U.S. patent number 3,927,193 [Application Number 05/361,718] was granted by the patent office on 1975-12-16 for localization of tumors by radiolabelled antibodies.
This patent grant is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Hans John Hansen, Frederick James Primus.
United States Patent |
3,927,193 |
Hansen , et al. |
December 16, 1975 |
Localization of tumors by radiolabelled antibodies
Abstract
A method of utilizing radiolabelled antibodies to
carcinoembryonic antigens for determining the site of tumors which
produce or are associated with carcinoembryonic antigen is
disclosed.
Inventors: |
Hansen; Hans John (Allendale,
NJ), Primus; Frederick James (Waldwick, NJ) |
Assignee: |
Hoffmann-La Roche Inc. (Nutley,
NJ)
|
Family
ID: |
23423179 |
Appl.
No.: |
05/361,718 |
Filed: |
May 18, 1973 |
Current U.S.
Class: |
424/1.49;
250/303 |
Current CPC
Class: |
A61K
51/1048 (20130101); A61K 2123/00 (20130101) |
Current International
Class: |
A61K
51/02 (20060101); A61K 51/10 (20060101); A61K
043/00 (); G01T 001/161 () |
Field of
Search: |
;424/1,9,12 ;23/23B
;250/303 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Nuclear Science Abstracts, Vol. 24, No. 23, Dec. 15, 1970, p. 4722,
Item No. 48,360. .
Nuclear Science Abstracts, Vol. 26, No. 22, Nov. 30, 1972, p. 5345,
Item No. 55,505. .
Chemical Abstracts, Vol. 77, No. 1, July 3, 1972, p. 342, Item No.
3636n. .
Chemical Abstracts, Vol. 77, No. 23, Nov. 30, 1972, p. 274, Item
No. 150403. .
Chemical Abstracts, Vol. 78, No. 5, Feb. 5, 1973, p. 338, Item No.
27647p. .
Chemical Abstracts, Vol. 78, No. 15, Apr. 16, 1973, p. 330, Item
No. 95838b..
|
Primary Examiner: Padgett; Benjamin R.
Assistant Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Welt; Samuel L. Saxe; Jon S. Rosen;
Gerald S.
Claims
We claim:
1. A method for determining the location of a tumor which either
produces or is associated with carcinoembryonic antigen which
comprises injecting a subject parenterally with an antibody
radiolabelled with a pharmacologically inert radioisotope having a
half-life up to about 8 days and a gamma emission of 10 to 5000 Kev
said antibody being specific to carcinoembryonic antigen and
subsequently scanning the subject with a photoscanning device to
determine the location of the resulting uptake of radiolabelled
antibody by said tumor.
2. The method of claim 1 wherein the antibody is radiolabelled with
iodine-131.
3. The method of claim 1 wherein the subject is injected
intravenously.
Description
BACKGROUND
Carcinoembryonic antigen (CEA) is a mixture of several components,
at least two of which have antigenic activity which is associated
with human carcinoma. Methods such as those described in U.S. Pat.
Nos. 3,663,684 and 3,697,638 have been developed for detecting the
presence of CEA circulating in human blood. These known processes,
however, do not provide a means for pinpointing the location and
the source of the CEA. There is thus a need for a diagnostic method
which will be satisfactory for locating the site of the tumor which
produces or is associated with CEA.
DESCRIPTION OF THE INVENTION
We have discovered that when a measured amount of radiolabelled
antibody specific to carcinoembryonic antigen is administered
parenterally, preferably by intravenous injection, the location of
carcinoma tumors which either produce or are associated with
carcinoembryonic antigen can be determined. As used herein
carcinoembryonic antigen (CEA) includes both a mixture of antigenic
components with CEA activity or a single such component.
In order to localize the tumor, radiolabelled antibody specific to
CEA is injected into the subject and during the following eight
days the subject is scanned with a photoscanning device which
locates the sites at which the radiolabelled antibody is complexed
with CEA. It is at these sites where the tumors associated with CEA
are located.
The radioisotope suitable for use in this invention is an atom
which does not interfere with the acitivity of the antibody, is
sufficiently stable to be detected after complexation with CEA, has
a half-life of sufficient duration to enable detection within 8
days and is pharmaceutically inert, i.e., non-toxic and without
pharmacological effect, in the amounts used and has a sufficient
gamma intensity to be detected readily by the photoscanning
techniques presently in use, i.e., 100 to 500 Kev (thousand
electron volts). It has been found that while I.sup.125 or
I.sup.131 are suitable for use in localizing tumors, I.sup.131 is
preferred for use in humans because of its favorable half-life. As
a practical matter, any radioisotope with a half-life up to about
eight days and a gamma emission of 10 to 5000 Kev is suitable for
use.
When radioactive iodine is used, the antibody is radio-iodinated by
the procedure of Greenwood, Hunter and Glover, Biochem J. 89
114-123, (1963) as modified by McConahy and Dixon, Int. Arch.
Allergy 29 185-189, 1966. The reaction is effected, for example, by
using a 100 .mu.l reaction mixture containing 50 .mu.l. of
chloramine-T (sodium p-toluenesulfochloramine); 40 .mu.l. of goat
anti-CEA Immunoglobulin G in a phosphate buffer of pH 7.5 (10
mg./ml. 0.05 M phosphate, pH 7.5) and 2 mCi of I.sup.125 or
I.sup.131 in the form of NaI. The reaction takes place in about 1.5
minutes at room temperature and is stopped by the addition of
sodium metabisulfite. The radioiodinated product can be separated
from the unreacted radioisotope by chromatography in a cross-linked
dextran gel column, e.g., Sephadex G-25 or Sephadex G-100 by
eluting with Tris-NaCl or PBS. The labelled antibody is stabilized
by mixing with a carrier protein such as serum or aqueous human
albumin. The resulting product has a specific activity of about 4
to 6 .mu.Ci/.mu.g. This specific activity can be modified by
altering the reaction conditions. The level of the specific
activity to be used should be below that which would interfere with
the immunological activity of the anti-CEA antibody. It has been
found that about 4 to 50 .mu.Ci/.mu.g. are suitable with 4 to 6
.mu.Ci/.mu.g preferred.
The antibody utilized is important to the operability of the
process since it must be CEA specific and must be amenable to
radiolabelling with sufficient activity per unit weight to result
in a readily demonstrable effect.
It has been found that animal sources used for producing the
antibodies vary greatly from species to species and between animals
within a species in their ability to produce large concentrations
of highly specific CEA antibodies per unit volume of serum. It has
been found that rabbits and goats are suitable, with goats
preferred. The antibodies are made by immunizing the animals
against purified CEA, such as the CEA produced by the process
disclosed in U.S. Pat. No. 3,697,638, and after a suitable time
interval obtaining serum from the animal. The antibodies are
isolated from the serum by column chromatography using, for
example, Sephadex G-200 (Pharmacia, Uppsala, Sweden) and
diethylaminoethyl (DEAE) cellulose (Whatman DE 52, H. Reeve Angel
Inc., Clifton, N.J.). The purity and specificity of the antibodies
is checked by immunoelectrophoresis and immunodiffusion.
The radiolabelled antibody is administered to the subject
parenterally, preferably I.V., in an amount sufficient to provide
radioactivity which can be readily detected, but not so much that
the radioactivity is unsafe for the patient. This is accomplished
utilizing a parenteral formulation containing about 300 to 1000
.mu.Ci per ml. Generally about 1 to 2 ml. are administered. A
suitable formulation for injection contains a phosphate buffer at
pH 7.0 and a stabilizer for the radiolabelled antibody.
In order to demonstrate that the radiolabelled antibody does
localize tumors which produce or are associated with CEA, hamsters
having such tumors implanted are simultaneously injected with an
equal mixture of anti-CEA antibody labelled with iodine-125 and
normal non-specific immunoglobulin G labelled with iodine-131.
After a suitable time, generally from 1 to 8 days, the hamsters are
sacrificed and various organs as well as blood are tested for
radioactivity and the percent of injected dose found per gram of
tissue is calculated for each isotope. By means of this technique,
i.e., the paired-labelled antibody technique, there is demonstrated
that tumors are preferentially radiolabelled by the labelled CEA
antibody whereas the non-specific antibody is distributed randomly.
This is shown by the localization ratio.
The following examples illustrate the invention.
EXAMPLE 1
Radioiodination of Antibodies to CEA
a. Two mCi of .sup.125 I in the form of NaI are mixed with 40
.mu.l. of goat anti-CEA IgG (10 mg./ml. of 0.05 M phosphate, pH
7.5). Fifty .mu.l. of Chloramine-T (3 mg./ml. phosphate buffer) are
added and the mixture allowed to react for 1.5 minutes at room
temperature. Fifty .mu.l. of Na.sub.2 S.sub.2 O.sub.5 (6 mg./ml.
phosphate buffer) is added to stop the reaction. The unbound
radioisotpe is separated by filtration over a 2.6 .times. 35 cm
Sephadex G-100 column employing Tris-NaCl as the eluting buffer.
The iodinated antibody is collected and stabilized in the carrier
protein 5% aqueous human albumin (Albuspan - Parke, Davis).
The specific activity of the product is 4-6 .mu.Ci/.mu.g.
b. Two mCi of .sup.131 I in the form of NaI are mixed with 40
.mu.l. of goat anti-CEA IgG (10 mg./ml. 0.05 M phosphate, pH 7.5).
Fifty .mu.l. of Chloramine-T (3 mg./ml. phosphate buffer) are added
and the mixture allowed to react for 1.5 minutes at room
temperature. The unbound radioisotope is separated by filtration
over a 2.6 .times. 35 cm. Sephadex G-100 column employing Tris-NaCl
as the eluting buffer. The iodinated antibody is collected and
stabilized in the carrier protein 5% aqueous human albumin
(Albuspan - Parke, Davis).
The specific activity of the product is 4-6 .mu.Ci/.mu.g. For use
in the paired labelled antibody technique normal goat IgG is
radioiodinated in the same manner using it in place of the anti-CEA
IgG.
EXAMPLE 2
CEA isolated from liver metastasis originating as adenocarcinoma of
the colon was purified by column chromatography as described in
U.S. Pat. No. 3,697,638. Goat antiserum to the thus prepared CEA
was prepared by the s.c. injection into 2 sites of 500 .mu.gm. of
CEA in 1 ml. phosphate-buffered saline (PBS) containing 1 mg. of
methylated bovine serum albumin and an equal volume of complete
Freund's adjuvant. CEA was injected every 2 weeks for a total of 5
injections. The animals were terminally bled 10 days following the
final injection.
Twenty ml. of the resulting goat anti-CEA antiserum is dialyzed
against 0.1 M Tris-HCl, pH 7.0, containing 0.15 M NaCl and 0.02%
sodium azide and applied to a 5 .times. 90 cm cross-linked dextran
gel column (Sephadex G-200) equilibrated with the same buffer and
maintained at 4.degree.C. Isolated fractions are tested for
anti-CEA activity by the radioimmunoassay of Hansen (U.S. Pat. No.
3,697,638) and the material eluting between 690 and 930 ml. of
elution volume is pooled and concentrated by pressure filtration
over a Diaflo UM 100 ultrafilter (retains 100,000 MW or greater).
The concentrated material was dialyzed against 0.01 M phosphate
buffer, pH 8.0, and applied to a 2.6 .times. 25 cm.
diethylaminoethyl (DEAE)-cellulose column equilibrated with the
same buffer. Following elution with 1 liter of starting buffer, the
anti-CEA IgG appearing between 260 to 600 ml. of elution volume was
pooled, dialyzed against distilled water, and lyophilized.
Immunoelectrophoresis and immunodiffusion were performed to
evaluate the purity of the anti-CEA IgG.
EXAMPLE 3
An injectable formulation was prepared containing 0.200 gms. of
radiolabelled anti-CEA antibody with an activity of 400 .mu.Ci/ml.
of a phosphate buffered saline solution at pH 7.0 containing, by
weight, 0.5 to 2.5% hepatitis free-human albumin.
EXAMPLE 4
Demonstration of Localization of Tumors
Male Syrian hamsters weighing 50-60 gms., were heterografted
intramuscularly with a CEA producing human signet-ring cell
carcinoma of the colon, designated in the laboratory as GW-39. At
the time of transplantation, 2.5 mg. of cortisone acetate was given
subcutaneously. The resulting tumor-bearing hamsters were
administered an intracardial injection of the radiolabelled
antibody and normal IgG mixture 1-4 weeks after implantation of
tumor. The anti-CEA antibody and normal IgG were labelled with
.sup.125 I and .sup.131 I, respectively. The radioactivities were
mixed 1:1 on an activity basis and sterilely filtered. 10-12 .mu.Ci
of each were given in a final volume of 0.2-0.3 ml. Animals were
administered Lugol's solution (a stable KI--I.sub.2 aqueous
solution) in their drinking water to block thyroid uptake. At timed
intervals, each animal was exsanguinated by cardiac puncture and
various organs (liver, spleen, kidney, lung, stomach, muscle,
tumor) were removed and radioactivity was determined in each organ
as well as in 1 ml. of blood using a gamma scintillation counter.
Using appropriately diluted injection mixture standards, the
percent of injected dose found per gram of tissue was calculated
for each radioisotope. In addition, a localization ratio was
derived using the formula: ##EQU1##
The level of significance between tumors and reference tissues was
calculated by the Student's t-test.
The results showing the tumor localization ratio at different time
intervals after injection as set forth in the following Table:
TABLE I ______________________________________ Localization Ratio
Obtained in Tumor Following Injection of Anti-CEA -- Normal IgG
Mixture Tumor .+-. Standard Tumor Day After Localization Error Wt.
Gms. Injection Ratio (S.E.) .+-. S.E.
______________________________________ 1 1.6151 .0355 0.49 .+-.
0.33 2 1.9607 .0826 0.58 .+-. 0.32 4 2.4825 .1289 0.94 .+-. 0.46 6
3.5392 .2156 1.2 .+-. 0.72 8 4.1434 .6506 2.23 .+-. 1.2
______________________________________
The tumor localization ratio indicates that the anti-CEA antibody
is localized on the tumor when compared to the nonspecific
antibody. The data show that this ratio increases with time, at
least up to 8 days.
The measurement of the ratio of labelled anti-CEA antibody in a
tumor compared to the amount in blood increased with time while
labelled normal antibodies show a relatively stable ratio. This
indicates that at day 1 after injection of the labelled anti-CEA
antibody, a tumor can be localized by photoscanning.
The following Table shows the ratios:
Table 2 ______________________________________ Tumor/Blood Ratios
Obtained After Injection of I.sup.125 Labelled Anti-CEA Antibody
and I.sup.131 Labelled Normal Antibody Antibody Anti-CEA Antibody
Normal Antibody Days After Tumor/Blood Tumor/Blood Injection Ratio
Ratio ______________________________________ 1 .8132 .4866 2 .9960
.5380 4 1.1420 .5080 6 1.7516 .5800 8 2.3016 .5350
______________________________________
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