
between the blood-group-A trisaccharide carried on a Type-1 or Type-2
backbone structure, for the A-tetra- and hexasaccharides were equally
active, both being 3 times more active than the trisaccharide. Fig. 1
a l s o s h o w s t h a t the i n h i b i t o r y a c t i v i t y of the b l o o d - g r o u p - A
trisaccharide sequence is impaired in the presence of a second fucose
residue joined by c~1-4 or ~1-3 linkage to N-acetylglucosamine of the
Type-1 or Type-2 backbones, respectively, as in the A-penta- and
heptasaccharides.
This phenomenon has been noted previously with
c o n v e n t i o n a l a n t i - A antibodies and the Type-2-based difucosylated
structure (34).
10~8
GOOI
I00
ET
AL.
m
80
i
93 .
oo-
'-~
40
-
20
-
0
-]0
-
0.01
1
I
I
1
I
1
0.I
I
tO
tO0
I000
I0,000
nmoles inhibitor added
Fig. l. Inhibition of binding of TL5 antibody to radioiodinated
blood-group-A-active glycoprotein by oligosaccharides.
GalNAc
9
A-disaccharide
GalNAc~I-3Gal
9
A-trisaccharide
GalNAc~l-3Oal
[ i~2
Fuc~
A-tetrasaccharide
GalNAcel-3GalSI-4GIc
I 1,2
Fuc~
G 9 v A-pentasaccharide
(GS)(HS)
9
A-hexasaccharide
GalNAcal-3Gal ~I-4GI c
11,2
I 1,3
Fuc~
Fuc~
GalNAc~I-3GalBI-3GIcNAcSI-3GaI61-4GIc
I I~2
Fuc~
O
A-heptasaccharide
GalNAc~I-3Ga1131-3GI cNAc 81-3Ga i BI-4GI c
It,2
FucO~
11,4
Fuc~
In the batchwise absorption studies (Table I) it was found that the
Synsorb adsorbent containing the Forssman disaccharide, like those with
the blood-group-A disaccharide and trisaccharide abolished the binding
of TL5 antibody to the blood-group-A-active glycoprotein, while the
corresponding adsorbents with the blood-group-B trisaccharide and the
d i s a c c h a r i d e G a l N A c c ~ I - 3 G I c N A c had no e f f e c t on t h e binding.
T L 5 a n t i b o d y r e s e m b l e s c o n v e n t i o n a l a n t i - A a n t i b o d i e s (35)
a b i l i t y to r e a c t with t h e F o r s s m a n s t r u c t u r e .
Thus
in its
MONOCLONAL ANTIBODY TO EGF RECEPTOR
10#9
Table i. Inhibition of binding of TL5 antibody to
radioiodinated blood-group-A-active glycoprotein by adsorbents
containing the blood-group-A-related disaccharide
and trisaccharide and the Forssman disaccharide
TL5 antibody
(80 ~i containing 6 ~g of antibody per ml) was
absorbed with 4 mg of adsorbent and the binding of the supernatant
to radioiodinated blood-group-A-active glycoprotein was measured
as described in the Materials and Methods section.
Radioactivity
bound was calculated as the mean of duplicate experiments minus
the counts in the control tubes from which TL5 antibody was
omitted.
Carbohydrate sequence
on adsorbents
Designation
No adsorbent
C.p.m.
bound
%
inhibition
1231
GalNAc~I-3Gal
A-disaccharide
44
97
GalNAcal-3Gal
A-trisaccharide
4"4
97
I 1~2
Fuc~
GalNAcal-3GalNAc
Forssman
disaccharide
Gal~l-3Gal
B-trisaccharide
131
89
1402
0
1404
0
I 1~2
Fuc~
GalNAc~I-3GIcNAc
Discussion
These observations clearly show that antibody TLS, which precipit a t e s the EGF r e c e pt or of the A#31 epidermoid carcinoma cell line,
recognizes the blood-group-A trisaccharide st ruct ure.
The com pl et e
trisaccharide is not essential for reaction as the disaccharide sequence
G a l N A c ~ I - 3 G a l and e v e n t he F o r s s m a n - t y p e sequence GaINAc~I3GalNAc- can r e a c t with this antibody. To our knowledge, this is the
first monoclonal anti-A antibody whose combining specificity has been
described in detail.
Since this hybridoma antibody precipitates the EGF r e c e p t o r from
A#31 e p i d e r m o i d c a r c i n o m a cells (15), it is likely that a bloodgroup-A-related structure
GalNAc~I-3Gal (orGalNAc)
-+ I 1,2
Fucc~
o c c u r s as a terminal sequence on the carbohydrate chains of the
g l y c o p r o t e i n r e c e p t o r for EGF.
Alternatively, the blood-group-A
antigen may be expressed on other glycoproteins and/or glycolipids that
r e m a i n a s s o c i a t e d with the Triton-X-t00-solubilized receptor.
The
1050
GOOI
ET
AL.
170K g t y c o p r o t e i n r e c e p t o r for EGF was the main radioactive
component that was immune-precipitated by the anti-A antibody from
A431 cells which had been metabolically labelled with [35S]methionine,
but the i m m u n e p r e c i p i t a t e contained in addition some diffusely
migrating components of lower molecular weight (15).
The o c c u r r e n c e of the blood-group antigens A and H on the
EGF-receptor glycoprotein of A431 ceils would not be surprising since
in i m m u n o f l u o r e s c e n c e s t u d i e s with c o n v e n t i o n a l anti-A and -H
antibodies we (S. Thorpe and T. Feizi, unpublished observations) have
observed that both the blood-group-A and -H antigens are strongly
e x p r e s s e d on this cell line.
Moreover, several other carbohydrate
antigens recognized by monoclonal antibodies such as the FC10.2 (36)
and the I,i (37) antigens that are associated with Type-I and Type-2
b a c k b o n e regions, and the SSEA-I (23) and C14 (38) antigens,
associated with peripheral regions of oligosaccharides of the lacto-Nglycosyl series, are also expressed to varying degrees in this cell line.
It will be of interest to investigate whether these various carbohydrate
antigens are also associated with the EGF receptor.
Although the TL5 antibody binds to a determinant that is distinct
from the EGF-combining site [the antibody does not inhibit the binding
of EGF to the receptor (15)], it is of special interest that the
antibody stimulates DNA synthesis in human foreskin fibroblasts when
cross-linked by a second (anti-mouse Ig) antibody (15).
Presumably
this e f f e c t is a n a l o g o u s to the well-known mitogenic effects of
carbohydrate-binding proteins, lectins (39), on animal ceils. We have
s i m i l a r l y o b s e r v e d a small but consistent stimulation of tritiated
t h y m i d i n e u p t a k e by human p e r i p h e r a l - b l o o d l y m p h o c y t e s upon
t r e a t m e n t with monoclonal anti-I or -i sera followed by anti-human
IgM (Y. Katagiri, R. A. Childs, S. Knight, and T. Feizi, unpublished
observations).
While r e c e n t s t u d i e s have established that blood-group-related
structures serve as receptors for microbial agents, e.g. for certain
strains of Escherichia coli (t40) and Mycoplasma pneumoniae (41), the
roles of these carbohydrate structures in vivo are not yet known. It
is possible that the above observations with monoclonal antibodies are
p o i n t i n g to long-sought roles for these structures as receptors for
endogenous ligands.
C e l l - s u r f a c e c a r b o h y d r a t e s , including the blood-group-antigens,
change during differentiation and oncogenesis (~2,5,~3), and it is now
clear that these changing structures are associated with glycoproteins
such as the EGF receptor, the 'T200' glycoproteins of lymphocytes
(g,10), and other high-molecular-weight glycoproteins (2,12). With the
availability of monoclonal antibodies which can be used to specifically
perturb these carbohydrate structures, the way is open to investigate
w h e t h e r the c a r b o h y d r a t e moieties per se have roles in growthregulating processes.
Acknowledgements
HCG is supported by the Arthritis and Rheumatism Council. The
a u t h o r s are most grateful to Drs. E. A. Kabat, A. Lundblad, H.
S a b h a r w a l , G. Strecker, and W. M. Watkins for providing valuable
reference compounds, to Dr. R. A. Childs and W. M. Watkins for
helpful discussions, and to Mrs. M. Runnicles for the preparation of
the manuscript.
MONOCLONAL ANTIBODY TO EGF RECEPTOR
1051
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