U.S. patent application number 12/480033 was filed with the patent office on 2010-01-07 for cln101 antibody compositions and methods of use alone and in combination with prostate specific antigen and other cancer markers.
Invention is credited to Xiaozhu Duan, Nam Kim, Robert L. Wolfert.
Application Number | 20100003705 12/480033 |
Document ID | / |
Family ID | 34278340 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100003705 |
Kind Code |
A1 |
Duan; Xiaozhu ; et
al. |
January 7, 2010 |
Cln101 Antibody Compositions and Methods of Use Alone and in
Combination with Prostate Specific Antigen and Other Cancer
Markers
Abstract
This invention relates to a method for assessing risk of
prostate and/or ovarian cancer. Specifically, in one embodiment it
relates to utilizing both Cln101 and Prostate Specific Antigen
(PSA) in combination to determine the risk of prostate cancer. In
an alternative embodiment, the invention is relates to utilizing
Cln101 alone or in combination with CA125 to determine the risk of
ovarian cancer. The invention further provides isolated
anti-prostate or ovarian cancer antigen (Cln101) antibodies that
bind to Cln101 in vivo. The invention also encompasses compositions
comprising an anti-Cln101 antibody and a carrier. These
compositions can be provided in an article of manufacture or a kit.
Another aspect of the invention is an isolated nucleic acid
encoding an anti-Cln101 antibody, as well as an expression vector
comprising the isolated nucleic acid. Also provided are cells that
produce the anti-Cln101 antibodies. The invention encompasses a
method of producing the anti-Cln101 antibodies. Other aspects of
the invention are a method of killing an Cln101-expressing cancer
cell, comprising contacting the cancer cell with an anti-Cln101
antibody and a method of alleviating or treating an
Cln101-expressing cancer in a mammal, comprising administering a
therapeutically effective amount of the anti-Cln101 antibody to the
mammal.
Inventors: |
Duan; Xiaozhu; (Alameda,
CA) ; Kim; Nam; (Santa Clara, CA) ; Wolfert;
Robert L.; (Palo Alto, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
34278340 |
Appl. No.: |
12/480033 |
Filed: |
June 8, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10558543 |
Oct 23, 2006 |
7560531 |
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PCT/US2004/016969 |
Jun 1, 2004 |
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12480033 |
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60556239 |
Mar 25, 2004 |
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60474110 |
May 29, 2003 |
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/57449 20130101;
G01N 33/57434 20130101; C07K 16/3069 20130101 |
Class at
Publication: |
435/7.23 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Claims
1. A method for assessing risk of prostate cancer in a patient
which comprises measuring levels of both Cln101 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
Cln101, and using the combined risks to assess the risk of prostate
cancer in the patient.
2-7. (canceled)
8. A method for assessing risk of ovarian cancer in a patient which
comprises measuring levels of Cln101 in the patient to assess the
risk of ovarian cancer in the patient.
9. A method for assessing risk of ovarian cancer in a patient which
comprises measuring levels of both Cln101 and CA125 in the patient,
analyzing a risk associated with the level of CA125 and a risk
associated with the level of Cln101, and using the combined risks
to assess the risk of ovarian cancer in the patient.
10-83. (canceled)
84. A method for detecting Cln101 overexpression in a subject in
need thereof comprising, (a) combining a bodily fluid sample of a
subject with an isolated Cln101 antibody that binds to mammalian
Cln101 in vivo or in vitro under conditions suitable for specific
binding of the Cln101 antibody to Cln101 in said bodily fluid
sample (b) determining the level of Cln101 in the bodily fluid
sample, (c) comparing the level of Cln101 determined in step b to
the level of Cln101 in a control, wherein an increase in the level
of Cln101 in the bodily fluid sample from the subject as compared
to the control is indicative of Cln101 overexpression in the
subject.
85-94. (canceled)
Description
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/558,543, filed Oct. 23, 2006, which is the
National Stage of International Application No. PCT/US2004/016969
filed Jun. 1, 2004, which claims benefit of priority to U.S.
Provisional Patent Application Ser. No. 60/556,239, filed Mar. 25,
2004 and U.S. Provisional Patent Application Ser. No. 60/474,110,
filed May 29, 2003, teachings of each of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-Cln101 antibody
compositions and methods for assessing risk of prostate and/or
ovarian cancer. Specifically, it relates to utilizing both Cln101
(also known as Regenerating Protein IV or Reg IV) and Prostate
Specific Antigen (PSA) in combination to detect prostate cancer. In
addition, this invention is related to the use of Cln101 alone or
in combination to detect ovarian cancer.
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 with the extension 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; Burdefte,
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 .alpha.-reductase Type 2 gene, the gene
which codes for the enzyme that converts testosterone into
dihydrotestosterone. Id. at 30. Dihydrotestosterone has greater
affinity for the AR than testosterone, resulting in increased
transactivation of genes responsive to androgens. Id. While studies
have reported differences among the races in the length of a TA
dinucleotide repeat in the 3' untranslated region, no link has been
established between the length of that repeat and prostate cancer.
Id. Interestingly, while ras gene mutations are implicated in
numerous other cancers, such mutations appear not to play a
significant role in prostate cancer, at least among Caucasian
males. Augustus, supra at 52.
[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).
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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).
Current Therapeutics
[0014] Radical Prostatectomy
[0015] This operation removes the entire prostate gland plus some
tissue around it and is used most often if the cancer is thought
not to have spread outside of the gland. There are two main types
of radical prostatectomy: radical retropubic prostatectomy and
radical perineal prostatectomy. In the retropubic operation, the
surgeon makes a skin incision in the lower abdomen. The surgeon can
remove lymph nodes during this operation through the same incision.
A nerve-sparing radical retropubic prostatectomy is a modification
of this operation.
[0016] The radical peritoneal prostatectomy removes the prostate
through an incision in the skin between the scrotum and anus.
Nerve-sparing operations are more difficult by this approach and
lymph nodes cannot be removed through this incision. If lymph node
examination is needed for men having a radical peritoneal
prostatectomy, the surgeon can remove some lymph nodes through a
very small skin incision in the abdomen or by using a laparoscope.
A laparoscope is a long slender tube through which a surgeon can
view and remove lymph nodes near the prostate gland.
[0017] Radiation Therapy
[0018] Radiation is sometimes used to treat prostate cancer that is
still confined to the prostate gland, or has spread to nearby
tissue. If the disease is more advanced, radiation may be used to
reduce the size of the tumor. The two main types of radiation
therapy are external beam radiation and brachytherapy (internal
radiation) and internal radiation therapy (brachytherapy). Internal
radiation therapy uses small radioactive pellets (each about the
size of a grain of rice) that are directly implanted (permanently
or temporarily) into the prostate.
[0019] Hormone Therapy
[0020] This treatment is often used for patients whose prostate
cancer has spread beyond the prostate or has recurred after
treatment. The goal of hormone therapy is to lower levels of the
male hormones, androgens. The main androgen is called testosterone.
Androgens are produced mainly in the testicles and cause prostate
cancer cells to grow. Lowering androgen levels can make prostate
cancers shrink or grow more slowly. But, hormone therapy does not
cure the cancer. There are several methods used for hormone
therapy.
[0021] Some prostate cancers do not respond to hormone therapy, and
are called androgen independent cancers. Some prostate cancers
respond to hormonal therapy for a few years before becoming
androgen independent. Less often, prostate cancers may be androgen
independent at the time they are diagnosed.
[0022] Orchiectomy: This operation removes the testicles. Although
it is a surgical treatment, orchiectomy is considered hormonal
therapy because it works by removing the main source of male
hormones.
[0023] Luteinizing hormone-releasing hormone (LHRH) analogs: These
drugs can decrease the amount of testosterone produced by a man's
testicles, as effectively as surgical removal of the testicles.
LHRH analogs (also called LHRH agonists) are injected either
monthly or every three months. The two LHRH analogs currently
available in the United States are leuprolide (Lupron.RTM.) and
goserelin (Zoladex.RTM.).
[0024] Anti-androgens: Even after orchiectomy or during treatment
with LHRH analogs, a small amount of androgen is still produced by
the adrenal glands. Anti-androgens block the body's ability to use
androgens. Drugs of this type, such as flutamide (Eulexin.RTM.),
bicalutamide (Casodex.RTM.), and nilutamide (Nilandron.RTM.), are
taken as pills, once or three times a day. Anti-androgens are often
used in combination with orchiectomy or LHRH analogs. This
combination is called total androgen blockade.
[0025] Other hormonal drugs: Megestrol acetate (Megace.RTM.) and
medroxyprogesterone (Depo-Provera.RTM.) are sometimes used if
"first-line" hormonal treatments lose effectiveness. Ketoconazole
(Nizoral.RTM.), initially used for treating fungal infections and
later found to also work as an anti-androgen, is another drug for
"second line" hormonal therapy.
[0026] Chemotherapy
[0027] Chemotherapy is an option for patients whose prostate cancer
has spread outside of the prostate gland and for whom hormone
therapy has failed. It is not expected to destroy all of the cancer
cells, but it may slow tumor growth and reduce pain.
[0028] Some of the chemotherapy drugs used in treating prostate
cancer that has returned or continued to grow and spread after
treatment with hormonal therapy include doxorubicin (Adriamycin),
estramustine, etoposide, mitoxantrone, vinblastine, and paclitaxel.
Two or more drugs are often given together to reduce the likelihood
of the cancer cells becoming resistant to chemotherapy. Small cell
carcinoma is a rare type of prostate cancer that is more likely to
respond to chemotherapy than to hormonal therapy. Small cell
carcinoma develops more often in the lungs than in the prostate.
Since small cell lung cancer often responds to chemotherapy with
cisplatin and etoposide, these drugs are recommended for treating
small cell cancers that develop in the prostate.
[0029] 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
[0030] 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 with the
extension 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. J.
Gynecol. Pathol. 20(1): 48-63 (2001). Although our understanding of
the etiology of ovarian cancer is incomplete, the results of
extensive research in this area point to a combination of age,
genetics, reproductive, and dietary/environmental factors. Age is a
key risk factor in the development of ovarian cancer: while the
risk for developing ovarian cancer before the age of 30 is slim,
the incidence of ovarian cancer rises linearly between ages 30 to
50, increasing at a slower rate thereafter, with the highest
incidence being among septagenarian women. Jeanne M. Schilder et
al., Hereditary Ovarian Cancer. Clinical Syndromes and Management,
in Ovarian Cancer 182 (Stephen C. Rubin & Gregory P. Sutton
eds., 2d ed. 2001).
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] Other markers of interest are HE4 and mesothelin, see Urban
et al. Ovarian cancer screening Hematol Oncol Clin North Am. August
2003;17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is a
biomarker for ovarian carcinoma, Cancer Res. Jul. 1,
2003;63(13):3695-700; Ordonez, Application of mesothelin
immunostaining in tumor diagnosis, Am J Surg Pathol. November
2003;27(11):1418-28.
[0037] 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.
[0038] Stage II ovarian cancer refers to tumor growth involving one
or both ovaries, along with pelvic extension. Id. Substage IIA
involves extension and/or implants on the uterus and/or fallopian
tubes, with no malignant cells in the ascites or peritoneal
washings, while substage IIB involves extension into other pelvic
organs and tissues, again with no malignant cells in the ascites or
peritoneal washings. Id. Substage IIC involves pelvic extension as
in IIA or IIB, but with malignant cells in the ascites or
peritoneal washings. Id.
[0039] 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.
[0040] 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.
[0041] The treatment of ovarian cancer typically involves a
multiprong attack, with surgical intervention serving as the
foundation of treatment. Dennis S. Chi & William J. Hoskins,
Primary Surgical Management of Advanced Epithelial Ovarian Cancer,
in Ovarian Cancer 241 (Stephen C. Rubin & Gregory P. Sutton
eds., 2d ed. 2001). For example, in the case of epithelial ovarian
cancer, which accounts for .about.90% of cases of ovarian cancer,
treatment typically consists of: (1) cytoreductive surgery,
including total abdominal hysterectomy, bilateral
salpingo-oophorectomy, omentectomy, and lymphadenectomy, followed
by (2) adjuvant chemotherapy with paclitaxel and either cisplatin
or carboplatin. Eltabbakh, G. H. & Awtrey, C. S., Expert Op.
Pharmacother. 2(10): 109-24. Despite a clinical response rate of
80% to the adjuvant therapy, most patients experience tumor
recurrence within three years of treatment. Id. Certain patients
may undergo a second cytoreductive surgery and/or second-line
chemotherapy. Memarzadeh & Berek, supra.
[0042] 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.
[0043] 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.
Angiogenesis in Cancer
[0044] 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.
[0045] 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).
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] The present invention provides alternative methods of
detecting prostate and ovarian cancer that overcome the limitations
of conventional diagnostic methods as well as offer additional
advantages that will be apparent from the detailed description
below. Furthermore, the present invention provides alternative
methods of treating prostate and ovarian 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
[0051] This invention is directed to a method for assessing risk of
prostate cancer in a patient which comprises measuring levels of
both Cln101 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 Cln101, 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 Cln101 levels are done
simultaneously. In another aspect of the invention the measuring of
PSA and Cln101 are done sequentially.
[0052] In yet another aspect of the invention, the respective
levels of PSA and Cln101 are based on dividing a patient population
dataset into borderline levels of PSA and elevated levels of Cln101
and a patient having both borderline PSA and high Cln101 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.
[0053] In addition, the invention is directed to a method of
detecting ovarian cancer.
[0054] 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 Cln101 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.
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 Cln101 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.
[0055] Lastly the invention is directed to a kit for diagnosing a
patient's susceptibility to prostate cancer comprising both a
suitable assay for measuring Cln101 levels and a suitable assay for
measuring Prostate Specific Antigen (PSA) levels wherein the levels
of both PSA and Cln101 are determined.
[0056] This invention is directed to an isolated Cln101 antibody
that binds to Cln101 on a mammalian cell in vivo. The invention is
further directed to an isolated Cln101 antibody that internalizes
upon binding to Cln101 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-5877 and PTA-5876.
[0057] 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-5877 and
PTA-5876.
[0058] 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, maytansin, maytansinoids, saporin, gelonin, ricin
or calicheamicin.
[0059] The mammalian cell may be a cancer cell. Preferably, the
anti-Cln101 monoclonal antibody that inhibits the growth of
Cln101-expressing cancer cells in vivo.
[0060] The antibody may be produced in bacteria. Alternatively, the
antibody may be a humanized form of an anti-Cln101 antibody
produced by a hybridoma selected from the group of hybridomas
having ATCC accession number PTA-5877 and PTA-5876.
[0061] Preferably, the cancer is selected from the group consisting
of prostate and ovarian 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.
[0062] 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.
[0063] The invention is also directed to a method of killing a
Cln101-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 and ovarian cancer cell.
[0064] The prostate or ovarian cancer may be ovarian serous
adenocarcinoma or metastatic cancer.
[0065] The invention is also directed to a method of alleviating a
Cln101-expressing cancer in a mammal, comprising administering a
therapeutically effective amount of the antibodies to the
mammal.
[0066] 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 or ovarian cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0067] FIG. 1 shows the Anti-Cln101 MAb Epitope Map.
[0068] FIG. 2 shows Detection of Cln101 in Normal and Cancer Serum
Samples (primary panel).
[0069] FIG. 3 shows Detection of Cln101 in Normal and Cancer Serum
Samples (primary panel), Expanded Scale.
[0070] FIG. 4 shows Detection of Cln101 in Normal and Cancer Serum
Samples (multiple panels).
[0071] FIG. 5 shows Detection of Cln101 in Normal and Cancer Serum
Samples (multiple panels), Expanded Scale.
[0072] FIG. 6 shows Detection of Cln101 in Normal and Cancer Serum
Samples (master panel).
[0073] FIG. 7 shows Detection of Cln101 in Normal and Cancer Serum
Samples (master panel), Expanded Scale.
[0074] FIG. 8 shows Detection of Cln101 in Normal, Prostate Cancer
and Prostate Benign Serum Samples.
[0075] FIG. 9 shows Detection of PSA in Normal, Prostate Cancer and
Prostate Benign Serum Samples.
[0076] FIG. 10 shows Detection of Cln101 in Normal, Ovarian Cancer
and Ovarian Benign Serum Samples
[0077] FIG. 11 shows ROC Curves for Cln101 and PSA in Prostate
Cancer vs. Normal Serum Samples.
[0078] FIG. 12 shows ROC Curves for Cln101 and PSA in Prostate
Cancer and Benign Disease vs. Normal Serum Samples.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
[0079] Human "Cln101" as used herein, refers to a protein of 158
amino acids that is secreted from cells. The nucleotide and amino
acid sequences of Cln101 have been disclosed, e.g., WO200020640-A1,
DIADEXUS, Human cancer specific protein, CC-2; WO9639541-A1 HUMAN
GENOME SCIENCES, Human colon specific protein; and J. C. Hartupee
et al. Isolation and characterization of a cDNA encoding a novel
member of the human regenerating protein family: Reg IV. Biochin
Biophys Acta. Apr. 16, 2001; 1518(3):287-93. Most of the amino
acids of Cln101 are understood to be secreted from cells. Cln101
has also been disclosed in the REFSEQ database as:
NM.sub.--032044.2 (GI: 36054181) Homo sapiens regenerating
islet-derived family, member 4 (REG4, REG IV). Cln101 (REG IV)
protein contains N-terminal signal peptide and the following
domains: C-type lectin domain (InterPro Accession: IPR001304) and
Pancreatitis-associated domain (InterPro Accession: IPR003990). The
InterPro database is accessible at the European Bioinformatics
Institute (EBI) website, ebi with the extension .ac.uk of the world
wide web, the contents of which are incorporated by reference.
[0080] Cln101 as used herein include allelic variants and
conservative substitution mutants of the protein which have Cln101
biological activity.
[0081] Recently, a series of independent publications have linked
Cln101 to colorectal and gastric cancer. See Violette, S. et al.
Reg IV, a new member of the regenerating gene family, is
overexpressed in colorectal carcinomas. Int. J. Cancer 103:185-193
(2003); Kamarainen, M. et al. RELP, a novel human REG-like protein
with up-regulated expression in inflammatory and metaplastic
gastrointestinal mucosa. Am. J. Patlol. 163:11-20 (2003); Yonemura,
Y. et al. REG gene expression is associated with the infiltrating
growth of gastric carcinoma. Cancer 98:1394-1400 (2003); Zhang, Y.
et al. Overexpression of Reg IV in colorectal adenoma. Cancer Lett.
200:69-76 (2003).
[0082] Our findings that Cln101 is apparently associated to
prostate and ovarian cancers make this antigen an attractive target
for detection and immunotherapy of these and possibly other tumor
types.
[0083] 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.
[0084] 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.
[0085] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (VH) followed by three constant domains (CH) for each of the
.alpha., .delta. and .gamma. chains and four CH domains for .mu.
and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (VL) followed by a constant domain (CL) at its
other end.
[0086] 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.
[0087] 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.
[0088] 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).
[0089] 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)).
[0090] 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.
[0091] 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.
[0092] An "intact" antibody is one which comprises an
antigen-binding site as well as a CL and at least heavy chain
constant domains, CHI, 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.
[0093] 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.
[0094] 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.
[0095] "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.
[0096] "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.
[0097] 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).
[0098] 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.
[0099] 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 Cln101 will possess at least about 70%
homology with the native sequence Cln101, 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.
[0100] 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.
[0101] "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).
[0102] "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).
[0103] As used herein, an anti-Cln101 antibody that "internalizes"
is one that is taken up by (i.e., enters) the cell upon binding to
Cln101 on a mammalian cell (i.e. cell surface Cln101). 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 a Cln101-expressing cell, especially a Cln101-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.
[0104] Whether an anti-Cln101 antibody internalizes upon binding
Cln101 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 Cln101
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 Cln101-expressing tumor
transplant or xenograft, or a mouse into which cells transfected
with human Cln101 have been introduced, or a transgenic mouse
expressing the human Cn101 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
Cln101-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.
[0105] The faster the rate of internalization of the antibody upon
binding to the Cln101-expressing cell in vivo, the faster the
desired killing or growth inhibitory effect on the target
Cln101-expressing cell can be achieved, e.g., by a cytotoxic
immunoconjugate. Preferably, the kinetics of internalization of the
anti-Cln101 antibodies are such that they favor rapid killing of
the Cln101-expressing target cell. Therefore, it is desirable that
the anti-Cln101 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-Cln101 antibody in vivo. The antibody will
preferably be internalized into the cell within a few hours upon
binding to Cln101 on the cell surface, preferably within 1 hour,
even more preferably within 15-30 minutes.
[0106] To determine if a test antibody can compete for binding to
the same epitope as the epitope bound by the anti-Cln101 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, Cln101-coated wells of a microtiter plate,
or Cln101-coated sepharose beads, are pre-incubated with or without
candidate competing antibody and then a biotin-labeled anti-Cln101
antibody of the invention is added. The amount of labeled
anti-Cln101 antibody bound to the Cln101 antigen in the wells or on
the beads is measured using avidin-peroxidase conjugate and
appropriate substrate.
[0107] Alternatively, the anti-Cln101 antibody can be labeled,
e.g., with a radioactive or fluorescent label or some other
detectable and measurable label. The amount of labeled anti-Cln101
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-Cln101 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-Cln101 antibody
of the invention if the candidate competing antibody can block
binding of the anti-Cln101 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.
[0108] An antibody having a "biological characteristic" of a
designated antibody, such as any ofthe monoclonal antibodies
Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1,
Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16,
Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,
Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1,
Cln101.A41.1, Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3,
Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25,
Cln101.C36, Cln101.C43 and Cln101.C47, 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,
Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1,
Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16,
Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,
Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1,
Cln101.A41.1, Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3,
Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25,
Cln101.C36, Cln101.C43 and Cln101.C47 will bind the same epitope as
that bound by Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1,
Cln101.A8.1, Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1,
Cln101.A16, Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,
Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1,
Cln101.A41.1, Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln10.C3,
Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25,
Cln101.C36, Cln101.C43 and Cln101.C47 (e.g. which competes for
binding or blocks binding of monoclonal antibody Cln101.A1.1,
Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1, Cln101.A9.1,
Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16, Cln101.A17.1,
Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1,
Cln101.A35.1, Cln101.A37.1, Cln101.A38.1, Cln101.A41.1,
Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3, Cln101.C6,
Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25, Cln101.C36,
Cln101.C43 and Cln101.C47 to Cln101), be able to target an
Cln101-expressing tumor cell in vivo and will bind to Cln101 on a
mammalian cell in vivo. It is noted that the nomenclature format
for anti-Cln101 antibodies may also be represented without the
first punctuation point ".". For example Cln101.A9.1 and
Cln101.A46.1 may be represented as Cln101 A9.1 and Cln101 A46.1,
respectively.
[0109] Furthermore, an antibody with the biological characteristic
of the Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1,
Cln101.A8.1, Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1,
Cln101.A16, Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,
Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1,
Cln101.A41.1, Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3,
Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25,
Cln101.C36, Cln101.C43 and Cln101.C47 antibody will internalize
upon binding to Cln101 on a mammalian cell in vivo.
[0110] Likewise, an antibody with the biological characteristic of
the Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1,
Cln101.A8.1, Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1,
Cln101.A16, Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,
Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1,
Cln101.A41.1, Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3,
Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25,
Cln101.C36, Cln101.C43 and Cln101.C47 antibody will have the same
epitope binding, targeting, internalizing, tumor growth inhibitory
and cytotoxic properties of the antibody.
[0111] 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 Cln101
protein disclosed herein. Methods for identifying antagonists of an
Cln101 polypeptide may comprise contacting an Cln101 polypeptide or
a cell expressing Cln101 on the cell surface, with a candidate
antagonist antibody and measuring a detectable change in one or
more biological activities normally associated with the Cln101
polypeptide.
[0112] An "antibody that inhibits the growth of tumor cells
expressing Cln101" or a "growth inhibitory" antibody is one which
binds to and results in measurable growth inhibition of cancer
cells expressing or overexpressing Cln101. Alternatively, an
antibody that inhibits the growth of tumor cells expressing Cln101
or a growth inhibitory antibody is one which binds to and inhibits
the function of Cln101 which results in measurable growth
inhibition of cancer cells expressing or overexpressing Cln101.
Preferred growth inhibitory anti-Cln101 antibodies inhibit growth
of Cln101-expressing tumor cells e.g., prostate or ovarian 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-Cln101 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.
[0113] 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 Cln101. Preferably the cell is a tumor cell, e.g. an
prostate or ovarian 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.
[0114] 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: C1q 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.
[0115] "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. Nos. 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).
[0116] "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.RI1B 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)).
[0117] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.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.
[0118] "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 (C1q) 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.
[0119] 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.
[0120] A "Cln101-expressing cell" is a cell which expresses
secreted endogenous or transfected Cln101. A "Cln101-expressing
cancer" is a cancer comprising cells that secrete Cln101 protein
from the cells. A "Cln101-expressing cancer" produces sufficient
levels of Cln101 such that an anti-Cln101 antibody can bind thereto
and have a therapeutic effect with respect to the cancer. A cancer
which "overexpresses" Cln101 is one which has significantly higher
levels of Cln101 compared to a noncancerous cell of the same tissue
type. Such overexpression may be caused by gene amplification or by
increased transcription or translation. Cln101 overexpression may
be determined in a diagnostic or prognostic assay by evaluating
increased levels of the Cln101 protein (e.g. via an
immunohistochemistry assay; FACS analysis). Alternatively, or
additionally, one may measure levels of Cln101-encoding nucleic
acid or mRNA in the cell, e.g. via fluorescent in situ
hybridization; (FISH; see W098/45479 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 Cln101 overexpression by measuring the 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;
W091/05264 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)). Biological or bodily fluids further include blood, serum,
salvia, urine, sputum, seminal fluids, and other bodily excretions
such as stool. Aside from the above assays, various in vivo assays
are available to the skilled practitioner. For example, one may
expose cells or tissues 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 cells 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 Cln101-expressing cancer
includes ovarian, pancreatic, lung or breast cancer.
[0121] 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.
[0122] "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
Cln101-expressing cancer if, after receiving a therapeutic amount
of an anti-Cln101 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-Cln101 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.
[0123] 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).
[0124] 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.
[0125] "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.
[0126] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0127] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0128] "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.
[0129] 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..
[0130] 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.
[0131] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
an Cln101-expressing cancer cell, either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of Cln101-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 ara-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.
(W B 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.
[0132] "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.
[0133] The term "epitope tagged" used herein refers to a chimeric
polypeptide comprising an anti-Cln101 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).
[0134] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0135] 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.
[0136] 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.
[0137] "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 ColEl 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.
[0138] The cell that produces an anti-Cln101 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.
[0139] 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.
[0140] 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).
[0141] 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).
[0142] 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'-O 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.
[0143] 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.
[0144] 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
[0145] The invention provides anti-Cln101 antibodies. Preferably,
the anti-Cln101 antibodies bind to Cln101 in vivo or in a mammalian
bodily fluid. Alternatively, the anti-Cln101 antibodies internalize
upon binding to cell surface Cln101 on a mammalian cell. The
anti-Cln101 antibodies may also destroy or lead to the destruction
of tumor cells bearing or secreting Cln101.
[0146] It was not apparent that Cln101 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 Cln101 is
internalization competent upon binding by the anti-Cln101
antibodies of the invention. Additionally, it was demonstrated that
the anti-Cln101 antibodies of the present invention can
specifically target Cln101-expressing tumor cells in vivo and
inhibit or kill these cells. These in vivo tumor targeting,
internalization and growth inhibitory properties of the anti-Cln101
antibodies make these antibodies very suitable for therapeutic
uses, e.g., in the treatment of various cancers including prostate
or ovarian cancer. Internalization of the anti-Cln101 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.
[0147] The anti-Cln101 antibodies of the invention also have
various non-therapeutic applications. The anti-Cln101 antibodies of
the present invention can be useful for diagnosis and staging of
Cln101-expressing cancers (e.g., in radioimaging). They may be used
alone or in combination with other ovarian cancer markers,
including, but not limited to, CA125, HE4 and mesothelin. The
anti-Cln101 antibodies of the present invention may also be used
alone or in combination with other prostate cancer markers,
including, but not limited to Prostate Specific Antigen (PSA). The
antibodies are also useful for purification or immunoprecipitation
of Cln101 from cells or bodily fluids, for detection and
quantitation of Cln101 in vitro, e.g. in an ELISA or a Western
blot, to kill and eliminate Cln101-expressing cells from a
population of mixed cells as a step in the purification of other
cells. The anti-Cln101 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.
[0148] 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-Cln101 antibodies of the invention are also contemplated,
e.g., an anti-Cln101 antibody which has the biological
characteristics of a monoclonal antibody produced by the hybridomas
accorded ATCC accession numbers PTA-5877 and PTA-5876, specifically
including the in vivo tumor targeting, internalization and any cell
proliferation inhibition or cytotoxic characteristics. Specifically
provided are anti-Cln101 antibodies that bind to an epitope present
in amino acids 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,
100-110, 110-120, 120-130, 130-140, 140-150, 150-158 of human
Cln101.
[0149] Methods of producing the above antibodies are described in
detail below.
[0150] The present anti-Cln101 antibodies are useful for treating a
Cln101-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal. Such a cancer includes prostate or ovarian
cancer, cancer of the urinary tract, breast cancer, lung cancer,
and colon cancer. Such a cancer includes 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. 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 or
ovarian cancer metastases. The antibody is able to bind to at least
a portion of the cancer cells that express Cln101 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
Cln101-expressing tumor cells or inhibit the growth or
proliferation of such tumor cells, in vitro or in vivo, upon
binding to Cln101 on the cell. Alternatively, the antibody is
effective to destroy or kill Cln101-expressing tumor cells or
inhibit the growth or proliferation of such tumor cells, in vitro
or in vivo, upon binding to secreted Cln101 in proximity to such
tumor cells. Such an antibody includes a naked anti-Cln101 antibody
(not conjugated to any agent). Such naked anti-Cln101 antibodies
may inhibit Cln101 function or activity upon binding. Naked
anti-Cln101 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-Cln101 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.
[0151] The invention provides a composition comprising an
anti-Cln101 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-Cln101 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-Cln101 antibody of the invention, and a carrier.
The formulation may be a therapeutic formulation comprising a
pharmaceutically acceptable carrier.
[0152] Another aspect of the invention is isolated nucleic acids
encoding the anti-Cln101 antibodies. 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.
[0153] The invention also provides methods useful for treating a
Cln101-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal, comprising administering a therapeutically
effective amount of an anti-Cln101 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
Cln101 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-Cln101 antibody of
this invention that binds to Cln101 in vivo or in a mammalian
bodily fluid or at least one internalizing anti-Cln101 antibody of
this invention. Kits containing anti-Cln101 antibodies find use in
detecting Cln101 expression, or in therapeutic or diagnostic
assays, e.g., for Cln101 cell killing assays or for purification
and/or immunoprecipitation of Cln101 from cells. For example, for
isolation and purification of Cln101, the kit can contain an
anti-Cln101 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
Cln101 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-Cln101 Antibodies
[0154] 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 Cln101
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 Cln101 lacking the signal peptide, a soluble form of
recombinant Cln101 with a signal peptide or synthetic peptides to
selected portions of the protein.
[0155] Alternatively, cells expressing Cln101 at their cell surface
(e.g. CHO or NIH-3T3 cells transformed to overexpress Cln101;
ovarian, pancreatic, lung, breast or other Cln101-expressing tumor
cell line), or membranes prepared from such cells can be used to
generate antibodies. The nucleotide and amino acid sequences of
human and murine Cln101 are available as provided above. Cln101 can
be produced recombinantly in and isolated from, prokaryotic cells,
e.g., bacterial cells, or eukaryotic cells using standard
recombinant DNA methodology. Cln101 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.
[0156] Antibodies or binding proteins that bind to various tags and
fusion sequences are available as elaborated below. Other forms of
Cln101 useful for generating antibodies will be apparent to those
skilled in the art.
[0157] Tags
[0158] 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 etal., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)).
[0159] Polyclonal Antibodies
[0160] 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), glutaraidehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1 N.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.
[0161] 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.
[0162] Monoclonal Antibodies
[0163] 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)).
[0164] 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.
[0165] 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)).
[0166] 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).
[0167] 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.
[0168] 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.
[0169] 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).
[0170] 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.
[0171] 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.
[0172] Humanized Antibodies
[0173] 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.
[0174] 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)).
[0175] 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.
[0176] 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.
[0177] Various forms of a humanized anti-Cln101 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.
[0178] Human Antibodies
[0179] 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); U.S. Pat. No. 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).
[0180] Antibody Fragments
[0181] 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)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.
[0182] Bispecific Antibodies
[0183] 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
Cln101 protein. Other such antibodies may combine an Cln101 binding
site with a binding site for another protein. Alternatively, an
anti-Cln101 .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
Cln101-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express Cln101. These
antibodies possess an Cln101-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.
[0184] 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).
[0185] 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.
[0186] 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).
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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).
[0193] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0194] Multivalent Antibodies
[0195] 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, XI 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.
[0196] Otlier Amino Acid Sequence Modifications
[0197] Amino acid sequence modification(s) of the anti-Cln101
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-Cln101 antibody are prepared by introducing appropriate
nucleotide changes into the anti-Cln101 antibody nucleic acid, or
by peptide synthesis.
[0198] Such modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the anti-Cln101 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-Cln101 antibody,
such as changing the number or position of glycosylation sites.
[0199] A useful method for identification of certain residues or
regions of the anti-Cln101 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-Cln101
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 Cln101 antigen.
[0200] 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-Cln101
antibody variants are screened for the desired activity.
[0201] 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-Cln101 antibody
with an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the
anti-Cln101 antibody molecule include the fusion to the N- or
C-terminus of the anti-Cln101 antibody to an enzyme (e.g. for
ADEPT) or a fusion to a polypeptide which increases the serum
half-life of the antibody.
[0202] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-Cln101 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-00001 TABLE I Amino Acid Substitutions Preferred Original
Exemplary Substitutions 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; ile ala;
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; set Phe Val (V)
ile; leu; met; phe; ala; leu
[0203] 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.
[0204] 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 confonnation of the
anti-Cln101 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).
[0205] 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 Cln101. 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.
[0206] 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).
[0207] Nucleic acid molecules encoding amino acid sequence variants
of the anti-Cln101 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-Cln101 antibody.
[0208] 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 cytotoxicity (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).
[0209] 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
[0210] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0211] The growth inhibitory effects of an anti-Cln101 antibody of
the invention may be assessed by methods known in the art, e.g.,
using cells which express Cln101 either endogenously or following
transfection with the Cln101 gene. For example, the tumor cell
lines and Cln101-transfected cells provided in Example 1 below may
be treated with an anti-Cln101 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-Cln101 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 Cln101. Preferably, the
anti-Cln101 antibody will inhibit cell proliferation of a
Cln101-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-Cln101 antibody at about 1
.mu.kg/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.
[0212] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., propidium iodide (PI),
trypan 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. Cln101-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.
[0213] To screen for antibodies which bind to an epitope on Cln101
bound by an antibody of interest, e.g., the Cln101 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-Cln101 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 Cln101 can
be used in competition assays with the test antibodies or with a
test antibody and an antibody with a characterized or known
epitope.
[0214] 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 Cln101-containing sample with a test antibody
and an antibody of this invention to form a mixture, the level of
Cln101 antibody bound to Cln101 in the mixture is then determined
and compared to the level of Cln101 antibody bound in the mixture
to a control mixture, wherein the level of Cln101 antibody binding
to Cln101 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-Cln101 antibody of this invention. The level of Cln101
antibody bound to Cln101 is determined by ELISA. The control may be
a positive or negative control or both. For example, the control
may be a mixture of Cln101, Cln101 antibody of this invention and
an antibody known to bind the epitope bound by the Cln101 antibody
of this invention. The anti-Cln101 antibody labeled with a label
such as those disclosed herein. The Cln101 may be bound to a solid
support, e.g., a tissue culture plate or to beads, e.g., sepharose
beads.
Immunoconjugates
[0215] 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.
[0216] 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.
[0217] Maytansine and Maytansinoids
[0218] Preferably, an anti-Cln101 antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0219] 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.
[0220] Maytansinoid-Antibody Conjugates
[0221] 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.
[0222] Anti-Cln101 Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0223] Anti-Cln101 antibody-maytansinoid conjugates are prepared by
chemically linking an anti-Cln101 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.
[0224] 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 B 1,
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-pyridyidithio)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 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.
[0225] 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.
[0226] Calicheamicin
[0227] Another immunoconjugate of interest comprises an anti-Cln101
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
[0228] Other antitumor agents that can be conjugated to the
anti-Cln101 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, 15 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).
[0229] 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-Cln101 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.
[0230] The radio- or other labels may be incorporated in the
conjugate in known ways.
[0231] 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.123, 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.
[0232] 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.
[0233] Alternatively, a fusion protein comprising the anti-Cln101
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.
[0234] 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 Mediated Prodrug Therapy (ADEPT)
[0235] 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 W081/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0236] 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-Cln101 antibodies by techniques well
known in the art such as the use of the heterobifunctional
crosslinking reagents discussed above.
[0237] 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
[0238] 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).
[0239] The anti-Cln101 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 W097/38731 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
[0240] The invention also provides isolated nucleic acid molecule
encoding the humanized anti-Cln101 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.
[0241] Signal Sequence Component
[0242] The anti-Cln101 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-Cln101 antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, lpp, 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-Cln101 antibody.
[0243] Origin of Replication
[0244] 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).
[0245] Selection Gene Component
[0246] 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.
[0247] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-Cln101 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).
[0248] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-Cln101 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.
[0249] 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.
[0250] 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).
[0251] Promoter Component
[0252] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-Cln101 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-Cln101 antibody.
[0253] 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.
[0254] 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.
[0255] Anti-Cln101 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.
[0256] 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
HindIll 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.
[0257] Enhancer Element Component
[0258] Transcription of a DNA encoding the anti-Cln101 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-Cln101 antibody-encoding sequence, but is preferably located
at a site 5' from the promoter.
[0259] Transcription Termination Component
[0260] 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-Cln101 antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO 94/11026 and the expression vector disclosed therein.
[0261] Selection and Transformation of Host Cells
[0262] 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.
[0263] 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.
[0264] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-Cln101 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.
[0265] Suitable host cells for the expression of glycosylated
anti-Cln101 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
Spodopterafrugiperda (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.
[0266] 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.
[0267] 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 (CVI 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).
[0268] Host cells are transformed with the above-described
expression or cloning vectors for anti-Cln101 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0269] Culturing Host Cells
[0270] The host cells used to produce the anti-Cln101 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. 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.
[0271] Purification of anti-Cln101 Antibody
[0272] 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.
[0273] 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 .gamma.3 (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.
[0274] 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
[0275] 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.
[0276] 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-Cln101 antibody which internalizes, it may be desirable to
include in the one formulation, an additional antibody, e.g. a
second anti-Cln101 antibody which binds a different epitope on
Cln101, 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.
[0277] 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).
[0278] 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.
[0279] 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-Cln101 Antibodies
[0280] According to the present invention, the anti-Cln101 antibody
that binds to Cln101 in vivo or internalizes upon binding Cln101 on
a cell surface is used to treat a subject in need thereof having a
cancer characterized by Cln101-expressing cancer cells, in
particular, ovarian, pancreatic, lung or breast cancer, such as
ovarian serous adenocarcinoma or breast infiltrating ductal
carcinoma cancer, and associated metastases.
[0281] The cancer will generally comprise Cln101-expressing cells,
such that the anti-Cln101 antibody is able to bind thereto. While
the cancer may be characterized by overexpression of the Cln101
molecule, the present application further provides a method for
treating cancer which is not considered to be a
Cln101-overexpressing cancer.
[0282] This invention also relates to methods for detecting cells
which overexpress Cln101 and to diagnostic kits useful in detecting
cells expressing Cln101 or in detecting Cln101 in serum from a
patient or other bodily fluids. 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
Cln101 overexpressing cells. A level of Cln101 binding higher than
that of such a control sample would be indicative of the test
sample containing cells that overexpress Cln101. Alternatively the
control may be a sample of cells known to contain cells that
overexpress Cln101. In such a case, a level of Cln101 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 Cln101.
[0283] Cln101 overexpression may be detected with a various
diagnostic assays. For example, over expression of Cln101 may be
assayed by immunohistochemistry (IHC). Parrafin embedded tissue
sections from a tumor biopsy may be subjected to the IHC assay and
accorded an Cln101 protein staining intensity criteria as
follows.
[0284] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0285] 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.
[0286] Score 2+ a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0287] Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0288] Those tumors with 0 or 1+ scores for Cln101 expression may
be characterized as not overexpressing Cln101, whereas those tumors
with 2+ or 3+ scores may be characterized as overexpressing
Cln101.
[0289] 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 Cln101 overexpression in
the tumor. Cln101 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 Cln101 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.
[0290] A sample suspected of containing cells expressing or
overexpressing Cln101 is combined with the antibodies of this
invention under conditions suitable for the specific binding of the
antibodies to Cln101. Binding and/or internalizing the Cln101
antibodies of this invention is indicative of the cells expressing
Cln101. The level of binding may be determined and compared to a
suitable control, wherein an elevated level of bound Cln101 as
compared to the control is indicative of Cln101 overexpression. The
sample suspected of containing cells overexpressing Cln101 may be a
cancer cell sample, particularly a sample of an ovarian cancer,
e.g. ovarian serous adenocarcinoma, or a breast cancer, e.g., a
breast infiltrating ductal carcinoma. A serum sample from a subject
may also be assayed for levels of Cln101 by combining a serum
sample from a subject with an Cln101 antibody of this invention,
determining the level of Cln101 bound to the antibody and comparing
the level to a control, wherein an elevated level of Cln101 in the
serum of the patient as compared to a control is indicative of
overexpression of Cln101 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.
[0291] Currently, depending on the stage of the cancer, ovarian,
pancreatic, lung or breast 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-Cln101 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 and internalizing
anti-Cln101 antibodies of the invention are useful to alleviate
Cln101-expressing cancers, e.g., ovarian, pancreatic, lung or
breast cancers upon initial diagnosis of the disease or during
relapse. For therapeutic applications, the anti-Cln101 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-Cln101 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-Cln101 antibody in conjunction with treatment
with the one or more of the preceding chemotherapeutic agents. In
particular, combination therapy with paclitaxel and modified
derivatives (see, e.g., EP0600517) is contemplated. The anti-Cln101
antibody will be administered with a therapeutically effective dose
of the chemotherapeutic agent. The anti-Cln101 antibody may also be
administered in conjunction with chemotherapy to enhance the
activity and efficacy of the chemotherapeutic agent, e.g.,
paclitaxel. The Physicians3 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.
[0292] Particularly, an immunoconjugate comprising the anti-Cln101
antibody conjugated with a cytotoxic agent may be administered to
the patient. Preferably, the immunoconjugate bound to the Cln101
protein is internalized by the cell, resulting in increased
therapeutic efficacy of the immunoconjugate in killing the cancer
cell to which it binds. 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.
[0293] The anti-Cln101 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-Cln101 antibody.
[0294] 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.
[0295] It may also be desirable to combine administration of the
anti-Cln101 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 Cln101-expressing tumor cells. The
cocktail may also comprise antibodies that are directed to other
epitopes of Cln101. Preferably the other antibodies do not
interfere with the binding and or internalization of the antibodies
of this invention.
[0296] The antibody therapeutic treatment method of the present
invention may involve the combined administration of an anti-Cln101
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).
[0297] 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-Cln101 antibody (and optionally other agents
as described herein) may be administered to the patient.
[0298] 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-Cln101
antibody.
[0299] 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-Cln101 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.
[0300] 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.
[0301] 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.
[0302] 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-Chol, 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
[0303] The invention also relates to an article of manufacture
containing materials useful for the detection for Cln101
overexpressing cells and/or the treatment of Cln101 expressing
cancer, in particular prostate or ovarian 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 Cln101 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-Cln101 antibody of the invention. The label or package
insert indicates that the composition is used for detecting Cln101
expressing cells and/or for treating prostate or ovarian 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 or ovarian 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.
[0304] Kits are also provided that are useful for various purposes
, e.g., for Cln101 cell killing assays, for purification or
immunoprecipitation of Cln101 from cells or for detecting the
presence of Cln101 in a serum sample or other bodily fluids or
detecting the presence of Cln101-expressing cells in a cell sample.
For isolation and purification of Cln101, the kit can contain an
anti-Cln101 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 Cln101 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
[0305] The following MAb/hybridomas of the present invention are
described below: Cln101.A1.1, Cln101.A3.1, Cln101.A6.1,
Cln101.A7.1, Cln101.A8.1, Cln101.A9.1, Cln101.A10.1, Cln101.A11.1,
Cln101.A15.1, Cln101.A16, Cln101.A17.1, Cln101.A18.1, Cln101.A23.1,
Cln101.A26.1, Cln101.A28.1, Cln101.A35.1, Cln101.A37.1,
Cln101.A38.1, Cln101.A41.1, Cln101.A42.1, Cln101.A46.1,
Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12, Cln101.C17,
Cln101.C18, Cln101.C25, Cln101.C36, Cln101.C43 and Cln101.C47.
[0306] If the MAb has been cloned, it will get the nomenclature
"X.1," e.g., the first clone of A2 will be referred to as A2.1, the
second clone of A2 will be referred to as A2.2, etc. For the
purposes of this invention, a reference to MAb A2 will include all
clones, e.g., A2.1, A2.2, etc.
Immunogens and Antigens (Recombinant Proteins, HA & His Tags
& Transfected Cells)
[0307] For immunization of mice and production of MAbs, two Cln101
recombinant proteins were generated. The first was a bacterial (E.
coli) expressed soluble Cln101 recombinant protein.
TABLE-US-00002 SEQ ID NO:1
MASRSMRLLLLLSCLAKTGVLGDIIMRPSCAPGWFYHKSNCYGYFRKLRN
WSDAELECQSYGNGAHLASILSLKEASTIAEYISGYQRSQPIWIGLHDPQ
KRQQWQWIDGAMYLYRSWSGKSMGGNKHCAEMSSNNNFLTWSSNECNKRQ HFLCKYRP,.
[0308] A second Cln101 construct was cloned with a histidine tag
immediately downstream of codon Ser161. For production of the
C-series of MAbs this protein was cloned into a standard expression
vector and expressed in mammalian cells using standard technology
known to those of skill in the art.
TABLE-US-00003 SEQ ID NO:2
MLQNSAVLLVLVISASATHEAEQDIIMRPSCAPGWFYHKSNCYGYFRKLR
NWSDAELECQSYGNGAHLASILSLKEASTIAEYISGYQRSQPIWIGLHDP
QKRQQWQWIDGAMYLYRSWSGKSMGGNKHCAEMSSNNNFLTWSSNECNKR
QHFLCKYRPASHHHHHHHHHH,.
Immunizations
[0309] For generation of the A series MAbs mice were immunized with
bacterial expressed soluble Cln101 recombinant protein, SEQ ID
NO:1. Groups of 8 BALB/c mice were immunized intradernally in both
rear footpads. All injections were 25 uL per foot. The first
injection (day 1) of 10 ug of antigen per mouse was in Dulbecco's
phosphate buffered saline (DPBS) mixed in equal volume to volume
ratio with Titermax gold adjuvant (Sigma, Saint Louis, Mo.).
Subsequent injections of 10 ug of antigen 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. The final boost
injection on day 33 consisted of antigen diluted in DPBS alone.
Fusion occurred on Day 37.
[0310] For generation of the C-series MAbs mice were immunized as
above with soluble mammalian expressed Cln101 recombinant protein,
SEQ ID NO:2.
Hybridoma Fusions
[0311] 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 by pressing
through a sterile sieve into DMEM and removing T-cells via
anti-CD90 (Thy1.2) coated magnetic beads (Miltenyl Biotech,
Baraisch-Gladbach, Germany).
[0312] These primary B-cell enriched lymph node cells were then
immortilized 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) containing selection medium (DMEM/10% FBS). These fusion
cultures were immediately distributed, 10 million cells per plate,
into wells of 96 well culture plates. 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 Cln101 protein.
[0313] Monoclonal cultures, consisting of the genetically uniform
progeny from single cells, were established after the screening
procedure above, by sorting of single viable cells into wells of
two 96 well plates, using flow cytometry (Coulter Elite). 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
[0314] Hybridoma cell lines were selected for production of Cln101
specific antibody by enzyme linked solid phase immunoassay (ELISA).
Cln101 or Pro104 proteins were nonspecifically adsorbed to wells of
96 well polystyrene EIA plates (VWR). Fifty .mu.L of Cln101 or
Pro104 protein at 0.91 mg/mL in (DPBS) was 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, 50 .mu.L of hybridoma culture medium samples was added to
the wells and incubated for 1 hour at RT. The wells were then
washed 3 times with (TBST). One hundred .mu.L 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 .mu.L of alkaline phosphatase
substrate para-nitrophenylphosphate (pNPP) (Sigma) at 1 mg/mL in 1
M Diethanolamine buffer pH 8.9 (Sigma) was then added to each well
and incubated for 20 min. at RT. Bound alkaline phosphatase
activity was indicated by the development of a visible yellow
color. The enzymatic reaction was quantitated by measuring the
solution's absorbance at 405 nm wavelength. Cultures producing the
highest absorbance values are chosen for expansion and further
evaluation.
Western Blots
[0315] Protein extracts for western blot analysis were prepared in
cell lysis buffer (1% NP-40, 10 mM Sodium Phosphate pH 7.2, 150 mM
Sodium Chloride) from Cln101 expressing cell lines. Proteins were
separated by electrophoresis on NuPAGE 4-12% Bis-Tris gels
(Invitrogen Life Technologies, Carlsbad, Calif.) under denaturing
conditions in Novex-XCell II Minicell gel apparatus (Invitrogen,
Life Tech) and subsequently transferred to PVDF membranes using an
XCell II Blot Module (Invitrogen Life Technologies). Following the
transfer of proteins, the membranes were blocked in 1% blocking
reagent (Roche Diagnostic Corp., Indianapolis, Ind.) and incubated
overnight at 4.degree. C. with purified primary antibodies (Cln101
monoclonal antibodies: A46.1, A17.1, A9.1, A6.1 A26.1 and A38.1)
and then with horseradish-peroxidase conjugated goat anti-mouse IgG
(Jackson Immunoresearch Laboratories, Inc.) and finally visualized
by chemiluminescence using an ECL advance western blotting
detection kit (Amersham Biosciences, Piscataway, N.J.).
[0316] The preferred antibodies were Anti Cln101 Mab A46.1, A17.1,
A9.1, A6.1 A26.1 and A38.1
ProteinChip SELDI Immunoassay for Cln101.
[0317] The ProteinChip SELDI mass spectrometry biosystem was
obtained from Ciphergen Biosystems, Inc., 6611 Dumbarton Circle,
Fremont, Calif. 94555. Preactivated surface ProteinChip arrays PS20
(Ciphergen Biosystems) have carbonyl diimidazole moieties that can
react covalently with their amine groups. 0.4 ug of purified MAB
A16B recognizing the Cln101 was applied on each spot of a PS20 chip
and incubated for 16 h at 4.degree. C. or 1 h at 37.degree. C. in a
humidity chamber. An irrelevant MAB was used as a control. Residual
active sites were then blocked by incubating the array in a 15-ml
conical tube with 8 ml of 1 M ethanolamine, for 30 min, on a
shaking platform. After three washes of 5 min each with 0.05%
Triton X-100 in PBS (pH 7.4) followed by 3 washes for 5 min with
PBS (pH 7.4) in a 15-ml conical tube on a shaking platform, the
chip was incubated for 4 h in a humidity chamber with 100 .mu.l of
normal and breast serum sample, 100 ul of A549, HT29, PC-3 cell
line medium at 100 ng/ml using bioprocessor (Ciphergen Biosystems).
The chip was washed as previously with 0.05% Triton X-100 in PBS
(pH 7.4) and PBS (pH 7.4). Sinapinic acid was applied on each spot
and mass spectrometry analysis was performed in a PBS-II mass
reader (Ciphergen Biosystems). Spectra were collected using an
average 80 nitrogen laser shots with a laser intensity of 280 and
290 and a detector sensitivity of 10. Spectrum analysis was
performed using the ProteinChip software version 2.1 (Ciphergen
Biosystems). The result of ProteinChip SELDI Immunoassay for Cln101
showed Mab 16B could capture a unique protein from HT29 medium
about 16 kd, well other control antibodies could not. The size of
captured protein is very close to the full length of Cln101 17 Kd,
suggested that the captured protein in serum is Cln101. The
capturing of Cln101 from serum was not successful.
[0318] Mab/Mab ELISA. A sequential sandwich ELISA assay was used to
measure levels of Cln101 in serum. Assay consisted of monoclonal Ab
capture, monoclonal Ab-biotin and Alk Phos detector. High binding
polystyrene strip-wells were obtained from Corning Life Sciences
(MA). The plates were coated overnight at 4.degree. C. with 0.5
.mu.g/well of anti-Cln101 MAb. The coating solution was then
aspirated off and blocked with 300 .mu.l/well of Superblock-TBS
(Pierce Biotechnology, Illinois) for 1 hour at room temperature
under shaking conditions. The blocking solution was then aspirated
off and 20 .mu.l of Cln101 Standards diluted in 100% heat
inactivated fetal bovine serum (Hyclone, UT) at concentrations of
6.25, 1.25, 0.5, 0.25, 0.1 and 0 ng/ml were then added to the
wells. 20 .mu.l of serum was also added to the other wells. 100
.mu.l of Assay buffer containing 1% mouse serum, 10% FBS, and
1.times. TBS (Teknova, Calif.) was then added to the 20 .mu.l of
samples and standards, and incubated at RT, shaking for 1 hour. The
plate was washed four times with 360 .mu.l 1.times. TBS+0.05%
Tween-20 (Teknova, Calif.). Biotinylated Mab anti-Cln101 was
diluted as 0.75 ug/ml in 1.times. TBS pH 7.4 (Teknova, Calif.) in
Assay Buffer and 100 .mu.l was added to each well and then
incubated for 1 hour at room temperature while shaking. The plate
was washed four times with 360 .mu.l 1.times. TBS+0.05% Tween-20
(Teknova, Calif.). 100 .mu.l of Alkaline Phosphatase conjugated
Streptavidin, 1:2000 diluted in 1.times. TBS pH 7.4 (Jackson
ImmunoResearch Laboratories, PA) was added to each well and
incubated for 30 minutes at RT while shaking. The plate was washed
four times with 360 .mu.l 1.times. TBS+0.05% Tween-20 (Teknova,
Calif.). The plate was developed using pNPP substrate in 1.times.
DEA buffer (Pierce Biotechnology, Illinois) for 20 minutes at RT
while shaking. The reaction was stopped using 100 .mu.l/well 1N
NaOH, and read at 405 nm using a Spectramax 190 plate reader
(Molecular Devices, Calif.).
Example 2
Monoclonal Sandwich ELISA Detection of Cln101
[0319] High binding polystyrene plates (Corning Life Sciences (MA))
were coated overnight at 4.degree. C. with 0.8 .mu.g/well of
anti-Cln101 MAb. The coating solution was aspirated off and free
binding sites were blocked with 300 .mu.l/well Superblock-TBS
(Pierce Biotechnology, Illinois) plus 100% calf serum for 1 hour at
room temperature (RT). After washing 4.times. with TBS+0.1%Tween20,
50 .mu.l of Assay Buffer (TBS, 1% BSA, 1% mouse Serum, 1% Calf
Serum, 0.1% Tween20) was added to each well and then 50 .mu.l of
antigen was added for 90 minutes incubation. For the checkerboard
experiment, each pair was tested on 50 ng/ml and 0 ng/ml of
recombinant Cln101. For each sandwich ELISA, standards of 10, 2.5,
0.5, 0.25, 0.1 and 0 ng/ml Cln101 were run in parallel with the
test samples. Standards and test samples were diluted in Assay
Buffer. For the detection, 100 .mu.l of 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 horseradish
peroxidase conjugated streptavidin (1 mg/ml, Jackson ImmunoResearch
Laboratories, PA) at a 1:20.000 dilution was added to each well and
incubated for 30 minutes at RT while shaking. After washing, the
plate was then developed using DAKO TMB Plus substrate (DAKO,
Denmark) for 30 minutes at RT. The reaction was stopped using 100
.mu.l/well 1N HCL, and the plates were read at 450 nm using a
Spectramax 190 plate reader (Molecular Devices, CA).
[0320] The results of the checkerboard ELISA for the anti-Cln101
A-series MAbs using bacterial recombinant Cln101 are shown in the
table below.
Identification of Anti-Cln101 Antibody A-Series Pairs
TABLE-US-00004 [0321] Coating Detecting MAb MAb A1.1 A3.1 A6.1 A7.1
A8.1 A9.1 A10.1 A11.1 A12.1 A15.1 A16.1 A17.1 A18.1 A1.1 1 1 1 1 1
1 1 1 1 1 1 1 1 A3.1 1 1 1 1 1 1 1 1 1 1 1 1 0 A6.1 1 1 1 1 1 1 9 1
1 1 18 1 1 A7.1 1 1 1 1 1 1 9 1 1 1 16 1 1 A8.1 1 1 1 1 1 1 1 1 1 1
1 1 1 A9.1 1 1 1 1 1 1 21 1 1 1 1 1 1 A10.1 1 1 41 41 1 45 1 1 1 1
20 43 1 A11.1 1 1 1 1 1 4 1 1 1 1 1 1 1 A12.1 1 1 1 1 1 1 1 1 1 1 1
1 1 A15.1 1 1 1 1 1 1 1 1 1 1 1 1 1 A16.1 1 1 35 19 1 1 3 1 1 1 1 1
1 A17.1 1 1 1 1 1 1 5 1 1 1 1 1 1 A18.1 0 1 1 1 1 1 1 1 1 1 1 1 1
A23.1 1 1 1 10 3 50 1 1 1 5 13 31 32 A26.1 1 1 27 24 1 1 2 5 1 10 1
20 27 A28.1 1 1 1 1 1 1 1 1 1 1 1 1 1 A35.1 1 1 1 1 1 1 1 1 1 1 1 1
1 A37.1 1 1 9 1 1 1 1 1 1 1 1 14 1 A38.1 1 1 34 33 1 1 12 7 1 17 1
27 25 A41.1 1 1 1 1 1 1 1 1 1 1 1 1 1 A42.1 1 1 1 1 1 2 1 1 1 1 1 1
1 A46.1 1 1 1 1 1 40 1 1 1 2 27 32 14 A47.1 1 1 1 1 1 1 1 1 1 1 1 1
1 Coating Detecting MAb MAb A23.1 A26.1 A28.1 A35.1 A37.1 A38.1
A41.1 A42.1 A46.1 A47.1 A1.1 1 1 1 1 1 1 1 1 1 1 A3.1 1 1 0 1 1 1 1
1 1 1 A6.1 3 33 1 1 1 33 1 1 1 1 A7.1 24 18 1 1 1 36 1 1 1 1 A8.1
20 1 1 1 1 28 1 1 2 1 A9.1 31 1 1 1 1 1 1 2 36 1 A10.1 1 28 1 1 1
47 1 1 1 1 A11.1 26 24 1 1 1 31 1 1 3 1 A12.1 1 1 1 1 1 1 1 1 1 1
A15.1 19 18 1 1 1 30 1 1 3 1 A16.1 30 1 1 1 1 1 1 1 32 1 A17.1 23
11 1 1 1 23 1 1 30 1 A18.1 27 23 1 1 1 28 1 1 16 1 A23.1 1 22 1 1 1
41 1 45 1 1 A26.1 22 1 1 1 1 1 1 29 28 1 A28.1 1 1 1 2 1 1 1 1 1 1
A35.1 1 1 1 1 1 1 1 1 1 1 A37.1 5 1 1 1 1 1 1 1 2 1 A38.1 27 1 1 1
1 1 1 28 30 1 A41.1 1 1 1 1 1 1 1 1 1 1 A42.1 28 23 1 1 1 30 1 1 27
1 A46.1 1 33 1 1 1 42 1 40 1 1 A47.1 1 1 1 1 1 1 1 1 1 1
The results of the checkerboard ELISA for the anti-Cln101 C-series
MAbs using mammalian recombinant Cln101 are shown in the table
below.
Identification of Anti-Cln101 Antibody C-Series Pairs
TABLE-US-00005 [0322] detecting Ab coating Ab A46 A9 C3 C6 C12 C17
C18 C25 C36 C43 C47 control A46 2 52 46 5 33 12 52 47 53 52 43 1 A9
43 1 1 44 41 49 3 46 3 8 3 1 C3 46 1 1 50 45 50 9 50 9 48 8 1 C6 3
54 45 2 3 4 49 1 52 1 44 1 C12 5 2 12 2 1 2 9 1 17 1 11 1 C17 23 37
20 1 1 6 44 1 33 1 43 1 C18 47 1 2 44 51 45 16 44 16 40 12 1 C25 49
45 44 1 1 2 45 1 48 1 43 1 C36 43 1 2 37 45 49 8 44 7 45 6 1 C43 45
1 50 2 1 3 44 2 48 1 42 1 C47 42 1 2 45 47 50 13 50 16 50 14 1
[0323] The results of the checkerboard ELISA for the anti-Cln101
A-series and C-series MAbs using mammalian recombinant Cln101 are
shown in the table below.
Identification of Anti-Cln101 Antibody A and C-Series Pairs
TABLE-US-00006 [0324] Detecting Ab Coating Ab A6 A9 A10 A26 A38 A42
A46 C6 C17 C25 C36 control A6 1 1 13 10 13 1 1 1 1 1 2 1 A9 1 1 18
1 1 1 44 21 20 22 2 1 A10 5 21 1 2 5 1 1 1 1 1 48 1 A26 3 1 2 1 1 2
4 4 4 3 1 1 A38 4 1 3 1 1 5 11 9 9 9 2 1 A42 1 1 1 2 2 1 6 1 1 1 8
1 A46 1 51 1 10 23 16 1 1 1 13 50 1 C6 1 19 1 6 17 1 1 1 1 1 48 1
C17 1 9 1 4 18 1 1 1 1 1 48 1 C25 1 5 1 2 8 1 6 1 1 1 51 1 C36 1 1
24 1 1 20 51 52 48 49 5 1 control 1 1 1 1 1 1 1 1 1 1 1 1
[0325] For the checkerboard ELISA, all possible combination of
antibodies, were tested for efficiency as coating or detecting
reagents. The pair A9.1 and A46.1 demonstrated an excellent
signal/noise ratio and were further evaluated in sandwich ELISA
assays to analyze the efficiency of detection of endogenous Cln101
in lysates from cancer cell lines and body fluids. Additional
antibody pairs for detecting Cln101 include A10/C36, A46/C25,
A46/C36, C6/A9, C6/C36, C36/A46, C36/C17, A9/C17 and A9/C6. Binding
results are graphically represented in FIG. 1.
Example 3
Cln101 Assays
Human Serum Samples
[0326] The human cancer serum samples in primary #1, primary #2 and
secondary panel #1 were obtained from IMPATH-BCP, Inc.(Los Angeles,
Calif.) and Diagnostic Support Service, Inc. (West Barnstable,
Mass.). The human normal serum samples were obtained from ProMedDx,
LLC. (Norton, Mass.). The human disease serum samples, primary
panel #3 (cancer serum), colon, prostate, lung and breast extended
panels (cancer and benign serum samples) and all the samples in
legacy panel (cancer and normal serum samples) were obtained from
Diagnostic Support Service, Inc., (West Barnstable, Mass).
Additional ovarian cancer samples were obtained from DIAGNOSTIC
ONCOLOGY CRO, Inc. (DOCRO) (Seymour, Conn.). The ELISA assays were
done in primary panel #1, primary panel #2, primary panel #3,
benign panel, ProMedDx normal panel, bioavailability panel, colon
and prostate extended panels and DOCRO ovarian panel. The primary
#1 and #2 panel has 23 cancer serum samples for breast, Colon,
Lung, Ovarian and prostate cancer each, plus 45 normal serums. The
25 cancer serum samples in each type covered different age (from 40
to 70) and mixed stage (stage 1 to 4). All the normal serum samples
were from ProMedDx, they covered a wide range of age (from 20 to
80) and both genders. The benign panels covered 16 disease, they
are: Renal; Cystitis; Hypertension; BPH; Hepatic Toxicity;
Prostatitis; Cirrhosis; Colon polyps; Diabetes; Ulcerative Colitis;
Crohns; Nephrolithaisis; Polycystic; Glomerulonephritis;
Pancreatitis; Diverticulitis. The bioavailability panel consists of
49 individuals with 6 to 9 multiple draws in one month of period
time. The colon extended panel has 50 cancer serum samples and 200
benign serum samples (50 of each disease: Ulcerative Colitis;
Crohns; Colon polyps; Diverticulitis). The prostate extended panel
has 200 cancer serum samples and 100 benign serum samples (50 BPH;
50 Prostatitis). The DOCRO panel has 57 ovarian cancer serum
samples and 24 Endometriosis serum samples.
[0327] FIGS. 2-8 show Cln101 in the all cancer panel, the prostate
cancer panel, and the prostate cancer panel with benign diseases.
FIG. 9 shows PSA in the prostate cancer panel with benign diseases.
FIG. 10 shows Cln101 in ovarian cancer (DOCRO panel). For
determination of PSA levels, the serum samples were tested with PSA
ELISA kit from Biocheck Inc (Burlingame, Calif.) following the
manufacturer's protocol.
Example 4
ROC Analysis of Cln101 and PSA
[0328] 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).
[0329] 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).
[0330] 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).
[0331] ROC Analysis for Prostate Cancer (normal vs. cancer)
analysis for individual markers for differentiation of normal males
(n=121) from males with prostate cancer (n=138).
TABLE-US-00007 Statistic Cln101 PSA AUC (95% CI) 0.933 0.801
(0.895-0.960) (0.747-0.848) Cutoff for best combination of Sens/
0.306 3.7 Spec. Sens./Spec. at best cutoff 89%/92% 82%/67% Sens. (@
90% Spec.) (Cutoff) 90% (0.291) 34% (9.9)
[0332] FIG. 11 shows the area under the curve for Cln101 and PSA
(normal vs. cancer).
[0333] ROC Analysis for Prostate Cancer Synergistic effects (normal
vs. cancer) Logistic regression results for differentiation of
normal males (n=121) from males with prostate cancer
(n=138)--Synergistic effects
TABLE-US-00008 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.933
0.940 0.801 Wald p-value <0.0001 Cln101: <0.0001 <0.0001
PSA: 0.002 Odds Ratio (OR) 1.2E-13 Cln101: 2.2E-12 8.3E-12 PSA:
5.7E-8: R2 0.5093 0.5523 0.1843 Sens. (@ 90% Spec.) 90% 85% 34%
[0334] ROC Analysis for Prostate Cancer (normals+benigns vs.
cancer) Analysis for individual markers for differentiation of
normal males and males with benign conditions (n=268) from males
with prostate cancer (n=138)
TABLE-US-00009 Statistic Cln101 PSA AUC (95% CI) 0.741 0.762
(0.695-0.783) (0.718-0.803) Cutoff for best combination 0.35 3.7 of
Sens/Spec. Sens./Spec. at best cutoff 87%/50% 82%/60% Sens. (@ 90%
Spec.) (Cutoff) 36% (1.08) 31% (10.8)
[0335] FIG. 12 shows the area under the curve for Cln101 and PSA
(normals+benigns vs. cancer).
[0336] ROC Analysis for Prostate Cancer Synergistic effects
(normals+benigns vs. cancer) Logistic regression for
differentiation of normal males and males with benign conditions
(n=268) from males with prostate cancer (n=138)--Synerqistic
effects
TABLE-US-00010 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.741
0.800 0.762 Wald p-value <0.0001 Cln101: <0.0001 <0.0001
PSA: <0.0001 Odds Ratio (OR) 0.0011 Cln101: 0.004 1.4E-9 PSA:
1.5E-8 R2 0.1251 0.2374 0.1611 Sens. 36% 44% 31% (@ 90% Spec.)
[0337] Number of Serum Samples in PSA "Grey Zone"
TABLE-US-00011 PSA PSA PSA Samples 2-4 ng/mL 4-10 ng/mL 2-10 ng/mL
Normal 47 44 91 (n = 121) Prostate Cancer 20 52 72 (n = 138)
Prostate Benign 40 40 80 (n = 147)
[0338] ROC Analysis for Prostate Cancer Synergistic effects (normal
vs. cancer, 4-10 ng/mL PSA) Logistic regression results for
differentiation of normal males (n=44) from males with prostate
cancer (n=52)
[0339] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 4.0-10.0 ng/ml
TABLE-US-00012 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.937
0.950 0.526 Wald p-value <0.0001 Cln101: <0.0001 0.6997 PSA:
0.0700 Odds Ratio (OR) 4.7E-13 Cln101: 2.6E-15 0.736 PSA: 11.8 R2
0.5252 0.5617 0.0014 Sens. (@ 90% Spec.) 88% 92% 6%
[0340] ROC Analysis for Prostate Cancer Synergistic effects
(norm/ben vs. cancer, 4-10 ng/mL PSA) Logistic regression results
for differentiation of normal males and males with benign
conditions (n=84) from males with prostate cancer (n=52)
[0341] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 4.0-10.0 ng/ml
TABLE-US-00013 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.657
0.657 0.522 Wald p-value 0.0041 Cln101: 0.0044 0.6634 PSA: 0.9937
Odds Ratio (OR) 0.041 Cln101: 0.041 0.769 PSA: 0.995 R2 0.0547
0.0547 0.0011 Sens. (@ 90% Spec.) 25% 25% 5%
[0342] ROC Analysis for Prostate Cancer Synergistic effects (normal
vs. cancer, 2-10 ng/mL PSA) Logistic regression results for
differentiation of normal males (n=91) from males with prostate
cancer (n=72)
[0343] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 2.0-10.0 ng/ml
TABLE-US-00014 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.920
0.921 0.640 Wald p-value <0.0001 Cln101: <0.0001 0.0105 PSA:
0.3272 Odds Ratio (OR) 5.6E-10 Cln101: 1.2E-9 0.213 PSA: 0.440 R2
0.4613 0.4667 0.0390 Sens. (@ 90% Spec.) 88% 81% 9%
[0344] ROC Analysis for Prostate CancerSynergistic effects
(norm/ben vs. cancer, 2-10 ng/mL PSA) Logistic regression for
differentiation of normal males and males with benign conditions
(n=171) from males with prostate cancer (n=72)
[0345] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 2.0-10.0 ng/ml
TABLE-US-00015 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.694
0.714 0.627 Wald p-value <0.0001 Cln101: <0.0001 0.0027 PSA:
0.0123 Odds Ratio (OR) 0.019 Cln101: 0.024 0.245 PSA: 0.292 R2
0.0838 0.1063 0.0328 Sens. (@ 90% Spec.) 26% 28% 9%
[0346] ROC Analysis for Prostate Cancer Synergistic effects (normal
vs. cancer, 2-4 ng/mL PSA) Logistic regression results for
differentiation of normal males (n=47) from males with prostate
cancer (n=20)
[0347] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 2.0-4.0 ng/ml
TABLE-US-00016 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.905
0.921 0.575 Wald p-value 0.0007 Cln101: 0.0010 0.4335 PSA: 0.1778
Odds Ratio (OR) 8.3E-7 Cln101: 1.4E-7 0.47 PSA: 0.14 R2 0.4137
0.4448 0.0095 Sens. (@ 90% Spec.) 62% 62% 24%
[0348] ROC Analysis for Prostate Cancer Synergistic effects
(norm/ben vs. cancer, 2-4 ng/mL PSA) Logistic regression for
differentiation of normal males and males with benign conditions
(n=87) from males with prostate cancer (n=20)
[0349] Synergistic effects in the diagnostic "grey zone"
sub-population of patients with PSA 2.0-4.0 ng/ml
TABLE-US-00017 Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.733
0.733 0.500 Wald p-value 0.0024 Cln101: 0.0023 0.9739 PSA: 0.8370
Odds Ratio (OR) 0.008 Cln101: 0.008 1.03 PSA: 1.21 R2 0.1368 0.1373
0.000 Sens. (@ 90% Spec.) 57% 52% ~2%
Example 5
ROC Analysis of Cln101 and CA125
[0350] 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).
[0351] 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).
[0352] 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).
[0353] ROC Analysis for Ovarian Cancer (normal+benign disease vs.
cancer) analysis for individual markers for differentiation of
normal (n=31) or benign disease (endometriosis n=24) patients from
patients with ovarian cancer (n=57).
TABLE-US-00018 Statistic Cln101 CA125 ROC AUC 0.947 0.772 Sens. @
90% Spec. 88% 67% Spec. @ 90% Sens. 87% 4%
[0354] ROC Analysis for Ovarian Cancer synergistic effects
(normal+benign disease vs. cancer) analysis for individual markers
for differentiation of normal (n=31) or benign disease
(endometriosis n=24) patients from patients with ovarian cancer
(n=57).
TABLE-US-00019 Statistic Cln101 CA125 CA125 + Cln101 ROC AUC 0.947
0.772 0.969 Sens. @ 90% Spec. 88% 67% 93% Spec. @ 90% Sens. 87% 4%
95%
[0355] ROC Analysis for Stage 1 and 2 Ovarian Cancer (normal+benign
disease vs. cancer) analysis for individual markers for
differentiation of normal (n=31) or benign disease (endometriosis
n=24) patients from patients with stage 1 and 2 ovarian cancer
(n=28).
TABLE-US-00020 Statistic Cln101 CA125 ROC AUC 0.921 0.696 Sens. @
90% Spec. 79% 61% Spec. @ 90% Sens. 75% 4%
[0356] ROC Analysis for Stage 1 and 2 Ovarian Cancer synergistic
effects (normal+benign disease vs. cancer) analysis for individual
markers for differentiation of normal (n=31) or benign disease
(endometriosis n=24) patients from patients with stage 1 and 2
ovarian cancer (n=28).
TABLE-US-00021 Statistic Cln101 CA125 CA125 + Cln101 ROC AUC 0.947
0.772 0.958 Sens. @ 90% Spec. 88% 67% 89% Spec. @ 90% Sens. 87% 4%
85%
[0357] Cln101 showed efficacy in detecting ovarian cancer in
samples where CA125 levels were not indicative of ovarian cancer.
In addition to detecting ovarian cancer with CA125 levels below 30
U/ml, Cln101 is useful in detecting ovarian cancer where CA125
values are between 30 and 40 U/ml or between 30-35 U/ml.
[0358] ROC Analysis for CA125 negative (<30 U/mL) Ovarian Cancer
(normal+benign disease vs. cancer) analysis for individual markers
for differentiation of normal (n=31) or benign disease
(endometriosis n=16) patients from patients with CA125 negative
(<30 U/mL) Ovarian Cancer (n=19).
TABLE-US-00022 Statistic Cln101 ROC AUC 0.940 Sens. @ 90% Spec. 84%
Spec. @ 90% Sens. 83%
Results
[0359] ROC analysis results demonstrated Cln101 is an excellent
marker for detecting ovarian cancer compared to known marker CA125.
High Cln101 AUC scores indicate Cln101 is useful for detecting
Stage 1 & 2 ovarian cancer alone or in combination with imown
marker CA125. Additionally, high Cln101 AUC scores indicate Cln101
is useful for detecting ovarian cancer in patients where known
markers fail. Specifically, Cln101 detects ovarian cancer in
patients with what is considered normal CA125 levels, CA125 <30
U/ml. Furthermore, multivariate (Cln101+CA125) analysis may improve
sensitivity and specificity for detecting ovarian cancer.
[0360] In summary, Cln101 is useful for detecting early stage
ovarian cancer, ovarian cancer in general and discriminating
between ovarian cancer and benign ovarian disease. Only 25% of all
ovarian cancer is found in stage 1. If ovarian cancer is found in
stage 1 therapy (e.g. surgery) is very effective and the 5-year
survival rate is 90%.
Example 6
Deposits
Deposit of Cell Lines and DNA
[0361] 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.
[0362] The following hybridoma cell lines were deposited with ATCC,
Cln101.A9.1 and Cln101.A46.1. The names of the deposited hybridoma
cell lines above may be shortened for convenience of reference.
E.g. A01.1 corresponds to Cln101.A01.1. These hybridomas correspond
to the clones (with their full names) deposited with the ATCC. The
table below lists the hybridoma clone deposited with the ATCC, the
accorded ATCC accession number, and the date of deposit.
ATCC Deposits
TABLE-US-00023 [0363] Hybridoma ATCC Accession No. Deposit Date
Cln101.A9.1 PTA-5877 Mar. 23, 2004 Cln101.A46.1 PTA-5876 Mar. 23,
2004
[0364] 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 37 CFR .sctn.1.14 with particular reference to
886 OG 638).
[0365] 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.
REFERENCES
[0366] Balk S P, Ko Y J, Bubley G J. Biology of prostate-specific
antigen. J Clin Oncol. Jan. 15, 2003;21(2):383-91. Review. [0367]
Barry M J Prostate-Specific-Antigen Testing for Early Diagnosis of
Prostate Cancer N Engl J Med 344(18):1373-1377. Review. [0368]
Canto E I, Shariat S F, Slawin K M. Biochemical staging of prostate
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[0370] Frankel S, Smith G D, Donovan J, Neal D. Screening for
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Review. [0372] Gann P H, Hennekens C H, Stampfer M J. A prospective
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Mayewski R J, Mushlin A I, Greenland P (1981) Selection and
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Shappell S B, Coffey C S, Chang S S, Cookson M S. [0375] The
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predictor of tumor involvement in the radical prostatectomy
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September-October 2002;7(5):195-8. [0376] Hanley J A, McNeil B J
(1982) The meaning and use of the area under a receiver operating
characteristic (ROC) curve. Radiology, 143, 29-36. [0377] Hanley J
A, McNeil B J (1983) A method of comparing the areas under receiver
operating characteristic curves derived from the same cases.
Radiology, 148, 839-843. [0378] Hartupee J C, Zhang H, Bonaldo M F,
Soares M B, Dieckgraefe B K. Isolation and characterization of a
cDNA encoding a novel member of the human regenerating protein
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[0379] Jain S, Bhojwani A G, Mellon J K. Improving the utility of
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Postgrad Med J. November 2002;78(925):646-50. Review. [0381]
Kobayashi S, Akiyama T, Nata K, Abe M, Tajima M, Shervani N J, Unno
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Web Sites:
[0386] [0387] cancer with the extension
gov/cancer_information/cancer_type/prostate/ of the world wide web
Sequence CWU 1
1
21158PRTArtificial sequenceSynthetic 1Met Ala Ser Arg Ser Met Arg
Leu Leu Leu Leu Leu Ser Cys Leu Ala1 5 10 15Lys Thr Gly Val Leu Gly
Asp Ile Ile Met Arg Pro Ser Cys Ala Pro 20 25 30Gly Trp Phe Tyr His
Lys Ser Asn Cys Tyr Gly Tyr Phe Arg Lys Leu 35 40 45Arg Asn Trp Ser
Asp Ala Glu Leu Glu Cys Gln Ser Tyr Gly Asn Gly 50 55 60Ala His Leu
Ala Ser Ile Leu Ser Leu Lys Glu Ala Ser Thr Ile Ala65 70 75 80Glu
Tyr Ile Ser Gly Tyr Gln Arg Ser Gln Pro Ile Trp Ile Gly Leu 85 90
95His Asp Pro Gln Lys Arg Gln Gln Trp Gln Trp Ile Asp Gly Ala Met
100 105 110Tyr Leu Tyr Arg Ser Trp Ser Gly Lys Ser Met Gly Gly Asn
Lys His 115 120 125Cys Ala Glu Met Ser Ser Asn Asn Asn Phe Leu Thr
Trp Ser Ser Asn 130 135 140Glu Cys Asn Lys Arg Gln His Phe Leu Cys
Lys Tyr Arg Pro145 150 1552171PRTArtificial sequenceSynthetic 2Met
Leu Gln Asn Ser Ala Val Leu Leu Val Leu Val Ile Ser Ala Ser1 5 10
15Ala Thr His Glu Ala Glu Gln Asp Ile Ile Met Arg Pro Ser Cys Ala
20 25 30Pro Gly Trp Phe Tyr His Lys Ser Asn Cys Tyr Gly Tyr Phe Arg
Lys 35 40 45Leu Arg Asn Trp Ser Asp Ala Glu Leu Glu Cys Gln Ser Tyr
Gly Asn 50 55 60Gly Ala His Leu Ala Ser Ile Leu Ser Leu Lys Glu Ala
Ser Thr Ile65 70 75 80Ala Glu Tyr Ile Ser Gly Tyr Gln Arg Ser Gln
Pro Ile Trp Ile Gly 85 90 95Leu His Asp Pro Gln Lys Arg Gln Gln Trp
Gln Trp Ile Asp Gly Ala 100 105 110Met Tyr Leu Tyr Arg Ser Trp Ser
Gly Lys Ser Met Gly Gly Asn Lys 115 120 125His Cys Ala Glu Met Ser
Ser Asn Asn Asn Phe Leu Thr Trp Ser Ser 130 135 140Asn Glu Cys Asn
Lys Arg Gln His Phe Leu Cys Lys Tyr Arg Pro Ala145 150 155 160Ser
His His His His His His His His His His 165 170
* * * * *