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Cancer Patent Abstract
This invention relates to a method for assessing risk of prostate
cancer. Specifically, it relates to utilizing both Pro108 and Prostate
Specific Antigen (PSA) in combination to determine the risk of prostate
cancer. In addition, it is directed to a method for assessing risk
of ovarian, colon, breast or stomach cancer utilizing Pro108 or
specific antibodies to Pro108. The invention provides isolated anti-prostate,
ovarian, colon, breast or stomach cancer antigen (Pro108) antibodies
that bind to Pro108 on a mammalian cell in vivo. The invention also
encompasses compositions comprising an anti-Pro108 antibody and
a carrier. These compositions can be provided in an article of manufacture
or a kit. Another aspect of the invention is an isolated nucleic
acid encoding an anti-Pro108 antibody, as well as an expression
vector comprising the isolated nucleic acid. Also provided are cells
that produce the anti-Pro108 antibodies. The invention encompasses
a method of producing the anti-Pro108 antibodies. Other aspects
of the invention are a method of killing an Pro108-expressing cancer
cell, comprising contacting Pro108 present in the ECM with an anti-Pro108
antibody and a method of alleviating or treating an Pro108-expressing
cancer in a mammal, comprising administering a therapeutically effective
amount of the anti-Pro108 antibody to the mammal.
Cancer Patent Claims
We claim:
1. An isolated monoclonal antibody specific for Pro108 produced
by the hybridoma selected from the group consisting of ATCC accession
number PTA-5885 and PTA-5886, or an antigen binding fragment of
said monoclonal antibody wherein Pro108 comprises residues 1-331
of SEQ ID NO:1 or SEQ ID NO:2.
2. An isolated monoclonal antibody or antibody fragment which competes
for binding to the same epitope of Pro108 recognized, by the monoclonal
antibody produced by a hybridoma selected from the group consisting
of ATCC accession number PTA-5885 and PTA-5586, wherein said Pro108
comrrises residues 1-331 of SEQ ID NO:1 or SEQ ID NO:2.
3. A chimeric or a humanized antibody or antibody fragment thereof
of the monoclonal antibody specific for Pro108 produced by the hybridoma
selected from the group consisting of ATCC accession number PTA-5885
and PTA-5886, wherein Pro108 comprises residues 1-331 of SEQ ID
NO:1 or SEQ ID NO:2.
4. The antibody or antibody fragment of claim 2 which is a chimeric,
a human or a humanized antibody or antibody fragment thereof.
5. The antibody or antibody fragment of claim 2 that binds to said
Pro108 expressed by a mammalian cell.
6. The antibody or antibody fragment of claim 1 or 2 which is conjugated
to a growth inhibitory agent.
7. The antibody or antibody fragment of claim 1 or 2 which is conjugated
to a cytotoxic agent.
8. The antibody or antibody fragment of claim 7 wherein the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
9. The antibody or antibody fragment of claim 8 wherein the cytotoxic
agent is a toxin.
10. The antibody or antibody fragment of claim 9, wherein the toxin
is selected from the group consisting of ricin, saponin, maytansinoid
and calicheamicin.
11. The antibody or antibody fragment of claim 10, wherein the
toxin is a maytansinoid.
12. The antibody or antibody fragment of claim 5, wherein the mammalian
cell is a cancer cell.
13. The antibody or antibody fragment of claim 12 wherein the cancer
cell is from a cancer selected from the group consisting of prostate,
ovarian, colon, breast and stomach cancer.
14. The antibody or antibody fragment of claim 13 wherein the cancer
is prostate, ovarian or colon cancer.
15. The antibody or antibody fragment of claim 12 that inhibits
the growth of Pro108-expressing cancer cells in vivo.
16. The antibody or antibody fragment of claim 15 which is produced
in bacteria.
17. The antibody or antibody fragment of claim 15, wherein the
cancer cells are from a cancer selected from the group consisting
of prostate, ovarian, colon, breast and stomach cancer.
18. The antibody or antibody fragment of claim 17, wherein the
cancer is prostate, ovarian or colon cancer.
19. An isolated cell that produces the antibody or antibody fragment
of claim 2.
20. The cell of claim 19, wherein the cell is selected from the
group consisting of hybridoma cells deposited under ATCC accession
number PTA-5885 and PTA5586.
21. A composition comprising the antibody or antibody fragment
of claim 2, and a carrier.
22. The composition of claim 21, wherein the antibody or antibody
fragment is conjugated to a cytotoxic agent.
23. The composition of claim 22, wherein the cytotoxic agent is
a maytansinoid.
24. The composition of claim 21, wherein the antibody or antibody
fragment is a human or humanized antibody or antibody fragment and
the carrier is a pharmaceutical carrier.
25. The composition of claim 24, wherein the humanized antibody
or antibody fragment is a humanized antibody or antibody fragment
thereof of the monoclonal antibody specific for Pro108 produced
by a hybridoma selected from the group consisting of ATCC accession
number PTA-5885 and PTA-5886.
26. An article of manufacture comprising a container and a composition
contained therein, wherein the composition comprises an antibody
or antibody fragment of claim 1 or 2.
27. The article of manufacture of claim 26 further comprising a
package insert indicating that the composition can be used to treat
prostate, ovarian, colon, breast or stomach cancer.
28. The article of manufacture of claim 26 further comprising a
package insert indicating that the composition can beused to detect
prostate, ovarian, colon, breast or stomach cancer.
29. The article of manufacture of claim 26 further comprising a
package insert indicating that the composition can be used to monitor
prostate, ovarian, colon, breast or stomach cancer.
30. The antibody or antibody fragment of claim 1 or 2 which is
detectably labeled.
31. The antibody or antibody fragment of claim 30 wherein the label
is selected from the group consisting of radiolabels, fluorescent
labels, gold labels, biotin and enzymatic labels.
32. A composition comprising the antibody or antigen binding fragment
thereof of claim 1, and a carrier.
Cancer Patent Description
FIELD OF THE INVENTION
This invention relates to a method for assessing risk for cancer.
Specifically, it relates to utilizing both Pro108 (also known as
Spondin 2) Prostate Specific Antigen (PSA) in combination to detect
prostate cancer. In addition, it is directed to a method for assessing
risk of ovarian, colon, breast or stomach cancer utilizing Pro108
or anti-Pro108 antibodies specific to Pro108. Furthermore, the present
invention relates to anti-Pro108 antibody compositions and methods
of inhibiting production and function or killing Pro108-expressing
prostate, ovarian, colon, breast or stomach cancers cells.
BACKGROUND OF THE INVENTION
Prostate Cancer
Prostate cancer is the most prevalent cancer in men and is the
second leading cause of death from cancer among males in the United
States. AJCC Cancer Staging Handbook 203 (Irvin D. Fleming et al.
eds., 5.sup.th ed. 1998); Walter J. Burdette, Cancer: Etiology Diagnosis,
and Treatment 147 (1998). In 1999, it was estimated that 37,000
men in the United States would die as result of prostate cancer.
Elizabeth A. Platz et al., & Edward Giovannucci, Epidemiology
of and Risk Factors for Prostate Cancer, in Management of Prostate
Cancer 21 (Eric A Klein, ed. 2000). More recently, the American
Cancer Society estimated there will be 230,110 new cases of prostate
cancer and 29,900 deaths in 2004. American Cancer Society website:
cancer.org of the world wide web. Cancer of the prostate typically
occurs in older males, with a median age of 74 years for clinical
diagnosis. Burdette, supra at 147. A man's risk of being diagnosed
with invasive prostate cancer in his lifetime is one in six. Platz
et al., supra at 21.
Although our understanding of the etiology of prostate cancer is
incomplete, the results of extensive research in this area point
to a combination of age, genetic and environmental/dietary factors.
Platz et al., supra at 19; Burdette, supra at 147; Steven K. Clinton,
Diet and Nutrition in Prostate Cancer Prevention and Therapy, in
Prostate Cancer: a Multidisciplinary Guide 246-269 (Philip W. Kantoff
et al. eds. 1997). Broadly speaking, genetic risk factors predisposing
one to prostate cancer include race and a family history of the
disease. Platz et al., supra at 19, 28-29, 32-34. Aside from these
generalities, a deeper understanding of the genetic basis of prostate
cancer has remained elusive. Considerable research has been directed
to studying the link between prostate cancer, androgens, and androgen
regulation, as androgens play a crucial role in prostate growth
and differentiation. Meena Augustus et al., Molecular Genetics and
Markers of Progression, in Management of Prostate Cancer 59 (Eric
A Klein ed. 2000). While a number of studies have concluded that
prostate tumor development is linked to elevated levels of circulating
androgen (e.g., testosterone and dihydrotestosterone), the genetic
determinants of these levels remain unknown. Platz et al., supra
at 29-30.
Several studies have explored a possible link between prostate
cancer and the androgen receptor (AR) gene, the gene product of
which mediates the molecular and cellular effects of testosterone
and dihydrotestosterone in tissues responsive to androgens. Id.
at 30. Differences in the number of certain trinucleotide repeats
in exon 1, the region involved in transactivational control, have
been of particular interest. Augustus et al., supra at 60. For example,
these studies have revealed that as the number of CAG repeats decreases
the transactivation ability of the gene product increases, as does
the risk of prostate cancer. Platz et al., supra at 30-31. Other
research has focused on the .alpha.-reductase Type 2 gene, the gene
which codes for the enzyme that converts testosterone into dihydrotestosterone.
Id. at 30. Dihydrotestosterone has greater affinity for the AR than
testosterone, resulting in increased transactivation of genes responsive
to androgens. Id. While studies have reported differences among
the races in the length of a TA dinucleotide repeat in the 3' untranslated
region, no link has been established between the length of that
repeat and prostate cancer. Id. Interestingly, while ras gene mutations
are implicated in numerous other cancers, such mutations appear
not to play a significant role in prostate cancer, at least among
Caucasian males. Augustus, supra at 52.
Environmental/dietary risk factors which may increase the risk
of prostate cancer include intake of saturated fat and calcium.
Platz et al., supra at 19, 25-26. Conversely, intake of selenium,
vitamin E and tomato products (which contain the carotenoid lycopene)
apparently decrease that risk. Id. at 19, 26-28 The impact of physical
activity, cigarette smoking, and alcohol consumption on prostate
cancer is unclear. Platz et al., supra at 23-25.
Periodic screening for prostate cancer is most effectively performed
by digital rectal examination (DRE) of the prostate, in conjunction
with determination of the serum level of prostate-specific antigen
(PSA). Burdette, supra at 148. While the merits of such screening
are the subject of considerable debate, Jerome P. Richie & Irving
D. Kaplan, Screening for Prostate Cancer. The Horns of a Dilemma,
in Prostate Cancer: A Multidisciplinary Guide 1-10 (Philip W. Kantoff
et al. eds. 1997), the American Cancer Society and American Urological
Association recommend that both of these tests be performed annually
on men 50 years or older with a life expectancy of at least 10 years,
and younger men at high risk for prostate cancer. Ian M. Thompson
& John Foley, Screening for Prostate Cancer, in Management of
Prostate Cancer 71 (Eric A Klein ed. 2000). If necessary, these
screening methods may be followed by additional tests, including
biopsy, ultrasonic imaging, computerized tomography, and magnetic
resonance imaging. Christopher A. Haas & Martin I. Resnick,
Trends in Diagnosis, Biopsy, and Imaging, in Management of Prostate
Cancer 89-98 (Eric A Klein ed. 2000); Burdette, supra at 148.
Once the diagnosis of prostate cancer has been made, treatment
decisions for the individual are typically linked to the stage of
prostate cancer present in that individual, as well as his age and
overall health. Burdette, supra at 151. One preferred classification
system for staging prostate cancer was developed by the American
Urological Association (AUA). Id. at 148. The AUA classification
system divides prostate tumors into four broad stages, A to D, which
are in turn accompanied by a number of smaller substages. Burdette,
supra at 152-153; Anthony V. D+Amico et al., The Staging of Prostate
Cancer, in Prostate Cancer: A Multidisciplinary Guide 41 (Philip
W. Kantoff et al. eds. 1997).
Stage A prostate cancer refers to the presence of microscopic cancer
within the prostate gland. D+Amico, supra at 41. This stage is comprised
of two substages: A1, which involves less than four well-differentiated
cancer foci within the prostate, and A2, which involves greater
than three well-differentiated cancer foci or alternatively, moderately
to poorly differentiated foci within the prostate. Burdette, supra
at 152; D+Amico, supra at 41. Treatment for stage A1 preferentially
involves following PSA levels and periodic DRE. Burdette, supra
at 151. Should PSA levels rise, preferred treatments include radical
prostatectomy in patients 70 years of age and younger, external
beam radiotherapy for patients between 70 and 80 years of age, and
hormone therapy for those over 80 years of age. Id.
Stage B prostate cancer is characterized by the presence of a palpable
lump within the prostate. Burdette, supra at 152-53; D+Amico, supra
at 41. This stage is comprised of three substages: B1, in which
the lump is less than 2 cm and is contained in one lobe of the prostate;
B2, in which the lump is greater than 2 cm yet is still contained
within one lobe; and B3, in which the lump has spread to both lobes.
Burdette, supra, at 152-53. For stages B1 and B2, the treatment
again involves radical prostatectomy in patients 70 years of age
and younger, external beam radiotherapy for patients between 70
and 80 years of age, and hormone therapy for those over 80 years
of age. Id. at 151. In stage B3, radical prostatectomy is employed
if the cancer is well-differentiated and PSA levels are below 15
ng/mL; otherwise, external beam radiation is the chosen treatment
option. Id.
Stage C prostate cancer involves a substantial cancer mass accompanied
by extraprostatic extension. Burdette, supra at 153; D'Amico, supra
at 41. Like stage A prostate cancer, Stage C is comprised of two
substages: substage C1, in which the tumor is relatively minimal,
with minor prostatic extension, and substage C2, in which the tumor
is large and bulky, with major prostatic extension. Id. The treatment
of choice for both substages is external beam radiation. Burdette,
supra at 151.
The fourth and final stage of prostate cancer, Stage D, describes
the extent to which the cancer has metastasized. Burdette, supra
at 153; D'Amico, supra at 41. This stage is comprised of four substages:
(1) D0, in which acid phophatase levels are persistently high, (2)
D1, in which only the pelvic lymph nodes have been invaded, (3)
D2, in which the lymph nodes above the aortic bifurcation have been
invaded, with or without distant metastasis, and (4) D3, in which
the metastasis progresses despite intense hormonal therapy. Id.
Treatment at this stage may involve hormonal therapy, chemotherapy,
and removal of one or both testes. Burdette, supra at 151.
Despite the need for accurate staging of prostate cancer, current
staging methodology is limited. The wide variety of biological behavior
displayed by neoplasms of the prostate has resulted in considerable
difficulty in predicting and assessing the course of prostate cancer.
Augustus et al., supra at 47. Indeed, despite the fact that most
prostate cancer patients have carcinomas that are of intermediate
grade and stage, prognosis for these types of carcinomas is highly
variable. Andrew A Renshaw & Christopher L. Corless, Prognostic
Features in the Pathology of prostate Cancer, in Prostate Cancer:
A Multidisciplinary Guide 26 (Philip W. Kantoff et al. eds. 1997).
Techniques such as transrectal ultrasound, abdominal and pelvic
computerized tomography, and MRI have not been particularly useful
in predicting local tumor extension. D+Amico, supra at 53 (editors'
comment). While the use of serum PSA in combination with the Gleason
score is currently the most effective method of staging prostate
cancer, id., PSA is of limited predictive value, Augustus et al.,
supra at 47; Renshaw et al., supra at 26, and the Gleason score
is prone to variability and error, King, C. R. & Long, J. P.,
Int'l. J. Cancer 90(6): 326-30 (2000). As such, the current focus
of prostate cancer research has been to obtain biomarkers to help
better assess the progression of the disease. Augustus et al., supra
at 47; Renshaw et al., supra at 26; Pettaway, C. A., Tech. Urol.
4(1): 35-42 (1998).
Accordingly, there is a great need for more sensitive and accurate
methods for predicting whether a person is likely to develop prostate
cancer, for diagnosing prostate cancer, for monitoring the progression
of the disease, for staging the prostate cancer, for determining
whether the prostate cancer has metastasized and for imaging the
prostate cancer. There is also a need for better treatment of prostate
cancer.
Ovarian Cancer
Cancer of the ovaries is the fourth-most common cause of cancer
death in women in the United States, with more than 23,000 new cases
and roughly 14,000 deaths predicted for the year 2001. Shridhar,
V. et al., Cancer Res. 61(15): 5895-904 (2001); Memarzadeh, S. &
Berek, J. S., J. Reprod. Med. 46(7): 621-29 (2001). The American
Cancer Society estimates that there will be about 25,580 new cases
of ovarian cancer in 2004 in the United States alone. Ovarian cancer
will cause about 16,090 deaths in the United States in the same
year. ACS Website: cancer.org of the world wide web. The incidence
of ovarian cancer is of serious concern worldwide, with an estimated
191,000 new cases predicted annually. Runnebaum, I. B. & Stickeler,
E., J. Cancer Res. Clin. Oncol. 127(2): 73-79 (2001). Unfortunately,
women with ovarian cancer are typically asymptomatic until the disease
has metastasized. Because effective screening for ovarian cancer
is not available, roughly 70% of women diagnosed have an advanced
stage of the cancer with a five-year survival rate of 25-30%. Memarzadeh,
S. & Berek, J. S., supra; Nunns, D. et al., Obstet. Gynecol.
Surv. 55(12): 746-51. Conversely, women diagnosed with early stage
ovarian cancer enjoy considerably higher survival rates. Werness,
B. A. & Eltabbakh, G. H., Int'l. J. Gynecol. Pathol. 20(1):
48-63 (2001). Although our understanding of the etiology of ovarian
cancer is incomplete, the results of extensive research in this
area point to a combination of age, genetics, reproductive, and
dietary/environmental factors. Age is a key risk factor in the development
of ovarian cancer: while the risk for developing ovarian cancer
before the age of 30 is slim, the incidence of ovarian cancer rises
linearly between ages 30 to 50, increasing at a slower rate thereafter,
with the highest incidence being among septagenarian women. Jeanne
M. Schilder et al., Hereditary Ovarian Cancer: Clinical Syndromes
and Management, in Ovarian Cancer 182 (Stephen C. Rubin & Gregory
P. Sutton eds., 2d ed. 2001).
With respect to genetic factors, a family history of ovarian cancer
is the most significant risk factor in the development of the disease,
with that risk depending on the number of affected family members,
the degree of their relationship to the woman, and which particular
first degree relatives are affected by the disease. Id. Mutations
in several genes have been associated with ovarian cancer, including
BRCA1 and BRCA2, both of which play a key role in the development
of breast cancer, as well as hMSH2 and hMLH1, both of which are
associated with hereditary non-polyposis colon cancer. Katherine
Y. Look, Epidemiology, Etiology, and Screening of Ovarian Cancer,
in Ovarian Cancer 169, 171-73 (Stephen C. Rubin & Gregory P.
Sutton eds., 2d ed. 2001). BRCA1, located on chromosome 17, and
BRCA2, located on chromosome 13, are tumor suppressor genes implicated
in DNA repair; mutations in these genes are linked to roughly 10%
of ovarian cancers. Id. at 171-72; Schilder et al., supra at 185-86.
hMSH2 and hMLH1 are associated with DNA mismatch repair, and are
located on chromosomes 2 and 3, respectively; it has been reported
that roughly 3% of hereditary ovarian carcinomas are due to mutations
in these genes. Look, supra at 173; Schilder et al., supra at 184,
188-89.
Reproductive factors have also been associated with an increased
or reduced risk of ovarian cancer. Late menopause, nulliparity,
and early age at menarche have all been linked with an elevated
risk of ovarian cancer. Schilder et al., supra at 182. One theory
hypothesizes that these factors increase the number of ovulatory
cycles over the course of a woman's life, leading to "incessant
ovulation," which is thought to be the primary cause of mutations
to the ovarian epithelium. Id.; Laura J. Havrilesky & Andrew
Berchuck, Molecular Alterations in Sporadic Ovarian Cancer, in Ovarian
Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed.
2001). The mutations may be explained by the fact that ovulation
results in the destruction and repair of that epithelium, necessitating
increased cell division, thereby increasing the possibility that
an undetected mutation will occur. Id. Support for this theory may
be found in the fact pregnancy, lactation, and the use of oral contraceptives,
all of which suppress ovulation, confer a protective effect with
respect to developing ovarian cancer. Id.
Among dietary/environmental factors, there would appear to be an
association between high intake of animal fat or red meat and ovarian
cancer, while the antioxidant Vitamin A, which prevents free radical
formation and also assists in maintaining normal cellular differentiation,
may offer a protective effect. Look, supra at 169. Reports have
also associated asbestos and hydrous magnesium trisilicate (talc),
the latter of which may be present in diaphragms and sanitary napkins.
Id. at 169-70.
Current screening procedures for ovarian cancer, while of some
utility, are quite limited in their diagnostic ability, a problem
that is particularly acute at early stages of cancer progression
when the disease is typically asymptomatic yet is most readily treated.
Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 166
(1998); Memarzadeh & Berek, supra; Runnebaum & Stickeler,
supra; Werness & Eltabbakh, supra. Commonly used screening tests
include biannual rectovaginal pelvic examination, radioimmunoassay
to detect the CA-125 serum tumor marker, and transvaginal ultrasonography.
Burdette, supra at 166.
Pelvic examination has failed to yield adequate numbers of early
diagnoses, and the other methods are not sufficiently accurate.
Id. One study reported that only 15% of patients who suffered from
ovarian cancer were diagnosed with the disease at the time of their
pelvic examination. Look, supra at 174. Moreover, the CA-125 test
is prone to giving false positives in pre-menopausal women and has
been reported to be of low predictive value in post-menopausal women.
Id. at 174-75. Although transvaginal ultrasonography is now the
preferred procedure for screening for ovarian cancer, it is unable
to distinguish reliably between benign and malignant tumors, and
also cannot locate primary peritoneal malignancies or ovarian cancer
if the ovary size is normal. Schilder et al., supra at 194-95. While
genetic testing for mutations of the BRCA1, BRCA2, hMSH2, and hMLH1
genes is now available, these tests may be too costly for some patients
and may also yield false negative or indeterminate results. Schilder
et al., supra at 191-94.
Other markers of interest are HE4 and mesothelin, see Urban et
al. Ovarian cancer screening Hematol Oncol Clin North Am. 2003 August;
17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is a biomarker
for ovarian carcinoma, Cancer Res. 2003 Jul. 1;63(13):3695-700;
Ordonez, Application of mesothelin immunostaining in tumor diagnosis,
Am J Surg Pathol. 2003 November; 27(11):1418-28.
The staging of ovarian cancer, which is accomplished through surgical
exploration, is crucial in determining the course of treatment and
management of the disease. AJCC Cancer Staging Handbook 187 (Irvin
D. Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh
& Berek, supra; Shridhar et al., supra. Staging is performed
by reference to the classification system developed by the International
Federation of Gynecology and Obstetrics. David H. Moore, Primary
Surgical Management of Early Epithelial Ovarian Carcinoma, in Ovarian
Cancer 203 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed.
2001); Fleming et al. eds., supra at 188. Stage I ovarian cancer
is characterized by tumor growth that is limited to the ovaries
and is comprised of three substages. Id. In substage IA, tumor growth
is limited to one ovary, there is no tumor on the external surface
of the ovary, the ovarian capsule is intact, and no malignant cells
are present in ascites or peritoneal washings. Id. Substage IB is
identical to A1, except that tumor growth is limited to both ovaries.
Id. Substage IC refers to the presence of tumor growth limited to
one or both ovaries, and also includes one or more of the following
characteristics: capsule rupture, tumor growth on the surface of
one or both ovaries, and malignant cells present in ascites or peritoneal
washings. Id.
Stage II ovarian cancer refers to tumor growth involving one or
both ovaries, along with pelvic extension. Id. Substage IIA involves
extension and/or implants on the uterus and/or fallopian tubes,
with no malignant cells in the ascites or peritoneal washings, while
substage IIB involves extension into other pelvic organs and tissues,
again with no malignant cells in the ascites or peritoneal washings.
Id. Substage IIC involves pelvic extension as in IIA or IIB, but
with malignant cells in the ascites or peritoneal washings. Id.
Stage III ovarian cancer involves tumor growth in one or both ovaries,
with peritoneal metastasis beyond the pelvis confirmed by microscope
and/or metastasis in the regional lymph nodes. Id. Substage IIIA
is characterized by microscopic peritoneal metastasis outside the
pelvis, with substage IIIB involving macroscopic peritoneal metastasis
outside the pelvis 2 cm or less in greatest dimension. Id. Substage
IIIC is identical to IIIB, except that the metastasis is greater
than 2 cm in greatest dimension and may include regional lymph node
metastasis. Id. Lastly, Stage IV refers to the presence distant
metastasis, excluding peritoneal metastasis. Id.
While surgical staging is currently the benchmark for assessing
the management and treatment of ovarian cancer, it suffers from
considerable drawbacks, including the invasiveness of the procedure,
the potential for complications, as well as the potential for inaccuracy.
Moore, supra at 206-208, 213. In view of these limitations, attention
has turned to developing alternative staging methodologies through
understanding differential gene expression in various stages of
ovarian cancer and by obtaining various biomarkers to help better
assess the progression of the disease. Vartiainen, J. et al., Int'l
J. Cancer, 95(5): 313-16 (2001); Shridhar et al. supra; Baekelandt,
M. et al., J. Clin. Oncol. 18(22): 3775-81.
The treatment of ovarian cancer typically involves a multiprong
attack, with surgical intervention serving as the foundation of
treatment. Dennis S. Chi & William J. Hoskins, Primary Surgical
Management of Advanced Epithelial Ovarian Cancer, in Ovarian Cancer
241 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).
For example, in the case of epithelial ovarian cancer, which accounts
for .about.90% of cases of ovarian cancer, treatment typically consists
of: (1) cytoreductive surgery, including total abdominal hysterectomy,
bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy,
followed by (2) adjuvant chemotherapy with paclitaxel and either
cisplatin or carboplatin. Eltabbakh, G. H. & Awtrey, C. S.,
Expert Op. Pharmacother. 2(10): 109-24. Despite a clinical response
rate of 80% to the adjuvant therapy, most patients experience tumor
recurrence within three years of treatment. Id. Certain patients
may undergo a second cytoreductive surgery and/or second-line chemotherapy.
Memarzadeh & Berek, supra.
From the foregoing, it is clear that procedures used for detecting,
diagnosing, monitoring, staging, prognosticating, and preventing
the recurrence of ovarian cancer are of critical importance to the
outcome of the patient. Moreover, current procedures, while helpful
in each of these analyses, are limited by their specificity, sensitivity,
invasiveness, and/or their cost. As such, highly specific and sensitive
procedures that would operate by way of detecting novel markers
in cells, tissues, or bodily fluids, with minimal invasiveness and
at a reasonable cost, would be highly desirable.
Accordingly, there is a great need for more sensitive and accurate
methods for predicting whether a person is likely to develop ovarian
cancer, for diagnosing ovarian cancer, for monitoring the progression
of the disease, for staging the ovarian cancer, for determining
whether the ovarian cancer has metastasized, for imaging the ovarian
cancer and for better treatment of ovarian cancer.
Colon Cancer
Colorectal cancer is the second most common cause of cancer death
in the United States and the third most prevalent cancer in both
men and women. M. L. Davila & A. D. Davila, Screening for Colon
and Rectal Cancer, in Colon and Rectal Cancer 47 (Peter S. Edelstein
ed., 2000). The American Cancer Society estimates that there will
be about 106,370 new cases of colon cancer and 40,570 new cases
of rectal cancer in the 2004 in the United States alone. Colon cancer
and rectal cancer will cause about 56,730 deaths combined in the
United States. ACS Website: cancer.org of the world wide web. Nearly
all cases of colorectal cancer arise from adenomatous polyps, some
of which mature into large polyps, undergo abnormal growth and development,
and ultimately progress into cancer. Davila at 55-56. This progression
would appear to take at least 10 years in most patients, rendering
it a readily treatable form of cancer if diagnosed early, when the
cancer is localized. Davila at 56; Walter J. Burdette, Cancer: Etiology,
Diagnosis, and Treatment 125 (1998).
Although our understanding of the etiology of colon cancer is undergoing
continual refinement, extensive research in this area points to
a combination of factors, including age, hereditary and nonhereditary
conditions, and environmental/dietary factors. Age is a key risk
factor in the development of colorectal cancer, Davila at 48, with
men and women over 40 years of age become increasingly susceptible
to that cancer, Burdette at 126. Incidence rates increase considerably
in each subsequent decade of life. Davila at 48. A number of hereditary
and nonhereditary conditions have also been linked to a heightened
risk of developing colorectal cancer, including familial adenomatous
polyposis (FAP), hereditary nonpolyposis colorectal cancer (Lynch
syndrome or HNPCC), a bowel disease, diabetes mellitus, and obesity.
Id. at 47; Henry T. Lynch & Jane F. Lynch, Hereditary Nonpolyposis
Colorectal Cancer (Lynch Syndromes), in Colon and Rectal Cancer
67-68 (Peter S. Edelstein ed., 2000).
Environmental/dietary factors associated with an increased risk
of colorectal cancer include a high fat diet, intake of high dietary
red meat, and sedentary lifestyle. Davila at 47; Reddy, B. S., Prev.
Med. 16(4): 460-7 (1987). Conversely, environmental/dietary factors
associated with a reduced risk of colorectal cancer include a diet
high in fiber, folic acid, calcium, and hormone-replacement therapy
in post-menopausal women. Davila at 50-55. The effect of antioxidants
in reducing the risk of colon cancer is unclear. Davila at 53.
Because colon cancer is highly treatable when detected at an early,
localized stage, screening should be a part of routine care for
all adults starting at age 50, especially those with first-degree
relatives with colorectal cancer. One major advantage of colorectal
cancer screening over its counterparts in other types of cancer
is its ability to not only detect precancerous lesions, but to remove
them as well. Davila at 56. The key colorectal cancer screening
tests in use today are fecal occult blood test, sigmoidoscopy, colonoscopy,
double-contrast barium enema, and the carcinoembryonic antigen (CEA)
test. Burdette at 125; Davila at 56.
The fecal occult blood test (FOBT) screens for colorectal cancer
by detecting the amount of blood in the stool, the premise being
that neoplastic tissue, particularly malignant tissue, bleeds more
than typical mucosa, with the amount of bleeding increasing with
polyp size and cancer stage. Davila at 56-57. While effective at
detecting early stage tumors, FOBT is unable to detect adenomatous
polyps (premalignant lesions), and, depending on the contents of
the fecal sample, is subject to rendering false positives. Davila
at 56-59. Sigmoidoscopy and colonoscopy, by contrast, allow direct
visualization of the bowel, and enable one to detect, biopsy, and
remove adenomatous polyps. Davila at 59-60, 61. Despite the advantages
of these procedures, there are accompanying downsides: sigmoidoscopy,
by definition, is limited to the sigmoid colon and below, colonoscopy
is a relatively expensive procedure, and both share the risk of
possible bowel perforation and hemorrhaging. Davila at 59-60. Double-contrast
barium enema (DCBE) enables detection of lesions better than FOBT,
and almost as well a colonoscopy, but it may be limited in evaluating
the winding rectosigmoid region. Davila at 60. The CEA blood test,
which involves screening the blood for carcinoembryonic antigen,
shares the downside of FOBT, in that it is of limited utility in
detecting colorectal cancer at an early stage. Burdette at 125.
Once colon cancer has been diagnosed, treatment decisions are typically
made in reference to the stage of cancer progression. A number of
techniques are employed to stage the cancer (some of which are also
used to screen for colon cancer), including pathologic examination
of resected colon, sigmoidoscopy, colonoscopy, and various imaging
techniques. AJCC Cancer Staging Handbook 84 (Irvin D. Fleming et
al. eds., 5.sup.th ed. 1998); Montgomery, R. C. and Ridge, J. A.,
Semin. Surg. Oncol. 15(3): 143-150 (1998). Moreover, chest films,
liver functionality tests, and liver scans are employed to determine
the extent of metastasis. Fleming at 84. While computerized tomography
and magnetic resonance imaging are useful in staging colorectal
cancer in its later stages, both have unacceptably low staging accuracy
for identifying early stages of the disease, due to the difficulty
that both methods have in (1) revealing the depth of bowel wall
tumor infiltration and (2) diagnosing malignant adenopathy. Thoeni,
R. F., Radiol. Clin. N. Am. 35(2): 457-85 (1997). Rather, techniques
such as transrectal ultrasound (TRUS) are preferred in this context,
although this technique is inaccurate with respect to detecting
small lymph nodes that may contain metastases. David Blumberg &
Frank G. Opelka, Neoadjuvant and Adjuvant Therapy for Adenocarcinoma
of the Rectum, in Colon and Rectal Cancer 316 (Peter S. Edelstein
ed., 2000).
Several classification systems have been devised to stage the extent
of colorectal cancer, including the Dukes' system and the more detailed
International Union against Cancer-American Joint Committee on Cancer
TNM staging system, which is considered by many in the field to
be a more useful staging system. Burdette at 126-27. The TNM system,
which is used for either clinical or pathological staging, is divided
into four stages, each of which evaluates the extent of cancer growth
with respect to primary tumor (T), regional lymph nodes (N), and
distant metastasis (M). Fleming at 84-85. The system focuses on
the extent of tumor invasion into the intestinal wall, invasion
of adjacent structures, the number of regional lymph nodes that
have been affected, and whether distant metastasis has occurred.
Fleming at 81.
Stage 0 is characterized by in situ carcinoma (Tis), in which the
cancer cells are located inside the glandular basement membrane
(intraepithelial) or lamina propria (intramucosal). In this stage,
the cancer has not spread to the regional lymph nodes (N0), and
there is no distant metastasis (M0). In stage I, there is still
no spread of the cancer to the regional lymph nodes and no distant
metastasis, but the tumor has invaded the submucosa (T1) or has
progressed further to invade the muscularis propria (T2). Stage
II also involves no spread of the cancer to the regional lymph nodes
and no distant metastasis, but the tumor has invaded the subserosa,
or the nonperitonealized pericolic or perirectal tissues (T3), or
has progressed to invade other organs or structures, and/or has
perforated the visceral peritoneum (T4). Stage III is characterized
by any of the T substages, no distant metastasis, and either metastasis
in 1 to 3 regional lymph nodes (N1) or metastasis in four or more
regional lymph nodes (N2). Lastly, stage IV involves any of the
T or N substages, as well as distant metastasis. Fleming at 84-85;
Burdette at 127.
Currently, pathological staging of colon cancer is preferable over
clinical staging as pathological staging provides a more accurate
prognosis. Pathological staging typically involves examination of
the resected colon section, along with surgical examination of the
abdominal cavity. Fleming at 84. Clinical staging would be a preferred
method of staging were it at least as accurate as pathological staging,
as it does not depend on the invasive procedures of its counterpart.
Turning to the treatment of colorectal cancer, surgical resection
results in a cure for roughly 50% of patients. Irradiation is used
both preoperatively and postoperatively in treating colorectal cancer.
Chemotherapeutic agents, particularly 5-fluorouracil, are also powerful
weapons in treating colorectal cancer. Other agents include irinotecan
and floxuridine, cisplatin, levamisole, methotrexate, interferon-.alpha.,
and leucovorin. Burdette at 125, 132-33. Nonetheless, thirty to
forty percent of patients will develop a recurrence of colon cancer
following surgical resection, which in many patients is the ultimate
cause of death. Wayne De Vos, Follow-up After Treatment of Colon
Cancer, Colon and Rectal Cancer 225 (Peter S. Edelstein ed., 2000).
Accordingly, colon cancer patients must be closely monitored to
determine response to therapy and to detect persistent or recurrent
disease and metastasis.
The next few paragraphs describe the some of molecular bases of
colon cancer. In the case of FAP, the tumor suppressor gene APC
(adenomatous polyposis coli), chromosomally located at 5q21, has
been either inactivated or deleted by mutation. Alberts et al.,
Molecular Biology of the Cell 1288 (3d ed. 1994). The APC protein
plays a role in a number of functions, including cell adhesion,
apoptosis, and repression of the c-myc oncogene. N. R. Hall &
R. D. Madoff, Genetics and the Polyp-Cancer Sequence, Colon and
Rectal Cancer 8 (Peter S. Edelstein, ed., 2000). Of those patients
with colorectal cancer who have normal APC genes, over 65% have
such mutations in the cancer cells but not in other tissues. Alberts
et al., supra at 1288. In the case of HPNCC, patients manifest abnormalities
in the tumor suppressor gene HNPCC, but only about 15% of tumors
contain the mutated gene. Id. A host of other genes have also been
implicated in colorectal cancer, including the K-ras, N-ras, H-ras
and c-myc oncogenes, and the tumor suppressor genes DCC (deleted
in colon carcinoma) and p53. Hall & Madoff, supra at 8-9; Alberts
et al., supra at 1288.
Abnormalities in Wg/Wnt signal transduction pathway are also associated
with the development of colorectal carcinoma. Taipale, J. and Beachy,
P. A. Nature 411: 349-354 (2001). Wnt1 is a secreted protein gene
originally identified within mouse mammary cancers by its insertion
into the mouse mammary tumor virus (MMTV) gene. The protein is homologous
to the wingless (Wg) gene product of Drosophila, in which it functions
as an important factor for the determination of dorsal-ventral segmentation
and regulates the formation of fly imaginal discs. Wg/Wnt pathway
controls cell proliferation, death and differentiation. Taipal (2001).
There are at least 13 members in the Wnt family. These proteins
have been found expressed mainly in the central nervous system (CNS)
of vertebrates as well as other tissues such as mammary and intestine.
The Wnt proteins are the ligands for a family of seven transmembrane
domain receptors related to the Frizzled gene product in Drosophila.
Binding Wnt to Frizzled stimulates the activity of the downstream
target, Dishevelled, which in turn inactivates the glycogen synthesase
kinase 3.beta. (GSK3.beta.). Taipal (2001). Usually active GSK3.beta.
will form a complex with the adenomatous polyposis coli (APC) protein
and phosphorylate another complex member, .beta.-catenin. Once phosphorylated,
.beta.-catenin is directed to degradation through the ubiquitin
pathway. When GSK3.beta., or APC activity is down regulated, .alpha.-catenin
is accumulated in the cytoplasm and binds to the T-cell factor or
lymphocyte excitation factor (Tcf/Lef) family of transcriptional
factors. Binding of .alpha.-catenin to Tcf releases the transcriptional
repression and induces gene transcription. Among the genes regulated
by .beta.-catenin are a transcriptional repressor Engrailed, a transforming
growth factor-.beta., (TGF-.beta.) family member Decapentaplegic,
and the cytokine Hedgehog in Drosophila. .beta.-Catenin also involves
in regulating cell adhesion by binding to .alpha.-catenin and E-cadherin.
On the other hand, binding of .beta.-catenin to these proteins controls
the cytoplasmic .beta.-catenin level and its complexing with TCF.
Taipal (2001). Growth factor stimulation and activation of c-src
or v-src also regulate .beta.-catenin level by phosphorylation of
.alpha.-catenin and its related protein, p120.sup.cas. When phosphorylated,
these proteins decrease their binding to E-cadherin and .beta.-catenin
resulting in the accumulation of cytoplasmic .beta.-catenin. Reynolds,
A. B. et al. Mol. Cell Biol. 14: 8333-8342 (1994). In colon cancer,
c-src enzymatic activity has been shown increased to the level of
v-src. Alternation of components in the Wg/Wnt pathway promotes
colorectal carcinoma development. The best known modifications are
to the APC gene. Nicola S et al. Hum. Mol. Genet 10:721-733 (2001).
This germline mutation causes the appearance of hundreds to thousands
of adenomatous polyps in the large bowel. It is the gene defect
that accounts for the autosomally dominantly inherited FAP and related
syndromes. The molecular alternations that occur in this pathway
largely involve deletions of alleles of tumor-suppressor genes,
such as APC, p53 and Deleted in Colorectal Cancer (DCC), combined
with mutational activation of proto-oncogenes, especially c-Ki-ras.
Aoki, T. et al. Human Mutat. 3: 342-346 (1994). All of these lead
to genomic instability in colorectal cancers.
Another source of genomic instability in colorectal cancer is the
defect of DNA mismatch repair (MMR) genes. Human homologues of the
bacterial mutHLS complex (hMSH2, hMLH1, hPMS1, hPMS2 and hMSH6),
which is involved in the DNA mismatch repair in bacteria, have been
shown to cause the HNPCC (about 70-90% HNPCC) when mutated. Modrich,
P. and Lahue, R. Ann Rev. Biochem. 65: 101-133 (1996); and Peltomaki,
P. Hum. Mol. Genet 10: 735-740 (2001). The inactivation of these
proteins leads to the accumulation of mutations and causes genetic
instability that represents errors in the accurate replication of
the repetitive mono-, di-, tri- and tetra-nucleotide repeats, which
are scattered throughout the genome (microsatellite regions). Jass,
J. R. et al. J. Gastroenterol Hepatol 17: 17-26 (2002). Like in
the classic FAP, mutational activation of c-Ki-ras is also required
for the promotion of MSI in the alternative HNPCC. Mutations in
other proteins such as the tumor suppressor protein phosphatase
PTEN (Zhou, X. P. et al. Hum. Mol. Genet 11: 445-450 (2002)), BAX
(Buttler, L. M. Aus. N. Z. J. Surg. 69: 88-94 (1999)), Caspase-5
(Planck, M. Cancer Genet Cytogenet. 134: 46-54 (2002)), TGF.beta.-RII
(Fallik, D. et al. Gastroenterol Clin Biol. 24: 917-22 (2000)) and
IGFII-R (Giovannucci E. J. Nutr. 131: 3109S-20S (2001)) have also
been found in some colorectal tumors possibly as the cause of MMR
defect.
Some tyrosine kinases have been shown up-regulated in colorectal
tumor tissues or cell lines like HT29. Skoudy, A. et al. Biochem
J. 317 (Pt 1): 279-84 (1996). Focal adhesion kinase (FAK) and its
up-stream kinase c-src and c-yes in colonic epithelia cells may
play an important role in the promotion of colorectal cancers through
the extracellular matrix (ECM) and integrin-mediated signaling pathways.
Jessup, J. M. et al., The molecular biology of colorectal carcinoma,
in: The Molecular Basis of Human Cancer, 251-268 (Coleman W. B.
and Tsongalis G. J. Eds. 2002). The formation of c-src/FAK complexes
may coordinately deregulate VEGF expression and apoptosis inhibition.
Recent evidences suggest that a specific signal-transduction pathway
for cell survival that implicates integrin engagement leads to FAK
activation and thus activates PI-3 kinase and akt. In turn, akt
phosphorylates BAD and blocks apoptosis in epithelial cells. The
activation of c-src in colon cancer may induce VEGF expression through
the hypoxia pathway. Other genes that may be implicated in colorectal
cancer include Cox enzymes (Ota, S. et al. Aliment Pharmacol. Ther.
16 (Suppl 2): 102-106 (2002)), estrogen (al-Azzawi, F. and Wahab,
M. Climacteric 5: 3-14 (2002)), peroxisome proliferator-activated
receptor-.gamma. (PPAR-.gamma.) (Gelman, L. et al. Cell Mol. Life
Sci. 55: 932-943 (1999)), IGF-I (Giovannucci (2001)), thymine DNA
glycosylase (TDG) (Hardeland, U. et al. Prog. Nucleic Acid Res.
Mol. Biol. 68: 235-253 (2001)) and EGF (Mendelsohn, J. Endocrine-Related
Cancer 8: 3-9 (2001)).
Gene deletion and mutation are not the only causes for development
of colorectal cancers. Epigenetic silencing by DNA methylation also
accounts for the lost of function of colorectal cancer suppressor
genes. A strong association between MSI and CpG island methylation
has been well characterized in sporadic colorectal cancers with
high MSI but not in those of hereditary origin. In one experiment,
DNA methylation of MLH1, CDKN2A, MGMT, THBS1, RARB, APC, and p14ARF
genes has been shown in 80%, 55%, 23%, 23%, 58%, 35%, and 50% of
40 sporadic colorectal cancers with high MSI respectively. Yamamoto,
H. et al. Genes Chromosomes Cancer 33: 322-325 (2002); and Kim,
K. M. et al. Oncogene. 12;21(35): 5441-9 (2002). Carcinogen metabolism
enzymes such as GST, NAT, CYP and MTHFR are also associated with
an increased or decreased colorectal cancer risk. Pistorius, S.
et al. Kongressbd Dtsch Ges Chir Kongr 118: 820-824 (2001); and
Potter, J. D. J. Natl. Cancer Inst. 91: 916-932 (1999).
From the foregoing, it is clear that procedures used for detecting,
diagnosing, monitoring, staging, prognosticating, and preventing
the recurrence of colorectal cancer are of critical importance to
the outcome of the patient. Moreover, current procedures, while
helpful in each of these analyses, are limited by their specificity,
sensitivity, invasiveness, and/or their cost. As such, highly specific
and sensitive procedures that would operate by way of detecting
novel markers in cells, tissues, or bodily fluids, with minimal
invasiveness and at a reasonable cost, would be highly desirable.
Accordingly, there is a great need for more sensitive and accurate
methods for predicting whether a person is likely to develop colorectal
cancer, for diagnosing colorectal cancer, for monitoring the progression
of the disease, for staging the colorectal cancer, for determining
whether the colorectal cancer has metastasized, and for imaging
the colorectal cancer. Following accurate diagnosis, there is also
a need for less invasive and more effective treatment of colorectal
cancer.
Gastric Cancer
The American Cancer Society estimates that there will be about
22,710 new cases of stomach cancer in 2004 in the United States
alone. Stomach cancer will cause about 11,780 deaths in the United
States. ACS Website: cancer.org of the world wide web. As recent
as 2001 gastric cancer was estimated to rank as the thirteenth most
common and the eighth most deadly cancer in the United States. AJCC
Cancer Staging Handbook 71 (Irvin D. Fleming et al. eds., 5.sup.th
ed. 1998). Due to a dramatic decline in the United States over the
last four decades, stomach cancer was estimated to account for 2.5%
of deaths from cancer in the United States in 1997, with roughly
22,000 new cases and 14,000 deaths estimated for that year. Roderich
E. Schwarz, Surgical Management of Gastric Cancer: The Western Experience,
in Management of Upper Gastrointestinal Cancer 83-84 (John M. Daly
et al. eds. 1999). However, stomach cancer persists in being responsible
for considerable mortality rates in Asia, Europe and South America.
Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 91
(1998). In Japan for example, gastric cancer accounts for roughly
one-half of the cancer deaths in men and one-third of those in women.
Id. Overall, patients diagnosed with gastric cancer have an approximate
5-year survival rate of around 25-30%. J. Rudiger Siewert et al.,
Early Gastric Cancer, in Management of Upper Gastrointestinal Cancer
136 (John M. Daly et al. eds. 1999).
Although our understanding of the etiology of gastric cancer is
undergoing continual refinement, research in this area points to
several risk factors, including various stomach diseases, diet,
occupation, and genetic factors. Burdette, supra at 91. In the case
of stomach diseases, stomach polyps, atrophic gastritis and metaplasia,
hyperplasia related to Menetrier's disease, Helicobacter pylori
infection, ulcers, and operations to the stomach have all been associated
with an increased incidence of stomach cancer. Id. Dietary nitrate
ingestion, which results in nitrosamine production in the stomach,
as well as the intake of smoked meats, are also suspected as contributing
factors. Id.; Fleming et al. eds., supra at 71. From an occupational
standpoint, those who work in the metalworking, painting, fishing,
ceramic, and printing industries all appear to have an elevated
risk of acquiring stomach cancer. Burdette, supra at 91. From a
genetic standpoint, gastric carcinomas are believed to occur through
two genetic pathways: (1) chromosomal deletions that involve tumor
suppressor genes and (2) microsatellite instability which targets
the mononucleotide segments in coding regions of genes related to
cancer. Rhyu, M. G., J. Korean Med. Sci. 13(4): 339-49 (1998). A
variation in the N-acetyltransferase 1 gene has also been linked
to elevated risk of gastric cancer. Boissy, R. J. et al., Int'l
J. Cancer 87(4): 507-11 (2000).
Like many cancers, gastric cancer is more readily treatable when
detected early. Patients diagnosed with early gastric cancer that
follow proper treatment have survival rates that match healthy control
patients of the same age. Siewert, supra at 136. Unfortunately,
the symptoms and clinical manifestations of gastric cancer typically
do not appear early in the course of the disease, and the majority
of patients have symptoms of the disease for six months or more
prior to diagnosis. Burdette, supra at 93. Accordingly, effective
screening devices are crucial in diagnosing the disease early and
in effecting proper treatment.
Following an initial assessment of a potential gastric cancer patient's
symptoms, which may include, inter alia, indigestion, abdominal
discomfort, dysphagia, nausea, anorexia, flatulence, weight loss,
melena, the presence of a palpable mass, anemia, and enlarged lymph
nodes, id., a physician may perform various screening tests. These
tests include scanning for the presence of elevated levels of carcinoembryonic
and oncofetal antigens, achlorhydria, blood in the stool, and cytologic
analysis of gastric washings. Id. Unfortunately, in the case of
the first three tests, positive results are not necessarily obtained
when gastric cancer is present, or false positives may result due
to the presence of other conditions. Id. A certain diagnosis is
typically achieved by way of endoscopy and/or radiography using
barium contrast medium. Id.; Schwarz, supra at 87. Ultrasonography,
computed tomography (CT), and magnetic resonance imaging (MRI) are
additionally useful in determining the extent of metastasis. Burdette,
supra at 94.
Once gastric cancer has been diagnosed, treatment decisions are
made in reference to the stage of cancer progression. Iain G. Martin,
Staging of Esophageal and Gastric Cancer, in Management of Upper
Gastrointestinal Cancer 3 (John M. Daly et al. eds. 1999). Accurate
staging has become even more vital to a successful treatment regimen
in view of the present trend toward multi-modal therapy for gastric
cancer, and particularly neoadjuvant therapy. Id.
A number of techniques are employed to stage gastric cancer (some
of which are also used to screen for gastric cancer), including
endoscopic ultrasonography (EUS), CT, and MRI. Id. at 24-31. EUS
is the only method of staging capable of providing accurate data
regarding the tumor stage (T stage) of gastric cancer, and its overall
accuracy for gathering data regarding the lymph nodal stage of gastric
cancer is about 70% Id. at 27-28. EUS, however, is limited for several
reasons: (1) roughly 15% of patients present with non-traversable
lesions, (2) there are regions of the stomach in which it is difficult
to obtain high quality images, and (3) it has difficulty in discerning
particular types of cancerous lesions. Id. at 27. CT scanning is
of some utility when used in combination with other techniques,
but it is too inaccurate to be used alone for several reasons: (1)
it is limited in its ability to assess the tumor stage due to its
inability to distinguish between the individual layers of the gastric
wall, (2) it is highly inaccurate in assessing lymph node metastasis,
and (3) it is generally unhelpful in assessing peritoneal or liver
metastasis. Id. at 24, 26-27. MRI, by contrast, is able to distinguish
between muscle layers in the stomach, and one study suggests that
MRI is able to assist in determining the extent of tumor and serosal
invasion with considerable accuracy. Id. at 27. Nonetheless, other
studies have indicated that MRI has little to offer to supplement
a CT assessment. Id.
The development of staging through the techniques of molecular
biology is still in its infancy, but some progress in this area
has been made. For example, researchers have found that Thomsen-Friedenreich
(TF) and MUC 1-TF immunoreactivity characterizes a high-risk Stage
I subgroup of gastric cancer patients. Baldus, S. E. et al., Oncology
61(2): 147-55 (2001). Elevated serum levels of interleukin-2 and
tumor necrosis factor-alpha have been studied as possibly useful
markers for advanced gastric cancer. Forones, N. M. et al., Hepatogastroenterology
48(40): 1199-201 (2001). Likewise, elevated levels of serum soluble
E-cadherin may also serve as a useful prognostic marker for stomach
cancer. Chan, A. O. et al., Gut 48(6): 808-11 (2001).
The two major classification systems for staging gastric cancer
are the Union Internationale Contre le Cancer's TNM system, and
the system devised by the Japanese Research Society for Gastric
Cancer. Id. at 18-23. The TNM system is a rather simple, and in
some cases arbitrary system, which is divided into several stages,
each of which evaluates the extent of cancer growth with respect
to primary tumor (T), regional lymph nodes (N), and distant metastasis
(M). Id. at 18, 20, 22; Fleming et al. eds., supra at 3. The Japanese
system is considerably more detailed, but in some cases may be overly
complex and time consuming. Martin, supra at 18-20, 22-23. Because
most countries other than Japan have adopted the TNM system, id.
at 23, that system will be discussed further here.
Stage 0 is characterized by carcinoma in situ (Tis, an intra-epithelial
tumor that has not invaded the lamina propria), and stage IA involves
tumor invasion of the lamina propria or submucosa (T1); neither
stage involves metastasis to the regional lymph nodes (N0) nor distant
metastasis (MO). Fleming et al. eds., supra at 73. Stage IB is the
same as stage IA except that either (1) regional lymph node metastasis
has occurred in 1 to 6 lymph nodes (N1) or (2) the tumor has invaded
the muscularis propria or subserosa (T2). Id. Stage II gastric cancer
is a bit more complex than the previous stages, involving one of
three scenarios, none of which involve distant metastasis: (1) tumor
category T1 and metastasis into 7 to 15 regional lymph nodes (N2),
(2) tumor category T2 and nodal category N1, or (3) tumor invasion
into serosa without invasion into adjacent structures (i.e., spleen,
liver, transverse colon, diaphragm, adrenal gland, kidney, pancreas,
small intestine, retroperitoneum, and abdominal wall) and nodal
category N0. Id. Stage IIIA likewise involves one of three possible
scenarios: (1) tumor category T2 and nodal category N2, (2) tumor
category T3 and nodal category N3, or (3) tumor invasion into adjacent
structures (T4) and nodal category N0. Id. at 73-74. Stage IIIB,
however, involves tumor category T3 and nodal category N2. Id. Neither
stage IIIA nor stage IIIB involves distant metastasis. Id. Stage
IV is characterized by a variety permutations of tumor and nodal
categories, with or without distant metastasis. Id.
Turning to the treatment of gastric cancer, surgical resection
is the "mainstay" of treating gastric carcinomas but is
only an option for 50% to 60% of patients. David Kelsen, Combined
Modality Therapy, in Management of Upper Gastrointestinal Cancer
123 (John M. Daly et al. eds. 1999). While radiotherapy is sometimes
used in conjunction with resection with some effect, gastric carcinomas
are typically more resistant to radiation than are other carcinomas.
Burdette, supra at 97. Likewise, chemotherapy has generally been
of limited utility in treating gastric carcinomas, although neoadjuvant
and adjuvant chemotherapy have been used with some success. Id.
at 98; Schuhmacher, C. P. et al., Cancer 91(5): 918-27 (2001). Pre-
or postoperative adjuvant therapy is currently being studied due
to the considerable risk for reoccurrence, as well as the fact that
systemic metastasis is commonplace. Kelsen, supra at 123. When chemotherapy
is used, combinations of chemotherapeutic agents yield better results
than single agents; agents used in successful combinations include
5-fluoruracil, leucovorin, adriamycin, cisplatin, mitomycin, etoposide,
and semustine. Burdette, supra at 98.
From the foregoing, it is clear that procedures used for detecting,
diagnosing, monitoring, staging, prognosticating, treating and preventing
the recurrence of gastric cancer are of critical importance to the
outcome of the patient. Moreover, current procedures, while helpful
in each of these areas, are limited by their specificity, sensitivity,
invasiveness, and/or their cost. As such, highly specific and sensitive
procedures that would operate by way of detecting novel markers
in cells, tissues, or bodily fluids, with minimal invasiveness and
at a reasonable cost, would be highly desirable.
Angiogenesis in Cancer
Growth and metastasis of solid tumors are also dependent on angiogenesis.
Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman, J., 1989,
Journal of the National Cancer Institute, 82, 4-6. It has been shown,
for example, that tumors which enlarge to greater than 2 mm must
obtain their own blood supply and do so by inducing the growth of
new capillary blood vessels. Once these new blood vessels become
embedded in the tumor, they provide a means for tumor cells to enter
the circulation and metastasize to distant sites such as liver,
lung or bone. Weidner, N., et al., 1991, The New England Journal
of Medicine, 324(1), 1-8.
Angiogenesis, defined as the growth or sprouting of new blood vessels
from existing vessels, is a complex process that primarily occurs
during embryonic development. The process is distinct from vasculogenesis,
in that the new endothelial cells lining the vessel arise from proliferation
of existing cells, rather than differentiating from stem cells.
The process is invasive and dependent upon proteolysis of the extracellular
matrix (ECM), migration of new endothelial cells, and synthesis
of new matrix components. Angiogenesis occurs during embryogenic
development of the circulatory system; however, in adult humans,
angiogenesis only occurs as a response to a pathological condition
(except during the reproductive cycle in women).
Under normal physiological conditions in adults, angiogenesis takes
place only in very restricted situations such as hair growth and
wounding healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol
Ther. 63(3):265-3 11; Ribatti et al., 1991, Haematologica 76(4):3
11-20; Risau, 1997, Nature 386(6626):67 1-4. Angiogenesis progresses
by a stimulus which results in the formation of a migrating column
of endothelial cells. Proteolytic activity is focused at the advancing
tip of this "vascular sprout", which breaks down the ECM
sufficiently to permit the column of cells to infiltrate and migrate.
Behind the advancing front, the endothelial cells differentiate
and begin to adhere to each other, thus forming a new basement membrane.
The cells then cease proliferation and finally define a lumen for
the new arteriole or capillary.
Unregulated angiogenesis has gradually been recognized to be responsible
for a wide range of disorders, including, but not limited to, cancer,
cardiovascular disease, rheumatoid arthritis, psoriasis and diabetic
retinopathy. Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation
99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh,
1999, Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997,
Nat Med 3(2): 158-64.
Of particular interest is the observation that angiogenesis is
required by solid tumors for their growth and metastases. Folkman,
1986 supra; Folkman 1990, J. Natl. Cancer Inst., 82(1) 4-6; Folkman,
1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24.
A tumor usually begins as a single aberrant cell which can proliferate
only to a size of a few cubic millimeters due to the distance from
available capillary beds, and it can stay `dormant` without further
growth and dissemination for a long period of time. Some tumor cells
then switch to the angiogenic phenotype to activate endothelial
cells, which proliferate and mature into new capillary blood vessels.
These newly formed blood vessels not only allow for continued growth
of the primary tumor, but also for the dissemination and recolonization
of metastatic tumor cells. The precise mechanisms that control the
angiogenic switch is not well understood, but it is believed that
neovascularization of tumor mass results from the net balance of
a multitude of angiogenesis stimulators and inhibitors Folkman,
1995, supra.
One of the most potent angiogenesis inhibitors is endostatin identified
by O'Reilly and Folkman. O'Reilly et al., 1997, Cell 88(2):277-85;
O'Reilly et al., 1994, Cell 79(2):315-28. Its discovery was based
on the phenomenon that certain primary tumors can inhibit the growth
of distant metastases. O'Reilly and Folkman hypothesized that a
primary tumor initiates angiogenesis by generating angiogenic stimulators
in excess of inhibitors. However, angiogenic inhibitors, by virtue
of their longer half life in the circulation, reach the site of
a secondary tumor in excess of the stimulators. The net result is
the growth of primary tumor and inhibition of secondary tumor. Endostatin
is one of a growing list of such angiogenesis inhibitors produced
by primary tumors. It is a proteolytic fragment of a larger protein:
endostatin is a 20 kDa fragment of collagen XVIII (amino acid H1132-K1315
in murine collagen XVIII). Endostatin has been shown to specifically
inhibit endothelial cell proliferation in vitro and block angiogenesis
in vivo. More importantly, administration of endostatin to tumor-bearing
mice leads to significant tumor regression, and no toxicity or drug
resistance has been observed even after multiple treatment cycles.
Boehm et al., 1997, Nature 390(6658):404-407. The fact that endostatin
targets genetically stable endothelial cells and inhibits a variety
of solid tumors makes it a very attractive candidate for anticancer
therapy. Fidler and Ellis, 1994, Cell 79(2):185-8; Gastl et al.,
1997, Oncology 54(3):177-84; Hinsbergh et al., 1999, Ann Oncol 10
Suppl 4:60-3. In addition, angiogenesis inhibitors have been shown
to be more effective when combined with radiation and chemotherapeutic
agents. Klement, 2000, J. Clin Invest, 105(8) R15-24. Browder, 2000,
Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279(5349):377-80;
Mauceri et al., 1998, Nature 394(6690):287-91.
The present invention provides alternative methods of assessing
risk of, detecting or treating prostate, ovarian, colon, breast
and stomach cancer that overcome the limitations of conventional
therapeutic methods as well as offer additional advantages that
will be apparent from the detailed description below.
SUMMARY OF THE INVENTION
This invention is directed to a method for assessing risk of prostate
cancer in a subject which comprises measuring levels of both Pro108
and Prostate Specific Antigen (PSA) in the subject, analyzing a
risk associated wit the level of PSA and a risk associated with
the level of Pro108, and using the combined risks to assess the
risk of prostate cancer in the subject. In one aspect of the invention
the measuring of PSA and Pro108 levels are done simultaneously.
In another aspect of the invention the measuring of PSA and Pro108
are done sequentially. In addition, the invention is directed to
specific antibody pairs directed to Pro108 for detection of prostate,
ovarian, colon, breast or stomach cancer. Preferably, the antibodies
are used alone or in combination to detect prostate, ovarian, colon,
breast or stomach cancer.
In yet another aspect of the invention, the respective levels of
PSA and Pro108 are based on dividing a subject population dataset
into borderline levels of PSA and elevated levels of Pro108 and
a subject having both borderline PSA and high Pro108 levels is indicative
of heightened risk of prostate cancer. The borderline levels of
PSA may be between about 2 ng/mL and about 10 ng/mL. The borderline
levels of PSA may also between about 4 ng/mL and about 10 ng/mL
or between about 2 ng/mL and about 4 ng/mL.
The invention is also directed to a method for treating a subject
with elevated risk of a prostate cancer, comprising: selecting a
subject who has borderline levels of Prostate Specific Antigen (PSA)
and elevated levels of Pro108 and treating the subject with a therapy
selected from the group consisting of surgery, radiation therapy,
hormone therapy or chemotherapy so as to alleviate the elevated
risk of prostate cancer in the subject.
This invention is further directed to an isolated Pro108 antibody
that binds to Pro108 on a mammalian cell in vivo. The invention
is further directed to an isolated Pro108 antibody that internalizes
upon binding to Pro108 on a mammalian cell in vivo. The antibody
may be a monoclonal antibody. Alternatively, the antibody is an
antibody fragment or a chimeric or a humanized antibody. The monoclonal
antibody may be produced by a hybridoma selected from the group
of hybridomas deposited under American Type Culture Collection accession
number PTA-5885 and PTA-5886.
The antibody may compete for binding to the same epitope as the
epitope bound by the monoclonal antibody produced by a hybridoma
selected from the group of hybridomas deposited under the American
Type Culture Collection accession number PTA-5885 and PTA-5886.
The invention is also directed to conjugated antibodies. They may
be conjugated to a growth inhibitory agent or a cytotoxic agent.
The cytotoxic agent may be selected from the group consisting of
toxins, antibiotics, radioactive isotopes and nucleolytic enzymes
and toxins. Examples of toxins include, but are not limited to,
auristatin, maytansin, maytansinoids, saporin, gelonin, ricin or
calicheamicin.
The mammalian cell may be a cancer cell. Preferably, the anti-Pro108
monoclonal antibody that inhibits the growth of Pro108-expressing
cancer cells in vivo.
The antibody may be produced in bacteria. Alternatively, the antibody
may be a humanized form of an anti-Pro108 antibody produced by a
hybridoma selected from the group of hybridomas having ATCC accession
number PTA-5885 and PTA-5886.
Preferably, the cancer is selected from the group consisting of
prostate, ovarian, colon, breast and stomach cancer. The invention
is also directed to a method of producing the antibodies comprising
culturing an appropriate cell and recovering the antibody from the
cell culture.
The invention is also directed to compositions comprising the antibodies
and a carrier. The antibody may be conjugated to a cytotoxic agent.
The cytotoxic agent may be a radioactive isotope or other chemotherapeutic
agent.
The invention is also directed to a method of killing an Pro108-expressing
cancer cell, comprising contacting the cancer cell with the antibodies
of this invention, thereby killing the cancer cell. The cancer cell
may be selected from the group consisting of prostate, ovarian,
colon, breast and stomach cancer cell.
The ovarian or breast cancer may be ovarian serous or mucinous
adenocarcinoma or breast infiltrating ductal carcinoma or metastatic
cancer. The breast cancer may be HER-2 negative breast cancer.
The invention is also directed to a method of alleviating a Pro108-expressing
cancer in a mammal, comprising administering a therapeutically effective
amount of the antibodies to the mammal.
This invention is further directed to a method for assessing risk
of ovarian cancer in a patient which comprises measuring levels
of both Pro108 and CA125 in the patient, analyzing a risk associated
with the level of CA125 and a risk associated with the level of
Pro108, and using the combined risks to assess the risk of Ovarian
Cancer in the patient. In one aspect of the invention the measuring
of CA125 and Pro108 levels are done simultaneously. In another aspect
of the invention the measuring of CA125 and Pro108 are done sequentially.
In yet another aspect of the invention, the respective levels of
CA125 and Pro108 are based on dividing a patient population dataset
into low levels of CA125 and elevated levels of Pro108 and a patient
having both low CA125 and high Pro108 levels is indicative of heightened
risk of Ovarian Cancer. The low levels of CA125 may be below about
30 U/mL.
The invention is also directed to a method for treating a subject
with elevated risk of a Ovarian Cancer, comprising: selecting a
subject who has low levels of CA125 and elevated levels of Pro108
and treating the subject with a therapy selected from the group
consisting of surgery, radiation therapy, hormone therapy or chemotherapy
so at to treat the subject with the elevated risk of Ovarian Cancer.
The invention is also directed to a method for selecting a patient
for ovarian biopsy comprising measuring levels of both Pro108 and
CA125 in the patient, analyzing a risk associated with the level
of CA125 and a risk associated with the level of Pro108, and based
on the combined levels of both Pro108 and CA125 selecting the patient
for ovarian biopsy.
Moreover, the invention is directed to a kit for determining the
likelihood of a patient having Ovarian Cancer which comprises both
a suitable assay for measuring Pro108 levels and a suitable assay
for measuring CA125 levels wherein the levels of both CA125 and
Pro108 are determined using the combined results.
This invention is further directed to a method for assessing risk
of prostate cancer in a patient which comprises measuring levels
of both Pro108 and Prostate Specific Antigen (PSA) in the patient,
analyzing a risk associated with the level of PSA and a risk associated
with the level of Pro108, and using the combined risks to assess
the risk of prostate cancer in the patient. In one aspect of the
invention the measuring of PSA and Pro108 levels are done simultaneously.
In another aspect of the invention the measuring of PSA and Pro108
are done sequentially.
In yet another aspect of the invention, the respective levels of
PSA and Pro108 are based on dividing a patient population dataset
into borderline levels of PSA and elevated levels of Pro108 and
a patient having both borderline PSA and high Pro108 levels is indicative
of heightened risk of prostate cancer. The borderline levels of
PSA may be between about 2 ng/mL and about 10 ng/mL. The borderline
levels of PSA may also between about 4 ng/mL and about 10 ng/mL
or between about 2 ng/mL and about 4 ng/mL.
The invention is also directed to a method for treating a subject
with elevated risk of a prostate cancer, comprising: selecting a
subject who has borderline levels of Prostate Specific Antigen (PSA)
and elevated levels of Pro108 and treating the subject with a therapy
selected from the group consisting of surgery, radiation therapy,
hormone therapy or chemotherapy so at to treat the subject with
the elevated risk of prostate cancer.
The invention is also directed to a method for selecting a patient
for prostate biopsy comprising measuring levels of both Pro108 and
Prostate Specific Antigen (PSA) in the patient, analyzing a risk
associated with the level of PSA and a risk associated with the
level of Pro108, and based on the combined levels of both Pro108
and PSA selecting the patient for prostate biopsy.
The invention also involves comparing the level of Pro108 or PSA
for the individual with a predetermined value. The predetermined
value can take a variety of forms. It can be single cut-off value,
such as a median or mean. It can be established based upon comparative
groups, such as where the risk in one defined group is double the
risk in another defined group. It can be a range, for example, where
the tested population is divided equally (or unequally) into groups,
e.g., tertiles, such as-a low-risk group, a medium-risk group and
a high-risk group, or into quadrants, the lowest quadrant being
individuals with the lowest risk and the highest quadrant being
individuals with the highest risk.
There presently are commercial sources which produce reagents for
assays for PSA. These include, but are not limited to, Abbott Pharmaceuticals
(Abbott Park, Ill.); Fujirebio Inc. (Tokyo, Japan), Biocheck Inc.
(Burlingame, Calif.), Dade Behring (Deerfield, Ill.), Beckman Coulter
Inc. (Chaska, Minn.); Roche Diagnostics (Indianapolis, Ind.). In
preferred embodiments the invention provides novel kits or assays
which are specific for, and have appropriate sensitivity with respect
to, predetermined values selected on the basis of the present invention.
The preferred kits, therefore, would differ from those presently
commercially available, by including, for example, different cut-offs,
different sensitivities at particular cut-offs as well as instructions
or other printed material for characterizing risk based upon the
outcome of the assay.
As discussed herein the invention provides methods for evaluating
the likelihood that an individual will benefit from treatment with
an agent for reducing risk of prostate, ovarian, colon, breast or
stomach cancer. This method has important implications for patient
treatment and also for clinical development of new therapeutics.
Physicians select therapeutic regimens for patient treatment based
upon the expected net benefit to the patient. The net benefit is
derived from the risk to benefit ratio. The present invention permits
selection of individuals who are more likely to benefit by intervention,
thereby aiding the physician in selecting a therapeutic regimen.
This might include using drugs with a higher risk profile where
the likelihood of expected benefit has increased. Likewise, clinical
investigators desire to select for clinical trials a population
with a high likelihood of obtaining a net benefit. The present invention
can help clinical investigators select such individuals. It is expected
that clinical investigators now will use the present invention for
determining entry criteria for clinical trials.
Moreover, the invention is directed to a kit for determining the
likelihood of a patient having prostate cancer which comprises both
a suitable assay for measuring Pro108 levels and a suitable assay
for measuring Prostate Specific Antigen (PSA) levels wherein the
levels of both PSA and Pro108 are determined using the combined
results.
In addition, the invention is directed to an article of manufacture
comprising a container and a composition contained therein, wherein
the composition comprises an antibody as described herein. The article
of manufacture may also comprise an additional component, e.g.,
a package insert indicating that the composition can be used to
treat prostate, ovarian, colon, breast or stomach cancer.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the anti-Pro108 antibody epitope mapping.
FIG. 2 shows Pro108 serum levels in healthy subjects and subjects
with various cancers.
FIG. 3 shows Pro108 levels in prostate cancer and benign prostate
disease.
FIG. 4 shows Pro108 levels in ovarian cancer and benign ovarian
disease.
FIG. 5 shows Pro108 levels in serous and mucinous ovarian cancer
and in benign ovarian disease.
FIG. 6 shows Pro108 levels in colon cancer and benign colon disease.
FIG. 7 shows Pro108 levels in stomach cancer.
FIG. 8 shows detection of Pro108 in the lysate of normal somatic
and cancer tissues.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
Human Pro108 as used herein, refers to a protein of 331 amino acids,
the nucleotide and amino acid sequences were previously disclosed
in WO200023108-A1 as Cancer specific gene Pro108; EP1130094-A2 as
Human polypeptide SEQ ID NO: 2847; WO200229038-A2 as Human Spondin
2-like protein NOV6; DE10050274-A1 as Human spondin 2; WO200230268-A2
as Prostate cancer-associated protein #7, WO2003009814-A2 as Prostate
cancer marker protein; WO200153312-A1 as Human polypeptide SEQ ID
NO 5589; U.S. 2003104998-A1 as Human secreted/transmembrane protein,
PRO866; and WO0144291-A2 as RG1.
Human Pro108 has also been identified as Spondin 2. The RefSeq
database identifies Spondin 2 as "Homo sapiens spondin 2, extracellular
matrix protein (SPON2)" and references the nucleotide and amino
acid sequences as NM.sub.--012445 and NP.sub.--036577, respectively.
Pro108 as used herein include allelic variants and conservative
substitution mutants of the protein which have Pro108 biological
activity.
Spondin 2 (Pro108) has been described as a gene differentially
expressed in cancerous and non-cancerous lung cells, with higher
mRNA expression in normal lung. Manda, R. et al., 1999, Genomics,
61: 5-14. The gene encodes a protein of 331 amino acids with a calculated
molecular mass of 35 kD. Sequence analysis indicates the existence
of a signal sequence within the first 27 amino acids therefore amino
acids 27-331 are presumably secreted from cells. In addition, sequence
analysis identifies Spondin 2 as a human homologue of the zebrafish
genes, Mindin1 and Mindin2, which are members of the F-spondin superfamily
genes. The F-spondin superfamily genes encode proteins with two
conserved domains, FS1 and FS2, near the amino terminus. Additionally,
at least one thrombospondin type I repeat is present at the carboxy-terminus.
The F-Spondin genes products are secreted and are likely to be extracellular
matrix molecules (ECM). ECM molecules are known to play a role in
cell adhesion which is critical for maintaining tissue architecture,
cellular differentiation, cellular function, growth and apoptosis.
ECM molecules have also been implicated in human carcinogenesis,
tumor invasion and malignant transformation. Disruption of maintenance
of cell-ECM adhesion is a well know indicator of tumor progression
and malignant transformation. Variations in levels of other ECM
molecules such as fibronectin (FM) have been associated with cancerous
and malignant tissues compared to normal tissues. Chakrabarty, S.
et al. Chapter 36 Adhesion Molecules as Tumor Markers, Tumor Markers,
Diamandis, E. Ed. (2002). Likewise, variations in Pro108 levels
in the ECM and in plasma or serum is anticipated to be involved
with, and indicate changes in maintenance of tissue architecture,
cellular differentiation, cellular function, growth, apoptosis,
and promotion of carcinogenesis, tumor invasion and malignant transformation.
It has been shown that the Trombospondin type I repeat, present
in Pro108, has the ability to inhibit angiogenesis and it also inhibits
the growth of several melanoma cell lines. Tolsma, S. et al., 1993,
J. Cell Biol. 122; 497-511; Terai, Y. et al., 2001, J Cell Physiol,
188: 394-402; Guo, N. H. et al., 1997, J. Peptide Res. 50: 210-221.
Breakdown of the ECM allows for angiogenesis to occur which is required
for tumor growth and progression. Therefore, maintenance of ECM
molecule function and levels, such as Pro108, is essential in inhibiting
angiogenesis and tumor growth and progression.
The closest human homolog of Spondin 2, F-Spondin, (or VSPG; M-Spondin
in drosophila; SCO-Spondin in bovine) is a secreted adhesion molecule
that is expressed at high level in the developing floor plate. Klar,
A. et al., 1992, Cell, 69: 95-110. F-Spondin is required for accurate
pathfinding of commissural axons and inhibits the outgrowth of embryonic
motor neurons. Burstyn-Cohen, T. et al., 1999, Neuron, 23: 233-246;
Tzarfati-Majar, V. et al., 2001, Proc Natl Acad Sci USA, 98: 4722-4727.
The exact function of Pro108 is not known yet but a recent publication
from He et al. describe the Pro108 mouse homologue mindin as pattern-recognition
molecule involved in the innate immune response to microbial pathogens
He, Y-W. et al., 2004, Nature Immunology 5, 88-97.
Our findings that Pro108 is associated with aggressive prostate,
ovarian, colon, breast and stomach cancers make this extracellular
matrix antigen an attractive target for detection, risk assessment,
monitoring or immunotherapy of these and possibly other tumor types.
Prostate Specific Antigen (PSA) has also been described widely,
for a recent review see Barry 2001. It is a glycoprotein produced
in the epithelium of the prostate. A variety of diseases both benign
and cancerous may cause elevated levels of PSA. The Physicians Health
Study found subjects with a PSA level of greater than 4.0 ng/mL
had a 46% specificity with to identify subjects that would have
prostate cancer within the next 10 years. Gann 1995. Others have
reported the following:
TABLE-US-00001 PSA levels (ng/mL) Probability of Prostate Cancer
0-2.4 Uncertain 2.5-4.0 12-23% 4.1-10.0 25% >10.0 >50%
See Barry 2001 and the references cited therein.
Methods for treating prostate cancer have been discussed in the
background section above. The level of the markers of this invention
may be obtained by a variety of recognized methods. Typically, the
level is determined by measuring the level of the marker in a body
fluid, for example, blood, lymph, saliva, urine and the like. The
preferred body fluid is blood. The level can be determined by ELISA,
or immunoassays or other conventional techniques for determining
the presence of the marker. Conventional methods include sending
samples of a patient's body fluid to a commercial laboratory for
measurement. For the measurement of PSA enzymatic assays may also
be used, see U.S. Pat. No. 6,361,955 (Roche), U.S. Pat. No. 6,300,088
(Duke), U.S. Pat. No. 6,107,049 (Bayer) U.S. Pat. No. 5,939,533
(Lilja), U.S. Pat. No. 5,928,878 (Bayer), U.S. Pat. No. 5,856,182
(Beckman Coulter), U.S. Pat. No. 5,672,480 (Abbott Laboratories),
U.S. Pat. No. 5,474,903 (Huland) or U.S. Pat. No. 5,242,802 (Hybritech),
the contents of which are hereby incorporated by reference into
the subject application.
The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.
bispecific antibodies), and antibody fragments, so long as they
exhibit the desired biological activity. The term "immunoglobulin"
(Ig) is used interchangeably with "antibody" herein.
An "isolated antibody" is one which has been identified
and separated and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials
which would interfere with diagnostic or therapeutic uses for the
antibody, and may include enzymes, hormones, and other proteinaceous
or nonproteinaceous solutes. Preferably, the antibody will be purified
(1) to greater than 95% by weight of antibody as determined by the
Lowry method, and most preferably more than 99% by weight, (2) to
a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup sequenator,
or (3) to homogeneity by SDS-PAGE under reducing or non-reducing
conditions using Coomassie blue or, preferably, silver stain. Isolated
antibody includes the antibody in situ within recombinant cells
since at least one component of the antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will
be prepared by at least one purification step.
The basic 4-chain antibody unit is a heterotetrameric glycoprotein
composed of two identical light (L) chains and two identical heavy
(H) chains (an IgM antibody consists of 5 of the basic heterotetramer
unit along with an additional polypeptide called J chain, and therefore
contain 10 antigen binding sites, while secreted IgA antibodies
can polymerize to form polyvalent assemblages comprising 2-5 of
the basic 4-chain units along with J chain). In the case of IgGs,
the 4-chain unit is generally about 150,000 daltons. Each L chain
is linked to an H chain by one covalent disulfide bond, while the
two H chains are linked to each other by one or more disulfide bonds
depending on the H chain isotype. Each H and L chain also has regularly
spaced intrachain disulfide bridges. Each H chain has at the N-terminus,
a variable domain (VH) followed by three constant domains (CH) for
each of the .alpha., .delta. and .gamma. chains and four CH domains
for .mu. and .epsilon. isotypes. Each L chain has at the N-terminus,
a variable domain (VL) followed by a constant domain (CL) at its
other end.
The VL is aligned with the VH and the CL is aligned with the first
constant domain of the heavy chain (CHI). Particular amino acid
residues are believed to form an interface between the light chain
and heavy chain variable domains. The pairing of a VH and VL together
forms a single antigen-binding site. For the structure and properties
of the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Teff and Tristram
G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page
71 and Chapter 6.
The L chain from any vertebrate species can be assigned to one
of two clearly distinct types, called kappa and lambda, based on
the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy chains
(CH), immunoglobulins can be assigned to different classes or isotypes.
There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, having heavy chains designated .alpha., .delta., .epsilon.,
.gamma. and .mu., respectively. The .gamma. and .alpha. classes
are further divided into subclasses on the basis of relatively minor
differences in C.sub.H sequence and function, e.g., humans express
the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among antibodies.
The V domain mediates antigen binding and define specificity of
a particular antibody for its particular antigen. However, the variability
is not evenly distributed across the 1-10-amino acid span of the
variable domains. Instead, the V regions consist of relatively invariant
stretches called framework regions (FRs) of 15-30 amino acids separated
by shorter regions of extreme variability called "hypervariable
regions" that are each 9-12 amino acids long. The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a P-sheet configuration, connected by three hypervariable
regions, which form loops connecting, and in some cases forming
part of, the P-sheet structure. The hypervariable regions in each
chain are held together in close proximity by the FRs and, with
the hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in binding an antibody
to an antigen, but exhibit various effector functions, such as participation
of the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term "hypervariable region" when used herein refers
to the amino acid residues of an antibody which are responsible
for antigen-binding. The hypervariable region generally comprises
amino acid residues from a "complementarity determining region"
or "CDR" (e.g. around about residues 24-34 (L1), 5056
(L2) and 89-97 (L3) in the VL, and around about 1-35 (H1), 50-65
(H2) and 95-102 (113) in the VH; Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12)
and 96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)).
The term "monoclonal antibody" as used herein refers
to an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population
are identical except for possible naturally occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations which
include different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they may be synthesized
uncontaminated by other antibodies. The modifier "monoclonal"
is not to be construed as requiring production of the antibody by
any particular method. For example, the monoclonal antibodies useful
in the present invention may be prepared by the hybridoma methodology
first described by Kohler et al., Nature, 256:495 (1975), or may
be made using recombinant DNA methods in bacterial, eukaryotic animal
or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
The monoclonal antibodies herein include "chimeric" antibodies
in which a portion of the heavy and/or light chain is identical
with or homologous to corresponding sequences in antibodies derived
from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical
with or homologous to corresponding sequences in antibodies derived
from another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (see U.S. Pat. No. 4,816,567; and
Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Chimeric antibodies of interest herein include "primatized"
antibodies comprising variable domain antigen-binding sequences
derived from a non-human primate (e.g. Old World Monkey, Ape etc),
and human constant region sequences.
An "intact" antibody is one which comprises an antigen-binding
site as well as a CL and at least heavy chain constant domains,
CH1, CH2 and CH3. The constant domains may be native sequence constant
domains (e.g. human native sequence constant domains) or amino acid
sequence variant thereof. Preferably, the intact antibody has one
or more effector functions.
An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments. Papain digestion of antibodies
produces two identical antigen-binding fragments, called "Fab"
fragments, and a residual "Fc" fragment, a designation
reflecting the ability to crystallize readily. The Fab fragment
consists of an entire L chain along with the variable region domain
of the H chain (VH), and the first constant domain of one heavy
chain (CHI). Each Fab fragment is monovalent with respect to antigen
binding, i.e., it has a single antigen-binding site. Pepsin treatment
of an antibody yields a single large F(ab').sub.2 fragment which
roughly corresponds to two disulfide linked Fab fragments having
divalent antigen-binding activity and is still capable of cross-linking
antigen. Fab' fragments differ from Fab fragments by having additional
few residues at the carboxy terminus of the CH1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s)
of the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of 8 Fab' fragments
which have hinge cysteines between them. Other chemical couplings
of antibody fragments are also known.
The Fc fragment comprises the carboxy-terminal portions of both
H chains held together by disulfides. The effector functions of
antibodies are determined by sequences in the Fc region, which region
is also the part recognized by Fc receptors (FcR) found on certain
types of cells.
"Fv" is the minimum antibody fragment which contains
a complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable region
domain in tight, non-covalent association. From the folding of these
two domains emanate six hypervariable loops (3 loops each from the
H and L chain) that contribute the amino acid residues for antigen
binding and confer antigen binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to recognize
and bind antigen, although at a lower affinity than the entire binding
site.
"Single-chain Fv" also abbreviated as "sFv"
or "scFv" are antibody fragments that comprise the VH
and VL antibody domains connected into a single polypeptide chain.
Preferably, the sFv polypeptide further comprises a polypeptide
linker between the VH and VL domains which enables the sFv to form
the desired structure for antigen binding. For a review of sFv,
see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.
113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra.
The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the VH and VL domains
such that inter-chain but not intra-chain pairing of the V domains
is achieved, resulting in a bivalent fragment, i.e., fragment having
two antigen-binding sites. Bispecific diabodies are heterodimers
of two "crossover" sFv fragments in which the VH and VL
domains of the two antibodies are present on different polypeptide
chains. Diabodies are described more fully in, for example, EP 404,097;
WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448
(1993).
A "native sequence" polypeptide is one which has the
same amino acid sequence as a polypeptide (e.g., antibody) derived
from nature. Such native sequence polypeptides can be isolated from
nature or can be produced by recombinant or synthetic means. Thus,
a native sequence polypeptide can have the amino acid sequence of
a naturally occurring human polypeptide, murine polypeptide, or
polypeptide from any other mammalian species.
The term "amino acid sequence variant" refers to a polypeptide
that has amino acid sequences that differ to some extent from a
native sequence polypeptide. Ordinarily, amino acid sequence variants
of Pro108 will possess at least about 70% homology with the native
sequence Pro108, preferably, at least about 80%, more preferably
at least about 85%, even more preferably at least about 90% homology,
and most preferably at least 95%. The amino acid sequence variants
can possess substitutions, deletions, and/or insertions at certain
positions within the amino acid sequence of the native amino acid
sequence.
The phrase "functional fragment or analog" of an antibody
is a compound having qualitative biological activity in common with
a full-length antibody. For example, a functional fragment or analog
of an anti-IgE antibody is one which can bind to an IgE immunoglobulin
in such a manner so as to prevent or substantially reduce the ability
of such molecule from having the ability to bind to the high affinity
receptor, Fc.epsilon.R1.
"Homology" is defined as the percentage of residues in
the amino acid sequence variant that are identical after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent homology. Methods and computer programs for the
alignment are well known in the art. Sequence similarity may be
measured by any common sequence analysis algorithm, such as GAP
or BESTFIT or other variation Smith-Waterman alignment. See, T.
F. Smith and M. S. Waterman, J. Mol. Biol. 147:195-197 (1981) and
W. R. Pearson, Genomics 11:635-650 (1991).
"Humanized" forms of non-human (e.g., rodent) antibodies
are chimeric antibodies that contain minimal sequence derived from
the non-human antibody. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by residues
from a hypervariable region of a non-human species (donor antibody)
such as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are replaced
by corresponding non-human residues. Furthermore, humanized antibodies
may comprise residues that are not found in the recipient antibody
or in the donor antibody. These modifications are made to further
refine antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the hypervariable
loops correspond to those of a non-human immunoglobulin and all
or substantially all of the FRs are those of a human immunoglobulin
sequence. The humanized antibody optionally also will comprise at
least a portion of an immunoglobulin constant region (Fc), typically
that of a human immunoglobulin. For further details, see Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
As used herein, an anti-Pro108 antibody that binds Pro108 in mammalian
tissue in vivo is one that detectably (i.e. qualitative or quantitatively
measurable) binds mammalian tissues expressing Pro108 in vivo. Specifically,
the anti-Pro108 antibody will bind Pro108 in the Extra Cellular
Matrix (ECM) of a mammalian tissue in vivo. The anti-Pro108 antibody
may bind free Pro108 or Pro108 bound to a receptor molecule. Said
receptor molecule may be located in the ECM or serum or on the surface
of cells. Anti-Pro108 antibodies may be internalized when bound
to Pro108 which is bound to a receptor on the cell surface. Said
antibody includes antibody fragments, human or humanized antibodies
and antibody conjugates. For therapeutic applications, inhibition
of Pro108 activity or deliver |