of Biological Chemistry, Sciences, Kyoto University,
of Biotechnology, University,
Yamashina*' Faculty Kyoto
of Pharmaceutical 606, Japan
of Engineering, 603, Japan
.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
markers[l, 21. glycoproteins,
and Siaa2-6GalNAc-Ser(Thr)(sialyl cancer-associated antigens. the
We have developed antibodies directed
to an anti-Tn antibody. and characterization of
such as GalNAcu-Ser(Thr)(Tn) Tn)
of monoclonal One of
are known to
against glycoas MLS 128, was
This paper reports this antibody.
MATERIALS AND METHODS Immunization and Establishment performed using Balb/c nude (nu/nu) 'To whom correspondence Abbreviations* A albumin.
of MLS 128. Immunization miceeach bearing a solid
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Cell fusion and the 180 tumor according to Hirohashi et al. [PI. hybridoma screening were performedas-described previously . Other anti-Tn antibodies. NCC-LU-35 (anti-Tn, IgM) 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 . 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.
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
to OSM or to Tn erythrocytes,
erythrocytes. The agglutination caused by CA 3239 or NCC-LU-35
MLS 128. Inhibition
an anti-Tn antibody was and by mutual inhibition G-501. but
Fig. 1 normal
however, weaker than due to the difference
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
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
and asialo 3239
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.
E 6 54 5 2 8
'= 5 p 100 a
m $ ln s L B
50 0 Reciprocal
Q .g p
03 of MLS
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-
Inhibition to OSM (100
of pronase nglwell).
digest of -a-, 034;
OSM to the binding -0-, pronase digest
These findings ri's
CA 3239 bound only
those to asialo
to OSM [71.
MLS 128 bound to rin
M and N types) whereas it did sialidase treated glycophorin
intact and properties were the
same as those
treated glycophobind at all to
A. These binding anti-Tn antibodies
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 to anti-Tn antibodies. of N-acetylgalactosamine was
capacity of OSM markedly, as shown in Fig. 4. that a certain peptide region with N-ace(non-sialylated)
involved in the Involvement
OSM is confirmed
(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
rides also the binding
supports the involvement since galactose, fucose
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.
bound to blood
 showed --et al. isotype which they raised against not bind to blood group A glycolipid.
MLS 128 epitope
IgM nature that
a cancer-associated Studies in progress.
This work research
was supported in from the Ministry
a Grant-in-Aid We wish
by a Grant-in-Aid Education, Science for
scienCuland Research, and by
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