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Cancer Patent Abstract
A method for the isolation and purification of a cancer recognition
(CARE) antigen about 66,000 Dalton molecular weight with a pH between
4.5 and 6.5, and detection of antibodies to this CARE antigen (CARE
antibody). The CARE antigen and CARE antibody are cancer markers
for a broad range of cancers and can be detected in fluids or tissues
of cancer patients with simple ELISA assays. The combined use of
these assays for both the CARE antigen and the CARE antibody provide
improved sensitivity and selectivity for cancer detection compared
to assaying for the antigen or antibody alone. The CARE antigen
and antibodies to this antigen have therapeutic usefulness.
Cancer Patent Claims
I claim:
1. An ELISA assay for the detection of a CARE antibody, prepared
by a process comprising the steps of: a) coating a surface with
CARE antigen having the amino acid sequence shown in SEQ ID NO:
2; b) adding biological samples from a patient; c) adding conjugated
anti-human IgM antibody; and d) detecting anti-human IgM antibody
bound to said CARE antigen at SEQ ID NO: 2.
2. The assay according to claim 1 wherein said step of detecting
is performed by adding substrate for conjugated anti-human IgM antibody
and measuring product produced from said substrate by said conjugated
anti-human IgM antibody.
3. The assay according to claim 2 wherein said surface is a microtitre
plate, said anti-human IgM antibody is goat anti-human IgM antibody
conjugated with horseradish peroxidase, said substrate is chromogen,
and said biological samples are sera.
4. A method for detecting cancer comprising the steps of: a) isolating
and purifying a CARE antigen which has the amino acid sequence of
SEQ ID NO:1; b) incorporating said antigen into an ELISA assay;
and c) measuring the level of a human IgM CARE antibody in serum
which specifically binds to said antigen at SEQ ID NO:1, wherein
an increase in the level of binding relative to the level of binding
in normal serum is indicative of cancer.
5. A method for detecting cancer comprising the steps of: a) isolating
and purifying a CARE antigen which has the amino acid sequence of
SEQ ID NO:2; b) incorporating said antigen into an ELISA assay;
and c) measuring the level of a human IgM CARE antibody in serum
which specifically binds to said antigen at SEQ ID NO:2, wherein
an increase in the level of binding relative to the level of binding
in normal serum is indicative of cancer.
6. The method of claim 4 wherein said ELISA assay is prepared comprising
the steps of: a) coating a surface with said CARE antigen; b) adding
serum from a patient; c) adding conjugated anti-human IgM antibody;
and d) detecting anti-human IgM antibody bound to said CARE antigen.
7. The method of claim 4 wherein said detecting is produced by
adding substrate for conjugated anti-human IgM antibody and measuring
product produced from said substrate by said conjugated anti-human
IgM antibody.
8. The method of claim 4 wherein said surface is a microtiter plate,
said anti-human IgM antibody is goat anti-human IgM antibody conjugated
with horseradish peroxidase and said substrate is chromogen.
9. The method of claim 5 wherein said ELISA assay is prepared comprising
the steps of: a) coating a surface with said CARE antigen; b) adding
serum from a patient; c) adding conjugated anti-human IgM antibody;
and d) detecting anti-human IgM antibody bound to said CARE antigen.
10. The method of claim 5 wherein said detecting is produced by
adding substrate for conjugated anti-human IgM antibody and measuring
product produced from said substrate by said conjugated anti-human
IgM antibody.
11. The method of claim 5 wherein said surface is a microtiter
plate, said anti-human IgM antibody is goat anti-human IgM antibody
conjugated with horseradish peroxidase and said substrate is chromogen.
Cancer Patent Description
BACKGROUND OF INVENTION
1. Field of Invention
This invention relates generally to the detection of cancer using
cancer markers and, more particularly, to a unique antigenic cancer
protein and its related specific IgM antibody for the detection
of cancers.
2. Technical Background
Cancer is a leading cause of death in men and women throughout
the world. However, cancer represents a very heterogeneous group
of neoplasms. Because of this heterogeneity, it is difficult to
detect or diagnose the presence of cancer in a patient in early
stages and the diagnosis of cancer has remained a very difficult
task. It is known that cancer occurs because of some abnormality
in a gene. A change or abnormality in a gene at its transcriptional
level often can lead to the production of proteins which are relatively
unique for the cancer cell. These proteins sometimes are useful
as markers that are assayable from blood or tissue samples of a
patient. A marker is any property which can be used to distinguish
cancer from normal tissue and from other disease states. Most markers
are antigenic proteins, for example, carcenoembryonic antigen (CEA),
Alpha Fetoprotein (AFP), or prostatic specific antigen (PSA). Others
include CA 125, CA 15-3, and CA 19-9. These antigens are usually
detected by using the antibody which is relatively specific for
these antigenic markers. The assays using these markers have not
to date been markedly predictive of the presence of cancer in patients.
The sensitivity and specificity of these antigenic marker assays
has been relatively low. For any screening test to have sufficient
utility in a clinical setting, it must display adequate sensitivity
and specificity. Sensitivity is defined as the probability that
a test will be positive given that the patient has the disease.
Specificity of the test refers to the probability that the test
will be negative provided that the disease is absent. In addition,
many of the commercially available tests are only applicable to
a narrow range of cancer types and these tests suffer not only from
the disadvantage that other types of cancer may be missed, but also
from the disadvantage that a narrow applicability of the tests requires
running multiple tests on a single patient. The ideal marker is
one that is specific and universal, resulting from the expression
of the unique gene product in all kinds of transformed cancer cells.
Although, most cancer markers are antigenic proteins, some cancer
markers are antibodies. For example, an anti-GAD2 antibody has been
used in conjunction with the GAD2 antigenic protein to measure GAD2
antibody which occurs in hepatic cancer. The method of detecting
the presence of autoantibodies on the fetalphosphal protein in patients
having cancer is also known. U.S. Pat. No. 4,840,915 issued to Bogosh
in 1989 describes the use of a tumor antigen called malignin to
measure antibody as a cancer marker. In U.S. Pat. No. 5,866,690
Bogosh disclosed that malignin antibodies were IgM antibodies. In
2000, Thornthwaite published the results of a clinical study using
the malignin antigen to measure antimalignin antibodies as cancer
markers in breast cancer patients. Other cancer antigen markers
were used for comparison in this clinical study which demonstrated
that measurement of the malignin antibody was more sensitive (97%)
in detecting breast cancer than the measurement of the antigens,
CEA 0%, CA 15-3 (10%), CA 19-5 (5%), or CA 125 (16%).
Antibodies are immunoglobulins which combine antigens specifically.
The use of antibodies and immunoassays for the detection of minute
amounts of substances in physiological fluids is well known in the
art. The assay basically depends on the binding interaction between
an antigen and an antibody therefor. In the great majority of systems
described in the prior art, the antibody is usually immunoglobulin
G (IgG). Both polyclonal and monoclonal IgG's have been used. IgM
is a well known 19S immunoglobulin which comprises about 7% of the
immunoglobulins found in man. IgM antibodies are the earliest antibodies
generated in the immune response. Although IgM antibodies tend to
be very effective, especially in combating bacterial infections,
they have a relatively short in vivo half life of about 5 days.
The use of IgM antibodies has received relatively less attention
in the immunoassay art in the diagnosis of cancer. IgM antibodies
tend to aggregate and are relatively difficult to stabilize, especially
in the purified form. They are very sensitive to reducing agents,
they self-aggregate and precipitate out of solution, and have been
found to bind in a nonspecific manner. Also, they are considered
to have substantially less binding affinities for antigens than
IgG antibodies. For these reasons, IgMs have not been utilized in
immunoassays except in rare instances. One would not expect an IgM
antibody to be useful to measure the presence of an antigen cancer
marker.
The present invention provides new cancer markers utilizing an
IgM antibody that is highly specific for a new cancer related antigen
that appears to be common in a wide range of cancers. This cancer
recognition system of the present invention provides a means for
the detection of cancer in a broad range of cancers with relatively
high selectivity and specificity. Furthermore, this novel antigen
and its related antibody may also have utility in the treatment
of cancers.
SUMMARY OF INVENTION
The present invention provides for the isolation and purification
of a cancer recognition protein obtained from cancer cells using
iso-electric focusing, size exclusion chromatography of fractions
within a pH range of 5.5 to 7.5, and concentration centrifugation.
The cancer recognition (CARE) protein (antigen) material so isolated
has molecular weights ranging from between 2,000 to 70,000 Daltons,
a pKa ranging from 4.5 6.5, and an IgM binding region. An antibody
to the CARE antigen can be produced by inoculating chickens with
the CARE antigen and extracting IgY antibody from the egg yolks
produced by the inoculated chickens. An ELISA test incorporating
the CARE antigen can be used to measure antibody to the CARE antigen
(CARE antibody) in the tissues or fluids of patients for a large
variety of cancers. Antibodies to the CARE antigen can also be produced
in rabbits and mice. Using this assay, patients with a history of
cancer were shown to have 7 times higher serum CARE antibody levels
than normal subjects. Likewise, an ELISA test incorporating the
IgY antibody to the CARE antigen can be used to measure the CARE
antigen in tissues or fluids of patients for a large variety of
cancers. Using this assay, patients with various cancers were shown
to have 2.6 times higher serum CARE antigen levels than normal subjects.
This increased CARE antigen level was associated with an 11 times
higher CARE antibody level. The present invention further provides
a combination ELISA CARE antigen/antibody test to measure both cancer
markers to further increase the sensitivity and selectivity of cancer
detection.
An advantage of the present invention is that it provides a method
for detecting disease utilizing an assay to detect an antigenic
substance relatively specific for that disease and to detect an
IgM antibody specific for that antigenic substance.
Another advantage of the present invention is that it provides
a method for detecting cancer utilizing an assay to detect an antigenic
substance relatively specific for that cancer and an assay to detect
an antibody specific for that antigenic substance.
Another advantage of the present invention is that it provides
an antigenic protein or a cancer recognition antigen found in a
large variety of cancers which is useful in the detection of cancer
as a cancer marker.
Another advantage of the present invention is that it provides
a specific IgM antibody to the cancer recognition antigen which
is useful in the detection of cancer as a cancer marker.
Another advantage of the present invention is that it provides
a simple ELISA assay for the cancer recognition CARE antigen for
the detection of cancer.
Another advantage of the present invention is that it provides
a simple ELISA assay for the antibody to the CARE antigen (CARE
antibody) for the detection of cancer.
Another advantage of the present invention is that it provides
a simple method for the isolation and purification of the CARE antigen.
Another advantage of the present invention is that it provides
a simple method for the production of an antibody to the CARE antigen
(CARE antibody).
Another advantage of the present invention is that it provides
a therapeutic treatment of cancer using the CARE antigen as a vaccine.
Another advantage of the present invention is that it provides
a therapeutic treatment of cancer using an IgY antibody to the anti-CARE
antigen to induce an immune response against cancer cells.
Another advantage of the present invention is that it provides
cancer marker assays for the detection of cancer which are compact,
simple, easy to use, rapid, and inexpensive.
Another advantage of the present invention is that it provides
an antigenic protein specific for a wide range of cancers which
can be isolated and purified from any source of cancer or tumor
cells.
Another advantage of the present invention is that it provides
cancer marker assays for the detection of cancer which are safe
and meet all regulatory standards including those of the FDA, the
EPA, and OSHA.
Another advantage of the present invention is that it provides
a method for producing CARE antibodies from any biological source
capable of producing antibodies both in vivo and in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the method for isolation and purification of the CARE
antigenic protein from cancer tissue.
FIG. 2 shows the amino acid sequence of two fragments of the CARE
antigen which bind to CARE antibody (SEQ ID NOs: 1 & 2).
FIG. 3 shows the ELISA method for the detection of the CARE antibody
in serum.
FIG. 4 shows the ELISA assay for the detection of the CARE antigen
in serum.
FIG. 5 shows the ELISA assay used to determine the amount of cancer
antigen present in serum of patients.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
While the following description details the preferred embodiments
of the present invention, it is to be understood that the invention
is not limited in its application to the details of construction
and arrangement of the parts illustrated in the accompanying drawings,
since the invention is capable of other embodiments and of being
practiced in various ways.
1. Isolation and Purification of the CARE Antigen
The method for the isolation and purification of the CARE antigen
is shown in FIG. 1. Ten ml of commercially available MCF-7 human
breast cancer cells were combined with 10 ml of 0.2% triton X-100
in water and homogenized on ice for 30 seconds. Homogenate was then
sonicated on ice for 2 one minute intervals separated by a one minute
rest period. Cell debris was removed by centrifugation at 2,135.times.G
and then the supernatant was removed and recentrifuged at 10,000.times.G
for 10 minutes Final supernatant was incorporated into a preparative
granulated gel for isoelectric focusing and separated under 8 W
constant power overnight at 10.degree. C. Power was increased to
20 W for the final hour of separation. Fractions were collected
and combined with water. Those fractions having a pH in the range
of 3.5 to 7.5 were transferred to elution columns for size exclusion
chromatography and the liquid portion was allowed to drain by gravity.
The filtrates were transferred to 10,000 nominal molecular weight
micron concentrator tubes and centrifuged at 14,000.times.G for
30 minutes in a fixed angle centrifuge. The aqueous portion above
the concentrated material was removed and the concentrated material
was then diluted in water and recentrifuged. The final protein concentrate
was collected, and it provided 100 micrograms of CARE antigen protein.
The drained gel portions can be washed by resuspension in water
and in turn drained again using the filtrates as described above
to increase the yield of CARE antigenic protein. To identify the
proteins comprising the antigenic substance of the present invention,
a portion of the final concentrate was applied to a 10 20% gradient
SDS-PAGE gel under denaturing conditions. The gel was stained with
Coomassie R-250 and then destained until the bands were adequately
visible. The appearance of the Dalton bands in the fractions which
range in pH from 3.5 to 7.5 are the preferred antigenic cancer marker
proteins of the present invention. These proteins range in Daltons
from 2,000 to 70,000. The pKa of these proteins ranges between 4.0
8.0, preferably 4.5 6.5. These proteins have an IgM binding region.
The preferred protein comprising these CARE antigens of the present
invention occurs in fractions that have a pH of 5.5 and a pKa ranging
between 4.5 6.5. This is about a 66,000 Dalton protein that represents
the preferred embodiment of the present invention. The 66,000 Dalton
band was subjected to fast protein liquid chromatography for performing
amino acid sequence analysis.
The CARE antigen was analyzed for amino acid sequences or fragments
that bind to the CARE antibody. Two polypeptides were discovered
which represent the binding site of the CARE antigen. The amino
acid sequences of those two polypeptides are shown in FIG. 2. The
following amino acid sequence:
Thr Arg Asp Gyl Lys Leu Val Ser Glu Ser Ser Asp Val Leu Pro Lys
1 5 10 15
is common to both polypeptides. Binding studies of the CARE antibody
to these polypeptides were performed. Binding of the CARE antibody
to the isolated polypeptides correlated well (correlation coefficient=0.88)
with CARE antibody biding to the whole tumor homogenate.
Other methods may be used for the purification and detection of
the CARE antigen of the present invention, including but not limited
to, thin layer chromatography, ion exchange chromatography, affinity
chromatography, ultra filtration, ultra centrifugation, formation
of antigen/antibody complexes, or a combination thereof. The use
of an antigen/antibody complex in the detection or purification
of the CARE protein may include developing a first antibody to the
CARE antigenic protein and then developing a second antibody to
the first antibody. The first antibody may be found in human blood
and/or any human fluid and/or cells, including tissues against the
CARE protein antigen wherein the CARE protein antigen is bound to
a surface. Competitive binding assays between the patient's antibody
and an antibody from any source may be used wherein the CARE antigenic
protein antibody can be bound to the inside of a multiwell plate.
The antibodies and the CARE antigenic protein may be fragmented.
The fragment of the CARE antigenic protein may be an amino acid
sequence that is the active binding sight for antibody. The CARE
antigenic protein may be bound to a dipstick and antigen/antibody
complexes can be precipitated with metal complexes, a gold bound
antibody or a dye bound antibody. The antigen/antibody complex can
be detected by reflection, color, fluorescence, chemiluminescence,
conductivity, electromagnetic frequency vibration, and light scatter
differences in the antigen/antibody complex.
The antigen/antibody complexes can also be detected in a western
blot assay. This assay was performed, for example, as follows. A
15 pl aliquot of the CARE Antigen Protein was subjected to SDS-PAGE,
and then transferred to Costar Bio-blot nitrocellulose using an
LKB semi-dry transfer apparatus. The membrane was blocked with 5%
dry milk in PBS for one hour. The blot was then incubated overnight
at room temperature in one of the three solutions. In the first
solution, the blot was incubated with a 1:100 dilution of primary
patient serum (diluted in PBS containing 3% BSA). In the second
solution the blot was incubated with a 1:100 dilution of our negative
serum. In the third solution the blot was incubated in dilution
buffer alone (no primary serum). After incubation the blots were
rinsed exhaustively with PBS, and then one of two sample lanes on
each blot were cut apart and incubated in either anti-human IgG
or anti-human IgM, both of which were conjugated to alkaline phosphatase.
These secondary antisera were diluted 1:10,000 in antibody buffer.
The blots were then incubated for one hour at room temperature,
washed extensively, and then developed with substrate (BCIP) for
alkaline phosphatase.
In order to demonstrate the presence of the CARE antigen on cancer
cells, a study was performed in patients having ovarian carcinoma.
Cancerous ovarian tissue was removed from these patients and analyzed
for the presence of the CARE antigen using histologic methods. The
tissues were exposed to IgY, a purified IgG from chickens immunized
with the CARE whole antigen preparation. This IgY antibody binds
specifically to the CARE antigen in tissues. A secondary antibody
to the chicken IgY, derived from goat and having horseradish peroxidase
(HRP) attached to it, was then added. The HRP was then reacted with
a dye which stains brown in the presence of the HRP. The brain stain
reflects the region where the CARE antigen resides. Normal ovarian
tissue did not stain while staining was consistently present in
cancerous tissue, as shown in FIG. 3.
2. CARE Antibody ELISA Test.
The CARE antibody test is an indirect assay used to detect the
presence of the CARE antibody in human serum against the cancer-expressed
CARE antigen shown in FIG. 4. Indirect ELISA (enzyme linked immunosorbent
assay) surprisingly was extremely effective in detecting and quantifying
the CARE antibodies in serum. The wells of a 96-well plate are coated
with the CARE antigen that had been obtained as described above
(Step 30). The plate is allowed to incubate overnight at 4.degree.
C. to facilitate efficient adsorption of the antigen to the plate
(Step 31). The plate is washed three times in a 96-well plate washer
with plate washing buffer (Step 32). Washing is a necessary and
crucial step because it separates bound versus free antigen. After
washing, a blocking reagent containing bovine serum albumin is added
to prevent nonspecific adsorption of the CARE antibody to the exposed
plastic sites where antigen failed to bind. Blocking reagents are
immunologically inert and therefore, do not interfere with the subsequent
antigen or antibody reactions. After the wells have been blocked,
they are allowed to incubate for one hour at room temperature (Step
32). During incubation, serum samples from test patients and reference
serum are diluted into buffer. The reference serum or standard is
serially diluted by one third concentrations to obtain a standard
or calibration curve. After washing, the serum samples and the reference
standard are added to the wells. The serum contains the CARE antigen
specific antibody, the primary antibody. Upon adding the serum containing
CARE antibody to the CARE antigen coated plate, the CARE antibody
will bind to the CARE antigen adsorbed to the plate (Step 34). After
incubation for one hour at room temperature and subsequent washing,
goat anti-human IgM antibody conjugated with horseradish peroxidase
(HRP) is added to the wells (Step 34). After washing, chromogen
substrate is then added (Step 35), followed by stop solution (Step
36) to determine calorimetrically the product generated by HRP from
the chromogen substrate, which provides the mean relative concentration
of CARE antibody in the patient serum.
Blood was sampled from healthy volunteers, 130 males and 176 females.
The serum mean relative concentration (MRC) was determined for the
CARE antibody using the ELISA assay. The mean MRC.+-. the standard
deviation (SD) for the CARE antibody for these healthy volunteers
was 38.6.+-.41.3 SD. 105 patients with many different types of cancers
(breast, melanoma, lymphoma, rectum, CLL, colon, cervical, mylodisplastic,
gastric, head and neck, chronic myeloginous leukemia, lung, tonsil)
were also studied for the mean relative concentration of antibody
in the blood. The MRC for the CARE antibody for these patients with
cancer was 352.+-.262 SD. 60 patients with a history of cancer but
without any evidence of current cancer were studied and had a mean
MRC of 49.+-.60 SD. The ELISA assay of the present invention was
able to demonstrate in patients with a broad range of cancers seven
times higher serum CARE antibody levels than for normal subjects.
In addition, the ELISA test for the CARE antibody also was able
to distinguish patients who had cancer but were free of cancer,
these patients having a CARE antibody level in the normal range.
3. CARE Antigen ELISA Test.
The CARE antigen test is an enzyme-linked immunosorbent assay (ELISA)
used to detect the amount of cancer antigen present in serum of
patients, shown in FIG. 5. The antigen may be free or bound to the
antibody in the form of an immune complex. Total antigen is free
plus bound. The CARE antigen test determines the total and bound
antigen with the difference being the free antigen. To trap the
free and bound antigen from serum, a 96-well microtiter plate is
coated overnight with chicken anti-CARE antigen IgY antibody (Step
40). This antibody is derived from the yolk of eggs from chickens
innoculated with CARE antigen. The chick antibody adheres to a 96-well
plate overnight at 2 4.degree. C. (Step 41). The following day,
the plate is washed and coated with a blocking agent and incubated
for one hour at room temperature as described previously in the
CARE antibody test description (Step 42). After washing, patient
sera are added to the plate to incubate for one hour at room temperature.
(Step 43). The assay allows both the bound and total antigen to
be determined (Step 44). The bound antigen is coated with a solution
that will not react with the complex. At the same time, the total
antigen is determined in parallel samples where the free antigen
is bound by a reference positive patients' CARE antibody against
the CARE antigen (Step 45). Standard methods are used to prepare
IgM from the pooled serum of cancer patients. IgM (MW 700K) is much
larger than any other serum protein component and is purified by
size exclusion chromotagraphy. The plate is allowed to incubate
for one hour at room temperature after which it is washed (Step
46). Goat anti-human IgM antibody conjugated to horseradish peroxidase
(HRP) is added to all the wells. Chromogen is added to all the wells
and reacts with the horseradish peroxidase linked to the antibody
(Step 48). Stop solution is then added (Step 49). Colormetric readings
are made to determine the mean relative concentration of antigen,
bound and total, present in the patient's serum (Step 50). The amount
of free antigen is obtained by subtracting bound antigen from total
antigen.
The ELISA test incorporating the chicken IgY antibody to the CARE
antigen was used to measure the CARE antigen in the serum of patients
having a large variety of cancers. In this study, the bound antigen
and total antigen were measured in 108 healthy volunteers and, in
addition, the serum CARE antibody to the CARE antigen was also measured.
In the healthy volunteers (n=108), bound antigen was 209 mean relative
concentration (MRC).+-.262 SD and total antigen was 297.+-.328.
Antibody was 39.+-.41. The apparent false negative was 5%. In 7
patients diagnosed with a variety of cancers, including breast cancer,
lymphoma, myelodysplastic disease, colon cancer, melanoma, lung
cancer, rectal cancer, and chronic myelogenous leukemia, the MRC
for total CARE antigen was 776.+-.259 and serum CARE antibody was
525.+-.364. Thus, using this assay, patients with various cancers
were shown to have 2.6 times higher serum CARE antigen levels than
the normal subject and this increased CARE antigen level was associated
with an 11 times higher CARE antibody level. Three patients were
also studied who were successfully treated for cancer. Two of these
patients had breast cancer and one had malignant lymphoma. After
treatment for their cancers, these patients had no detectable serum
CARE antibody and their serum CARE antigen levels were 124.+-.54.
These studies assaying for the serum CARE antibody and serum CARE
antigen clearly show the ability of these assays in combination
to detect the presence of cancer in a broad range of cancers. They
further show the consistency between measuring the antibody and
the antigen and strongly indicate the usefulness of combining the
tests to get a complete detection of cancer with a high degree of
sensitivity and selectivity.
The results of these studies demonstrate that the present invention
provides a method and system to increase the diagnostic sensitivity
and selectivity not only for cancer but for any disease, compared
to existing methods. Application of the present invention by testing
both antigen and antibody in fluids or tissues of patients substantially
increases the probability of correctly and accurately detecting
not only cancer, but any disease, compared to testing only an antigen
or only an antibody. The system and method of the present invention
will be useful in a variety of cancers, including, for example,
leukemia and related blood disorders, lung cancer, breast cancer,
colon cancer, prostate cancer, anal cancer, cancer of the bile duct,
bladder cancer, bone cancer, bone metastasis, brain and spinal cord
cancers, cervical cancer, non-Hodgkin lymphoma, rectal cancer, endometrial
cancer, esophageal cancer, gallbladder cancer, gastrointestinal
carcinoid tumors, Kaposi's carcinoma, kidney cancer, renal cell
carcinoma, laryngeal cancer, hyphopharyngeal cancer, liver cancer,
malignant mesothelioma, melanoma, metastatic cancer, multiple myeloma,
mylodysplastic syndrome, neuroblastoma, oral cavity cancer, oral
pharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,
penile cancer, pituitary cancer, retinblastoma, rhabdomyosarcoma,
salivary gland cancer, soft tissue cancer, skin cancer, stomach
cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal
cancer, vulvar cancer, and Wilm's tumor, and in other diseases,
such as, for example, autoimmune disease, heart disease, graft-versus-host
disease, and in diseases involving the process of apoptosis.
4. Serum CARE Antibody.
The serum CARE antibody was determined to be an IgM antibody by
using a secondary antibody specific for binding to IgM antibodies.
As noted above, CARE antigen or CARE antigen fragment can be bound
to a microtitre well plate and the CARE IgM antibody can then be
bound to the CARE antigen or fragment. The secondary antibody can
then be used to detect the CARE IgM antibody. Secondary antibodies
against IgG or IgA will not detect the IgM CARE antibody.
Normally, in the primary response to an antigen, there is a sharp
increase in serum IgM; however, the concentration of IgM eventually
drops as a rise in IgG concentration is seen to increase. This is
the process of immunoglobulin class switching or seroconversion.
This process is typically irreversible because of deletions of genes
within the DNA of memory B cells. This class switch from a high-valency,
low affinity immunoglobulin M to a low-valency, high affinity IgG
may provide a mechanism for more specific recognition and response
upon a second challenge by the antigen. However, the CARE antigen
is IgM specific and the CARE IgM antibody reflects no seroconversion
to IgG. This is unexpected, and if the CARE IgM antibody were converted
to IgG, the CARE antibody test would not be of benefit because IgG
is not specific to the CARE antigen. Therefore, it would be impossible
to effectively measure CARE antibody concentration specific for
the CARE antigen if CARE IgM were converted to IgG. In 13 patients
with elevated CARE antibody concentrations, the concentrations of
IgG were also measured. The concentration of IgG was only 7.3.+-.4.8%
of the CARE antibody concentration.
5. Therapeutic Uses
Both the CARE antigen and the anti-CARE chicken IgY antibodies
will have therapeutic utility as vaccines to a large number of cancers.
Administration of the CARE antigen will induce an immune response
of IgM CARE antibodies which will bind to CARE antigen bound to
cancer cells, inducing an immune response to destroy the cancer
cells. Also, the anti-CARE chicken IgY antibodies may be administered
to a patient and will bind to CARE antigen on cancer cells. The
CARE antigen IgY antibody complex will be recognized as foreign
and the patient's body will mount an immune response that will destroy
the cancer cells. The human IgM CARE antibody, and any one or more
types of proteins produced using the CARE antigen, can also be administered
directly to patients to bind to CARE antigen and thereby destroy
cancer cells. These proteins can be administered individually or
in combination.
The foregoing description has been limited to specific embodiments
of this invention. It will be apparent, however, that variations
and modifications may be made by those skilled in the art to the
disclosed embodiments of the invention, with the attainment of some
or all of its advantages and without departing from the spirit and
scope of the present invention. For example, the polypeptides shown
in FIG. 2 may be used in the ELISA antibody assay to substitute
for the CARE antigen, and can be used to inoculate chickens to produce
the chicken IgY anti-CARE antigen antibody for use in the ELISA
CARE antigen assay. They may also be used as vaccines to induce
an immune response to the CARE antigen.
It will be understood that various changes in the details, materials,
and arrangements of the parts which have been described and illustrated
above in order to explain the nature of this invention may be made
by those skilled in the art without departing from the principle
and scope of the invention as recited in the following claims.
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2umanCHAIN(2)No Other Relevant Information on this Sequence l Val
Val Lys Lys Ile Glu Thr Arg Asp Gly Lys Leu Val Ser 5 u Ser Ser
Asp Val Leu Pro Lys 2Human 2Cys Thr Arg Asp Gly Lys Leu Val Ser
Glu Ser Ser Asp Val Leu Pro 5 s |