Vol. August
170,
No.
3, 1990
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
16, 19190
A MONOCLONAL
Yoshito
Numata,
Kitagawa,
Department
Hiroshi
DIRECTED
Nakada*,
Keiichi
Ozaki,
Mizue
Ikuo
Funakoshi*
TO Tn
Shigeyuki Inoue*,
and Ikuo
of Biological Chemistry, Sciences, Kyoto University,
*Department
Received
ANTIBODY
of Biotechnology, University,
ANTIGEN
Fukui,
Toshisuke
Hiroshi Kawasaki,
Yamashina*' Faculty Kyoto
Faculty Kyoto
981-985
of Pharmaceutical 606, Japan
of Engineering, 603, Japan
Kyoto
Sangyo
.June 23, 1990
SUMMARY: A murine monoclonal antibody, MLS 128, that was assigned tom an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly. "1990 Academic Press,Inc.
Carbohydrate-directed useful tumor of mucin-type
monoclonal
markers[l, 21. glycoproteins,
and Siaa2-6GalNAc-Ser(Thr)(sialyl cancer-associated antigens. the
production
the
are regarded
some core
[5,
6,
71,
We have developed antibodies directed
peptides
[8].
antibodies,
assigned lishment
to an anti-Tn antibody. and characterization of
designated
as
structures
such as GalNAcu-Ser(Thr)(Tn) Tn)
of monoclonal One of
antibodies
In particular,
[3,
41
are known to
be
procedure
for
a
against glycoas MLS 128, was
This paper reports this antibody.
the
estab-
MATERIALS AND METHODS Immunization and Establishment performed using Balb/c nude (nu/nu) 'To whom correspondence Abbreviations* A albumin.
should
OSM, ovine
of MLS 128. Immunization miceeach bearing a solid
was LS
be addressed.
submaxillary
mucin;
BSA, bovine
serum
0006-291x/90
981
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell fusion and the 180 tumor according to Hirohashi et al. [PI. hybridoma screening were performedas-described previously [8]. Other anti-Tn antibodies. NCC-LU-35 (anti-Tn, IgM)[3] and CA 3239 (anti-Tn, IgM)[lO] were gifts from Dr. S. Hirohashi of the National Cancer Center Institute (Japan) and from Dr. G. F. Sprinqer of Northwestern University, respectively. Preparation of antigens and antibody binding assay. Tn ervthrocvtes weredonated from Dr. Y. Ohkubo of Osaka Red Cross Blood Center. Mucin glycopeptides, G-501, and asialo G-501 were prepared from LS 180 cells as described previously [8]. OSM prepared according to Tettamanti and Pigman [ll] and that donated from Dr. S. Hakomori of the Biomembrane Institute were used sialithroughout. Asialo OSM was prepared by treating OSM with dase -from Arthrobacter ureafaciens (nacarai tesque, Kyoto). Glvcophorin A was obtained from Siqma, St. Louis. Blood qroup A glycoiipid was obtained from BioCarb Chemicals, Lund. -Asialoagalacto-glycophorin A in the wells was prepared using sialidase that from Vibrio cholerae (Calbiochem-Behring Corp., San Diego), then .8-galactosidase from from Arthrobacter ureafaciens and bovine testis (Boehringer Mannheim, Mannheim). Finally, the plates were washed with 1 % BSA 3 times. Antibody binding assay was performed as follows. On wells OSM of the microtiter plates, G-501, asialo G-501, OSM or asialo and were adsorbed with the aid of glutaraldehyde and polylysine, otherglycophorin A was adsorbed without modification. Unless antibody bound to the antigens in wells of the wise stated, plates was determined by direct binding of lz51-Protein A. Details of the procedure are described in a previous paper [81. To 25 1.11 of a 2 % suspension oferythroHemagglutination. The cvtes. an euual volume of the antibody solution was added. mixture was-incubated for 2 h at room-temperature and then hemagglutination was examined under a microscope.
RESULTS Isotype
of
chain. Assignment agglutination of binding of shows that
MLS 128 was
AND DISCUSSION identified
of MLS 128 to Tn erythrocytes
the anti-Tn antibodies MLS 128 agglutinated
as
to OSM or to Tn erythrocytes,
erythrocytes. The agglutination caused by CA 3239 or NCC-LU-35
was, probably
the
isotype:
are
of
MLS 128. Inhibition
these
antibodies
IgG3
with
the
K light
an anti-Tn antibody was and by mutual inhibition G-501. but
made of
Fig. 1 normal
not
however, weaker than due to the difference
IgM in
contrast
to
by the
IgG
that in isotype
using OSM as the test antigen showed MLS 128 to OSM was completely inhibited by CA and -~ vice versa. Fig. 2 shows the results of inhibition of the binding of MLS 128 to OSM by other antibodies. Fig. 3b shows that MLS 128 reacted with G-501 and asialo G501 to the same extent in contrast to an anti-sialyl Tn antibody, that 3239
experiments
the binding of and NCC-LU-35,
MLS 102 (Fig. 3a) [5, 61. The same was true with OSM (Figs. 3d and 3~). Other anti-Tn antibodies, 982
OSM CA
and asialo 3239
and
Vol.
170,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig. 1. Agglutination of Tn erythrocytes caused by MLS 128. MLS 128 produced in the spent medium was used, and agglutination was discernible with 2-fold dilution of the spent medium. A, Normal erythrocytes; B, Tn erythrocytes.
? 2
E 6 54 5 2 8
'= 5 p 100 a
m $ ln s L B
02Fig.
50 0 Reciprocal
Dilution
of Antibody
Q .g p
4 3
? $r
1
03 of MLS
2
Reciprocal
Dilution
of Antibody
128 to OSM by other 2. Inhibition of the binding 5F-10 is a control anti-Tr; antibodies (CA 3239 and NCC-LU-35). For MLS 128, NCC-LU-35 and 5F-10 spent medium was IgM antibody. '251-MLs used and for CA 3239 loo-fold diluted ascites fluid. [S)) was used as the reference 128 (prepared as described in Ref. antibody, and its binding to OSM (100 rig/well) was determined in 0, MLS 128; q , NCC-LU-35; 5, the presence of other antibodies. CA 3239; 0, 5F-10. 3. Reactivities of MLS 128 and MLS 102 with mucin glycopepFig. -L tides from LS 180 cells and their asialo forms. As antibodies, a and b, G-501; c and d, OSM. -o- G-501, spent medium was used. asialo G-501 or asialo OSM. or OSM; -O-
983
vol.
170,
No.
BIOCHEMICAL
3, 1990
0.1
1
AND BIOPHYSICAL
10 GalNAc
Fig. MIS
4. 128
Inhibition to OSM (100
of pronase nglwell).
100
1000
RESEARCH COMMUNICATIONS
loo00
(pl)
digest of -a-, 034;
OSM to the binding -0-, pronase digest
of
of
OSM.
NCC-LU-35,
were similar
These findings ri's
group
no binding
are,
with
however,
reporting
that
to these
inconsistent
binding
with
CA 3239 bound only
properties. Hakomoof
those to asialo
OSM with
to OSM [71.
MLS 128 bound to rin
respect
A (both
sialidase-f3-galactosidase
M and N types) whereas it did sialidase treated glycophorin
intact and properties were the
same as those
of
treated glycophobind at all to
not
other
A. These binding anti-Tn antibodies
171.
On treating
a-N-acetylgalactosaminidase
OSM with
(chicken
OSM lost the capacity to liver, Sigma, St. Louis), 128 as well as to CA 3239 and NCC-LU-35. Digestion reduced the These facts
binding suggest
tylgalactosamine
by the
fact
attached
binding to anti-Tn antibodies. of N-acetylgalactosamine was
that
to
with
pronase
MLS
capacity of OSM markedly, as shown in Fig. 4. that a certain peptide region with N-ace(non-sialylated)
involved in the Involvement
bind
the
treatment
of
OSM with
in
intact
further
OSM is confirmed
galactose
oxidase
(Dactylium dendroides, Sigma, St. Louis) abolished the binding capacity of OSM. This treatment had no effect on the binding to Tn-antigen directed antibody. MLS 102, sialyl Inhibition
of
the
antigen-antibody
rides also the binding
supports the involvement since galactose, fucose
et
reported
of
binding by monosacchaN-acetylgalactosamine in
and N-acetylglucosamine at 100 mM, had no effect to the binding, but N-acetylgalactosamine, a- and t3-methyl-N-acetylgalactosaminides at 100 mM inhibited the binding about 70 %. MLS 128 did not bind to blood group A glycolipid. Hirohashi al.
[3]
that
NCC-LU-35 984
bound to blood
group
A
glyco-
Vol.
170,
No.
3, 1990
lipid.
This
since
Takahashi
BIOCHEMICAL
is
probably
due
AND
to
the
[4] showed --et al. isotype which they raised against not bind to blood group A glycolipid.
structure
of
the
BIOPHYSICAL
MLS 128 epitope
RESEARCH
IgM nature that
of
anti-Tn
COMMUNICATIONS
this
antibody
a cancer-associated Studies in progress.
are
antibody of
IgG
mucin to
determine
did exact
ACKNOWLEDGMENTS
tific
This work research
was supported in from the Ministry
ture
of
by the
Japan,
a Grant-in-Aid We wish
to
from thank
Miss
the
Fugaku
Trust
Tokyo Tomoko
part of
by a Grant-in-Aid Education, Science for
Medicinal
Biochemical Saito
for
scienCuland Research, and by
Research her
excellent
for
Foundation. secretarial
assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Hakomori, S. (1985) Cancer Res. 45, 2405-2414. Feizi, T. (1985) Nata4,3-57. Hirohashi, S., Clausen, H., Yamada, T., Shimosato, Y., and Hakomori, S. (1985) Proc. Natl. Acad. Sci . USA 82, 70397043. Takahashi, H., Metoki, R., and Hakomori, S. (1988) Cancer Res.48, 4361-4367. Kurosaka, A., Fukui, S., Kitagawa, H., Nakada, H., Numata, Y Funakoshi, I., Kawasaki, T., and Yamashina, I. (1987) F&3 -Lett. 215, 137-139. Kurosaka, A., Kitagawa, H., Fukui, S., Numata, Y., Nakada, H Funakoshi, I., Kawasaki, T., Ogawa, T., Iijima, H., and Yamashina, I. (1988) J. Biol. Chem. 263, 8724-8726. Kjelden, T., Clausen,H.,ohashi, S., Ogawa, T., Iijima, H and Hakomori, S. (1988) Cancer Res. 48, 2214-2220. Fuiui, S., Numata, Y., Kurosaka,,Kitagawa, H., Nakada, H ., Funakoshi, I., Kawasaki, T., Takahashi, Y., Hayashi, K., and Yamashina, I. (1988) Jpn. J. Cancer Res. 79, 1119-1129. Hirohashi, S., Watanabe, M., Sh%osato,Y.,and Sekine, T. (1984) Jpn. J. Cancer Res. 75, 485-488. Springer, G.F.,drasekaran, E. V., Desai, P. R., and Tegtmeyer, H. (1988) Carbohydr. Res. 178, 271-292. Biochem. Biophys. Tettamanti, G. and Pigman, W. (1968) Arch. 124, 45-50.
985
170,
No.
3, 1990
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
16, 19190
A MONOCLONAL
Yoshito
Numata,
Kitagawa,
Department
Hiroshi
DIRECTED
Nakada*,
Keiichi
Ozaki,
Mizue
Ikuo
Funakoshi*
TO Tn
Shigeyuki Inoue*,
and Ikuo
of Biological Chemistry, Sciences, Kyoto University,
*Department
Received
ANTIBODY
of Biotechnology, University,
ANTIGEN
Fukui,
Toshisuke
Hiroshi Kawasaki,
Yamashina*' Faculty Kyoto
Faculty Kyoto
981-985
of Pharmaceutical 606, Japan
of Engineering, 603, Japan
Kyoto
Sangyo
.June 23, 1990
SUMMARY: A murine monoclonal antibody, MLS 128, that was assigned tom an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly. "1990 Academic Press,Inc.
Carbohydrate-directed useful tumor of mucin-type
monoclonal
markers[l, 21. glycoproteins,
and Siaa2-6GalNAc-Ser(Thr)(sialyl cancer-associated antigens. the
production
the
are regarded
some core
[5,
6,
71,
We have developed antibodies directed
peptides
[8].
antibodies,
assigned lishment
to an anti-Tn antibody. and characterization of
designated
as
structures
such as GalNAcu-Ser(Thr)(Tn) Tn)
of monoclonal One of
antibodies
In particular,
[3,
41
are known to
be
procedure
for
a
against glycoas MLS 128, was
This paper reports this antibody.
the
estab-
MATERIALS AND METHODS Immunization and Establishment performed using Balb/c nude (nu/nu) 'To whom correspondence Abbreviations* A albumin.
should
OSM, ovine
of MLS 128. Immunization miceeach bearing a solid
was LS
be addressed.
submaxillary
mucin;
BSA, bovine
serum
0006-291x/90
981
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell fusion and the 180 tumor according to Hirohashi et al. [PI. hybridoma screening were performedas-described previously [8]. Other anti-Tn antibodies. NCC-LU-35 (anti-Tn, IgM)[3] and CA 3239 (anti-Tn, IgM)[lO] were gifts from Dr. S. Hirohashi of the National Cancer Center Institute (Japan) and from Dr. G. F. Sprinqer of Northwestern University, respectively. Preparation of antigens and antibody binding assay. Tn ervthrocvtes weredonated from Dr. Y. Ohkubo of Osaka Red Cross Blood Center. Mucin glycopeptides, G-501, and asialo G-501 were prepared from LS 180 cells as described previously [8]. OSM prepared according to Tettamanti and Pigman [ll] and that donated from Dr. S. Hakomori of the Biomembrane Institute were used sialithroughout. Asialo OSM was prepared by treating OSM with dase -from Arthrobacter ureafaciens (nacarai tesque, Kyoto). Glvcophorin A was obtained from Siqma, St. Louis. Blood qroup A glycoiipid was obtained from BioCarb Chemicals, Lund. -Asialoagalacto-glycophorin A in the wells was prepared using sialidase that from Vibrio cholerae (Calbiochem-Behring Corp., San Diego), then .8-galactosidase from from Arthrobacter ureafaciens and bovine testis (Boehringer Mannheim, Mannheim). Finally, the plates were washed with 1 % BSA 3 times. Antibody binding assay was performed as follows. On wells OSM of the microtiter plates, G-501, asialo G-501, OSM or asialo and were adsorbed with the aid of glutaraldehyde and polylysine, otherglycophorin A was adsorbed without modification. Unless antibody bound to the antigens in wells of the wise stated, plates was determined by direct binding of lz51-Protein A. Details of the procedure are described in a previous paper [81. To 25 1.11 of a 2 % suspension oferythroHemagglutination. The cvtes. an euual volume of the antibody solution was added. mixture was-incubated for 2 h at room-temperature and then hemagglutination was examined under a microscope.
RESULTS Isotype
of
chain. Assignment agglutination of binding of shows that
MLS 128 was
AND DISCUSSION identified
of MLS 128 to Tn erythrocytes
the anti-Tn antibodies MLS 128 agglutinated
as
to OSM or to Tn erythrocytes,
erythrocytes. The agglutination caused by CA 3239 or NCC-LU-35
was, probably
the
isotype:
are
of
MLS 128. Inhibition
these
antibodies
IgG3
with
the
K light
an anti-Tn antibody was and by mutual inhibition G-501. but
made of
Fig. 1 normal
not
however, weaker than due to the difference
IgM in
contrast
to
by the
IgG
that in isotype
using OSM as the test antigen showed MLS 128 to OSM was completely inhibited by CA and -~ vice versa. Fig. 2 shows the results of inhibition of the binding of MLS 128 to OSM by other antibodies. Fig. 3b shows that MLS 128 reacted with G-501 and asialo G501 to the same extent in contrast to an anti-sialyl Tn antibody, that 3239
experiments
the binding of and NCC-LU-35,
MLS 102 (Fig. 3a) [5, 61. The same was true with OSM (Figs. 3d and 3~). Other anti-Tn antibodies, 982
OSM CA
and asialo 3239
and
Vol.
170,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig. 1. Agglutination of Tn erythrocytes caused by MLS 128. MLS 128 produced in the spent medium was used, and agglutination was discernible with 2-fold dilution of the spent medium. A, Normal erythrocytes; B, Tn erythrocytes.
? 2
E 6 54 5 2 8
'= 5 p 100 a
m $ ln s L B
02Fig.
50 0 Reciprocal
Dilution
of Antibody
Q .g p
4 3
? $r
1
03 of MLS
2
Reciprocal
Dilution
of Antibody
128 to OSM by other 2. Inhibition of the binding 5F-10 is a control anti-Tr; antibodies (CA 3239 and NCC-LU-35). For MLS 128, NCC-LU-35 and 5F-10 spent medium was IgM antibody. '251-MLs used and for CA 3239 loo-fold diluted ascites fluid. [S)) was used as the reference 128 (prepared as described in Ref. antibody, and its binding to OSM (100 rig/well) was determined in 0, MLS 128; q , NCC-LU-35; 5, the presence of other antibodies. CA 3239; 0, 5F-10. 3. Reactivities of MLS 128 and MLS 102 with mucin glycopepFig. -L tides from LS 180 cells and their asialo forms. As antibodies, a and b, G-501; c and d, OSM. -o- G-501, spent medium was used. asialo G-501 or asialo OSM. or OSM; -O-
983
vol.
170,
No.
BIOCHEMICAL
3, 1990
0.1
1
AND BIOPHYSICAL
10 GalNAc
Fig. MIS
4. 128
Inhibition to OSM (100
of pronase nglwell).
100
1000
RESEARCH COMMUNICATIONS
loo00
(pl)
digest of -a-, 034;
OSM to the binding -0-, pronase digest
of
of
OSM.
NCC-LU-35,
were similar
These findings ri's
group
no binding
are,
with
however,
reporting
that
to these
inconsistent
binding
with
CA 3239 bound only
properties. Hakomoof
those to asialo
OSM with
to OSM [71.
MLS 128 bound to rin
respect
A (both
sialidase-f3-galactosidase
M and N types) whereas it did sialidase treated glycophorin
intact and properties were the
same as those
of
treated glycophobind at all to
not
other
A. These binding anti-Tn antibodies
171.
On treating
a-N-acetylgalactosaminidase
OSM with
(chicken
OSM lost the capacity to liver, Sigma, St. Louis), 128 as well as to CA 3239 and NCC-LU-35. Digestion reduced the These facts
binding suggest
tylgalactosamine
by the
fact
attached
binding to anti-Tn antibodies. of N-acetylgalactosamine was
that
to
with
pronase
MLS
capacity of OSM markedly, as shown in Fig. 4. that a certain peptide region with N-ace(non-sialylated)
involved in the Involvement
bind
the
treatment
of
OSM with
in
intact
further
OSM is confirmed
galactose
oxidase
(Dactylium dendroides, Sigma, St. Louis) abolished the binding capacity of OSM. This treatment had no effect on the binding to Tn-antigen directed antibody. MLS 102, sialyl Inhibition
of
the
antigen-antibody
rides also the binding
supports the involvement since galactose, fucose
et
reported
of
binding by monosacchaN-acetylgalactosamine in
and N-acetylglucosamine at 100 mM, had no effect to the binding, but N-acetylgalactosamine, a- and t3-methyl-N-acetylgalactosaminides at 100 mM inhibited the binding about 70 %. MLS 128 did not bind to blood group A glycolipid. Hirohashi al.
[3]
that
NCC-LU-35 984
bound to blood
group
A
glyco-
Vol.
170,
No.
3, 1990
lipid.
This
since
Takahashi
BIOCHEMICAL
is
probably
due
AND
to
the
[4] showed --et al. isotype which they raised against not bind to blood group A glycolipid.
structure
of
the
BIOPHYSICAL
MLS 128 epitope
RESEARCH
IgM nature that
of
anti-Tn
COMMUNICATIONS
this
antibody
a cancer-associated Studies in progress.
are
antibody of
IgG
mucin to
determine
did exact
ACKNOWLEDGMENTS
tific
This work research
was supported in from the Ministry
ture
of
by the
Japan,
a Grant-in-Aid We wish
to
from thank
Miss
the
Fugaku
Trust
Tokyo Tomoko
part of
by a Grant-in-Aid Education, Science for
Medicinal
Biochemical Saito
for
scienCuland Research, and by
Research her
excellent
for
Foundation. secretarial
assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Hakomori, S. (1985) Cancer Res. 45, 2405-2414. Feizi, T. (1985) Nata4,3-57. Hirohashi, S., Clausen, H., Yamada, T., Shimosato, Y., and Hakomori, S. (1985) Proc. Natl. Acad. Sci . USA 82, 70397043. Takahashi, H., Metoki, R., and Hakomori, S. (1988) Cancer Res.48, 4361-4367. Kurosaka, A., Fukui, S., Kitagawa, H., Nakada, H., Numata, Y Funakoshi, I., Kawasaki, T., and Yamashina, I. (1987) F&3 -Lett. 215, 137-139. Kurosaka, A., Kitagawa, H., Fukui, S., Numata, Y., Nakada, H Funakoshi, I., Kawasaki, T., Ogawa, T., Iijima, H., and Yamashina, I. (1988) J. Biol. Chem. 263, 8724-8726. Kjelden, T., Clausen,H.,ohashi, S., Ogawa, T., Iijima, H and Hakomori, S. (1988) Cancer Res. 48, 2214-2220. Fuiui, S., Numata, Y., Kurosaka,,Kitagawa, H., Nakada, H ., Funakoshi, I., Kawasaki, T., Takahashi, Y., Hayashi, K., and Yamashina, I. (1988) Jpn. J. Cancer Res. 79, 1119-1129. Hirohashi, S., Watanabe, M., Sh%osato,Y.,and Sekine, T. (1984) Jpn. J. Cancer Res. 75, 485-488. Springer, G.F.,drasekaran, E. V., Desai, P. R., and Tegtmeyer, H. (1988) Carbohydr. Res. 178, 271-292. Biochem. Biophys. Tettamanti, G. and Pigman, W. (1968) Arch. 124, 45-50.
985
