U.S. patent application number 13/438427 was filed with the patent office on 2012-12-27 for pro108 antibody compositions and methods of use and use of pro108 to assess cancer risk.
This patent application is currently assigned to diaDexus, Inc.. Invention is credited to Laura Corral, Nam Kim, Charis Lawrenson, Glenn Pilkington, Iris Simon, Robert L. Wolfert.
Application Number | 20120329079 13/438427 |
Document ID | / |
Family ID | 34637140 |
Filed Date | 2012-12-27 |
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United States Patent
Application |
20120329079 |
Kind Code |
A1 |
Simon; Iris ; et
al. |
December 27, 2012 |
Pro108 Antibody Compositions and Methods of Use and Use of Pro108
to Assess Cancer Risk
Abstract
A method for assessing risk of prostate cancer utilizing both
Pro108 and Prostate Specific Antigen (PSA) in combination is
provided. Also provided is a method for assessing risk of cancer
utilizing Pro108 or specific antibodies to Pro108. Antibodies that
bind to Pro108 on a mammalian cell in vivo and compositions
comprising an anti-Pro108 antibody and a carrier which can be
provided in an article of manufacture or a kit are also provided.
An isolated nucleic acid encoding an anti-Pro108 antibody, an
expression vector comprising the isolated nucleic acid, cells that
produce the anti-Pro108 antibodies and a method of producing the
anti-Pro108 antibodies as well as methods for use of the antibodies
in killing an Pro108-expressing cancer cell and alleviating or
treating an Pro108-expressing cancer in a mammal are also
provided.
Inventors: |
Simon; Iris; (San Francisco,
CA) ; Corral; Laura; (San Francisco, CA) ;
Lawrenson; Charis; (San Jose, CA) ; Kim; Nam;
(Santa Clara, CA) ; Pilkington; Glenn; (Sorrento,
AU) ; Wolfert; Robert L.; (Palo Alto, CA) |
Assignee: |
diaDexus, Inc.
South San Francisco
CA
|
Family ID: |
34637140 |
Appl. No.: |
13/438427 |
Filed: |
April 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11866475 |
Oct 3, 2007 |
8148093 |
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13438427 |
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10919215 |
Aug 16, 2004 |
7294704 |
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11866475 |
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60556465 |
Mar 25, 2004 |
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60495759 |
Aug 15, 2003 |
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Current U.S.
Class: |
435/7.94 ;
435/7.92; 530/388.2 |
Current CPC
Class: |
G01N 33/57449 20130101;
A61P 35/00 20180101; G01N 33/57415 20130101; G01N 33/57434
20130101; G01N 33/57446 20130101; G01N 33/57419 20130101; C07K
16/3069 20130101; C07K 16/30 20130101 |
Class at
Publication: |
435/7.94 ;
435/7.92; 530/388.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method for assessing risk of cancer in a patient which
comprises measuring levels of Pro108 in the patient to assess the
risk of cancer in the patient, wherein an elevated level of Pro108
as compared to a control is indicative of heightened risk for
cancer wherein Pro108 comprises residues 1-331 of SEQ ID NO:1 or
SEQ ID NO:2 and Pro108 is measured with an antibody selected from:
(i) an isolated antibody or antigen binding fragment specific for
Pro108 produced by a hybridoma selected from the group consisting
of ATCC Accession Number PTA-5885 and PTA-5886; or (ii) an isolated
antibody or antigen binding fragment which competes for binding to
the same epitope of Pro108 recognized by the antibody produced by a
hybridoma selected from the group consisting of ATCC Accession
Number PTA-5885 and PTA-5886.
2. The method of claim 1 wherein the cancer is selected from the
group consisting of prostate, ovarian, colon, breast and stomach
cancer.
3. The method of claim 2 wherein the cancer is prostate, ovarian or
colon cancer.
4-7. (canceled)
8. A kit for determining the likelihood of a patient having cancer
which comprises a suitable assay for measuring Pro108 levels with
an antibody selected from: (i) an isolated antibody or antigen
binding fragment specific for Pro108 produced by a hybridoma
selected from the group consisting of ATCC Accession Number
PTA-5885 and PTA-5886; or (ii) an isolated antibody or antigen
binding fragment which competes for binding to the same epitope of
Pro108 recognized by the antibody produced by a hybridoma selected
from the group consisting of ATCC Accession Number PTA-5885 and
PTA-5886.
9. The kit of claim 8 wherein the cancer is selected from the group
consisting of prostate, ovarian, colon, breast and stomach
cancer.
10. The kit of claim 9 wherein the cancer is prostate, ovarian or
colon cancer.
11-95. (canceled)
96. The method of claim 1, wherein the antibody that competes for
binding to the same epitope is a monoclonal antibody, humanized
antibody or human antibody.
97. The method of claim 96 wherein the antibody is a labeled
antibody.
98. The method of claim 1 wherein PRO108 is measured in a sample
selected from the group consisting of cells, tissues, blood, serum,
plasma, urine, stool, salvia and sputum.
99. The kit of claim 8, wherein the antibody that competes for
binding to the same epitope is a monoclonal antibody, humanized
antibody or human antibody.
100. The kit of claim 99 wherein the antibody is a labeled
antibody.
Description
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 11/866,475 filed Oct. 3, 2007, which is a
divisional of U.S. patent application Ser. No. 10/919,215 filed
Aug. 16, 2004, now issued as U.S. Pat. No. 7,294,704, which claims
the benefit of priority of U.S. Provisional Patent Application Ser.
No. 60/556,465, filed Mar. 25, 2004 and U.S. Provisional Patent
Application Ser. No. 60/495,759, filed Aug. 15, 2003, each of which
is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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.
[0005] 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 .A-inverted.-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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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).
[0013] 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
[0014] 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 .about.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. 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 septuagenarian 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).
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 HA or IIB, but with malignant cells in the ascites or peritoneal
washings. Id.
[0023] 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.
[0024] 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.
[0025] The treatment of ovarian cancer typically involves a
multiprong attack, with surgical intervention serving as the
foundation of treatment, Dennis S. CM & 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.
[0026] 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.
[0027] 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
[0028] 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).
[0029] 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
personal and/or family history of colorectal cancer or adenomatous
polyps, inflammatory 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).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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).
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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 synthesise 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, .beta.-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 .beta.-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.
[0040] 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.
[0041] 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)).
[0042] 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).
[0043] 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.
[0044] 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
[0045] 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. R.eta.diger
Siewert et al., Early Gastric Cancer, in Management of Upper
Gastrointestinal Cancer 136 (John M. Daly et al. eds. 1999).
[0046] 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).
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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 MM 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.
[0051] 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 MUC1-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).
[0052] 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.
[0053] Stage 0 is characterized by carcinoma in situ (T is, 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 (M0). 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.
[0054] 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.
[0055] 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
[0056] 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.
[0057] 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).
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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):3 15-28. 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.
[0062] 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
[0063] 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 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 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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
[0091] FIG. 1 shows the anti-Pro108 antibody epitope mapping.
[0092] FIG. 2 shows Pro108 serum levels in healthy subjects and
subjects with various cancers.
[0093] FIG. 3 shows Pro108 levels in prostate cancer and benign
prostate disease.
[0094] FIG. 4 shows Pro108 levels in ovarian cancer and benign
ovarian disease.
[0095] FIG. 5 shows Pro108 levels in serous and mucinous ovarian
cancer and in benign ovarian disease.
[0096] FIG. 6 shows Pro108 levels in colon cancer and benign colon
disease.
[0097] FIG. 7 shows Pro108 levels in stomach cancer.
[0098] FIG. 8 shows detection of Pro108 in the lysate of normal
somatic and cancer tissues.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
[0099] 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; US2003104998-A1
as Human secreted/transmembrane protein, PRO866; and WO0144291-A2
as RG1.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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%.sup. 4.1-10.0 25% >10.0
>50%
[0106] See Barry 2001 and the references cited therein.
[0107] 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. Nos. 6,361,955
(Roche), 6,300,088 (Duke), 6,107,049 (Bayer) 5,939,533 (Lilja),
5,928,878 (Bayer), 5,856,182 (Beckman Coulter), 5,672,480 (Abbott
Laboratories), 5,474,903 (Huland) or 5,242,802 (Hybritech), the
contents of which are hereby incorporated by reference into the
subject application.
[0108] 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.
[0109] 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.
[0110] 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 a isotypes. Each L chain has at the N-terminus, a variable
domain (VL) followed by a constant domain (CL) at its other
end.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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 (LI), 5056 (L2) and 89-97 (L3) in
the VL, and around about 1-35 (HI), 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 (LI), 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)).
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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')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')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 CHI 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')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.
[0119] 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.
[0120] "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.
[0121] "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.
[0122] 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).
[0123] 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.
[0124] 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.
[0125] 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.RI.
[0126] "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).
[0127] "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).
[0128] 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
delivery of toxin in vivo is contemplated. The number of antibody
molecules bound to Pro108 in the ECM will be sufficient or adequate
to kill a Pro108-expressing cell, especially a Pro108-expressing
cancer cell. Depending on the potency of the antibody or antibody
conjugate, in some instances, binding of a single antibody molecule
to Pro108 in the ECM is sufficient to kill the target
Pro108-expressing cell. Loss of Pro108 function in the ECM or
delivery of toxins to tissues with a Pro108-expressing cell is
sufficient to kill a Pro108-expressing cell. For example, as stated
above, ECM molecules are known to regulate critical cellular
processes, such as growth, differentiation, and apoptosis and some
are believed to be involved in human carcinogenesis. Inhibition of
these functions by binding of an anti-Pro108 antibody to Pro108 is
sufficient to kill tumor cells. Additionally, certain toxins are
highly potent in killing such that internalization of one molecule
of the toxin is sufficient to kill the tumor cell.
[0129] As used herein, an anti-Pro108 antibody that "internalizes"
is one that is taken up by (i.e., enters) the cell upon binding to
Pro108 on a mammalian cell (i.e. cell surface Pro108). The
internalizing antibody will of course include antibody fragments,
human or humanized antibody and antibody conjugate. For therapeutic
applications, internalization in vivo is contemplated. The number
of antibody molecules internalized will be sufficient or adequate
to kill an Pro108-expressing cell, especially an Pro108-expressing
cancer cell. Depending on the potency of the antibody or antibody
conjugate, in some instances, the uptake of a single antibody
molecule into the cell is sufficient to kill the target cell to
which the antibody binds. For example, certain toxins are highly
potent in killing such that internalization of one molecule of the
toxin conjugated to the antibody is sufficient to kill the tumor
cell.
[0130] Whether an anti-Pro108 antibody internalizes upon binding
Pro108 on a mammalian cell can be determined by various assays
including those described in the experimental examples below. For
example, to test internalization in vivo, the test antibody is
labeled and introduced into an animal known to have Pro108
expressed on the surface of certain cells. The antibody can be
radiolabeled or labeled with fluorescent or gold particles, for
instance. Animals suitable for this assay include a mammal such as
a NCR nude mouse that contains a human Pro108-expressing tumor
transplant or xenograft, or a mouse into which cells transfected
with human Pro108 have been introduced, or a transgenic mouse
expressing the human Pro108 transgene. Appropriate controls include
animals that did not receive the test antibody or that received an
unrelated antibody, and animals that received an antibody to
another antigen on the cells of interest, which antibody is known
to be internalized upon binding to the antigen. The antibody can be
administered to the animal, e.g., by intravenous injection. At
suitable time intervals, tissue sections of the animal can be
prepared using known methods or as described in the experimental
examples below, and analyzed by light microscopy or electron
microscopy, for internalization as well as the location of the
internalized antibody in the cell. For internalization in vitro,
the cells can be incubated in tissue culture dishes in the presence
or absence of the relevant antibodies added to the culture media
and processed for microscopic analysis at desired time points. The
presence of an internalized, labeled antibody in the cells can be
directly visualized by microscopy or by autoradiography if
radiolabeled antibody is used. Alternatively, in a quantitative
biochemical assay, a population of cells comprising
Pro108-expressing cells are contacted in vitro or in vivo with a
radiolabeled test antibody and the cells (if contacted in vivo,
cells are then isolated after a suitable amount of time) are
treated with a protease or subjected to an acid wash to remove
uninternalized antibody on the cell surface. The cells are ground
up and the amount of protease resistant, radioactive counts per
minute (cpm) associated with each batch of cells is measured by
passing the homogenate through a scintillation counter. Based on
the known specific activity of the radiolabeled antibody, the
number of antibody molecules internalized per cell can be deduced
from the scintillation counts of the ground-up cells. Cells are
"contacted" with antibody in vitro preferably in solution form such
as by adding the cells to the cell culture media in the culture
dish or flask and mixing the antibody well with the media to ensure
uniform exposure of the cells to the antibody. Instead of adding to
the culture media, the cells can be contacted with the test
antibody in an isotonic solution such as PBS in a test tube for the
desired time period. In vivo, the cells are contacted with antibody
by any suitable method of administering the test antibody such as
the methods of administration described below when administered to
a patient.
[0131] The faster the rate of internalization of the antibody upon
binding to the Pro108-expressing cell in vivo, the faster the
desired killing or growth inhibitory effect on the target
Pro108-expressing cell can be achieved, e.g., by a cytotoxic
immunoconjugate. Preferably, the kinetics of internalization of the
anti-Pro108 antibodies are such that they favor rapid killing of
the Pro108-expressing target cell. Therefore, it is desirable that
the anti-Pro108 antibody exhibit a rapid rate of internalization
preferably, within 24 hours from administration of the antibody in
vivo, more preferably within about 12 hours, even more preferably
within about 30 minutes to 1 hour, and most preferably, within
about 30 minutes. The present invention provides antibodies that
internalize as fast as about 15 minutes from the time of
introducing the anti-Pro108 antibody in vivo. The antibody will
preferably be internalized into the cell within a few hours upon
binding to Pro108 on the cell surface, preferably within 1 hour,
even more preferably within 15-30 minutes.
[0132] To determine if a test antibody can compete for binding to
the same epitope as the epitope bound by the anti-Pro108 antibodies
of the present invention including the antibodies produced by the
hybridomas deposited with the ATCC, a cross-blocking assay e.g., a
competitive ELISA assay can be performed. In an exemplary
competitive ELISA assay, Pro108-coated wells of a microtiter plate,
or Pro108-coated sepharose beads, are pre-incubated with or without
candidate competing antibody and then a biotin-labeled anti-Pro108
antibody of the invention is added. The amount of labeled
anti-Pro108 antibody bound to the Pro108 antigen in the wells or on
the beads is measured using avidin-peroxidase conjugate and
appropriate substrate.
[0133] Alternatively, the anti-Pro108 antibody can be labeled,
e.g., with a radioactive or fluorescent label or some other
detectable and measurable label. The amount of labeled anti-Pro108
antibody that binds to the antigen will have an inverse correlation
to the ability of the candidate competing antibody (test antibody)
to compete for binding to the same epitope on the antigen, i.e.,
the greater the affinity of the test antibody for the same epitope,
the less labeled anti-Pro108 antibody will be bound to the
antigen-coated wells. A candidate competing antibody is considered
an antibody that binds substantially to the same epitope or that
competes for binding to the same epitope as an anti-Pro108 antibody
of the invention if the candidate competing antibody can block
binding of the anti-Pro108 antibody by at least 20%, preferably by
at least 20-50%, even more preferably, by at least 50% as compared
to a control performed in parallel in the absence of the candidate
competing antibody (but may be in the presence of a known
noncompeting antibody). It will be understood that variations of
this assay can be performed to arrive at the same quantitative
value.
[0134] An antibody having a "biological characteristic" of a
designated antibody, such as any of the monoclonal antibodies
Pro108A2, Pro108.A5, Pro108.B1, Pro108.B2, Pro108.B3, Pro108.B4,
Pro108.B5, Pro108.B6, Pro108.B7, Pro108.B8, Pro108.B9, Pro108.B10,
Pro108.B11, Pro108.B12, Pro108.B13, Pro108.B14, Pro108.B15,
Pro108.B16, Pro108.B17, Pro108.B18, Pro108.B19, Pro108.B20,
Pro108.B21, Pro108.B22, Pro108.B23, Pro108.B24, Pro108.B25,
Pro108.B26, Pro108.B27, Pro108.B28, Pro108.B29, Pro108.B30,
Pro108.B31, Pro108.B32, Pro108.B33, Pro108.B34, Pro108.B35,
Pro108.B36, Pro108.B37, Pro108.B38, Pro108.B39, Pro108.B40,
Pro108.B41, Pro108.B42, Pro108.B43, Pro108.B44 or Pro108.B45, is
one which possesses one or more of the biological characteristics
of that antibody which distinguish it from other antibodies that
bind to the same antigen, Pro108.A2, Pro108.A5, Pro108.B1,
Pro108.B2, Pro108.B3, Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7,
Pro108.B8, Pro108.B9, Pro108.B10, Pro108.B11, Pro108.B12,
Pro108.B13, Pro108.B14, Pro108.B15, Pro108.B16, Pro108.B17,
Pro108.B18, Pro108.B19, Pro108.B20, Pro108.B21, Pro108.B22,
Pro108.B23, Pro108.B24, Pro108.B25, Pro108.B26, Pro108.B27,
Pro108.B28, Pro108.B29, Pro108.B30, Pro108.B31, Pro108.B32,
Pro108.B33, Pro108.B34, Pro108.B35, Pro108.B36, Pro108.B37,
Pro108.B38, Pro108.B39, Pro108.B40, Pro108.B41, Pro108.B42,
Pro108.B43, Pro108.B44 or Pro108.B45 will bind the same epitope as
that bound by Pro108.A2, Pro108.A5, Pro108.B1, Pro108.B2,
Pro108.B3, Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7, Pro108.B8,
Pro108.B9, Pro108.B10, Pro108.B11, Pro108.B12, Pro108.B13,
Pro108.B14, Pro108.B15, Pro108.B16, Pro108.B17, Pro108.B18,
Pro108.B19, Pro108.B20, Pro108.B21, Pro108.B22, Pro108.B23,
Pro108.B24, Pro108.B25, Pro108.B26, Pro108.B27, Pro108.B28,
Pro108.B29, Pro108.B30, Pro108.B31, Pro108.B32, Pro108.B33,
Pro108.B34, Pro108.B35, Pro108.B36, Pro108.B37, Pro108.B38,
Pro108.B39, Pro108.B40, Pro108.B41, Pro108.B42, Pro108.B43,
Pro108.B44 or Pro108.B45 (e.g. which competes for binding or blocks
binding of monoclonal antibody Pro108.A2, Pro108.A5, Pro108.B1,
Pro108.B2, Pro108.B3, Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7,
Pro108.B8, Pro108.B9, Pro108.B10, Pro108.B11, Pro108.B12,
Pro108.B13, Pro108.B14, Pro108.B15, Pro108.B16, Pro108.B17,
Pro108.B18, Pro108.B19, Pro108.B20, Pro108.B21, Pro108.B22,
Pro108.B23, Pro108.B24, Pro108.B25, Pro108.B26, Pro108.B27,
Pro108.B28, Pro108.B29, Pro108.B30, Pro108.B31, Pro108.B32,
Pro108.B33, Pro108.B34, Pro108.B35, Pro108.B36, Pro108.B37,
Pro108.B38, Pro108.B39, Pro108,B40, Pro108.B41, Pro108.B42,
Pro108.B43, Pro108344 or Pro108.B45 to Pro108), be able to target
an Pro108-expressing tumor cell in vivo and will bind to Pro108 on
a mammalian cell in vivo.
[0135] Furthermore, an antibody with the biological characteristic
of the Pro108.A2, Pro108.A5, Pro108.B1, Pro108.B2, Pro108.B3,
Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7, Pro108.B8, Pro108.B9,
Pro108.B10, Pro108.B11, Pro108.B12, Pro108.B13, Pro108.B14,
Pro108.B15, Pro108.B16, Pro108.B17, Pro108.B18, Pro108.B19,
Pro108.B20, Pro108.B21, Pro108.B22, Pro108.B23, Pro108.B24,
Pro108.B25, Pro108.B26, Pro108.B27, Pro108.B28, Pro108.B29,
Pro108.B30, Pro108.B31, Pro108.B32, Pro108.B33, Pro108.B34,
Pro108.B35, Pro108.B36, Pro108.B37, Pro108.B38, Pro108.B39,
Pro108.B40, Pro108.B41, Pro108.B42, Pro108.B43, Pro108.B44 or
Pro108.B45 antibody will bind to Pro108 in mammalian tissue in
vivo.
[0136] Likewise, an antibody with the biological characteristic of
the Pro108.A2, Pro108.A5, Pro108.B1, Pro108.B2, Pro108.B3,
Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7, Pro108.B8, Pro108.B9,
Pro108310, Pro108.B11, Pro108.B12, Pro108.B13, Pro108.B14,
Pro108.B15, Pro108.B16, Pro108.B17, Pro108.B18, Pro108.B19,
Pro108.B20, Pro108.B21, Pro108.B22, Pro108.B23, Pro108.B24,
Pro108.B25, Pro108.B26, Pro108.B27, Pro108.B28, Pro108.B29,
Pro108330, Pro108.B31, Pro108.B32, Pro108.B33, Pro108.B34,
Pro108.B35, Pro108336, Pro108.B37, Pro108.B38, Pro108.B39,
Pro108.B40, Pro108.B41, Pro108342, Pro108.B43, Pro108.B44 or
Pro108.B45 antibody will have the same epitope binding, targeting,
tissue staining, ECM localization, internalizing, tumor growth
inhibitory and cytotoxic properties of the antibody.
[0137] The term "antagonist" antibody is used in the broadest
sense, and includes an antibody that partially or fully blocks,
inhibits, or neutralizes a biological activity of a native Pro108
protein disclosed herein. Methods for identifying antagonists of a
Pro108 polypeptide may comprise contacting an Pro108 polypeptide or
a cell expressing Pro108 on the cell surface, with a candidate
antagonist antibody and measuring a detectable change in one or
more biological activities normally associated with the Pro108
polypeptide.
[0138] An "antibody that inhibits the growth of tumor cells
expressing Pro108" or a "growth inhibitory" antibody is one which
binds to Pro108 and results in measurable growth inhibition of
cancer cells expressing or overexpressing Pro108. Preferred growth
inhibitory anti-Pro108 antibodies inhibit growth of
Pro108-expressing tumor cells e.g., prostate, ovarian, colon,
breast and stomach cancer cells) by greater than 20%, preferably
from about 20% to about 50%, and even more preferably, by greater
than 50% (e.g. from about 50% to about 100%) as compared to the
appropriate control, the control typically being tumor cells not
treated with the antibody being tested. Growth inhibition can be
measured at an antibody concentration of about 0.1 to 30 pg/ml or
about 0.5 nM to 200 nM in cell culture, where the growth inhibition
is determined 1-10 days after exposure of the tumor cells to the
antibody. Growth inhibition of tumor cells in vivo can be
determined in various ways such as is described in the Experimental
Examples section below. The antibody is growth inhibitory in vivo
if administration of the anti-Pro108 antibody at about 1 pg/kg to
about 100 mg/kg body weight results in reduction in tumor size or
tumor cell proliferation within about 5 days to 3 months from the
first administration of the antibody, preferably within about 5 to
30 days.
[0139] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is usually one which
overexpresses Pro108. Preferably the cell is a tumor cell, e.g. a
prostate, ovarian, colon, breast or stomach cell. Various methods
are available for evaluating the cellular events associated with
apoptosis. For example, phosphatidyl serine (PS) translocation can
be measured by annexin binding; DNA fragmentation can be evaluated
through DNA laddering; and nuclear/chromatin condensation along
with DNA fragmentation can be evaluated by any increase in
hypodiploid cells. Preferably, the antibody which induces apoptosis
is one which results in about 2 to 50 fold, preferably about 5 to
50 fold, and most preferably about 10 to 50 fold, induction of
annexin binding relative to untreated cells in an annexin binding
assay.
[0140] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: Clq binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor); and B cell activation.
[0141] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0142] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126.330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer, of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0143] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc'RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g. from blood.
[0144] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may be
performed.
[0145] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, multiple myeloma and B-cell lymphoma, brain, as well as
head and neck cancer, and associated metastases.
[0146] A "Pro108-expressing cell" is a cell which expresses
endogenous or transfected Pro108. Pro108 is typically secreted
outside the cell (e.g. in the Extra Cellular Matrix, ECM), but may
be transiently localized internally (e.g. in the cytoplasm or
secretory organelles) or on the cell surface. A "Pro108-expressing
cancer" is a cancer comprising cells that have Pro108 protein
predominately present in the Extra Cellular Matrix (ECM). A
"Pro108-expressing cancer" produces sufficient levels of Pro108 in
the ECM of cells thereof, such that an anti-Pro108 antibody can
bind thereto and have a therapeutic effect with respect to the
cancer. A cancer which "overexpresses" Pro108 is one which has
significantly higher levels of Pro108 in the ECM thereof, compared
to a noncancerous cell of the same tissue type. Such overexpression
may be caused by gene amplification or by increased transcription
or translation. Pro108 overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the Pro108 protein present in the ECM (e.g. via an
immunohistochemistry assay, ELISA, cell capture, FACS analysis).
Alternatively, or additionally, one may measure levels of
Pro108-encoding nucleic acid or mRNA in the cell, e.g. via
fluorescent in situ hybridization; (FISH; see WO98/45479 published
October, 1998), Southern blotting, Northern blotting, or polymerase
chain reaction (PCR) techniques, such as real time quantitative PCR
(RT-PCR). One may also study Pro108 overexpression by measuring
shed antigen in a biological fluid such as serum, e.g., using
antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294
issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat.
No. 5,401,638 issued Mar. 28, 1995; and Sias et al. J. Immunol.
Methods 132: 73-80 (1990)). Aside from the above assays, various in
vivo assays are available to the skilled practitioner. For example,
one may expose cells within the body of the patient to an antibody
which is optionally labeled with a detectable label, e.g. a
radioactive isotope, and binding of the antibody to Pro108 in
tissues in the patient can be evaluated, e.g. by external scanning
for radioactivity or by analyzing a biopsy taken from a patient
previously exposed to the antibody. A Pro108-expressing cancer
includes prostate, ovarian, colon, breast or stomach cancer.
[0147] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Ariz.) or PATHVISION.TM. (VySiS, Ill.)
may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of Pro108 overexpression in
the tumor. Pro108 overexpression or amplification may be evaluated
using an in vivo diagnostic assay, e.g. by administering a molecule
(such as an antibody) which binds the molecule to be detected and
is tagged with a detectable label (e.g. a radioactive isotope or a
fluorescent label) and externally scanning the patient for
localization of the label.
[0148] A "mammal" for purposes of treating a cancer or alleviating
the symptoms of cancer, refers to any mammal, including--humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
Preferably, the mammal is human.
[0149] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for an
Pro108-expressing cancer if, after receiving a therapeutic amount
of an anti-Pro108 antibody according to the methods of the present
invention, the patient shows observable and/or measurable reduction
in or absence of one or more of the following: reduction in the
number of cancer cells or absence of the cancer cells; reduction in
the tumor size; inhibition (i.e., slow to some extent and
preferably stop) of cancer cell infiltration into peripheral organs
including the spread of cancer into soft tissue and bone;
inhibition (i.e., slow to some extent and preferably stop) of tumor
metastasis; inhibition, to some extent, of tumor growth; and/or
relief to some extent, one or more of the symptoms associated with
the specific cancer; reduced morbidity and mortality, and
improvement in quality of life issues. To the extent the
anti-Pro108 antibody may prevent growth and/or kill existing cancer
cells, it may be cytostatic and/or cytotoxic. Reduction of these
signs or symptoms may also be felt by the patient.
[0150] The above parameters for assessing successful treatment and
improvement in the disease are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
[0151] The term "therapeutically effective amount" refers to an
amount of an antibody or a drug effective to "treat" a disease or
disorder in a subject or mammal. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. See preceding definition of "treating".
To the extent the drug may prevent growth and/or kill existing
cancer cells, it may be cytostatic and/or cytotoxic.
[0152] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time.
[0153] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0154] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0155] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed.
[0156] Often the physiologically acceptable carrier is an aqueous
pH buffered solution. Examples of physiologically acceptable
carriers include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid; low molecular
weight (less than about 10 residues) polypeptide; proteins, such as
serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN.TM., polyethylene glycol
(PEG), and PLURONICS.TM..
[0157] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, e.g., gelonin, ricin, saporin, and the
various antitumor or anticancer agents disclosed below. Other
cytotoxic agents are described below. A tumoricidal agent causes
destruction of tumor cells.
[0158] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a Pro108-expressing cancer cell, either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of Pro108-expressing cells in S phase. Examples of
growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce GI arrest and M-phase arrest Classical M-phase blockers
include the vincas (vincristine and vinblastine), taxanes, and
topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest GI
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and am-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0159] "Label" as used herein refers to a detectable compound or
composition which is conjugated directly or indirectly to the
antibody so as to generate a "labeled" antibody. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition which is
detectable.
[0160] The term "epitope tagged" used herein refers to a chimeric
polypeptide comprising an anti-Pro108 antibody polypeptide fused to
a "tag polypeptide". The tag polypeptide has enough residues to
provide an epitope against which an antibody can be made, yet is
short enough such that it does not interfere with activity of the
Ig polypeptide to which it is fused. The tag polypeptide is also
preferably fairly unique so that the antibody does not
substantially cross-react with other epitopes. Suitable tag
polypeptides generally have at least six amino acid residues and
usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino acid residues).
[0161] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0162] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0163] An "isolated nucleic acid molecule" is a nucleic acid
molecule, e.g., an RNA, DNA, or a mixed polymer, which is
substantially separated from other genome DNA sequences as well as
proteins or complexes such as ribosomes and polymerases, which
naturally accompany a native sequence. The term embraces a nucleic
acid molecule which has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogues or analogues biologically
synthesized by heterologous systems. A substantially pure nucleic
acid molecule includes isolated forms of the nucleic acid
molecule.
[0164] "Vector" includes shuttle and expression vectors and
includes, e.g., a plasmid, cosmid, or phagemid. Typically, a
plasmid construct will also include an origin of replication (e.g.,
the ColE1 origin of replication) and a selectable marker (e.g.,
ampicillin or tetracycline resistance), for replication and
selection, respectively, of the plasmids in bacteria. An
"expression vector" refers to a vector that contains the necessary
control sequences or regulatory elements for expression of the
antibodies including antibody fragment of the invention, in
prokaryotic, e.g., bacterial, or eukaryotic cells. Suitable vectors
are disclosed below.
[0165] The cell that produces an anti-Pro108 antibody of the
invention will include the parent hybridoma cell e.g., the
hybridomas that are deposited with the ATCC, as well as bacterial
and eukaryotic host cells into which nucleic acid encoding the
antibodies have been introduced. Suitable host cells are disclosed
below.
[0166] RNA interference refers to the process of sequence-specific
post transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The
corresponding process in plants is commonly referred to as post
transcriptional gene silencing or RNA silencing and is also
referred to as quelling in fungi. The process of post
transcriptional gene silencing is thought to be an evolutionarily
conserved cellular defense mechanism used to prevent the expression
of foreign genes which is commonly shared by diverse flora and
phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection
from foreign gene expression may have evolved in response to the
production of double stranded RNAs (dsRNA) derived from viral
infection or the random integration of transposon elements into a
host genome via a cellular response that specifically destroys
homologous single stranded RNA or viral genomic RNA. The presence
of dsRNA in cells triggers the RNAi response though a mechanism
that has yet to be fully characterized. This mechanism appears to
be different from the interferon response that results from dsRNA
mediated activation of protein kinase PKR and 2',5'-oligoadenylate
synthetase resulting in non-specific cleavage of mRNA by
ribonuclease L.
[0167] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNA) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from dicer
activity are typically about 21-23 nucleotides in length and
comprise about 19 base pair duplexes. Dicer has also been
implicated in the excision of 21 and 22 nucleotide small temporal
RNAs (stRNA) from precursor RNA of conserved structure that are
implicated in translational control (Hutvagner et al., 2001,
Science, 293, 834). The RNAi response also features an endonuclease
complex containing a siRNA, commonly referred to as an RNA-induced
silencing complex (RISC), which mediates cleavage of single
stranded RNA having sequence complementary to the antisense strand
of the siRNA duplex. Cleavage of the target RNA takes place in the
middle of the region complementary to the antisense strand of the
siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
[0168] Short interfering RNA mediated RNAi has been studied in a
variety of systems. Fire et al., 1998, Nature, 391, 806, were the
first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Nature
Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse
embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in
Drosophila cells transfected with dsRNA. Elbashir et al., 2001,
Nature, 411, 494, describe RNAi induced by introduction of duplexes
of synthetic 21-nucleotide RNAs in cultured mammalian cells
including human embryonic kidney and HeLa cells. Recent work in
Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20,
6877) has revealed certain requirements for siRNA length,
structure, chemical composition, and sequence that are essential to
mediate efficient RNAi activity. These studies have shown that 21
nucleotide siRNA duplexes are most active when containing two
nucleotide 3'-overhangs. Furthermore, complete substitution of one
or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl
nucleotides abolishes RNAi activity, whereas substitution of the
3'-terminal siRNA overhang nucleotides with deoxy nucleotides
(2'-H) was shown to be tolerated. Single mismatch sequences in the
center of the siRNA duplex were also shown to abolish RNAi
activity. In addition, these studies also indicate that the
position of the cleavage site in the target RNA is defined by the
5'-end of the siRNA guide sequence rather than the 3'-end (Elbashir
et al., 2001, EMBO J., 20, 6877). Other studies have indicated that
a 5'-phosphate on the target-complementary strand of a siRNA duplex
is required for siRNA activity and that ATP is utilized to maintain
the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell,
107, 309).
[0169] Studies have shown that replacing the 3'-overhanging
segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs
with deoxyribonucleotides does not have an adverse effect on RNAi
activity. Replacing up to 4 nucleotides on each end of the siRNA
with deoxyribonucleotides has been reported to be well tolerated
whereas complete substitution with deoxyribonucleotides results in
no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In
addition, Elbashir et al., supra, also report that substitution of
siRNA with 2'-O-methyl nucleotides completely abolishes RNAi
activity. Li et al., International PCT Publication No. WO 00/44914,
and Beach et al., International PCT Publication No. WO 01/68836
both suggest that siRNA "may include modifications to either the
phosphate-sugar back bone or the nucleoside to include at least one
of a nitrogen or sulfur heteroatom", however neither application
teaches to what extent these modifications are tolerated in siRNA
molecules nor provide any examples of such modified siRNA. Kreutzer
and Limmer, Canadian Patent Application No. 2,359,180, also
describe certain chemical modifications for use in dsRNA constructs
in order to counteract activation of double stranded-RNA-dependent
protein kinase PKR, specifically 2'-amino or 2'-O-methyl
nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene
bridge. However, Kreutzer and Limmer similarly fail to show to what
extent these modifications are tolerated in siRNA molecules nor do
they provide any examples of such modified siRNA.
[0170] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested
certain chemical modifications targeting the unc-22 gene in C.
elegans using long (>25 nt) siRNA transcripts. The authors
describe the introduction of thiophosphate residues into these
siRNA transcripts by incorporating thiophosphate nucleotide analogs
with T7 and T3 RNA polymerase and observed that "RNAs with two
(phosphorothioate) modified bases also had substantial decreases in
effectiveness as RNAi triggers (data not shown); (phosphorothioate)
modification of more than two residues greatly destabilized the
RNAs in vitro and we were not able to assay interference
activities." Id. at 1081. The authors also tested certain
modifications at the 2'-position of the nucleotide sugar in the
long siRNA transcripts and observed that substituting
deoxynucleotides for ribonucleotides "produced a substantial
decrease in interference activity", especially in the case of
Uridine to Thymidine and/or Cytidine to deoxy-Cytidine
substitutions. Id. In addition, the authors tested certain base
modifications, including substituting 4-thiouracil, 5-bromouracil,
5-iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for
guanosine in sense and antisense strands of the siRNA, and found
that whereas 4-thiouracil and 5-bromouracil were all well
tolerated, inosine "produced a substantial decrease in interference
activity" when incorporated in either strand. Incorporation of
5-iodouracil and 3-(aminoallyl)uracil in the antisense strand
resulted in substantial decrease in RNAi activity as well.
[0171] Beach et al., International PCT Publication No. WO 01/68836,
describes specific methods for attenuating gene expression using
endogenously derived dsRNA. Tuschl et al., International PCT
Publication No. WO 01/75164, describes a Drosophila in vitro RNAi
system and the use of specific siRNA molecules for certain
functional genomic and certain therapeutic applications; although
Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be
used to cure genetic diseases or viral infection due "to the danger
of activating interferon response". Li et al., International PCT
Publication No. WO 00/44914, describes the use of specific dsRNAs
for use in attenuating the expression of certain target genes.
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646, describes certain methods for inhibiting the expression
of particular genes in mammalian cells using certain dsRNA
molecules. Fire et al., International PCT Publication No. WO
99/32619, describes particular methods for introducing certain
dsRNA molecules into cells for use in inhibiting gene expression.
Plaetinck et al., International PCT Publication No. WO 00/01846,
describes certain methods for identifying specific genes
responsible for conferring a particular phenotype in a cell using
specific dsRNA molecules. Mello et al., International PCT
Publication No. WO 01/29058, describes the identification of
specific genes involved in dsRNA mediated RNAi. Deschamps
Depaillette et al., International PCT Publication No. WO 99/07409,
describes specific compositions consisting of particular dsRNA
molecules combined with certain anti-viral agents. Driscoll et al.,
International PCT Publication No. WO 01/49844, describes specific
DNA constructs for use in facilitating gene silencing in targeted
organisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087,
describes specific chemically modified siRNA constructs targeting
the unc-22 gene of C. elegans. Tuschl et al., International PCT
Publication No. WO 02/44321, describe certain synthetic siRNA
constructs.
Compositions and Methods of the Invention
[0172] The invention provides anti-Pro108 antibodies. Preferably,
the anti-Pro108 antibodies bind to Pro108 in mammalian tissue in
vivo. The anti-Pro108 antibodies may also inhibit the growth,
destroy or lead to the destruction of tumor cells expressing
Pro108.
[0173] It was not apparent that Pro108 was ECM localized in the
extracellular matrix. In addition the ability of an antibody to
bind Pro108 in the ECM depends on several factors including the
affinity, avidity, and isotype of the antibody, and the epitope
that it binds. We have demonstrated herein that Pro108 is localized
in the ECM of tissue upon binding by the anti-Pro108 antibodies of
the invention. Additionally, it was demonstrated that the
anti-Pro108 antibodies of the present invention can specifically
target Pro108-expressing tumor cells or tissues in vivo and inhibit
or kill these cells. These in vivo tumor targeting, and growth
inhibitory properties of the anti-Pro108 antibodies make these
antibodies very suitable for therapeutic uses, e.g., in the
treatment of various cancers including prostate, ovarian, colon,
breast or stomach cancer. Internalization of the anti-Pro108
antibody is preferred, e.g., if the antibody or antibody conjugate
has an intracellular site of action and if the cytotoxic agent
conjugated to the antibody does not readily cross the plasma
membrane (e.g., the toxin calicheamicin). Internalization is not
necessary if the antibodies or the agent conjugated to the
antibodies do not have intracellular sites of action, e.g., if the
antibody can kill the tumor cell by ADCC or some other
mechanism.
[0174] It was not apparent that Pro108 was
internalization-competent. In addition the ability of an antibody
to internalize depends on several factors including the affinity,
avidity, and isotype of the antibody, and the epitope that it
binds. We have demonstrated herein that the cell surface Pro108 is
internalization competent upon binding by the anti-Pro108
antibodies of the invention. Additionally, it was demonstrated that
the anti-Pro108 antibodies of the present invention can
specifically target Pro108-expressing tumor cells in vivo and
inhibit or kill these cells. These in vivo tumor targeting,
internalization and growth inhibitory properties of the anti-Pro108
antibodies make these antibodies very suitable for therapeutic
uses, e.g., in the treatment of various cancers including prostate,
ovarian, colon, breast or stomach cancer. Internalization of the
anti-Pro108 antibody is preferred, e.g., if the antibody or
antibody conjugate has an intracellular site of action and if the
cytotoxic agent conjugated to the antibody does not readily cross
the plasma membrane (e.g., the toxin calicheamicin).
Internalization is not necessary if the antibodies or the agent
conjugated to the antibodies do not have intracellular sites of
action, e.g., if the antibody can kill the tumor cell by ADCC or
some other mechanism.
[0175] The anti-Pro108 antibodies of the invention also have
various non-therapeutic applications. The anti-Pro108 antibodies of
the present invention can be useful for diagnosis, staging or
monitoring of Pro108-expressing cancers (e.g., IHC, radioimaging).
They may be used alone or in combination with other ovarian cancer
markers, including, but not limited to, CA125, HE4 and mesothelin.
The antibodies are further useful in predicting outcome or response
to a therapy. In predicting outcome or response to therapy
anti-Pro108 antibodies are used to determine levels of Pro108, and
Pro108 levels are associated with subjects who had a defined
outcome or response to a therapy. Preferably, the antibodies are
used to predict the outcome or response to therapy for a subject
with a Pro108 expressing cancer.
[0176] Additionally, the anti-Pro108 antibodies can be useful for
monitoring a subject's response to therapy. The therapy may be
directed at Pro108 or Pro108 may act as a surrogate marker of
response to a therapy. The antibodies are used to determine Pro108
levels, and as a marker for response to therapy a decrease in
Pro108 expression in a Pro108 expressing cancer is indicative of a
positive response to therapy. No change or an increase in Pro108
expression in a Pro108 expressing cancer is indicative of no
response to therapy.
[0177] The anti-Pro108 antibodies are also useful for purification
or immunoprecipitation of Pro108 from cells, for detection and
quantitation of Pro108 in vitro, e.g. in an ELISA or a Western
blot, to kill and eliminate Pro108-expressing cells from a
population of mixed cells as a step in the purification of other
cells. The internalizing anti-Pro108 antibodies of the invention
can be in the different forms encompassed by the definition of
"antibody" herein. Thus, the antibodies include full length or
intact antibody, antibody fragments, native sequence antibody or
amino acid variants, humanized, chimeric or fusion antibodies,
immunoconjugates, and functional fragments thereof. In fusion
antibodies, an antibody sequence is fused to a heterologous
polypeptide sequence. The antibodies can be modified in the Fc
region to provide desired effector functions. As discussed in more
detail in the sections below, with the appropriate Fc regions, the
naked antibody bound on the cell surface can induce cytotoxicity,
e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by
recruiting complement in complement dependent cytotoxicity, or some
other mechanism. Alternatively, where it is desirable to eliminate
or reduce effector function, so as to minimize side effects or
therapeutic complications, certain other Fc regions may be
used.
[0178] The antibody may compete for binding, or binds substantially
to, the same epitope bound by the antibodies of the invention.
Antibodies having the biological characteristics of the present
anti-Pro108 antibodies of the invention are also contemplated,
e.g., an anti-Pro108 antibody which has the biological
characteristics of a monoclonal antibody produced by the hybridomas
accorded ATCC accession numbers PTA-5885 and PTA-5886, specifically
including the in vivo tumor targeting, internalization and any cell
proliferation inhibition or cytotoxic characteristics. Specifically
provided are anti-Pro108 antibodies that bind to an epitope present
in amino acids 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,
90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160,
160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230,
230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300,
300-310, 310-320, 320-331 of human Pro108, SEQ ID NO: 1-2.
[0179] Methods of producing the above antibodies are described in
detail below.
[0180] The present anti-Pro108 antibodies are useful for treating a
Pro108-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal. Such a cancer includes prostate, ovarian,
colon, breast and stomach cancer, cancer of the urinary tract, lung
cancer and pancreatic cancer. Such a cancer includes more
specifically, ovarian serous and mucinous adenocarcinoma, breast
infiltrating ductal carcinoma, prostate adenocarcinoma, renal cell
carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung
squamous cell carcinomas, and pleural mesothelioma. The breast
cancer may be HER-2 negative or positive breast cancer. The cancers
encompass metastatic cancers of any of the preceding, e.g.,
prostate, ovarian, colon, breast and stomach cancer metastases. The
antibody is able to bind to at least a portion Pro108 in tissues
with cancer cells that express Pro108 in the mammal and preferably
is one that does not induce or that minimizes HAMA response.
Preferably, the antibody is effective to destroy or kill
Pro108-expressing tumor cells or inhibit the growth of such tumor
cells, in vitro or in vivo, upon binding to Pro108 in the Extra
Cellular Matrix. Such an antibody includes a naked anti-Pro108
antibody (not conjugated to any agent). Naked anti-Pro108
antibodies having tumor growth inhibition properties in vivo
include the antibodies described in the Experimental Examples
below. Naked antibodies that have cytotoxic or cell growth
inhibition properties can be further conjugated with a cytotoxic
agent to render them even more potent in tumor cell destruction.
Cytotoxic properties can be conferred to an anti-Pro108 antibody
by, e.g., conjugating the antibody with a cytotoxic agent, to form
an immunoconjugate as described below. The cytotoxic agent or a
growth inhibitory agent is preferably a small molecule. Toxins such
as maytansin, maytansinoids, saporin, gelonin, ricin or
calicheamicin and analogs or derivatives thereof, are
preferable.
[0181] The invention provides a composition comprising an
anti-Pro108 antibody of the invention, and a carrier. For the
purposes of treating cancer, compositions can be administered to
the patient in need of such treatment, wherein the composition can
comprise one or more anti-Pro108 antibodies present as an
immunoconjugate or as the naked antibody. Further, the compositions
can comprise these antibodies in combination with other therapeutic
agents such as cytotoxic or growth inhibitory agents, including
chemotherapeutic agents. The invention also provides formulations
comprising an anti-Pro108 antibody of the invention, and a carrier.
The formulation may be a therapeutic formulation comprising a
pharmaceutically acceptable carrier.
[0182] Another aspect of the invention is isolated nucleic acids
encoding the anti-Pro108 antibodies of this invention. Nucleic
acids encoding both the H and L chains and especially the
hypervariable region residues, chains which encode the native
sequence antibody as well as variants, modifications and humanized
versions of the antibody, are encompassed.
[0183] The invention also provides methods useful for treating a
Pro108-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal, comprising administering a therapeutically
effective amount of an anti-Pro108 antibody to the mammal. The
antibody therapeutic compositions can be administered short term
(acute) or chronic, or intermittent as directed by physician. Also
provided are methods of inhibiting the growth of, and killing a
Pro108 expressing cell. Finally, the invention also provides kits
and articles of manufacture comprising at least one antibody of
this invention, preferably at least one anti-Pro108 antibody of
this invention that binds to Pro108 in tissue in vivo or at least
one anti-Pro108 antibody which binds Pro108 in the ECM, of this
invention. Kits containing anti-Pro108 antibodies find use in
detecting Pro108 expression, or in therapeutic or diagnostic
assays, e.g., for Pro108 cell killing assays or for purification
and/or immunoprecipitation of Pro108 from cells, tissues or bodily
fluids. Additionally, kits containing anti-Pro108 antibodies find
use in monitoring Pro108 expression over time to determine
progression or regression of a cancer. For example, for isolation
and purification of Pro108, the kit can contain an anti-Pro108
antibody coupled to a solid support, e.g., a tissue culture plate
or beads (e.g., sepharose beads). Kits can be provided which
contain antibodies for detection and quantitation of Pro108 in
vitro, e.g. in an ELISA or a Western blot. Such antibody useful for
detection may be provided with a label such as a fluorescent or
radiolabel.
Production of Anti-Pro108 Antibodies
[0184] The following describes exemplary techniques for the
production of the antibodies useful in the present invention. Some
of these techniques are described further in Example 1. The Pro108
antigen to be used for production of antibodies may be, e.g., the
full length polypeptide or a portion thereof, including a soluble
form of Pro108 lacking the signal peptide sequence, or synthetic
peptides to selected portions of the protein.
[0185] Alternatively, cells expressing Pro108 (e.g. CHO, NIH-3T3 or
other cell lines transformed to overexpress Pro108; prostate,
ovarian, colon, breast, stomach or other Pro108-expressing tumor
cell line), or secretory organelles prepared from such cells can be
used to generate antibodies. The nucleotide and amino acid
sequences of human and murine Pro108 are available as provided
above or in public databases. Pro108 can be produced recombinantly
in and isolated from, prokaryotic cells, e.g., bacterial cells, or
eukaryotic cells using standard recombinant DNA methodology. Pro108
can be expressed as a tagged (e.g., epitope tag) or other fusion
protein to facilitate its isolation as well as its identification
in various assays.
[0186] Antibodies or binding proteins that bind to various tags and
fusion sequences are available as elaborated below. Other forms of
Pro108 useful for generating antibodies will be apparent to those
skilled in the art.
[0187] Tags
[0188] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)). The FLAG-peptide (Hopp et al.,
BioTechnology, 6:1204-1210 (1988)) is recognized by an anti-FLAG M2
monoclonal antibody (Eastman Kodak Co., New Haven, Conn.).
Purification of a protein containing the FLAG peptide can be
performed by immunoaffinity chromatography using an affinity matrix
comprising the anti-FLAG M2 monoclonal antibody covalently attached
to agarose (Eastman Kodak Co., New Haven, Conn.). Other tag
polypeptides include the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
(Skinner et al., J. Biol. Chenz., 266:15163-15166 (1991)); and the
T7 gene protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)).
[0189] Polyclonal Antibodies
[0190] Polyclonal antibodies are preferably raised in animals,
preferably non-human animals, by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen
(especially when synthetic peptides are used) to a protein that is
immunogenic in the species to be immunized. For example, the
antigen can be conjugated to keyhole limpet hemocyanin (KLH),
serum, bovine thyroglobulin, or soybean trypsin inhibitor, using a
bifunctional or derivatizing agent, e.g., maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups. Conjugates also can be made
in recombinant cell culture as protein fusions. Animals are
immunized against the antigen, immunogenic conjugates, or
derivatives by combining, e.g., 5-100 pg of the protein or
conjugate (for rabbits or mice, respectively) with 3 volumes of
Freund's complete adjuvant and injecting the solution intradermally
at multiple sites. One month later, the animals are boosted with
1/5 to 1/10 the original amount of peptide or conjugate in Freund's
complete adjuvant by subcutaneous injection at multiple sites.
Seven to 14 days later, the animals are bled and the serum is
assayed for antibody titer. Animals are boosted until the titer
plateaus. Also, aggregating agents such as alum are suitably used
to enhance the immune response.
[0191] Monoclonal Antibodies
[0192] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567). In the
hybridoma method, a mouse or other appropriate host animal, such as
a hamster, is immunized as described above to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. After
immunization, lymphocytes are isolated and then fused with a
"fusion partner", e.g., a myeloma cell line using a suitable fusing
agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies. Principles and Practice, pp 103
(Academic Press, 1986)).
[0193] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
fusion partner, e.g., the parental myeloma cells. For example, if
the parental myeloma cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the selective culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which substances prevent
the growth of HGPRT-deficient cells.
[0194] Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-II mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Rockville, Md. USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0195] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0196] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980). Once hybridoma cells
that produce antibodies of the desired specificity, affinity,
and/or activity are identified, the clones may be subcloned by
limiting dilution procedures and grown by standard methods (Goding,
Monoclonal Antibodies: Principles and Practice, pp 103 (Academic
Press, 1986)). Suitable culture media for this purpose include, for
example, D-MEM or RPMI-1640 medium. In addition, the hybridoma
cells may be grown in vivo as ascites tumors in an animal e.g., by
i.p. injection of the cells into mice.
[0197] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0198] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transformed or transfected into prokaryotic or eukaryotic host
cells such as, e.g., E. coli cells, simian COS cells, Chinese
Hamster Ovary (CHO) cells, or myeloma cells, that do not otherwise
produce antibody protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. Review articles on
recombinant expression in bacteria of DNA encoding the antibody
include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993)
and Phickthun, Immunol. Revs., 130:151-188 (1992).
[0199] Further, the monoclonal antibodies or antibody fragments can
be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0200] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain (CH
and CL) sequences for the homologous murine sequences (U.S. Pat.
No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA,
81:6851 (1984)), or by fusing the immunoglobulin coding sequence
with all or part of the coding sequence for a non-immunoglobulin
polypeptide (heterologous polypeptide). The nonimmunoglobulin
polypeptide sequences can substitute for the constant domains of an
antibody, or they are substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for an antigen and another antigen-combining site having
specificity for a different antigen.
[0201] Humanized Antibodies
[0202] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
nonhuman. These non-human amino acid residues are often referred to
as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0203] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0204] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art.
[0205] Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0206] Various forms of a humanized anti-Pro108 antibody are
contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0207] Human Antibodies
[0208] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno.,
7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and Alternatively, phage display technology
(McCafferty et al., Nature 348:552-553 (1990)) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B-cell. Phage
display can be performed in a variety of formats, reviewed in,
e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in
Structural Biology 3:564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature,
352:624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.
12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905. As discussed above, human antibodies may also be
generated by in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275).
[0209] Antibody Fragments
[0210] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors. Various techniques have been
developed for the production of antibody fragments. Traditionally,
these fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods 24:107-117 (1992); and Brennan et al., Science,
229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. Fab, Fv and ScFv antibody
fragments can all be expressed in and secreted from E. coli, thus
allowing the facile production of large amounts of these fragments.
Antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab).sub.2 fragments (Carter et al., Bio/Technology 10: 163-167
(1992)). According to another approach, F(ab)2 fragments can be
isolated directly from recombinant host cell culture. Fab and
F(ab)2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues are described in U.S.
Pat. No. 5,869,046. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. The
antibody of choice may also be a single chain Fv fragment (scFv).
See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. Fv and sFv are the only species with intact combining
sites that are devoid of constant regions; thus, they are suitable
for reduced nonspecific binding during in vivo use. sFv fusion
proteins may be constructed to yield fusion of an effector protein
at either the amino or the carboxy terminus of an sFv. See Antibody
Engineering, ed. Borrebaeck, supra. The antibody fragment may also
be a "linear antibody", e.g., as described in U.S. Pat. No.
5,641,870 for example. Such linear antibody fragments may be
monospecific or bispecific.
[0211] Bispecific Antibodies
[0212] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
Pro108 protein. Other such antibodies may combine an Pro108 binding
site with a binding site for another protein. Alternatively, an
anti-Pro108,Arm may be combined with an arm which binds to a
triggering molecule on a leukocyte such as a Tcell receptor
molecule (e.g. C133), or Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16),
so as to focus and localize cellular defense mechanisms to the
Pro108-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express Pro108. These
antibodies possess an Pro108-binding arm and an arm which binds the
cytotoxic agent (e.g. saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab)2 bispecific
antibodies). WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0213] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0214] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (CHI) containing the site necessary for light chain bonding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0215] Preferably, the bispecific antibodies in this approach are
composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. It was found that this asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details of generating
bispecific antibodies see, for example, Suresh et al., Methods in
Enzymology, 121:210 (1986).
[0216] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain. In this method, one or
more small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0217] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0218] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent, sodium arsenite, to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0219] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0220] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers.
[0221] The "diabody" technology described by Hollinger et al.,
Proc. Natl, Acad. Sci. USA, 90:6444-6448 (1993) has provided an
alternative mechanism for making bispecific antibody fragments. The
fragments comprise a VH connected to a VL by a linker which is too
short to allow pairing between the two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to
pair with the complementary VL and VH domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0222] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0223] Multivalent Antibodies
[0224] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1(X1n-VD2-(X2)n-Fc, wherein VDI is a first variable
domain, VD2 is a second variable domain, Fc is one polypeptide
chain of an Fc region, X1 and X2 represent an amino acid or
polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fc region
chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0225] Other Amino Acid Sequence Modifications
[0226] Amino acid sequence modification(s) of the anti-Pro108
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the anti-Pro108 antibody are prepared by introducing appropriate
nucleotide changes into the anti-Pro108 antibody nucleic acid, or
by peptide synthesis.
[0227] Such modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the anti-Pro108 antibody. Any
combination of deletion, insertion, and substitution is made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the anti-Pro108 antibody,
such as changing the number or position of glycosylation sites.
[0228] A useful method for identification of certain residues or
regions of the anti-Pro108 antibody that are preferred locations
for mutagenesis is called "alanine scanning mutagenesis" as
described by Cunningham and Wells in Science, 244:1081-1085 (1989).
Here, a residue or group of target residues within the anti-Pro108
antibody are identified (e.g., charged residues such as arg, asp,
his, lys, and glu) and replaced by a neutral or negatively charged
amino acid (most preferably alanine or polyalanine) to affect the
interaction of the amino acids with Pro108 antigen.
[0229] Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at a target codon or region and the expressed anti-Pro108
antibody variants are screened for the desired activity.
[0230] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-Pro108 antibody
with an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the
anti-Pro108 antibody molecule include the fusion to the N- or
C-terminus of the anti-Pro108 antibody to an enzyme (e.g. for
ADEPT) or a fusion to a polypeptide which increases the serum
half-life of the antibody.
[0231] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-Pro108 antibody molecule replaced by a different residue. The
sites of greatest interest for substitutional mutagenesis include
the hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in Table I under
the heading of "preferred substitutions". If such substitutions
result in a change in biological activity, then more substantial
changes, denominated "exemplary substitutions" in Table 1, or as
further described below in reference to amino acid classes, may be
introduced and the products screened for a desired
characteristic.
TABLE-US-00002 TABLE I Amino Acid Substitutions Original Exemplary
Substitutions Preferred Substitutions Ala (A) val; leu; ile Val Arg
(R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D)
glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp;
gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;
val; met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ile
Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser Phe Val (V)
ile; leu; met; phe; ala; leu
[0232] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn,
gin, his, lys, arg; (5) residues that influence chain orientation:
gly, pro; and (6) aromatic: trp, tyr, phe.
[0233] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Any cysteine
residue not involved in maintaining the proper conformation of the
anti-Pro108 antibody also may be substituted, generally with
serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[0234] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino acid
substitutions at each site. The antibody variants thus generated
are displayed in a monovalent fashion from filamentous phage
particles as fusions to the gene III product of M13 packaged within
each particle. The phage-displayed variants are then screened for
their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region
sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and human Pro108. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0235] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody. Glycosylation of antibodies is
typically either N-linked or O-linked. N-linked refers to the
attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine
and asparagine-X-threonine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used. Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0236] Nucleic acid molecules encoding amino acid sequence variants
of the anti-Pro108 antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
nucleic acid molecule encoding a variant or a non-variant version
of the anti-Pro108 antibody.
[0237] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0238] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of the
antibody.
Screening for Antibodies with the Desired Properties
[0239] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0240] The growth inhibitory effects of an anti-Pro108 antibody of
the invention may be assessed by methods known in the art, e.g.,
using cells which express Pro108 either endogenously or following
transfection with the Pro108 gene. For example, the tumor cell
lines and Pro108-transfected cells provided in Example 1 below may
be treated with an anti-Pro108 monoclonal antibody of the invention
at various concentrations for a few days (e.g., 2-7) days and
stained with crystal violet or MTT or analyzed by some other
colorimetric assay. Another method of measuring proliferation would
be by comparing .sup.3H-thymidine uptake by the cells treated in
the presence or absence an anti-Pro108 antibody of the invention.
After antibody treatment, the cells are harvested and the amount of
radioactivity incorporated into the DNA quantitated in a
scintillation counter. Appropriated positive controls include
treatment of a selected cell line with a growth inhibitory antibody
known to inhibit growth of that cell line. Growth inhibition of
tumor cells in vivo can be determined in various ways such as is
described in the Experimental Examples section below. Preferably,
the tumor cell is one that over-expresses Pro108. Preferably, the
anti-Pro108 antibody will inhibit cell proliferation of a
Pro108-expressing tumor cell in vitro or in vivo by about 25-100%
compared to the untreated tumor cell, more preferably, by about
30-100%, and even more preferably by about 50-100% or 70-100%, at
an antibody concentration of about 0.5 to 30 .mu.g/ml. Growth
inhibition can be measured at an antibody concentration of about
0.5 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, where
the growth inhibition is determined 1-10 days after exposure of the
tumor cells to the antibody. The antibody is growth inhibitory in
vivo if administration of the anti-Pro108 antibody at about 1
.mu.g/kg to about 100 mg/kg body weight results in reduction in
tumor size or tumor cell proliferation within about 5 days to 3
months from the first administration of the antibody, preferably
within about 5 to 30 days.
[0241] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., propidium iodide (PD,
tryptan blue or 7AAD uptake may be assessed relative to a control.
A PI uptake assay can be performed in the absence of complement and
immune effector cells. Pro108-expressing tumor cells are incubated
with medium alone or medium containing of the appropriate
monoclonal antibody at e.g., about 10 .mu.g/ml. The cells are
incubated for a 3 day time period. Following each treatment, cells
are washed and aliquoted into 35 mm strainer-capped 12.times.75
tubes (1 ml per tube, 3 tubes per treatment group) for removal of
cell clumps. Tubes then receive PI (10 .mu.g/ml). Samples may be
analyzed using a FACSCAN.TM. flow cytometer and FACSCONVERT.TM.
CellQuest software (Becton Dickinson). Those antibodies which
induce statistically significant levels of cell death as determined
by PI uptake may be selected as cell death-inducing antibodies.
[0242] To screen for antibodies which bind to an epitope on Pro108
bound by an antibody of interest, e.g., the Pro108 antibodies of
this invention, a routine cross-blocking assay such as that
describe in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed. This
assay can be used to determine if a test antibody binds the same
site or epitope as an anti-Pro108 antibody of the invention.
Alternatively, or additionally, epitope mapping can be performed by
methods known in the art. For example, the antibody sequence can be
mutagenized such as by alanine scanning, to identify contact
residues. The mutant antibody is initially tested for binding with
polyclonal antibody to ensure proper folding. In a different
method, peptides corresponding to different regions of Pro108 can
be used in competition assays with the test antibodies or with a
test antibody and an antibody with a characterized or known
epitope.
[0243] For example, a method to screen for antibodies that bind to
an epitope which is bound by an antibody this invention may
comprise combining an Pro108-containing sample with a test antibody
and an antibody of this invention to form a mixture, the level of
Pro108 antibody bound to Pro108 in the mixture is then determined
and compared to the level of Pro108 antibody bound in the mixture
to a control mixture, wherein the level of Pro108 antibody binding
to Pro108 in the mixture as compared to the control is indicative
of the test antibody's binding to an epitope that is bound by the
anti-Pro 108 antibody of this invention. The level of Pro108
antibody bound to Pro108 is determined by ELISA. The control may be
a positive or negative control or both. For example, the control
may be a mixture of Pro108, Pro108 antibody of this invention and
an antibody known to bind the epitope bound by the Pro108 antibody
of this invention. The anti-Pro108 antibody labeled with a label
such as those disclosed herein. The Pro108 may be bound to a solid
support, e.g., a tissue culture plate or to beads, e.g., sepharose
beads.
Immunoconjugates
[0244] The invention also pertains to therapy with immunoconjugates
comprising an antibody conjugated to an anti-cancer agent such as a
cytotoxic agent or a growth inhibitory agent.
[0245] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Conjugates of an
antibody and one or more small molecule toxins, such as a
calicheamicin, maytansinoids, a trichothene, and CC1065, and the
derivatives of these toxins that have toxin activity, are also
contemplated herein.
[0246] Maytansine and Maytansinoids
[0247] Preferably, an anti-Pro108 antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0248] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the cast African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
[0249] Maytansinoid-Antibody Conjugates
[0250] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci, USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DMI linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10 5 HER-2 surface antigens per
cell. The drug conjugate achieved a degree of cytotoxicity similar
to the free maytansonid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0251] Anti-Pro108 Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0252] Anti-Pro108 antibody-maytansinoid conjugates are prepared by
chemically linking an anti-Pro108 antibody to a maytansinoid
molecule without significantly diminishing the biological activity
of either the antibody or the maytansinoid molecule. An average of
3-4 maytansinoid molecules conjugated per antibody molecule has
shown efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0253] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al. Cancer Research 52: 127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred. Conjugates of the antibody and
maytansinoid may be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl (2-pyridyldithio) propionate
(SPDP), succinimidyl-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as his (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include N-succinimidyl
(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J.
173:723-737 [1978]) and N-succinimidyl (2-pyridylthio)pentanoate
(SPP) to provide for a disulfide linkage.
[0254] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group.
Preferably, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0255] Calicheamicin
[0256] Another immunoconjugate of interest comprises an anti-Pro108
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sub.1.sup.I, (Hinman et al. Cancer Research 53: 3336
(1993), Lode et al. Cancer Research 5 8: 2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
Other Cytotoxic Agents
[0257] Other antitumor agents that can be conjugated to the
anti-Pro108 antibodies of the invention include BCNU,
streptozoicin, vincristine and 5-fluorouracil, the family of agents
known collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296). Enzymatically active toxins and fragments thereof which
can be used include diphtheria A chain, 1 5 nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin
and the tricothecenes. See, for example, WO 93/21232 published Oct.
28, 1993. The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0258] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-Pro108 antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, In.sup.111, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, and radioactive isotopes of Lu.
When the conjugate is used for diagnosis, it may comprise a
radioactive atom for scintigraphic studies, for example Tc.sup.99M
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0259] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
Tc.sup.99M, I.sup.12, In.sup.111, Re.sup.186, Re.sup.188, can be
attached via a cysteine residue in the peptide. Yttrium-90 can be
attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine "Monoclonal Antibodies in Immunoscintigraphy"
(Chatal, CRC Press 1989) describes other methods in detail.
[0260] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl
(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCL), active esters (such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as
bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives
(such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates
(such as tolyene 2,6diisocyanate), and bis-active fluorine
compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a
ricin immunotoxin can be prepared as described in Vitetta et al.
Science 238: 1098 (1987). Carbon labeled 1-isothiocyanatobenzyl
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary
chelating agent for conjugation of radionucleotide to the antibody.
See WO 94/11026. The linker may be a "cleavable linker"
facilitating release of the cytotoxic drug in the cell. For
example, an acid-labile linker, peptidase-sensitive linker,
photolabile linker, dimethyl linker or disulfide-containing linker
(Chari et al. Cancer Research 52: 127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0261] Alternatively, a fusion protein comprising the anti-Pro108
antibody and cytotoxic agent may be made, e.g. by recombinant
techniques or peptide synthesis. The length of DNA may comprise
respective regions encoding the two portions of the conjugate
either adjacent one another or separated by a region encoding a
linker peptide which does not destroy the desired properties of the
conjugate.
[0262] In addition, the antibody may be conjugated to a "receptor"
(such streptavidin) for utilization in tumor pre-targeting wherein
the antibody-receptor conjugate is administered to the patient,
followed by removal of unbound conjugate from the circulation using
a clearing agent and then administration of a "ligand" (e.g.
avidin) which is conjugated to a cytotoxic agent (e.g. a
radionucleotide).
Antibody Dependent Enzyme Prodrug Therapy (ADEPT)
[0263] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0264] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form. Enzymes that
are useful in the method of this invention include, but are not
limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as O-galactosidase and neuraminidase useful for converting
glycosylated prodrugs into free drugs; P-lactamase useful for
converting drugs derivatized with P-lactams into free drugs; and
penicillin amidases, such as penicillin V amidase or penicillin G
amidase, useful for converting drugs derivatized at their amine
nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,
into free drugs. Alternatively, antibodies with enzymatic activity,
also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g.,
Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can
be prepared as described herein for delivery of the abzyme to a
tumor cell population. The enzymes of this invention can be
covalently bound to the anti-Pro108 antibodies by techniques well
known in the art such as the use of the heterobifunctional
crosslinking reagents discussed above.
[0265] Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature, 312: 604-608
(1984).
Other Antibody Modifications
[0266] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly(methylmethacylate) microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,
Ed., (1980).
[0267] The anti-Pro108 antibodies disclosed herein may also be
formulated as immunoliposomes. A "liposome" is a small vesicle
composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
Vectors, Host Cells, and Recombinant Methods
[0268] The invention also provides isolated nucleic acid molecule
encoding the humanized anti-Pro108 antibody, vectors and host cells
comprising the nucleic acid, and recombinant techniques for the
production of the antibody. For recombinant production of the
antibody, the nucleic acid molecule encoding it is isolated and
inserted into a replicable vector for further cloning
(amplification of the DNA) or inserted into a vector in operable
linkage with a promoter for expression. DNA encoding the monoclonal
antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to nucleic acid molecules encoding the
heavy and light chains of the antibody). Many vectors are
available. The vector components generally include, but are not
limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence.
Signal Sequence Component
[0269] The anti-Pro108 antibody of this invention may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the native anti-Pro 108 antibody signal sequence, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group of the alkaline phosphatase,
penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, oc factor leader (including
Saccharomyces and Kluyveromyces cc-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the
signal described in WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available. The DNA for
such precursor region is ligated in reading frame to DNA encoding
the anti-Pro108 antibody.
[0270] Origin of Replication
[0271] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu., plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0272] Selection Gene Component
[0273] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. One example of a selection scheme
utilizes a drug to arrest growth of a host cell. Those cells that
are successfully transformed with a heterologous gene produce a
protein conferring drug resistance and thus survive the selection
regimen. Examples of such dominant selection use the drugs
neomycin, mycophenolic acid and hygromycin.
[0274] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-Pro108 antibody nucleic acid, such as DHFR,
thymidine kinase, metallothionein-I and -11, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc. For example, cells transformed with the DHFR
selection gene are first identified by culturing all of the
transformants in a culture medium that contains methotrexate (Mtx),
a competitive antagonist of DHFR. An appropriate host cell when
wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell
line deficient in DHFR activity (e.g., ATCC CRL-9096).
[0275] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-Pro108 antibody, wild-type DHFR protein,
and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0276] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4 Jones, Genetics, 85:12 (1977).
The presence of the trp1 lesion in the yeast host cell genome then
provides an effective environment for detecting transformation by
growth in the absence of tryptophan. Similarly, Leu2-deficient
yeast strains (ATCC 20,622 or 38,626) are complemented by known
plasmids bearing the Leu2 gene.
[0277] In addition, vectors derived from the 1.6 pm circular
plasmid pKDI can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0278] Promoter Component
[0279] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-Pro108 antibody nucleic acid. Promoters suitable for use
with prokaryotic hosts include the phoA promoter, P-lactamase and
lactose promoter systems, alkaline phosphatase promoter, a
tryptophan (trp) promoter system, and hybrid promoters such as the
tac promoter. However, other known bacterial promoters are
suitable. Promoters for use in bacterial systems also will contain
a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the anti-Pro108 antibody.
[0280] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors. Examples of
suitable promoter sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase or other glycolytic
enzymes, such as enolase, glyceraldehyde phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose
phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase.
[0281] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein, glyceraldehyde phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and promoters for use in yeast
expression are further described in EP 73,657. Yeast enhancers also
are advantageously used with yeast promoters.
[0282] Anti-Pro108 antibody transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
most preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0283] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human P-interferon cDNA
in mouse cells under the control of a thymidine kinase promoter
from herpes simplex virus. Alternatively, the Rous Sarcoma Virus
long terminal repeat can be used as the promoter.
[0284] Enhancer Element Component
[0285] Transcription of a DNA encoding the anti-Pro108 antibody of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
anti-Pro108 antibody-encoding sequence, but is preferably located
at a site 5' from the promoter.
[0286] Transcription Termination Component
[0287] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
anti-Pro108 antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO 94/11026 and the expression vector disclosed therein.
[0288] Selection and Transformation of Host Cells
[0289] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W31 10 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0290] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation region
(TIR) and signal sequences for optimizing expression and secretion,
these patents incorporated herein by reference. After expression,
the antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g., in
CHO cells.
[0291] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-Pro108 antibody-encoding vectors. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used
among lower eukaryotic host microorganisms. However, a number of
other genera, species, and strains are commonly available and
useful herein, such as Schizosaccharomyces pombe; Kluyveromyces
hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,
and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP
183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora
crassa; Schwanniomyces such as Schwanniomyces occidentalis; and
filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans and A.
niger.
[0292] Suitable host cells for the expression of glycosylated
anti-Pro108 antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0293] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, Arabidopsis and tobacco can also be utilized as
hosts. Cloning and expression vectors useful in the production of
proteins in plant cell culture are known to those of skill in the
art. See e.g. Hiatt et al., Nature (1989) 342: 76-78, Owen et al.
(1992) Bio/Technology 10: 790-794, Artsaenko et al. (1995) The
Plant J 8: 745-750, and Fecker et al. (1996) Plant Mol Biol 32:
979-986.
[0294] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0295] Host cells are transformed with the above-described
expression or cloning vectors for anti-Pro108 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0296] Culturing Host Cells
[0297] The host cells used to produce the anti-Pro108 antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's FIO (Sigma), Minimal Essential Medium
(MEM)(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium (DMEM)(Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0298] Purification of anti-Pro108 antibody
[0299] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10: 163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0300] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human 73 (Guss et al.,
EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand
is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SIDS-PAGE, and
ammonium sulfate precipitation are also available depending on the
antibody to be recovered.
[0301] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
Pharmaceutical Formulations
[0302] Pharmaceutical formulations of the antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as acetate, Tris, phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol, and mcresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyllolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; tonicifiers such as
trehalose and sodium chloride; sugars such as sucrose, mannitol,
trehalose or sorbitol; surfactant such as polysorbate; salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.TM.,
PLURONICS.TM. or polyethylene glycol (PEG). The antibody preferably
comprises the antibody at a concentration of between 5-200 mg/ml,
preferably between 10-100 mg/ml.
[0303] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, in addition to the
anti-Pro108 antibody which internalizes, it may be desirable to
include in the one formulation, an additional antibody, e.g. a
second anti-Pro108 antibody which binds a different epitope on
Pro108, or an antibody to some other target such as a growth factor
that affects the growth of the particular cancer. Alternatively, or
additionally, the composition may further comprise a
chemotherapeutic agent, cytotoxic agent, cytokine, growth
inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
Such molecules are suitably present in combination in amounts that
are effective for the purpose intended.
[0304] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0305] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-) hydroxybutyric acid.
[0306] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
Methods and Treatment Using Anti-Pro108 Antibodies
[0307] According to the present invention, the anti-Pro108 antibody
that binds to Pro108 in a mammalian tissue in vivo is used to treat
a subject in need thereof having a cancer characterized by
Pro108-expressing cancer cells, in particular, ovarian, pancreatic,
lung or breast cancer, such as ovarian serous or mucinous
adenocarcinoma or breast infiltrating ductal carcinoma cancer, and
associated metastases.
[0308] The cancer will generally comprise Pro108-expressing cells,
such that the anti-Pro 108 antibody is able to bind thereto. The
cancer may be characterized by overexpression of Pro108 in the
Extra Cellular Matrix (ECM) within a tissue and bodily fluids.
While the cancer may be characterized by overexpression of the
Pro108 molecule, the present application further provides a method
for treating cancer which is not considered to be an
Pro108-overexpressing cancer.
[0309] This invention also relates to methods for detecting cells
which overexpress Pro108 and to diagnostic kits useful in detecting
cells expressing Pro108 or in detecting Pro108 in serum from a
patient. The methods may comprise combining a cell-containing test
sample with an antibody of this invention, assaying the test sample
for antibody binding to cells in the test sample and comparing the
level of antibody binding in the test sample to the level of
antibody binding in a control sample of cells. A suitable control
is, e.g., a sample of normal cells of the same type as the test
sample or a cell sample known to be free of Pro108 overexpressing
cells. A level of Pro108 binding higher than that of such a control
sample would be indicative of the test sample containing cells that
overexpress Pro108. Alternatively the control may be a sample of
cells known to contain cells that overexpress Pro108. In such a
case, a level of Pro108 antibody binding in the test sample that is
similar to, or in excess of, that of the control sample would be
indicative of the test sample containing cells that overexpress
Pro108.
[0310] Pro108 overexpression may be detected with a various
diagnostic assays. For example, over expression of Pro108 may be
assayed by immunohistochemistry (IHC). Parrafin embedded tissue
sections from a tumor biopsy may be subjected to the IHC assay and
accorded an Pro108 protein staining intensity criteria as
follows.
[0311] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0312] Score 1+a faint/barely perceptible membrane staining is
detected in more than 10% of the tumor cells. The cells are only
stained in part of their membrane.
[0313] Score 2+a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0314] Score 3+a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0315] Those tumors with 0 or 1+scores for Pro108 expression may be
characterized as not overexpressing Pro108, whereas those tumors
with 2+ or 3+ scores may be characterized as overexpressing
Pro108.
[0316] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Ariz.) or PATHVISION.TM. (VySiS, Ill.)
may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of Pro108 overexpression in
the tumor. Pro108 overexpression or amplification may be evaluated
using an in vivo diagnostic assay, e.g. by administering a molecule
(such as an antibody of this invention) which binds Pro108 and
which is labeled with a detectable label (e.g. a radioactive
isotope or a fluorescent label) and externally scanning the patient
for localization of the label.
[0317] A sample suspected of containing cells expressing or
overexpressing Pro108 is combined with the antibodies of this
invention under conditions suitable for the specific binding of the
antibodies to Pro108. Binding of Pro108 antibodies of this
invention is indicative of the cells expressing Pro108. The level
of binding may be determined and compared to a suitable control,
wherein an elevated level of bound Pro108 as compared to the
control is indicative of Pro108 overexpression. The sample
suspected of containing cells overexpressing Pro108 may be a cancer
cell sample, particularly a sample of prostate, ovarian, colon,
breast or stomach cancer, e.g. ovarian serous or mucinous
adenocarcinoma or a breast infiltrating ductal carcinoma. A serum
sample from a subject may also be assayed for levels of Pro108 by
combining a serum sample from a subject with an Pro108 antibody of
this invention, determining the level of Pro108 bound to the
antibody and comparing the level to a control, wherein an elevated
level of Pro108 in the serum of the patient as compared to a
control is indicative of overexpression of Pro108 by cells in the
patient. The subject may have a cancer such as e.g., an ovarian
cancer, e.g. ovarian serous adenocarcinoma, or a breast cancer,
e.g., a breast infiltrating ductal carcinoma.
[0318] Currently, depending on the stage of the cancer, prostate,
ovarian, colon, breast or stomach cancer treatment involves one or
a combination of the following therapies: surgery to remove the
cancerous tissue, radiation therapy, androgen deprivation (e.g.,
hormonal therapy), and chemotherapy. Anti-Pro108 antibody therapy
may be especially desirable in elderly patients who do not tolerate
the toxicity and side effects of chemotherapy well, in metastatic
disease where radiation therapy has limited usefulness, and for the
management of prostatic carcinoma that is resistant to androgen
deprivation treatment. The tumor targeting anti-Pro108 antibodies
of the invention are useful to alleviate Pro108-expressing cancers,
e.g., prostate, ovarian, colon, breast or stomach cancers upon
initial diagnosis of the disease or during relapse. For therapeutic
applications, the anti-Pro108 antibody can be used alone, or in
combination therapy with, e.g., hormones, antiangiogens, or
radiolabelled compounds, or with surgery, cryotherapy, and/or
radiotherapy, notably for ovarian, pancreatic, lung or breast
cancers, also particularly where shed cells cannot be reached.
Anti-Pro108 antibody treatment can be administered in conjunction
with other forms of conventional therapy, either consecutively
with, pre- or post-conventional therapy, Chemotherapeutic drugs
such as Taxotere.RTM. (docetaxel), Taxol.RTM. (paclitaxel),
estramustine and mitoxantrone are used in treating metastatic and
hormone refractory ovarian, pancreatic, lung or breast cancer, in
particular, in good risk patients. In the present method of the
invention for treating or alleviating cancer, in particular,
androgen independent and/or metastatic ovarian, pancreatic, lung or
breast cancer, the cancer patient can be administered anti-Pro108
antibody in conjunction with treatment with the one or more of the
preceding chemotherapeutic agents. In particular, combination
therapy with palictaxel and modified derivatives (see, e.g.,
EP0600517) is contemplated. The anti-Pro108 antibody will be
administered with a therapeutically effective dose of the
chemotherapeutic agent. The anti-Pro108 antibody may also be
administered in conjunction with chemotherapy to enhance the
activity and efficacy of the chemotherapeutic agent, e.g.,
paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages
of these agents that have been used in treatment of various
cancers. The dosing regimen and dosages of these aforementioned
chemotherapeutic drugs that are therapeutically effective will
depend on the particular cancer being treated, the extent of the
disease and other factors familiar to the physician of skill in the
art and can be determined by the physician.
[0319] Particularly, an immunoconjugate comprising the anti-Pro108
antibody conjugated with a cytotoxic agent may be administered to
the patient. Preferably, the immunoconjugate bound to Pro108 in the
Extra Cellular Matrix (ECM) results in therapeutic efficacy of the
immunoconjugate in killing the Pro108-expressing cancer cell.
Alternatively, the immunoconjugate bound to the Pro108 protein is
internalized by the cell, resulting in increased therapeutic
efficacy of the immunoconjugate in killing the Pro108-expressing
cancer cell. Preferably, the cytotoxic agent targets or interferes
with the nucleic acid in the cancer cell. Examples of such
cytotoxic agents are described above and include maytansin,
maytansinoids, saporin, gelonin, ricin, calicheamicin,
ribonucleases and DNA endonucleases.
[0320] The anti-Pro108 antibodies or immunoconjugates are
administered to a human patient, in accord with known methods, such
as intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The antibodies or
immunoconjugates may be injected directly into the tumor mass.
Intravenous or subcutaneous administration of the antibody is
preferred. Other therapeutic regimens may be combined with the
administration of the anti-Pro108 antibody.
[0321] The combined administration includes co-administration,
using separate formulations or a single pharmaceutical formulation,
and consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Preferably such
combined therapy results in a synergistic therapeutic effect.
[0322] It may also be desirable to combine administration of the
anti-Pro108 antibody or antibodies, with administration of an
antibody directed against another tumor antigen associated with the
particular cancer. As such, this invention is also directed to an
antibody "cocktail" comprising one or more antibodies of this
invention and at least one other antibody which binds another tumor
antigen associated with the Pro108-expressing tumor cells. The
cocktail may also comprise antibodies that are directed to other
epitopes of Pro108. Preferably the other antibodies do not
interfere with the binding and or internalization of the antibodies
of this invention.
[0323] The antibody therapeutic treatment method of the present
invention may involve the combined administration of an anti-Pro108
antibody (or antibodies) and one or more chemotherapeutic agents or
growth inhibitory agents, including co-administration of cocktails
of different chemotherapeutic agents. Chemotherapeutic agents
include, e.g., estramustine phosphate, prednimustine, cisplatin,
5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and
hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or
anthracycline antibiotics. Preparation and dosing schedules for
such chemotherapeutic agents may be used according to
manufacturers' instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0324] The antibody may be combined with an anti-hormonal compound;
e.g., an anti-estrogen compound such as tamoxifen; an
anti-progesterone such as onapristone (see, EP 616 812); or an
anti-androgen such as flutamide, in dosages known for such
molecules. Where the cancer to be treated is androgen independent
cancer, the patient may previously have been subjected to
anti-androgen therapy and, after the cancer becomes androgen
independent, the anti-Pro108 antibody (and optionally other agents
as described herein) may be administered to the patient.
[0325] Sometimes, it may be beneficial to also co-administer a
cardioprotectant (to prevent or reduce myocardial dysfunction
associated with the therapy) or one or more cytokines to the
patient. In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy, before, simultaneously with, or post antibody
therapy. Suitable dosages for any of the above co-administered
agents are those presently used and may be lowered due to the
combined action (synergy) of the agent and anti-Pro108
antibody.
[0326] For the prevention or treatment of disease, the dosage and
mode of administration will be chosen by the physician according to
known criteria. The appropriate dosage of antibody will depend on
the type of disease to be treated, as defined above, the severity
and course of the disease, whether the antibody is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, and the discretion
of the attending physician. The antibody is suitably administered
to the patient at one time or over a series of treatments.
Preferably, the antibody is administered by intravenous infusion or
by subcutaneous injections. Depending on the type and severity of
the disease, about 1 pg/kg to about 50 mg/kg body weight (e.g.
about 0.1-15 mg/kg/dose) of antibody can be an initial candidate
dosage for administration to the patient, whether, for example, by
one or more separate administrations, or by continuous infusion. A
dosing regimen can comprise administering an initial loading dose
of about 4 mg/kg, followed by a weekly maintenance dose of about 2
mg/kg of the anti-Pro108 antibody. However, other dosage regimens
may be useful. A typical daily dosage might range from about 1
pg/kg to 100 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of disease symptoms occurs. The progress of
this therapy can be readily monitored by conventional methods and
assays and based on criteria known to the physician or other
persons of skill in the art.
[0327] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy. Such administration of a nucleic acid
molecule encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, WO 96/07321 published Mar. 14, 1996 concerning
the use of gene therapy to generate intracellular antibodies.
[0328] There are two major approaches to introducing the nucleic
acid molecule (optionally contained in a vector) into the patient's
cells; in vivo and ex vivo. For in vivo delivery the nucleic acid
molecule is injected directly into the patient, usually at the site
where the antibody is required. For ex vivo treatment, the
patient's cells are removed, the nucleic acid molecule is
introduced into these isolated cells and the modified cells are
administered to the patient either directly or, for example,
encapsulated within porous membranes which are implanted into the
patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). There
are a variety of techniques available for introducing nucleic acid
molecules into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. A commonly used vector for ex vivo
delivery of the gene is a retroviral vector.
[0329] The currently preferred in vivo nucleic acid molecule
transfer techniques include transfection with viral vectors (such
as adenovirus, Herpes simplex I virus, or adeno-associated virus)
and lipid-based systems (useful lipids for lipid-mediated transfer
of the gene are DOTMA, DOPE and DC-Cho1, for example). For review
of the currently known gene marking and gene therapy protocols see
Anderson et at., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
Articles of Manufacture and Kits
[0330] The invention also relates to an article of manufacture
containing materials useful for the detection for Pro108
overexpressing cells and/or the treatment of Pro108 expressing
cancer, in particular prostate, ovarian, colon, breast and stomach
cancer. The article of manufacture comprises a container and a
composition contained therein comprising an antibody of this
invention. The composition may further comprise a carrier. The
article of manufacture may also comprise a label or package insert
on or associated with the container. Suitable containers include,
for example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for detecting
Pro108 expressing cells and/or treating a cancer condition and may
have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an anti-Pro108 antibody of the invention. The label
or package insert indicates that the composition is used for
detecting Pro108 expressing cells and/or for treating prostate,
ovarian, colon, breast or stomach cancer, or more specifically
ovarian serous adenocarcinoma, breast infiltrating ductal
carcinoma, prostate adenocarcinoma, renal cell carcinomas,
colorectal adenocarcinomas, lung adenocarcinomas, lung squamous
cell carcinomas, and pleural mesothelioma, in a patient in need
thereof. The breast cancer may be HER-2 negative or positive breast
cancer. The cancers encompass metastatic cancers of any of the
preceding, e.g., prostate, ovarian, colon, breast or stomach cancer
metastases. The label or package insert may further comprise
instructions for administering the antibody composition to a cancer
patient. Additionally, the article of manufacture may further
comprise a second container comprising a substance which detects
the antibody of this invention, e.g., a second antibody which binds
to the antibodies of this invention. The substance may be labeled
with a detectable label such as those disclosed herein. The second
container may contain e.g., a pharmaceutically-acceptable buffer,
such as bacteriostatic water for injection (BWFI),
phosphate-buffered saline, Ringer's solution and dextrose solution.
The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0331] Kits are also provided that are useful for various purposes,
e.g., for Pro108 cell killing assays, for purification or
immunoprecipitation of Pro108 from cells or for detecting the
presence of Pro108 in a serum sample or detecting the presence of
Pro108-expressing cells in a cell sample. For isolation and
purification of Pro108, the kit can contain an anti-Pro108 antibody
coupled to a solid support, e.g., a tissue culture plate or beads
(e.g., sepharose beads). Kits can be provided which contain the
antibodies for detection and quantitation of Pro108 in vitro, e.g.
in an ELISA or a Western blot. As with the article of manufacture,
the kit comprises a container and a composition contained therein
comprising an antibody of this invention. The kit may further
comprise a label or package insert on or associated with the
container. The kits may comprise additional components, e.g.,
diluents and buffers, substances which bind to the antibodies of
this invention, e.g., a second antibody which may comprise a label
such as those disclosed herein, e.g., a radiolabel, fluorescent
label, or enzyme, or the kit may also comprise control antibodies.
The additional components may be within separate containers within
the kit. The label or package insert may provide a description of
the composition as well as instructions for the intended in vitro
or diagnostic use.
EXAMPLES
Example 1
Production and Isolation of Monoclonal Antibody Producing
Hybridomas
[0332] The following MAb/hybridomas of the present invention are
described below: Pro108.A2, Pro108.A5, Pro108.B1, Pro108.B2,
Pro108.B3, Pro108.B4, Pro108.B5, Pro108.B6, Pro108.B7, Pro108.B8,
Pro108.B9, Pro108.B10, Pro108.B11, Pro108.B12, Pro108.B13,
Pro108.B14, Pro108.B15, Pro108.B16, Pro108.B17, Pro108.B18,
Pro108.B19, Pro108.B20, Pro108.B21, Pro108.B22, Pro108.B23,
Pro108.B24, Pro108.B25, Pro108.B26, Pro108.B27, Pro108.B28,
Pro108.B29, Pro108.B30, Pro108.B31, Pro108.B32, Pro108.B33,
Pro108.B34, Pro108.B35, Pro108.B36, Pro108.B37, Pro108.B38,
Pro108.B39, Pro108.B40, Pro108.B41, Pro108.B42, Pro108.B43,
Pro108.B44, Pro108.B45.
[0333] If the MAb has been cloned, it will get the nomenclature
"X.1," e.g., the first clone of A7 will be referred to as A7.1, the
second clone of A7 will be referred to as A7.2, etc. For the
purposes of this invention, a reference to A7 will include all
clones, e.g., A7.1, A7.2, etc. An alternative nomenclature format
without the "period" (.) punctuation between "Pro108" and the
hybridoma may be employed and denotes the same MAb/hybridoma as one
with the "period" (.) punctuation.
Immunogens and Antigens (Recombinant Proteins, HA Tag &
Transfected Cells)
[0334] Pro108 Expressed Sequence & Protein Production
[0335] A PCR fragment of Pro108 cDNA encoding Met1 to Val331 was
introduced into an expression vector via recombination. The
construct was cloned in-frame to a six-histidine tag, located at
the C-terminal end, so that the Pro108 construct would be expressed
as a six-histidine tagged protein of 349 amino acids. The
recombinant plasmid was used to transform competent cells for
generation of the infection vector by transposition. A Pro108
recombinant vector was expressed in suitable cell lines.
[0336] Construct Sequence (underlined recombination site; bold six
histidine tag) (SEQ ID NO:1):
TABLE-US-00003
MENPSPAAALGKALCALLLATLGAAGQPLGGESICSARAPAKYSITFTGKWSQTAFPKQYPLFRPP
AQWSSLLGAAHSSDYSMWRKNQYVSNGLRDFAERGEAWALMKEIEAAGEALQSVHEVFSAPAVPSG
TGQTSAELEVQRRHSLVSFVVRIVPSPDWFVGVDSLDLCDGDRWREQAALDLYPYDAGTDSGFTFS
SPNFATIPQDTVTEITSSSPSHPANSFYYPRLKALPPIARVTLVRLRQSPRAFIPPAPVLPSRDNE
IVDSASVPETPLDCEVSLWSSWGLCGGHCGRLGTKSRTRYVRVQPANNGSPCPELEEEAECVPDNC
VDPAFLYKVVRWAHHHHHH
[0337] Cells expressing Pro108 were lysed in a buffer containing
0.4 M NaCl, 100 mM Na2HPO3/NaH2PO3, 10% glycerol, 1% Triton X-100,
and 10 mM imidazole, pH 8.0. The extracts were centrifuged at about
40,000 g and the recovered pellets were dissolved in a strong
chaotropic buffer containing 8 M urea, 1 M NaCl, 0.1 M
Na2HPO3/NaH2PO3, pH 8.1. The suspended samples were stirred
overnight at room temperature and then clarified by centrifugation
and filtration. The supernatants were loaded onto a Ni-NTA column,
equilibrated with a buffer containing 8 M urea, 5 mM .beta.-3-ME,
and 10 mM imidazole, pH 8.0. The columns were then washed with the
same buffers with increasing concentration of imidazole. The most
stringent wash contained 100 mM imidazole. Following the elution,
proteins were precipitated by dialysis against PBS, pH 7.2, and
used as a homogenized suspension.
[0338] Pro108 293T Cell Expressed Sequence & Protein
Production
[0339] A PCR fragment of Pro108 cDNA encoding Met1 to Val331 was
introduced in an expression vector via recombination. The construct
was cloned in-frame to a V5 epitope and six-histidine tag, located
at the C-terminal end, so that the Pro108 construct would be
expressed as a V5 epitope/six-histidine tagged protein of 371 amino
acids. The resulted plasmid was used to transfect a 293T suspension
culture and the recombinant Pro108 protein was recovered from
culture media for purification.
[0340] Construct Sequence (SEQ ID NO:2):
TABLE-US-00004
MENPSPAAALGKALCALLLATLGAAGQPLGGESICSARAPAKYSITFTGKWSQTAFPKQYPLFRPP
AQWSSLLGAAHSSDYSMWRKNQYVSNGLRDFAERGEAWALMKEIEAAGEALQSVHEVFSAPAVPSG
TGQTSAELEVQRRHSLVSFVVRIVPSPDWFVGVDSLDLCDGDRWREQAALDLYPYDAGTDSGFTFS
SPNFATIPQDTVTEITSSSPSHPANSFYYPRLKALPPIARVTLLRLRQSPRAFIPPAPVLPSRDNE
IVDSASVPETPLDCEVSLWSSWGLCGGHCGRLGTKSRTRYVRVQPANNGSPCPELEEEAECVPDNC
VDPAFLYKVVDLEGPRFEGKPIPNPLLGLDSTRTGHHHHHH
[0341] Recombinant mammalian Pro108 was harvested from both the
media and cells of a transiently transfected 293T suspension
culture. Concentrated culture media were exchanged into PBS, pH
7.9, by diafiltration and cells were lysed in 100 mM
Na2HPO3/NaH2PO3, pH 8.0, containing 0.4 M NaCl, 10% glycerol, 1%
Triton X-100, and 10 m M imidazole. Following the buffer exchange
or lysis, the sample was centrifuged and the supernatant was
filtered through a 10.quadrature. filter. The filtered sample was
then loaded onto a Ni-NTA column and the intended Pro108 was bound
on the column efficiently. The column was washed with the buffer
containing 0.4 M NaCl, 100 mM Na2HPO3/NaH2PO3, 10% glycerol, and 50
mM imidazole, pH 8.0. The protein was then eluted in the same
buffer containing 1 M imidazole. Following the elution, Pro108 was
dialyzed into a buffer containing 0.1 M sodium phosphate, 0.5 M
NaCl, 10% glycerol, pH 8.0.
[0342] Pro111 Expressed Sequence & Protein Production
[0343] Pro111 (human prostate-specific transglutaminase) protein
was used as the control for Pro108 antibody screening. The
recombinant construct encoding Met1 to Lys684 was generated by
introduction of a cDNA fragment into an expression vector via
recombination. The construct was cloned in-frame to a six-histidine
tag, located at the C-terminal end, so that Pro111 would be
expressed as a six-histidine tagged protein of 690 amino acids. The
recombinant plasmid was used to transform competent cells. A Pro111
expressing recombinant vector was expressed in suitable cells.
[0344] Construct Sequence (Bold Six Histidine Tag) (SEQ ID
NO:3):
TABLE-US-00005
MMDASKELQVLHIDFLNQDNAVSHHTWEFQTSSPVFRRGQVFHLRLVLNQPLQSYHQLKLEFSTGP
NPSIAKHTLVVLDPRTPSDHYNWQATLQNESGKEVTVAVTSSPNAILGKYQLNVKTGNHILKSEEN
ILYLLFNPWCKEDMVFMPDEDERKEYILNDTGCHYVGAARSIKCKPWNFGQFEKNVLDCCISLLTE
SSLKPTDRRDPVLVCRAMCAMMSFEKGQGVLIGNWTGDYEGGTAPYKWTGSAPILQQYYNTKQAVC
FGQCWVFAGILTTVLRALGIPARSVTGFDSAHDTERNLTVDTYVNENGEKITSMTHDSVWNFHVWT
DAWMKRPDLPKGYDGWQAVDATPQERSQGVFCCGPSPLTAIRKGDIFIVYDTRFVFSEVNGDRLIW
LVKMVNGQEELHVISMETTSIGKNISTKAVGQDRRRDITYEYKYPEGSSEERQVMDHAFLLLSSER
EHRRPVKENFLHMSVQSDDVLLGNSVNFTVILKRKTAALQNVNILGSFELQLYTGKKMAKLCDLNK
TSQIQGQVSEVTLTLDSKTYINSLAILDDEPVIRGFIIAEIVESKEIMASEVFTSFQYPEFSTELP
NTGRIGQLLVCNCIFKNTLAIPLTDVKFSLESLGISSLQTSDHGTVQPGETIQSQIKCTPIKTGPK
KFIVKLSSKQVKEINAQKIVLITKHHHHHH
[0345] Cells producing recombinant Pro111 were lysed in a buffer
containing 0.4 M NaCl, 0.1 M Na2HPO3/NaH2PO3, 1% Triton X-100, and
10 mM imidazole, pH 8.0, with protease inhibitor cocktail and
DNase. After one hour stirring on ice, the sample was centrifuged
and the supernatant was filtered and passed through a Ni-NTA
column. The column was washed with buffers containing 0.4 M NaCl,
0.1 M Na2HPO3/NaH2PO3, pH 8.0, with a linear increase of imidazole
concentration to 100 mM. The intended Pro111 was then eluted from
the column with the same buffer containing 1 M imidazole. Following
the elution, the protein was dialyzed into a buffer containing 0.1
M sodium phosphate, 0.5 M NaCl and 10% glycerol, pH 8.0.
Immunizations
[0346] For generation of both the A and B series MAbs mice were
immunized with insect expressed Pro108 recombinant protein,
encoding a region of Pro108 from Met1 to Val331 of the full length
protein. Groups of 8 BALB/c mice were immunized intradermally in
both rear footpads. All injections were 25 uL per foot. The first
injection (day 1) of 10 ug of insect expressed Pro108 per mouse was
in Dulbecco's phosphate buffered saline (DPBS) mixed in equal
volume to volume ratio with Titermax gold adjuvant (Sigma, Saint
Louis, Miss.). Subsequent injections of 10 ug of insect expressed
Pro108 per mouse occurred on days 5, 9, 12, 16, 19, 23, 26, 29, 30
and consisted of antigen in 20 uL of DPBS plus 5 uL of Adju-phos
adjuvant (Accurate Chemical & Scientific Corp., Westbury, N.Y.)
per mouse. For the A series MAbs the final boost injection on day
33 consisted of 10 ug of insect cell expressed Pro108 diluted in
DPBS alone. For the B series MAbs the final boost injection on day
33 consisted of 4.8 ug of mammalian expressed Pro108 diluted in
DPBS alone. Fusion occurred on Day 37.
Hybridoma Fusions
[0347] Mice were sacrificed at the completion of the immunization
protocol and draining lymph node (popliteal) tissue was collected
by sterile dissection. Lymph node cells were dispersed using a
Tenbroeck tissue grinder (Wheaton #357426, VWR, Brisbane, Calif.)
followed by pressing through a sterile 40 uM sieve (VWR) into DMEM
and removing T-cells via anti-CD90 (Thy1.2) coated magnetic beads
(Miltenyl Biotech, Baraisch-Gladbach, Germany).
[0348] These primary B-cell enriched lymph node cells were then
immortalized by electro-cell fusion (BTX, San Diego, Calif.) with
the continuous myeloma cell line P3x63Ag8.653 (Kearney, J. F. et
al., J. Immunology 123: 1548-1550, 1979). Successfully fused cells
were selected by culturing in standard Hypoxanthine, Azaserine (HA)
(Sigma, St. Louis, Mo.) containing selection medium (DMEM/15%
FBS/0.5 ng/mL rIL-6 (Sigma)/10% P388D.sub.1 (ATCC, Manassas, Va.)
conditioned medium). These fusion cultures were immediately
distributed, 2 million cells per plate, into wells of 96 well
culture plates (Costar Cat. #3585, VWR). Distributing the culture
in 96 well culture plates, immediately following fusion,
facilitated selection of a larger diversity of hybridoma clones
producing single, specific antibodies. Supernatants from wells were
screened by ELISA, for reactivity against Pro108 E. coli expressed
protein, Pro108 insect expressed protein, and for no
cross-reactivity with the serine protease Pro111 extracellular
domain (insect expressed).
[0349] Monoclonal cultures, consisting of the genetically uniform
progeny from single cells, were established after the screening
procedure above, by limiting dilution (Coller, H and Coller, B.
Hybridoma 2: 91-6, 1983), or cell sorting of single viable cells
into wells of two 96 well plates (VWR), using flow cytometry
(Coulter Elite, Beckman Coulter, Miami, Fla.). The resulting murine
B-cell hybridoma cultures were expanded using standard tissue
culture techniques. Selected hybridomas were cryopreserved in fetal
bovine serum (FBS) with 10% DMSO and stored in Liquid Nitrogen at
-196.degree. C. to assure maintenance of viable clone cultures.
Screening & Selection of Antibody Producing Hybridomas
[0350] Hybridoma cell lines were selected for production of Pro108
specific antibody by enzyme linked solid phase immunoassay (ELISA).
Pro108 or Pro111 proteins were nonspecifically adsorbed to wells of
96 well polystyrene EIA plates (VWR). One hundred uL volumes of
Pro108 or Pro111 proteins at approximately 1 ug/mL in (DPBS) were
incubated overnight at 4.degree. C. in wells of 96 well polystyrene
EIA plates. Plates were washed twice with Tris buffered saline with
0.05% Tween 20, pH 7.4 (TBST). The plate wells were then emptied
and nonspecific binding capacity was blocked by completely filling
the assay wells with TBST/0.5% bovine serum albumin (TBST/BSA) and
incubating for 30 minutes at room temperature (RT). The plate wells
were then emptied, 100 uL of hybridoma culture medium samples
diluted 1:1 with TBST/BSA was added to the wells and incubated for
1 hour at RT. The wells were then washed 3 times with (TBST). One
hundred uL of alkaline phosphatase conjugated goat anti-mouse IgG
(Fc) (Pierce Chemical Co., Rockford, Ill.), diluted 1:5000 in
TBST/BSA, was then added to each well and incubated for 1 hour at
RT. The wells were then washed 3 times with TBST. One hundred uL of
alkaline phosphatase substrate para-nitrophenylphosphate (pNPP)
(Sigma) at 1 mg/mL in 1 M Diethanolamine buffer pH 8.9 (Pierce,
Rockford, Ill.) was then added to each well and incubated for 20
min. at RT. Color development was stopped by addition of 50 uL of
2N NaOH/well. Bound alkaline phosphatase activity was indicated by
the development of a visible yellow color. The enzymatic reaction
was quantified by measuring the solution's absorbance at 405 nm
wavelength. Cultures producing the highest absorbance values were
chosen for expansion and further evaluation. Selected ELISA
positive cultures from the original 96 well plates were transferred
to new 96 well tissue culture plates (VWR).
ELISA Screening of Pro108 MAbs
[0351] After 1 week in culture, the 3 A series hybridomas and 59 B
series hybridomas specific for Pro108 (negative with Pro111) were
retested to confirm continued production of Pro108 specific MAbs.
Two of the original three A series (designated A2 and A5) and
forty-five of the original 59 B series hybridoma cultures
(designated B1 through B45) with supernatants retaining ELISA
absorbance values greater than 1.0 with Pro108 and less than 0.2
with Pro111 were expanded in tissue culture and cryopreserved, as
described above. Selected Pro108 specific cultures were subcloned
by limiting dilution or single cell sorting (Coulter Elite) to
ensure genetically stable and uniform progeny.
Results from ELISA Screening of Cloned Pro108 MAbs
[0352] The 45 B series hybridomas remaining specific for Pro108
were ranked according to the ELISA results. The clones were tested
for reactivity on Pro108 and Pro111. Pro108.B12 had an OD 405 nm of
3.9255 and 0.1018 against Pro108 and Pro111, respectively.
Additionally, Pro108.B23 had an OD 405 nm of 3.7632 and 0.0901
against Pro108 and Pro111, respectively. Pro108.B12 received a rank
of 2 and Pro108.B23 received a rank of 6.
[0353] Based on the ELISA ranking, Pro108.A2, Pro108.A5,
Pro108.B10, Pro108.B12, Pro108.B16, Pro108.B23, Pro108.B24,
Pro108.B29, Pro108.B30, Pro108.B33, and Pro108.B38 were selected
for subcloning. ELISA checker board results (see below) determined
that Pro108.A5 paired with Pro108.B12, Pro108.B16, Pro108.B23,
Pro108.B24, Pro108.B29, Pro108.B30, Pro108.B33 and Pro108.B38; and
that Pro108.B12 and Pro108.B23 paired well; and that Pro108.B12 and
Pro108.B10 paired well with each other by sandwich ELISA. The
clones obtained from limiting dilution were tested for reactivity
on Pro108. Cloned Pro108.B12.1 had an OD 405 nm of 2.8767 and
cloned Pro108.B23.1 had an OD 405 nm of 2.6713.
[0354] Pro108.A2, Pro108.A5, Pro108.B10, Pro108.B12, Pro108.B16,
Pro108.B23, Pro108.B24, Pro108.B29, Pro108.B30, Pro108.B33, and
Pro108.B38 MAb clones were scaled up for further characterization
by western blot and ELISA.
[0355] The isotypes of the B series MAbs were determined using
commercially available mouse monoclonal antibody isotyping
immunoassay test kits (IsoStrip, Roche Diagnostic Corp.,
Indianapolis, Ind.). Results of the isotyping are listed in Table
2.
TABLE-US-00006 TABLE 2 Pro108 MAb Isotypes MAb Isotype Pro108.B12.1
IgG.sub.1 kappa Pro108.B10.1 IgG.sub.1 kappa
Example 2
Tissue Distribution and Detection of Pro108 in Serum
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
[0356] To detect the presence and tissue distribution of Pro108
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was
performed using cDNA generated from a panel of tissue RNAs. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d
ed., Cold Spring Harbor Laboratory Press (1989) and; Kawasaki E S
et al., PNAS 85(15):5698 (1988). Total RNA was extracted from a
variety of tissues or cell lines and first strand cDNA is prepared
with reverse transcriptase (RT). Each tissue panel includes 23
cDNAs from five cancer types (lung, ovary, breast, colon, and
prostate) and normal samples of testis, placenta and fetal brain.
Each cancer set is composed of three cancer cDNAs from different
donors and one normal pooled sample. Using a standard enzyme kit
from BD Bioscience Clontech (Mountain View, Calif.), Pro108 was
detected with sequence-specific primers designed to only amplify
Pro108. The PCR reaction was run on the GeneAmp PCR system 9700
(Applied Biosystem, Foster City, Calif.) thermocycler under optimal
conditions. One of ordinary skill can design appropriate primers
and determine optimal conditions. The amplified product was
resolved on an agarose gel to detect a band of equivalent size to
the predicted RT-PCR product. A band indicated the presence of
Pro108 a sample. FIG. 1A shows the RT-PCR results.
Western Blots
[0357] SDS-PAGE was performed according to the method of Laemmli.
All samples were reduced with 20 mM DTT in 1.times.LDS Sample
Buffer (Invitrogen) and heated at 70 C for 10 min. 15 ug of each
cell lysate and supernatant and 20 and 50 ng recombinant Spondin2
were loaded onto a 4-12% Bis Tris gel (Invitrogen). The gel was
transferred gel onto a PVDF membrane (Invitrogen) according to the
manufacture's guideline. After blocking with 5% milk in TBST (10 mM
Tris pH7.4, 150 mM NaCl, 0.05% Tween20), the membrane was incubate
with MAb A5.1, B23.1 or B12.1 (1 ug/ml) for 1 hour at room
temperature. After washes with TBST, the blot was incubated with
donkey anti-mouse--HRP 1:10,000 (Jackson) for 1 hr at room
temperature while shaking. The blot was incubated with ECL Plus
developer (Amersham) for 5 min at room temperature and exposed to
film for 20 seconds, following manufacture's guidelines.
RT-PCR and Western Blot Results
[0358] A comparison of mRNA and protein expression in various
cancer cell lines was preformed and is outlined in Table 3 below.
For the Western Blot, cells were harvested and lysed in a CHAPS
buffer as described above. All lysates were adjusted to a final
concentration of 1.2 mg/ml and 15 ul of each lysate was loaded onto
the SDS PAGE gel. Recombinant Pro108 (50 ng) was loaded as a
positive control.
[0359] RT-PCR analysis showed Pro108 mRNA expression in
hormone-dependent prostate cancer lines but no indication of Pro108
expression in any other tested cell line. Additionally, the Western
Blot results indicate protein expression is in very good agreement
with the mRNA expression. Table 3 indicates native Pro108 was
detected in the detergent lysate of various cancer cell lines. In
the lysate of the androgen-dependent prostate cancer cell line
LNCap and MDA PCa2b a band of the predicted molecular weight of
Pro108 (35 kD) was detected. The expression of Pro108 is not
hormone-dependent as the results with LNCaps plus and minus
stimulation with 5-alpha-dihydrotestosterone indicate. No Pro108
was detected in the prostate cancer cell line PC3, the colon cancer
line HT29, the breast cancer cell line MDA MB453, the cervical
cancer cell line HeLa, the ovarian cancer cell line CaOV3, the lung
cancer cell line A549 or 293 cells which were used for
transfection. See Table 3 below.
TABLE-US-00007 TABLE 3 Comparison of Pro108 mRNA and protein
expression in various cancer cell lines MDA LnCAP+ PC3 MB453 HeLa
LnCAP- A549 HT29 293 CaOV3 PCa2b RT-PCR + - - - + - - - - + Western
+ - - - + - - - - + Blot at ~35 kD
[0360] For the western bolts detecting Pro108 in cell lysate and
supernatant, 15 ul of cell lysate and cell supernatant (1.2 mg/ml
each) was loaded onto a 4-12% SDS Page gel, blotted and developed
using 1 ug/ml Pro108.B23 and Pro108.A5, respectively. Recombinant
Pro108 (with a 10 His-tag) was loaded as a positive control. HeLa
cells are negative while LNCap cells are positive for Pro108.
Intensity of the Pro108 western blot is indicated in Table 4 below
as high intensity (+++), moderate intensity (++), low intensity (+)
and no Pro108 detected (-).
[0361] As shown in Table 4 below, Pro108 was not only expressed in
LNCap cells but was also very efficiently secreted into the medium.
For this experiment, LNCap cells were switched to serum-free medium
48 hours prior to harvest the cell supernatant. The total
concentration of cell lysate and supernatant was adjusted to 1.2
mg/ml and equal amounts were loaded on the SDS PAGE gel. Pro108 was
detected using the Pro108.A5 antibody which has been in used in IHC
and the antibody Pro108.B23 which was used as detecting antibody in
the sandwich ELISA. The antibody Pro108.B23 reacted with a single
band of 35 kD in the LNCap lysate and supernatant. The antibody
Pro108.A5 reacted with full length Pro108 and a.about.30 kd
breakdown product of Pro108 with low intensity (+). Neither
antibodies showed reactivity towards any protein in the medium of
the RT-PCR-negative cell line HeLa, indicating that both antibodies
are very specific.
TABLE-US-00008 TABLE 4 Detection of Pro108 in cell lysate and
supernatant Recombinant Pro108.His Cell Lysate Cell Supernatant 50
ng 20 ng LnCAP+ LnCAP- HeLa LnCAP+ LnCAP- HeLa Pro108.B23 +++ ++
+++ +++ - ++ +++ - Pro108.A5 +++ ++ +++ +++ - ++ +++ -
Detection of Pro108 in Serum Samples by Immunoprecipitation
[0362] For the immunoprecipitation experiment the coating antibody
of the ELISA was bound to CnBr-Sephaose beads and used to capture
Pro108 from serum. Samples used included recombinant Pro108
(rPro108) as a positive control, calf serum, normal human serum
(Nrm hum serum), LnCAP supernatant (LnCAP supe), HeLa supernatant
(HeLa supe), prostate cancer serum (Pro can serum), lung cancer
serum (Lng can), breast cancer serum (Mam can), colon cancer serum
(Cln can) and ovarian cancer serum (Ovr can). Anti-Pro108 mAb
Pro108.B12.1 was covalently attached to CnBr-Separose beads
following the manufacturer's protocol (Amersham Pharmacia). 1 ml of
serum was incubated with 100 ul of beads overnight at 4.degree. C.
After washing three times with TBS+Tween20, the bound antigen was
eluted using gentle elution buffer pH 3.0 (Pierce). The eluate was
reduced to a volume of 50 ul and loaded onto a 4-12% Bis-Tris gel
(Invitrogen) as described above. The antibody Pro108.B23, used for
detection in the ELISA, was used for the detection on the Western
Blot. Intensity of the Pro108 western blot is indicated in Table 5
below as high intensity (+++), moderate intensity (++), low
intensity (+) and no Pro108 detected (-).
[0363] Results from the Western Blot are summarized in Table 5
below. Immunoprecipitation results show Pro108 can be isolated from
serum of healthy individuals as well as from serum of subjects with
cancer. The antibody Pro108.B23 reacted with a band of 40 kD in the
recombinant Pro108, normal human serum, LnCAP supernatant, prostate
cancer serum, lung cancer serum, breast cancer serum, colon cancer
serum and ovarian cancer serum samples. Additionally, Pro108.B23
reacted with a dimer product at .about.80 kD in the prostate, lung
breast, colon and ovarian cancer serum samples. In agreement with
the ELISA data below, more Pro108 is present in serum of subjects
with cancer than in healthy individuals. Additionally, the
over-expression of Pro led to increased dimerization of the
protein. We tested if the dimeric form of Pro108 is an independent
predictor for cancer but found that the dimerization is
concentration dependent but not disease dependent.
TABLE-US-00009 TABLE 5 Immunoprecipitation of Pro108 from normal
and cancer serum samples Normal Samples Cancer Serum Samples Calf
Nrm hum LNCaP HeLa Pro Lng Mam Cln Ovr rPro108 Serum Serum supe
supe can can can can can Pro108.B23 +++ - + + - + ++ ++ + ++
Example 3
Sandwich and Checkerboard ELISA of Pro108
[0364] High binding polystyrene plates (Corning Life Sciences (MA))
were coated overnight at 4.degree. C. with 8 ug/ml of anti-Pro108
MAb (note: later experiments used 4 ug/ml). The coating solution
was aspirated off and free binding sites were blocked with 300
.mu.l/well Superblock-TBS (Pierce Biotechnology, Illinois) for 1
hour at room temperature. After washing 4.times. with TBS+0.1%
Tween20, 25 ul (note: later experiments used 20 ul) of antigen was
added to each well for 90 minutes incubation. For the checkerboard
experiment, each pair was tested on 50 ng/ml and 0 ng/ml of
recombinant Pro108-decaHis. For each Sandwich ELISA, a standard
curve of 250, 100, 50, 10, 1 and 0 ng/ml Pro108 was run in parallel
with the samples. Standard Curve and samples were diluted in Assay
Buffer (TBS, 1% BSA, 1% Mouse Serum, 1% Calf Serum, 0.1% Tween20)
to a final volume of 100 ul. For the detection, 1000 Biotinylated
MAb (1 .mu.g/ml) were added to each well and incubated for 1 hour
at room temperature while shaking. After washing, 100 .mu.l of
Alkaline Phosphatase conjugated Streptavidin (Jackson
ImmunoResearch Laboratories, PA) was added to each well and
incubated for 30 minutes at RT while shaking. After washing, the
plate was then developed using pNPP substrate in 1.times.DEA buffer
(Pierce Biotechnology, Illinois) for 30 minutes at RT. The reaction
is stopped using 100 .mu.l/well 1N NaOH, and the plate was read at
405 nm using a Spectramax 190 plate reader (Molecular Devices,
CA).
Pro108 Checkerboard ELISA
[0365] For the checkerboard ELISA, all possible combination of
antibodies used as coating and detecting antibody were tested. The
pairs B12/B10 and B12/B23 performed best (highest signal/noise
ratio) in the Sandwich ELISA and B12/B23 was used in following
Sandwich ELISA to analyze native Pro108 in cell lines and serum
samples.
[0366] The results of the checkerboard ELISA using the Pro108 MAbs
are shown in Tables 6A and 6B below. The binding results and
epitope map are graphically represented in FIG. 1.
TABLE-US-00010 TABLE 6A Results of Checkerboard Analysis (numbers
represent signal/noise ratio) detecting Mab coating Mab A5.1 A2.3
B12 B16 B23 B24 B29 B30 B33 control mAb A5.1 1 1 2.44 3.1 4.1 2.3
1.4 3.8 1.7 1.1 A2.3 1.5 1.2 4.7 7.5 17 7.5 2.8 11.2 2.3 1 B12 10
4.6 1.1 1 38 11 1 1.2 10 1 B16 14.8 7.7 1.3 1.4 24 9.9 1 2.2 7.2 1
B23 11 5.3 7 15.4 1 1 3.7 21 1 1 B24 12.7 6.3 6.5 13.7 1.8 1.1 4.1
18.5 1 1 B29 17.4 9.6 1.4 1.7 30.8 9.5 1 2.8 8.2 1 B30 22 11.9 1.2
1.2 41.7 17.5 1 2.6 14.4 1 B33 15 8.5 14.4 30 9.4 2 9.35 36 1.1 1
B39 19.7 8.5 1.7 3.5 24.7 7 1.2 6 6.1 1
TABLE-US-00011 TABLE 6B Results of Checkerboard Analysis (numbers
represent signal/noise ratio) Detecting Mab coating Mab B1.1 B6.1
B7.1 B10.1 B12.1 B20.1 B23.1 B26.1 B27.1 A5.1 control B1.1 2.3 1.5
3.9 14.7 1.5 1.1 9.3 2.4 6.8 1.7 1.1 B6.1 13.2 1.0 3.2 13.0 9.3 1.0
12.0 6.6 3.8 17.6 1.0 B7.1 10.0 1.0 1.5 3.8 4.9 1.0 5.7 1.7 1.4
13.9 1.1 B10.1 9.3 0.9 1.3 2.0 7.0 1.0 3.8 1.3 1.1 12.1 1.0 B12.1
13.1 8.5 20.6 41.3 1.3 2.6 49.9 13.7 36.9 20.7 1.0 B20.1 10.5 1.2
3.9 10.6 7.3 1.1 11.2 5.8 3.8 6.6 1.1 B23.1 17.0 1.2 1.1 2.4 6.0
1.0 3.1 1.3 1.1 13.7 0.9 B26.1 1.6 1.0 1.4 1.5 1.5 1.1 1.4 0.8 1.3
1.4 1.0 B27.1 10.3 0.4 1.6 4.4 3.1 1.5 5.9 1.9 1.5 10.6 0.9
Pro108 Checkerboard ELISA
[0367] To establish a sensitive Sandwich-ELISA assay, hybridoma
clones with a high binding affinity in direct ELISA were selected
and antibodies were purified and tested in the checkerboard ELISA.
Each antibody was used as a coating as well as a detecting antibody
in all possible combinations. During the incubation with detecting
antibody, a 10-fold higher concentration of coating antibody was
added to the wells to prevent self-pairing. Self-pairing may be
observed when antigens are partly multimerized and may confound MAb
pairing results. Performing the ELISA assay under competitive
conditions ensures that antibodies cannot bind to the same or
proximal epitopes even when the antigen is partly aggregated.
[0368] Using the described method, antibodies against three
distinct epitopes have been identified. Several different
combinations of antibody sandwiches were tested to establish an
ELISA assay for the detection of native Pro108 in cancer cell
lines, transfected cell lines and serum. The pairs B12/B23 and
B12/B10 showed the highest sensitivity and specificity. The
sensitivity of both B12/B23 and B12/B10 pairs for recombinant
Pro108 is 1 ng/ml. The B12/B23 pair did not react with Spondin I or
human thrombospondin by ELISA or in Western Blots. The pair B12/B23
reacted positively with lysate and supernatant from transfected 293
cells but not with those from untransfected cells. In good
agreement with the Western Blot and RT-PCR results, the ELISA
detected Pro108 expressed in LNCap and MDA PCa2b cell lines. The
protein was detected in the supernatant of these androgen-dependent
cell lines but not in the supernatant of other cells.
[0369] To compare the performance of antibody pair B12/B23 with the
B12/B10 pair, 160 serum samples (35 of healthy male and female, 25
serum samples each from subjects with colon, breast, ovarian,
prostate or lung cancer) were run in parallel with the two assay
formats. The inter-assay CV for this sample set was 6% and the data
correlated very well (R2-value=0.95) indicating an ELISA with
antibody pairs B12/B23 and B12/B10 are comparable to one
another.
Example 4
Pro108 and Tumor Marker Assays
Patient Population
[0370] A total of 555 (281 males and 274 females) normal serum
samples, collected from healthy donors with age ranging from 19
years to 81 years old (median of 54 years) in addition to cancer
panels consisting of 1023 subjects with cancer and 997 subjects
with related benign diseases were obtained from the following
commercial sources: IMPATH-BCP, Inc. (Los Angeles, Calif.),
ProMedDx, LLC (Norton, Mass.) and Diagnostic Support Service, Inc.,
(West Barnstable, Mass.). Additional ovarian cancer samples were
obtained from DIAGNOSTIC ONCOLOGY CRO, Inc. (DOCRO). The human
serum samples from subjects with stomach cancer were received from
University of Pittsburgh, Medical Center. (Seymour, Conn.). All
cancer samples were collected prior to treatment, and provided with
age, gender, histology and stage information. The benign group
included subjects with BPH and prostatitis (n=143) for the prostate
cancer analysis; subjects with endometriosis, enlarged ovaries and
ovarian cysts (n=146) for the ovarian cancer study; subjects with
fibroadenoma, atypical hyperplasia and fibrocystic disease (n=179)
for the breast cancer study; subjects with chronic bronchitis,
emphysema, asthma, interstitial lung disease and pulmonary
hypertension (n=246) in the lung cancer study and subjects with
Crohn's disease, diverticulitis, ulcerative colitis and colon
polyps (n=283) in the colon cancer study.
Results
[0371] The cell line results above indicated that Pro108 is
secreted from prostate cancer tissue and possibly from normal
prostate and therefore detectable in serum. To test this
hypothesis, we screened the sera from healthy subjects and compared
the Pro108 concentration with Pro108 values found in subjects with
prostate cancer. Since the mRNA profiling also indicated expression
in other cancers, we tested also sera of subjects with other forms
of cancer as well as subjects with benign diseases. See Table 4 for
the summary of all sera samples that were used in our study.
TABLE-US-00012 TABLE 7 List of all Serum Samples tested for Pro108
concentration Sample Type Number of Samples Normal 315 (195 Male,
120 Female) Breast Cancer 235 Breast Benign 180 Colon Cancer 125
(56 Male, 69 Female) Colon Benign 296 (151 Male, 145 Female) Lung
Cancer 298 (210 Male, 88 Female) Lung Benign 250 (130 Male, 120
Female) Ovarian Cancer 225 Ovarian Benign 150 Prostate Cancer 138
Prostate Benign 147
[0372] Pro108 was detected in the sera of female and male subjects
with no significant difference between genders. However, the median
Pro108 concentration in sera of healthy subjects was significantly
lower than the Pro108 concentration of subjects with cancer.
[0373] The elevated level of Pro108 in the sera of subjects with
cancer confirms the RT-PCR results which showed over-expression of
Pro108 in prostate cancer tissue.
[0374] FIG. 2 shows Pro108 detection in breast, lung, ovarian,
colon and prostate cancer samples in comparison to Pro108
concentration in healthy subjects (female and male). The ELISA
plates were coated with 4 ug/ml mAb Pro108.B12 and after blocking
and washing steps, incubated with 20 ul of serum sample. Pro108 was
detected with 1 ug/ml biotinylated Pro108.B23 followed by
Streptavidin-HRP and pNpp substrate for chromogenic reaction.
[0375] In an alternative to the assay described above Pro108.B23
was replaced with Pro108.B10 as the detecting antibody.
Tumor Marker Immunoassays
[0376] To compliment and contrast Pro108, Prostate Specific Antigen
(PSA), Carcinoembryonic Antigen (CEA), CA15.3, CA19.9 and CA125
levels were measured on the Lumipulse bioanalyzer (Fujirebio,
Tokyo, Japan) using commercially available reagents according to
the manufacturer's protocol.
[0377] Additionally, Regenerative Protein IV (RegIV) levels were
measured. PCT application PCT/US2004/016969, which is hereby
incorporated by reference in its entirety, discloses the
development of mouse monoclonal antibodies (mAbs) to recombinant
Reg IV (also know as Cln101) protein and the development of a
sequential sandwich ELISA using two Reg IV-specific mAbs. High
binding polystyrene plates (Corning Life Sciences (MA) were coated
with capture mAb Cln101.A46.1. Twenty uL of serum samples were used
in the assay. Calibration was accomplished by using recombinant
RegIV standards at concentrations of 10, 5, 1, 0.5, 0.05 and 0
ng/mL. Antigen was detected by biotinylated Cln101.A9.1 mAb,
followed by streptavidin-alkaline phosphatase, and pNPP
substrate.
Example 5
Detection and ROC Analysis of Pro108 and PSA in Prostate Cancer
[0378] The ability of a test to discriminate diseased cases from
normal cases is evaluated using Receiver Operating Characteristic
(ROC) curve analysis (Metz, 1978; Zweig & Campbell, 1993). ROC
curves can also be used to compare the diagnostic performance of
two or more laboratory or diagnostic tests (Griner et al.,
1981).
[0379] ROC curve is generated by plotting sensitivity against
specificity for each value. From the plot, the area under the curve
(AUC) can be determined. The value for the area under the ROC curve
(AUC) can be interpreted as follows: an area of 0.84, for example,
means that a randomly selected positive result has a test value
larger than that for a randomly chosen negative result 84% of the
time (Zweig & Campbell, 1993). When the variable under study
can not distinguish between the two result groups, i.e. where there
is no difference between the two distributions, the area will be
equal to 0.5 (the ROC curve will coincide with the diagonal). When
there is a perfect separation of the values of the two groups, i.e.
there no overlapping of the distributions, the area under the ROC
curve equals 1 (the ROC curve will reach the upper left corner of
the plot).
[0380] The 95% confidence interval for the area can be used to test
the hypothesis that the theoretical area is 0.5. If the confidence
interval does not include the 0.5 value, then there is evidence
that the laboratory test does have an ability to distinguish
between the two groups (Hanley & McNeil, 1982; Zweig &
Campbell, 1993).
Detection of Pro108 in Prostate Cancer
[0381] FIG. 3 shows Pro108 detection in subjects with prostate
cancer in comparison to healthy subjects (female and male) and
subjects with benign prostate diseases (BPH and Prostatitis). The
ELISA plates were coated with 8 ug/ml mAb Pro108.B12 and after
blocking and washing steps, incubated with 25 ul of serum sample.
Pro108 was detected with 1 ug/ml biotinylated mAb Pro108.B23
followed by Streptavidin-HRP and pNpp substrate for chromogenic
reaction.
ROC Analysis of Pro108 Alone and in Combination in Prostate
Cancer
[0382] Analysis of Pro108, Reg IV and PSA levels in blood serum was
preformed on normal males, men with prostate benign disease (BPH
and prostatitis), and subjects with prostate cancer. The data were
analyzed by Receiver Operating Characteristic (ROC) curves to
determine and compare the sensitivity and specificity of each
marker in detecting cancer as described above. The analyses showed
that the levels of Reg IV and Pro108 are elevated in serum samples
from subjects with prostate cancer compared to normal control, BPH,
and prostatitis samples.
[0383] Area Under the Curve (AUC) values from ROC analysis of
prostate cancer versus normal and benign samples showed that RegIV
and Pro108 have sensitivities and specificities that are comparable
to PSA in detecting prostate cancer. Interestingly, in the PSA
"grey zone" of 4-10 ng/mL, Pro108 showed significantly higher
sensitivity and specificity in detecting prostate cancer than PSA.
Furthermore, the increased sensitivity and specificity of RegIV and
Pro108 over PSA were even more dramatic in the 2-4 ng/mL PSA range,
where RegIV and Pro108 were able to stratify 57% and 33% of
prostate cancer samples, respectively, with 90% specificity.
Application of multiple markers in "synergistic effect" analyses
showed slight improvement in AUC, where the combination of all
three markers in the PSA 2-4 ng/mL range showed an AUC=0.823.
Tables 8-10 demonstrate various ROC analyses of Pro108 alone or in
combination with other markers in prostate cancer. Tables 11-12
demonstrate results from ROC analyses of synergistic effects of
Pro108 with other markers in a PSA range of 4-10 ng/ml and 2-4
ng/ml, respectively.
TABLE-US-00013 TABLE 8 ROC analysis for Pro108 for differentiation
of normal males and males with benign conditions from males with
prostate cancer Pro108 Pro108 Statistic Original N = 406 Current N
= 431 AUC (95% CI) 0.681 (0.633-0.726) 0.701 (0.656-0.744) Cutoff
for best combination 49.2 49.2 of Sens/Spec. Sens./Spec. at best
cutoff 61%/74% 63%/74% Sens. @ 90% Spec. (Cutoff) 34% 35% p-value
vs. PSA ROC 0.021 (PSA ROC is Not applicable higher [0.765]) due to
nonequivalent N
TABLE-US-00014 TABLE 9 ROC analysis for Pro108 for differentiation
of normal males from males with prostate cancer Pro108 Pro108
Statistic Original N = 259 Current N = 284 AUC (95% CI) 0.767
(0.711-0.817) 0.787 (0.734-0.833) Cutoff for best combination 47.0
47.0 of Sens/Spec. Sens./Spec. at best cutoff 63%/83% 65%/83% Sens.
@ 90% Spec. (Cutoff) 45% 45% p-value vs. PSA ROC 0.292 Not
applicable due to nonequivalent N
TABLE-US-00015 TABLE 10 Logistic regression for differentiation of
normal males and males with benign conditions from males with
prostate cancer for Pro108, RegIV and PSA. RegIV + Pro108 + Pro108
+ Statistics PSA RegIV Pro108 PSA RegIV PSA ROC 0.762 0.741 0.686
0.800 0.768 0.814 AUC Sens. @ 31% 36% 34% 44% 37% 46$ 90% Spec.
Spec. @ 44% 44% 20% 47% 46% 55% 90% Sens.
TABLE-US-00016 TABLE 11 Analysis of synergistic effects in PSA
range of 4-10 ng/ml. Logistic regression for differentiation of
normal males and males with benign conditions from males with
prostate cancer for Pro108, RegIV and PSA. Pro108 + RegIV + Pro108
+ Pro108 + RegIV + Statistics PSA RegIV Pro108 PSA RegIV PSA PSA
ROC AUC 0.522 0.657 0.652 0.657 0.698 0.657 0.699 Sens. @ 5% 25%
33% 25% 35% 33% 42% 90% Spec. Spec. @ 10% 36% 14% 36% 38% 18% 35%
90% Sens.
TABLE-US-00017 TABLE 12 Analysis of synergistic effects in PSA
range of 2-4 ng/ml. Logistic regression for differentiation of
normal males and males with benign conditions from males with
prostate cancel for Pro108, RegIV and PSA. Pro108 + RegIV + Pro108
+ Pro108 + RegIV + Statistics PSA RegIV Pro108 PSA RegIV PSA PSA
ROC AUC 0.500 0.733 0.812 0.733 0.823 0.811 0.823 Sens. @ 2% 57%
33% 52% 33% 33% 33% 90% Spec. Spec. @ 10% 40% 66% 42% 67% 65% 67%
90% Sens.
[0384] From the statistical analysis can be concluded that Pro108
adds sensitivity and specificity to PSA, and can detect cancers
missed by PSA. Additionally, RegIV also adds sensitivity and
specificity to PSA, and can help detect cancers missed by PSA.
Pro108 and RegIV especially increase the detection rate of prostate
cancer in the PSA "grey zone", both 2-4 ng/mL and 4-10 ng/mL.
[0385] The combinations of Pro108, RegIV and PSA are not
significantly better than Pro108 alone in the PSA range of 2-4
ng/ml but in the PSA range of 4-10 ng/ml the combinations of Pro108
and RegIV may improve detection of cancer. Both, Pro108 and RegIV
have potential clinical applications in the PSA 2-4 ng/mL and 4-10
ng/ml range.
Example 6
Detection and ROC Analysis of Pro108 and CA125 in Ovarian
Cancer
[0386] The ability of Pro108 to detect and discriminate ovarian
cancer from normal samples and benign ovarian diseases was
evaluated using Receiver Operating Characteristic (ROC) curve
analysis as described above.
Detection of Pro108 in Ovarian Cancer
[0387] FIG. 4 shows Pro108 detection in subjects with ovarian
cancer in comparison to subjects with benign ovarian diseases
(polycystic ovaries, endometriosis or enlarged ovaries=Edema) and
healthy subjects (male and female). For the ELISA, plates were
coated with 4 ug/ml mAb Pro108.B12 and after blocking and washing
steps, incubated with 10 .mu.l of serum sample. Pro108 was detected
with 1 ug/ml biotinylated Pro108.B23 followed by Streptavidin-HRP
and pNpp substrate for chromogenic reaction.
[0388] FIG. 5 shows Pro108 detection in subjects with various forms
of ovarian cancer. The median values are compared to median Pro108
values of subjects with endometriosis and to values of healthy
women. The ELISA plates were coated with 4 ug/ml mAb Pro108.B12 and
after blocking and washing steps, incubated with 10 ul of serum
sample Pro108 was detected with 1 ug/ml biotinylated Pro108.B23
followed by Streptavidin-HRP and pNpp substrate for chromogenic
reaction.
[0389] The Pro108 concentration in serum from subjects with ovarian
cancer was elevated when compared with Pro108 values in healthy
women and women with benign ovarian diseases. The median Pro108
concentration was nearly two-fold higher in women with ovarian
cancer (54.1 ng/ml) than in healthy women (29.8 ng/ml).
Interestingly, the Pro108 serum concentration was elevated in all
tested ovarian cancer types. The median Pro108 concentration in
serous cancer patients was comparable to values in patients with
mucinous cancer. This of special interest since the currently used
marker CA125 is up-regulated only in serous cancer. In addition,
CA125 can also be elevated in patients with benign conditions as
endometriosis, benign ovarian cysts, uterine fibroids, pregnancy,
or pelvic inflammatory disease while Pro108 seemed to be present in
normal concentration in women with benign ovarian diseases. These
results show that Pro108 was detectable in all ovarian cancer
patients and distinguish healthy individuals from cancer patients,
demonstrating it's usefulness as a ovarian cancer marker.
ROC Analysis of Pro108 Alone and in Combination in Ovarian
Cancer
[0390] Analysis of Pro108 and CA125 levels in blood serum was
preformed on normal females, women with benign ovarian disease
(endometriosis), and subjects with ovarian cancer. The data were
analyzed by Receiver Operating Characteristic (ROC) curves to
determine and compare the sensitivity and specificity of each
marker in detecting cancer as described above. The analyses showed
that the levels Pro108 are elevated in serum samples from subjects
with ovarian cancer compared to normal control and benign disease
samples.
[0391] Area Under the Curve (AUC) values from ROC analysis of
ovarian cancer versus normal and benign samples showed that Pro108
sensitivity and specificity is comparable to CA125 in detecting
ovarian cancer. Furthermore, the increased sensitivity and
specificity of Pro108 over CA125 was even more dramatic in the
CA125 negative (<30 U/mL) range, where Pro108 was able to
stratify 26% of ovarian cancer samples, with 90% specificity.
Application of multiple markers in "synergistic effect" analyses
showed improvement in AUC, where the combination of Pro108 and
CA125 markers in stage 1 and 2 ovarian cancer showed an AUC=0.837.
Tables 13-14 demonstrate various ROC analyses of Pro108 alone or in
combination with other markers in ovarian cancer. Table 15
demonstrate results from ROC analyses of the ability to detect
cancers that are CA125 negative (<30 U/mL).
TABLE-US-00018 TABLE 13 ROC analysis of Pro108 and CA125 to
differentiate alone or synergistically normal (n = 31) or benign
disease (endometriosis n = 24) subjects from subjects with ovarian
cancer (n = 57). (normal + benign disease vs. cancer) Statistic
Pro108 CA125 Pro108 + CA125 ROC AUC 0.659 0.772 0.816 Sens. @ 90%
Spec. 33% 67% 61% Spec. @ 90% Sens. 8% 4% 27%
TABLE-US-00019 TABLE 14 ROC analysis of Pro108 and CA125 to
differentiate alone or synergistically normal (n = 31) or benign
disease (endometriosis n = 24) subjects from subjects with stage 1
and stage 2 ovarian cancer (n = 28). (normal + benign disease vs.
cancer) Statistic Pro108 CA125 Pro108 + CA125 ROC AUC 0.754 0.696
0.837 Sens. @ 90% Spec. 32% 61% 64% Spec. @ 90% Sens. 40% 4%
38%
TABLE-US-00020 TABLE 15 ROC analysis of Pro108 to differentiate
normal (n = 31) or benign disease (endometriosis n = 16) subjects
from subjects with CA125 negative (<30 U/mL) ovarian cancer (n =
19). (normal + benign disease vs. cancer) Statistic Pro108 ROC AUC
0.61 Sens. @ 90% Spec. 26% Spec. @ 90% Sens. 0%
[0392] ROC analysis results for Pro108 in ovarian cancer are at
least equal to or better compared to known marker CA125. Pro108 AUC
scores are good in subjects with Stage 1 & 2 ovarian cancer.
Additionally, Pro108 AUC scores are high even in CA125<30 U/ml.
Multivariate (Pro108+CA125) analysis indicates that the use of
CA125 and Pro108 in combination improves sensitivity and
specificity.
[0393] To confirm the performance of Pro108 as a diagnostic for
ovarian cancer, Pro108 and CA125 and CEA were measured in a second
study using a different sample set from Johns Hopkins (Baltimore,
Md.). The study consisted of healthy women (n=50), individuals with
benign endometrial and ovarian disease (n=45) and subjects with
ovarian cancer (n=50), The ROC analysis (cancer versus
normal+benign) resulted in an AUC=0.81 for Pro108 while the AUC for
CA125 in this study was 0.89. The combination of Pro108+CA125
improved sensitivity and specificity even further (AUC=0.91).
[0394] Pro108 is useful as an early stage ovarian cancer
diagnostic. Only 25% of all ovarian cancer is found in stage I. If
ovarian cancer is found in stage 1 surgery is very effective and
the 5-year survival rate is 90%.
Example 7
Detection and ROC Analysis of Pro108, CEA and CA19.9 in Colon
Cancer
Detection of Pro108 in Colon Cancer
[0395] FIG. 6 shows Pro108 detection in the serum of subjects with
colon cancer, Crohn's diseases, Diverticulitis, Ulcerative Colitis,
colon polyps, in comparison to Pro108 in serum of healthy
individuals. The median values are compared to median Pro108 values
of healthy individuals (male and female). The ELISA plates were
coated with 4 ug/ml mAb Pro108.B12 and after blocking and washing
steps, incubated with 10 ul of serum sample. Pro108 was detected
with 1 ug/ml biotinylated Pro108.B23 followed by Streptavidin-HRP
and pNpp substrate for chromogenic reaction.
ROC Analysis of Pro108 Alone and in Combination in Colon Cancer
[0396] Analysis of Pro108, CEA and CA19.9 levels in blood serum was
preformed on normal subjects, subjects with benign colon disease
(Crohn's diseases, Diverticulitis, etc.), and subjects with colon
cancer. The data were analyzed by Receiver Operating Characteristic
(ROC) curves to determine and compare the sensitivity and
specificity of each marker in detecting cancer as described above.
The analyses showed that the levels Pro108 are elevated in serum
samples from subjects with colon cancer compared to normal control
and benign disease samples.
[0397] Area Under the Curve (AUC) values from ROC analysis of colon
cancer versus normal and benign samples showed that Pro108
sensitivity and specificity is at least comparable to CEA and
CA19.9 in detecting colon cancer. Furthermore, the sensitivity and
specificity of Pro108 compared to CEA and CA19.9 was even more
dramatic in the stage 1 and stage 2 cancer sample set. Tables 16-17
demonstrate various ROC analyses of Pro108 alone or in combination
with other markers in colon cancer.
TABLE-US-00021 TABLE 16 ROC analysis of Pro108, CEA and CA19.9 to
differentiate normal or benign disease (n = 833) subjects from
subjects with colon cancer (n = 142). (normal + benign disease vs.
cancer) Statistic Pro108 CEA CA19.9 ROC AUC 0.77 0.65 0.58 Sens. @
90% Spec. 33% 17% 67% Spec. @ 90% Sens. 8% 25% 4%
[0398] To confirm the performance of Pro108 as a diagnostic for
colon cancer, Pro108, CA19.9 and CEA were measured in a second
study using a different sample set from Johns Hopkins (Baltimore,
Md.), The study consisted of healthy individuals (n=99),
individuals with benign colon diseases (n=22) and subjects with
colon cancer (n=49). The ROC analysis (cancer versus normal+benign)
resulted in an AUC=0.78 for Pro108 while the AUC for CA19.9 and CEA
in this study were 0.7 and 0.8, respectively.
TABLE-US-00022 TABLE 17 ROC analysis of Pro108, CEA and CA19.9 to
differentiate normal (n = 99) or benign disease (n = 22) subjects
from subjects with colon cancer (n = 49) or from subjects with
stage 1 or 2 colon cancer (n = 25). (normal + benign disease vs.
cancer) Statistic Sample Set Pro108 CEA CA19.9 ROC AUC All stages
0.78 0.8 0.7 Stage I + II 0.65 0.7 0.58
[0399] ROC analysis results for Pro108 in colon cancer are at least
equal to or better compared to known markers CEA and CA19.9. Pro108
AUC scores are good in subjects with Stage 1 & 2 colon cancer.
It is contemplated that multivariate use of Pro108, CEA and/or
CA19.9 in combination improves sensitivity and specificity for
detection of colon cancer.
[0400] Pro108 is useful as an early stage colon cancer diagnostic.
It is well known that if colon cancer is found in stage 1 surgery
is very effective and the 5-year survival rate increases
dramatically.
Example 8
Detection of Pro108 in Stomach Cancer
[0401] FIG. 7 shows Pro108 detection in stomach cancer and prostate
cancer samples. The median values are compared to median Pro108
values of healthy individuals (male and female). The ELISA plates
were coated with 4 ug/ml mAb B12 and after blocking and washing
steps, incubated with 10 ul of serum sample. Pro108 was detected
with 1 ug/ml biotinylated Pro108.B23 followed by Streptavidin-HRP
and pNpp substrate for chromogenic reaction. The median Pro108
level in subjects with stomach cancer was 3.4 times higher than in
healthy individuals. The sensitivity to detect stomach cancer at
95% specificity was 82% in this sample set.
Example 9
Multivariate ROC Analysis of Pro108 and Known Cancer Markers in
Various Cancers
[0402] In addition to increasing sensitivity and specificity for
detecting the cancers shown above, ROC analysis, as described
above, indicated Pro108 increases sensitivity and specificity for
detection of breast and lung cancer alone or in combination with
known markers. Detection of Pro108 and other markers was performed
as described above. Table 18 below summarizes Receiver Operating
Characteristic (ROC) curve analysis for Pro108 alone in a
combination with traditional markers for each cancer type. AUC
scores are reported for Pro108, each traditional marker and
multivariate analysis of Pro108 and a traditional marker.
TABLE-US-00023 TABLE 18 Multivariate ROC analysis with Pro108 and
traditional markers in various cancers. Pro108 Traditional Marker
Multivariate Cancer AUC AUC AUC Breast 0.62 0.58 (CEA) 0.63 (P108 +
CEA) 0.6 (CA15.3) 0.64 (P108 + CA15.3) Colon 0.77 0.65 (CEA) 0.78
(P108 + CEA) 0.58 (CA19.9) 0.78 (P108 + CA19.9) Lung 0.69 0.61
(CEA) 0.71 (P108 + CEA) Ovary 0.72 0.48 (CEA) 0.72 (P108 + CEA)
0.81 (CA125) 0.82 (P108 + CA125) Prostate 0.73 0.78 (PSA) 0.86
(P108 + PSA) 0.7 (% F/T PSA) * * (% F/T PSA) indicates the Percent
Free/Total PSA assay.
Example 10
Detection of Pro108 in Tissue by ELISA and IHC
Immunohistochemical (IHC) Staining
[0403] Formalin-fixed, paraffin-embedded tissue blocks were
sectioned to Sum and mounted on charged glass slides (Superfrost
Plus, Fisher Scientific, Pittsburgh, Pa.). Endogenous peroxidase
activity was blocked with 3.0% hydrogen peroxide for 15 minutes.
Antigen retrieval was performed in a citrate buffer (20 mmol/L, pH
6.0) at 120.degree. C. for 10 minutes Staining was conducted on a
DAKO autostainer (DakoCytomation, Carpinteria, Calif.) using an
indirect avidin-biotin immunoperoxidase method (Vector Labs,
Burlingame, Calif.). Sections were incubated at 25.degree. C. for
60 minutes with the Pro108.B23.1 antibody (1 .mu.g/ml). Negative
controls were run on all sections at 1 .mu.g/ml of a
subclass-matched IgG1 gamma (BD PharMingen, San Diego, Calif.),
generated against unrelated antigens. Pro108 staining was
visualized using 3,3'-diaminobenzidine (DakoCytomation,
Carpinteria, Calif.). Specificity of Pro108 staining was confirmed
by a blocking experiment with preincubation of the Pro108.B23.1
antibody with the full-length Pro108 protein (8 ng/ml) at
25.degree. C. for 60 minutes, prior to immunohistochemical
processing.
Results
[0404] The ELISA assay described above was used to test cytosolic
detergent extracts from somatic tissue and cancer tissue. Results
are presented in FIG. 8. Pro108 protein was found in low amounts in
several tissues including lung, muscles, small intestines, adrenal
and pituitary gland and lymph nodes. These results are in good
agreement with data from our mRNA profiling as well as with
published northern blot experiments (Manda et al., 1999). The
highest amount of Pro108 was found in normal prostate tissue and in
cancer tissue. The highest amount of Pro108 (up to 200 ng/mg total
protein) was consistently found in prostate cancer tissue. Since
Pro108 can be readily detected in sera of healthy female and male
persons with no significant difference between genders, the normal
level of Pro108 in blood must result from expression in normal
somatic tissues other than prostate. This indicates that the high
detectable Pro108 concentration in normal prostate tissue may not
reflect high protein expression but low secretion efficiency.
[0405] The results from the ELISA of tissue extracts were confirmed
by IHC experiments. Pro108 was detected in prostate cancer and
other cancer but not in most of the normal somatic tissues. In
prostate cancer, the Pro108 staining intensity and the percentage
of positive tissue increased with tumor grade. In addition, a
significant Pro108 staining in prostate cancer tissue indicated a
higher relative risk of capsular extension while organ confined
tumors showed less significant staining. These results show that
Pro108 is useful as diagnostic, staging and prognostic marker in
cancer.
[0406] Table 19 below summarize IHC staining results for Pro108 in
various cancer stages (Gleason Score) and assigns an Index Score
for each group based on the intensity of staining and percent of
the tissue that was stained. The increase in the Index Score with
progression of the cancer Gleanson Score indicates that Pro108 is
useful in staging cancer and monitoring progression of cancers.
Pro108 is contemplated to be useful as a marker for determining
response to a therapy, where a decrease in Pro108 levels is
indicative of a reduction of the Gleason Score and effectiveness of
the therapy. The target of therapy may be Pro108 or Pro108 may
serve as a surrogate marker for various other therapies.
TABLE-US-00024 TABLE 19 Summary of IHC staining in Cancer Tissues
by Gleason Sum. # No # +++ Avg. % Stain # + # ++ Stain of Ca
Gleason (% No Stain Stain (% Avg. tissue Index Score n stain) (% +)
(% ++) +++) staining stained Score* 2-4 7 2 (29) 2 (29) 3 (43) 0
(0) 1.14(+) 38% 2.7 5-6 88 23 (26) 29 (33) 30 (34) 6 (7) 1.22(+)
34.11% 2.59 (s.d. 1.84) 7-10 60 7 (12) 7 (12) 19 (32) 27 (45)
2.12(+) 61.92% 4.27 (s.d. 2.00) Predominant stain intensity used
for calculations *Index Score--Composite of tissue staining
intensity and % of tissue stained Score awarded as follows: + Stain
1 ++ Stain 2 +++ Stain 3 0% of Tissue Stained 0 1-33% of Tissue
Stained 1 34-66% of Tissue Stained 2 67-100% of Tissue Stained
3
[0407] Table 20 summarizes Pro108 IHC staining in prostate cancers
with capsular extension or that are organ confined. Capsular
extension in prostate cancer is common indicator of poor prognosis
for the disease. Significant staining is defined as ++ or greater
staining intensity and greater than 20% of cells are stained. The
results below indicate that capsular extension samples showed more
significant staining than organ confined samples and more capsular
extension samples showed significant staining than not.
Furthermore, there is a 1.676 relative risk of capsular extension
if significant staining is present in a sample (two-tailed p
value=0.002). These results indicate that Pro108 is useful as a
prognostic indicator of severity and potentially outcome of
prostate cancer.
TABLE-US-00025 TABLE 20 Comparison of capsular extension vs. organ
confined Pro108 staining in prostate cancer. Capsular Extension
Organ Confined Samples Samples Total Significant Staining 44 19 63
No Significant Staining 25 35 60 Total 69 54 123
[0408] Table 21 summarizes Pro108 IHC staining various cancer and
normal somatic tissues. The results indicate that Pro108 is
detected in pancreatic, colon, urinary bladder and gastric cancer
tissues. No Pro108 was detected in the kidney or lung cancer
tissues. Pro108 was not detected in the majority of normal somatic
tissues, but was present in samples of adrenal gland and Ileum.
These results are in agreement with results above that Pro108
expression is limited in normal tissues and is elevated in
cancerous tissues as well as serum in subjects with cancer.
TABLE-US-00026 TABLE 21 Pro108 expression in various cancer types
and normal somatic tissue. N samples with % samples with positive
staining positive staining Cancer Tissues Pancreatic Cancer 5/5 100
Colon Cancer 4/5 80 Urinary Bladder Cancer 2/3 67 Gastric Cancer
1/5 20 Kidney Cancer 0/5 0 Lung Cancer 0/9 0 Normal Somatic Tissues
Adrenal Gland 1/1 100 Bone Marrow 0/1 0 Colon 0/1 0 Esophagus 0/1 0
Gallbladder 0/1 0 Heart 0/1 0 Ileum 1/1 100 Kidney 0/1 0 Liver 0/1
0 Lung 0/1 0 Pancreas 1/1 100 Peritoneum 0/1 0 Spleen 0/1 0 Stomach
0/1 0 Thymus 0/1 0 Thyroid 0/1 0 Urinary Bladder 0/1 0
Example 11
Deposits
Deposit of Cell Lines and DNA
[0409] Hybridoma cell lines were deposited with the American Type
Culture Collection (ATCC) located at 10801 University Boulevard,
Manassas, Va. 20110-2209, U.S.A., and accorded accession
numbers.
[0410] The following hybridoma cell lines were deposited with ATCC,
Pro108.B10.1 and Pro108.B12.1. The names of the deposited hybridoma
cell lines above may be shortened for convenience of reference.
E.g. A01.1 corresponds to Pro108.A01.1. These hybridomas correspond
to the clones (with their full names) deposited with the ATCC.
Table 22 lists the hybridoma clone deposited with the ATCC, the
accorded ATCC accession number, and the date of deposit.
TABLE-US-00027 TABLE 22 ATCC deposits Hybridoma ATCC Accession No.
Deposit Date Pro108.B10.1 PTA-5885 23 Mar. 2004 Pro108.B12.1
PTA-5886 23 Mar. 2004
[0411] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations there under (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of deposit. The
organisms will be made available by ATCC under the terms of the
Budapest Treaty, and subject to an agreement between diaDexus, Inc.
and ATCC, which assures permanent and unrestricted availability of
the progeny of the cultures to the public upon issuance of the
pertinent U.S. patent or upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC .sctn.122 and the Commissioner's rules pursuant
thereto (including 3 7 CFR .sctn.1.14 with particular reference to
886 OG 638).
[0412] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strains are not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws. The making of these deposits is by
no means an admission that deposits are required to enable the
invention
Sequence CWU 1
1
31349PRTArtificial sequenceSynthetic 1Met Glu Asn Pro Ser Pro Ala
Ala Ala Leu Gly Lys Ala Leu Cys Ala1 5 10 15Leu Leu Leu Ala Thr Leu
Gly Ala Ala Gly Gln Pro Leu Gly Gly Glu 20 25 30Ser Ile Cys Ser Ala
Arg Ala Pro Ala Lys Tyr Ser Ile Thr Phe Thr 35 40 45Gly Lys Trp Ser
Gln Thr Ala Phe Pro Lys Gln Tyr Pro Leu Phe Arg 50 55 60Pro Pro Ala
Gln Trp Ser Ser Leu Leu Gly Ala Ala His Ser Ser Asp65 70 75 80Tyr
Ser Met Trp Arg Lys Asn Gln Tyr Val Ser Asn Gly Leu Arg Asp 85 90
95Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu Met Lys Glu Ile Glu Ala
100 105 110Ala Gly Glu Ala Leu Gln Ser Val His Glu Val Phe Ser Ala
Pro Ala 115 120 125Val Pro Ser Gly Thr Gly Gln Thr Ser Ala Glu Leu
Glu Val Gln Arg 130 135 140Arg His Ser Leu Val Ser Phe Val Val Arg
Ile Val Pro Ser Pro Asp145 150 155 160Trp Phe Val Gly Val Asp Ser
Leu Asp Leu Cys Asp Gly Asp Arg Trp 165 170 175Arg Glu Gln Ala Ala
Leu Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp 180 185 190Ser Gly Phe
Thr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp 195 200 205Thr
Val Thr Glu Ile Thr Ser Ser Ser Pro Ser His Pro Ala Asn Ser 210 215
220Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile Ala Arg Val
Thr225 230 235 240Leu Val Arg Leu Arg Gln Ser Pro Arg Ala Phe Ile
Pro Pro Ala Pro 245 250 255Val Leu Pro Ser Arg Asp Asn Glu Ile Val
Asp Ser Ala Ser Val Pro 260 265 270Glu Thr Pro Leu Asp Cys Glu Val
Ser Leu Trp Ser Ser Trp Gly Leu 275 280 285Cys Gly Gly His Cys Gly
Arg Leu Gly Thr Lys Ser Arg Thr Arg Tyr 290 295 300Val Arg Val Gln
Pro Ala Asn Asn Gly Ser Pro Cys Pro Glu Leu Glu305 310 315 320Glu
Glu Ala Glu Cys Val Pro Asp Asn Cys Val Asp Pro Ala Phe Leu 325 330
335Tyr Lys Val Val Arg Trp Ala His His His His His His 340
3452371PRTArtificial sequenceSynthetic 2Met Glu Asn Pro Ser Pro Ala
Ala Ala Leu Gly Lys Ala Leu Cys Ala1 5 10 15Leu Leu Leu Ala Thr Leu
Gly Ala Ala Gly Gln Pro Leu Gly Gly Glu 20 25 30Ser Ile Cys Ser Ala
Arg Ala Pro Ala Lys Tyr Ser Ile Thr Phe Thr 35 40 45Gly Lys Trp Ser
Gln Thr Ala Phe Pro Lys Gln Tyr Pro Leu Phe Arg 50 55 60Pro Pro Ala
Gln Trp Ser Ser Leu Leu Gly Ala Ala His Ser Ser Asp65 70 75 80Tyr
Ser Met Trp Arg Lys Asn Gln Tyr Val Ser Asn Gly Leu Arg Asp 85 90
95Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu Met Lys Glu Ile Glu Ala
100 105 110Ala Gly Glu Ala Leu Gln Ser Val His Glu Val Phe Ser Ala
Pro Ala 115 120 125Val Pro Ser Gly Thr Gly Gln Thr Ser Ala Glu Leu
Glu Val Gln Arg 130 135 140Arg His Ser Leu Val Ser Phe Val Val Arg
Ile Val Pro Ser Pro Asp145 150 155 160Trp Phe Val Gly Val Asp Ser
Leu Asp Leu Cys Asp Gly Asp Arg Trp 165 170 175Arg Glu Gln Ala Ala
Leu Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp 180 185 190Ser Gly Phe
Thr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp 195 200 205Thr
Val Thr Glu Ile Thr Ser Ser Ser Pro Ser His Pro Ala Asn Ser 210 215
220Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile Ala Arg Val
Thr225 230 235 240Leu Leu Arg Leu Arg Gln Ser Pro Arg Ala Phe Ile
Pro Pro Ala Pro 245 250 255Val Leu Pro Ser Arg Asp Asn Glu Ile Val
Asp Ser Ala Ser Val Pro 260 265 270Glu Thr Pro Leu Asp Cys Glu Val
Ser Leu Trp Ser Ser Trp Gly Leu 275 280 285Cys Gly Gly His Cys Gly
Arg Leu Gly Thr Lys Ser Arg Thr Arg Tyr 290 295 300Val Arg Val Gln
Pro Ala Asn Asn Gly Ser Pro Cys Pro Glu Leu Glu305 310 315 320Glu
Glu Ala Glu Cys Val Pro Asp Asn Cys Val Asp Pro Ala Phe Leu 325 330
335Tyr Lys Val Val Asp Leu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile
340 345 350Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His
His His 355 360 365His His His 3703690PRTArtificial
sequenceSynthetic 3Met Met Asp Ala Ser Lys Glu Leu Gln Val Leu His
Ile Asp Phe Leu1 5 10 15Asn Gln Asp Asn Ala Val Ser His His Thr Trp
Glu Phe Gln Thr Ser 20 25 30Ser Pro Val Phe Arg Arg Gly Gln Val Phe
His Leu Arg Leu Val Leu 35 40 45Asn Gln Pro Leu Gln Ser Tyr His Gln
Leu Lys Leu Glu Phe Ser Thr 50 55 60Gly Pro Asn Pro Ser Ile Ala Lys
His Thr Leu Val Val Leu Asp Pro65 70 75 80Arg Thr Pro Ser Asp His
Tyr Asn Trp Gln Ala Thr Leu Gln Asn Glu 85 90 95Ser Gly Lys Glu Val
Thr Val Ala Val Thr Ser Ser Pro Asn Ala Ile 100 105 110Leu Gly Lys
Tyr Gln Leu Asn Val Lys Thr Gly Asn His Ile Leu Lys 115 120 125Ser
Glu Glu Asn Ile Leu Tyr Leu Leu Phe Asn Pro Trp Cys Lys Glu 130 135
140Asp Met Val Phe Met Pro Asp Glu Asp Glu Arg Lys Glu Tyr Ile
Leu145 150 155 160Asn Asp Thr Gly Cys His Tyr Val Gly Ala Ala Arg
Ser Ile Lys Cys 165 170 175Lys Pro Trp Asn Phe Gly Gln Phe Glu Lys
Asn Val Leu Asp Cys Cys 180 185 190Ile Ser Leu Leu Thr Glu Ser Ser
Leu Lys Pro Thr Asp Arg Arg Asp 195 200 205Pro Val Leu Val Cys Arg
Ala Met Cys Ala Met Met Ser Phe Glu Lys 210 215 220Gly Gln Gly Val
Leu Ile Gly Asn Trp Thr Gly Asp Tyr Glu Gly Gly225 230 235 240Thr
Ala Pro Tyr Lys Trp Thr Gly Ser Ala Pro Ile Leu Gln Gln Tyr 245 250
255Tyr Asn Thr Lys Gln Ala Val Cys Phe Gly Gln Cys Trp Val Phe Ala
260 265 270Gly Ile Leu Thr Thr Val Leu Arg Ala Leu Gly Ile Pro Ala
Arg Ser 275 280 285Val Thr Gly Phe Asp Ser Ala His Asp Thr Glu Arg
Asn Leu Thr Val 290 295 300Asp Thr Tyr Val Asn Glu Asn Gly Glu Lys
Ile Thr Ser Met Thr His305 310 315 320Asp Ser Val Trp Asn Phe His
Val Trp Thr Asp Ala Trp Met Lys Arg 325 330 335Pro Asp Leu Pro Lys
Gly Tyr Asp Gly Trp Gln Ala Val Asp Ala Thr 340 345 350Pro Gln Glu
Arg Ser Gln Gly Val Phe Cys Cys Gly Pro Ser Pro Leu 355 360 365Thr
Ala Ile Arg Lys Gly Asp Ile Phe Ile Val Tyr Asp Thr Arg Phe 370 375
380Val Phe Ser Glu Val Asn Gly Asp Arg Leu Ile Trp Leu Val Lys
Met385 390 395 400Val Asn Gly Gln Glu Glu Leu His Val Ile Ser Met
Glu Thr Thr Ser 405 410 415Ile Gly Lys Asn Ile Ser Thr Lys Ala Val
Gly Gln Asp Arg Arg Arg 420 425 430Asp Ile Thr Tyr Glu Tyr Lys Tyr
Pro Glu Gly Ser Ser Glu Glu Arg 435 440 445Gln Val Met Asp His Ala
Phe Leu Leu Leu Ser Ser Glu Arg Glu His 450 455 460Arg Arg Pro Val
Lys Glu Asn Phe Leu His Met Ser Val Gln Ser Asp465 470 475 480Asp
Val Leu Leu Gly Asn Ser Val Asn Phe Thr Val Ile Leu Lys Arg 485 490
495Lys Thr Ala Ala Leu Gln Asn Val Asn Ile Leu Gly Ser Phe Glu Leu
500 505 510Gln Leu Tyr Thr Gly Lys Lys Met Ala Lys Leu Cys Asp Leu
Asn Lys 515 520 525Thr Ser Gln Ile Gln Gly Gln Val Ser Glu Val Thr
Leu Thr Leu Asp 530 535 540Ser Lys Thr Tyr Ile Asn Ser Leu Ala Ile
Leu Asp Asp Glu Pro Val545 550 555 560Ile Arg Gly Phe Ile Ile Ala
Glu Ile Val Glu Ser Lys Glu Ile Met 565 570 575Ala Ser Glu Val Phe
Thr Ser Phe Gln Tyr Pro Glu Phe Ser Ile Glu 580 585 590Leu Pro Asn
Thr Gly Arg Ile Gly Gln Leu Leu Val Cys Asn Cys Ile 595 600 605Phe
Lys Asn Thr Leu Ala Ile Pro Leu Thr Asp Val Lys Phe Ser Leu 610 615
620Glu Ser Leu Gly Ile Ser Ser Leu Gln Thr Ser Asp His Gly Thr
Val625 630 635 640Gln Pro Gly Glu Thr Ile Gln Ser Gln Ile Lys Cys
Thr Pro Ile Lys 645 650 655Thr Gly Pro Lys Lys Phe Ile Val Lys Leu
Ser Ser Lys Gln Val Lys 660 665 670Glu Ile Asn Ala Gln Lys Ile Val
Leu Ile Thr Lys His His His His 675 680 685His His 690
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