Note: Descriptions are shown in the official language in which they were submitted.
1339340
TITLE OF T~E lN Vh~ llON
~OD FOR T~HOR DETECTION AND TR~M~T
BACRGROUND OF THE lN V~ llON
Field of the Invention
This invention is directed to a method of
diagnosing a tumor site in an individual by a non-
invasive technique, and treating such tumor by a similar
non-invasive technique.
DescriPtion of the Bach~L~-~ Art
Currently available non-invasive diagnostic methods
of tumor localization include isotope scans (e.g. liver
scan, gallium scan), radiology (e.g. plain X-ray, barium
meal, computerized tomography), and ultrasonography.
Such methods vary in their sensitivity, depending upon
the size, site, and histological type of cancer, but all
of them are nonspecific. Zalcberg, J.R., Am. J. Clin.
Oncol. 8:481-9 (1985).
Antibodies have long been recognized as potential
target-specific imaging agents. Pressman et al. tJ.
Immunol. 59:141-6 (1948)) were the first to demonstrate
conclusively the localization of radiolabeled antibodies
~.'
-2- 1339340
in specific target organs in vivo. Since then,
antibodies have been used for detection and visualiza-
tion of various malignant tissues in experimental
animals. (For a review, see Zalcberg, supra, and
Carrasquillo, J.A., et al., Cancer Treatment Reports
68:317-28 (1984).)
Early studies used the immunoglobulin fractions of
tumor-specific antisera for detection of tumors, or non-
specific markers such as anti-fibrinogen, usually
detectably labeled with radioactive 131I. More recent-
ly, antibodies to oncofetal proteins, also labeled with
131I, were used as immunologic tumor imaging agents;
however, the amount of radiolabeled antibody localized
in the tumor was low compared with that localized in the
blood and other organs, particularly the liver, thus
limiting the clinical utility of such methods.
Advances in the production of tumor-specific or
tumor-associated antibodies led to the advent of mono-
clonal antibody technology to provide a source of large
quantities of a specific antibody directed against a
single epitope. Kohler, G., et al., Nature 256:495-7
(1975); Mach, J.R., et al., Immunol. Today 2:239-49
(1981).
Improvements in radiolabeling now permit the forma-
tion of stable, pharmacologically inert complexes of
antibody with isotopes such as t~chn i tium-99m or indium-
111. These radiolabels, which have desirably short
half-lives, allow high quality images to be recorded by
scintigraphy with low radiation burden to the patient.
Khaw, B.A., et al., J. Nucl. Med. 25:592-603 (1984).
Radiolabels have generally been attached to
antibody proteins by two general techniques: oxidation
methods, and coupling with cross-linkers. Oxidation
y
~'~
1339340
methods include chloramine-T, lactoperoxidase, and
chloramide iodogen (see, for example: Zalutzsky, M., et
al., Int. J. Nucl. Med. Biol. 12:227-33 (1985);
Sternthal, E., et al., New Engl. J. Med. 303:1083-8
(1980); and Marchalonis, J.J., et al., Biochem. J.
113:299-20S (1969)). Coupling of radiolabels to
antibody proteins using cross-linkers such as
diethylenetriaminepentacetic acid cyclic anhydride in
the presence of SnC12 and citrate in the presence of
SnC12 are the current methods of choice. (See, for
example, Krejcarek, G.E., et al., Biochem. Biophys. Res.
Commun. 77:581-5 (1977); Khaw, B.A., et al., Science
2-09:205-7 (1980); Khaw, B.A., et al., J. Nucl. Med.
23:1011-19 (1982); Wong, D.W., U.S. Pat. No. 4,636,380;
and Gansow, O.A., et al., U.S. Pat. No. 4,472,509.)
Generally such labeling procedures produce a labeled
immunoprotein which retains its physicobiological pro-
perties, is pharmacologically inert, and is suitable for
imaging tumors.
Wong, U.S. patent supra, has disclosed the use of
indium-111-labeled, tumor-specific autologous polyclonal
antibodies for tumor imaging by scintigraphy. 111In-
labeled human fibrinogen was also disclosed by this
patent for neoplasm imaging.
Detectably labeled monoclonal antibodies directed
to specific tumor antigens have achieved prominence in
imaging specific tumors in vivo by scintigraphy (see,
for example, Carrasquillo, supra; Khaw, supra; Zalcberg,
supra; Nelp, W.B., et al., J. Nucl. Med. 28:34-41
(1987); Hayes, D.F., et al., Cancer Res. 46:3157-63
(1986); Murray, J.L., et al., J. Nucl. Med. 28:28-33
(1987); Larson, S.M., J. Nucl. Med. 26:538-45 (1985);
Hwang, K.M., et al., J. Natl. Canc. Inst. 76:849-55
_4- 1 339 3~ o
(1986); Goldenberg, M.D., U.S. Pat. No. 4,624,846; and
Wong, D.W., U.S. Pat., supra).
Anti-tumor monoclonal antibodies (TMoAb) are
directed against antigenic determinants that are selec-
tively expressed on the surfaces of tumor cells. How-
ever, before such monoclonal antibodies can be used for
imaging purposes, two essential criteria must be ful-
filled. Hwang, supra. The first criterion is the
target antigen specificity of the individual tumor
monoclonal antibody. This is established by preliminary
experiments on both tissue sections and cultured cells
using a variety of techniques to establish both surface
and intracellular antigen expression. A second
criterion, which also requires examination in a
systematic manner, is the pharmacokinetics of the puta-
tive TMoAb; this involves a comparison of the uptake by
tumor relative to that by normal tissue, determination
of the extent of degradation of the antibody to inert
species by the host's enzymes, and excretion of the
antibody or fragments thereof by the kidney.
The preparation of a specific TMoAb that meets all
necessary criteria for tumor imaging is a difficult,
laborious and expensive process. Larson, supra. The
overall procedure entails: (1) isolation and
identification of the specific tumor antigen; (2)
immunizing a mouse against this antigen; (3) preparing
spleen cells from such an immunized mouse and fusing
these cells with an immortal human cell line (e.g.
myeloma cells); (4) establishing hybridoma colonies; (5)
identifying hybridomas that secrete the antibody of
interect, particularly those that produce large
quantities; (6) cloning the hybridomas of interest; (7)
isolating and purifying all of the different monoclonal
~,
1339340
antibodies; and (8) determining whether single or
multiple TMoAb's are required for tumor imaging, as each
monoclonal antibody is directed to a specific epitope on
the tumor antigen.
Another complication in the use of TMoAb arises
from its frequently inadequate concentration at tumor
sites, and from its tendency to form immune complexes
that are poorly excreted by the kidney. This, in turn,
has created a need to use fragments derived from the
monoclonal antibody for tumor imaging. It has been
reported that antibody fragments such as F(ab')2 or Fab,
perhaps because of their relatively small size, diffuse
m;ore easily into tumors and are excreted more rapidly by
the kidney. Thus, the tumor to blood ratio might be
increased as a result of these two concurrent events.
Zalcberg, supra at 484; Mach et al., supra; Larson et
al., supra; Khaw et al., supra; Carrasquillo et al.,
supra. The F(ab')2 and Fab fragments of the immuno-
globulin IgG represent the specific, variable N-terminal
heavy and light chain domains of the immunoglobulin.
These fragments must be prepared from the parent protein
by proteolysis with specific proteinases, followed by
isolation and rigorous purification.
Immunoglobulin molecules can bind to the surfaces
of tumor cells by two mechAni~ms. The first requires
the presence of a specific antigenic determinant on the
cell surface, which interacts with an immunological site
found in the variable region of the antibody. This
region contains the tumor-specific F(ab)2 and Fab frag-
ments previously used in tumor imaging (see supra). The
second mechAnism requires a cell-surface receptor that
binds to a non-specific constant (Fc) region of homolo-
gous and heterologous immunoglobulins. Such receptors
' ~'~
-6- 1339340
are termed the Fc receptors. Various cells of the
reticuloendothelial and lymphatic tissues (monocytes,
macrophages, T and B lymphocytes), as well as malignan-
cies from these cells (such as lymphoma, sarcomas, and
leukemias), as well as certain types of breast and lung
cancer, possess Fc receptors on their surfaces. The
amino acid sequence of the heavy chain C-terminal domain
of the immunoglobulin (FC) fragment remains relatively
stable, regardless of the antigenic stimulus.
Thus, there is a distinct difference between the
preparation of an antibody that is capable of reacting
specifically via its Fab region with a particular tumor-
specific epitope, and a non-specific immunoglobulin that
interacts with the Fc receptor on cells at the tumor
site. A tumor imaging approach that can employ the
latter mP~h~n;sm would be highly desirable because of
its simplicity and inexpensive nature.
SUMMARY OF THE lNvhNllON
The present invention relates to a substantially
non-invasive method of diagnosing sites of certain
tumors in patients and of treating such tumors.
The present inventors have discovered that, when an
intact human immunoglobulin is allowed to contact both a
normal and a tumor site expressing Fc receptors in an
individual, the intact immunoglobulin tends to accumu-
late at the tumor site.
Further, it was surprisingly discovered that the
accumulation of immunoglobulin at the site of the Fc
receptor-bearing tumor is not dependent upon the
epitopic specificity of the immunoglobulins, and that
non-specific immunoglobulins and the Fc, but not
.
-7- 1339340
F(ab' )2~ portions of the immunoglobulin will accumulate at
the site of the tumor.
This effect, the concentration of polyclonal
immunoglobulin and the Fc fragment derived therefrom at
tumor sites expressing Fc receptors, but no concentration
of the F(ab' )2 fragment derived therefrom, and the use of
this property in non-invasive diagnostic imaging and
therapeutic treatment of such tumors, has not been previ-
ously recognized. In other words, non-specific immuno-
globulins or mixtures thereof, or non-specific Fc or Fc'
fragments derived therefrom, can be used for imaging of Fc
receptor-expressing tumors.
The present invention thus relates to an in vivo
method of detecting an Fc receptor-expressing tumor site in
an individual, this method comprising administering to the
individual a detectably labeled non-specific immunoglobulin
or an Fc or Fc' fragment thereof; allowing the contact of
the immunoglobulin or Fc or Fc' fragment thereof with Fc
receptors at the tumor site; and detecting the detectably
labeled immunoglobulin or Fc or Fc' fragment thereof.
The present invention also relates to the use of a
diagnostically effective amount of a detectably labeled
non-specific immunoglobulin or Fc or Fc' fragment thereof
in conjunction with a therapeutically effective amount of
an anti-tumor agent bound to an immunoglobulin or fragment
thereof in the treatment of an Fc receptor-expressing tumor
site in an individual.
In one embodiment, the invention relates to an
immunoglobulin comprising pooled, human, polyclonal IgG
conjugated to a diagnostically detectable label, wherein
the immunoglobulin is not IgG conjugated to EDTA and
labeled with 111In and the immunoglobulin is not IgG labeled
with ~mTc. The immunoglobulin accumulates at a site of
inflammation but has substantially no epitopic specificity
for the site of inflammation.
_,.
-7a- 1 3 3 9 3 1 0
In another embodiment, the invention relates to an
immunoglobulin fragment comprising an Fc fragment of one or
more monoclonal antibodies, wherein the Fc fragment is
conjugated to a diagnostically detectable label, and the Fc
fragment is not an Fc fragment labeled with l25I, and wherein
the Fc fragment accumulates at a site of inflammation.
In yet another embodiment, the invention relates to an
immunoglobulin comprising Fc fragments of IgG conjugated to
a diagnostically detectable label, wherein the Fc fragments
of IgG are not Fc fragments labeled with l25I, and wherein
the Fc fragments accumulate at a site of inflammation.
In a further alternative embodiment, the invention
relates to an immunoglobulin comprising pooled, human,
polyclonal IgG conjugated to DTPA and labeled with a
radioisotope label, wherein the immunoglobulin accumulates
at a site of inflammation and the immunoglobulin has
substantially no epitopic specificity for the site of
inflammation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to a method of detecting a tumor
site in vivo in an individual which comprises administering
to such an individual a diagnostically effective amount of
a detectably labeled immunoglobulin or an Fc fragment
thereof, wherein such immunoprotein
f~
~ .~ ,,.
-8- 1 3393~0
substantially accumulates at such a tumor site when the
site is an Fc receptor-bearing tumor. The invention
also relates to an antibody-based method of treating
such Fc receptor-bearing tumors.
By the term "individual" is meant to include both
animals and humans.
By the term "tumor" is intenAPA abnormal growth of
cells that may result in the invasion of normal tissue
sites or in the spread through distant organs. Tumors
may be malignant or benign. By the term "malignant" is
intended those abnormal cells that exhibit the propen-
sity for invasion and distant spread. By "benign" is
intended those abnormal cell growths that are not
invasive in character.
By the term "Fc receptor-expressing tumor" is
intended those tumors, particularly of hematopoietic or
reticuloendothelial cell origin, that produce and insert
into their cell surfaces special proteins termed "Fc
receptors," that recognize and bind with high affinity
to the non-specific, constant heavy chain portion of
circulating immunoglobulins, termed "Fc region" or "Fc
fragment." Examples of cell types that express large
numbers of Fc receptors include monoclonal phagocytes,
granulocytes, lymphocytes, basophils, and mast cells.
Examples of tumors of such cell types include melanomas,
lymphomas, sarcomas, leukemias, as well as certain types
of breast and lung cancer.
By the term "non-specific immunoglobulin or Fc or
Fc' fragment thereof" is intended any intact non-speci-
fic immunoglobulin or Fc or Fc' fragment thereof,
whether monoclonally or polyclonally derived, that has
no epitopic specificity for the tumor site and that can
be directed against any antigen, including tumor bind-
. .
-9- 13393~0
ing sites necessary to effect binding of said non-speci-
fic immunoglobulin or Fc or Fc' fragment thereof to
tumors and to accumulate at the site thereof.
Polyclonal immunoglobulin preparations can be
derived directly from the blood of the desired animal
species. Thus, in the case of humans, polyclonal
immunoglobulin preparations can be prepared from out-
dated units of blood utilizing protocols known or
readily ascertA~n~hle to those of ordinary skill in the
art. Such products are commercially available (Sandoz
Limited; Cutter Laboratories; Hyland Laboratories) and
are conventionally used in the treatment of immunodefi-
ciency states, but not in diagnosis.
In addition, if desired, polyclonal immunoglobulin
preparations may be prepared from the blood of immunized
individuals of the desired species following immuniza-
tion with any of a variety of antigens, followed by
harvesting of the blood and processing it according to
defined techniques. A distinctive advantage of non-
specific, immunoglobulin preparations is that by prepar-
ing immunoglobulin from the same species into which it
will be injected, immune reactions across species bar-
riers are prevented and repeated injections of the same
product are less likely to cause side-effects. It
should be emphasized that cross-species injections can
be done. However, their use might increase the in-
cidence of untoward reactions such as anaphylactic reac-
tions, febrile reactions, and/or the generation of an
immune response to the foreign immunoglobulin protein
that will block its effective use, as well as endanger
the health of the patient. The avoidance of such reac-
tions adds greatly to the appeal of using an im-
~'
-lO- 1339340
munoglobulin preparation which is from the same species
as that being diagnosed.
Monoclonal immunoglobulins which can be used
according to the method of the invention can be prepared
using hybridoma fusion techniques (Kohler et al.,
European Journal of Immunoloqy 6:292, 1976) or can be
derived from known secreting myeloma cell lines such as
those available from depositories such as the American
Type Culture Collection. As with the polyclonal
immunoglobulin preparation, no antigenic or epitopic
specificity is needed for the monoclonal immunoglobulin
preparation to function effectively in this method. As
a consequence, monoclonal antibodies of any specificity
can be used.
In detecting an in vivo Fc receptor-expressing
tumor site in an individual, the detectably labeled
immunoglobulin is advantageously given in a dose which
is diagnostically effective. The term "diagnostically
effective" means that the amount of detectably labeled
immunoglobulin administered is sufficient to enable
detection of the tumor site when compared to the back-
ground signal. It is preferred that the non-specific
immunoglobulin exhibit no non-tumor binding. However,
to the extent that non-tumor binding of the im-
munoglobulin does occur, one with ordinary skill will be
able to differentiate over tumor-bound immunoglobulin
based on location, intensity of the image, and the like.
Generally, the dosage of detectably labeled
immunoglobulin for diagnosis will vary depending on
considerations such as age, condition, sex, and extent
of disease in the patient, counterindications, if any,
and other variables, to be adjusted by the individual
,. .
~1
-11- 1339340
physician. Dosage can vary from 0.0003 mg/kg to 0.3
mg/kg.
The term "Fc fragment or part thereof" as used in
this invention is meant to include intact Fc fragments
as well as portions of the Fc fragment capable of accum-
ulating at the site of the tumor. Fc fragments, which
contain the CHl, CH2 and CH3 domains of the IgG mole-
cule, are produced by proteolytic methods (i.e., use of
the proteinase papain) well known to those of ordinary
skill in the art. A fragment of the Fc domain, termed
"Fc'," consists of the separated C-terminal CH3 domain
of the IgG molecule. By the term "Fc' fragment" as used
;herein is intended a detectably-labeled CH3 fragment of
the IgG molecule that is capable of substantially ac-
cumulating at a tumor site. Fc' fragments are prepared
by a combination of proteinases, employed sequentially,
namely, papain, pepsin and subtilisin.
The term "detectably labeled" means that the im-
munoglobulin has attached to it a diagnostically detec-
table label.
There are many different labels and methods of
labeling known to those of ordinary skill in the art.
Examples of the types of labels which can be used in the
present invention include radioactive isotopes and
paramagnetic isotopes. Those of ordinary skill in the
art will know of other suitable labels for binding to
the immunoglobulins used in the invention, or will be
able to ascertain such, using routine experimentation.
Furthermore, the binding of these labels to the
immunoglobulin can be done using st~n~rd t~chn;ques
common to those of ordinary skill in the art.
For diagnostic in vivo imaging, the type of detec-
tion instrument available is a major factor in selecting
-12- 1339340
a given radionuclide. The radionuclide chosen must have
a type of decay which is detectable for a given type of
instrument. In general, any conventional method for
visualizing diagnostic imaging can be utilized in
accordance with this invention.
Another important factor in selecting a radio-
nuclide for in vivo diagnosis is that the half-life of a
radionuclide be long enough so that it is still detec-
table at the time of maximum uptake by the target, but
short enough so that deleterious radiation upon the host
is minimized. Ideally, a radionuclide used for in vivo
imaging will lack a particulate emission, but produce a
large number of photons in a 140-200 keV range, which
may be readily detected by conventional gamma cameras.
For in vivo diagnosis, radionuclides may be bound
to immunoglobulin either directly or indirectly by using
an intermediary functional group. Intermediary func-
tional groups which are often used to bind radioisotopes
which exist as metallic ions to immunoglobulins are
diethylenetriaminepentaacetic acid (DTPA) and ethylene-
diaminetetracetic acid (EDTA). Typical examples of
metallic ions which can be bound to immunoglobulins are
Tc, I, 111In 131I, 97Ru 67CU 67Ga 125I 68
72AS 89zr, and 20iTl.
The immunoglobulins used in the method of the
invention can also be labeled with paramagnetic isotopes
for purposes of in vivo diagnosis. Elements which are
particularly useful (as in Magnetic Resonance Imaging
(MRI) techni~ues) in this manner include 157Gd, 55Mn,
162Dy 52cr, and 56Fe.
Alternatively, the method of the in~ention can be
used to monitor the course of tumor invasiveness or
metastasis in an individual. Thus, by measuring the
-13- 1339340
increase or decrease in the size or number of tumor
sites by serial imaging it would be possible to deter-
mine whether a particular therapeutic regimen aimed at
ameliorating the cause of the tumorigenic process, or
the tumor itself, is effective.
Another embodiment of the invention includes a
method for tumor therapy wherein the non-specific im-
munoglobulin or Fc fragment therefrom of the invention
is modified, prior to administration to an individual,
by coupling to it covalently a cytotoxic material such
as the lectin ricin which has a cell-destructive capa-
bility. Thus, the ricin, delivered by the immunopro-
;teins of the invention, will destroy the tumor cells,when delivered in therapeutically effective concentra-
tions.
The invention also embodies another method for
tumor therapy. In this method, an individual suspected
of having an Fc receptor-bearing tumor site is first
a~inictered a diagnostically effective amount of non-
specific immunoglobulin or Fc fragment thereof, as
previously described. This detectably labeled immuno-
protein may be of the same or different species as the
individual to whom it is being administered. The indi-
vidual suspected of having a tumor site is then imaged
to determine the presence of such a site. If the indi-
vidual is found to have a tumor site, the individual is
then given an antibody preparation(s) specific for the
tumor which is suspected. This specific antibody can be
from an individual of the same, or a different, species
to that of the individual having the tumor site. After
determining the specific tumor type it is then possible
to administer a therapeutic agent, such as therapeuti-
cally conjugated antibody specific for the tumor or
1339340
-14-
tumorigenic tissue at the tumor site. By "treating" is
intended the administration to an individual of an agent
that has an ameliorative, curative or prophylactic
effect upon the tumor or tumorigenesis.
The term "therapeutically conjugated" means that a
non-specific or specific immunoglobulin or fragment
thereof used in the just-described preferred method of
the invention is conjugated to a therapeutic agent. The
therapeutic agents used in this conjugate act directly
upon the tumor or upon the underlying cause of the tumor
site. Examples of therapeutic agents that can be
coupled to the specific antibodies used according to the
method of the invention are anti-tumor drugs, DNA alkyl-
ating agents, analogs of nucleotides and nucleosides,
DNA intercalcating drugs, antimetabolites, radioiso-
topes, lectins, toxins, and antibiotics. Many anti-
tumor chemicals are known in the art. A requirement of
the present invention is that the therapeutic means
should not be conjugated to the Fc portion of the im-
munoglobulin so as not to block binding of the thera-
peutic immunoprotein to the Fc receptor-bearing tumor.
By the term "lectins" is intended a glycoprotein,
usually isolated from plant material, which bind to
specific sugar moieties. Many lectins are also able to
agglutinate cells and stimulate lymphocytes. Certain
lectins are extremely toxic to animal cells. For ex-
ample, ricin is a toxic lectin which has been used
immunotherapeutically. This is accomplished by binding
the alpha-peptide chain of ricin, which is responsible
for toxicity, to the antibody molecule to enable site-
specific delivery of the toxic effect.
Toxins are poisonous substances produced by plants,
animals, or microorganisms that, in sufficient dose, are
. _
yi
-15- 133 93~0
often lethal. One such toxin is diphtheria toxin, a
protein produced by Corynebacterium diphtheriae that
inhibits protein synthesis in various cell types. This
toxin consists of an alpha and beta subunit which, under
proper conditions, can be separated. The toxic compo-
nent can be bound to antibody and used for site-specific
delivery to the primary or metastisizing tumor sites.
Examples of radioisotopes which can be bound to
specific antibody for therapeutic purposes, used accord-
ing to the method of the invention, are 125I, 131I, 90Y,
Cu, Bi, At, 212pb, 47Sc, 109Pd, and 184Re-
Antibiotics are substances which inhibit suchinfectious microorganisms as bacteria, viruses, fungi,
and parasites. These antibiotics can be any of those
known to those of ordinary skill in the art.
Other therapeutic agents which can be coupled to
immunoproteins used according to the method of the
invention are known, or can be easily ascertained, by
those of ordinary skill in the art.
Preparations of the imaging immunoglobulins for
parenteral administration include sterile aqueous or
non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil such as olive oil,
and injectable organic esters such as ethyl oleate.
Aqueous carriers include water, alcoholic/aqueous solu-
tions, emulsions or suspensions, including saline and
buffered media, parenteral vehicles including sodium
chloride solution, Ringer's dextrose, dextrose and
sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient repleni-
shers, electrolyte replenishers, such as those based on
Ringer's dextrose, and the like. Preservatives and
~,
. ~
~,,
1339340
-16-
other additives may also be present, such as, for ex-
ample, antimicrobials, anti-oxidants, chelating agents,
and inert gases and the like. See, generally, Reminq-
ton's Pharmaceutical Science, 16th ed., A. Osol, ed.,
Mack, 1980.
The above disclosure generally describes the
present invention. A more complete underst~n~;ng can be
obtained by reference to the following specific example
which is provided herein for purposes of illustration
only, and is not intended to be limiting unless other-
wise specified.
; EXAMPL~ 1
Murine ovarian reticulum cell sarcoma of monocytic
origin (M5076), an Fc receptor-bearing tumor, was im-
planted intramuscularly in the right thigh of syngeneic
C57BL/6 in-bred mice. Five days post-implantation of a
10 mg tumor, each mouse was injected with 40-50 ug (60-
80 uCi) of one of the following lllIn-labeled types of
antibodies: intact polyclonal human IgG, or its puri-
fied Fc or F(ab')2 fragment. lllInCl was coupled to
immunoproteins by the mixed and cyclic anhydride-DTPA
methods, as described, for example, by B. Khaw et al.,
Science 209:295 (1980) and Hnatowich et al., Jour. of
Appl. Radiat. Isot. 33:327 (1982). At least 6 mice per
group were used. Scintigraphic imaging was performed on
a gamma camera with pinhole collimation immediately
post-injection of the radiolabeled material, and again
at 24 and 48 hours thereafter. The animals were imaged
for ten minutes each. Whole body, organ, and tumor
uptake of the radiopharmaceuticals was calculated
according to the following formula: (organ counts-
.~. , .~-
r~ ,
-17- 1339340
background counts/standard counts) * (standard dose/
injected dose). Biodistribution of radiolabel in tis-
sues (Table 1 infra) was done at 48 hours. At the time
of sacrifice, tumor size was about 20 mg.
In order to compare tumor uptake of Fc to that of
Fab, l11In-labeled antibody modified by cyclic DTPA was
used, and one group of mice received radiolabeled Fc
fragments and the second group radiolabeled Fab frag-
ments.
In order to compare tumor localization with 125I-
labeled antibody to that of lllIn-labeled antibody, six
animals were injected with doubly-labeled intact poly-
;clonal IgG, and both l11In and 25I peaks were visualizedby posterior pinhole imagery.
As seen by the data of Table 1, tumor localization was
seen with 111In-labeled intact polyclonal IgG and its Fc
fragment, but not with the F(ab')2 fragment. The bio-
distribution data revealed that about 32~ of the injec-
ted dose of l11In-labeled Fc was retained per gram of
tumor at 48 hours post-injection, which can be explained
by the considerably higher blood activity in the IgG
group (Table 1). The tumor-to-background ratio was 12:1
in the IgG group and 10:1 in the Fc group.
The results of imaging experiments with 111In-
labeled Fab(A) and Fc(B) modified by cyclic anhydride-
DTPA are shown in Figure 1. In these experiments, tumor
was detected with Fc fragments only.
It can be concluded that it is possible to detect
Fc receptor-bearing tumors with non-specific polyclonal
IgG in both its intact and Fc forms, but not in its
F(ab)2 form. As tumor detection appears to depend on
the presence of intact Fc fragments, it is likely to be
mediated by the Fc receptor. In comparison with the
13393 10
-18-
biodistribution of F(ab')2 at 48 hours post-injection,
there was no evidence that Fc fragment was significantly
retained in tissues of the reticuloendothelial septem.
These data indicate that employment of Fc receptor
imaging provides a new approach to early non-specific
tumor detection.
~''
1~- 1339340
a ~1 ~
0 '~ ~ ~ ~ O
~i ~ N N
o ~ ~ o ~ ~ a~
c~ ,i a~ ~ ~ t~ o
o ~ ~ ~r ~q ~ o ~
O O N CO O ~ O O
~ . .... ... .. .... . . . . .
~ ~ ~0 ~ ~ ~ O ~ ~ O
?: W ~ o ~ O O O ~ ~ O
o ~ ~ o o ,,~ ~ o
d~
a. ~ g l~o) ~ ~
o o ~p ~ o o ~ ~
L
