Document:

United States Patent                                                   5,310,653
Hanausek-Walaszek ,   et al.                                        May 10, 1994

Tumor marker protein and antibodies thereto for cancer risk assessment or
diagnosis

                                    Abstract
A tumor-associated marker protein was purified and antibodies thereto developed
for cancer diagnosis and assessment of cancer risk associated with the long-term
use of synthetic steroid hormones, both contraceptive and non-contraceptive, and
other drugs that exhibit tumor promotional properties. The marker protein and
antibodies thereto provided are interspecies immunologically cross-reactive. In
summary, the marker p65 tumor-associated factor of the present invention has the
following characteristics: (a) binds substantially completely to a phenyl
hydrophobic interaction column in a buffer containing 20% ammonium sulfate and
eluted at ca. 16% ammonium sulfate; (b) localized primarily in the nuclear
envelopes with only small amounts present in the cytoplasm from where is
released to the blood circulation in vivo or cell culture medium in vitro; (c)
induced in normal, adult tissues by chemical carcinogens (initiators) but not by
tumor promoters, the carcinogen-induced production being enhanced by the latter.
Also disclosed herein are processes for purifying the 65 kDa tumor marker from
plasma, tumor cytosol or ascitic fluid of carcinoma bearing animals; processes
for producing antisera and purified antibody preparations to the 65 kDa tumor
marker; and methods using antibody to the 65 kDa to diagnose or assess the
likelihood of cancer.

Inventors:     Hanausek-Walaszek; Margaret (Bastrop, TX); Slaga; Thomas J.
               (Austin, TX); Walaszek; Zbigniew (Bastrop, TX)
Assignee:      Board of Regents, The University of Texas System (Austin, TX)
Appl. No.:     012972
Filed:         February 2, 1993
Current U.S. Class:                        435/7.23; 435/7.92; 530/350; 530/358;
                                     530/387.7; 530/388.8; 530/388.85; 530/389.7
Intern'l Class:                          G01N 033/574; A61K 039/00; A61K 037/08;
                                                                     C07K 015/18
Field of Search:                  435/7.23,7.92 424/88 530/387.7,388.8,389.7,358

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                        References Cited [Referenced By]
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                              U.S. Patent Documents
4448890           May., 1984            Smetana et al.                 436/508.
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4746539           May., 1988            Webb et al.                     424/88.
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                                Other References

Hamausek-Walaszek et al., Progress in Clinical and Biological Research (1989).
Hanausek-Walaszek et al., "Carcinogenesis", Proceedings of the American Associa-
tion for Cancer Research 30:190, Abstract 754 (1989).

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Larroya et al., "Immunology", Proceedings of the American Association for Cancer
Research 30:349, Abstract 1385 (1989). Hanausesk-Walaszek et al.,
"Carcinogenesis", Proceedings of AACR 29:167, Abstract 665 (1988).
Hanausek-Walaszek et al., "Correspondence Between Biochemical and Antigenic
Activity of a 60 Kilodalton Oncofetal Protein During Carcinogenesis and
Tumorigenesis," Cancer Letters 33:55-61 (1986).
Hanausek-Walaszek et al., "A 60 Kilodalton Oncofetal Protein As Tumor Marker,"
J. Med. 17:13-23 (1986).
Schroder et al., "Proteins from rat liver cytosol which stimulate mRNA
transport," Eur. J. Biochem. 159:51-59 (1986).
Hanausek-Walaszek et al., "Immunological Identity of a 60 KD Oncofetal Protein
Induced in Rats by Chemical Carcinogens and Released by Transformed Cells," BBRC
127:779-785 (1985). Hanausek-Walaszek et al., "Chemical carcinogens as specific
inducers of a 60-kilodalton oncofetal protein in rats," Carcinogenesis
7:1725-1730 (1985).
Schumm et al., "Absence of the Cancer-associated Factor with a Molecular Weight
of 60,000 from the Plasma of Patients with a Spectrum of Nonneoplastic
Conditions," Cancer Res. 44:401-406 (1984).
Hanausek-Walaszek et al., "Characterization of a 60,000-dalton Oncofetal Protein
from the Plasma of Tumor-Bearing Rats," Cancer Invest. 2:433-441 (1984). French
et al., "Nucleocytoplasmic Release of Pepetitive DNA Transcripts in
Carcinogenesis Correlates with a 60 Kilodalton Cytoplasmic Protein," Cancer
Letters 23:45-52 (1984). Walaszek et al., "An Oncofetal 60-Kilodalton Protein in
the Plasma of Tumor-Bearing and Carcinogen-Treated Rats," Cancer Letters
20:277-282 (1983).
Hanausek-Walaszek et al., "Structural and Immunological Identity of p, 65
Tumor-Associated Factors from Rat and Mouse Hepatocarcinomas," Progress in
Clinical and Biological Ressearch (1989).

Primary Examiner: Nucker; Christine M.
Assistant Examiner: Woodward; M. P.
Attorney, Agent or Firm: Arnold, White & Durkee

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                               Goverment Interests
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GOVERNMENT RIGHTS

The United States Government may have certain rights to this invention pursuant
to National Institutes of Health grant RR 5511-23.

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                                Parent Case Text
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This application is a continuation of application Ser. No. 07/426,408, filed
Oct. 24, 1989 now abandoned.

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                                     Claims
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What is claimed is:

1. A process for preparing an immunogen of a tumor-associated protein comprising
the steps of:

(a) collecting from plasma, tumor cytosol or ascitic fluid of carcinoma-bearing
mammals or from culture medium in which cancer cells were grown, the protein
fraction which is precipitated from plasma, tumor, cytosol or ascitic fluid
between 30% and 60% saturation of an aqueous ammonium sulfate solution or from
culture medium at 90% saturation of an aqueous ammonium sulfate solution,
respectively;

(b) chromatographing the protein fraction on a molecular sieving column and
collecting a protein fraction having a molecular weight in the range of about 50
to 90 kilodaltons;

(c) applying the collected chromatographed protein fraction to a high
performance liquid chromatography (HPLC) phenyl hydrophobic interaction column
and allowing protein to bind to the column;

(d) eluting the bound protein on the phenyl hydrophobic interaction column with
a buffer comprising about 16% ammonium sulfate;

(e) collecting the first distinct protein peak eluted from the column;

(f) electrophoresing the collected protein;

(g) transblotting the electrophoresed protein to a nitro-cellulose sheet;

(h) cutting from the nitro-cellulose sheet that portion which contains a band of
protein corresponding to about 65 kD.

2. An antibody preparation comprising antibodies specific to the immunogen of
claim 1 and substantially free of antibodies to normal plasma proteins.

3. The antibody preparation of claim 2 which is comprised of monoclonal anti-
bodies.

4. A method for determining the presence in a mammal of a 65 kD tumor-associated
protein; the method comprising:

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providing antibodies specific for immunogen of claim 1,

contacting said antibodies with biological material of said mammal; and

determining the presence of an immunological reaction product between said
antibodies and said protein.

5. The method of claim 4 wherein the presence of said reaction product is
determined by radioimmunoassay, enzyme linked immunosorbent assay,
immunoblotting or immunohistochemical staining.

6. The method of claim 4 wherein the biological material is plasma, serum,
cytosol fluid, ascites or tissue.

7. The method of claim 4 wherein the biological material is liver tissue or
skin.

8. A method for diagnosing a cancer which produces a 65 kD tumor-associated
protein in a mammal, comprising:

providing a sample of biological material from said mammal wherein the
biological material is plasma, serum, cytosol fluid, ascites or tissue;

contacting said biological material with antibodies specific or the immunogen of
claim 1;

determining the presence or absence of an immunological reaction product between
said antibodies and the 65 kD tumor-associated protein, the presence of an
immunological reaction product being indicative of or an early indication of
cancer.

9. The method of claim 8 wherein the biological material is liver tissue or
skin.

10. The method of claim 8 wherein the cancer subject to detection is liver
cancer, breast cancer, skin cancer or squamous cell carcinoma.

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                                   Description
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FIELD OF THE INVENTION

This invention relates to the isolation and identification of a cancer
associated protein, preparation of antibodies thereto, and methods of cancer
risk assessment or diagnosis.

<PAGE>

BACKGROUND OF THE INVENTION

In spite of improved treatments for certain forms of cancer, it is still a
leading cause of death in the United States. Since the chance for complete
remission of cancer is, in most cases, greatly enhanced by early diagnosis, it
is very desirable that physicians be able to detect cancers before a substantial
tumor develops. Also, in cases where the primary tumor has been substantially
removed by surgery or destroyed by other means, it is important that the
physician be capable of detecting any trace of cancer in the patient (either in
the form of residues of the primary tumor or of secondary tumors caused by
metastasis), in order that the physician can prescribe appropriate subsequent
treatment, such as chemotherapy.

The quantities of cancer cells that must be detected for early diagnosis or
following removal or destruction of the primary tumor are so small that the
physician cannot rely upon physical examination of the cancer site. Moreover, in
many cases the cancer site is of course not susceptible to direct visual
observation and it is almost always impractical to detect secondary tumors by
visual observation, since it is not possible to predict exactly where they are
likely to occur. Accordingly, sensitive tests have to rely upon detection of
cancer-associated materials, usually proteins, present in body fluids of
patients who have, or are about to develop, cancer cells in their bodies.
Several diagnostic materials for detection of cancer-associated proteins are
available commercially. Tests for alpha-fetoprotein are used to detect primary
liver cancer and teratocarcinoma in humans; and carcinoembryonic antigen is used
for digestive system cancers, as well as lung and breast carcinomas; chorionic
gonadotropin is employed to detect trophoblast and germ cell cancers; calcitonin
is used for thyroid gland cancers; and prostatic acid phosphatase is used to
detect prostate carcinoma. These markers are detectable in advanced rather than
in early cancer.

Unfortunately, many of the commercially available tests are only applicable to a
narrow range of cancer types, and therefore these tests suffer not only from the
disadvantage that other types of cancer may be missed but also from the
disadvantage that the narrow applicability of the tests means that it may be
necessary to run multiple tests on a single patient for diagnostic purposes, a
procedure which not only increases the expense of the diagnostic testing but
also increases the risk that one or other of the tests may give a false positive
result. Accordingly, there is a need for a single diagnostic test able to detect
the presence of very small amounts of cells of a wide variety of different
cancers. The ideal marker would be one that is specific and universal. Such a
marker may exist if malignant transformation is associated with the expression
of a unique gene product in all kinds of transformed cells.

It is already known that serum from the blood of animals suffering from a wide
variety of cancers contains an oncofetal protein having a molecular weight of
approximately 60,000 and having the capacity to increase the release of
ribonucleic acid (RNA) from cell nuclei. This protein is referred to as
oncofetal RNA-transport protein (ORTP) or 60 kDa cancer-associated protein.

ORTP is localized in the cytoplasm of tumors of humans and experimental animals
and small amounts are released into the host circulatory system. The 60 kDa ORTP

<PAGE>

is notably absent from the nuclei of rat liver and rat liver tumors. It has been
shown to be present in fetal rats at 18 days of gestation and in human and rat
amniotic fluid, but not in maternal blood. It has not been detected in adult
rats. Nor is it present in detectable concentrations in the blood of normal
human subjects or those with a variety of non-neoplastic conditions or diseases,
including benign tumors and other non-neoplastic proliferative diseases. In
contrast, of more than 200 cancer patients with confirmed active disease, all
tested positive for the factor. It was also present in all of about 200
tumor-bearing rats tested. Unfortunately, antibodies to a rat ORTP preparation
purified as described in the prior art do not cross-react with human ORTP. Thus,
the 60 kDa cancer marker proteins from different species are not immunologically
equivalent, e.g., an antibody to the rat cancer marker protein does not
cross-react with a human cancer marker protein. Thus, when the purified 60 kDa
cancer marker protein preparation is to be used for production of antibodies for
diagnostic purposes, it is necessary to begin the preparation process with
plasma from the species in which the diagnosis is to be used.

We have recently identified and characterized another RNA-transport-stimulating
oncofetal protein with a molecular weight of 65 kDa (p65) which exhibits certain
properties which strongly favor its candidacy as a general tumor marker, as well
as a marker of cancer risk associated with the prolonged use of drugs, such as
androgenic and estrogenic hormones, that have tumor promotional potential.

SUMMARY OF THE INVENTION

This invention provides a protein preparation containing a relatively pure form
of an oncofetal RNA-transport-stimulating cancer marker protein. More
specifically, the invention provides a protein preparation comprising a
cancer-associated protein having the following characteristics:

(a) not being precipitated by 30% saturated aqueous ammonium sulfate solution at
25.degree. C.;

(b) molecular weight ca. 65,000.+-.5,000, with some variation, as measured by
electrophoresis on 12.5% SDS polyacrylamide slab gels;

(c) binds completely to a phenyl hydrophobic interaction column in a buffer
containing 20% ammonium sulfate in 50 mM TrisC1, pH 7.5 with 1 mM EDTA and 10 mM
2-mercaptoethanol and is eluted by gradient when the ammonium sulfate
concentration drops to ca. 16%;

(d) having a cyanogen bromide (CNBr) cleavage pattern of six major peptides with
molecular weights of about 6, 9, 27, 39, 43 and 47 kDa;

(e) exhibiting interspecies immunological cross-reactivity, specifically the
human p65 reacts with antibodies raised in rabbits directed against the rat p65
protein;

(f) present in only some lesions characterized by altered enzyme expression
(i.e., those assumed to be precursors of malignant tumors) and presumably all

<PAGE>

malignant tumors but substantially absent from normal tissue, specifically
absent from the maternal blood of non-cancerous normal mammals;

(g) localized primarily in the nuclear envelopes (but not within the nucleoli or
intranuclear structures) with only small amounts present in the cytoplasm from
where is released to the blood circulation in vivo or cell culture medium in
vitro;

(h) induced in normal, adult tissues by chemical carcinogens (initiators) but
not by tumor promoters, the carcinogen induced production being enhanced by the
latter; and

(i) having an RNA-releasing activity of at least about 10.0 units per milligram
of total protein when assayed by the procedure set forth in column 4 of U.S.
Pat. No. 4,746,539.

As used herein, the term "substantially" is a relative term meaning largely but
not absolutely wholly as specified. The term allows for trace deviance from the
absolute.

This invention also provides a process for preparing a purified
cancer-associated protein, this process comprising:

(a) collecting from plasma, tumor cytosol or ascitic fluid of carcinoma-bearing
animals or from culture medium in which cancer cells were grown, the protein
fraction which is precipitated from plasma, tumor cytosol or ascitic fluid
between 30% and 60% saturation of the aqueous ammonium sulfate solution or from
culture medium at 90% saturation of the aqueous ammonium sulfate;

(b) dissolving the precipitate in a buffer at pH 7.5, chromatographing on a
molecular sieving column and collecting a protein fraction having a molecular
weight of about 65,000 daltons;

(c) separating the chromatographed fraction on a high performance liquid chroma-
tography (HPLC) phenyl hydrophobic interaction column;

(d) purifying the collected 65,000 dalton protein fraction by electrophoresis on
12.5% SDS polyacrylamide gels; and

(e) excising the protein fraction which appears at a mobility corresponding to a
molecular weight of about 65,000 daltons.

This invention also provides an antibody preparation substantially free of
antibodies to normal plasma fraction, and which antibody preparation is capable
of forming an immunoconjugate with the instant p65 cancer-associated protein.
The antibody preparation is capable of forming a visible precipitate with the
cancer marker protein p65 when each is diffused toward one another in agar gel
but is not capable of forming a conjugate with the 60 kDa ORTP derived from

<PAGE>

cytoplasm of tumors. Further, the antibody preparation exhibits interspecies
cross-reactivity with other species derived 65 kDa tumor markers of this
invention.

Finally, this invention provides a method for assessing the likelihood of cancer
which involves immunoassays to detect the presence of the instant p65 tumor
marker protein in biological material of a host suspected of developing cancer
or being at high risk for developing cancer as the result of treatment with
drug(s) known to have a tumor promotion potential .

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. p65 tumor-associated protein production during 7,12-dimethylbenz[a]
anthracene (DMBA)-induced, and 12-O-tetradecanoylphorbol-13-acetate
(TPA)-promoted skin carcinogenesis in SENCAR mice.

FIG. 1A shows the time course of papilloma appearance following carcinogenesis
promotion and FIG. 1B shows the time course of p65 accumulation int he blood
plasma following the same carcinogenesis promotion.

FIG. 2. Enhancement by synthetic steroid hormone of p65 tumor-associated protein
production during chemical carcinogenesis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods for Diagnosing or Assessing the Likelihood of Cancer

Samples of blood plasma or serum tissue are obtained from human cancer patients
or human subjects prior to and at different times in the course of treatment
with synthetic steroid hormones or other drugs known or suspected of having a
tumor promotion potential. Alternatively, samples of blood plasma, serum or
tissue are obtained from other mammals suspected of suffering from cancer or
treated with substances suspected of causing cancer.

Briefly, samples of the blood plasma or serum containing the same amount of
protein are mixed with an electrophoresis sample buffer, boiled for three
minutes and subjected to 12.5% SDS-PAGE. Subsequently, the gel slabs are
prepared for transfer by equilibrating for one hour in 0.025M Tris, 0.192M
glycine, 20% (v/v) methanol, pH 8.3, and then transblotted onto nitrocellulose
sheets. To conduct the immunoassay, the nitrocellulose sheets are treated with
appropriate blocking solution to block unspecific binding sites and incubated
overnight, either with pre-immune serum (controls) or antibody against the p65
protein diluted 1:200. Secondary biotinylated antibody is applied next and the
color is developed using ABC Elite Kit from Vector Laboratories, Burlingame,
Calif. The highly specific polyclonal antibody against p65 is obtained by
immunizing rabbit with pure human or rat p65 preparations. As an alternative,
mouse monoclonal antibodies to p65 may be prepared using the techniques
generally described in "Hybridoma Techniques" Cold Spring Harbor, N.Y., 1980,
ISBN 0-87969-143-3.

<PAGE>

The immunoblots are photographed and the bands of p65 immune complexes on the
film are quantitated using a laser densitometer coupled to an integrator.
Alternatively, following probing with the antibodies to p65 or pre-immune serum,
nitrocellulose sheets are labeled with .sup.125 I-protein A. The labeled sheets
are subjected to autoradiography followed by scanning the film with a laser
densitometer coupled to an integrator. Another alternative is to use an ELISA
procedure.

To interpret the plasma or serum or tissue samples, the relative quantity of p65
in the sample is indicated by the intensity of a band at nominal molecular
weight about 65,000 as measured by laser scanning. Comparison with the
corresponding area of the control (derived with the use of pre-immune serum)
permits distinguishing response specific for p65 above non-specific background
response. Specific activity of the marker band from clinical samples taken in
the course of treatment is compared with the samples taken prior to treatment
and/or with the average sample from a normal healthy pool. The clinical response
can be then expressed as a factor against the response prior to treatment or
against the response of the normal healthy pool, respectively. Steady increases
in the value of clinical response over a period of time are indicative of an
increased cancer risk associated with the long-term treatment with a given
synthetic steroid or other drug that has tumor promotional properties. The high
initial response is indicative of an existing cancer.

The following examples are given by way of illustration, without intent to limit
the scope of the invention, to show details of particularly preferred reagents
and techniques utilized in the processes of the instant invention.

EXAMPLE I

Animal Models for Determination of the Presence of Cancer Cells

This example illustrates the instant process for purification of the p65
tumor-associated protein and preparation of antibodies thereto as source of p65.

Rat and Mouse Liver Tumors

Rat hepatoma cell lines McA-RH7777 and McA-RH8994 were purchased from the
American Type Culture Collection (Rockville, Md.) and carried as cell cultures
in Swim's S77 medium with 4 mM L-glutamine and supplemented with 5% fetal calf
serum and 20% horse serum (Gibco, Grand Island, N.Y.). After several passages in
cell culture, rat hepatoma cells (1.times.10.sup.6 /0.2 ml phosphate-buffered
saline) were inoculated subcutaneously into a hind leg of male Buffalo rats
(120-150 g) (Harlan Labs, Indianapolis, Ind.) and were carried as solid tumors
according to conventional procedures.

Transplantable hepatocellular carcinoma (THC) 1682C was derived from primary
hepatic tumors in ACI rats maintained for four months on the choline-deficient
diet of Shinozuka containing 0.2% ethionine and eight months on a

<PAGE>

choline-supplemented (0.8%) diet without ethionine. THC T52 was established by
intraperitoneal transplantation of growing tumors induced in ACI rats by
2-acetylaminofluorene (AAF). THC cultures were maintained in vitro and also
carried as solid (1682C) and ascitic (T52) tumors in male ACI rats (The
University of Texas M.D. Anderson Cancer Center Science Park-Veterinary
Resources Division, Bastrop, Tex.).

The Reuber hepatoma cell culture H35 was provided by Dr. Andrew P. Butler,
University of Texas M.D. Anderson Cancer Center Carcinogenesis Department,
Science Park, Smithville, Tex.

Mouse liver carcinoma CRL 6421 (formerly NBL #MM45T.Li) was obtained from
American Type Culture Collection and carried as cell cultures as described above
for rat hepatoma cell lines.

Purification of p65

Preparation of the antibody involves purification of the p65 marker protein from
the tissue culture medium of tumor cells of human or animal origin.
Specifically, the cells were grown to confluence at 37.degree. C. in the
presence of media and a serum or serum-like supplement, or completely defined
medium containing salts and hormones. Growing cells to confluence was a purely
economical step which ascertains a high yield of the p65 marker protein. The
preferred medium used was Dulbecco's Modified Eagle's Medium (DMEM) with 5%
fetal calf serum and cells were grown in 5% CO.sub.2. The confluent cells were
washed three times in serum-free DMEM medium and incubated in this medium for
16-24 hours (the optimal time being 24 hours). At this point, medium was
collected by centrifugation at 10,000.times.g for 10-15 minutes and treated with
ammonium sulfate solution at 90% saturation of aqueous solution for 30 minutes
at 4.degree. C.

Protein precipitate was collected by 30-minute centrifugation at 10,000.times.g,
dissolved in a small volume of 50 mM TrisC1 buffer, pH 7.5, with 50 mM NaC1, 10
mM 2-mercaptoethanol and 1 mM EDTA, and dialyzed overnight against the same
buffer. After dialysis, proteins were loaded on an LKB TSK 3000 SW molecular
sieving column and separated according to the molecular weight using an LKB HPLC
system. Fractions containing proteins with molecular weight in the range of
50-90 kDa were collected and dialyzed overnight against buffer A, containing 20%
ammonium sulfate in 50 mM Tris-C1, pH 7.5 with 10 mM 2-mercaptoethanol and 1 mM
EDTA.

Following dialysis, the 50-90 kDa proteins were loaded on a phenyl hydrophobic
interaction column (PHI) (LKB, Pharmacia) equilibrated with buffer A. Generally,
800-1,000 ml of conditioned medium was processed as described above. The p65
marker protein weakly binds to the PHI column and is eluted in the first
distinct peak by a gradient of 80% to 0% of buffer A, in combination with buffer
B (50 mM Tris-C1, pH 7.5 with 10 mM 2-mercaptoethanol and 1 mM EDTA,

<PAGE>

supplemented with 50% ethylene glycol). Fractions containing the p65 marker
protein are then combined and dialyzed against buffer A devoid of ammonium
sulfate, concentrated by lyophilization and electrophoresed on 12.5% SDS-PAGE at
constant current of a 10 mA for two hours at room temperature.

Proteins are transblotted to a 0.22.mu. nitrocellulose sheet as described by
Towbin et al. (Proc. Natl. Acad. Sci. U.S.A., Vol. 76, pages 4350-4353, 1979). A
reversible ponceau A stain was used to visualize the nitrocellulose bound
proteins according to the procedure of Salinovich and Montellaro (Anal.
Biochem., Vol. 156, pages 341-347, 1986). The band of the p65 protein was cut
out of the nitrocellulose sheets and used for immunization of rabbits.

Mouse p65 was isolated from the mouse liver carcinoma cell line CRL 6421 (MM45)
and purified as described above for the rat factor.

Preparation of Antibodies to p65

Antisera to the purified rat p65 preparation were raised in rabbits, as follows.

Specific Pathogen Free (Pasteurella) male New Zealand white rabbits of
approximately four kilograms body weight were used (Myrtle's Rabbitry).
Pre-immune (day 0) and test blood samples (10 days following each of four
immunizations on day 1, 14, 28 and 42) were obtained from the central artery of
the ear or a lateral ear vein. The anesthetized rabbits were placed in sternal
recumbency, the dorsal fur was removed with surgical clippers, and the surgical
site was aseptically prepared with provodine-iodine (Wescodyne, West Chemical)
followed by 70% ethanol. Six incisions about 1.0 cm each were made in an
anterioposterior direction through the skin and subcutis with a scalpel. The
incisions were undermined with blunt and sharp dissection to allow implant
placement over the superficial epaxial musculature. The nitrocellulose strip
containing the rat p65 protein was cut into six pieces (each approximately 0.5
cm.times.1.5 cm) which were then formed into rolls for insertion. Following
placement, the skin edges were opposed with tissue forceps and closed with
surgical adhesive (Vetbond, 3M). Adequate spacing (about 2.5 cm) between
insertion fields will allow subsequent immunization incisions to be made
adjacently. Terminal blood collection was made in Alsever's solution in
anesthetized rabbits by the use of a vacuum assisted collection device to
provide the serum which is the polyclonal antibody source. The antisera were
absorbed with normal plasma proteins immobilized on nitrocellulose sheets
following its electrophoresis on 10% SDS-PAGE and transblotting. An ELISA assay
or immunoblotting analysis was conducted to determine the potency and
specificity of the antisera obtained.

A standard ELISA procedure was used for detection of specific antibodies in
serum. Ninety-six well microtiter plates designed for ELISA were used (Immunol
2, Dynatech). For detection of rabbit anti-p65 antibodies, the ELISA plates were
pre-coated with several different concentrations of antigen. To test sera, a
positive reference serum and a negative pre-immune serum were added to the wells
in five-fold dilutions in PBS. Anti-p65 antibodies were detected by goat
anti-rabbit IgG conjugated with horse radish peroxidase (Bio-Rad). After the
substrate reaction, plates were read on an ELISA plate reader at 405 nm (Litton

<PAGE>

Bionetics, Laboratory Product Division, S.C.). A serum sample was considered
positive when it read 0.05 units or more above the background.

Probing of Western blots with prepared antibody was carried as follows.

The purified rat tumor-associated protein, p65, was separated by PAGE and
electrophoretically transferred to nitrocellulose as described above. Free
binding sites on the nitrocellulose sheets were then blocked overnight using 1%
normal goat serum in TTBs buffer (0.5% Tween, 0.1 mM Tris-HCl, pH 7.1, 09%
saline). Antisera obtained from immunized rabbits were diluted serially in the
blot buffer (TTBs) and incubated with the nitrocellulose strips for one hour at
room temperature. The blots were then washed with several changes of TTBs buffer
containing 1% goat serum

Bound antibody was detected using biotinylated second antibody (goat anti-rabbit
IgG, biotinylated) and the avidinbiotin-peroxidase method (Vectastain ABC Elite,
Vector, Burlingame, Calif.). To develop blots, 0.02% hydrogen peroxide was used,
mixed with 0.1% diaminobenzidine tetrachloride (DAB) made in 0.1M Tris-HCl
buffer, pH 7.2. Color generally developed within five to ten minutes; blots were
rinsed with distilled water and air dried to preserve color.

Alternatively, bound antibody was detected by incubating the nitrocellulose
strips with .sup.125 I-Protein A (1.times.10.sup.6 cpm/ml of blot buffer). After
a 60-minute incubation, unbound label was removed by repeated washes of the
blots with PBS buffer containing 0.5% Tween. The bound antigen .sup.125
I-Protein A complex was detected by overnight autoradiography using Kodak
X-OMAT-AR film.

In addition, monoclonal antibody preparations can be made to the 65 kDa cancer
marker by employing conventional techniques well known to the art.

EXAMPLE II

SDS-PAGE and Immunoblotting Analyses

Carefully dissected hepatomas (see Example I) and liver fragments from normal
rats and mice were rinsed with an ice-cold saline solution and processed for
immunochemical determination of p65. Liver tumor cells grown in culture, as
described in Example I, were separated from medium by centrifugation, rinsed
with the cold saline solution, and processed for immunoassay of the p65
tumor-associated factor. Briefly, small pieces of tissues or tumor cell pellets
were homogenized in TMK-sucrose buffer, pH 7.2, and samples of the total
homogenate containing the same amount of protein were mixed with electrophoresis
sample buffer, boiled for three minutes, and subjected to 10% SDS-PAGE. The
proteins precipitated at 90% saturation of ammonium sulfate from the conditioned
medium of hepatocarcinoma cells were dissolved in TMK buffer and electrophoresed
in a similar manner. Subsequently, the gel slabs were prepared for transfer by
equilibration for one hour in 0.025M Tris, 0.192M glycine, 20% methanol
(vol/vol), pH 8.3, and then transblotted onto nitrocellulose sheets.

<PAGE>

For immunoassay, the nitrocellulose sheets were treated with the appropriate
blocking solution to block non-specific binding sites and incubated overnight,
either with pre-immune serum (controls) or antibody against rat p65 diluted
1:200. Secondary biotinylated antibody was applied next, and the color was
developed using an ABC Elite Kit (Vector Laboratories, Burlingame, Calif.). The
highly specific polyclonal antibody against p65 was obtained by immunizing
rabbits with the rat p65 preparation purified as described above.

Presence of p65 in Rat and Mouse Liver Tumors and its Absence from Normal Mouse
and Rat Liver

When total protein samples from rat and mouse liver carcinomas grown in vitro
were analyzed by SDS-PAGE and then anti-rat p65 antibody probing of western
blots, a single, prominent band was detected in the 65 kDa region of the blots.
Some weak bands seen in the 60-64 kDa region may represent degradation products
of the native species of p65. Other minor bands resulted from non-specific
staining. There is no band characteristic of p65 in immunoblots representing
normal adult rat or mouse liver.

All hepatocarcinoma cells grown in vitro as cell cultures and tumor tissues from
Morris hepatomas carried as solid tumors in vivo were positive for the p65
antigen. There was no reaction with normal liver cells of either rat or mouse
origin. Thus, immunoblotting analysis has demonstrated that p65 was specifically
produced by liver cancer cells but not by the cells of normal adult rat or mouse
liver.

EXAMPLE III

Interspecies Cross-reactivity of Antibodies Against p65

The p65 tumor-associated factors derived from different species are
immunologically cross-reactive.

                  TABLE 1
    --------------------------------------
    Immunoprecipitation of p65 from Different Sources
    by Antibodies Raised in Rabbits against Rat p65.
    Source of p65      Immunoprecipitation of p65.sup.a
    --------------------------------------
    Plasma:
    Morris Hepatoma 7777-bearing rats
                       +++
    Morris Hepatoma 8994-bearing rats
                       +++
    Pregnant Rats      -
    Normal Rats        -
    Cytosol:
    Morris Hepatoma 7777 tumors
                       +++
    Morris Hepatoma 8994 tumors
                       +++

<PAGE>

    Normal Rat Liver   -
    Conditioned culture medium:
    Morris Hepatoma 7777 cells
                       +++
    Morris Hepatoma 8994 cells
                       +++
    Rat THC 1682 cells +++
    Mouse squamous cell carcinoma
                       ++
    Human breast cancer cells
                       ++
    (MCF-7)
    Unconditioned culture medium
                       -
    --------------------------------------
     .sup.a +++ = over 90% precipitation; ++ = 50-90% precipitation; - = less
     than 0.5% precipitation.

The p65 activity as measured by the mRNA-transport assay was immunoprecipitated
using polyclonal anti-rat p65 antibodies from the serum of rabbits immunized
against the rate transplantable hepatocellular carcinoma THC 1682. This antibody
removed activity from cytosols derived from Morris Hepatomas 7777 and 8994 and
from cell culture media in which tumor cells were grown. Polyclonal anti-rat p65
antibodies reacted with human p65 secreted to the culture medium by the MCF-7
breast cancer cell line. The antibodies also cross-reacted with the mouse p65
factor secreted to medium by mouse squamous cell carcinoma. The p65
tumor-associated factor was not detected in the blood of pregnant rats. It was
neither detected in the blood of normal rats nor in unconditioned cell culture
medium.

EXAMPLE IV

CNBr Cleavage of p65

Cyanogen bromide (CNBr) cleavage maps of p65 purified from cell culture medium
of rat transplantable hepatocellular carcinoma cell line 1682C or mouse liver
carcinoma cell line CRL 6421 (MM45) were obtained as follows. p65 preparations
(20 .mu.g) purified from cell culture medium were subjected to 12.5% sodium
deodecyl sulfate (SDS)-PAGE. The p65 band located on slab gels by Coomassie blue
staining of parallel gel tracks was cut from appropriate gel tracks and
incubated at room temperature for 16 hours in 1 ml of 88% formic acid containing
20 mg/ml of CNBr. The gel slices were then rinsed five times with 1 ml of water
and washed several times for 10 minutes with 1 ml 120 mM Tris-HCl, pH 7.0, 20%
glycerol (vol/vol), 2% SDS until the pH of the slices reached 7.0. CNBr-treated
gel slices were placed onto 15% SDS-PAGE slabs, electrophoresed, and then
stained with silver. Slices of gel containing p65 treated with 88 % formic acid
served as controls.

<PAGE>

Structural Identity of Rat and Mouse p65

Rat and mouse p65 were purified to apparent homogeneity as described above. CNBr
cleavage maps were obtained as the first step toward the final characterization
of the amino acid composition and sequence of p65. The cleavage of p65 with CNBr
resulted in six major peptides identifiable by silver staining of the SDS-PAGE
gels. The peptides have molecular weights of about 6, 9, 27, 39, 43 and 47 kDa.
Identical cleavage maps were obtained for rat and mouse p65.

EXAMPLE V

Animal Models for Determination of Cancer Risk

The ultimate objective of animal carcinogenicity studies is the determination of
possible human risk. The available data show that known human carcinogens that
have been adequately studied are also carcinogenic in laboratory animals, often
at the same target site. Thus, the identification and elucidation of the
mechanisms underlying each stage of the carcinogenic process in animals may
offer testable hypothesis for the stages in human. The current tests for cancer
risk assessment focus mainly on markers of genetic damage, at the level of the
DNA or chromosome, as indicators of genotoxic exposure. These tests, however,
are not able to detect the risk associated with exposure to synthetic steroid
hormones and other tumor promoters.

It was recently indicated that the promotional status of human subpopulations
could be the dominant factor in determining the cancer risk. The development of
a more systematic analysis of possible tumorigenesis mechanism has also been
suggested. One of the approaches to study in a more systematic way the mechanism
of tumorigenesis, involves comparisons among different systems in which tumor
induction, or cell transformation has been optimized through the use of the most
effective system-specific agents and protocols. When each tumor system is
operating optimally, intersystem comparisons could be undertaken with respect to
carefully selected biochemical parameters. Tumor-associated proteins, such as
the one described herein which appears to be a general marker of preneoplastic
and neoplastic alterations, are good candidates to be used in intersystem
comparisons.

Monitoring Skin Carcinogenesis with p65 Tumor-Associated Factor

A multistage skin carcinogenesis model was used to monitor the carcinogenic
process using the p65 tumor-associated marker.

Skin tumors were induced on the back of SENCAR mice by a single dose of 10 nmol
of 7,12-dimethylbenz[a]anthracene (DMBA) and repetitive applications of 1 .mu.g
of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week. Blood samples were
randomly obtained from four mice at times indicated in FIG. 1. The p65 activity
was measured in the blood plasma by use of an ELISA assay.

In a modified ELISA inhibition procedure, 100 .mu.l of purified antigen of
predetermined dilution was added to Dynatech Immunon 2 plates and incubated at
37.degree. C. for one hour. This was followed by washing, then binding 1% bovine

<PAGE>

serum albumin in bicarbonate buffer to cover residual binding sites in the
wells. After washing the plates, 100 ml of antiserum plus test samples were
added and the incubation carried out for one hour at 37.degree. C. Finally, the
immune complex was detected by adding 100 .mu.l of goat/anti-rabbit
immunoglobulin conjugated to horseradish peroxidase. Then the substrate
2,2'-Azino-(3-ethylbenzthiazolinesulfonic acid) and 0.03% H.sub.2 O.sub.2 was
added to each well and incubated 10 minutes at room temperature. After
terminating the reaction with addition of 20 ml of 2.0 mM NaN.sub.3 to each
well, the absorbance (405 nm) was read on an ELISA reader.

For convenience of graphing, the inhibition values were changed to units where
the sample giving the greatest inhibition will be selected as the end point. The
percent inhibition=100-[(Absorbance inhibited/absorbance
uninhibited).times.100]. The percent inhibition of the sample was multiplied by
the reciprocal of the dilution to obtain units of the activity.

Shown in FIG. 1 was the time-course of (A) papilloma appearance and (B) p65
accumulation in the blood plasma. p65 was detected in the plasma at four weeks
of promotion, then its activity increased, first slowly up to 20 weeks, and then
more rapidly up to 30 weeks, when it began to plateau. p65 was not detected in
the blood of non-initiated mice treated with TPA as described above up to 20
weeks (data not shown).

By using a simple blood test for the presence of p65 factor, the skin cancer
risk from the tumor promoter TPA can be detected as early as four weeks of
treatment with the tumor promoter. A majority of skin papillomas are considered
to be non-malignant tumors at early stages of development. Conventional
histological and cytogenetic techniques are time consuming and are able to
detect skin cancers in mice only at later stages of papilloma development, i.e.,
at 30-40 weeks.

EXAMPLE VI

Monitoring Liver Carcinogenesis with p65 Tumor-Associated Factor

Altered hepatic foci (AHF) were induced in the course of 2-acetylaminofluorene
(AAF)-initiated, phenobarbital (PB)-promoted hepatocarcinogenesis in the rat.
Rats (male weanling albinos, Sprague Dawley strain) were purchased from Harlan
Labs, Indianapolis, Ind. The 0.06% (w/w) AAF diet and 0.05% PB diet were
prepared and pelleted by Altromin, Lage, FRG, and Dyets, Inc., Bethlehem, Pa.,
respectively. Each of two experimental groups of rats and a control group
consisted of 40 rats. All rats entered the experiment at 22 days of age. One
group received AAF diet for 18 days, then AIN-76A diet (Dyets, Inc.). The second
group received AAF diet for 18 days then AIN-76A diet for 24 days and then
AIN-76A diet supplemented with 0.05% phenobarbital. The third group (controls)
received AIN-76A diet for 42 days, then AIN-76A diet plus 0.05% phenobarbital.

<PAGE>

Immunohistochemical Procedures

The blood and livers of rats (4 animals per each time-point) sacrificed at
different times in the course of hepato-carcinogenesis experiment were used for
immunochemical studies. Paraffin liver sections were prepared and stained with
specific antibodies. Polyclonal antibodies to the rat p65, purified to apparent
homogeneity, were raised in rabbits as described herein. The p65 was visualized
in the liver sections using the avidinbiotin-peroxidase complex (Vectastain ABC
Kit, Vector Laboratories, Burlingame, Calif.). Appropriate controls with
non-immune serum were performed routinely. Antiserum to p65 was diluted 1:200 in
PBS with 1% goat serum for use in the staining protocol. The blood plasma was
assayed for the presence of p65 using an LISA assay or PAGE followed by
immunoblotting analyses.

The p65 tumor-associated protein was detected in rats fed AAF and PB diets as
early as two weeks of feeding with the tumor promoter PB. The p65 marker was
predominantly present in the cells of putative preneoplastic foci found at 24
weeks of trial in livers of rats fed AAF and PB diets. The p65 marker was highly
concentrated in the foci with little or none being detected in the surrounding
cells. Either no staining or weak positive staining was found in the areas known
for oval and ductular proliferation. No positive staining was found in control
livers from normal rats or rats fed only the PB diet. Most of p65 activity
appears to be associated with the nuclei of the p65-positive hepatocytes and
more precisely with the nuclear envelopes, with relatively little being detected
in the cytoplasm. Immunohistochemical staining of the cross-sectioned nuclei
revealed that only the periphery of the nuclei, i.e., nuclear envelopes, were
stained. These results also affirm the putative role of p65 in the
nucleocytoplasmic transport of mRNA.

EXAMPLE VII

Enhancement of p65 Production During Sex Hormone Promotion

Shown in FIG. 2 is the effect of the contraceptive steroid ethynylestradiol
(EE), a known tumor promoter in the rat, on the p65 production in female rats
initiated with N-methylnitrosourea (MNU), using a protocol designed to induce
mammary gland tumors.

A short (one week) exposure to EE at six to seven weeks post-carcinogen
treatment, i.e., when the MNU-induced production of p65 was relatively low,
caused three-fold increase of the p65 level in the blood plasma. MNU was shown
to induce pre-neoplastic foci in the liver; however, they were not detected by
the use of the gamma-glutamyl transpeptidase (GGT) assay, even after 23-week
promotion with EE. Thus, the p65 production in the rat appears to be extremely
sensitive to hormonal stimulation. The high sensitivity of the p65 synthesis
during chemical carcinogenesis to sex hormone and phenobarbital promotion
indicate that p65 can be used not only as a tumor marker, but also for early
assessment of cancer risk associated with the use of synthetic steroids and
other drugs that exhibit tumor promotion properties.

It will be apparent to those skilled in the art that numerous changes and
modifications can be made in the preferred embodiments of the invention

<PAGE>

described above without departing from the scope of the invention. Accordingly,
the whole of the foregoing description is to be construed in an illustrative and
not in a limiting sense, the scope of the invention being defined solely by the
appended claims.

<PAGE>United States Patent                                                   5,773,215
Hanausek-Walaszek ,   et al.                                       June 30, 1998

--------------------------------------------------------------------------------
Tumor marker protein for cancer risk assessment

                                    Abstract

This invention relates to the isolation, identification and sequencing of a
cancer associated protein, preparation of hybridization probes therefrom,
preparation of antibodies thereto, and methods of cancer risk assessment and
diagnosis.

--------------------------------------------------------------------------------
Inventors:     Hanausek-Walaszek; Margaret (Bastrop, TX); Slaga; Thomas J.
               (Austin, TX); Walaszek;
               Zbigniew (Bastrop, TX)
Assignee:      Board of Regents, The University of Texas System (Austin, TX)
Appl. No.:     405648
Filed:         March 17, 1995
Current U.S. Class:                 435/6; 435/7.23; 435/69.3; 530/352; 530/358;
                                                                      530/388.8;
                                        530/388.85; 530/389.7; 530/828; 536/23.5
Intern'l Class:                                        C12Q 001/68; G01N 033/574
Field of Search:              435/6,69.3,7.23 530/352,358,828,388.8,388.85,389.7
                                                                        536/23.5

--------------------------------------------------------------------------------
                        References Cited [Referenced By]
--------------------------------------------------------------------------------

                              U.S. Patent Documents
4448890            May., 1984              Smetana et al.           436/508.
-------
4746539            May., 1988              Webb et al.                435/7.
-------
                            Foreign Patent Documents
0 462 623 A1       Dec., 1991                 EP.
0 582 477 A1       Feb., 1994                 EP.

                                Other References

Result 7, "Human Retinoic Acid Receptor . . . " M57707.
Hanausek-Walaszk et al., "Carcinogenesis," Proceedings of the American Associa-
tion for Cancer Research 30:190, Abstract 754 (1989).
Larroya et al., "Immunology," Proceedings of the American Association for Cancer
Research 30:349, Abstract 1385 (1989).
Hanausek-Walaszek et al., "Carcinogenesis," Proceedings of AACR 29:167, Abstract
665 (1988). Hanausek-Walaszek et al., "Correspondence Between Biochemical and
Antigenic Activity of a 60 Kilodalton Oncofetal Protein During Carcinogenesis
and Tumorigenesis," Cancer Letters 33:55-61 (1986).
Hanausek-Walaszek et al., "A 60 Kilodalton Oncofetal Protein As Tumor Marker,"
J. Med. 17:13-23 (1986).
Schroder et al., "Proteins from rat lover cytosol which stimulate mRNA
transport," Eur. J. Biochem. 159:51-59 (1986).

<PAGE>

Hanausek-Walaszek et al., "Immunological Identity of a 60 KD Onmcofetal Protein
Induced in Rats by Chemical Carcinogens and Released by Transformed Cells," BBRC
127:779-785 (1985). Hanusek-Walaszek et al., "Chemical carcinogens as specific
inducers of a 60-kilodalton oncofetal protein in rats," Carcinogenesis
7:1725-1730 (1985).
Schumm et al., "Abscence of the Cancer-Associated Factor with a Molecular Weight
of 60,000 from the Plasma of Patients with a Spectrum of Nonneoplastic
Conditions," Cancer Res. 44:401-406 (1984).
Hanausek-Walaszek et al., "Characterization of a 60,000-dalton Oncofetal Protein
from the Plasma of Tumor-Bearing Rats," Cancer Invest. 2:433-441 (1984). French
et al., "Nucleocytoplasmic Release of Repetitive DNA Transcripts in
Carcinogenesis Correlates with a 60 Kilodalton Cytoplasmic Protein," Cancer
Letters 23:45-52 (1984). Walaszek et al., "An Oncofetal 60-Kilodalton Protein in
the Plasma of Tumor-Bearing and Carcinogen-Treated Rats," Cancer Letters
20:277-282 (1983).
Hanausek-Walaszek et al., "Structural and Immunological Identity of p65 Tumor-
Associated Factors from Rat and mouse Hepatocarcinomas," Progress in Clinical
and Biological Research (1989). Mirowski et al., "Purification and Characteriza-
tion of a 65-kDa Tumor-Associated Phosphoprotein from rat Transplantable
Hepatocellular Carcinoma 1682C Cell Line," Protein Exp. and Pur. 3:196-203
(1992).
Wang et al., "Monoclonal Antibodies Against a 65-kDa Tumor-Associated
Phosphoprotein: Development and Use in Cancer Detection," Hybridoma 12:167-176
(1993). Mirowski et al., "Demonstration of a 65 kDa Tumor-Specific
Phospho-protein in Urine and Serum of Rats with N-methyl-N-nitrosourea-induced
Mammary Adenocarcinomas," Carcinogenesis 14:1659-1664 (1993).
Mirowski et al., "Comparative Structural Analysis of Human and Rat 65 kDa Tumor-
Associated Phosphoproteins," Int. J. Biochem. 25:1865-1871 (1993).
Del Rio et al., "Expression of a 65 kDa Oncofetal Phosphoprotein in the Altered
Hepaic Foci of Rats Fed 2-Acetylaminofluorene Followed by Phenobarbital," Int.
J. Oncol. 5:259-265 (1994). Mirowski et al., "Serological and Immunohisto-
chemical Detection of a 65-kDa Oncofetal Protein in Breast Cancer," European
Journal of Cancer 30A:1108-1113 (1994).
Hanausek et al., "The Oncofetal Protein p65 as a New Member of the Steroid/
Thyroid Receptor Superfamily," Cancer Detection and Prevention 19:1995, Abstract
118/307 307 (1995).
Mirowski et al., "Isolation of the 65 kDa Oncofetal Phosphoprotein from Fetal
Bovine Serum, Polyclonal Antibody Production and use in Cancer Detection," AACR
(/toronto, Mar. 20, 1995). Hanausek et al., "The Oncofetal Protein p65 as a
Member of the Steroid/Thyroid Receptor Superfamily: Relevance to Breast Cancer,"
AACR (Toronto, Mar. 20, 1995).

<PAGE>

Primary Examiner: Woodward; Miichael P.
Attorney, Agent or Firm: Arnold, White & Durkee

--------------------------------------------------------------------------------
                               Goverment Interests
--------------------------------------------------------------------------------

GOVERNMENT RIGHTS

The United States Government may have certain rights to this invention pursuant
to National Institutes of Health grants RR 5511-23 and CA 54296.

--------------------------------------------------------------------------------
                                Parent Case Text
--------------------------------------------------------------------------------

RELATED APPLICATION

The present application is a continuation-in-part of U.S. patent application
Ser. No. 08/236,547, filed May 2, 1994, abandoned, which was a divisional
application of U.S. patent application Ser. No. 08/012,972, filed Feb. 2, 1993,
issued as a U.S. Pat. No. 5,310,653 on May 10, 1994, which was a continuation of
U.S. patent application Ser. No. 07/426,408, filed Oct. 24, 1989, abandoned.

--------------------------------------------------------------------------------
                                     Claims
--------------------------------------------------------------------------------

What is claimed is:

1. A nucleic acid molecule of less than 2200 base pairs (DNA) or less than 2200
bases (RNA) substantially free of other proteinaceous and nucleic acid
materials, wherein said molecule is selected from the group consisting of:

a. a DNA segment comprising a sequence region having the sequence of SEQ ID NO:
1; and

b. an RNA segment comprising a sequence region having the sequence of SEQ ID NO:
1 with a proviso that in said RNA molecule, the deoxynucleotides A, G, C and T
of SEQ ID NO: 1 are replaced by ribonucleotides A, G, C and U, respectively.

2. The nucleic acid molecule of claim 1 substantially free of other
proteinaceous and nucleic acid materials, wherein said molecule is selected from
the group consisting of:

a. a DNA molecule having a sequence of SEQ ID NO: 1; and

<PAGE>

b. an RNA molecule having a sequence of SEQ ID NO: 1 with a proviso that said
RNA molecule, the deoxynocleotides A, G, C and T of SEQ ID NO: 1 are replaced by
ribonucleotides A, G, C and U, respectively.

3. The nucleic acid molecule of claim 1, wherein said molecule is a DNA segment
and is under the control of a promoter.

4. The nucleic acid molecule of claim 1, wherein said molecule is a DNA segment
and is incorporated into a DNA plasmid.

5. The nucleic acid molecule of claim 1, wherein said molecule is a DNA segment
incorporated into a host cell to form a recombinant host cell.

6. The host cell of claim 5, further defined as a prokaryotic host cell.

7. The host cell of claim 6, wherein the prokaryotic cell is E. coli.

8. The host cell of claim 5, further defined as a eukaryotic host cell.

9. A protein molecule substantially free of other proteinaceous and nucleic acid
materials, said protein molecule having the amino acid sequence of SEQ ID NO: 2.

10. A method of using a DNA segment having the sequence of SEQ ID NO: 1
comprising the steps of:

a. preparing a recombinant vector in which the sequence region having the
sequence of SEQ ID NO: 1 is positioned under the control of a promoter;

b. introducing said recombinant vector into a host cell to generate a
recombinant host cell;

c. culturing the recombinant host cell under conditions effective to allow
expression of the protein encoded by the sequence region having the sequence of
SEQ ID NO: 1; and

d. collecting the protein encoded by SEQ ID NO: 1.

11. A recombinant p65 protein prepared by expressing SEQ ID NO: 1 in a
recombinant host cell in accordance with claim 10 and purifying the expressed
protein away from total recombinant host cell components.

12. A nucleic acid molecule comprising a sequence region that consists of at
least a 20 nucleotide long continuous sequence that hybridizes under stringent
conditions to a contiguous sequence of SEQ ID NO: 1 from nucleic acid position 1
to nucleic acid position 1035.

<PAGE>

13. A method for detecting the presence of nucleic acids encoding for the p65
oncofetal protein in a mammalian sample, comprising the steps of:

a. obtaining nucleic acids from a sample suspected of containing the p65
oncofetal protein;

b. isolating nucleic acids from said sample;

c. contacting said nucleic acids with a nucleic acid segment of at least a 20
nucleotide long continuous sequence that hybridizes under stringent conditions
to a contiguous sequence of SEQ ID NO: 1 from nucleic acid position 1 to nucleic
acid position 1035 under conditions effective to allow hybridization of
substantially complementary nucleic acids; and

d. detecting the hybridized complementary nucleic acids thus formed.

14. The method of claim 13, wherein the sample nucleic acids contacted are
located within a cell.

15. The method of claim 13, wherein the sample nucleic acids are separated from
a cell prior to contact.

16. The method of claim 15, wherein the sample nucleic acids are DNA.

17. The method of claim 15, wherein the sample nucleic acids are RNA.

18. A method to aid in the diagnosis of a cancer which produces a 65 kD tumor-
associated protein in a mammal comprising:

a. providing a sample of nucleic acids from the biological material of said
mammal wherein the biological material is plasma, serum, urine, saliva, cystol
fluid, ascites or tissue;

b. contacting said sample nucleic acids with a nucleic acid segment of at least
a 20 nucleotide long continuous sequence that hybridizes under stringent
conditions to a contiguous sequence of SEQ ID NO: 1 from nucleic acid position 1
to nucleic acid position 1035 under conditions effective to allow hybridization
of substantially complementary nucleic acids; and

c. detecting the hybridized complementary nucleic acids thus formed.

19. A method for detecting p65 oncofetal protein in a mammalian sample
comprising the steps of:

a. preparing an antibody preparation to a purified protein p65 of claim 9;

<PAGE>

b. selecting an antibody set that is immunoreactive to p65 protein, and not
immunoreactive to estrogen receptor protein;

c. providing a mammalian sample comprising plasma, serum, urine, saliva, cystic
fluid, ascites, or tissue;

d. contacting said sample and said antibody set to form an immunoconjugate; and

e. detecting said immunoconjugate.

20. The method of claim 19, wherein said antibody preparation is polyclonal,
monoclonal, or monospecific.

21. The nucleic acid molecule of claim 12, further defined as having the
sequence of SEQ ID NO: 1.

22. The method of claim 13, wherein the nucleic acid segment has the sequence of
SEQ ID NO:1.

23. A method for detecting p65 oncofetal protein in a mammalian sample
comprising the steps of:

a. preparing an antibody preparation to the N-terminal half of the p65 protein;

b. providing a mammalian sample comprising plasma, serum, urine, saliva, cystic
fluid, ascites, or tissue;

c. contacting said sample and said antibody preparation to form an
immunoconjugate; and

d. detecting said immunoconjugate.

24. The method of claim 23, wherein said antibody preparation is polyclonal,
monoclonal, or monospecific.

--------------------------------------------------------------------------------
                                   Description
--------------------------------------------------------------------------------

FIELD OF THE INVENTION

This invention relates to the isolation, identification and sequencing of a
cancer associated protein, preparation of hybridization probes therefrom,
preparation of antibodies thereto, and methods of cancer risk assessment and
diagnosis.

<PAGE>

BACKGROUND OF THE INVENTION

In spite of improved treatments for certain forms of cancer, it is still a
leading cause of death in the United States. Since the chance for complete
remission of cancer is, in most cases, greatly enhanced by early diagnosis, it
is very desirable that physicians be able to detect cancers before a substantial
tumor develops. Also, in cases where the primary tumor has been substantially
removed by surgery or destroyed by other means, it is important that the
physician be capable of detecting any trace of cancer in the patient (either in
the form of residues of the primary tumor or of secondary tumors caused by
metastasis), in order that the physician can prescribe appropriate subsequent
treatment, such as chemotherapy.

The quantities of cancer cells that must be detected for early diagnosis or
following removal or destruction of the primary tumor are so small that the
physician cannot rely upon physical examination of the cancer site. Moreover, in
many cases the cancer site is of course not susceptible to direct visual
observation and it is almost always impractical to detect secondary tumors by
visual observation, since it is not possible to predict exactly where they are
likely to occur. Accordingly, sensitive tests have to rely upon detection of
cancer-associated materials, usually proteins, present in body fluids of
patients who have, or are about to develop, cancer cells in their bodies.
Several diagnostic materials for detection of cancer-associated proteins are
available commercially. Tests for alpha-fetoprotein are used to detect primary
liver cancer and teratocarcinoma in humans; and carcinoembryonic antigen is used
for digestive system cancers, as well as lung and breast carcinomas; chorionic
gonadotropin is employed to detect trophoblast and germ cell cancers; calcitonin
is used for thyroid gland cancers; and prostatic acid phosphatase or prostate
specific antigen are used to detect prostate carcinoma. These markers are
detectable in advanced rather than in early cancer.

Unfortunately, many of the commercially available tests are only applicable to a
narrow range of cancer types, and therefore these tests suffer not only from the
disadvantage that other types of cancer may be missed but also from the
disadvantage that the narrow applicability of the tests means that it may be
necessary to run multiple tests on a single patient for diagnostic purposes, a
procedure which not only increases the expense of the diagnostic testing but
also increases the risk that one or other of the tests may give a false positive
result. Accordingly, there is a need for a single diagnostic test able to detect
the presence of very small amounts of cells of a wide variety of different
cancers. The ideal marker would be one that is specific and universal. Such a
marker may exist if malignant transformation is associated with the expression
of a unique gene product in all kinds of transformed cells.

It is already known that serum from the blood of animals suffering from a wide
variety of cancers contains an oncofetal protein having a molecular weight of
approximately 60,000 and having the capacity to increase the release of
ribonucleic acid (RNA) from cell nuclei. This protein is referred to as
oncofetal RNA-transport protein (ORTP) or 60 kDa cancer-associated protein.

<PAGE>

ORTP is localized in the cytoplasm of tumors of humans and experimental animals
and small amounts are released into the host circulatory system. The 60 kDa ORTP
is notably absent from the nuclei of rat liver and rat liver tumors. It has been
shown to be present in fetal rats at 18 days of gestation and in human and rat
amniotic fluid, but not in maternal blood. It has not been detected in adult
rats. Nor is it present in detectable concentrations in the blood of normal
human subjects or those with a variety of non-neoplastic conditions or diseases,
including benign tumors and other non-neoplastic proliferative diseases. In
contrast, of more than 200 cancer patients with confirmed active disease, all
tested positive for the factor. It was also present in all of about 200
tumor-bearing rats tested. Unfortunately, antibodies to a rat ORTP preparation
purified as described in the prior art do not cross-react with human ORTP. Thus,
the 60 kDa cancer marker proteins from different species are not immunologically
equivalent, e.g., an antibody to the rat cancer marker protein does not
cross-react with a human cancer marker protein. Thus, when the purified 60 kDa
cancer marker protein preparation is to be used for production of antibodies for
diagnostic purposes, it is necessary to begin the preparation process with
plasma from the species in which the diagnosis is to be used.

We have recently identified, characterized in terms of its physical properties,
and sequenced another oncofetal protein with a molecular weight of 65 kDa (p65)
which exhibits certain properties which strongly favor its candidacy as a
general tumor marker, as well as a marker of cancer risk associated with the
prolonged use of drugs, such as androgenic and estrogenic hormones, that have
tumor promotional potential.

This newly isolated and sequenced 65 kDa oncofetal protein, termed p65, exhibits
properties desirable for a marker of pre-malignant and malignant alterations.
The p65 protein is a novel protein that appears to have some homology to the
steroid receptor superfamily of genes. Therefore, the p65 gene may belong to a
family of genes which encode nuclear receptors, composed of several domains
important in hormone binding, DNA-binding, dimerization, and activation of
transcription for various hydrophobic ligands such as steroids, vitamin D,
retinoic acid and thyroid hormones. More support for this theory is evident by
the discovery that the p65 protein is located in nuclei of tumor cells and has
DNA-binding properties. Alterations in hormone receptors such as estrogen (ER)
and progesterone receptor (PR) may be of prognostic significance in breast
cancer; therefore, elucidation of whether p65 is related to ER or PR is of
utmost importance. Elevated serum levels of p65 are detectable by ELISA in 90%
of patients with stage I-IV of breast cancer and limited immunohistochemical
studies have shown nuclear and cytoplasmic expression in 80% of breast cancer
biopsies. In depth study of the p65 protein is thus helpful in understanding the
mechanisms by which hormones and their respective receptors regulate the
metabolism of normal and malignant breast and prostate cancer as well as other
cancers.

The present invention, specifically the elucidation of p65's sequence can be
utilized for the detection, diagnosis and hopefully, the future treatment of
breast, prostate, ovarian and possibly other cancers that relate to the steroid
receptor superfamily of genes.

<PAGE>

SUMMARY OF THE INVENTION

This invention provides a protein preparation containing a pure form of a newly
discovered oncofetal cancer marker protein encoded by the nucleic acid sequence
SEQ ID NO: 1 and having the amino acid sequence of SEQ ID NO: 2. More
specifically, the invention provides the sequence of and methods of using the
nucleic acid sequence SEO ID NO: 1 and the rat p65 cancer-associated protein
having the amino acid sequence SEQ ID NO:2 for the preparation of hybridization
probes and antibodies to be used for the detection of cancer.

One embodiment of the present invention provides a nucleic acid molecule of less
than 2200 base pairs in length coding for the rat p65 protein. An aspect of this
embodiment provides the DNA segment comprising a sequence region having the
sequence of SEQ ID NO: 1. Another aspect provides the analogous RNA segment. And
yet a further aspect provides the amino acid sequence having the sequence of SEQ
ID NO: 2.

The invention also provides that the DNA segment comprising a sequence region
having the sequence of SEQ ID NO: 1 be placed under the control of a promoter.
The DNA segment can also be placed into a plasmid and additionally into a
recombinant host cell.

The present invention further provides a method of using the DNA segment having
the sequence of SEQ ID NO: 1 to make the encoded rat p65 comprising:

(a) preparing a recombinant vector in which the sequence of SEQ ID NO: 1 is
positioned under the control of a promoter;

(b) introducing the recombinant vector into a host cell to form a recombinant
host cell;

(c) culturing the recombinant host cell under conditions effective to allow
expression of the protein; and

(d) collecting the expressed protein.

The invention additionally provides a recombinant rat p65 protein prepared by
expressing the rat p65 protein in a recombinant host cell and purifying the
protein.

In another embodiment, the present invention provides a sequence region
complementary to a region of at least 10-14 contiguous nucleotides of SEQ ID NO:
1.

The invention further provides a method for detecting p65 and oncofetal protein
in a sample of mammalian origin comprising:

(a) obtaining nucleic acids from a sample suspected of containing the p65
protein;

(b) contacting the sample nucleic acids with a nucleic acid segment of at least
10-14 nucleotide long continuous sequence that is complementary to a contiguous
sequence of SEQ ID NO: 1 under appropriate conditions for hybridization; and

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(c) detecting the hybridized nucleic acids thus formed.

In one aspect of the aforementioned embodiment the sample nucleic acids are
located within a cell such as in the case of in situ hybridization.
Alternatively, the sample nucleic acids are separated from a cell and then
tested for the presence of a sequence complementary to the 10-14 nucleotide long
sequence. The nucleic acids to be tested can be of DNA (Southern blotting) or
RNA (Northern blotting).

Finally, this invention provides a method for assessing the likelihood of cancer
which involves immunoassays and hybridization assays to detect the presence of
the instant p65 tumor marker protein in biological material of a host suspected
of developing cancer or being at high risk for developing cancer as the result
of treatment with drug(s) known to have a tumor promotion potential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows p65 tumor-associated protein production during 7,12-dimethylbenz>
ajanthracene (DMBA)-induced, and 12-0-tetra-decanoylphorbol-13-acetate (TPA)-
promoted skin carcinogenesis in SENCAR mice.

FIG. 1A shows the time course of papilloma appearance following carcinogenesis
promotion and FIG. 1B shows the time course of p65 accumulation in the blood
plasma following the same carcinogenesis promotion.

FIG. 2 shows enhancement by synthetic steroid hormone of p65 tumor-associated
protein production during chemical carcinogenesis.

FIG. 3 shows zinc finger structure within p65. The amino acids abbreviated with
three letters represent conserved regions of zinc fingers among family members.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods for Diagnosing or Assessing the Likelihood of Cancer

Samples of blood plasma or serum tissue are obtained from human cancer patients
or human subjects prior to and at different times in the course of treatment
with synthetic steroid hormones or other drugs known or suspected of having a
tumor promotion potential. Alternatively, samples of blood plasma, serum or
tissue are obtained from other mammals suspected of suffering from cancer or
treated with substances suspected of causing cancer.

Briefly, samples of the blood plasma or serum containing the same amount of
protein are mixed with an electrophoresis sample buffer, boiled for three
minutes and subjected to 12.5% SDS-PAGE. Subsequently, the gel slabs are
prepared for transfer by equilibrating for one hour in 0.025M Tris, 0.192M
glycine, 20% (v/v) methanol, pH 8.3, and then transblotted onto nitrocellulose
sheets. To conduct the immunoassay, the nitrocellulose sheets are treated with
appropriate blocking solution to block unspecific binding sites and incubated

<PAGE>

overnight, either with pre-immune serum (controls) or antibody against the p65
protein diluted 1:200. Secondary biotinylated antibody is applied next and the
color is developed using ABC Elite Kit from Vector Laboratories, Burlingame,
Calif. The highly specific polyclonal antibody against p65 is obtained by
immunizing rabbit with pure human or rat p65 preparations. As an alternative,
mouse monoclonal antibodies to p65 may be prepared using the techniques
generally described in "Hybridoma Techniques" Cold Spring Harbor, N.Y., 1980,
ISBN 0-87969-143-3.

The immunoblots are photographed and the bands of p65 immune complexes on the
film are quantitated using a laser densitometer coupled to an integrator.
Alternatively, following probing with the antibodies to p65 or pre-immune serum,
nitrocellulose sheets are labeled with .sup.125 I-protein A. The labeled sheets
are subjected to autoradiography followed by scanning the film with a laser
densitometer coupled to an integrator. Another alternative is to use an ELISA
procedure.

To interpret the plasma or serum or tissue samples, the relative quantity of p65
in the sample is indicated by the intensity of a band at nominal molecular
weight about 65,000 as measured by laser scanning. Comparison with the
corresponding area of the control (derived with the use of pre-immune serum)
permits distinguishing response specific for p65 above non-specific background
response. Specific activity of the marker band from clinical samples taken in
the course of treatment is compared with the samples taken prior to treatment
and/or with the average sample from a normal healthy pool. The clinical response
can be then expressed as a factor against the response prior to treatment or
against the response of the normal healthy pool, respectively. Steady increases
in the value of clinical response over a period of time are indicative of an
increased cancer risk associated with the long-term treatment with a given
synthetic steroid or other drug that has tumor promotional properties. The high
initial response is indicative of an existing cancer.

EXAMPLE I

Animal Models for Determination of the Presence of Cancer Cells

This example illustrates the instant process for purification of the p65
tumor-associated protein and preparation of antibodies thereto as source of p65.

Rat and Mouse Liver Tumors

Rat hepatoma cell lines McA-RH7777 and McA-RH8994 were purchased from the
American Type Culture Collection (Rockville, Md.) and carried as cell cultures
in Swim's S77 medium with 4 mM L-glutamine and supplemented with 5% fetal calf
serum and 20% horse serum (Gibco, Grand Island, N.Y.). After several passages in
cell culture, rat hepatoma cells (1.times.10.sup.6 /0.2 ml phosphate-buffered
saline) were inoculated subcutaneously into a hind leg of male Buffalo rats
(120-150 g) (Harlan Labs, Indianapolis, Ind.) and were carried as solid tumors
according to conventional procedures.

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Transplantable hepatocellular carcinoma (THC) 1682C was derived from primary
hepatic tumors in ACI rats maintained for four months on the choline-deficient
diet of Shinozuka containing 0.2% ethionine and eight months on a
choline-supplemented (0.8%) diet without ethionine. THC T52 was established by
intraperitoneal transplantation of growing tumors induced in ACI rats by
2-acetylaminofluorene (AAF). THC cultures were maintained in vitro and also
carried as solid (1682C) and ascitic (T52) tumors in male ACI rats (The
University of Texas M.D. Anderson Cancer Center Science Park-Veterinary
Resources Division, Bastrop, Tex.).

The Reuber hepatoma cell culture H35 was provided by Dr. Andrew P. Butler,
University of Texas M.D. Anderson Cancer Center Carcinogenesis Department,
Science Park, Smithville, Tex.

Mouse liver carcinoma CRL 6421 (formerly NBL #MM45T.Li) was obtained from
American Type Culture Collection and carried as cell cultures as described above
for rat hepatoma cell lines.

Purification of p65

Preparation of the antibody involves purification of the p65 marker protein from
the tissue culture medium of tumor cells of human or animal origin.
Specifically, the cells were grown to confluence at 37.degree. C. in the
presence of media and a serum or serum-like supplement, or completely defined
medium containing salts and hormones. Growing cells to confluence was a purely
economical step which ascertains a high yield of the p65 marker protein. The
preferred medium used was Dulbeccols Modified Eagle's Medium (DMEM) with 5%
fetal calf serum and cells were grown in 5% CO.sub.2. The confluent cells were
washed three times in serum-free DMEM medium and incubated in this medium for
16-24 hours (the optimal time being 24 hours). At this point, medium was
collected by centrifugation at 10,000 x g for 10-15 minutes and treated with
ammonium sulfate solution at 90% saturation of aqueous solution for 30 minutes
at 4.degree. C.

Protein precipitate was collected by 30-minute centrifugation at 10,000x g,
dissolved in a small volume of 50 mM Tris-Cl buffer, pH 7.5, with 50 mM NaCl, 10
mM 2-mercaptoethanol and 1 mM EDTA, and dialyzed overnight against the same
buffer. After dialysis, proteins were loaded on an LKB TSK 3000 SW molecular
sieving column and separated according to the molecular weight using an LKB HPLC
system. Fractions containing proteins with molecular weight in the range of
50-90 kDa were collected and dialyzed overnight against buffer A, containing 20%
ammonium sulfate in 50 mM Tris-Cl, pH 7.5 with 10 mM 2-mercaptoethanol and 1 mM
EDTA.

Following dialysis, the 50-90 kDa proteins were loaded on a phenyl hydrophobic
interaction column (PHI) (LKB, Pharmacia) equilibrated with buffer A. Generally,
800-1,000 ml of conditioned medium was processed as described above. The p65
marker protein weakly binds to the PHI column and is eluted in the first

<PAGE>

distinct peak by a gradient of 80% to 0% of buffer A, in combination with buffer
B (50 mM Tris-Cl, pH 7.5 with 10 mM 2-mercaptoethanol and 1 mM EDTA,
supplemented with 50% ethylene glycol). Fractions containing the p65 marker
protein are then combined and dialyzed against buffer A devoid of ammonium
sulfate, concentrated by lyophilization and electrophoresed on 12.5% SDS-PAGE at
constant current of a 10 mA for two hours at room temperature.

Proteins are transblotted to a 0.22.mu. nitrocellulose sheet as described by
Towbin et al. (Proc. Natl. Acad. Sci. U.S.A., Vol. 76, pages 4350-4353, 1979).
A reversible Ponceau A stain was used to visualize the nitrocellulose bound
proteins according to the procedure of Salinovich and Montellaro (Anal.
Biochem., Vol. 156, pages 341-347, 1986). The band of the p65 protein was cut
out of the nitrocellulose sheets and used for immunization of rabbits.

Mouse p65 was isolated from the mouse liver carcinoma cell line CRL 6421 (MM45)
and purified as described above for the rat factor.

Preparation of Antibodies to p65

Antisera to the purified rat p65 preparation were raised in rabbits, as follows.

Specific Pathogen Free (Pasteurella) male New Zealand white rabbits of
approximately four kilograms body weight were used (Myrtle Is Rabbitry).
Pre-immune (day 0) and test blood samples (10 days following each of four
immunizations on day 1, 14, 28 and 42) were obtained from the central artery of
the ear or a lateral ear vein. The anesthetized rabbits were placed in sternal
recumbency, the dorsal fur was removed with surgical clippers, and the surgical
site was aseptically prepared with provodine-iodine (Wescodyne, West Chemical)
followed by 70% ethanol. Six incisions about 1.0 cm each were made in an
anteroposterior direction through the skin and subcutis with a scalpel. The
incisions were undermined with blunt and sharp dissection to allow implant
placement over the superficial epaxial musculature. The nitrocellulose strip
containing the rat p65 protein was cut into six pieces (each approximately 0.5
cm.times.1.5 cm) which were then formed into rolls for insertion. Following
placement, the skin edges were opposed with tissue forceps and closed with
surgical adhesive (Vetbond, 3M). Adequate spacing (about 2.5 cm) between
insertion fields will allow subsequent immunization incisions to be made
adjacently. Terminal blood collection was made in Alsever's solution in
anesthetized rabbits by the use of a vacuum assisted collection device to
provide the serum which is the polyclonal antibody source. The antisera were
absorbed with normal plasma proteins immobilized on nitrocellulose sheets
following its electrophoresis on 10% SDS-PAGE and transblotting. An ELISA assay
or immunoblotting analysis was conducted to determine the potency and
specificity of the antisera obtained.

A standard ELISA procedure was used for detection of specific antibodies in
serum. Ninety-six well microliter plates designed for ELISA were used (Immunol
2, Dynatech). For detection of rabbit anti-p65 antibodies, the ELISA plates were

<PAGE>

pre-coated with several different concentrations of antigen. To test sera, a
positive reference serum and a negative pre-immune serum were added to the wells
in five-fold dilutions in PBS. Anti-p65 antibodies were detected by goat
anti-rabbit IgG conjugated with horse radish peroxidase (Bio-Rad). After the
substrate reaction, plates were read on an ELISA plate reader at 405 nm (Litton
Bionetics, Laboratory Product Division, South Carolina). A serum sample was
considered positive when it read 0.05 units or more above the background.

Probing of Western blots with prepared antibody was carried as follows.

The purified rat tumor-associated protein, p65, was separated by PAGE and
electrophoretically transferred to nitrocellulose as described above. Free
binding sites on the nitro-cellulose sheets were then blocked overnight using 1
% normal goat serum in TTBS buffer (0.5% Tween, 0.1 mM Tris-HCl, pH 7.1, 09%
saline). Antisera obtained from immunized rabbits were diluted serially in the
blot buffer (TTBS) and incubated with the nitro- cellulose strips for one hour
at room temperature. The blots were then washed with several changes of TTBS
buffer containing 1% goat serum.

Bound antibody was detected using biotinylated second antibody (goat anti-rabbit
IgG, biotinylated) and the avidin- biotin-peroxidase method (Vectastain ABC
Elite, Vector, Burlingame, Calif.). To develop blots, 0.02% hydrogen peroxide
was used, mixed with 0.1% diaminobenzidine tetrachloride (DAB) made in 0.1M
Tris-HCl buffer, pH 7.2. Color generally developed within five to ten minutes;
blots were rinsed with distilled water and air dried to preserve color.

Alternatively, bound antibody was detected by incubating the nitrocellulose
strips with .sup.125 I-Protein A (1.times.10.sup.6 cpm/ml of blot buffer). After
a 60-minute incubation, unbound label was removed by repeated washes of the
blots with PBS buffer containing 0.5% Tween. The bound antigen .sup.125
I-Protein A complex was detected by overnight autoradiography using Kodak
X-OMAT-AR film.

In addition, monoclonal antibody preparations can be made to the 65 kDa cancer
marker by employing conventional techniques well known to the art.

EXAMPLE II

SDS-PAGE and Immunoblotting Analyses

Carefully dissected hepatomas (see Example I) and liver fragments from normal
rats and mice were rinsed with an ice-cold saline solution and processed for
immunochemical determination of p65. Liver tumor cells grown in culture, as
described in Example I, were separated from medium by centrifugation, rinsed
with the cold saline solution, and processed for immunoassay of the p65
tumor-associated factor. Briefly, small pieces of tissues or tumor cell pellets
were homogenized in TMK-sucrose buffer, pH 7.2, and samples of the total

<PAGE>

homogenate containing the same amount of protein were mixed with electrophoresis
sample buffer, boiled for three minutes, and subjected to 10% SDS-PAGE. The
proteins precipitated at 90% saturation of ammonium sulfate from the conditioned
medium of hepatocarcinoma cells were dissolved in TMK buffer and electrophoresed
in a similar manner. Subsequently, the gel slabs were prepared for transfer by
equilibration for one hour in 0.025M Tris, 0.192M glycine, 20% methanol
(vol/vol), pH 8.3, and then transblotted onto nitrocellulose sheets.

For immunoassay, the nitrocellulose sheets were treated with the appropriate
blocking solution to block non-specific binding sites and incubated overnight,
either with pre-immune serum (controls) or antibody against rat p65 diluted
1:200. Secondary biotinylated antibody was applied next, and the color was
developed Kit (Vector Laboratories, Burlingame, Calif.). The highly specific
polyclonal antibody against p65 was obtained by immunizing rabbits with the rat
p65 preparation purified as described above.

Presence of p65 in Rat and Mouse Liver Tumors and its Absence from Normal Mouse
and Rat Liver

When total protein samples from rat and mouse liver carcinomas grown in vitro
were analyzed by SDS-PAGE and then anti-rat p65 antibody probing of Western
blots, a single, prominent band was detected in the 65 kDa region of the blots.
Some weak bands seen in the 60-64 kDa region may represent degradation products
of the native species of p65. Other minor bands resulted from non-specific
staining. There is no band characteristic of p65 in immunoblots representing
normal adult rat or mouse liver.

All hepatocarcinoma cells grown in vitro as cell cultures and tumor tissues from
Morris hepatomas carried as solid tumors in vivo were positive for the p65
antigen. There was no reaction with normal liver cells of either rat or mouse
origin. Thus, immunoblotting analysis has demonstrated that p65 was specifically
produced by liver cancer cells but not by the cells of normal adult rat or mouse
liver.

EXAMPLE III

Interspecies cross-reactivity of Antibodies Against p65

The p65 tumor-associated factors derived from different species are
immunologically cross-reactive.

                  TABLE 1
    --------------------------------------
    Immunoprecipitation of p65 from Different Sources by Antibodies
    Raised in Rabbits against Rat p65.
    Source of p65     Immunoprecipitation of p65.sup.a
    --------------------------------------
    Plasma:
    Morris Hepatoma 7777-bearing Rats
                      +++
    Morris Hepatoma 8994-bearing Rats
                      +++

<PAGE>

    Pregnant Rats     -
    Normal Rats       -
    Cytosol:
    Morris Hepatoma 7777 Tumors
                      +++
    Morris Hepatoma 8994 Tumors
                      +++
    Normal Rat Liver  -
    Conditioned Culture Medium:
    Morris Hepatoma 7777 Cells
                      +++
    Morris Hepatoma 8994 Cells
                      +++
    Rat THC 1682 Cells
                      +++
    Mouse Squamous Cell Carcinoma
                      ++
    Human Breast Cancer Cells (MCF-7)
                      ++
    Unconditioned Culture Medium
                      -
    --------------------------------------
     *+++ = over 90% precipitation; ++ = 50-90% precipitation
     - = less than 25% precipitation

The p65 activity as measured by the mRNA-transport assay was immunoprecipitated
using polyclonal anti-rat p65 antibodies from the serum of rabbits immunized
against the rat transplantable hepatocellular carcinoma THC 1682. This antibody
removed activity from cytosols derived from Morris Hepatomas 7777 and 8994 and
from cell culture media in which tumor cells were grown. Polyclonal anti-rat p65
antibodies reacted with human p65 secreted to the culture medium by the MCF-7
breast cancer cell line. The anti-bodies also cross-reacted with the mouse p65
factor secreted to medium by mouse squamous cell carcinoma. The p65
tumor-associated factor was not detected in the blood of pregnant rats. It was
neither detected in the blood of normal rats nor in unconditioned cell culture
medium.

EXAMPLE IV

CNBR Cleavage of p65

Cyanogen bromide (CNBR) cleavage maps of p65 purified from cell culture medium
of rat transplantable hepatocellular carcinoma cell line 1682C or mouse liver
carcinoma cell line CRL 6421 (MM45) were obtained as follows. p65 preparations
(20.mu.) purified from cell culture medium were subjected to 12.5% sodium
deodecyl sulfate (SDS)-PAGE. The p65 band located on slab gels by Coomassie blue
staining of parallel gel tracks was cut from appropriate gel tracks and
incubated at room temperature for 16 hours in 1 ml of 88% formic acid containing
20 mg/ml of CNBR. The gel slices were then rinsed five times with 1 ml of water
and washed several times for 10 minutes with 1 ml 120 mM Tris-HCl, pH 7.0, 20%
glycerol (vol/vol), 2% SOS until the pH of the slices reached 7.0. CNBr-treated
gel slices were placed onto 15% SDS- PAGE slabs, electrophoresed, and then

<PAGE>

stained with silver. Slices of gel containing p65 treated with 88% formic acid
served as controls.

Structural Identity of Rat and Mouse p65

Rat and mouse p65 were purified to apparent homogeneity as described above. CNBR
cleavage maps were obtained as the first step toward the final characterization
of the amino acid composition and sequence of p65. The cleavage of p65 with CNBR
resulted in six major peptides identifiable by silver staining of the SDS-PAGE
gels. The peptides have molecular weights of about 6, 9, 270, 39, 43 and 47 kDa.
Identical cleavage maps were obtained for rat and mouse p65.

EXAMPLE V

Animal Models for Determination of Cancer Risk

The ultimate objective of animal carcinogenicity studies is the determination of
possible human risk. The available data show that known human carcinogens that
have been adequately studied are also carcinogenic in laboratory animals, often
at the same target site. Thus, the identification and elucidation of the
mechanisms underlying each stage of the carcinogenic process in animals may
offer testable hypothesis for the stages in human. The current tests for cancer
risk assessment focus mainly on markers of genetic damage, at the level of the
DNA or chromosome, as indicators of genotoxic exposure. These tests, however,
are not able to detect the risk associated with exposure to synthetic steroid
hormones and other tumor promoters.

It was recently indicated that the promotional status of human subpopulations
could be the dominant factor in determining the cancer risk. The development of
a more systematic analysis of possible tumorigenesis mechanism has also been
suggested. One of the approaches to study in a more systematic way the mechanism
of tumorigenesis, involves comparisons among different systems in which tumor
induction, or cell transformation has been optimized through the use of the most
effective system-specific agents and protocols. When each tumor system is
operating optimally, intersystem comparisons could be undertaken with respect to
carefully selected biochemical parameters. Tumor-associated proteins, such as
the one described herein which appears to be a general marker of preneoplastic
and neoplastic alterations, are good candidates to be used in intersystem
comparisons.

Monitoring Skin Carcinogenesis with p65 Tumor-Associated Factor

A multistage skin carcinogenesis model was used to monitor the carcinogenic
process using the p65 tumor-associated marker.

Skin tumors were induced on the back of SENCAR mice by a single dose of 10 nmol
of 7,12-dimethylbenz>a!anthracene (DMBA) and repetitive applications of 1 .mu.g
of 12-0-tetradecanoylphorbol-13-acetate (TPA) twice a week. Blood samples were

<PAGE>

randomly obtained from four mice at times indicated in FIG. 1. The p65 activity
was measured in the blood plasma by use of an ELISA assay.

In a modified ELISA inhibition procedure, 100 .mu.l of purified antigen of
predetermined dilution was added to Dynatech Immunon 2 plates and incubated at
37.degree. C. for one hour. This was followed by washing, then binding 1% bovine
serum albumin in bicarbonate buffer to cover residual binding sites in the
wells. After washing the plates, 100 .mu.l of antiserum plus test samples were
added and the incubation carried out for one hour at 37.degree. C. Finally, the
immune complex was detected by adding 100 .mu.l of goat/anti-rabbit
immunoglobulin conjugated to horseradish peroxidase. Then the substrate
2,21-Azino-(3-ethyl- benzthiazolinesulfonic acid) and 0.03% H 202 was added to
each well and incubated 10 minutes at room temperature. After terminating the
reaction with addition of 20 ml of 2.0 mM NaN3 to each well, the absorbance (405
nm) was read on an ELISA reader.

For convenience of graphing, the inhibition values were changed to units where
the sample giving the greatest inhibition will be selected as the end point. The
percent inhibition=100->(Absorbance inhibited/absorbance
uninhibited).times.100!. The percent inhibition of the sample was multiplied by
the reciprocal of the dilution to obtain units of the activity.

Shown in FIG. 1 was the time-course of (A) papilloma appearance and (B) p65
accumulation in the blood plasma. p65 was detected in the plasma at four weeks
of promotion, then its activity increased, first slowly up to 20 weeks, and then
more rapidly up to 30 weeks, when it began to plateau. p65 was not detected in
the blood of non-initiated mice treated with TPA as described above up to 20
weeks (data not shown).

By using a simple blood test for the presence of p65 factor, the skin cancer
risk from the tumor promoter TPA can be detected as early as four weeks of
treatment with the tumor promoter. A majority of skin papillomas are considered
to be non- malignant tumors at early stages of development. Conventional
histological and cytogenetic techniques are time consuming and are able to
detect skin cancers in mice only at later stages of papilloma development, i.e.,
at 30-40 weeks.

EXAMPLE VI

Monitoring Liver Carcinogenesis with p65 Tumor-Associated Factor

Altered hepatic foci (AHF) were induced in the course of 2-acetylaminofluorene
(AAF)-initiated, phenobarbital (PB)-promoted hepatocarcinogenesis in the rat.
Rats (male weanling albinos, Sprague Dawley strain) were purchased from Harlan
Labs, Indianapolis, Ind. The 0.06% (w/w) AAF diet and 0.05% PB diet were
prepared and pelleted by Altromin, Lage, FRG, and Dyets, Inc., Bethlehem, Pa.,
respectively. Each of two experimental groups of rats and a control group
consisted of 40 rats. All rats entered the experiment at 22 days of age. One

<PAGE>

group received AAF diet for 18 days, then AIN-76A diet (Dyets, Inc.) The second
group received AAF diet for 18 days then AIN-76A diet for 24 days and then
AIN-76A diet supplemented with 0.05% phenobarbital. The third group (controls)
received AIN-76A diet for 42 days, then AIN- 76A diet plus 0.05% phenobarbital.

Immunohistochemical Procedures

The blood and livers of rats (4 animals per each time-point) sacrificed at
different times in the course of hepato-carcinogenesis experiment were used for
immunochemical studies. Paraffin liver sections were prepared and stained with
specific antibodies. Polyclonal antibodies to the rat p65, purified to apparent
homogeneity, were raised in rabbits as described herein. The p65 was visualized
in the liver sections using the avidin- biotin-peroxidase complex (Vectastain
ABC Kit, Vector Laboratories, Burlingame, Calif.). Appropriate controls with
non-immune serum were performed routinely. Antiserum to p65 was diluted 1:200 in
PBS with 1% goat serum for use in the staining protocol. The blood plasma was
assayed for the presence of p65 using an ELISA assay or PAGE followed by
immunoblotting analyses.

The p65 tumor-associated protein was detected in rats fed AAF and PB diets as
early as two weeks of feeding with the tumor promoter PB. The p65 marker was
predominantly present in the cells of putative preneoplastic foci found at 24
weeks of trial in livers of rats fed AAF and PB diets. The p65 marker was highly
concentrated in the foci with little or none being detected in the surrounding
cells. Either no staining or weak positive staining was found in the areas known
for oval and ductular proliferation. No positive staining was found in control
livers from normal rats or rats fed only the PB diet. Most of p65 activity
appears to be associated with the nuclei of the p65-positive hepatocytes and
more precisely with the nuclear envelopes, with relatively little being detected
in the cytoplasm. Immunohistochemical staining of the cross-sectioned nuclei
revealed that only the periphery of the nuclei, i.e., nuclear envelopes, were
stained.

EXAMPLE VII

Enhancement of p65 Production During Sex Hormone Promotion

Shown in FIG. 2 is the effect of the contraceptive steroid ethynylestradiol
(EE), a known tumor promoter in the rat, on the p65 production in female rats
initiated with N-methyl- nitrosourea (MNU), using a protocol designed to induce
mammary gland tumors.

A short (one week) exposure to EE at six to seven weeks post-carcinogen
treatment, i.e., when the MNU-induced production of p65 was relatively low,
caused three-fold increase of the p65 level in the blood plasma. MNU was shown
to induce pre-neoplastic foci in the liver; however, they were not detected by
the use of the gamma-glutamyl transpeptidase (GGT) assay, even after 23-week
promotion with EE. Thus the 65 production in the rat appears to be extremely
sensitive to hormonal stimulation. The high sensitivity of the p65 synthesis
during chemical carcinogenesis to sex and phenobarbital promotion indicate that
p65 can be used not only as a tumor marker, but also for early assessment of

<PAGE>

cancer risk associated with the use of synthetic steroids and other drugs that
exhibit tumor promotion properties.

Evidence of resemblance of p65 to Steroid Receptors

p65 amino acid sequence (SEQ ID NO: 2) deduced from the cDNA sequence. Blocks of
zinc figure structures are underlined and SV40 translocation domain is double
underlined. Fragments of the sequence in bold are peptide sequences obtained
from the purified protein after CNBr cleavage. ##STR1##

Additional evidence that p65 may indeed belong to a superfamily of steroid
receptors comes from the cloning and sequencing of rat p65 cDNA. The C-terminal
end of p65 cDNA contains two sequences homologous to zinc fingers. Specifically,
the sequence of the Cl region of p65 is very similar to several other steroid
hormones. FIG. 3 depicts a proposed zinc finger structure of p65.

Post-translational modification of receptors is a possible mechanism by which
receptor function can be regulated. Glucocorticoid, progesterone, estrogen and
vitamin D receptors as well as p65 are phosphoproteins. The
phosphorylation/dephosphorylation process is most likely a control mechanism for
the regulation of transformation, hormone binding, DNA binding and
transactivation. In addition, a prenylation site was found not very far from the
C-terminal end. This is also post-translational modification by the attachment
of either a farnesyl or geranyl/geranyl group to cysteine residues located at
C-terminal extremity. Such modification is characteristic for several Ras and
Ras-like proteins and nuclear lamins A and B as well as a number of G-proteins
(transducins).

The intracellular localization of steroid receptors after synthesis has been
studied extensively and it is proposed that cytoplasmic receptors bind hormone
and rapidly translocate to the nucleus. This fact may explain immunostainings
showing the presence of p65 not only in nuclei, but also in the cytoplasm in
breast cancer tissues. Nuclear localization of proteins may occur by two
different mechanisms. One is the diffusion of proteins through nuclear membrane;
second, an interaction of proteins with the nuclear pores. This process is
mediated by a translocation signal in the protein. Amino acid sequences having
strong homology to the nuclear translocation signal of SV 40 T antigen have been
found in several steroid receptors. The sequence above shows comparisons of
highly homologous domains of translocation signal from human steroid receptors
like GR, MR, AR, PR, ER and p65. These sequences, in the case of p65, are
located close to the C-terminal site of the DNA-binding domain (Cl region),
similarly to other receptor proteins in this family. It is still unknown, but is
being actively studied, as to what serves as a ligand for p65.

The following examples describe in detail the protocols utilized to identify and
sequence the p65 gene as well as to make hybridization probes for the
identification of p65 protein in a sample.

<PAGE>

EXAMPLE VIII

Cloning and Sequencing of the p65 gene

An H-35 Reuber hepatoma rat cDNA library was screened using rat anti-p65
monoclonal antibodies and have found to have several clones containing sequences
homologous to p65, having approximately 1200 bp. Several hundred nucleotides
(reverse and forward) from both sides of this cDNA was sequenced. p65 cDNA was
cloned in plasmid pTA65Hum and used to transform E. coli JM 105 strains.
Single-stranded templates were prepared and sequenced by the dideoxy
chain-termination procedure (Messing J., Methods in Enzymology, 101: 20-78,
1983). The sequencing reaction was primed with either the universal primers or
specific oligonucleotides complementary to sequence already determined (Strauss,
E. C. et al., Anal . Biochem. 154: 353-360, 1986). The DNA sequences were
analyzed with the GCG DNA Sequence Analysis Program. Initially, an 800-900 bp
sequence data for the C-terminal end of the rat p65 cDNA (open reading frame)
was obtained. Two polyadenylation signals (TTTAATT in p65 cDNA or AAATTAA in
mRNA) and the poly A tail containing 18 As were found. Next, in the 1200 bp p65
cDNA fragment of p65 two fragments homologous to oligonucleotides backtranslated
from the amino acid sequence of the p39 and p51 peptides of p65 were found. This
was a strong confirmation that the proper cDNA fragment of p65 had been
isolated. The nucleotide sequences were next assembled and analyzed with
GeneJockey and DNA Strider softwares (Biosoft, Cambridge, UK). The sequence
similarities were searched by GCG package through GeneBank and Owl data bases.

Hybrid p65-like proteins encoded by recombinant clones were induced with IPTG.
Lysates were prepared and analyzed by SDS-PAGE gel electrophoresis and Western
blot assay. The anti-p65 MAbs revealed immunoreactivity with respective
..beta.-galactosidase/p65 fusion proteins, as indicated by immunoblot analysis on
bacterial cell extracts. MAbs were unreactive with other antigens present in
different bacterial lysates and with .beta.-galactosidase alone in pBluescript
plasmid. The p65-like bacterial product was found to have molecular mass of 36
kDa as determined by Coomassie-blue-stained SDS-PAGE gel. These findings
supported the theory that this cDNA clone coded for a fragment of the desired
antigen.

It should be emphasized that the p65-like products, reacted identically with
both monoclonal and polyclonal anti-p65 antibodies in immunoblotting
experiments.

Sequencing efforts were continued in order to obtain the full sequence of the
1200 bp cDNA fragment. Because p65 has a molecular weight 65 kDa, the expected
length of the clone containing rat p65 cDNA was estimated to be approximately
2000 bp. As discussed above, initial screening attempts, using monoclonal
antibodies, to obtain the full cDNA sequence were not successful. Specifically,
only fragments of the p65 gene with the C-terminal end or close to the
C-terminal end were determinable. The N-terminal end (approximately 800 bp) was
always missing. Therefore, a RACE PCR protocol was used to obtain the missing
sequences. This protocol eventually resulted in the determination of the full
cDNA sequence of rat p65 shown in SEQ ID NO: 1. The p65 amino acid sequence
deduced from the cDNA sequence is shown in SEQ ID NO: 2.

<PAGE>

PCR amplification of 5'-end p65 cDNA

5' AmpliFINDER anchor primer (CLONTECH Laboratories, Inc.) and specific to p65
cDNA antisense primer 5'CAGGTCCAGCTAGGACCGGG3' was used for amplification of the
5' end of p65 cDNA. Maximum amplification specificity was obtained using "hot
start " PCR (D'Aquila, R. T., Nucleic Acids Res. 19: 3749, 1991). PCR reactions
were carried out as described (Sambrook J. et al., Molecular Cloning : A Labora-
tory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
Amplification product was analyzed by agarose electrophoresis and Southern
hybridization. Selected PCR product was purified from the acrylamide gel and
cloned into TA vector, using TA cloning kit from Invitrogen Co. p65 inserts
after EcoRI digestion recombined TAH10 plasmid were recloned into Bluscript II
KS at EcoRI site.

Southern blot analysis

Amplification products were fractionated by electrophoresis in 1% agarose gel
containing 1.times.TAE buffer (40 mM Tris-HCl, 18 mM NaCl, 20 mM sodium acetate,
and 2 mM EDTA) after alkaline denaturation (Freeman M. R., Cancer Res., 49:
6221-6225 , 1989). DNA was transferred from agarose gel to nitrocellulose
(Schleicher & Schuell) according to Southern (Southern, E. M., J . Mol. Biol.,
98 : 503-518, 1975). The membrane was baked for 2 h at 80.degree. C. and pre-
hybridized in a hybridization buffer (5.times.SSC, 5.times.Denhardt's solution,
10% dextran sulfate, 1% SDS, 20 .mu.g/ml of salmon sperm DNA in 50% formamide)
in 42.degree. C., then hybridized with >.sup.32 P!-random primed cDNA probe over
-night at the same temperature. After hybridization, the membrane was washed in
2.times.SSC at room temperature for 30 min. and then the membrane was washed
under high stringency conditions (0.5.times.SSC, 1% SDS ) at 65.degree. C. for
15 min. Autoradiography was performed using Hyperfilm.TM.-MP from Amersham with
the intensifying screen at -80.degree. C.

Expression of the p65 Gene in Recombinant Vectors

After identifying and isolating the p65 DNA molecule, it may be inserted into
any one of the many vectors currently known in the art and transferred to a
prokaryotic or eukaryotic host cell where it will direct the expression and
production of the so-called recombinant version of the protein.

A technique often employed by those skilled in the art of protein production
today is to obtain a so-called "recombinant" version of the protein, to express
it in a recombinant cell and to obtain the protein from such cells. To achieve
this, a specific oligonucleotide based upon the sequence of the desired peptide,
as is known to those of skill in the art and described herein is prepared. The
oligonucleotide is then inserted into an expression vector, such as any one of
the many expression vectors currently available commercially. A prokaryotic or
eukaryotic host cell is then transformed with the vector, where it will direct
the expression of the so-called recombinant version of the peptide, which may
then be purified from the recombinant host cell. The preparation of
oligonucleotide, vector and transformation of the host cell are within the skill
of the ordinary artisan and is described in detail in Sambrook et al. (1989).

<PAGE>

Particularly useful vectors are contemplated to be those vectors in which the
coding portion of the DNA segment, whether encoding a full length protein or
smaller peptide, is positioned under the control of a promoter. The promoter may
be in the form of the promoter that is naturally associated with a p65 gene,
e.g., in p65 tumor associated cells, as may be obtained by isolating the 5'
non-coding sequences located upstream of the coding segment or exon, for
example, using recombinant cloning and/or PCR technology, in connection with the
compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will be gained
by positioning the coding DNA segment under the control of a recombinant, or
heterologous, promoter. As used herein, a recombinant or heterologous promoter
is intended to refer to a promoter that is not normally associated with a rat
p65 tumor marker gene in its natural environment. Naturally, it will be
important to employ a promoter that effectively directs the expression of the
DNA segment in the cell type chosen for expression. The use of promoter and cell
type combinations for protein expression is generally known to those of skill in
the art of molecular biology, for example, see Sambrook et al. (1989). The
promoters employed may be constitutive, or inducible, and can be used under the
appropriate conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of recombinant
proteins or peptides.

As mentioned above, in connection with expression embodiments to prepare
recombinant rat p65 proteins and peptides, it is contemplated that longer DNA
segments will most often be used, with DNA segments encoding the entire p65
protein being most preferred. However, it will be appreciated that the use of
shorter DNA segments to direct the expression of shorter peptides or epitopic
core regions, such as may be used to generate antibodies, also falls within the
scope of the invention. Single stranded DNA molecules coding for the p65 protein
can then be created and subsequently sequenced by the dideoxy chain termination
procedure as described in Messing (1983). In addition, fragments may be expanded
through the use of PCR technology of U.S. Pat. No. 4,603,102 (incorporated
herein by reference).

Nucleic Acid Sequence of the p65 Molecule

Important aspects of the present invention concern isolated DNA segments and
recombinant vectors encoding the 65 kDa oncofetal protein, and the creation and
use of recombinant host cells through the application of DNA technology, that
express this 65 kDa tumor marker protein.

As used herein, the term "DNA segment" refers to a DNA molecule that has been
isolated free of total genomic DNA of a particular species. Therefore, a DNA
segment encoding the rat p65 oncofetal protein refers to a DNA segment that
contains p65 coding sequences yet is isolated away from, or purified free from,
total genomic DNA of the rat. Included within the term "DNA segment", are DNA

<PAGE>

segments and smaller fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified rat p65 gene refers
to a DNA segment including p65 coding sequences and, in certain aspects,
regulatory sequences, isolated substantially away from other naturally occurring
genes or protein encoding sequences. In this respect, the term "gene" is used
for simplicity to refer to a functional protein, polypeptide or peptide encoding
unit. As will be understood by those in the art, this functional term includes
both genomic sequences, cDNA sequences and smaller engineered gene segments that
express, or may be adapted to express proteins, polypeptides or peptides.

Isolated substantially away from other coding sequences" means that the gene of
interest, in this case p65, forms the significant part of the coding region of
the DNA segment, and that the DNA segment does not contain large portions of
naturally-occurring coding DNA, such as large chromosomal fragments or other
functional genes or cDNA coding regions. Of course, this refers to the DNA
segment as originally isolated, and does not exclude genes or coding regions
later added to the segment by the hand of man.

In preferred embodiments, the invention concerns isolated DNA segments and
recombinant vectors incorporating DNA sequences that encode the rat p65
oncofetal protein comprising a sequence region having the sequence of SEQ ID NO:
1

The term "comprising a sequence region having the sequence of SEQ ID NO: 1"
means that the sequence substantially corresponds to a portion of SEQ ID NO: 1
and has relatively few amino acids that are not identical to the amino acids of
SEQ ID NO: 1. Accordingly, sequences that have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more preferably,
between about 91% and about 99%; of amino acids that are identical or
functionally equivalent to the amino acids of SEQ ID NO: 1 will be sequences
that "comprising a sequence region having the sequence of SEQ ID NO: 1". The
most preferred embodiment of the present invention consists of a protein
molecule of SEQ ID NO: 1.

Naturally, where the DNA segment or vector encodes a full length p65 protein, or
is intended for use in expressing the p65 protein, the most preferred sequences
are those that are essentially as set forth in SEQ ID NO: 1 and that encode a
protein that retains immunologic activity, e.g., as may be determined by the
antibody assay, as disclosed herein.

Sequences Complementary to the p65 Molecule

Naturally, the present invention also encompasses DNA and RNA segments that are
complementary, or essentially complementary, to the sequence set forth in SEQ ID
NO: 1. Nucleic acid sequences that are "complementary" are those that are
capable of base-pairing according to the standard Watson-Crick complementarity
rules. As used herein, the term "complementary sequences" means nucleic acid
sequences that are substantially complementary, as may be assessed by the same
nucleotide comparison set forth above, or as defined as being capable of

<PAGE>

hybridizing to the nucleic acid segment of SEQ ID NO: 1 under relatively
stringent conditions such as those described herein in Example 10.

Amino Acid Sequence of the p65 Protein Molecule

In certain other embodiments, the invention concerns the rat p65 protein having
the sequence of SEQ ID NO: 2. The term "having the sequence of SEQ ID NO: 2" is
used in the same sense as described above and means that the nucleic acid
sequence substantially corresponds to a portion of SEQ ID NO: 2 and has
relatively few codons that are not identical, or functionally equivalent, to the
codons of SEQ ID NO: 2. The term "functionally equivalent codon" is used herein
to refer to codons that encode the same amino acid, such as the six codons for
arginine or serine, and also refers to codons that encode biologically
equivalent amino acids. The term "functionally equivalent codon" is well
understood in the art.

Certain amino acids can be substituted for other amino acids in a sequence
without appreciable loss of immunologic activity.

In making such changes, the hydropathic index of amino acids can be considered.
The importance of the hydropathic amino acid index in conferring interactive
biologic function on a peptide is generally understood in the art (Kyte &
Doolittle, J. Mol. Biol, 157:105-132, 1982). It is known that certain amino
acids can be substituted for other amino acids having a similar hydropathic
index or score and still result in a peptide with similar biological activity.
Each amino acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5);
valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).

It is understood in the art that the relative hydropathic character of the amino
acid determines the secondary structure of the resultant peptide, which in turn
defines the interaction of the peptide with other molecules, such as enzymes,
substrates, receptors, antibodies, antigens, and the like. It is known in the
art that an amino acid can be substituted by another amino acid having a similar
hydropathic index and still obtain a functionally equivalent peptide. In such
changes, the substitution of amino acids whose hydropathic indices are within
..+-.2 is preferred, those which are within .+-.1 are particularly preferred, and
those within .+-.0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydro-
philicity, particularly where the biological functional equivalent peptide
thereby created is intended for use in immunological embodiments. U.S. Pat. No.
4,554,101, incorporated herein by reference, states that the greatest local
average hydrophilicity of a peptide, as governed by the hydrophilicity of its
adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e.
with a biological property of the peptide.

<PAGE>

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have
been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine
(-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan
(-3.4). It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a biologically
equivalent, and in particular, an immunologically equivalent peptide. In such
changes, the substitution of amino acids whose hydrophilicity values are within
..+-.2 is preferred, those which are within .+-.1 are particularly preferred, and
those within .+-.0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the
relative similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary
substitutions which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and include: arginine
and lysine; glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine (See table below). The present
invention thus contemplates functional or biological equivalents of a peptide
inhibitor of plasminogen activator inhibitor-1 as set forth above.

It will also be understood that amino acid and nucleic acid sequences may
include additional residues, such as additional N- or C-terminal amino acids or
5' or 3' sequences, and yet still be essentially as set forth in one of the
sequences disclosed herein, so long as the sequence meets the criteria set forth
above, including the maintenance of biological protein activity where protein
expression is concerned. The addition of terminal sequences particularly applies
to nucleic acid sequences that may, for example, include various non-coding
sequences flanking either of the 5' or 3' portions of the coding region or may
include various internal sequences, i.e., introns, which are known to occur
within genes.

The following examples are given by way of illustration, without intent to limit
the scope of the invention, to show details of particularly preferred reagents
and techniques utilized in the processes of the instant invention.

EXAMPLE IX

Creation of Hybridization Probes

Another important aspect of this invention resulting from the sequencing of the
p65 protein is the ability to make and use hybridization probes for use in the
detection of cancer.

The use of a hybridization probe of about 10-14 nucleotides in length allows the
formation of a duplex molecule that is both stable and selective. One might also

<PAGE>

prefer to design nucleic acid molecules having gene-complementary stretches of
15 to 20 contiguous nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences
disclosed herein. All that is required is to review the sequence set forth in
SEQ ID NO: 1 and to select any continuous portion of the sequence, from about
10-14 nucleotides in length up to and including the full length sequence, that
one wishes to utilize as a probe or primer. The choice of probe and primer
sequences may be governed by various factors, such as, by way of example only,
one may wish to employ primers from towards the termini of the total sequence,
or from the ends of the functional domain-encoding sequences, in order to
amplify further DNA; one may also employ probes corresponding to regions of the
entire DNA that are homologous to genes from any species including human in
order to screen for cancer.

Accordingly, the nucleotide sequences of the invention may be used for their
ability to selectively form duplex molecules with complementary stretches of p65
genes or cDNAs. Depending on the application envisioned, one will desire to
employ varying conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications requiring high
selectivity, one will typically desire to employ relatively stringent conditions
to form the hybrids, e.g., one will select relatively low salt and/or high
temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of
50.degree. C. to 70.degree. C. Such selective conditions tolerate little, if
any, mismatch between the probe and the template or target strand, and would be
particularly suitable for isolating rat p65 genes.

In certain embodiments, it will be advantageous to employ nucleic acid sequences
of the present invention in combination with an appropriate means, such as a
label, for determining hybridization. A wide variety of appropriate indicator
means are known in the art, including fluorescent, radioactive, enzymatic or
other ligands, such as avidin/biotin, which are capable of giving a detectable
signal. In preferred embodiments, one will likely desire to employ a fluorescent
label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase,
instead of radioactive or other environmental undesirable reagents. In the case
of enzyme tags, colorimetric indicator substrates are known that can be employed
to provide a means visible to the human eye or spectrophotometrically, to
identify specific hybridization with complementary nucleic acid-containing
samples.

In general, it is envisioned that the hybridization probes described herein will
be useful both as reagents in solution hybridization as well as in embodiments
employing a solid phase. In embodiments involving a solid phase, the test DNA
(or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to specific hybridization
with selected probes under desired conditions. The selected conditions will
depend on the particular circumstances based on the particular criteria required
(depending, for example, on the G+C contents, type of target nucleic acid,

<PAGE>

source of nucleic acid, size of hybridization probe, etc.). Following washing of
the hybridized surface so as to remove nonspecifically bound probe molecules,
specific hybridization is detected, or even quantified, by means of the label.

p65 mRNA-specific oligonucleotide probes may be used for detecting proliferating
cells like cancerous cells in tissue/cell samples. The steps include (a)
preparation of tissue/cell sample; (b) hybridization with cocktail of
non-radioactively, or radioactively labeled oligonucleotide probes for p65 mRNA;
and (c) detecting the presence of p65 probe hybrids.

EXAMPLE X

Antisense Technology

Inhibition of gene function with antisense nucleotides may have some therapeutic
utility. Therefore, series of antisense phosphorothioate oligonucleotides
complementary to the p65 nucleotide sequence were prepared that can be used to
inhibit expression of the p65 gene in breast, ovary and prostate cancer cell
lines, as well as other cancer cell lines. The oligonucleotides are effective
only when complexed with Lipofectin and the level of expression may be reduced
by approximately 50-80%. In experiments with the antisense expression the
steady-state level of mRNA is reduced. Antisense therapy using small
oligonucleotide antisense molecules of p65 mRNA or DNA as well as or defective
retrovirus or adenovirus carrying the antisense ribo- or deoxy-sequences of p65,
respectively, may be very effective in the prevention and or treatment of
cancer.

REFERENCES CITED

1. Mirowski, M., Sherman, U., and Hanausek, M. Purification and characterization
of a 65-kDa tumor-associated phosphoprotein from rat transplantable hepatocell-
ular carcinoma 16820cell line. Protein Expression and Purification, 3:196-203,
1992.

2. Wang, S., Mirowski, M., Sherman, U., Walaszek, Z., and Hanausek, M.
Monoclonal antibodies against a 65 kDa tumor-associated phosphoprotein: develop-
ment and use in cancer detection. Hybridoma, 12:167-176, 1993.

3. Mirowski, M., Walaszek, Z., Sherman, U., Adams, A. K. and Hanausek, M. Demon-
stration of a 65 kDa tumor-specific phosphoprotein in urine and serum of rats
with N-methyl-N-nitrosourea-induced mammary adenocarcinoma. Carcinogenesis 14:8,
1659-1664, 1993.

4. Mirowski, M., Walaszek, Z., Sherman, U., Adams, A. K. and Hanausek, M.
Comparative structural analysis of human and rat 65 kDa phosphoprotein. Int. J.
Biochem., 25:1865-1871, 1993.

5. Del Rio, M., Hanausek M., Walaszek, Z. and Stoica, G. Expression of a 65 kDa
oncofetal phosphoprotein in the altered hepatic foci of rats fed
2-acetylaminofluorene followed by phenobarbital. Int. J. Oncology 5:259-265,
1994.

<PAGE>

6. Mirowski, M., Klijanienko, J., Wang, S., Vielh, P., Walaszek, Z. and
Hanausek, M. Serological and immunohistochemical detection of a 65 kDa protein
breast cancer. Eur. J. Cancer., 30A:1108-1113, 1994.

7. Southern, E. M. Detection of specific sequences among DNA fragments separated
by gel electrophoresis. J . Mol. Biol., 98:503-518, 1975.

8. Freeman M. R., Washecka, R., and Chung L. W. K. Aberrant expression of epider
-mal growth factor receptor and Her-2 (erbB-2) messenger RNAs in human renal
cancers. Cancer Res., 49:6221-6225, 1989.

9. Strauss, E. C. et al., Use of the universal primers or specific
oligonucleotides complementary to sequences already determined. Anal. Biochem.
154:353-360, 1986.

10. Messing J. A system for shotgun DNA sequencing. Methods in Enzymology,
101:20-78, 1983.

11. Sambrook J . et al., Molecular Cloning:A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor , N.Y., 1989.

--------------------------------------------------------------------------------
    SEQUENCE LISTING
    (1) GENERAL INFORMATION:
    (iii) NUMBER OF SEQUENCES: 2
    (2) INFORMATION FOR SEQ ID NO:1:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2070 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA (genomic)
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
    GACCCCGAGAACGTGAGAGCCGAGGGCGGGGACATGAGGGAGAAAGTGAGACTGTCCAGC60
    GCTAGACAGAGACTGCGATGCAGCACTAAGAATAGTCTTCTCGTTGCTAGAACATGCGTG120
    CTGAGGCTGGTCGATTGGATCGAGCAGGATCTCAAGAGCACTTGCGAAAAGGCTCACGTT180
    TCCCTCCGCGGGTACCTCGAATTGCACCTGCAATGCGGGAGACGAAAAGAGGTTGCCGGG240
    GCTCTGGGTGGGGATACCAGACCTGACCCCAAGAAGCCGCGGGGGGGCTCCAAAAAGAAC300
    GTGGAGGACACCCTTGTCAAAGACAAGCTGAGCCCAAGTAGCGCGGTTTGCGAGTCCCCC360
    GAGGTGTATGGTGATGACGTAGGCTCTCAGGCTGCGGACAGCCCGAGGAAACAGCTGGCC420
    GCGAAAGGGACATTCAGAGATAAGGACAAAATTGAAGCGCTGTTCAAGCTCGGGGAGCTG480
    GTGGCTAAAAAAGCCTTGTCCTCAGCAATTACTTGGTTCCCCAACAGCGTGTCGCCTCTA540
    CACGCCCATTATGGCGATGAAATCCTCTACAAGGATGAATCCGGGCTTGTCAACATTAGC600
    GAGGGTGGCAAAAGAGGCGTGGAGATCCACCCCCCAGATAACTTCGGCATCACAACCCTT660
    GATGAAGATCTTGGCTTTCCCCAAATAATTGTGATTAACGTGAAGCCTCAAACCGAGGAA720
    GCCAACACTTGGTAAAGACAGGATCTGAAATATCATAACAGTGCAAACGAGGCCGGGTAC780
    TCCGACGAGAATAAAACCTTTGTGAGAGGATGTAGAAAAGATGGGCATAGTGAGCGTAAC840
    AATATGACCACAGGTGACAGAAATTCAAAAAAGGCCCAGCCCGTAAACTTCTCCCTCATG900
    GCATCCCTCGCCCTGGATTCTAGGGGCAAAGCCGCGGGGCCCCGGCGCGGAGCGAGGCGC960
    CTGTGCCTGGTGTGTGAGGACTATGCCAGCTGTTCAAACACCTGTGTCTGGTCCTGTGAA1020
    GCCTACAAGGTCTTCTTTCGCCGAAGTCAGGGAAACACAGACTACTATTGTTTCACAAAC1080
    GATTGCAACATCTCTAAGAATAGATCTAAGTCTTGCCCAGCCTGCCTCCTTCGTTGCCTG1140
    CACCCTAGCATCAATGAGATCCGAAAAGACAAGCGAGCAGCGCTGAATGTGCGAGACAAC1200
    GTTGGTGAAGAGGTGGATATGACCGGTCCTAGCTGGACCTGCCTGAAGCTACTCTTTTCA1260
    GATGGAGAAAAAGTGATACCCAGATTGGCCCATGAACTTCCAGGGATCAAGCGTGGCCGG1320
    CAGGCACAACAGCAGTCCCACCGAGGAAGCCCCATTCCCAAAAAGAGGAAAGGTTGGCCT1380
    CCTGGACATGTCCTGTCAAATGACCGCGCAGCTGCTGGCACGGTATGGAAACCAAAATCC1440
    TGTGAACCAATTCGCCGAGAAGGCCCCAAGTGGGACGCTCGGCTGAATGAATCTACCACC1500
    TTTGTTTTGGGGTCTCGAGCCAACAAGGCCTTAGGGAAGGGAGGCACCAGAGGGAGGATT1560
    TACATCAAGCACCCACACCTCTTTAAGTATGCAGCAGATCCTCAGGACAAGCACTGGCTG1620
    GCTGAGCAGCATCATCGGCAGCGGTTCGCAGAATTGCTTCTCAAGATTAGCCATATTAGG1680
    CACATGGTTGAGGGAGTGGCTCATTGCTTGTACGACATGAAAGTTAAGGACAAAGTTCTG1740

<PAGE>

    CCATCCTGGAAGGTTGAGAAGTTGCGGAAATACGTGGAGACACTACGGACAGAAAATGAG1800
    CATCGTGTCGCTGAAGCAAGTCCCCAGACCTGAGCCGAGTGTCCTGGTCTACTACACTTG1860
    CAGTCTGCCTCCCAGACCCTCTTTCCCGGCCCGGCTGAGGCCATCATGGGGATGCGGTCT1920
    AGTTGGCTCTTAGCAGCAATCAAGCGTTACATGAGCTAGTTTGTAGTGACTCACTGCAGA1980
    GCCCCCAGACTGGCTTGTGGTTCTGTTTCTAAAGTTATTGGAATAAGAAGCAATTAAACA2040
    AGTTTGTAATTTAAAAAAAAAAAAAAAAAA2070
    (2) INFORMATION FOR SEQ ID NO:2:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 691 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 612
    (D) OTHER INFORMATION: /note= "Xaa = Unknown"
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 654
    (D) OTHER INFORMATION: /note= "Xaa = Unknown"
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 680
    (D) OTHER INFORMATION: /note= "Xaa = Unknown"
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 684
    (D) OTHER INFORMATION: /note= "Xaa = Unknown"
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
    MetAspProGluAsnValArgAlaGluGlyGlyAspMetArgGluLys
    151015
    ValArgLeuSerSerAlaArgGlnArgLeuArgCysSerThrLysAsn
    202530
    SerLeuLeuValAlaArgThrCysValLeuArgLeuValAspTrpIle
    354045
    GluGlnAspLeuLysSerThrCysGluLysAlaHisValSerLeuArg
    505560
    GlyTyrLeuGluLeuHisLeuGlnCysGlyArgArgLysGluValAla
    65707580
    GlyAlaLeuGlyGlyAspThrArgProAspProLysLysProArgGly
    859095
    GlySerLysLysAsnValGluAspThrLeuValLysAspLysLeuSer
    100105110
    ProSerSerAlaValCysGluSerProGluValTyrGlyAspAspVal
    115120125

<PAGE>

    GlySerGlnAlaAlaAspSerProArgLysGlnLeuAlaAlaLysGly
    130135140
    ThrPheArgAspLysAspLysIleGluAlaLeuPheLysLeuGlyGlu
    145150155160
    LeuValAlaLysLysAlaLeuSerSerAlaIleThrTrpPheProAsn
    165170175
    SerValSerProLeuHisAlaHisTyrGlyAspGluIleLeuTyrLys
    180185190
    AspGluSerGlyLeuValAsnIleSerGluGlyGlyLysArgGlyVal
    195200205
    GluIleHisProProAspAsnPheGlyIleThrThrLeuAspGluAsp
    210215220
    LeuGlyPheProGlnIleIleValIleAsnValLysProGlnThrGlu
    225230235240
    GluAlaAsnThrTrpLeuArgGlnAspLeuLysTyrHisAsnSerAla
    245250255
    AsnGluAlaGlyTyrSerAspGluAsnLysThrPheValArgGlyCys
    260265270
    ArgLysAspGlyHisSerGluArgAsnAsnMetThrThrGlyAspArg
    275280285
    AsnSerLysLysAlaGlnProValAsnPheSerLeuMetAlaSerLeu
    290295300
    AlaLeuAspSerArgGlyLysAlaAlaGlyProArgArgGlyAlaArg
    305310315320
    ArgLeuCysLeuValCysGluAspTyrAlaSerCysSerAsnThrCys
    325330335
    ValTrpSerCysGluAlaTyrLysValPhePheArgArgSerGlnGly
    340345350
    AsnThrAspTyrTyrCysPheThrAsnAspCysAsnIleSerLysAsn
    355360365
    ArgSerLysSerCysProAlaCysLeuLeuArgCysLeuHisProSer
    370375380
    IleAsnGluIleArgLysAspLysArgAlaAlaLeuAsnValArgAsp
    385390395400
    AsnValGlyGluGluValAspMetThrGlyProSerTrpThrCysLeu
    405410415
    LysLeuLeuPheSerAspGlyGluLysValIleProArgLeuAlaHis
    420425430
    GluLeuProGlyIleLysArgGlyArgGlnAlaGlnGlnGlnSerHis
    435440445
    ArgGlySerProIleProLysLysArgLysGlyTrpProProGlyHis
    450455460
    ValLeuSerAsnAspArgAlaAlaAlaGlyThrValTrpLysProLys
    465470475480
    SerCysGluProIleArgArgGluGlyProLysTrpAspAlaArgLeu
    485490495
    AsnGluSerThrThrPheValLeuGlySerArgAlaAsnLysAlaLeu
    500505510
    GlyLysGlyGlyThrArgGlyArgIleTyrIleLysHisProHisLeu
    515520525
    PheLysTyrAlaAlaAspProGlnAspLysHisTrpLeuAlaGluGln
    530535540
    HisHisArgGlnArgPheAlaGluLeuLeuLeuLysIleSerHisIle
    545550555560
    ArgHisMetValGluGlyValAlaHisCysLeuTyrAspMetLysVal
    565570575
    LysAspLysValLeuProSerTrpLysValGluLysLeuArgLysTyr
    580585590
<PAGE>

    ValGluThrLeuArgThrGluAsnGluHisArgValAlaGluAlaSer
    595600605
    ProGlnThrXaaAlaGluCysProGlyLeuLeuHisLeuGlnSerAla
    610615620
    SerGlnThrLeuPheProGlyProAlaGluAlaIleMetGlyMetArg
    625630635640
    SerSerTrpLeuLeuAlaAlaIleLysArgTyrMetSerXaaPheVal
    645650655
    ValThrHisCysArgAlaProArgLeuAlaCysGlySerValSerLys
    660665670
    ValIleGlyIleArgSerAsnXaaThrSerLeuXaaPheLysLysLys
    675680685
    LysLysLys
    690
--------------------------------------------------------------------------------

Source: [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00081-of-00352.parquet"}, [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00081-of-00352.parquet"}]]