(CANCER RESEARCH 49. 5689-5695. October 15.
1989|
Human IgG3 Monoclonal Antibody Directed to an Unbranched Repeating Type 2
Chain (Gal/S1^4GlcNAc/?l-^3Gal/S1^4GlcNAc/?l-*3Gal/?l-*R) Which Is
Highly Expressed in Colonie and Hepatocellular Carcinoma1
Masayuki Miyake,2 Nobuoki Kohno,2 Edward D. Nudelman, and Sen-itiroh Hakomori
The Biomembrane Institute and University of Washington, 201 Elliott Avenue West, Seattle, Washington
ABSTRACT
Previously established human monoclonal antibodies (MAbs) directed
to carbohydrate antigens are essentially all IgM class, and show relatively
low affinity and low reactivity at 37C.We report here the establishment
of a human IgG, MAb displaying high affinity antigen-binding activity
at 37Cand efficiently activating cellular cytotoxicity directed to human
tumor cell lines expressing the polylactosamine antigen. The IgC.i MAb
(MH21-134) reacted with the repeated unbranched polylactosamine
structure Gal/31-4GlcNAc/31-3Gal/31-4GlcNAc/31-3Gal/31-R, i.e., ingly, many of these human MAbs so far established have been
nLCft, nl,cn, etc., but did not react with sialyl 23
or 26
substituted
derivatives at the terminal Gal. This specificity differs from that of
several anti-i antibodies, or human anti-i-like MAbs which react with
sialyl 23
substituted structures. Directly biotinylated MH21-134 an
tibody was used in iminiinoli islorlicni irai staining of 154 formalin-fixed,
paraffin-embedded tissue sections to study distribution of the antigen.
High incidence of positive staining was found in colon cancer (11/17;
65%) and hepatocellular carcinoma (8/12; 67%), followed by large cell
and squamous cell carcinoma of lung cancer (10/13; 59%, and 14/26;
54%, respectively). TLC immunostaining of glycolipid extracts from a
variety of tumor tissues showed the presence of nLc6 and/or nLc8 in over
50% of cases. The antigens nI.<
and ill .i\ were found to be absent from
normal colonie epithelia, kidney, and pancreas. Only a weak band cor
responding to n I.(
and one corresponding to nI .r(,were found in liver and
spleen, although all these normal tissues, including gastrointestinal epi-
thelia, lung, liver, spleen, erythrocytes, and lymphocytes, were essentially
negative on immunohistology. However, the antigen was found to be
highly expressed in myelocytes and weakly in bronchial glands of lung
and pancreatic duct epithelia. Nevertheless, expression of unsubstituted,
unbranched polylactosamine antigen could be an important basis for
induction of humoral immune response against certain types of human
cancer, despite its limited expression in normal cells.
INTRODUCTION
The most important knowledge we have obtained in recent
years through the application of MAbs1 involves defining (in
chemical or physical terms) the exact epitope structure of
previously ambiguous or ill-defined antigens, e.g., tumor-asso
ciated or developmentally regulated antigens (1, 2). However,
our understanding of host immune response in human cancer
using the human MAb approach has lagged, primarily because
of technical difficulties involved in generating stable clones
secreting human MAbs. Although a number of technical prob
lems still exist, there have been many results published describ
ing established clones secreting human MAbs that have been
derived from EBV-transformed peripheral B-lymphocytes or
Received 1/30/89; revised 6/29/89; accepted 7/13/89.
The costs of publication 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 study was supported by funds from The Biomembrane Institute and in
part by Outstanding Investigator Grant (OIG) CA42505 from the National
Cancer Institute.
2 Senior Fellow. Department of Pathobiology, University of Washington, and
The Biomembrane Institute.
3The abbreviations used are: MAb. monoclonal antibody; BSA, bovine serum
albumin: CMW. chloroform-methanol-water; PCS, fetal calf serum; PBS. phos
phate-buffered saline: SPG, sialosylparagloboside; TLC. thin-layer chromatog-
raphy; HAT. hypoxanthine-aminopterin-thymidine; EBV. Epstein-Barr virus.
5689
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regional lymph node lymphocytes of patients with cancer (3-
6), human/mouse hybridomas derived from lymphocytes of
regional lymph nodes of patients with cancer (7-9), or a com
bination of these two approaches (6, 10). Although a number
of laboratories have been involved in establishing an efficient
human lymphocyte fusion partner to create human/human
hybridomas, successful results are few, and antibody productiv
ity in such systems has been unsatisfactory (11, 12). Interest
identified as being directed to glycosphingolipid antigens, par
ticularly gangliosides. Nevertheless, essentially all of these hu
man MAbs have been identified as IgM, and therefore their
antigen-binding affinity is low, particularly at 37C.
In this paper, we report (a) the establishment and character
ization of a human IgG, MAb directed to an unsubstituted
unbranched type 2 chain polylactosamine (Gal/314Glc-
NAc,ol-3Gal/31-4GlcNAc/31->3Gal/31^R);and (>) demon
stration of a strong expression of the structure defined by this
antibody in colonie and hepatocellular carcinoma.
MATERIALS AND METHODS
Cell Fusion and Hybridoma Cell Culture. The subcarinal lymph nodes
of a 72-year-old patient with squamous cell carcinoma of the lung were
resected and used for the human/mouse hybridoma. These lymph nodes
had no metastasis. Initially, lymphocytes from these lymph nodes were
prepared as a single cell suspension and incubated with 1:100 dilution
of pokeweed mitogen (G1BCO, Grand Island. NY) for 4 days. The
stimulated lymphocytes were then washed three times with RPMI 1640
and fused with mouse myeloma SP2 as follows: 1.0 ml of 50% poly-
ethyleneglycol 4000 (Sigma Chemical Co., St. Louis, MO) was added
dropwise to a dry cell pellet over a 2-min interval. At succeeding 2-min
intervals, increasing volumes (1, 1.5, 2, 3, 4, and 5 ml) of serum-free
RPMI 1640 were added. After addition of the final 5 ml volume of
medium, cells were spun at 700 x g for 5 min. After the fusion, the
cells were washed again with RPMI 1640 and seeded into 96-well plates
in a concentration of IO5 cells/well with HAT RPMI 1640 medium
supplemented with 10% FCS. The antibodies produced by the hybrid
oma were assayed for their binding to cancer cell lines by solid-phase
enzymoimmunoassay as described below. The positive hybridomas were
cloned by limiting dilution method as soon as possible.
Binding Assays of MAbs by Enzyme-linked Immunosorbent Assay.
The assays were performed after 3 weeks. PC-9 and KATO III cells
were used as targets. These cells were trypsinized and plated on 96-well
plates (Costar. Cambridge. MA) at a concentration of 4 x IO4 cells/
well to produce a subconfluent monolayer in the wells after overnight
culture. After washing five times in 50 mM Tris buffer (pH 7.4), the
plates were incubated for 2 h with biotinylated goat anti-human IgM
(p chain specific; Vectastatin, Burlingame, CA) and biotinylated goat
anti-human IgG (heavy and light chain specific; Vectastatin), washed
five times, and incubated for l h with avidin and biotinylated peroxidase
(Vectastatin). The plates were washed five times, and O-phenylenedi-
amine substrate was added. After 10 min, the absorbance was read in a
spectrophotometer at 450 nm.
Preparation of Standard Glycolipids. Globotriaosylceramide (Gb3;
CTH), globotetraosylceramide (Gb4; globoside) (13, 14), SPG (IV3-
NeuAcnLc4) (15), G6 (2>3sialyllactonorhexaosylceramide; Vl'Neu-
 HUMAN IgGj MAb DIRECTED TO AN UNBRANCHED
AcnLcft) (16), H2 (VI2FucnLc6) (17), H, (VI2FuclV>-Fuca2Gal/34Glc-
NAcnLc6) (17), and G, (VI'NeuAcIV('Fuc2Gal/i4GlcNAcnLc(,) (18)
were prepared from human O erythrocytes. nLc8 was purified from
rabbit muscle (19), and G,0 (VI1NeuAcIV<'Fuca2Gal/i4GlcNAcnLc6)
(19-21) was from human placenta. PG, nLc,,, and Gg., were prepared
by mild acid hydrolysis of SPG, sialosylnorhexaosylCer, and CM,
ganglioside, respectively. Lactoisooctaosylceramide (I antigen) was pre
pared from G10.
Assessment of the Reactivity of M \l> with Various Glycolipids by
Solid-Phase Enzymoimmunoassay. The enzymoimmunoassay was per
formed using various glycolipid antigens that were immobilized at the
bottom of 96-well culture plates (Costar, Cambridge, MA) as described
previously (22). Briefly, the plate was blocked with PBS containing 5%
BSA for 2 h and incubated for l h with medium containing the MAb.
After washing five times with PBS/0.5% BSA, the plate was incubated
for I h with medium containing peroxidase-conjugated goat anti-human
IgG (7 chain specific) (Cappel. Malvern, PA).
Preparation of Neutral Glycolipid Fraction from Various Kinds of
Tissues. Cancer tissues were removed surgically from patients with
various kinds of carcinomas. Neutral glycolipids of these cancer tissues
were prepared as follows. Glycolipids were extracted from cancer tissues
with isopropanol-hexane-water (55:20:25) (23) and partitioned accord
ing to Folch-Pi (24). The glycolipids in the upper layer fraction of the
Folch's partition were subjected to DEAE-Sephadex A-25 (Pharmacia,
Uppsala, Sweden) column chromatography (25). After the neutral
glycolipids were eluted with CMW (30:60:8), gangliosides were eluted
with chloroform-methanol-0.8 N sodium acetate (30:60:8). The neutral
glycolipid mixture in the former fraction was used for TLC immuno-
staining without further purification.
TLC Immunostaining. TLC immunostaining was performed with the
neutral glycolipids extracted from cancer tissues according to the
method originally introduced by Magnani et al. (26), with modifications
(22). Briefly, glycolipids were developed on a Baker high-performance
thin-layer chromatography plate (Si-HPF plate, 7011-3; Baker, Phil-
lipsburg, NJ) with a solvent system of CMW (50:40:10). The plate was
air dried and blocked with PBS/5% BSA for 2 h and exposed overnight
to the culture fluid containing respective MAbs (about 5 ^g/ml). After
washing five times with PBS/0.5% BSA. the plate was exposed for l h
to the solution of rabbit anti-human IgG (y chain specific, ICN Im-
munobiologicals. Lisle, IL). After five washings with PBS/0.5% BSA,
the plate was exposed to the PBS containing I25l-Protein A for l h and
washed 10 times with PBS. The plate was then air dried and subjected
to autoradiography.
Immunohistochemical Techniques and Fluorocytometric Analysis. The
avidin-biotin complex technique for the immunohistochemical study of for it did not react with KATO III cells treated with HIOj but
various kinds of cancers was performed as described below. Briefly,
tumor sections of 4-/jm thickness were freed of paraffin by soaking in of this MAb, MH21-134, was tested by solid-phase enzymoim
xylene and dehydrated in graded ethanol. The endogenous peroxidase
activity was blocked by treating the sections with 0.3% hydrogen
peroxide for 20 min. After washing for 5 min in 50 mM Tris buffer, pH
7.4, the sections were incubated for 2 h at room temperature with PBS/
5% BSA and then exposed overnight to the biotinylated MAb of the
MH21-134 hybridoma. MH21-134 hybridoma was propagated in nu/
nu mice as ascites, and the antibody was purified from the ascites using
Sepharose 4B affinity gels to human IgG (Cappel, Malvern, PA). One
mg MH21-134 antibody/ml 0.1 M sodium acetate buffer (pH 5.5) was and nLcx (Fig. 2). These results indicate that the epitope of
oxidized at 0Cfor 20 min with 10 m.Msodium periodate. The reaction
was stopped by addition of glycerol (final concentration, 15 mM) and
left for 5 min at 0"C. The reaction mixture was then dialyzed overnight
at room temperature against 0.1 M sodium acetate (pH 5.5). Oxidized
immunoglobulin preparations were placed in plastic screw-capped tubes
containing finely powdered biotin hydrazide (Vectastatin) to a final
concentration of 10 mM, mixed, and reacted with shaking for 2 h at
room temperature. Biotinylated MH21-134 was then dialyzed in PBS
overnight at 4C.After washing in 5 mM Tris buffer, pH 7.4, for 20
min, the sections were treated for l h with the PBS that contained the
avidin-biotinylated peroxidase complex. After washing in 5 mM Tris
buffer for 30 min, the sections were treated with the substrate that
contained 3,3'-diaminobenzidine (Dotile, Tokyo, Japan) for 60 s, (Fig. 3, a and A), breast cancer (Fig. 4, a and b), and lung cancer
washed with water, and weakly counterstained with hematoxylin. Sec
5690
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REPEATING TYPE 2 CHAIN
tions that had been incubated with SP2 culture supernatant served as a
negative control. Avidin and biotin were purchased from Vector (Vec
tastatin, Burlingame. CA).
Fluorocytometry was performed using fluorescein isothiocyanate-
conjugated anti-human IgG (goat, Tago, Burlingame, CA) in PICS
Profile (Coulter). The control value was that without the primary
antibody.
Antibody-dependent Cytotoxicity Assay and Complement-dependent
Cytotoxicity Assay. ADCC was studied by a 4-h chromium release assay
as previously described (27). Peripheral blood lymphocytes from a
healthy donor were separated by Ficoll Paque (Pharmacia, Piscataway,
NJ). The effectortarget cell ratios were 100:1, 50:1, and 25:1. HL-60
cells were labeled for 2 h with 100 ^Ci of MCr in RPMI 1640 medium
supplemented with 3% FCS at 37Cin a CO2 incubator and washed
three times and resuspended in medium. MCr-labeled HL-60 cells were
placed in 96-well round-bottom plates (Costar, Cambridge, MA) at a
density of 5 x 10' cells/well, and various numbers of effector cells (5 x
10', 2.5 x 10', and 1.25 x IO5) were added per well, followed by
addition of various concentrations of MH21-134 (50, 5, 0.5, and 0.05
Mg/ml) in RPMI 1640 supplemented with 20% FCS. For direct effector
cell lysis and total release by addition of 2% Triton X-100, medium
alone was added to some wells. The plate was then centrifuged at 400
x g for 3 min and incubated for 16 h at 37Cin a CO: incubator. After
2% Triton X-100 was added for total release wells, the plate was
centrifuged at 700 x g for 5 min, and radioactivity was measured in an
80-fil aliquot of each supernatant using a gamma counter. Spontaneous
MCr release was determined in wells that contained labeled HL-60 cells
and effector cells. There were three replicates per group. The % cytolysis
was calculated as follows:
Experimental release - spontaneous release
% Cytolysis = x 100
Total release - spontaneous release
Complement-dependent Cytotoxicity was studied in HL-60 cells using
rabbit complement with MCr release assay. The difference between
control (without complement) at 4 and 24 h was determined.
RESULTS
Specificity of MAb MH21-134. Among various hybridomas
secreting MAbs reacting with PC-9 and KATO III, stable clones
that could be propagated in nu/nu mice were selected. One of
these seven MAbs detected neutral glycolipids of KATO III,
did react with those treated with sialidase. When the specificity
munoassay using various standard glycolipids as antigens, a
significant reaction was obtained with nLc6 and nLcx, and no
reactivity was observed with LacCer, Gbj (CTH), Ggj (asialo
CM,), Lc4 (PG), Gb4 (globoside), SPG, Forssman, sialosyl-
nLc,,, H2, Hj, G,o, and lactoisooctaosylceramide (I antigen)
(Fig. 1, a and b). This specificity was confirmed by TLC
immunostaining, in which MH21-134 reacted only with nLc,,
MH21-134 is GaI/31-4GlcNAc/313Gal/314GlcNAc01->
3Gal/31R. The specificity of this MAb and the structures of
the glycolipids tested in this study are summarized in Table 1.
In the immunodiffusion test using sheep anti-human Igd,
IgG:, IgG,, and IgG., (ICN, Lisle, IL), MH21-134 was reactive
only with IgG, (data not shown).
TLC Immunostaining of Neutral Glycolipids Extracted from
Various Cancer Tissues by MH21-134. TLC immunostaining
of neutral glycolipids extracted from various cancer tissues by
MH21-134 indicated that nLc,, and nLcantigens are present
in the glycolipids extracted from some cases of colon cancer
(Fig. 4, c and d). As summarized in Table 2, the positive
 HUMAN IgGj MAb DIRECTED TO AN UNBRANCHED
20r
8
10
S
100 25 613 153 038 0045 4 16 64 256 1024
I/Antibody Dilution
Antigen Dilution (nq/well)
Fig. 1. Specificities of MAb MH21-I34 towards various kinds of glycolipid
antigens as ascertained by solid-phase enzymoimmunoassay. A, reactivity of
MH21-134 (5 Mg/ml) with various concentrations of glycolipid antigens; B,
reactivity of MH21-134 with glycolipid antigens (20 ng/well) at various concen
trations of MH21-134; initial concentration of MH21-134 (10 jg/ml)., nLc,,:
O. nLc;T, G6 (sialosyl nLc6): A. Hz; D. paragloboside; A. lacto-isooctaosylcer-
amidc (I antigen); H,: V. G,0.
a. b.
Orcinol-H2SC>4 Staining Immunostoining
2345 2345
Fig. 2. Specificities of MAb MH21-I34 towards various kinds of glycolipid
antigens as ascertained by TLC immunostaining. A, TLC stained with orcinol-
H2SO4; B, TLC immunostained with MH21-134. Lane I, the neutral glycolipid
mixture extracted from human type O erythrocytes serving as a control [lop to
bottom: LacCer. Gb3. Gb4 (major band). nLc4. H,, H2. H,|; lane 2, from top to
bottom. CDH. PG. nLc:lane 3. nLc6: lane 4. SPG. sialosyl-nLiv lane 5,
lactoisooctaosylceramide (I antigen). All quantitites = 1 <*g.
occurrence of nLc,, and nLcwas highest in colon cancer
(71.4%), followed by breast cancer (66.7%), and lung cancer
(50%).
In contrast, nLc6 and nLc* antigens were absent in extract of
normal colonie epithelia, and in extracts of normal pancreas
and normal kidney. A weak band corresponding to nLcwas
detected in liver extract, and nLc6 was found in spleen extract,
although these tissues showed negative immunohistology stain
ing with MAb MH21-134 (see below). A large quantity of both
antigens was detected in placenta extract, in agreement with
previous findings (21).
Immunohistochemical Examination of Various Kinds of Can
cers by MH21-134. Colon cancer and hepatocellular carcinoma
showed the highest incidence of expression of nLc6 and nLc*
(Fig. 5, b and d), followed by large cell carcinoma of the lung
(Fig. 5a), and cancer of the esophagus (Fig. 5c). These results
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REPEATING TYPE 2 CHAIN
are summarized in Table 3. The degree of incidence obtained
by immunohistochemical examination was lower than that ob
tained by TLC immunostaining. No staining was observed of
normal esophagus, stomach, colon, liver, or kidney, except in
the bronchial glands of lung and pancreatic duct (Table 3). In
cytofluorometric analysis, lymphocytes were negative, while
granulocytes showed strong positive reactivity (Table 3).
Antibody-dependent Cellular Cytotoxicity and Complement-
dependent Cytotoxicity. No differences were observed of 5'Cr
release between direct cell lysis with MH21-134 alone and the
negative control without MH21-134 and effector cells. There
fore, this antibody has no cytolytic activity by itself. Antibody-
dependent cellular Cytotoxicity was clearly demonstrated by a
lysis of target cells HL-60 at a high concentration of MH21-
134 and high effectontarget ratio (Fig. 6). The highest %
cytolysis (68%) was reached at a concentration of 50 fig/ml of
MH21-134 and an effectontarget ratio of 100. In contrast, the
antibody did not show cytotoxic effect on HL-60 cells in the
presence of rabbit complement for 4- and 24-h durations.
Comparative Reactivity of MAb MH21-134 and Classical
Anti-i Dench Antibody. Since MAb MH21-134 reacts with
unbranched repeating jV-acetyllactosamines nLc6 and nLc,
similarly to classically known anti-i IgM present in sera of
Waldenstrm's macroglobulinemia, the reactivities between
these proteins were compared. MAb MH21-134 reacted equally
well at 4Cand 37C,in contrast to anti-i Dench, which reacts
with both sialyl 23norhexaosyl and norhexaosyl structures
(28), and reacts only at low temperature (e.g., 4C)(see Fig.
71). MAb MH21-134 did not agglutinate umbilical cord eryth-
rocytes regardless of temperature, because it was IgG> It did
not bind to umbilical cord erythrocytes nor adult erythrocytes,
but did bind strongly to the former after sialidase treatment
(data not shown). A weak agglutination (titer 1:4) was observed
by Coombs' test with goat anti-human immunoglobulin at both
4 and 37C.On the other hand, anti-i Dench agglutinated such
erythrocytes strongly at 4C(titer 1:25,600) or at room tem
perature (titer 1:100) but did not agglutinate them at 37C(see
Fig. III).
DISCUSSION
The significance of the human MAb approach is several-fold.
One important aspect is the ability to dissect specific host
immune responses, particularly the B-cell repertoire, to various
types of human cancer. A second significant use of human
MAbs is to determine the exact epitope structure of human
cancer that is capable of inducing human B-cell response. This
knowledge will be important to develop effective vaccines
against human cancer. Third, the MAbs themselves will be
valuable reagents for imaging of human tumors and suppression
of tumor cell growth, in cases where antigen expression is highly
limited to human cancer. This third possibility has been re
stricted in its application because essentially all known human
MAbs detecting glycolipids on cell surface membranes are of
IgM class and have low antigen-binding affinity, particularly at
37C.
Despite major technical problems in establishing stable
clones that efficiently secrete human MAbs, a number of im
mortal clones secreting human antibodies directed to tumor-
associated antigens have been established (3-12). Studies have
mainly focused on (a) EBV-transformed B-cell clones from
cancer patients, (b) lymphocytes of cancer patients fused with
HAT-sensitive mouse myeloma cells, or (c) a combination of
these two approaches. Attempts to establish human/human
5691
 HUMAN IgGj MAb DIRECTED TO AN UNBRANCHED REPEATING TYPE 2 CHAIN

Table l Structures of various glycosphingolipid antigens used in this study, and their reactivities with human MAb MH2I-34

Name Structure ELISA" TLC*

CTH Gall-> 4Gal/3I -4Glcffl ICer
Globoside GalNAc/31 3Gall-4Gal/31 4Glc(il -> ICer
PC Gal/i I
4GlcN Ac/313Galtfl
4G le/il
ICer
SPG NeuAc2-3Galf)l -> 4GlcN Ac/31-> 3GaI|31 -4Glcpfl -. ICer
H, Fucal -> 2Gal/31 -4GlcNAc/31 3Gal/31 -> 4Glc/31 ICer
nLc6 Gal/31
4GlcNAc01 3Gal/31 -. 4GlcNAc/31 3Gal/31 -. 4Glcf31 ICer
G6 NeuAc2 3Gal/3l -4GlcNAc/31 -> 3Galf)l -> 4GlcNAc/31 -SGalffl 4Glcfil ICer
Hi Fucal -> 2Gal/31 4GlcNAc/31
3Gal/31 4GlcNAcf)l
3Gal/31 -. 4Glcf(l 1 Cer
nLc, Gali31 -. 4GlcNAc/31 3Gal/31
4GlcNAc--l
-3Gal/3l -. 4GlcNAc/31 3Galtfl
4Glc/31 -. ICer
lactoisooclaosylceramide Gal/31
4GlcNAc/31
(I antigen)
\
3Gal/31 -> 4GlcNAcffl
3Galrfl
4Glc/31 -ICer
,6
Gal/31 -.4GlcN Ac/31
H, Fucnl -2Gali)l -. 4GlcN Ac/11

Gal/31 -. 4GlcNAc-l 4Glcfil

ICer

Fucnl -. 2Gal/3l
G, NeuAc2-. 3Gal/3l
4GlcNAcf(l
"Gal/31 ^4GlcNAc01 -~ 3Gal/31
4Glc/31
ICer

Fucal -2Gal/3l -. 4GlcNAcf(l

<. NeuAc2-. 3GalM -. 4GlcNAc/ll
\
"Gal/31 -4GlcNAc-l
-3Galf)l
4Glc01 -> ICer

NeuAca2 -. 3Gal;31 4GlcNAc/3l

1ELISA (enzyme-linked immunosorbent assay), solid-phase antibody binding; * TLC, immunostaining.

O. Orcinol-H2S04 Staining loma cells resulted in stable mouse/human hybridomas directed

- =
234567 9 10 11 12 13 14 15
b. Immunostaining
nl_C6-
1 234 56789 10 12 13 14 15
Fig. 3. Presence of nLc6 and nLcantigens in the neutral glycolipids extracted
from colon cancer tissues. A, TLC stained with orcinol-H;SO4; B, TLC immu-
nostained with the MAb MH2I-134. Lane 1, the neutral glycolipid mixture
extracted from human type O erythrocytes serving as control; lanes 2-15, the
neutral glycolipid mixtures extracted from the cancer tissues of 14 patients with
colon cancer. The amount of the extract applied in each lane corresponds to the
glycolipids prepared from 100 mg (wet weight) of cancer tissue.
hybridomas based on HAT-sensitive human myeloma cells or
B-lymphoblastoid cells have so far resulted in only limited
success (11, 12). Many successfully established human MAbs
are directed to various types of gangliosides and recognize
carbohydrate residues including combinations of various sialic
acids (3-6, 29-32). Regional lymph node lymphocytes derived
from human lung cancer fused with HAT-sensitive mouse mye
5692
to sialyllactonorhexaosyl or sialyllactonoroctaosyl structure (8).
The same approach applied to lymphocytes from noncancer
patients previously allowed the establishment of stable hetero-
hybridoma clones secreting antibodies to Forssman antigen (33)
and to sialyl-i antigen (34) (see also below). All these antibodies
have been identified as IgM, and no immortal clones or hybrid
omas secreting IgGi antibodies with reasonable affinity have
been reported.
In all these studies, including the present work, it is uncertain
whether the tumors had anything to do with eliciting the
antibody response. The immunogenic stimulus could be inci
dental from normal antigens or could result simply from im
mortalized preexisting B-cells capable of secreting anticarbo-
hydrate antibodies. A control experiment based on normal
peripheral lymphocytes or normal lymph node lymphocytes
may also be necessary. Human/mouse hybridomas secreting
human anti-Forssman antibodies (33) and those secreting hu
man antibodies directed to sialyl polylactosamine (sialyl-i) (34)
were previously reported based on lymphocytes derived from
noncancer patients. EBV-transformed peripheral B-lympho-
cytes from a patient with an autoimmune disease (Hashimoto's
thyroiditis) secreted an antibody directed to sialyl Le\ a typical
tumor-associated antigen.4 Thus, a question still remains
whether the establishment of a human hybridoma secreting
human MAb directed to type 2 polylactosamine indeed reflects
host immune response. Nevertheless, blotting of glycolipids
extracted from various tumor tissues separated on TLC clearly
indicated that the unbranched type 2 chain antigens reacting
with this antibody are indeed accumulated in various types of
colonie and hepatocellular cancer. This glycolipid antigen was
absent in normal colonie epithelia, kidney, and pancreas, and
its expression was highly limited in various other human tissues
and cells, except placenta and granulocytes. Immunohistology
of 154 cases of human cancers clearly indicated that these
4 C. Berglund. I. Bernstein, E. Nudelman. and S. Hakomori. unpublished
observation.

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 HUMAN IgGj MAb DIRECTED TO AN UNBRANCHED REPEATING TYPE 2 CHAIN

O. Oronoi-HgSQ Staining b. Immunostainiminq

Fig. 4. Presence of nLc6 and nLc8 antigens nl_c6-

in the neutral glycolipids extracted from cancer
tissues of patients with breast cancer (A and B) nl_c8- nl_c8
and lung cancer (C and D). A and C, TLC
stained with orcinol-H2SO4; B and D. TLC
immunostained with the MAb MH21-134.
Lanes 1 and 8, the neutral glycolipid mixture 2 3 5 6 1234567
extracted from human type O erythrocytes
serving as a control; lanes 2-7, the neutral
glycolipid mixture extracted from the cancer
tissues of six patients with breast cancer: lanes C. Ordnol-H2SC>4 Staining
9-16. the neutral glycolipid mixture extracted u. Immunostaining
from the cancer tissues of eight patients with
lung cancer. The amount of the extract applied
in each lane corresponds to the glycolipids
prepared from 100 mg (wet weight) of cancer
tissues.

nl_c8~ nl_c8

8 9 10 11 12 13 14 15 16 8 9 10 11 12 13 14 15 16

Table 2 Results of TLC immunoslaining study of neutral glycosphingolipid 2 chain. It is important to note that synthesis of unbranched
fractions from various kinds of cancers using MH2I-134
type 2 chain is greatly enhanced due to enhanced l
nLc71%
and/or
3GlcNAc transferase, although \ 3 and l
4 Gal trans-
Colon cancer (8/14) (8/1 4) (10/14)
Lung cancer 50% (4/8) 38% (3/8) 50% (4/8)
ferases are essentially unchanged in colonie tumors (35). Per
Breast cancernLc,57%50% (3/6)nLc,57%67% (4/6)nLc,, 67% (4/6) haps the 01
oGlcNAc transferase, which is responsible for
synthesis of branched type 2 chain, could be competitively
suppressed by the predominance of the l 3GlcNAc trans
antigens are highly expressed in and associated with a variety ferase. The presence of the unbranched, unsubstituted type 2
of human cancers. Surprisingly, staining of normal tissues with chain not only in fetal erythrocytes but also in other fetal tissues
MH21-134 was very limited (Table 3) as compared with stain is assumed, since the structure is known to be reactive with one
ing with antibody NCC-LU-1004, which is also directed to class of anti-i antibodies, which is known to be oncodevelop-
unbranched type 2 chain but reacts strongly with the sialylated mentally regulated (36, 37). In our previous studies, i antigen
derivative (7). Differences in tissue stainability with the anti was detectable in semipurified glycoprotein prepared from six
bodies MH21-134 and NCC-LU-1004 seem to be due to the of eight cases of colonie cancer extracts but was undetectable
lack of reaction of MH21-134 with sialylated unbranched type in the same glycoprotein fraction prepared from five of eight

^'tv r
Fig. 5. Immunohistochemical staining us
ing the MAb MH2I-134. A, large cell carci
v
-- .
noma of the lung; B. well-differentiated ade-
nocarcinoma of the colon; C. poorly differen
tiated squamous cell carcinoma of the
esophagus: D. hepatocellular carcinoma, orig
inal magnification x 160. .

5693

Downloaded from cancerres.aacrjournals.org on June 28, 2017. 1989 American Association for Cancer Research.
 HUMAN IgG, MAb DIRECTED TO AN UNBRANCHF.DREPEATING TYPE 2 CHAIN
Table 3 Results of immunohistochemical study of various types of cancers, and normal tissues, using MAb MH2I-I34
tissuesLung
Cancer rate44.4% tissues"Brain rate0',
cancer
Adcnocarcinoma (12/27)
Squamous cell carcinoma 53.8% (14/26) Thyroid gland 0% (0/2)
Large cell carcinoma 58.8% (10/1 7)
carcinomaEsophagus
Small cell (2/11)53.3%
18.2% EsophagusLiverKidneyAdrenal 0%0%0%0%100%
(0/2)(0/2)(0/2)(0/1)(2/2)

cancerGastric (8/15)38.9%

cancerColon 8)64.7%(7/1

cancerHcpatocellular (11/17)66.7% glandPancreas

cancerPositive (8/1 2)Normal

Duct epithelia
Other areasPositive 0%(0/1)
(0/2)

Breast cancer 45.5% (5/11)

Lung
Bronchial gland epithelia 100% (2/2)
Other areas 0% (0/2)
Normal cells*
Lymphocytes 1.1% (1.3%)
Ciranulocytes 86.1% (5.1%)
" Cause of death in normal subjects was automobile accident.
* Analyzed with fluorescence-activated cell sorter; positive cell population expressed as percentage: numbers in parentheses, percentage positive cells in control
without antibody.

75r
I A. B.
MH21-134 antibody Anti-i(Dench) antibody

20
50 100:1

25 o
i? 1.0

5O 5 0.5 O.O5
Antibody Concentrotion (ug/ml)
Fig. 6. Antibody-dependent cytotoxicity of HL-60 cells by MAb MH2I-134. 5 6 1 23456
Percentage target cell lysis is indicated on the abscissa. The three lines represent
different effectortarget cell ratios. II
It-Dip

cases of normal adult colonie mucosa (38). Thus, association dirt-el

;i4-C25-C37-C('odili

of i antigen with various human cancers has been well estab

lished. MAb MH21-134, which does not react with sialyl-i (in Its'4-C37-C'lulin-itiiinn.a.n.a.n.a.H-sln.a.n.a.n.a.n.a.n.a.1:21:41:2,5001:100UM,1:204,811(11:1,4011n.a.

contrast to the majority of anti-i antibodies), is obviously a

useful reagent to use as a probe for tumor-associated i antigen.
On the other hand, the fact that i antigen is highly expressed
in granulocytes represents the major drawback to practical
application of this antibody in suppression of tumor growth in
vivo. However, the antibody could still be useful in probing
tumors and possibly in delivery of noncytotoxic metabolic mod
ulators or differentiation inducers (39).
Fig. 7. Comparative reactivity of MAb MH2l-134and classical anti-i antibody
(myeloma protein Dench): Dependence on temperature. /, antibody binding to
solid-phase antigen (nLcJ at 37 and 4'C. A, reactivity with MH2I-I34 antibody;
B. reactivity with anti-i (Dench) antibody. Numbers in abscissa, appropriate double
dilution starting with undiluted culture supernatant in I and lOOx diluted scrum
in li. Il, cord cry throcyte hemagglutination tiler. Sialidase. sialidase-treated cells;
n.a., no agglutination of undiluted hybridoma supernatant or 1:100 diluted anli-i
Dench serum, which reacts with both sialyl 23 norhexaosyl and norhexaosyl
structures (28).

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Human IgG3 Monoclonal Antibody Directed to an Unbranched
Repeating Type 2 Chain (Gal 14GlcNAc13Gal14GlcNAc
13Gal1R) Which Is Highly Expressed in Colonic and
Hepatocellular Carcinoma
Masayuki Miyake, Nobuoki Kohno, Edward D. Nudelman, et al.

Cancer Res 1989;49:5689-5695.

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