Vo1.187, No. 1,1992
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 404-412
August 31,1992
PRODUCTION AND PROPERTIES OF NOVEL HUMAN THYROID CANCER
SPECIFIC MONOCLONALANTIBODIES*
Hideo Sugawa, Masumi Miyamoto 1, Kayoko H i g u ch i 2, E r n e s t Smith, Toru Mori and H i r o o Imura 1
iDepartment
of Laboratory Medicine, Second Division of Internal Medicine,
and 2Department of
Anatomic Pathology ,
Kyoto University
Faculty of Medicine, 54-Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606, Japan
Received July 8, 1992
Monoclonal a n t i b o d i e s (TCM-7, -9 and -12) against human thyroid differentiated c a n c e r s were e s t a b l i s h e d by s c r e e n i n g w i t h human t h y r o i d c a n c e r s , normal and b e n i g n t h y r o i d t i s s u e , and normal human serum p r o t e i n . A m o n o e l o n a l a n t i b o d y (TCM-9) w i t h s t r o n g s p e c i f i c i t y f o r human t h y r o i d c a n c e r b u t n o t f o r G r a v e s ' d i s e a s e , adenoma or normal t h y r o i d , was shown t o r e c o g n i z e a 300 K p r o t e i n but not t o b i n d to n a t i v e or m a t u r e human thyroglobulin. W h e n TCM-9 was used in i m m u n o h i s t o c h e m i c a l s t a i n i n g tests on more than 30 t y p e s of n o n - t h y r o i d l e s i o n s , no r e a c t i v i t y o f TCM-9 was o b s e r v e d e x c e p t w i t h s k i n immature t e r a t o m a , l i p squamous c a r c i n o m a and stomach a d e n o c a r c i n o m a , which r e v e a l e d weak r e a c t i v i t i e s . TCM-9 also showed s t r o n g r e a c t i v i t y w i t h two u n d i f f e r e n t i a t e d thyroid cancer cell lines and one t i s s u e s p e c im e n . Thus TCM-9 i s a n o v e l m o n o c l o n a l a n t i b o d y against the thyroid cancer. ©1992Ao~d~miopress,~no.
Current palpation,
diagnosis
various
tomography,
of
differentiated thyroid
c a n c e r relies
on
imagingtechniques (ultrasonography, computed X-ray
radioisotope scintigraphy and/or magnetic resonance imaging)
and detection by needle or aspiration biopsy. Althoughthese methods have greatly improved the accuracy of diagnosis, i t is s t i l l quite d i f f i c u l t distinguish f o l l i c u l a r cancer from benign follicular adenoma. After development
of
hybridomatechnology, a variety of
to the
monoclonal antibodies
(MoAbs) are established and found to have wide applications including the diagnosis
of various cancers (1-3).
Our aim in this study was to
produce
MoAbs that were capable of distinguishing human thyroid cancer (especially f o l l i c u l a r cancer) from other benign thyroid nodules.
* This work was p a r t i a l l y s u p p o r t e d by g r a n t s in a i d from t h e M i n i s t r y of E d u c a t i o n (No. 62480252 and 63614512), t h e Japan s o c i e t y f o r t h e p r o m o t i o n of s c i e n c e for Japanese J u n i o r S c i e n t i s t s , t h e Mochida memorial f o u n d a t i o n f o r m e d i c a l and p h a r m a c e u t i c a l r e s e a r c h and f o u n d a t i o n o f Osaka Cancer Research Foundation. ooo6-291x/92 $4.00 Copyright © 1992 by Academic Press, lnc. All rights of reproduction in any form reserved.
404
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS 1. Membrane p r e p a r a t i o n V a r i o u s t h y r o i d t i s s u e s were o b t a i n e d a t s u r g e r y , homogenized i n 0.25M sucrose, 10 mM T r i s - H C 1 , pH 7 . 6 , 4 mM MgC12, t h e n gauze f i l t e r e d , and c e n t r i f u g e d a t 180 x g f o r 5 min a t 4°C. The s u p e r n a t a n t was r e c e n t r i f u g e d at 15,000 x g f o r 30 min and t h e p r e c i p i t a n t was suspended i n p h o s p h a t e b u f f e r e d s a l i n e and s t o r e d a t -80°C u n t i l u s e . 2. C e l l s Mouse myeloma c e l l s X63-Ag8.653 and human embryonal l u n g f i b r o b l a s t c e l l s (HEL) were p r o v i d e d by P r o f . T s u y o s h i Uchida ( I n s t i t u t e f o r M o l e c u l a r and C e l l u l a r B i o l o g y , Osaka U n i v e r s i t y ) . The human u n d i f f e r e n t i a t e d t h y r o i d c a n c e r c e l l l i n e (HTC/C3) was i s o l a t e d i n our l a b o r a t o r y ( 4 ) . O t h er human c a n c e r c e l l s of g a s t r i c cancer (AZ-251), human u n d i f f e r e n t i a t e d t h y r o i d c a n c e r ( 5 - C - 0 ) , r e n a l c e l l c a n c e r ( T h o m a s - W i r t s ) , and f i b r o s a r c o m a (HTI080) were p r o v i d e d by t h e J a p a n e s e Cancer R e s e a r c h R e s o u r c e s Bank. 3. T h y r o g l o b u l i n p r e p a r a t i o n Human t h y r o g l o b u l i n (hTg) was p r e p a r e d a c c o r d i n g t o t h e method d e s c r i b e d by B i l s t a d e t a l . (5) w i t h minor m o d i f i c a t i o n s . Human t h y r o i d t i s s u e was homogenized, and t h e n c e n t r i f u g e d a t 15,000 x g f o r 30 min. The supernatant was p r e c i p i t a t e d w i t h 1 . 4 - 1 . 8 M ammonium s u l f a t e and t h e precipitant was d i s s o l v e d in 2 x c o n den sed PBS (2 X PBS) and d i a l y z e d . Then t h e sample was a p p l i e d t o HPLC G3OOOSW column (Tosoh, J a p a n ) , and t h e v o i d f r a c t i o n was c o l l e c t e d and s t o r e d a t -80°C u n t i l u s e . 4. P r o d u c t i o n of hybridomas S p l e e n c e l l s were p r e p a r e d from B a l b / c mice immunized w i t h membrane f r a c t i o n s of the d i f f e r e n t i a t e d t h y r o i d c a n c e r , and f u s e d w i t h X63-Ag8.653 as d e s c r i b e d p r e v i o u s l y (6). ELISA p l a t e s were c o a t e d w i t h p u r i f i e d membranes' preparations from 25 c a n c e r s (17 p a p i l l a r y c a n c e r and 8 follicular cancer), 6 adenoma, 6 Graves' tissues and 5 normal human t h y r o i d s ; g u i n e a p i g f a t c e l l membrane, normal human serum p r o t e i n and hTg preparations and used f o r s c r e e n i n g . The c l a s s and s u b c l a s s o f each MoAb were i d e n t i f i e d by an i s o t y p i n g k i t f o r mouse m o n o e l o n a l a n t i b o d i e s ( S e r o t e c , Ox f o r d . U.K). 5. I m m u n o h i s t o c h e m i c a l s t a i n i n g I m m u n o h i s t o c h e m i c a l a n a l y s i s was p e r f o r m e d f o l l o w i n g t h e methods of De Micco e t al.(7). T i s s u e s e c t i o n s were i n c u b a t e d w i t h MoAb a t a 1:100 dilution of ascites with normal mouse serum used as a control, and ABealkaline phosphatase staining (Vector laboratories) was performed.
6.Thyroglobulin specificity of MoAbs Specificity of MoAb against thyroglobulin was evaluated by: i) antithyroglobulin and anti-thyroid microsomal antibody measurement using a p a s s i v ~ g g l u t i n a t i o nkit (Fujirebio, Tokyo, Japan), 2) Immunoprecipitation with Iz~I-labelled human thyroglobulin (Midorijuji, Osaka, Japan) by Affigel Protein A (Bio-Rad), and 3) absorption of standard thyroglobulin preparations in an IRMAkit (Midorijuji, Chiba, Japan). In the IRMA kit, various concentrations of standard hTg were incubated with or without monoclonal antibodies or controls in anti-hTg MoAb coated t u b e Then the solutions were removed and 1~Ol-MoAb directed against another hTg epitope was added, then the bound radioactivities were measured. RESULTS 1. I m m u n o h i s t o c h e m i c a l s t a i n i n g
of t h y r o i d g l a n d
E x t e n s i v e c l o n i n g was p e r f o r m e d u s i n g membranes from 25 t h y r o i d c a n c e r s , 6
adenoma,
clones
6
(TCM-7,
Graves' tissues
and 5 normal t h y r o i d s ,
-9 and -12) t h a t p r o d u c e d MoAbs w i t h 405
and
finally
reactivity
three against
Vol. 187, No. 1, 1992
thyroid
cancer
selected IgG2b. (Table
Further la).
level TCM-9
reacted
screened.
serum
protein)
follicular or not
cancer
adenoma
in colloid
staining
strongly TCM-7
tissue,
tissue.
was p e r f o r m e d
with
reacted
papillary The
nor interstitial
was c o n s i s t e n t l y
cancer,
staining tissues
seen in tests
with
was
HTC/C3;
as
and
be
described
strongly
with
exclusively
(Fig.l).
cancer
to
hut did not react
w i t h TCM-9.
the undifferentiated weakly
were
A l l 3 MoAbs w e r e i d e n t i f i e d
The MoAbs TCM-7, - 9 a n d - 1 2 w e r e f o u n d t o r e a c t
of binding
5-C-0;
58 c l o n e s
immunohistochemical
Graves'
cytoplasma,
and
m e m b r a n e ( b u t n o t a d e n o m a a n d human
from the
human t h y r o i d with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cell
The The
highest antibody
lines
TCM-12
in
HTC/C3
bound
Fig. 1. Immunohistochemical staining of t h y r o i d t i s s u e s e c t i o n s by TCM-9. 1, f o l l i c u l a r c a n c e r ; 2, p a p i l l a r y c a n c e r ; 3, G r a v e s ' d i s e a s e ; 4, adenoma; and 5, u n d i f f e r e n t i a t e d t h y r o i d c a n c e r c e l l l i n e HTC/C3. 406
to
VoI. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig.
i
-
Continued
407
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T a b l e 1. a) To t h y r o i d
lesions* Number o f positive staining
Thyroid disease Follicular
cancer
Papillary
disease
Adenoma
b) To o t h e r cell
line
+ +
+++
+
+ +
±
1
+
0
-
0
-
carcinoma cell
Reactivity TCM-9 TCM-12
TCM-7
2
cancer
Graves'
Immunohistochemical staining
NMS
lines**
origin
TCM-7
AZ521
gastric
.
5-C-0
thyroid
-
÷ +
-
-
Thomas-Wirts
renal
±
±
±
±
HTC/C3(ll)
thyroid
±
-
-
OG-90
lung
.
TCM-9 .
TCM-12
.
.
÷ ÷ .
NMS
.
.
Normal mouse s e r u m (NMS) was s i m u l t a n e o u s l y u s e d a s a c o n t r o l . *: Two human t h y r o i d f o l l i c u l a r c a n c e r s , one p a p i l l a r y c a n c e r , one G r a v e s ' t i s s u e a n d one adenoma were t e s t e d . * * : TCM-9 shows c r o s s - r e a c t i v i t y with undifferentiated thyroid cancer, but not with gastric and lung cancer cells.
neither(Table reduced
lb).
by
The reactivity
100% m e t h a n o l
temperature
for
30 m i n ( d a t a
m o u s e MoAb a g a i n s t result
2.
o f MoAbs w i t h
All reacted react the
the
with
with
gastric
non-thyroidal
they
were
teratoma
and
In reacted
skin
with
Reactivity
with
with
not
atrial
other
cell
lung
cancer did
cell
not
or malignant. squamous
react
cell
serum
mouse
TCM-9, of
carcinoma
nor
a
gave
tissues.
serum
regardless
stomach
did,
control
They did
As s h o w n i n T a b l e
with
room
(a-ANP)(6)
(Thomas-Wirts).
lines.
Some c a s e s
mouse
not
at
and non-thyroida]
normal
line
was
tested.
lines
and the cell
peptide
cells
cancer
treatment
normal
natriuretic
cancer
cancer
50mM p e r i o d a t e Neither
cancer
(TCM-7,-9,-12)
a renal
or
shown).
thyroid
Tg and thyroid
300 K under
shown).
microsomal
and electroblotting
TCM-9 a n d g a v e
TCM-9 w a s s t r o n g e r (data
not
thyroid
2,
however,
most
of
cancer,
not of
whether immature
exhibit
weak
TCM-9.
SDS-PAGE
approximately
the
tissues
benign
reactivity 3.
with
3 HoAbs
weakly
30 m i n o r
human alpha
a positive Binding
for
o f MoAbs w i t h
than
a single reducing
that
The lack
of of
antigen
assays, band
with
conditions
TCM-7, w h i c h positive 408
follicular relative (Fig.2).
cancer molecular
weight
The reactivity
showed a similar
immunostaining
membrane
of
of of
band
at
300 K
the
colloid,
Vol. 187, No. 1, 1992
Table 2.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R e a c t i o n of TCM-9 w i t h v a r i o u s b e n i g n and m a l i g n a n t l e s i o n s
Malignant l e s i o n s
Benign l e s i o n s
Uterine cervix Squamous c e l l ca. case1 case2
c e r v i c a l polyp
Ovary s e r o u s c y s t adenocarcinoma adenocarcinoma
+
Liver h e p a t o c e l l u l a r ca. e a s e l case2
cirrhosis
Soft tissue s y n o v i a l sarcoma immature t e r a t o m a epidermis serous epithelium sweat gland
neurofibroma easel case2 neurinoma e a s e l case2 case3 g r a n u l a r c e l l tumor angiolipoma hemangioma pyogenic granuloma leiomyoma dermatofibroma desmoid tumor
÷ ÷ ÷
Bone osteochondroma fibrous dysplasia
+?
Stomach adenocarcinoma e a s e l case2 case3
÷ ±
ATP inflammatory polyp
Large i n t e s t i n e adenocarcinoma case1 case2
£
adenoma e a s e l case2
Pituitary
gland adenoma
Skin squamous c e l l c a . ( l i p ) m a l i g n a n t melanoma Prostate adenocarcinoma Lymph node non Hodgkin's lymphoma
normal lymph node
Kidney r e n a l c e l l ca. Nasopharynx n a s o p h a r y n g e a l ca. e a s e l case2 The bone f i b r o u s d y s p l a s i a specimen c o n t a i n e d abundant c a l c i f i c a t i o n .
membrane these
preparation
MoAbs
likely
of Graves'
tissue,
do n o t b i n d t o hTg, o r a t
T h i s was f u r t h e r
c o n f i r m e d by s e v e r a l
not
latex
aggregate
antigen(data
and p u r i f i e d
not shown}.
beads
least
methods.
coated
with
When 1 2 5 I - l a b e l e d 409
hTg
hTg to
its
Antibodies or
indicated native
TCM-7 a n d 9
thyroid
hTg was i n c u b a t e d
that form. did
microsomal w i t h TCM-7 o r
V o l . 187, N o . 1, 1992
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
7 6 660
K
>
E Q.
330 K ~ 200
5
X
4
~-
3
-o (-
2
K~
0 r~
116K
>
1
®
0
Q
TCM-9
3
3 ~0
1'0
0
100 '
300 '
1000 '
Tg ( n g / m l )
Fig. 2. Western b l o t t i n g with TCM-9. SDS-PAGE of a follicular carcinoma membrane r e a c t e d with TC~4-9 showing a s i n g l e band (300 K). Lines i n d i c a t e the p o s i t i o n of s t a n d a r d molecular markers; 19S t h y r o g l o b u l i n (660 K), reduced thyroglobulin (330 K), myosin (200 K) and E. coli /3g a l a c t o s i d a s e (116 K). Fig. 3. E f f e c t of TCM-9 on the b i n d i n g of hTg with a n t i - h T g a n t i b o d i e s . Addition of TCM-7 (open c i r c l e ) or TCM-9 (closed c i r c l e ) did not i n f l u e n c e the s t a n d a r d b i n d i n g curve when compared to the a d d i t i o n of c o n t r o l MoAbs (anti-human A•P antibody) (closed t r i a n g l e ) or b u f f e r (open triangle).
-9
and p r e c i p i t a t e d
precipitated
radioactivity
MoAb (a n e g a t i v e data
not
TCM-7, {Fig.
by A f f i - G e l
3).
recognize
The
or polyclonal
Further,
-9 and c o n t r o l
hTg a n t i b o d y
the standard
epitopes
two
on t h e hTg m o l e c u l e .
result
were
not immunostained with anti
above, lines
thyroid
TCM-9 reacted and
one
cancer specific
tissue
with
c u r v e s of hTg
IRMA
MoAbs,
that
no
which
lesion).
lines
Thus, TCM-9 r e c o g n i z e s
of
antibody occurred.
(HTC/C3
thyroid
curve
inhibition
human Tg { d a t a n o t s h o w n ) .
with the undifferentiated
epitope
control,
I f TCM-7 o r TCM-9 b o u n d
indicates
cancer cell
the
~-ANP
as t h e IRMA a n t i - h T g MoAbs, t h e s t a n d a r d
The a b s e n c e of t h i s
undifferentiated
in
preparations
anti-hTg
t h e b i n d i n g o f hTg w i t h t h e p r o v i d e d m o n o c l o n a l a n t i - h T g The
increase
(a p o s i t i v e
o f s t a n d a r d hTg
IRMA s y s t e m i s composed o f
different
shift.
incubation
MoAb d i d n o t a f f e c t
t o t h e same or p r o x i m a l s i t e would
A, no s i g n i f i c a n t
was o b s e r v e d when compared t o a n t i - h u m a n
control),
shown).
Protein
and As
cancer a
5-C-0)
described (two
novel
cell
thyroid
n o t a m o l e c u l e of t h y r o g l o b u l i n .
DISCUSSION Immunohistochemical MoAbs
revealed
that
all
studies three
of various stained 410
thyroid
tissues
differentiated
with
cancer
these ceils.
VoI. 187, No. 1, 1992
Recently, unique of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
there
have been many r e p o r t s i n d i c a t i n g t h a t c a n c e r
cells
i n t h a t t h e y have v a r i o u s a b n o r m a l i t i e s i n t h e c a r b o h y d r a t e
the s t r u c t u r e
and m e t h a n o l binding
of p r o t e i n s t h a t can be d e t e c t e d by MoAb ( 8 ) .
t r e a t m e n t s of the membrane p r e p a r a t i o n d i d
of TCM-9.
molecule.
Periodate affect
the the
component of t h e a n t i g e n
Many MoAbs produced by i m m u n i z a t i o n with p u r i f i e d hTg have
reported (9-14),
and a c e r t a i n amount of s t r u c t u r a l
cancer
tissues
or s e r a of p a t i e n t s have been i n d i c a t e d
These
previous
results
moiety
These o b s e r v a t i o n s i n d i c a t e t h a t TCM-9 r e c o g n i z e s
p c p t i d e p o r t i o n b u t n o t t h e c a r b o h y d r a t e nor l i p i d
not
are
been
h e t e r o g e n e i t y of Tg by
r e p o r t e d MoAbs l i k e l y r e a c t w i t h n a t i v e hTg,
these
MoAbs.
whereas
our
i n d i c a t e t h a t TCM-7, -9 and -12 do n o t r e a c t w i t h n a t i v e hTg
benign thyroid tissues. previously
recognizes
in
from
A l l the Tg s p e c i f i c m o n o c l o n a l a n t i b o d i e s r e p o r t e d t h e p e p t i d e p o r t i o n of the
(15) or a c o n f o r m a t i o n a l s t r u c t u r e
of Tg (11).
molecule
predominantly
The m o n o c l o n a l
antibodies
i n t h e s e s t u d i e s were s e l e c t e d to r e c o g n i z e n a t i v e Tg, a l t h o u g h some showed different patients
reactivities
a g a i n s t serum Tg i n t h y r o i d
with non-malignant thyroid diseases
patients
knowledge,
t h e r e i s no r e p o r t about t h e m o n o c l o n a l a n t i b o d y a g a i n s t thyroid
cancer.
cancer specific antigen.
Tg.
Thus, TCM-9 i s a It will potentially
Furthermore,
novel
and
antibody
selected
thyroid
to r e c o g n i z e n a t i v e
cancer
The m o n o c l o n a l
TCM-9 was
undifferentiated
not
(9).
to
our human
MoAb a g a i n s t
have a p p l i c a t i o n
in
future studies. ACKNOWLEDGMENTS The a u t h o r s wish to t h a n k Ms. Yuko Yamada and Ms. s e c r e t a r i a l and t e c h n i c a l a s s i s t a n c e .
Ikuko
Okamoto
for
REFERENCES 1.
2.
3.
4.
B l a s z c z y k , M, Pak, KY, H e r l y n , M, L i n d g r e n , J, P e s s a n o , S, S t e p l e w s k , Z and Koprowsk, H (1984) C h a r a c t e r i z a t i o n of G a s t r o i n t e s t i n a l Tumorassociated carcinoembryonic A n t i g e n - r e l a t e d Antigens Defined by Monoclonal A n t i b o d i e s . Cancer Res. 44, 245-253. Murray, J . L . , Rosenblum, M.G., Sobol, R . E . , Bartholomew, R.M., P l a g e r , C . E . , Haynie, T . P . , J a h n s , M.F., G l e n n , H, H . J . , Lamki, L . , Benjamin, R.S., P a p a d o p o u l o s , N., Boddie, A.W., F r i n c k e , J . M . , David, G . S . , Carlo, n.J. and Hersh, E.M. (1985) Radioimmunoimaging i n m a l i g n a n t melanoma w i t h l l l l n - l a b e l e d m o n o c l o n a l a n t i b o d y 9 6 . 5 . Cancer R e s . , 45, 2376-2381. S p i t l e r , L . E . , D e l - R i o , M., K h e n t i g a n , A., Wedel, N . I . , Brophy, N.A., Miller, L.L., n a r k o n e n , W.S., R o s e n d o r f , L . L . , Lee, H.M., Mischak, R . P . , Kawahata, R . T . , S t o u d e m i r e , J . B . , F r a d k i n , L . B . , B a u t i s t a , E.E. and Seannon, R.3. (1987) Therapy of p a t i e n t s w i t h m a l i g n a n t melanoma u s i n g a m o n o c l o n a l a n t i m e l a n o m a a n t i b o d y - r i c i n A c h a i n immunotoxin. Cancer Res. 47, 1717-1723. Enomoto, T, Sugawa, H, I n o u e , D, Miyamoto, M, Kosugi, S, T a k a h a s h i , T, Kitamura, N, Yamamoto, I , Konoshi, J, Mori, T and Imura H. (1990) E s t a b l i s h m e n t of a human u n d i f f e r e n t i a t e d thyroid cancer cell l i n e p r o d u c i n g s e v e r a l growth f a c t o r s and c y t o k i n e s . Cancer, 65, 1971-1979. 411
Vol. 187, No. 1, 1992
5. 6.
7. 8. 9. 10.
11.
12.
13. 14. 15.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bilstad, JM, Edelhoch, H, L i p p o l d t , R, R a l l , JE, and S a l v a t o r e , G (1972) I s o l a t i o n and C h a r a c t e r i z a t i o n of d i s c r e t e f r a g m e n t s of 27S t h y r o i d i o d o p r o t e i n . Arch. Bioehem. Biophys. 151, 341-150. Mukoyama, M, Nakao, K, Sugawa, H, M o r i i , N, Sugawara, A, Yamada, T, I t o h , H, Shiono, S, S a i t o , Y, A r a i , H, Mori, T, Yamada, H, Sano, Y and Imura H (1988) A m o n o c l o n a l a n t i b o d y to a - h u m a n a t r i a l natriuretic polypeptide. H y p e r t e n s i o n 12, 117-121. De Micco, C, Ruf, J, Carayon, P, C h r e s t i a n , M, Henry, J, and Toga, M (1987) Immunohistochemica] s t u d y of t h y r o g l o b u l i n in thyroid c a r c i n o m a s with m o n o c l o n a l a n t i b o d i e s . Cancer, 59, 471-476. Hakomori, S (1984) T u m o r - a s s o c i a t e d c a r b o h y d r a t e a n t i g e n s . Ann. Rev. Immunol. 2, 103-126. H e i l i g , B, Hufner, M, Dorken, B and Schmidt-Gayk, H (1986) I n c r e a s e d h e t e r o g e n e i t y of serum t h y r o g l o b u l i n i n t h y r o i d c a n c e r p a t i e n t s as d e t e r m i n e d by m o n o c l o n a l a n t i b o d i e s . K l i n . Wochenschr., 64, 776-780. Kohno, Y, T a r u t a n i , O, S a k a t a , S and Nakajima, H {1985) Monoclonal a n t i b o d i e s to t h y r o g l o b u l i n e l u c i d a t e d i f f e r e n c e s i n p r o t e i n s t r u c t u r e of t h y r o g l o b u l i n i n h e a l t h y i n d i v i d u a l s and t h o s e with p a p i l l a r y adenocarcimoma. J. C l i n . E n d o c r i n o l . Mctab., 61, 343-350. Shimojo, N, S a i t o , K, Kohno, Y, S a s a k i , N, T a r u t a n i , 0 and Nakajima, H (1988) A n t i g e n i c d e t e r m i n a n t s on t h y r o g l o b u l i n : Comparison of the r e a c t i v i t i e s of d i f f e r e n t t h y r o g l o b u l i n p r e p a r a t i o n s with serum a n t i b o d i e s and T c e l l s of p a t i e n t s with c h r o n i c t h y r o i d i t i s . J. C l i n . E n d o c r i n o l . Metab., 66, 698-695. S c h u l z , R, B e t h a e u s e r , H, Stempka, L, H e i l i g , B, Moll, A and H u e f n e r , M (1989) Evidence f o r i m m u n o l o g i c a l d i f f e r e n c e s between c i r c u l a t i n g and t h y r o i d t i s s u e - d e r i v e d t h y r o g l o b u l i n i n men. Eur. J. Clinic I n v e s t . 19, 459-463. Narkar, AA, Shar, DH, Swaroop, VD, Velumani, A, Dandekar, SR and Sharma, SM. (1988) Monoclona] a n t i b o d i e s to human t h y r o g l o b u l i n : P r o d u c t i o n and c h a r a c t e r i z a t i o n . 7, 97-104. Bresler, HS, Lynne Burek, C and Rose, NR. {1990) A u t o a n t i g e n i c d e t e r m i n a n t s on human t h y r o g l o b u l i n . 1. D e t e r m i n a n t s p e c i f i c i t i e s of m u r i n e m o n o c l o n a l a n t i b o d i e s . 54, 64-75. Ruf, P . , Carayon, P. and L i s s i t z k y , S. (1985) An approach t o human thyroglobulin structure by i m m u n o l o g i c a l methods u s i n g m o n o c l o n a l antibodies. The t h y r o g l o b u l i n - t h e p r o t h y r o i d hormone. 13-20. Raven press.
412
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 404-412
August 31,1992
PRODUCTION AND PROPERTIES OF NOVEL HUMAN THYROID CANCER
SPECIFIC MONOCLONALANTIBODIES*
Hideo Sugawa, Masumi Miyamoto 1, Kayoko H i g u ch i 2, E r n e s t Smith, Toru Mori and H i r o o Imura 1
iDepartment
of Laboratory Medicine, Second Division of Internal Medicine,
and 2Department of
Anatomic Pathology ,
Kyoto University
Faculty of Medicine, 54-Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606, Japan
Received July 8, 1992
Monoclonal a n t i b o d i e s (TCM-7, -9 and -12) against human thyroid differentiated c a n c e r s were e s t a b l i s h e d by s c r e e n i n g w i t h human t h y r o i d c a n c e r s , normal and b e n i g n t h y r o i d t i s s u e , and normal human serum p r o t e i n . A m o n o e l o n a l a n t i b o d y (TCM-9) w i t h s t r o n g s p e c i f i c i t y f o r human t h y r o i d c a n c e r b u t n o t f o r G r a v e s ' d i s e a s e , adenoma or normal t h y r o i d , was shown t o r e c o g n i z e a 300 K p r o t e i n but not t o b i n d to n a t i v e or m a t u r e human thyroglobulin. W h e n TCM-9 was used in i m m u n o h i s t o c h e m i c a l s t a i n i n g tests on more than 30 t y p e s of n o n - t h y r o i d l e s i o n s , no r e a c t i v i t y o f TCM-9 was o b s e r v e d e x c e p t w i t h s k i n immature t e r a t o m a , l i p squamous c a r c i n o m a and stomach a d e n o c a r c i n o m a , which r e v e a l e d weak r e a c t i v i t i e s . TCM-9 also showed s t r o n g r e a c t i v i t y w i t h two u n d i f f e r e n t i a t e d thyroid cancer cell lines and one t i s s u e s p e c im e n . Thus TCM-9 i s a n o v e l m o n o c l o n a l a n t i b o d y against the thyroid cancer. ©1992Ao~d~miopress,~no.
Current palpation,
diagnosis
various
tomography,
of
differentiated thyroid
c a n c e r relies
on
imagingtechniques (ultrasonography, computed X-ray
radioisotope scintigraphy and/or magnetic resonance imaging)
and detection by needle or aspiration biopsy. Althoughthese methods have greatly improved the accuracy of diagnosis, i t is s t i l l quite d i f f i c u l t distinguish f o l l i c u l a r cancer from benign follicular adenoma. After development
of
hybridomatechnology, a variety of
to the
monoclonal antibodies
(MoAbs) are established and found to have wide applications including the diagnosis
of various cancers (1-3).
Our aim in this study was to
produce
MoAbs that were capable of distinguishing human thyroid cancer (especially f o l l i c u l a r cancer) from other benign thyroid nodules.
* This work was p a r t i a l l y s u p p o r t e d by g r a n t s in a i d from t h e M i n i s t r y of E d u c a t i o n (No. 62480252 and 63614512), t h e Japan s o c i e t y f o r t h e p r o m o t i o n of s c i e n c e for Japanese J u n i o r S c i e n t i s t s , t h e Mochida memorial f o u n d a t i o n f o r m e d i c a l and p h a r m a c e u t i c a l r e s e a r c h and f o u n d a t i o n o f Osaka Cancer Research Foundation. ooo6-291x/92 $4.00 Copyright © 1992 by Academic Press, lnc. All rights of reproduction in any form reserved.
404
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS 1. Membrane p r e p a r a t i o n V a r i o u s t h y r o i d t i s s u e s were o b t a i n e d a t s u r g e r y , homogenized i n 0.25M sucrose, 10 mM T r i s - H C 1 , pH 7 . 6 , 4 mM MgC12, t h e n gauze f i l t e r e d , and c e n t r i f u g e d a t 180 x g f o r 5 min a t 4°C. The s u p e r n a t a n t was r e c e n t r i f u g e d at 15,000 x g f o r 30 min and t h e p r e c i p i t a n t was suspended i n p h o s p h a t e b u f f e r e d s a l i n e and s t o r e d a t -80°C u n t i l u s e . 2. C e l l s Mouse myeloma c e l l s X63-Ag8.653 and human embryonal l u n g f i b r o b l a s t c e l l s (HEL) were p r o v i d e d by P r o f . T s u y o s h i Uchida ( I n s t i t u t e f o r M o l e c u l a r and C e l l u l a r B i o l o g y , Osaka U n i v e r s i t y ) . The human u n d i f f e r e n t i a t e d t h y r o i d c a n c e r c e l l l i n e (HTC/C3) was i s o l a t e d i n our l a b o r a t o r y ( 4 ) . O t h er human c a n c e r c e l l s of g a s t r i c cancer (AZ-251), human u n d i f f e r e n t i a t e d t h y r o i d c a n c e r ( 5 - C - 0 ) , r e n a l c e l l c a n c e r ( T h o m a s - W i r t s ) , and f i b r o s a r c o m a (HTI080) were p r o v i d e d by t h e J a p a n e s e Cancer R e s e a r c h R e s o u r c e s Bank. 3. T h y r o g l o b u l i n p r e p a r a t i o n Human t h y r o g l o b u l i n (hTg) was p r e p a r e d a c c o r d i n g t o t h e method d e s c r i b e d by B i l s t a d e t a l . (5) w i t h minor m o d i f i c a t i o n s . Human t h y r o i d t i s s u e was homogenized, and t h e n c e n t r i f u g e d a t 15,000 x g f o r 30 min. The supernatant was p r e c i p i t a t e d w i t h 1 . 4 - 1 . 8 M ammonium s u l f a t e and t h e precipitant was d i s s o l v e d in 2 x c o n den sed PBS (2 X PBS) and d i a l y z e d . Then t h e sample was a p p l i e d t o HPLC G3OOOSW column (Tosoh, J a p a n ) , and t h e v o i d f r a c t i o n was c o l l e c t e d and s t o r e d a t -80°C u n t i l u s e . 4. P r o d u c t i o n of hybridomas S p l e e n c e l l s were p r e p a r e d from B a l b / c mice immunized w i t h membrane f r a c t i o n s of the d i f f e r e n t i a t e d t h y r o i d c a n c e r , and f u s e d w i t h X63-Ag8.653 as d e s c r i b e d p r e v i o u s l y (6). ELISA p l a t e s were c o a t e d w i t h p u r i f i e d membranes' preparations from 25 c a n c e r s (17 p a p i l l a r y c a n c e r and 8 follicular cancer), 6 adenoma, 6 Graves' tissues and 5 normal human t h y r o i d s ; g u i n e a p i g f a t c e l l membrane, normal human serum p r o t e i n and hTg preparations and used f o r s c r e e n i n g . The c l a s s and s u b c l a s s o f each MoAb were i d e n t i f i e d by an i s o t y p i n g k i t f o r mouse m o n o e l o n a l a n t i b o d i e s ( S e r o t e c , Ox f o r d . U.K). 5. I m m u n o h i s t o c h e m i c a l s t a i n i n g I m m u n o h i s t o c h e m i c a l a n a l y s i s was p e r f o r m e d f o l l o w i n g t h e methods of De Micco e t al.(7). T i s s u e s e c t i o n s were i n c u b a t e d w i t h MoAb a t a 1:100 dilution of ascites with normal mouse serum used as a control, and ABealkaline phosphatase staining (Vector laboratories) was performed.
6.Thyroglobulin specificity of MoAbs Specificity of MoAb against thyroglobulin was evaluated by: i) antithyroglobulin and anti-thyroid microsomal antibody measurement using a p a s s i v ~ g g l u t i n a t i o nkit (Fujirebio, Tokyo, Japan), 2) Immunoprecipitation with Iz~I-labelled human thyroglobulin (Midorijuji, Osaka, Japan) by Affigel Protein A (Bio-Rad), and 3) absorption of standard thyroglobulin preparations in an IRMAkit (Midorijuji, Chiba, Japan). In the IRMA kit, various concentrations of standard hTg were incubated with or without monoclonal antibodies or controls in anti-hTg MoAb coated t u b e Then the solutions were removed and 1~Ol-MoAb directed against another hTg epitope was added, then the bound radioactivities were measured. RESULTS 1. I m m u n o h i s t o c h e m i c a l s t a i n i n g
of t h y r o i d g l a n d
E x t e n s i v e c l o n i n g was p e r f o r m e d u s i n g membranes from 25 t h y r o i d c a n c e r s , 6
adenoma,
clones
6
(TCM-7,
Graves' tissues
and 5 normal t h y r o i d s ,
-9 and -12) t h a t p r o d u c e d MoAbs w i t h 405
and
finally
reactivity
three against
Vol. 187, No. 1, 1992
thyroid
cancer
selected IgG2b. (Table
Further la).
level TCM-9
reacted
screened.
serum
protein)
follicular or not
cancer
adenoma
in colloid
staining
strongly TCM-7
tissue,
tissue.
was p e r f o r m e d
with
reacted
papillary The
nor interstitial
was c o n s i s t e n t l y
cancer,
staining tissues
seen in tests
with
was
HTC/C3;
as
and
be
described
strongly
with
exclusively
(Fig.l).
cancer
to
hut did not react
w i t h TCM-9.
the undifferentiated weakly
were
A l l 3 MoAbs w e r e i d e n t i f i e d
The MoAbs TCM-7, - 9 a n d - 1 2 w e r e f o u n d t o r e a c t
of binding
5-C-0;
58 c l o n e s
immunohistochemical
Graves'
cytoplasma,
and
m e m b r a n e ( b u t n o t a d e n o m a a n d human
from the
human t h y r o i d with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cell
The The
highest antibody
lines
TCM-12
in
HTC/C3
bound
Fig. 1. Immunohistochemical staining of t h y r o i d t i s s u e s e c t i o n s by TCM-9. 1, f o l l i c u l a r c a n c e r ; 2, p a p i l l a r y c a n c e r ; 3, G r a v e s ' d i s e a s e ; 4, adenoma; and 5, u n d i f f e r e n t i a t e d t h y r o i d c a n c e r c e l l l i n e HTC/C3. 406
to
VoI. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig.
i
-
Continued
407
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T a b l e 1. a) To t h y r o i d
lesions* Number o f positive staining
Thyroid disease Follicular
cancer
Papillary
disease
Adenoma
b) To o t h e r cell
line
+ +
+++
+
+ +
±
1
+
0
-
0
-
carcinoma cell
Reactivity TCM-9 TCM-12
TCM-7
2
cancer
Graves'
Immunohistochemical staining
NMS
lines**
origin
TCM-7
AZ521
gastric
.
5-C-0
thyroid
-
÷ +
-
-
Thomas-Wirts
renal
±
±
±
±
HTC/C3(ll)
thyroid
±
-
-
OG-90
lung
.
TCM-9 .
TCM-12
.
.
÷ ÷ .
NMS
.
.
Normal mouse s e r u m (NMS) was s i m u l t a n e o u s l y u s e d a s a c o n t r o l . *: Two human t h y r o i d f o l l i c u l a r c a n c e r s , one p a p i l l a r y c a n c e r , one G r a v e s ' t i s s u e a n d one adenoma were t e s t e d . * * : TCM-9 shows c r o s s - r e a c t i v i t y with undifferentiated thyroid cancer, but not with gastric and lung cancer cells.
neither(Table reduced
lb).
by
The reactivity
100% m e t h a n o l
temperature
for
30 m i n ( d a t a
m o u s e MoAb a g a i n s t result
2.
o f MoAbs w i t h
All reacted react the
the
with
with
gastric
non-thyroidal
they
were
teratoma
and
In reacted
skin
with
Reactivity
with
with
not
atrial
other
cell
lung
cancer did
cell
not
or malignant. squamous
react
cell
serum
mouse
TCM-9, of
carcinoma
nor
a
gave
tissues.
serum
regardless
stomach
did,
control
They did
As s h o w n i n T a b l e
with
room
(a-ANP)(6)
(Thomas-Wirts).
lines.
Some c a s e s
mouse
not
at
and non-thyroida]
normal
line
was
tested.
lines
and the cell
peptide
cells
cancer
treatment
normal
natriuretic
cancer
cancer
50mM p e r i o d a t e Neither
cancer
(TCM-7,-9,-12)
a renal
or
shown).
thyroid
Tg and thyroid
300 K under
shown).
microsomal
and electroblotting
TCM-9 a n d g a v e
TCM-9 w a s s t r o n g e r (data
not
thyroid
2,
however,
most
of
cancer,
not of
whether immature
exhibit
weak
TCM-9.
SDS-PAGE
approximately
the
tissues
benign
reactivity 3.
with
3 HoAbs
weakly
30 m i n o r
human alpha
a positive Binding
for
o f MoAbs w i t h
than
a single reducing
that
The lack
of of
antigen
assays, band
with
conditions
TCM-7, w h i c h positive 408
follicular relative (Fig.2).
cancer molecular
weight
The reactivity
showed a similar
immunostaining
membrane
of
of of
band
at
300 K
the
colloid,
Vol. 187, No. 1, 1992
Table 2.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R e a c t i o n of TCM-9 w i t h v a r i o u s b e n i g n and m a l i g n a n t l e s i o n s
Malignant l e s i o n s
Benign l e s i o n s
Uterine cervix Squamous c e l l ca. case1 case2
c e r v i c a l polyp
Ovary s e r o u s c y s t adenocarcinoma adenocarcinoma
+
Liver h e p a t o c e l l u l a r ca. e a s e l case2
cirrhosis
Soft tissue s y n o v i a l sarcoma immature t e r a t o m a epidermis serous epithelium sweat gland
neurofibroma easel case2 neurinoma e a s e l case2 case3 g r a n u l a r c e l l tumor angiolipoma hemangioma pyogenic granuloma leiomyoma dermatofibroma desmoid tumor
÷ ÷ ÷
Bone osteochondroma fibrous dysplasia
+?
Stomach adenocarcinoma e a s e l case2 case3
÷ ±
ATP inflammatory polyp
Large i n t e s t i n e adenocarcinoma case1 case2
£
adenoma e a s e l case2
Pituitary
gland adenoma
Skin squamous c e l l c a . ( l i p ) m a l i g n a n t melanoma Prostate adenocarcinoma Lymph node non Hodgkin's lymphoma
normal lymph node
Kidney r e n a l c e l l ca. Nasopharynx n a s o p h a r y n g e a l ca. e a s e l case2 The bone f i b r o u s d y s p l a s i a specimen c o n t a i n e d abundant c a l c i f i c a t i o n .
membrane these
preparation
MoAbs
likely
of Graves'
tissue,
do n o t b i n d t o hTg, o r a t
T h i s was f u r t h e r
c o n f i r m e d by s e v e r a l
not
latex
aggregate
antigen(data
and p u r i f i e d
not shown}.
beads
least
methods.
coated
with
When 1 2 5 I - l a b e l e d 409
hTg
hTg to
its
Antibodies or
indicated native
TCM-7 a n d 9
thyroid
hTg was i n c u b a t e d
that form. did
microsomal w i t h TCM-7 o r
V o l . 187, N o . 1, 1992
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
7 6 660
K
>
E Q.
330 K ~ 200
5
X
4
~-
3
-o (-
2
K~
0 r~
116K
>
1
®
0
Q
TCM-9
3
3 ~0
1'0
0
100 '
300 '
1000 '
Tg ( n g / m l )
Fig. 2. Western b l o t t i n g with TCM-9. SDS-PAGE of a follicular carcinoma membrane r e a c t e d with TC~4-9 showing a s i n g l e band (300 K). Lines i n d i c a t e the p o s i t i o n of s t a n d a r d molecular markers; 19S t h y r o g l o b u l i n (660 K), reduced thyroglobulin (330 K), myosin (200 K) and E. coli /3g a l a c t o s i d a s e (116 K). Fig. 3. E f f e c t of TCM-9 on the b i n d i n g of hTg with a n t i - h T g a n t i b o d i e s . Addition of TCM-7 (open c i r c l e ) or TCM-9 (closed c i r c l e ) did not i n f l u e n c e the s t a n d a r d b i n d i n g curve when compared to the a d d i t i o n of c o n t r o l MoAbs (anti-human A•P antibody) (closed t r i a n g l e ) or b u f f e r (open triangle).
-9
and p r e c i p i t a t e d
precipitated
radioactivity
MoAb (a n e g a t i v e data
not
TCM-7, {Fig.
by A f f i - G e l
3).
recognize
The
or polyclonal
Further,
-9 and c o n t r o l
hTg a n t i b o d y
the standard
epitopes
two
on t h e hTg m o l e c u l e .
result
were
not immunostained with anti
above, lines
thyroid
TCM-9 reacted and
one
cancer specific
tissue
with
c u r v e s of hTg
IRMA
MoAbs,
that
no
which
lesion).
lines
Thus, TCM-9 r e c o g n i z e s
of
antibody occurred.
(HTC/C3
thyroid
curve
inhibition
human Tg { d a t a n o t s h o w n ) .
with the undifferentiated
epitope
control,
I f TCM-7 o r TCM-9 b o u n d
indicates
cancer cell
the
~-ANP
as t h e IRMA a n t i - h T g MoAbs, t h e s t a n d a r d
The a b s e n c e of t h i s
undifferentiated
in
preparations
anti-hTg
t h e b i n d i n g o f hTg w i t h t h e p r o v i d e d m o n o c l o n a l a n t i - h T g The
increase
(a p o s i t i v e
o f s t a n d a r d hTg
IRMA s y s t e m i s composed o f
different
shift.
incubation
MoAb d i d n o t a f f e c t
t o t h e same or p r o x i m a l s i t e would
A, no s i g n i f i c a n t
was o b s e r v e d when compared t o a n t i - h u m a n
control),
shown).
Protein
and As
cancer a
5-C-0)
described (two
novel
cell
thyroid
n o t a m o l e c u l e of t h y r o g l o b u l i n .
DISCUSSION Immunohistochemical MoAbs
revealed
that
all
studies three
of various stained 410
thyroid
tissues
differentiated
with
cancer
these ceils.
VoI. 187, No. 1, 1992
Recently, unique of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
there
have been many r e p o r t s i n d i c a t i n g t h a t c a n c e r
cells
i n t h a t t h e y have v a r i o u s a b n o r m a l i t i e s i n t h e c a r b o h y d r a t e
the s t r u c t u r e
and m e t h a n o l binding
of p r o t e i n s t h a t can be d e t e c t e d by MoAb ( 8 ) .
t r e a t m e n t s of the membrane p r e p a r a t i o n d i d
of TCM-9.
molecule.
Periodate affect
the the
component of t h e a n t i g e n
Many MoAbs produced by i m m u n i z a t i o n with p u r i f i e d hTg have
reported (9-14),
and a c e r t a i n amount of s t r u c t u r a l
cancer
tissues
or s e r a of p a t i e n t s have been i n d i c a t e d
These
previous
results
moiety
These o b s e r v a t i o n s i n d i c a t e t h a t TCM-9 r e c o g n i z e s
p c p t i d e p o r t i o n b u t n o t t h e c a r b o h y d r a t e nor l i p i d
not
are
been
h e t e r o g e n e i t y of Tg by
r e p o r t e d MoAbs l i k e l y r e a c t w i t h n a t i v e hTg,
these
MoAbs.
whereas
our
i n d i c a t e t h a t TCM-7, -9 and -12 do n o t r e a c t w i t h n a t i v e hTg
benign thyroid tissues. previously
recognizes
in
from
A l l the Tg s p e c i f i c m o n o c l o n a l a n t i b o d i e s r e p o r t e d t h e p e p t i d e p o r t i o n of the
(15) or a c o n f o r m a t i o n a l s t r u c t u r e
of Tg (11).
molecule
predominantly
The m o n o c l o n a l
antibodies
i n t h e s e s t u d i e s were s e l e c t e d to r e c o g n i z e n a t i v e Tg, a l t h o u g h some showed different patients
reactivities
a g a i n s t serum Tg i n t h y r o i d
with non-malignant thyroid diseases
patients
knowledge,
t h e r e i s no r e p o r t about t h e m o n o c l o n a l a n t i b o d y a g a i n s t thyroid
cancer.
cancer specific antigen.
Tg.
Thus, TCM-9 i s a It will potentially
Furthermore,
novel
and
antibody
selected
thyroid
to r e c o g n i z e n a t i v e
cancer
The m o n o c l o n a l
TCM-9 was
undifferentiated
not
(9).
to
our human
MoAb a g a i n s t
have a p p l i c a t i o n
in
future studies. ACKNOWLEDGMENTS The a u t h o r s wish to t h a n k Ms. Yuko Yamada and Ms. s e c r e t a r i a l and t e c h n i c a l a s s i s t a n c e .
Ikuko
Okamoto
for
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