A radioactive diagnostic agent for imaging a breast cancer or tumor,
which comprises an anti-estriol-3-sulfate antibody labeled with
a radioisotope as an essential component.
What is claimed is:
1. A radioactive diagnostic composition for imaging breast cancer
or tumor, which comprises an anti-estriol-3-sulfate antibody labeled
with a radioisotope which composition does not contain estriol-3-sulfate.
2. The diagnostic composition according to claim 1, wherein the
anitbody is a monoclonal antibody.
3. The diagnostic composition according to claim 1, wherein the
radioisotope is chosen from iodine-131, iodine-123, gallium-67,
gallium-68, thallium-201, indium-111 and technetium-99m.
4. A method for imaging a breast cancer or tumor in a living body,
which comprises administering parenterally to the living body an
effective amount of a radioactive diagnostic composition which comprises
an anti-estriol-3-sulfate antibody labeled with a radioisotope allowing
the radioactive diagnostic composition to accumulate at the breast
cancer or tumor and subsequently imaging the breast cancer or tumor
to detect the radioactivity accumulated thereat.
5. The method according to claim 4, wherein the imaging is effected
The present invention relates to a diagnostic agent for cancer
or tumor. More particularly, it relates to a radioactive diagnostic
agent for imaging of breast cancer or tumor and an imaging method
of breast cancer or tumor with the same.
For the non-invading nuclear medical diagnosis of cancer or tumor,
there is ordinarily used gallium citrate .sup.67 Ga). While gallium
citrate (.sup.67 Ga) has an accumulating property on cancer or tumor
cells, it simultaneously possesses the following disadvantages:
(1) since its specificity to cancer or tumor cells is low and its
energy characteristics are not proper, clear and sharp scintigraphy
is hardly obtainable; (2) it takes a long time until the radioactivity
disappears from the entire body so that many days are needed for
the examination; and (3) its half life is so long as 78.1 hours,
and the amount of exposure dose against the patient can not be disregarded.
Because of the above reason, many researches have been made for
development of an imaging agent having a higher specificity to cancer
or tumor cells so as to make possible the quick examination.
One of the recent proposals is imaging of cancer or tumor by a
radioisotope-labeled antibody with a high specificity to any marker
related to cancer or tumor. Since the large scale production of
a monoclonal antibody having a high specificity to cancer or tumor
by cell culture of a hybridoma obtained from the cell fusion between
an antibody-producing cell and a myeloma cell, was reported by Milstein
et al. (Nature, Vol. 256, p. 495 (1975)), various antibodies specific
to cancer or tumor-related antigens have been produced, and imaging
of cancer or tumor using the thus produced monoclonal antibodies
has been made. The imaging technique by the use of said radioisotope-labeled
antibody is generally called a "radioimmunosintigraphy".
However, this technique still includes various problems. For instance,
the radioisotope-labeled antibody takes a long time for accumulation
on cancer or tumor and the up-take ratio is low. Further, for instance,
the accumulation is made not only on cancer or tumor but also on
other normal organs and tissues, and the disappearnce of its radioactivity
from such other organs and tissues takes a long time. Due to these
reasons, it could not be placed under the practical use.
On the other hand, studies on the diagnosis of breast cancer or
tumor have been made actively with such substances specific to steroid
hormone receptors as radioactive iodine-labeled estradiol derivatives
(Hanson et al.: American Chemical Society Meeting, Aug. 3-28, 1981,
Reference N.U.S.L. 56; Kabalka: Applications of Nuclear and Radiochemistry,
Lambrecht, R. M. Morcosn., Eds., Newark, N.J., Pergamon Press, 1981,
Chap. 17; JP-A-60-78995). In order to achieve a relaiable diagnosis
with these receptor-specific substances, those substances are required
to satisfy the following conditions: (1) they have high affinity
and specificity to the receptor; (2) their specific radioactivity
is sufficiently high; and (3) their labeling nuclide is not liberated
in living bodies. However, any radioactive receptor-specific substance
satisfying all these conditions has not been developed.
SUMMARY OF THE INVENTION
As a result of the extensive study, it has now been found that
an anti-estriol-3-sulfate antibody labeled with a radioisotope shows
good accumulation at breast cancer or tumor with a significantly
high up-take ratio relative to normal organs or tissues. This invention
is based on the above finding.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-4 are scintigrams referred to in Example 7.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, there is provided an imaging
agent for breast cancer or tumors, which comprises as an essential
agent an anti-estriol-3-sulfate antibody labeled with a radioisotope
(e.g. iodine .sup.123 I, iodine .sup.131 I, gallium-67, gallium-68,
thallium-201, indium-111, technetium-99m, etc.).
Estriol-3-sulfate (hereinafter referred to as "E.sub.3 -3S")
is a metabolite of estriol, and an anti-estriol-3-sulfate antibody
(hereinafter referred to as "E.sub.3 -3S antibody") can
be produced by administering parenterally a conjugate substance
comprising E.sub.3 -3S acting as a hapten to a living body for sensitization.
Labeling of the E.sub.3 -3S antibody with a radioisotope may be
made by a per se conventional procedure. Advantageously, the radioisotope-labeled
E.sub.3 -3S antibody to be used as the imaging agent in the invention
is not necessarily required to have a high specific radioactivity,
and therefore its production can be made with ease.
The E.sub.3 -3S antibody can be produced, for instance, by the
process as disclosed in U.S. Pat. No. 4,740,476. Namely, it may
be produced in a living body chosen from a vertebrate animal (e.g.
cattle, horse, sheep, goat, rabbit, rat, mouse) by parenteral administraiton
of a E.sub.3 -3S-protein conjugate of the formula: ##STR1## wherein
A is .dbd.N--O--or --O--CO--, n is an integer of 1 to 4 and --NH--P
is the residue of a protein excluding a hydrogen atom in the amino
form therefrom (hereinafter referred to as "ESP conjugate").
Then, a humor or body fluid (e.g. blood) is taken from the living
body, optionally followed by removal of impurities. Usually, a serum
containing the E.sub.3 -3S antibody, i.e. an anti-serum, is employed.
Also, the monoclonal antibody specific to E.sub.3 -3S as produced
by application of the Milstein et al. procedure for preparation
of a monoclonal antibody (Nature, Vol. 256, 495-497 (1975)) using
the ESP conjugate is usable. The chimeric antibody obtined from
any of the above antibodies as well as the human type antibody are
Labeling of the antibody with a radioisotope may be effected by
any appropriate conventional procedure depending on the kind of
the radioisotope. For instance, labeling with radioactive iodine
may be carried out by the chloramin T method, the iodine chloride
method, the lactoperoxidase method, the iodogen method or the like
(cf. "Experimental Procedures for Metabolism of Radio-isotope-labeled
Medicines", published by Maruzen on Jan. 30, 1981, pages 95-101).
Further, for instance, labeling with any other radioisotope may
be accomplished by first coupling the antibody with a bifunctional
chelate compound (e.g. diethylenetrimainepentaacetic acid cyclic
anhydride, ethylenediaminetetraacetic acid succinimide ester, deferoxamine
(JP-A- No. 56-125317), 1-(p-aminoalkyl)-phenylpropane-1,2-dione-bis(thiosemicarbazone)
derivative s (JP-A- No. 59-193870), etc.) by the carbodiimide method,
the acid anhydride method, the glutaraldehyde method or the like
and treating the resultant coupling product with the radioisotope.
On labeling with technetium-99m, a pertechnetate (.sup.99m TcO.sub.4.sup.-)
is to be treated with a reducing agent such as stannous chloride,
stannous fluoride, stannous nitrate) so as to lower the atomic valency
(e.g. III, IV or V) of technetium for formation of a stable chelate
The diagnostic agent of the invention may be administered parenterally,
particularly intravenously, to patients. The diagnostic agent should
have sufficient radioactivity and radioactivity concentration which
can assure reliable diagnosis. For instance, in case of the radioisotope
being technetium-99m, it may be included usually in an amount of
0.1 to 50 mCi in about 0.5 to 5.0 ml on administration. The diagnostic
agent may be administered immediately after the preparation, but
it is favorable to have such a stablility as can be stored for an
appropriate time after the preparation. When desired, the diagnostic
agent may contain any additive such as a pH controlling agent (e.g.
acid, base, buffer), a stabilizer (e.g. ascorbic acid) or an isotonizing
agent (e.g. sodium chloride).
Practical and presently preferred embodiments of the invention
are illustratively shown in the following Examples.
Preparation of the E.sub.3 -3S antibody:
The 6-oxoestriol-3-sulfate-O-carboxymethyloxime-BSA conjugate (I)
(0.5 mg) was dissolved in a sterilized isotonic saline solution
(0.25 ml) and emulsified with Freund complete adjuvant (0.25 ml).
The emulsion was subcutaneously injected into a guinea pig at each
thigh and below each shoulder blade. The subcutaneous injection
was repeated 14 and 28 days after the initial injection and every
30 days thereafter. Ten days after the booster injection, the blood
was taken from the animal. The serum was separated by centrifugation
at 3500 r.p.m. for 20 minutes and stored at -25.degree. C.
Purification of the E.sub.3 -35S antibody
To the anti-serum (300 .mu.l) as obtained in Example 1, a phosphate
buffer physiological saline solution (phosphate buffer solution,
0.01 M; pH, 7.5 (hereinafter referred to as PBS)) (300 .mu.l) was
added, and 50 % ammonium sulfate solution (pH, 7.5) (600 .mu.l)
was added thereto, followed by allowing to stand at 4.degree. C.
for 1 hour. The resultant mixture was subjected to centrifugation
at 8,000 rpm for 20 minutes. The supernatant was removed, and the
precipitate was collected. To the collected precipitate, 50 % ammonium
sulfate solution (600 .mu.l) was added, and the resultant mixture
was allowed to stand at 4.degree. C. for 1 hour and then centrifuged
under the same condition as above. This operation was repeatedly
carried out twice. To the precipitate, PBS (1 ml) was added, and
the resultant solution was dialyzed to PBS at 4.degree. C. overnight,
followed by freeze-drying to give the E.sub.3 -3S antibody in a
Preparation of the .sup.125 I-labeled E.sub.3 -3S antibody:
The E.sub.3 -3S antibody (5 .mu.g) as obtained in Example 2 was
dissolved in 0.05 M phosphate buffer (pH, 7.5) (25 .mu.l), Na.sup.125
I (200 .mu.Ci) and chloramin T (20 .mu.g) as an oxidizing agent
were added thereto, and the resultant mixture was stirred at 0.degree.
C. for 15 seconds, followed by addition of sodium metabisulfite
(200 .mu.g) and potassium iodide (2 mg). The resultant mixture was
subjected to gel filtration using Cephadex G-25, and the fraction
having radioactivity were collected to give the .sup.125 I-labeled
E.sub.3 -3S antibody.
Preparation of the .sup.131 I-labeled E -3S antibody
In the same manner as in Example 3 but using the E.sub.3 -3S antibody
(25 .mu.g), Na.sup.131 I (1 mCi), chloramin T (100 .mu.g), sodium
metabisulfite (1 mg) and potassium iodide (10 mg), there was obtained
the .sup.131 I-labeled E.sub.3 -3S antibody.
Preparation of the .sup.111 In-labeled E.sub.3 -3S antibody:
The E.sub.3 -3S antibody (5 mg) as obtained in Example 2 was dissolved
in 0.05 M phosphate buffer (pH, 7.5) (2 ml), diethylenetriaminepentaacetic
acid cyclic anhydride (hereinafter referred to as "DTPA")
in a an amount of 10 times by mol was added thereto, and the resultant
mixture was stirred at room temperature overnight. The resultant
solution was dialyzed to 1 M sodium chloride solution and 0.9 %
physiological saline solution and equilibrated with physiological
saline solution, followed by purification with Sephadex G-25. To
the thus obtained E.sub.3 -3S antibody-DTPA conjugate solution,
indium chloride (.sup.111 Incl.sub.3) (1 mCi) was added, and the
mixture was allowed to stand for 15 minutes to give a solution comprising
the .sup.111 IN-labeled E.sub.3 -3S antibody-DTPA conjugate.
Biodistribution of the .sup.125 I-labeled E.sub.3 -3S antibody
in breast cancer-induced rat
To each of SD strain female rats, 10 mg of a sesame oil suspension
of 7,12-dimethylbenzanthracene (hereinafter referred to as "DMBA")
(10 mg/ml) were orally administered, and one week thereafter, the
same amount of the suspension was again administered for induction
of breast cancer. About 60 to 90 days after the DMBA administration,
the breast cancer induction was confirmed, and the animals were
then used for the test. The .sup.125 I-labeled E.sub.3 -3S antibody
solution (1.5 .mu.Ci) as obtained in Example 1 was intravenously
administered to each of the breast cancer-induced rats (bodyweight,
about 159 g) as above through the tail vein. After 48 hours, the
animals were sacrificed, and various organs were taken out. The
radioactivity and the weight were measured for each organ, and the
radioactivity percentage per unit weight (% ID/g) was calculated.
To some of the breast cancer-induced rats 24 hours after said administration
of the .sup.125 I-labeled E.sub.3 -3S antibody, the 2nd antibody
to the E.sub.3 -3S antibody (the IgG fraction of the goat serum
to the anti-guinea pig IgG) (25 .mu.g) in a physiological saline
solution (200 .mu.l) was intravenously administered, and 24 hours
thereafter, they were sacrificed, and the organs taken from them
were subjected to examination in the same manner as above.
The results are shown in Table 1.
TABLE I ______________________________________ (Biodistribution
of .sup.125 I-labeled E.sub.3 -3S antibody in breast cancer-induced
rat, % ID/g .times. 10.sup.-1) Breast cancer- Breast cancer- induced
rat (2nd Organ induced rat antibody given) ______________________________________
Brain 0.2 .+-. 0 0.2 .+-. 0 Heart 1.1 .+-. 0.1 0.8 .+-. 0.2 Lung
1.9 .+-. 0.2 1.2 .+-. 0.5 Liver 2.4 .+-. 0.7 1.6 .+-. 0.2 Spleen
1.3 .+-. 0.4 1.3 .+-. 0.4 Adrenal gland 1.8 .+-. 0.5 1.3 .+-. 0.5
Kidney 3.1 .+-. 0 2.6 .+-. 0.4 Ovary 2.4 .+-. 0.1 2.4 .+-. 1.1 Uterus
2.0 .+-. 0.1 2.3 .+-. 0.6 Bone 1.0 .+-. 0.4 1.0 .+-. 0.2 Blood 4.6
.+-. 0.6 2.6 .+-. 0.6 Breast cancer 3.5 .+-. 0.8 4.3 .+-. 1.0 ______________________________________
Note: Mean .+-. standard deviation (n = 3)
As understood from the above, the .sup.125 I-labeled E.sub.3 -3S
antibody shows sufficiently higher accumulation in breast cancer
than in other organs, and it is extremely useful for the purpose
of nuclear medicine diagnosis.
When the 2nd antibody was administered, the accumulation rate per
gram tissue in other organs decreased, while that in breast cancer
increased. Namely, the breast cancer-to-blood ratio increased about
4 times, and the breast cancer-to-other organ ratio also increased
significantly. It is thus understood that the administration of
the 2nd antibody after administration of the .sup.125 I-labeled
E.sub.3 -3S antibody produces the increase of the breast cancer-to-other
organ ratio so that clear and sharp imaging is realized.
Scintigraphy of the .sup.131 I-labeled E.sub.3 -3S antibody in
breast cancer-induced rat
The .sup.131 I-labeled E -3S antibody (120 .mu.Ci) as obtained
in Example 2 was administered intravenously to normal rats and breast
cancer-induced rats as in Example 3. After 48 and 72 hours, scintigram
was obtained by a gamma camera ("LFOV" manufactured by
Shimadzu Seisakusho). After 24 hours from the administration of
the .sup.131 I-labeled E.sub.3 -3S antibody, the 2nd antibody (1.5
mg/200 ml physiological saline solution) was administered to the
breast cancer-induced rats. Also, a potassium iodide solution (10
mg/ml) was administered orally to the animals 24 hours before the
administration of .sup.131 I-labeled E.sub.3 -3S antibody for preventing
the accumulation of free .sup.131 I ion on the thyroid gland.
FIGS. 1, 2, 3 and 4 of the accompanying drawings show respectively
the whole body scintigram of the normal rat 48 hours after the administration,
the whole body scintigram of the breast cancer-incuded rat 48 hours
after the administration, the whole body scintigram of the normal
rat 72 hours after the administration and the whole body scintigram
of the breast cancer-incuded rat 72 hours after the administration.
Blacking at the lower parts of FIGS. 3 and 4 indicates the residual
radioactivity due to the mistake on the tail vein administration,
and it does not show any specific accumulation of .sup.125 I-labeled
E.sub.3 -3S antibody. Each arrowline indicates the existence of
a breast cancer.
Imaging of the breast cancer was recognized 48 hours after the
administration, and only the breast cancer could be imaged as a
hot spot after 72 hours to giva a very clear scintigram. From this
fact, it may be understood that the diagnostic agent of the invention
is very useful for the diagnosis of breast cancer.