U.S. patent application number 12/576313 was filed with the patent office on 2010-08-19 for ovr110 antibody compositions and methods of use.
Invention is credited to Laura Corral, Gilbert-Andre Keller, Wenlu Li, Jackie Papkoff, Glenn Pilkington, Iris Simon.
Application Number | 20100209438 12/576313 |
Document ID | / |
Family ID | 33458763 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100209438 |
Kind Code |
A1 |
Pilkington; Glenn ; et
al. |
August 19, 2010 |
OVR110 Antibody Compositions and Methods of Use
Abstract
The invention provides isolated anti-ovarian, pancreatic, lung
or breast cancer antigen (Ovr110) antibodies that internalize upon
binding to Ovr110 on a mammalian in vivo. The invention also
encompasses compositions comprising an anti-Ovr110 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-Ovr110 antibody, as well as
an expression vector comprising the isolated nucleic acid. Also
provided are cells that produce the anti-Ovr110 antibodies. The
invention encompasses a method of producing the anti-Ovr110
antibodies. Other aspects of the invention are a method of killing
an Ovr110-expressing cancer cell, comprising contacting the cancer
cell with an anti-Ovr110 antibody and a method of alleviating or
treating an Ovr110-expressing cancer in a mammal, comprising
administering a therapeutically effective amount of the anti-Ovr110
antibody to the mammal.
Inventors: |
Pilkington; Glenn; (Rye,
AU) ; Keller; Gilbert-Andre; (Belmont, CA) ;
Li; Wenlu; (South San Francisco, CA) ; Corral;
Laura; (Jamaica Plain, MA) ; Simon; Iris;
(Amsterdam, NL) ; Papkoff; Jackie; (San Francisco,
CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
33458763 |
Appl. No.: |
12/576313 |
Filed: |
October 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10557331 |
Nov 29, 2006 |
7619068 |
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PCT/US2004/014490 |
May 10, 2004 |
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12576313 |
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60556464 |
Mar 25, 2004 |
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60552959 |
Mar 12, 2004 |
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60469555 |
May 9, 2003 |
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Current U.S.
Class: |
424/174.1 ;
435/375; 436/501; 530/387.3; 530/388.8; 530/389.7; 530/391.7 |
Current CPC
Class: |
C07K 16/3015 20130101;
A61K 47/6869 20170801; C07K 16/3069 20130101; C07K 2317/92
20130101; A61P 35/00 20180101; C07K 2317/77 20130101; C07K 2317/73
20130101; A61K 47/6855 20170801 |
Class at
Publication: |
424/174.1 ;
530/389.7; 530/388.8; 530/387.3; 530/391.7; 435/375; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30; A61P 35/00 20060101
A61P035/00; G01N 33/53 20060101 G01N033/53 |
Claims
1. An isolated Ovr110 antibody that binds to Ovr110 on a mammalian
cell in vivo.
2. The antibody of claim 1 which internalizes upon binding to
Ovr110 on a mammalian cell in vivo.
3. The antibody of claim 1 which is a monoclonal antibody, an
antibody fragment, a chimeric antibody or a humanized antibody.
4-5. (canceled)
6. The antibody of claim 1 which is produced by a hybridoma
selected from the group consisting of American Type Culture
Collection accession number PTA-5180, PTA-5855, PTA-5856 and
PTA-5884 or which competes for binding to the same epitope as the
epitope bound by the monoclonal antibody produced by a hybridoma
selected from the group consisting of American Type Culture
Collection accession number PTA-5180, PTA-5855, PTA-5856 and
PTA-5884.
7. (canceled)
8. The antibody of claim 1 which is conjugated to a growth
inhibitory agent or a cytotoxic agent.
9-13. (canceled)
14. The antibody of claim 1, wherein the mammalian cell is a cancer
cell.
15. (canceled)
16. The Ovr110 antibody of claim 1 that inhibits the growth of
Ovr110-expressing cancer cells in vivo.
17. The antibody of claim 16 which is a humanized or human
antibody.
18. The antibody of claim 17 which is produced in bacteria.
19. (canceled)
20. The antibody of claim 16, wherein the cancer cells are from a
cancer selected from the group consisting of ovarian, pancreatic,
lung and breast cancer.
21. The antibody of claim 20, wherein the cancer is ovarian,
pancreatic, lung or breast cancer.
22-24. (canceled)
25. A composition comprising the antibody of claim 1, and a
carrier.
26-29. (canceled)
30. A method of killing an Ovr110-expressing cancer cell,
comprising contacting the cancer cell with the antibody of claim 1,
thereby killing the cancer cell.
31-48. (canceled)
49. An article of manufacture comprising a container and a
composition contained therein, wherein the composition comprises an
antibody of claim 1.
50. The article of manufacture of claim 49 further comprising a
package insert indicating that the composition can be used to treat
ovarian, pancreatic, lung or breast cancer.
51-60. (canceled)
61. A method for detecting Ovr110 overexpression in a subject in
need thereof comprising, (a) combining a serum sample of a subject
with an Ovr110 antibody of claim 1 under conditions suitable for
specific binding of the Ovr110 antibody to Ovr110 in said serum
sample (b) determining the level of Ovr110 in the serum sample, and
(c) comparing the level of Ovr110 determined in step b to the level
of Ovr110 in a control, wherein an increase in the level of Ovr110
in the serum sample from the subject as compared to the control is
indicative of Ovr110 overexpression in the subject.
62. The method of claim 61 wherein the subject has cancer.
63. The method of claim 62 wherein the subject has breast or
ovarian cancer.
64. The method of claim 63 wherein the ovarian cancer is an ovarian
serous adenocarcinoma and the breast cancer is a breast
infiltrating ductal carcinoma or metastatic cancer.
65. The method of claim 61 wherein the control is a serum sample
from a subject without a cancer overexpressing Ovr110.
66-71. (canceled)
72. The antibody of claim 1 which also binds to a rodent homolog of
Ovr110.
73. The antibody of claim 1 which binds to Ovr110 with a binding
affinity of 10.sup.-9 to 10.sup.-13 M.
74. The antibody of claim 1 which binds to deglycosylated Ovr110.
Description
[0001] This patent application is a continuation of U.S.
application Ser. No. 10/557,331. filed Nov. 29, 2006, which is the
U.S. National Stage of PCT Application PCT/US2004/014490, filed May
10, 2004, which claims the benefit of priority from U.S.
Provisional patent application Ser. No. 60/556,464, filed Mar. 25,
2004, U.S. Provisional patent application Ser. No. 60/552,959,
filed Mar. 12, 2004 and U.S. Provisional patent application Ser.
No. 60/469,555, filed May 9, 2003, each of which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-Ovr110 antibody
compositions and methods of killing Ovr110-expressing ovarian,
pancreatic, lung or breast cancers cells.
BACKGROUND OF THE INVENTION
[0003] 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 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., Heriditary Ovarian
Cancer: Clinical Syndromes and Management, in Ovarian Cancer 182
(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] Elevated serum CA125 levels have been associated with an
increased incidence of ovarian cancer in a prospective cohort
study. Jacobs, I. J., et al., Risk of diagnosis of ovarian cancer
after raised serum CA 125 concentration: a prospective cohort
study. Bmj, 1996. 313(7069): p. 1355-8. CA125 is a tumor-associated
antigen that has been used clinically to monitor patients with
epithelial ovarian carcinomas. About 9,320 postmenopausal women
underwent an initial screen and an average of 2.8 yearly screens
with the CA125 assay and were followed for an average of 6.8 years.
Forty-nine cancers were identified. A serum CA125 concentration of
at least 30 U/mL was associated with a relative risk of 35.9 (95%
confidence interval (CI) 18.3-70.4) during the first year after the
screen, and a relative risk of 14.3 (95% CI 8.5-24.4) during the 5
years after the screen. At a CA125 concentration of 100 U/mL, the
relative risks were 204.8 and 74.5, respectively. Women with CA125
levels below 30 U/mL had risks of 0.13 and 0.54, respectively.
[0010] Other markers of interest are HE4 and mesothelin, see Urban
et al. Ovarian cancer screening Hematol Oncol Clin North Am. 2003
August; 17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is
a biomarker for ovarian carcinoma, Cancer Res. 2003 Jul. 1;
63(13):3695-700; Ordonez, Application of mesothelin immunostaining
in tumor diagnosis, Am J Surg Pathol. 2003 November;
27(11):1418-28.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] Breast cancer, also referred to as mammary tumor cancer, is
the second most common cancer among women, accounting for a third
of the cancers diagnosed in the United States. One in nine women
will develop breast cancer in her lifetime and about 192,000 new
cases of breast cancer are diagnosed annually with about 42,000
deaths. Bevers, Primary Prevention of Breast Cancer, in Breast
Cancer, 20-54 (Kelly K Hunt et al., ed., 2001); Kochanek et al., 49
Nat'l. Vital Statistics Reports 1, 14 (2001). Breast cancer is
extremely rare in women younger than 20 and is very rare in women
under 30. The incidence of breast cancer rises with age and becomes
significant by age 50. White Non-Hispanic women have the highest
incidence rate for breast cancer and Korean women have the lowest.
Increased prevalence of the genetic mutations BRCA1 and BRCA2 that
promote breast and other cancers are found in Ashkenazi Jews.
African American women have the highest mortality rate for breast
cancer among these same groups (31 per 100,000), while Chinese
women have the lowest at 11 per 100,000. Although men can get
breast cancer, this is extremely rare. (American Cancer Society
Website: cancer with the extension.org of the world wide web). With
the exception of those cases with associated genetic factors,
precise causes of breast cancer are not known.
[0018] In the treatment of breast cancer, there is considerable
emphasis on detection and risk assessment because early and
accurate staging of breast cancer has a significant impact on
survival. For example, breast cancer detected at an early stage
(stage T0, discussed below) has a five-year survival rate of 92%.
Conversely, if the cancer is not detected until a late stage (i.e.,
stage T4 (IV)), the five-year survival rate is reduced to 13%. AJCC
Cancer Staging Handbook pp. 164-65 (Irvin D. Fleming et al. eds.,
5.sup.th ed. 1998). Some detection techniques, such as mammography
and biopsy, involve increased discomfort, expense, and/or
radiation, and are only prescribed only to patients with an
increased risk of breast cancer.
[0019] Current methods for predicting or detecting breast cancer
risk are not optimal. One method for predicting the relative risk
of breast cancer is by examining a patient's risk factors and
pursuing aggressive diagnostic and treatment regiments for high
risk patients. A patient's risk of breast cancer has been
positively associated with increasing age, nulliparity, family
history of breast cancer, personal history of breast cancer, early
menarche, late menopause, late age of first full term pregnancy,
prior proliferative breast disease, irradiation of the breast at an
early age and a personal history of malignancy. Lifestyle factors
such as fat consumption, alcohol consumption, education, and
socioeconomic status have also been associated with an increased
incidence of breast cancer although a direct cause and effect
relationship has not been established. While these risk factors are
statistically significant, their weak association with breast
cancer limited their usefulness. Most women who develop breast
cancer have none of the risk factors listed above, other than the
risk that comes with growing older. NIH Publication No. 00-1556
(2000).
[0020] Current screening methods for detecting cancer, such as
breast self exam, ultrasound, and mammography have drawbacks that
reduce their effectiveness or prevent their widespread adoption.
Breast self exams, while useful, are unreliable for the detection
of breast cancer in the initial stages where the tumor is small and
difficult to detect by palpation. Ultrasound measurements require
skilled operators at an increased expense. Mammography, while
sensitive, is subject to over diagnosis in the detection of lesions
that have questionable malignant potential. There is also the fear
of the radiation used in mammography because prior chest radiation
is a factor associated with an increase incidence of breast
cancer.
[0021] At this time, there are no adequate methods of breast cancer
prevention. The current methods of breast cancer prevention involve
prophylactic mastectomy (mastectomy performed before cancer
diagnosis) and chemoprevention (chemotherapy before cancer
diagnosis), which are drastic measures that limit their adoption
even among women with, increased risk of breast cancer. Bevers,
supra.
[0022] A number of genetic markers have been associated with breast
cancer. Examples of these markers include carcinoembryonic antigen
(CEA) (Mughal et al., JAMA 249:1881 (1983)), MUC-1 (Frische and
Liu, J. Clin. Ligand 22:320 (2000)), HER-2/neu (Haris et al., Proc.
Am. Soc. Clin. Oncology 15:A96 (1996)), uPA, PAI-1, LPA, LPC, RAK
and BRCA (Esteva and Fritsche, Serum and Tissue Markers for Breast
Cancer, in Breast Cancer, 286-308 (2001)). These markers have
problems with limited sensitivity, low correlation, and false
negatives, which limit their use for initial diagnosis. For
example, while the BRCA1 gene mutation is useful as an indicator of
an increased risk for breast cancer, it has limited use in cancer
diagnosis because only 6.2% of breast cancers are BRCA1 positive.
Malone et al., JAMA 279:922 (1998). See also, Mewman et al., JAMA
279:915 (1998) (correlation of only 3.3%).
[0023] There are four primary classifications of breast cancer
varying by the site of origin and the extent of disease
development. [0024] I. Ductal carcinoma in situ (DCIS): Malignant
transformation of ductal epithelial cells that remain in their
normal position. DCIS is a purely localized disease, incapable of
metastasis. [0025] II. Invasive ductal carcinoma (IDC): Malignancy
of the ductal epithelial cells breaking through the basal membrane
and into the supporting tissue of the breast. IDC may eventually
spread elsewhere in the body. [0026] III. Lobular carcinoma in situ
(LCIS): Malignancy arising in a single lobule of the breast that
fail to extend through the lobule wall, it generally remains
localized. [0027] IV. Infiltrating lobular carcinoma (ILC):
Malignancy arising in a single lobule of the breast and invading
directly through the lobule wall into adjacent tissues. [0028] By
virtue of its invasion beyond the lobule wall, ILC may penetrate
lymphatics and blood vessels and spread to distant sites.
[0029] For purpose of determining prognosis and treatment, these
four breast cancer types have been staged according to the size of
the primary tumor (T), the involvement of lymph nodes (N), and the
presence of metastasis (M). Although DCIS by definition represents
localized stage I disease, the other forms of breast cancer may
range from stage II to stage IV. There are additional prognostic
factors that further serve to guide surgical and medical
intervention. The most common ones are total number of lymph nodes
involved, ER (estrogen receptor) status, Her2/neu receptor status
and histologic grades.
[0030] Breast cancers are diagnosed into the appropriate stage
categories recognizing that different treatments are more effective
for different stages of cancer. Stage TX indicates that primary
tumor cannot be assessed (i.e., tumor was removed or breast tissue
was removed). Stage T0 is characterized by abnormalities such as
hyperplasia but with no evidence of primary tumor. Stage Tis is
characterized by carcinoma in situ, intraductal carcinoma, lobular
carcinoma in situ, or Paget's disease of the nipple with no tumor.
Stage T1 (I) is characterized as having a tumor of 2 cm or less in
the greatest dimension. Within stage T1, Tmic indicates
microinvasion of 0.1 cm or less, T1a indicates a tumor of between
0.1 to 0.5 cm, T1b indicates a tumor of between 0.5 to 1 cm, and
T1c indicates tumors of between 1 cm to 2 cm. Stage T2 (II) is
characterized by tumors from 2 cm to 5 cm in the greatest
dimension. Tumors greater than 5 cm in size are classified as stage
T3 (III). Stage T4 (IV) indicates a tumor of any size with
extension to the chest wall or skin. Within stage T4, T4a indicates
extension of the tumor to the chess wall, T4b indicates edema or
ulceration of the skin of the breast or satellite skin nodules
confined to the same breast, T4c indicates a combination of T4a and
T4b, and T4d indicates inflammatory 5.sup.th carcinoma. AJCC Cancer
Staging Handbook pp. 159-70 (Irvin D. Fleming et al. eds., ed.
1998). In addition to standard staging, breast tumors may be
classified according to their estrogen receptor and progesterone
receptor protein status. Fisher et al., Breast Cancer Research and
Treatment 7:147 (1986). Additional pathological status, such as
HER2/neu status may also be useful. Thor et al., J. Nat'l. Cancer
Inst. 90:1346 (1998); Paik et al., J. Nat'l. Cancer Inst. 90:1361
(1998); Hutchins et al., Proc. Am. Soc. Clin. Oncology 17:A2
(1998); and Simpson et al., J. Clin. Oncology 18:2059 (2000).
[0031] In addition to the staging of the primary tumor, breast
cancer metastasizes to regional lymph nodes may be staged. Stage NX
indicates that the lymph nodes cannot be assessed (e.g., previously
removed). Stage N0 indicates no regional lymph node metastasis.
Stage N1 indicates metastasis to movable ipsilateral axillary lymph
nodes. Stage N2 indicates metastasis to ipsilateral axillary lymph
nodes fixed to one another or to other structures. Stage N3
indicates metastasis to ipsilateral internal mammary lymph nodes.
Id.
[0032] Stage determination has potential prognostic value and
provides criteria for designing optimal therapy. Simpson et al., J.
Clin. Oncology 18:2059 (2000). Generally, pathological staging of
breast cancer is preferable to clinical staging because the former
gives a more accurate prognosis. However, clinical staging would be
preferred if it were as accurate as pathological staging because it
does not depend on an invasive procedure to obtain tissue for
pathological evaluation. Staging of breast cancer would be improved
by detecting new markers in cells, tissues, or bodily fluids that
could differentiate between different stages of invasion. Progress
in this field will allow more rapid and reliable method for
treating breast cancer patients.
[0033] Treatment of breast cancer is generally decided after an
accurate staging of the primary tumor. Primary treatment options
include breast conserving therapy (lumpectomy, breast irradiation,
and surgical staging of the axilla), and modified radical
mastectomy. Additional treatments include chemotherapy, regional
irradiation, and, in extreme cases, terminating estrogen production
by ovarian ablation.
[0034] Until recently, the customary treatment for all breast
cancer was mastectomy. Fonseca et al., Annals of Internal Medicine
127:1013 (1997). However, recent data indicate that less radical
procedures may be equally effective, in terms of survival, for
early stage breast cancer. Fisher et al., J. of Clinical Oncology
16:441 (1998). The treatment options for a patient with early stage
breast cancer (i.e., stage Tis) may be breast-sparing surgery
followed by localized radiation therapy at the breast.
Alternatively, mastectomy optionally coupled with radiation or
breast reconstruction may be employed. These treatment methods are
equally effective in the early stages of breast cancer.
[0035] Patients with stage I and stage II breast cancer require
surgery with chemotherapy and/or hormonal therapy. Surgery is of
limited use in Stage III and stage IV patients. Thus, these
patients are better candidates for chemotherapy and radiation
therapy with surgery limited to biopsy to permit initial staging or
subsequent restaging because cancer is rarely curative at this
stage of the disease. AJCC Cancer Staging Handbook 84, . 164-65
(Irvin D. Fleming et al. eds., 5.sup.th ed. 1998).
[0036] In an effort to provide more treatment options to patients,
efforts are underway to define an earlier stage of breast cancer
with low recurrence that could be treated with lumpectomy without
postoperative radiation treatment. While a number of attempts have
been made to classify early stage breast cancer, no consensus
recommendation on postoperative radiation treatment has been
obtained from these studies. Page et al., Cancer 75:1219 (1995);
Fisher et al., Cancer 75:1223 (1995); Silverstein et al., Cancer
77:2267 (1996).
[0037] As discussed above, each of the methods for diagnosing and
staging ovarian, pancreatic, lung or breast cancer is limited by
the technology employed. Accordingly, there is need for sensitive
molecular and cellular markers for the detection of ovarian,
pancreatic, lung or breast cancer. There is a need for molecular
markers for the accurate staging, including clinical and
pathological staging, of ovarian, pancreatic, lung or breast
cancers to optimize treatment methods. Finally, there is a need for
sensitive molecular and cellular markers to monitor the progress of
cancer treatments, including markers that can detect recurrence of
ovarian, pancreatic, lung or breast cancers following
remission.
[0038] The present invention provides alternative methods of
treating ovarian, pancreatic, lung or breast 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
[0039] This invention is directed to an isolated Ovr110 antibody
that binds to Ovr110 on a mammalian cell in vivo. The invention is
further directed to an isolated Ovr110 antibody that internalizes
upon binding to Ovr110 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-5180, PTA-5855, PTA-5856 and
PTA-5884.
[0040] 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-5180,
PTA-5855, PTA-5856 and PTA-5884.
[0041] 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.
[0042] The mammalian cell may be a cancer cell. Preferably, the
anti-Ovr110 monoclonal antibody inhibits the growth of
Ovr110-expressing cancer cells in vivo.
[0043] The antibody may be produced in bacteria. Alternatively, the
antibody may be a humanized form of an anti-Ovr110 antibody
produced by a hybridoma selected from the group of hybridomas
having ATCC accession number PTA-5180, PTA-5855, PTA-5856 and
PTA-5884.
[0044] Preferably, the cancer is selected from the group consisting
of ovarian, pancreatic, lung and breast 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.
[0045] 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.
[0046] The invention is also directed to a method of killing an
Ovr110-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 ovarian, pancreatic, lung and breast cancer cell.
[0047] The ovarian, or breast cancer may be ovarian serous
adenocarcinoma or breast infiltrating ductal carcinoma or
metastatic cancer. The breast cancer may be HER-2 negative breast
cancer. The invention is also directed to a method of alleviating
an Ovr110-expressing cancer in a mammal, comprising administering a
therapeutically effective amount of the antibodies to the
mammal.
[0048] 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 ovarian, pancreatic, lung or breast
cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0049] FIG. 1 shows the results of FACS Analysis of Ovr110
Transfected Mouse LMTK Cells.
[0050] FIG. 2 shows immunofluorescence with Ovr110-A57.1 in live
ovarian and breast cancer cells
[0051] FIG. 3 shows Ovr110-A57.1 binding and internalization in
live ovarian and breast cancer cells.
[0052] FIG. 4 shows immunohistochemistry with Ovr110-A57.1 in
ovarian serous adenocarcinoma.
[0053] FIG. 5 shows immunohistochemistry with Ovr110-A57.1 in
breast infiltrating ductal Adenocarcinoma.
[0054] FIG. 6 shows immunohistochemistry with Ovr110-A57.1 in
pancreas adenocarcinoma.
[0055] FIG. 7: A-F show expression of B7 family members on day 3 in
PHA stimulated T-CELLS CD3 FITC gated, and; G-I show binding of
BTLA-Fc fusion protein to Ovr110-293F cells.
[0056] FIG. 8 A-C show Western blot detection of Ovr110 protein
with mAb A57.1 in cell lines and human tumor tissues.
[0057] FIG. 9 shows Ovr110 protein is not detected in extracts of
major organs.
[0058] FIG. 10 shows specific knockdown of Ovr110 mRNA in SKBR3
breast cancer cells.
[0059] FIG. 11 shows down-regulation of Ovr110 protein by siRNA in
SKBR3 cells.
[0060] FIG. 12 shows that knockdown of Ovr110 mRNA induces
apoptosis in SKBR3 cells.
[0061] FIG. 13 shows that knockdown of Ovr110 mRNA induces caspase
activity in SKBR3 cells.
[0062] FIG. 14 shows that overexpression of Ovr110 enhances tumor
xenograft growth.
[0063] FIG. 15 shows that overexpression of Ovr110 protects from
apoptosis.
[0064] FIG. 16 shows the Ovr110 epitope map for the different
antibodies.
[0065] FIG. 17 shows Ovr110 detection in serum of healthy donors
and cancer patients.
[0066] FIG. 18 shows Ovr110 detection of different types of ovarian
cancer and benign disease samples.
[0067] FIG. 19 shows the Receiver Operator Characteristic (ROC)
curves for detecting Ovr110.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
[0068] Human "Ovr110" as used herein, refers to a protein of 282
amino acids that is expressed on the cell surface as glycoprotein,
whose nucleotide and amino acid sequence sequences are as disclosed
in e.g., WO 00/12758, Cancer specific gene (CSG) Ovr110; WO
99/63088, Membrane-bound protein PRO1291; WO00/36107, Human ovarian
carcinoma antigen; WO 02/02624-A2 Human B7-like protein (B7-L), the
disclosures of which are hereby expressly incorporated by
reference. The amino acids 30-282 are presumably on the cell
surface. Ovr110 as used herein includes allelic variants and
conservative substitution mutants of the protein, which have Ovr110
biological activity.
[0069] Recently, a series of three independent publications have
identified Ovr110 in mouse and human as new member of the T-cell B7
family of co-stimulatory molecules, an important class of molecules
that very tightly regulate the activation/inhibition of T-cell
function. Prasad et al., B7S1, a novel B7 family member that
negatively regulates T cell activation, Immunity 18:863-73 (2003);
Sica et al., B7-H4, a molecule of the B7 family, negatively
regulates T cell immunity, Immunity 18:849-61 (2003); and Zang et
al., B7x: a widely expressed B7 family member that inhibits T cell
activation, Proc. Natl. Acad. Sci. USA 100:10388-92 (2003). The
predicted amino acid sequence of the mouse gene for B7S1 (Prasad
2003) was highly homologous to our previously identified Ovr110
molecule, and the predicted sequence of the human B7-H4/B7.times.
(Sica 2003; Zang 2003) molecules were identical to Ovr110. Indirect
immunofluorescent analysis by flow cytometry further confirmed the
binding of our Ovr110 monoclonal antibodies to activated
T-lymphocyte populations, as described by these authors.
[0070] Our findings that Ovr110 is apparently restricted to the
more aggressive ovarian and breast cancers make this cell surface
antigen an attractive target for immunotherapy of these and
possibly other tumor types.
[0071] 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.
[0072] An "isolated antibody" is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that 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.
[0073] 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. and .gamma. chains and four CH domains for lambda and kappa
isotypes. Each 6 L chain has at the N-terminus, a variable domain
(VL) followed by a constant domain (CL) at its other end.
[0074] The VL is aligned with the VH and the CL is aligned with the
first constant domain of the heavy chain (CHI).
[0075] 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.
[0076] 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.
[0077] 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 defines
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).
[0078] 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 (L1), 50-52 (L2) and
91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and 96-101 (H3)
in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0079] 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.
[0080] 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.
[0081] An "intact" antibody is one that 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.
[0082] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. Papain
digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments, and a residual "Fe" fragment, a
designation reflecting the ability to crystallize readily. The Fab
fragment consists of an entire L chain along with the variable
region domain of the H chain (VH), and the first constant domain of
one heavy chain (CHI). Each Fab fragment is monovalent with respect
to antigen binding, i.e., it has a single antigen-binding site.
Pepsin treatment of an antibody yields a single large F(ab').sub.2
fragment which roughly corresponds to two disulfide linked Fab
fragments having divalent antigen-binding activity and is still
capable of cross-linking antigen. Fab' fragments differ from Fab
fragments by having additional few residues at the carboxy terminus
of the 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').sub.2 antibody fragments originally were produced as
pairs of 8 Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0083] 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.
[0084] "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.
[0085] "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.
[0086] 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).
[0087] 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.
[0088] 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 Ovr110 will possess at least about 70%
homology with the native sequence Ovr110, 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.
[0089] 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.
[0090] "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).
[0091] "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).
[0092] As used herein, an anti-Ovr110 antibody that "internalizes"
is one that is taken up by (i.e., enters) the cell upon binding to
Ovr110 on a mammalian cell (i.e. cell surface Ovr110). The
internalizing antibody will of course include antibody fragments,
human or humanized antibody and antibody conjugate. For therapeutic
applications, internalization in vivo is contemplated. The number
of antibody molecules internalized will be sufficient or adequate
to kill an Ovr110-expressing cell, especially an Ovr110-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.
[0093] Whether an anti-Ovr110 antibody internalizes upon binding
Ovr110 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 Ovr110
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 Ovr110-expressing tumor
transplant or xenograft, or a mouse into which cells transfected
with human Ovr110 have been introduced, or a transgenic mouse
expressing the human Ovr110 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
Ovr110-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.
[0094] The faster the rate of internalization of the antibody upon
binding to the Ovr110-expressing cell in vivo, the faster the
desired killing or growth inhibitory effect on the target
Ovr110-expressing cell can be achieved, e.g., by a cytotoxic
immunoconjugate. Preferably, the kinetics of internalization of the
anti-Ovr110 antibodies are such that they favor rapid killing of
the Ovr110-expressing target cell. Therefore, it is desirable that
the anti-Ovr110 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-Ovr110 antibody in vivo. The antibody will
preferably be internalized into the cell within a few hours upon
binding to Ovr110 on the cell surface, preferably within 1 hour,
even more preferably within 15-30 minutes.
[0095] To determine if a test antibody can compete for binding to
the same epitope as the epitope bound by the anti-Ovr110 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, Ovr110-coated wells of a microtiter plate,
or Ovr110-coated sepharose beads, are pre-incubated with or without
candidate competing antibody and then a biotin-labeled anti-Ovr110
antibody of the invention is added. The amount of labeled
anti-Ovr110 antibody bound to the Ovr110 antigen in the wells or on
the beads is measured using avidin-peroxidase conjugate and
appropriate substrate.
[0096] Alternatively, the anti-Ovr110 antibody can be labeled,
e.g., with a radioactive or fluorescent label or some other
detectable and measurable label. The amount of labeled anti-Ovr110
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-Ovr-110 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-Ovr110 antibody
of the invention if the candidate competing antibody can block
binding of the anti-Ovr110 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.
[0097] An antibody having a "biological characteristic" of a
designated antibody, such as any of the monoclonal antibodies
Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1,
Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1),
Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1,
Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2,
Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1,
Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1,
Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1 and Ovr110.C17.1,
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, Ovr110.A7.1,
Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1,
Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1,
Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1,
Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4,
Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8,
Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13,
Ovr110.C14, Ovr110.C15, Ovr110.C16.1 and Ovr110.C17.1 will bind the
same epitope as that bound by Ovr110.A7.1, Ovr110.A10.1,
Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously
identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1,
Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1,
Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,
Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,
Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,
Ovr110.C16.1 and Ovr110.C17.1 (e.g. which competes for binding or
blocks binding of monoclonal antibody Ovr110.A7.1, Ovr110.A10.1,
Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously
identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1,
Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1,
Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,
Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,
Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,
Ovr110.C16.1 and Ovr110.C17.1 to Ovr110), be able to target an
Ovr110-expressing tumor cell in vivo and will internalize upon
binding to Ovr110 on a mammalian cell in vivo. Likewise, an
antibody with the biological characteristic of the Ovr110.A7.1,
Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1,
Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1,
Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1,
Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4,
Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8,
Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13,
Ovr110.C14, Ovr110.C15, Ovr110.C16.1 and Ovr110.C17.1 antibody will
have the same epitope binding, targeting, internalizing, tumor
growth inhibitory and cytotoxic properties of the antibody.
[0098] 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 Ovr110
protein disclosed herein. Methods for identifying antagonists of an
Ovr110 polypeptide may comprise contacting an Ovr110 polypeptide or
a cell expressing Ovr110 on the cell surface, with a candidate
antagonist antibody and measuring a detectable change in one or
more biological activities normally associated with the Ovr110
polypeptide.
[0099] An "antibody that inhibits the growth of tumor cells
expressing Ovr110" or a "growth inhibitory" antibody is one which
binds to and results in measurable growth inhibition of cancer
cells expressing or overexpressing Ovr110. Preferred growth
inhibitory anti-Ovr110 antibodies inhibit growth of
Ovr110-expressing tumor cells e.g., ovarian, pancreatic, lung or
breast 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-Ovr110 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.
[0100] 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 Ovr110. Preferably the cell is a tumor cell, e.g. an
ovarian, pancreatic, lung or breast 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.
[0101] 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.
[0102] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0103] "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)).
[0104] "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.
[0105] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may be
performed.
[0106] 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.
[0107] A "Ovr110-expressing cell" is a cell which expresses
endogenous or transfected Ovr110 on the cell surface. A
"Ovr110-expressing cancer" is a cancer comprising cells that have
Ovr110 protein present on the cell surface. A "Ovr110-expressing
cancer" produces sufficient levels of Ovr110 on the surface of
cells thereof, such that an anti-Ovr110 antibody can bind thereto
and have a therapeutic effect with respect to the cancer. A cancer
which "overexpresses" Ovr110 is one which has significantly higher
levels of Ovr110 at the cell surface thereof, compared to a
noncancerous cell of the same tissue type. Such overexpression may
be caused by gene amplification or by increased transcription or
translation. Ovr110 overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the Ovr110 protein present on the surface of a cell (e.g. via an
immunohistochemistry assay; FACS analysis). Alternatively, or
additionally, one may measure levels of Ovr110-encoding nucleic
acid or mRNA in the cell, e.g. via fluorescent in situ
hybridization; (FISH; see WO98/45479 published October, 1998),
Southern blotting, Northern blotting, or polymerase chain reaction
(PCR) techniques, such as real time quantitative PCR(RT-PCR). One
may also study Ovr110 overexpression by measuring shed antigen in a
biological fluid such as serum, e.g., using antibody-based assays
(see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990;
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)). Aside from the above assays, various in vivo assays are
available to the skilled practitioner. For example, one may expose
cells within the body of the patient to an antibody which is
optionally labeled with a detectable label, e.g. a radioactive
isotope, and binding of the antibody to 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. An Ovr110-expressing cancer includes ovarian, pancreatic,
lung or breast cancer.
[0108] 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.
[0109] "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
Ovr110-expressing cancer if, after receiving a therapeutic amount
of an anti-Ovr110 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-Ovr110 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.
[0110] 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).
[0111] 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.
[0112] "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.
[0113] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0114] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0115] "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.
[0116] 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..
[0117] 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.
[0118] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
an Ovr110-expressing cancer cell, either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of Ovr110-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.
(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0119] "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.
[0120] The term "epitope tagged" used herein refers to a chimeric
polypeptide comprising an anti-Ovr110 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).
[0121] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0122] 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.
[0123] 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.
[0124] "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.
[0125] The cell that produces an anti-Ovr110 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.
[0126] 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.
[0127] 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).
[0128] 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).
[0129] 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.
[0130] 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.
[0131] 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
[0132] The invention provides anti-Ovr110 antibodies. Preferably,
the anti-Ovr110 antibodies internalize upon binding to cell surface
Ovr110 on a mammalian cell. The anti-Ovr110 antibodies may also
destroy or lead to the destruction of tumor cells bearing
Ovr110.
[0133] It was not apparent that Ovr110 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 Ovr110 is
internalization competent upon binding by the anti-Ovr110
antibodies of the invention. Additionally, it was demonstrated that
the anti-Ovr110 antibodies of the present invention can
specifically target Ovr110-expressing tumor cells in vivo and
inhibit or kill these cells. These in vivo tumor targeting,
internalization and growth inhibitory properties of the anti-Ovr110
antibodies make these antibodies very suitable for therapeutic
uses, e.g., in the treatment of various cancers including ovarian,
pancreatic, lung or breast cancer. Internalization of the
anti-Ovr110 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.
[0134] The anti-Ovr110 antibodies of the invention also have
various non-therapeutic applications. The anti-Ovr110 antibodies of
the present invention can be useful for diagnosis and staging of
Ovr110-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
antibodies are also useful for purification or immunoprecipitation
of Ovr110 from cells, for detection and quantitation of Ovr110 in
vitro, e.g. in an ELISA or a Western blot, to kill and eliminate
Ovr110-expressing cells from a population of mixed cells as a step
in the purification of other cells. The internalizing anti-Ovr110
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.
[0135] 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-Ovr110 antibodies of the invention are also contemplated,
e.g., an anti-Ovr110 antibody which has the biological
characteristics of a monoclonal antibody produced by the hybridomas
accorded ATCC accession numbers PTA-5180, PTA-5855, PTA-5856 and
PTA-5884, specifically including the in vivo tumor targeting,
internalization and any cell proliferation inhibition or cytotoxic
characteristics. Specifically provided are anti-Ovr110 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, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210,
210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-282 of
human Ovr110.
[0136] Methods of producing the above antibodies are described in
detail below.
[0137] The present anti-Ovr110 antibodies are useful for treating
an Ovr110-expressing cancer or alleviating one or more symptoms of
the cancer in a mammal. Such a cancer includes ovarian, pancreatic,
lung or breast cancer, cancer of the urinary tract, lung cancer,
breast cancer, colon cancer, pancreatic cancer, and ovarian cancer,
more specifically, prostate adenocarcinoma, renal cell carcinomas,
colorectal adenocarcinomas, lung adenocarcinomas, lung squamous
cell carcinomas, and pleural mesothelioma. The cancers encompass
metastatic cancers of any of the preceding, e.g., ovarian,
pancreatic, lung or breast cancer metastases. The antibody is able
to bind to at least a portion of the cancer cells that express
Ovr110 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 Ovr110-expressing tumor cells or inhibit the
growth of such tumor cells, in vitro or in vivo, upon binding to
Ovr110 on the cell. Such an antibody includes a naked anti-Ovr110
antibody (not conjugated to any agent). Naked anti-Ovr110
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-Ovr110 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.
[0138] The invention provides a composition comprising an
anti-Ovr110 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-Ovr110 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-Ovr110 antibody of the invention, and a carrier.
The formulation may be a therapeutic formulation comprising a
pharmaceutically acceptable carrier.
[0139] Another aspect of the invention is isolated nucleic acids
encoding the internalizing anti-Ovr110 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.
[0140] The invention also provides methods useful for treating an
Ovr110-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal, comprising administering a therapeutically
effective amount of an internalizing anti-Ovr110 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 an Ovr110 expressing cell. Finally, the invention also
provides kits and articles of manufacture comprising at least one
antibody of this invention, preferably at least one internalizing
anti-Ovr110 antibody of this invention. Kits containing anti-Ovr110
antibodies find use in detecting Ovr-110 expression, or in
therapeutic or diagnostic assays, e.g., for Ovr110 cell killing
assays or for purification and/or immunoprecipitation of Ovr110
from cells. For example, for isolation and purification of Ovr110,
the kit can contain an anti-Ovr110 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 Ovr110 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-Ovr110 Antibodies
[0141] 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 Ovr110
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 Ovr110 lacking the membrane spanning sequence, or synthetic
peptides to selected portions of the protein.
[0142] Alternatively, cells expressing Ovr110 at their cell surface
(e.g. CHO or NIH-3T3 cells transformed to overexpress Ovr110;
ovarian, pancreatic, lung, breast or other Ovr110-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 Ovr110 are available as provided above. Ovr110 can
be produced recombinantly in and isolated from, prokaryotic cells,
e.g., bacterial cells, or eukaryotic cells using standard
recombinant DNA methodology. Ovr110 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.
[0143] Antibodies or binding proteins that bind to various tags and
fusion sequences are available as elaborated below. Other forms of
Ovr110 useful for generating antibodies will be apparent to those
skilled in the art.
[0144] Tags
[0145] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)). The FLAG-peptide (Hopp et al.,
BioTechnology, 6:1204-1210 (1988)) is recognized by an anti-FLAG M2
monoclonal antibody (Eastman Kodak Co., New Haven, Conn.).
Purification of a protein containing the FLAG peptide can be
performed by immunoaffinity chromatography using an affinity matrix
comprising the anti-FLAG M2 monoclonal antibody covalently attached
to agarose (Eastman Kodak Co., New Haven, Conn.). Other tag
polypeptides include the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
(Skinner et al., J. Biol. Chenz., 266:15163-15166 (1991)); and the
T7 gene protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)).
[0146] Polyclonal Antibodies
[0147] Polyclonal antibodies are preferably raised in animals,
preferably non-human animals, by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen
(especially when synthetic peptides are used) to a protein that is
immunogenic in the species to be immunized. For example, the
antigen can be conjugated to keyhole limpet hemocyanin (KLH),
serum, bovine thyroglobulin, or soybean trypsin inhibitor, using a
bifunctional or derivatizing agent, e.g., maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups. Conjugates also can be made
in recombinant cell culture as protein fusions.
[0148] 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.
[0149] Monoclonal Antibodies
[0150] 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)).
[0151] 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.
[0152] 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)).
[0153] 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).
[0154] 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.
[0155] 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.
[0156] 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).
[0157] 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.
[0158] 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.
[0159] Humanized Antibodies
[0160] 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.
[0161] 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)).
[0162] 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.
[0163] 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.
[0164] Various forms of a humanized anti-Ovr110 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.
[0165] Human Antibodies
[0166] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno.,
7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and Alternatively, phage display technology
(McCafferty et al., Nature 348:552-553 (1990)) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B-cell. Phage
display can be performed in a variety of formats, reviewed in,
e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in
Structural Biology 3:564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature,
352:624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.
12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905. As discussed above, human antibodies may also be
generated by in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275).
[0167] Antibody Fragments
[0168] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors. Various techniques have been
developed for the production of antibody fragments. Traditionally,
these fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods 24:107-117 (1992); and Brennan et al., Science,
229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. Fab, Fv and ScFv antibody
fragments can all be expressed in and secreted from E coli, thus
allowing the facile production of large amounts of these fragments.
Antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab).sub.2 fragments (Carter et al., Bio/Technology 10: 163-167
(1992)). According to another approach, F(ab)2 fragments can be
isolated directly from recombinant host cell culture. Fab and
F(ab)2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues are described in U.S.
Pat. No. 5,869,046. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. The
antibody of choice may also be a single chain Fv fragment (scFv).
See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. Fv and sFv are the only species with intact combining
sites that are devoid of constant regions; thus, they are suitable
for reduced nonspecific binding during in vivo use. sFv fusion
proteins may be constructed to yield fusion of an effector protein
at either the amino or the carboxy terminus of an sFv. See Antibody
Engineering, ed. Borrebaeck, supra. The antibody fragment may also
be a "linear antibody", e.g., as described in U.S. Pat. No.
5,641,870 for example. Such linear antibody fragments may be
monospecific or bispecific.
[0169] Bispecific Antibodies
[0170] 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
Ovr110 protein. Other such antibodies may combine an Ovr110 binding
site with a binding site for another protein. Alternatively, an
anti-Ovr110.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 (CD 16),
so as to focus and localize cellular defense mechanisms to the
Ovr110-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express Ovr110. These
antibodies possess an Ovr110-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).sub.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/Fca antibody is shown in WO98/02463. U.S.
Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0171] 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).
[0172] 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.
[0173] 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).
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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).
[0180] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0181] Multivalent Antibodies
[0182] 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.
[0183] Other Amino Acid Sequence Modifications
[0184] Amino acid sequence modification(s) of the anti-Ovr110
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-Ovr110 antibody are prepared by introducing appropriate
nucleotide changes into the anti-Ovr110 antibody nucleic acid, or
by peptide synthesis.
[0185] Such modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the anti-Ovr110 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-Ovr110 antibody,
such as changing the number or position of glycosylation sites.
[0186] A useful method for identification of certain residues or
regions of the anti-Ovr110 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-Ovr110
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 Ovr110 antigen.
[0187] 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-Ovr110
antibody variants are screened for the desired activity.
[0188] 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-Ovr110 antibody
with an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the
anti-Ovr110 antibody molecule include the fusion to the N- or
C-terminus of the anti-Ovr110 antibody to an enzyme (e.g. for
ADEPT) or a fusion to a polypeptide which increases the serum
half-life of the antibody.
[0189] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-Ovr110 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 Exemplary Preferred
Original 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; ile met; 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; ser Phe Val (V)
ile; leu; met; phe; ala; leu
[0190] 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.
[0191] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Any cysteine
residue not involved in maintaining the proper conformation of the
anti-Ovr110 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).
[0192] 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 Ovr110. 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.
[0193] 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).
[0194] Nucleic acid molecules encoding amino acid sequence variants
of the anti-Ovr110 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-Ovr110 antibody.
[0195] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0196] 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
[0197] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0198] The growth inhibitory effects of an anti-Ovr110 antibody of
the invention may be assessed by methods known in the art, e.g.,
using cells which express Ovr110 either endogenously or following
transfection with the Ovr110 gene. For example, the tumor cell
lines and Ovr110-transfected cells provided in Example 1 below may
be treated with an anti-Ovr110 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-Ovr110 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 Ovr110. Preferably, the
anti-Ovr110 antibody will inhibit cell proliferation of an
Ovr110-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-Ovr110 antibody at about 1
.mu.g/kg to about 100 mg/kg body weight results in reduction in
tumor size or tumor cell proliferation within about 5 days to 3
months from the first administration of the antibody, preferably
within about 5 to 30 days.
[0199] 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. Ovr110-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.
[0200] To screen for antibodies which bind to an epitope on Ovr110
bound by an antibody of interest, e.g., the Ovr110 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-Ovr110 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 Ovr110 can
be used in competition assays with the test antibodies or with a
test antibody and an antibody with a characterized or known
epitope.
[0201] 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 Ovr110-containing sample with a test antibody
and an antibody of this invention to form a mixture, the level of
Ovr110 antibody bound to Ovr110 in the mixture is then determined
and compared to the level of Ovr110 antibody bound in the mixture
to a control mixture, wherein the level of Ovr110 antibody binding
to Ovr110 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-Ovr110 antibody of this invention. The level of Ovr110
antibody bound to Ovr110 is determined by ELISA. The control may be
a positive or negative control or both. For example, the control
may be a mixture of Ovr110, Ovr110 antibody of this invention and
an antibody known to bind the epitope bound by the Ovr110 antibody
of this invention. The anti-Ovr110 antibody labeled with a label
such as those disclosed herein. The Ovr110 may be bound to a solid
support, e.g., a tissue culture plate or to beads, e.g., sepharose
beads.
Immunoconjugates
[0202] 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.
[0203] 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.
[0204] Maytansine and Maytansinoids
[0205] Preferably, an anti-Ovr110 antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0206] Maytansinoids are mitotic 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.
[0207] Maytansinoid-Antibody Conjugates
[0208] 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.
[0209] Anti-Ovr110 Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0210] Anti-Ovr110 antibody-maytansinoid conjugates are prepared by
chemically linking an anti-Ovr110 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.
[0211] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al. Cancer Research 52: 127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred. Conjugates of the antibody and
maytansinoid may be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl (2-pyridyldithio) propionate
(SPDP), succinimidyl-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as 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.
[0212] 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.
[0213] Calicheamicin
[0214] Another immunoconjugate of interest comprises an anti-Ovr110
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
[0215] Other antitumor agents that can be conjugated to the
anti-Ovr110 antibodies of the invention include BCNU,
streptozoicin, vincristine and 5-fluorouracil, the family of agents
known collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296). Enzymatically active toxins and fragments thereof which
can be used include diphtheria A chain, 1 5 nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin
and the tricothecenes. See, for example, WO 93/21232 published Oct.
28, 1993. The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0216] 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-Ovr110 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.
[0217] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
Tc.sup.99M, I.sup.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.
[0218] 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.
[0219] Alternatively, a fusion protein comprising the anti-Ovr110
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.
[0220] 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)
[0221] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0222] 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; .beta.-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-Ovr110 antibodies by techniques well
known in the art such as the use of the heterobifunctional
crosslinking reagents discussed above.
[0223] 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
[0224] 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).
[0225] The anti-Ovr110 antibodies disclosed herein may also be
formulated as immunoliposomes. A "liposome" is a small vesicle
composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
Vectors, Host Cells, and Recombinant Methods
[0226] The invention also provides isolated nucleic acid molecule
encoding the humanized anti-Ovr110 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.
[0227] Signal Sequence Component
[0228] The anti-Ovr110 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-Ovr110 antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, oc factor leader (including
Saccharomyces and Kluyveromyces cc-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the
signal described in WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available. The DNA for
such precursor region is ligated in reading frame to DNA encoding
the anti-Ovr110 antibody.
[0229] Origin of Replication
[0230] 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).
[0231] Selection Gene Component
[0232] 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.
[0233] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-Ovr110 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).
[0234] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-Ovr110 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.
[0235] 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.
[0236] 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).
[0237] Promoter Component
[0238] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-Ovr110 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-Ovr110 antibody.
[0239] 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.
[0240] 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.
[0241] Anti-Ovr110 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.
[0242] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human P-interferon cDNA
in mouse cells under the control of a thymidine kinase promoter
from herpes simplex virus. Alternatively, the Rous Sarcoma Virus
long terminal repeat can be used as the promoter.
[0243] Enhancer Element Component
[0244] Transcription of a DNA encoding the anti-Ovr110 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-Ovr110 antibody-encoding sequence, but is preferably located
at a site 5' from the promoter.
[0245] Transcription Termination Component
[0246] 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-Ovr110 antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO 94/11026 and the expression vector disclosed therein.
[0247] Selection and Transformation of Host Cells
[0248] 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.
[0249] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fe 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.
[0250] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-Ovr110 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.
[0251] Suitable host cells for the expression of glycosylated
anti-Ovr110 antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0252] 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.
[0253] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0254] Host cells are transformed with the above-described
expression or cloning vectors for anti-Ovr110 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
Culturing Host Cells
[0255] The host cells used to produce the anti-Ovr110 antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's FIO (Sigma), Minimal Essential Medium
(MEM)(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium (DMEM)(Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0256] Purification of Anti-Ovr110 Antibody
[0257] 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.
[0258] 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.
[0259] 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
[0260] 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.
[0261] 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-Ovr110 antibody which internalizes, it may be desirable to
include in the one formulation, an additional antibody, e.g. a
second anti-Ovr110 antibody which binds a different epitope on
Ovr110, 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.
[0262] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatinmicrocapsules 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).
[0263] 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.
[0264] 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-Ovr110 Antibodies
[0265] According to the present invention, the anti-Ovr110 antibody
that internalizes upon binding Ovr110 on a cell surface is used to
treat a subject in need thereof having a cancer characterized by
Ovr110-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.
[0266] The cancer will generally comprise Ovr110-expressing cells,
such that the anti-Ovr110 antibody is able to bind thereto. While
the cancer may be characterized by overexpression of the Ovr110
molecule, the present application further provides a method for
treating cancer which is not considered to be an
Ovr110-overexpressing cancer.
[0267] This invention also relates to methods for detecting cells
which overexpress Ovr110 and to diagnostic kits useful in detecting
cells expressing Ovr110 or in detecting Ovr110 in serum from a
patient. The methods may comprise combining a cell-containing test
sample with an antibody of this invention, assaying the test sample
for antibody binding to cells in the test sample and comparing the
level of antibody binding in the test sample to the level of
antibody binding in a control sample of cells. A suitable control
is, e.g., a sample of normal cells of the same type as the test
sample or a cell sample known to be free of Ovr110 overexpressing
cells. A level of Ovr110 binding higher than that of such a control
sample would be indicative of the test sample containing cells that
overexpress Ovr110. Alternatively the control may be a sample of
cells known to contain cells that overexpress Ovr110. In such a
case, a level of Ovr110 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
Ovr110.
[0268] Ovr110 overexpression may be detected with a various
diagnostic assays. For example, over expression of Ovr110 may be
assayed by immunohistochemistry (IHC). Parrafin embedded tissue
sections from a tumor biopsy may be subjected to the IHC assay and
accorded an Ovr110 protein staining intensity criteria as
follows.
[0269] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0270] 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.
[0271] Score 2+ a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0272] Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0273] Those tumors with 0 or 1+ scores for Ovr110 expression may
be characterized as not overexpressing Ovr110, whereas those tumors
with 2+ or 3+ scores may be characterized as overexpressing
Ovr110.
[0274] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Arizona) or PATHVISION.TM. (VySiS,
Illinois) may be carried out on formalin-fixed, paraffin-embedded
tumor tissue to determine the extent (if any) of Ovr110
overexpression in the tumor. Ovr110 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 Ovr110 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.
[0275] A sample suspected of containing cells expressing or
overexpressing Ovr110 is combined with the antibodies of this
invention under conditions suitable for the specific binding of the
antibodies to Ovr110. Binding and/or internalizing the Ovr110
antibodies of this invention is indicative of the cells expressing
Ovr110. The level of binding may be determined and compared to a
suitable control, wherein an elevated level of bound Ovr110 as
compared to the control is indicative of Ovr110 overexpression. The
sample suspected of containing cells overexpressing Ovr110 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 Ovr110 by combining a serum
sample from a subject with an Ovr110 antibody of this invention,
determining the level of Ovr110 bound to the antibody and comparing
the level to a control, wherein an elevated level of Ovr110 in the
serum of the patient as compared to a control is indicative of
overexpression of Ovr110 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.
[0276] 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-Ovr110 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-Ovr110 antibodies of the invention are useful to alleviate
Ovr110-expressing cancers, e.g., ovarian, pancreatic, lung or
breast cancers upon initial diagnosis of the disease or during
relapse. For therapeutic applications, the anti-Ovr110 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-Ovr110 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. (palictaxel), 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-Ovr110 antibody in conjuction with treatment with
the one or more of the preceding chemotherapeutic agents. In
particular, combination therapy with palictaxel and modified
derivatives (see, e.g., EP0600517) is contemplated. The anti-Ovr110
antibody will be administered with a therapeutically effective dose
of the chemotherapeutic agent. The anti-Ovr110 antibody may also be
administered in conjunction with chemotherapy to enhance the
activity and efficacy of the chemotherapeutic agent, e.g.,
paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages
of these agents that have been used in treatment of various
cancers. The dosing regimen and dosages of these aforementioned
chemotherapeutic drugs that are therapeutically effective will
depend on the particular cancer being treated, the extent of the
disease and other factors familiar to the physician of skill in the
art and can be determined by the physician.
[0277] Particularly, an immunoconjugate comprising the anti-Ovr110
antibody conjugated with a cytotoxic agent may be administered to
the patient. Preferably, the immunoconjugate bound to the Ovr110
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.
[0278] The anti-Ovr110 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,
intracerobrospinal, 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-Ovr110 antibody.
[0279] 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.
[0280] It may also be desirable to combine administration of the
anti-Ovr110 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 Ovr110-expressing tumor cells. The
cocktail may also comprise antibodies that are directed to other
epitopes of Ovr110. Preferably the other antibodies do not
interfere with the binding and or internalization of the antibodies
of this invention.
[0281] The antibody therapeutic treatment method of the present
invention may involve the combined administration of an anti-Ovr110
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).
[0282] 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-Ovr110 antibody (and optionally other agents
as described herein) may be administered to the patient.
[0283] 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-Ovr110
antibody.
[0284] 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-Ovr110 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.
[0285] 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.
[0286] 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.
[0287] 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
[0288] The invention also relates to an article of manufacture
containing materials useful for the detection for Ovr110
overexpressing cells and/or the treatment of Ovr110 expressing
cancer, in particular ovarian, pancreatic, lung or breast 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
Ovr110 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-Ovr110 antibody of the invention. The label
or package insert indicates that the composition is used for
detecting Ovr110 expressing cells and/or for treating ovarian,
pancreatic, lung or breast cancer, or more specifically ovarian
serous adenocarcinoma or breast infiltrating ductal carcinoma
cancer, in a patient in need thereof. 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.
[0289] Kits are also provided that are useful for various purposes,
e.g., for Ovr110 cell killing assays, for purification or
immunoprecipitation of Ovr110 from cells or for detecting the
presence of Ovr110 in a serum sample or detecting the presence of
Ovr110-expressing cells in a cell sample. For isolation and
purification of Ovr110, the kit can contain an anti-Ovr110 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 Ovr110 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
[0290] The following MAb/hybridomas of the present invention are
described below:
[0291] Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1,
Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1),
Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1,
Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2,
Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1,
Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1,
Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1 and Ovr110.C17.1.
If the MAb has been cloned, it will get the nomenclature "X.1,"
e.g., the first clone of A7 will be referred to as A7.1, the second
clone of A7 will be referred to as A7.2, etc. For the purposes of
this invention, a reference to A7 will include all clones, e.g.,
A7.1, A7.2, etc.
Immunogens and Antigens (Recombinant Proteins, HA & His Tags
& Transfected Cells)
[0292] Ovr110A Sequence & Protein Production
[0293] A full length DNA encoding the entire immature Ovr110
protein sequence from Men to Lys282 (SEQ ID NO:1) was inserted into
a modified vector comprising a nucleotide sequence encoding a 17
amino acid secretion signal sequence from human stanniocalcin (STC)
and a sequence encoding a 6 His tag, to generate a vector encoding
a recombinant Ovr110 fusion protein having the secretion signal
fused to the N-terminus and the 6 His-tag fused to the C-terminus
of the Ovr110 protein. The resulting vector was used produce the
recombinant protein using standard methods. Briefly, cells
transformed with the resulting vectors were cultured under
conditions suitable for production of the recombinant Ovr110
protein. The transformed cells were washed with Dulbecco's
phosphate buffered saline (DPBS) and lysed in 5 volumes (5 ml/g
cells) of 50 mM sodium phosphate, pH 8.0, containing 0.8 M sodium
chloride, 0.3% Zwittergent 3-14 and 0.1% octyl phosphoglucoside by
sonication. Insoluble material was isolated as a precipitate and
the extraction was repeated twice. The isolated precipitate was
dissolved in 50 mM sodium phosphate buffer, pH 7.8, containing 6 M
guanidine hydrochloride (3 ml/g cells) and circulated through a
10-ml-Ni-NTA (Qiagen, Alameda, Calif.) column equilibrated with the
same buffer on an Akta-100 system (Amersham Biosciences,
Piscataway, N.J.) for about 40 column volumes (CV) at the flow rate
of 5 ml/min. The column was then washed with 2 CV of the same
phosphate-guanidine buffer, 2 CV of the 20 mM imidazole, 2 CV of 50
mM imidazole, and 4 CV of 100 mM imidazole in the above
phosphate-guanidine buffer.
[0294] Ovr110A was eluted with 4 CV of 500 mM imidazole in
phosphate-guanidine buffer and the column was further washed with 4
CV of 50 mM sodium phosphate, pH 7.6, containing 1 M imidazole and
6 M guanidine hydrochloride. Samples from collected fractions were
subjected to SDS-PAGE and Western blot analysis for assessing the
purity of Ovr110A. Purified fractions were pooled and dialyzed
against PBS. Precipitates were collected and re-suspended in
smaller volume of PBS by brief sonication.
##STR00001##
[0295] Ovr110B Sequence & Protein Production
[0296] For immunization of mice, a recombinant protein fragment of
Ovr110 was generated, which constituted only the predicted
extracellular portion of the molecule, in order to select for
monoclonal antibodies (MAb) that would bind to the exterior cell
surface. A DNA fragment encoding the Ovr110 sequence from Gly30
(underlined in the sequence below) to Lys282 (plus a Met at the
start codon position) of the immature protein, including the signal
peptide, was inserted into a modified vector, which contained a
nucleotide sequence encoding a 17 amino acid secretion signal
sequence from human stanniocalcin (STC) and a nucleotide sequence
encoding a 6 His tag such that the vector encoded a recombinant
Ovr110 fusion protein having the 17 amino acid secretion signal
sequence from human stanniocalcin (STC) fused to the N-terminus and
the 6 His-tag fused to the C-terminus of the Ovr110 protein
(Ovr110B). The resulting vector was used to transform DH10Bac
bacteria for generation of the infection vector by transposition.
Recombinant baculovirus were then generated by transfection of Sf9
cells with the transposed vector. Recombinant Ovr110B was expressed
by infection of Hi5 cell line with the amplified and harvested
virus particles.
[0297] Culture media from the recombinant Hi5 cells were harvested
at 48 hr post-infection. The media were concentrated 10 fold and
diafiltrated with 30 volumes of PBS, pH 7.9. The diafiltrated
material was then incubated with 10 ml of Ni-NTA fast-flow gel
(Qiagen) overnight at 4.degree. C. in the presence of
protease-inhibitor-cocktail. The gels were poured into a SK column
and washed with 2 CV of 50 mM sodium phosphate, pH7.8, containing
0.5 M sodium chloride. Ovr110B was eluted by step-increasing of
imidazole in the same phosphate-sodium chloride buffer (4 CV of 20
mM, 4 CV of 50 mM, 4 CV of 100 mM, 4 CV of 500 mM and 2 CV of 1000
mM). Samples from collected fractions were subjected to SDS-PAGE
and Western blot analysis for assessing the purity of Ovr110B.
Purified fractions were pooled and concentrated. Final products
were dialyzed in PBS.
##STR00002##
[0298] Sequence & Protein Production for Mammalian Cell
Expressed Ovr110:
[0299] A nucleic acid molecule encoding Ovr110 from Gly30 to Lys282
was generated from a shuttle vector containing a full length Ovr110
cDNA (pDONR201_Ovr110) by producing a PCR fragment using following
oligonucleotide primers:
TABLE-US-00002 ATN496: (SEQ ID NO: 3) 5'-CCA ATG CAT GGT ATT TCA
GGG AGA CAC TCC ATN552: (SEQ ID NO: 4) 5'-CG GCT AGC TTT TAG CAT
CAG GTA AGG GCT G.
[0300] The PCR fragment was digested with NsiI and NheI, and cloned
in-frame into a modified mammalian expression pCMV5His2 vector
comprising a nucleotide sequence encoding a human stanniocalcin 1
(STC-1) secretion signal and nucleotide sequence encoding a ten
histidine tag to produce the recombinant plasmid pCMV5jos2_Ovr110
which encoded a recombinant Ovr110 protein having the human
stanniocalcin 1 (STC-1) secretion signal fused to the NH2 terminus
and a ten histidine tag fused to the COOH terminus, respectively.
DNA sequence analysis was performed using an ABI Prism Big Dye
terminator cycle sequencing ready reaction kit from PE Applied
Biosystems (Foster City, Calif.).
[0301] The recombinant plasmid, pCMV5His2_Ovr110, was used to
transfect 293T cells in suspension culture (one liter serum free
medium) in a spinner flask.
[0302] Culture medium was harvested at 48 hours post-transfection.
Medium was concentrated 10-fold, and diafiltered with 100 mM sodium
phosphate, 400 mM NaCl, 10% glycerol, pH 8.0. Concentrated medium
containing Ovr110 was passed over a 5-mL nickel metal chelating
column (Ni-NTA fast flow, Qiagen Inc.), which had been previously
equilibrated with 100 mM sodium phosphate, 400 mM NaCl, 10%
glycerol, pH 8.0. Column was then washed with 6 column volume (CV)
of 100 mM sodium phosphate, 400 mM NaCl, 2 mM imidazole, 10%
glycerol, pH 8.0. Ovr110 was eluted from the column using 22 CV of
100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0
containing 5 mM imidazole and 500 mM imidazole, respectively.
Fractions containing Ovr110 were pooled and dialyzed in 100 mM
sodium phosphate, 400 mM NaCl, 5% glycerol, pH 7.5.
TABLE-US-00003 Ovr110 with STC-1 secretion signal (Ovr110 sequence
is underlined) (SEQ ID NO: 5)
MLQNSAVLLVLVISASATHEAEQSRMHGISGRHSTTVTTVASAGNIGE
DGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFR
GRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYK
TGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEV
SNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTE
SEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLKASHHHHHH HHHH
[0303] BTLA Sequence & Protein Production:
[0304] A nucleic acid molecule encoding a full length human BTLA
(hBTLA), from Met1 to Ser289, was cloned by PCR from the pituitary
gland and lymph node cDNA libraries using the following
oligonucleotide primers:
TABLE-US-00004 ATN551: (SEQ ID NO: 6) 5'-CTT TGT TTA AAC ATG AAG
ACA TTG CCT GCC ATG and ATN552: (SEQ ID NO: 7) 5'-CG GCT AGC ACT
CCT CAC ACA TAT GGA TGC.
[0305] A truncated hBTLA gene encoding Met1-Pro 152, encompassing
the surface immunoglobulin (Ig) domain, was cloned by PCR from a
Burkitt's lymphoma cDNA library using the following oligonucleotide
primers:
[0306] ATN551: (see sequence above) SEQ ID NO: 6 and
TABLE-US-00005 ATN554: (SEQ ID NO: 8) 5'-CG GCT AGC GGG TCT GCT TGC
CAC TTC GTC.
[0307] A nucleic acid molecule encoding a full length secreted
form, lacking the transmembrane domain, of hBTLA, from Met1-Ser241,
was cloned by PCR from a lymph node cDNA library using
oligonucleotide primers ATN551 and ATN552. The PCR fragments were
digested with PmeI and NheI and ligated either into pCMV5HIS2 or
pCMV5Fc1, which had been cut with the same enzymes, to generate
protein constructs that had a C-terminal extension AS-HHHHHHHHHH or
AS-mouse Fc domain (mFc), respectively. DNA sequence analysis was
performed using an ABI Prism BigDye terminator cycle sequencing
ready reaction kit from PE Applied Biosystems (Foster City,
Calif.).
[0308] The recombinant plasmid, pCMV5Fc1_BTLA5NT, which encoded
only the surface Ig domain of hBTLA fused to mFc (BTLA5NT_mFc), was
used to transfect 293T cells in suspension culture (one liter serum
free medium) in a spinner flask. Culture medium was harvested at 48
hours post-transfection. Sodium chloride was added to 3M final to
the harvested medium, and medium was adjusted to pH 8.0.
BTLA-containing medium was then passed over a 5-mL recombinant
protein A column, which had been previously equilibrated with 10
column volume (CV) of 50 mM borate, 4M NaCl, pH 8.0. Protein A
column was then washed with 30 CV of 50 mM borate, 4M NaCl, pH8.0.
BTLA5NT_mFc eluted from protein A column using 10 CV of 100 mM
citrate, pH 3.0. Fractions containing BTLA5NT-mFc was neutralized
with 1M Tris-HCl, pH 9.0, and dialyzed in 3 L PBS, pH 7.5.
TABLE-US-00006 BTLA sequence, full length (SEQ ID NO: 9)
MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSI
LAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNIS
FFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPS
KDEMASRPWLLYSLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTA
GREINLVDAHLKSEQTEASTRQNSQVLLSETGTYDNDPDLCFRMQEGS
EVYSNPCLEENKPGIVYASLNHSVIGLNSRLARNVKEAPTEYASICVR S BTLA, secreted
form (SEQ ID NO: 10)
MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSI
LAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNIS
FFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTGKQNELSDTA
GREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGS
EVYSNPCLEENKPGIVYASLNHSVIGLNSRLARNVKEAPTEYASICVR S BTLA5NT_mFc
(BTLA sequenced is underlined) (SEQ ID NO: 11)
MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSI
LAGDPFELECRVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNIS
FFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPS
KDEMASRPASENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPK
IKDVLMISLSPIVTCVVVDVSEDPDVQISWFVNNVEVHTAQTQTHRED
YNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGVR
APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYK
NTEPVLDSDGSYFMYSKLRVEKNWVERNSYSCSVVHEGLHNHHTTKSF SRTPGK
[0309] Ovr107 Sequence & Protein Production
[0310] A recombinant Ovr107 protein was used to screen out poly
reactive hybridoma clones. Ovr107 is upregulated widely in multiple
cancers and the recombinant Ovr107 used herein contains a
potentially cross reactive hexahistidine tag. Thus the recombinant
Ovr107 is useful for identifying polyreactive antibodies.
[0311] A full length cDNA encoding the Ovr107 sequence from Met1 to
Ile596 (WO 01/37864 Human Ovr107 ovarian cancer marker) was cloned
by PCR and inserted into a vector. The Ovr107 coding region was
then transferred by recombination into a vector comprising a
nucleotide sequence encoding a 6 His tag such that a Ovr107 fusion
protein having a 6 His-tag fused to its C-terminal was generated.
The resulting vector was used to transform DH10Bac bacteria for
generation of the infection vector by transposition. Recombinant
baculovirus was then generated by transfection of Sf9 cells with
the transposed vector. Recombinant Ovr107 was expressed by
infection of Sf9 or Hi5 cell lines with the amplified and harvested
recombinant baculovirus particles.
[0312] Recombinant baculovirus infected Hi5 cells were harvested 48
hr post-infection. The cells were washed with DPBS and lysed in (5
ml/g cells) 100 mM sodium phosphate, pH 8.0, containing 0.4 M
sodium chloride, 10% glycerol, 1% Triton X-100 and 10 mM imidazole
by sonication. The extract was incubated with 10 mg of DNase at
room temperature for 30 minutes and then centrifuged in a SS-34
rotor at 17,000 rpm for 30 minutes. The supernatant was further
filtered through a 45 nm filter and loaded onto a 5-ml-Ni-NTA
column (Qiagen) equilibrated with 0.1 M sodium phosphate, pH 8.1,
containing 0.4 M sodium chloride and 10% glycerol at the flow rate
of 3 ml/min. The column was washed with 15 column volumes (CV) of
the same equilibrating buffer and Ovr107 was eluted by
step-increasing of imidazole in the phosphate-sodium chloride
buffer (10 CV of 20 mM, 10 CV of 50 mM, 10 CV of 100 mM, 5 CV of
500 mM and 5 CV of 1000 mM). Fractions were collected in 5 ml/tube
and samples from collected fractions were subjected to SDS-PAGE and
Western analysis for assessing the purity of Ovr107. Purified
fractions were pooled and concentrated. Final products were
dialyzed in PBS.
TABLE-US-00007 Ovr107 Amino Acid Sequence with HA Tag (SEQ ID NO:
12) MNRTWPRRIWGSSQDEAELIREDIQGALHNYRSGRGERRAAALRATQE
ELQRDRSPAAETPPLQRRPSVRAVISTVERGAGRGRPQAKPIPEAEEA
QRPEPVGTSSNADSASPDLGPRGPDLVVLQAEREVDILNHVFDDVESF
VSRLQKSAEAARVLEHRERGRRSRRPAAGEGLLTLRAKPPSEAEYTDV
LQKIKYAFSLLARLRGNIADPSSPELLHFLFGPLQMIVNTSGGPEFAS
SVRRPHLTSDAVALLRDNVTPRENELWTSLGDSWTRPGLELSPEEGPP
YRPEFFSGWEPPVTDPQSRAWEDPVEKQLQHERRRRQQSAPQVAVNGH
RDLEPESEPQLESETAGKWVLCNYDFQARNSSELSVKQRDVLEVLDDS
RKWWKVRDPAGQEGYVPYNILTPYPGPRLHHSQSPARSLNSTPPPPPA
PAPAPPPALARPRWDRPRWDSCDSLNGLDPSEKEKFSQMLIVNEELQA
RLAQGRSGPSRAVPGPRAPEPQLSPGSDASEVRAWLQAKGFSSGTVDA
LGVLTGAQLFSLQKEELRAVSPEEGARVYSQVTVQRSLLEDKEKVSEL
EAVMEKQKKKVEGEVEMEVIDPAFLYKVVRWAHHHHHH
[0313] Generation of Stable LMTK Mouse Cell Lines
[0314] A mammalian vector encoding a HA-tagged Ovr110 was
transfected into mouse LMTK cells. Stable transfectants were
selected in Dulbecco's modified Eagle's medium (DMEM)/10% FBS, with
blastocidin at 10 ug/mL, for 7-10 days, followed by single cell
sorting (Coulter Elite, Beckmann Coulter, Sunnyvale, Calif.) based
on fluorescence, at 1 cell/well in 96 well plates. Transfected LMTK
cells were grown cells in 96 well plates (VWR, Brisbane, Calif.),
expanded into 24 well plates and subsequently into 6 well plates.
After one week in culture, individual clones were assayed for
expression of Ovr110 by Western blot using anti-HA antibody
(Covance, Richmond, Calif.). Two LMTK cell clones expressing the
highest level of Ovr110-HA were expanded into 75 cm.sup.2 flasks
(VWR) for screening of hybridomas, 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.
##STR00003##
[0315] Generation of Transient 293F Transfected Cells
[0316] A nucleic acid molecule encoding Ovr110 (SEQ ID NO: 13),
without the HA tag was cloned into the mammalian expression vector,
PcDNA3.1, and the recombinant vector was used to transfect human
293F cells (Invitrogen). Fifty ml of 293F cells cultured in
freestyle medium (GIBCO) at 10.sup.6 cells/ml were transfected
using 293fectin transfection reagent (Invitrogen), according to the
manufacturer's guidelines. DNA, cells and 293fectin were mixed in
OPTI-MEM medium (GIBCO). Cells were used for analysis 48 h after
transfection.
[0317] Immunization
[0318] For the A-series MAb fusion, mice were immunized with
soluble Ovr110B recombinant protein, which corresponds to the
extracellular domain of the native protein, in order to generate
MAbs of both in-vivo therapeutic and diagnostic utility. For the
C-series MAb fusion, mice were immunized with the mammalian
expressed extracellular domain. Groups of 8 BALB/c mice were
immunized intradermally in both rear footpads. All injections were
25 uL per foot. The first injection (day 1) of 10 ug of 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, Miss.). 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.
[0319] Hybridoma Fusion
[0320] 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).
[0321] These primary B-cell enriched lymph node cells were then
immortalized by electro-cell fusion (BTX, San Diego, Calif.) with
the continuous myeloma cell line P3x63Ag8.653 (Kearney, J. F. et
al., J. Immunology 123: 1548-1550, 1979). Successfully fused cells
were selected by culturing in standard Hypoxanthine, Azaserine (HA)
(Sigma) 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 Ovr110B, Ovr110A and no
cross-reactivity with an irrelevant protein (Ovr107).
[0322] 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.
[0323] Screening & Selection of Antibody Producing
Hybridomas
[0324] Hybridoma cell lines were selected for production of Ovr110
specific antibody by enzyme linked solid phase immunoassay (ELISA).
Ovr110B or Ovr107 proteins were nonspecifically adsorbed to wells
of 96 well polystyrene EIA plates (VWR). Fifty uL of Ovr110B
protein or peptide-BSA conjugate at 0.91 mg/mL in (DPBS) were
incubated overnight at 4.degree. C. in wells of 96 well polystyrene
EIA plates. Plates were washed twice with Tris buffered saline with
0.05% Tween 20, pH 7.4 (TBST). The plate wells were then emptied
and nonspecific binding capacity was blocked by completely filling
the 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 uL 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 uL of alkaline
phosphatase conjugated goat anti-mouse IgG (Fc) (Pierce Chemical
Co., Rockford, Ill.), diluted 1:5000 in TBST/BSA, was then added to
each well and incubated for 1 hour at RT. The wells were then
washed 3 times with TBST. One hundred uL of alkaline phosphatase
substrate para-nitrophenylphosphate (pNPP) (Sigma) at 1 mg/mL in 1
M Diethanolamine buffer pH 8.9 (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.
ELISA Screening of Ovr110 MAbs
[0325] After 2 weeks culture, hybridomas with supernatants
producing ELISA absorbance values greater than 1.0 with Ovr110B and
less than 0.2 with Ovr107, were re-arrayed from twenty-five 96 well
culture plates, into new 96 well culture plates and cultured for a
further week.
[0326] After a further week of culture, 12 hybridomas from the
A-series and 15 from the C-series, with supernatants producing
ELISA absorbance values greater than 1.0 with Ovr110B (Tables 1A
& 1B) and less than 0.2 with Ovr107, were selected for single
cell cloning into 96 well culture plates, by cell sorting (Coulter
Elite).
TABLE-US-00008 TABLE 1A RESULTS OF TESTING SINGLE CELL CLONES OF
Ovr110 A-SERIES MAbs ELISA OD Outgrowth (405 nm) (# clones/ Mab
Clone Original Original Plate 96 well Plating ELISA OD Clone # Well
# Well # Density plate) Method (405 nm) A7.1 2.3172 1 1 cell/well 3
Sorter 3.5388 A7.2 2.1940 3 1 cell/well Sorter 3.7160 A10.1 1.5391
1 1 cell/well 2 Sorter 3.1965 A10.2 3.9733 G10 1 cell/well Sorter
2.2502 A13.1 2.0736 2 1 cell/well 18 Sorter 3.3627 A13.2 2.0000 3 1
cell/well Sorter 3.5381 A31.1 2.7208 1 1 cell/well 8 Sorter 3.6109
A31.2 2.4506 2 1 cell/well Sorter 3.0818 A57.1 2.8313 1 1 cell/well
27 Sorter 3.6099 A57.2 2.7821 3 1 cell/well Sorter 3.9733 A72.1
2.6737 1 1 cell/well 13 Sorter 3.6999 A72.2 2.6059 5 1 cell/well
Sorter 4.0000 A77.1 1.6650 1 1 cell/well 2 Sorter 1.5370 A77.2
1.8328 4 1 cell/well 3 Sorter 1.6186 A102.1 2.1280 2 1 cell/well 4
Sorter 1.1054 A102.2 1.4710 3 1 cell/well Sorter 1.0121 A87.1
2.1396 3 1 cell/well 13 Sorter 1.8355 A87.2 1.9965 4 1 cell/well
Sorter 1.9795 A89.1 3.0326 7 1 cell/well 16 Sorter 1.9081 A89.2
3.0013 8 1 cell/well Sorter 1.9666 A99.1 3.2165 2 1 cell/well 4
Sorter 1.8815 A99.2 3.4925 4 1 cell/well Sorter 2.0927
TABLE-US-00009 TABLE 1B RESULTS OF TESTING SINGLE CELL CLONES OF
Ovr110 C-SERIES MAbs Plating Plate Clone # Method Density ELISA OD
C1 sorter 1 cell/well No positives C3.2 sorter 1 cell/well 1.9884
C4 sorter 1 cell/well No positives C5.3 sorter 5 cell/well 2.0032
C6.3 sorter 1 cell/well 1.9797 C7.1 sorter 1 cell/well 2.0218 C8
sorter 1 cell/well No positives C9.1 sorter 1 cell/well 2.5158
C10.1 sorter 1 cell/well 2.1172 C11.1 sorter 5 cell/well 2.3633
C12.1 sorter 5 cell/well 2.5522 C13 sorter 1 cell/well No positives
C14 sorter 1 cell/well No outgrowth C16.1 sorter 1 cell/well 2.0682
C17.1 sorter 1 & 5 cell/well 1.7183
Results from ELISA Screening of Cloned Ovr110 MAbs
[0327] After 2 weeks of culture, supernatants from 2 hybridoma
clones from each parent hybridoma were tested for production of
ELISA absorbance values greater than 1.5 with Ovr110B (Tables 1A
and B) or Ovr110 peptides and less than 0.2 with Ovr107. Clones
Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1,
Ovr110.A57.1, Ovr110.A72.1, Ovr110.A77.1, Ovr110.A87.1,
Ovr110.A89.1, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107.1,
Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.3,
Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,
Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,
Ovr110.C16.1 & Ovr110.C17.1 were all selected for scale up for
immunohistochemical, immunofluorescence and functional testing.
FACS Screening for Cell Surface Binding of Ovr110 MAbs
[0328] LMTK-Ovr110-HA stable transfectants and Ovr110 mRNA positive
(SKBR3) and mRNA negative (HT29) tumor cell lines were grown in
DMEM/10% FBS+P/S. One day prior to staining, the LMTK-Ovr110-HA
stable transfected cells were stimulated by adding sodium butyrate
to a 5 mM final concentration. For FACS analysis, LMTK-Ovr110-HA
cells or tumor cell lines were washed once with 10 ml
Ca.sup.+2/Mg.sup.+2 free DPBS and then 7 ml of warm (37.degree. C.)
Cellstripper (Mediatech, Herndon, Va.) was added per 150 cm.sup.2
flask. The cells were then incubated for 5 minutes at 37.degree. C.
with tapping of the flask to remove tightly attached cells. The
cells were removed and pipetted several times to break aggregates,
then immediately placed in DMEM/10% FBS/5 mM sodium butyrate. The
cells were then centrifuged down for 5 minutes at 1300 rpm and
resuspended in DMEM/10% FBS/5 mM sodium butyrate. The cells were
incubated at 37.degree. C. for a 30 min. recovery period. Prior to
staining, viability of the cells was measured using Guava Viacount
(Guava Cytometers, City, Calif.) and if >90% viable they were
distributed into 96-well v-bottom plates (VWR) for staining with
MAbs.
[0329] Cells were aliquoted at 0.5-1.0.times.10.sup.6 cells/well in
96-well v-bottom plates and centrifuged for 2 minutes at 1500 rpm.
Supernatants were aspirated and plates briefly shaken on a vortex
mixer to resuspend the cells, then 200 ul of DPBS/3% FBS/0.01% Na
Azide (FACS buffer) was added to each well. Centrifugation and
aspiration was repeated, then 25 uL of sequential dilutions of
hybridoma supernatant or purified MAb was added to the cells.
Plates were stored on ice for 15 min., then washed and centrifuged
as above, in 200 uL of FACS buffer. This washing procedure was
repeated a twice and then 25 uL of phycoerythrin (PE) conjugated
donkey anti-mouse IgG Fc antibody (Jackson Immunoresearch
Laboratories Inc., West Grove, Pa.) were added to cells. After 15
minutes on ice the cells were washed twice, as above and then
resuspended in 250 uL of FACS buffer for analysis on the cell
sorter or flow cytometer. In certain cases, for storage overnight
at 4.degree. C. prior to analysis, 133 ul of FACS buffer and 67 uL
of 1% paraformaldehyde/DPBS was added to each well, for fixation,
then the volume was increased to 250 uL with DPBS. Stained cells
were analyzed on an Elite fluorescent activated cell sorter (FACS)
(Beckman-Coulter, Miami, Fla.).
[0330] Results of a representative experiment demonstrating cell
surface expression by FACS analysis are depicted in FIG. 1. Binding
of the Ovr110 MAb A7.1, followed by binding of the donkey
anti-mouse Ig-PE conjugate (DAMPE) resulted in 49% of Ovr110
transfected mouse LMTK cells being positive, with a fluorescence
intensity (mean fluorescence intensity, MFI) 7.5-fold higher than
cells stained with DAMPE alone. Further FACS analysis data with
human tumor cell lines are presented in Table 2 below. As can be
seen from the results, Ovr110.C3.2, Ovr110.C5.3 and Ovr110.C6.3
each bound to greater than 80% of the fresh Ovr110 mRNA positive
SKBR3 cells, whereas the control negative MAb Pro104.D9.1 bound to
less than 2% of these same breast cancer derived cells.
Ovr110.C3.2, Ovr110.C5.3 and Ovr110.C6.3, similarly bound to less
than 2% of the Ovr110 mRNA negative cells of the colon cancer cell
line HT29.
TABLE-US-00010 TABLE 2 Ovr110 MAb BINDING TO VIABLE SKBR3 BREAST
CANCER CELL LINE SKBR3 HT29 MAb Clone % Cells Positive MFI % Cells
Positive MFI None 3.2 0.35 1.4 0.316 Ovr110.A57.1 9.8 0.82 1.2
0.385 Pro104.D9.1 1.9 0.395 1.3 0.354 Ovr110.C1 1.9 0.413 Ovr110.C3
83.8 4.08 2 0.373 Ovr110.C4 17.8 0.971 0.8 0.331 Ovr110.C5 86.5
4.34 1.5 0.356 Ovr110.C6 89.1 4.73 1.7 0.37 Ovr110.C7 5.2 0.641
Ovr110.C8 1.6 0.394 Ovr110.C9 22.3 0.936 1.5 0.342 Ovr110.C10 4.9
0.605 Ovr110.C11 2.3 0.442 Ovr110.C12 9.4 0.778 4.7 0.4 Ovr110.C13
1.6 0.399 Ovr110.C14 70.3 2.77 0.9 0.358 Ovr110.C16 3.4 0.479
Ovr110.C17 63.6 2.4 1.3 0.342
Ovr110 MAb Isotypes
[0331] The isotypes of the MAbs were determined using commercially
available mouse monoclonal antibody isotyping immunoassay test kits
(IsoStrip, Roche Diagnostic Corp., Indianapolis, Ind.). Results of
the isotyping are listed in Table 3. All MAbs were of the
IgG.sub.1/.kappa. isotype, except Ovr110 MAb A10.1, which was of
the IgG.sub.2b/.kappa. isotype.
TABLE-US-00011 TABLE 3 Ovr110 MAb ISOTYPES Clone Isotype A7.1 IgG1:
Kappa A10.1 IgG2.sub.b: Kappa A13.1 IgG1: Kappa A31.1 IgG1: Kappa
A57.1 IgG1: Kappa A72.1 IgG1: Kappa A77.1 IgG1: Kappa A87.1 IgG1:
Kappa A89.1 IgG1: Kappa A99.1 IgG1: Kappa A102.1 IgG1: Kappa A107.1
IgG1: Kappa C3.2 IgG1: Kappa C5.3 IgG1: Kappa C6.3 IgG1: Kappa C7.1
IgG1: Kappa C11.1 IgG1: Kappa C12.1 IgG1: Kappa C17.1 IgG1:
Kappa
Ovr110 MAb Affinity Analysis
[0332] Binding kinetics and affinity constants were calculated from
surface plasmon resonance measurements using a BIACORE 3000
instrument (Biacore, Piscataway, N.J.). Experiments were designed
to simultaneously generate on rate, off rate, and affinity values
for the Ovr110 MAbs.
[0333] Rabbit anti-mouse IgG Fc antibody (Biacore) was immobilized
on flow cells 2, 3, and 4 of a CM5 sensor chip (Biacore) by
standard amine coupling (Biacore). Flow cell one was used as a
blank surface for reference subtractions, and was activated and
then inactivated with ethanolamine. Ovr110 MAbs were captured on
the rabbit anti-mouse-IgG Fc coated chip, followed by binding of
the antigen. Therefore these measurements should represent real 1:1
affinities and not avidity effects that are observed with direct
antigen immobilizations, due to the divalent nature of IgG
antibodies. MAbs were diluted in HBS EP buffer (Biacore) to 15
ug/mL and were divided into multiple tubes to minimize evaporation
between cycles. The MAbs were passed through the flow cells for 2
minutes at 20 uL/minute. The MAb capture level ranged between 200
and 300 response units (RU) per flow cell. Following MAb capture
the surface was allowed to stabilize for 3 minutes. Ovr110B (1.56
mg/mL) antigen was then flowed over the captured MAbs at 20
uL/minute in flow cells and through the blank flow cell, for 4
minutes, at successive concentrations of 144, 72, 36, 18, 9, 4.5
ug/mL. Since the Ovr110B molecular weight is 35 kD these antigen
concentrations correspond to 4.11, 2.06, 1.03, 0.514, 0.257, 0.129
uM. Two replicate cycles were performed for each antigen
concentration or buffer. A dissociation time of 420 seconds was
allowed between cycles and regeneration of the chip surfaces to
anti-mouse IgG Fc antibody or blank surface, were performed by
flowing 100 mM Glycine pH 1.75 through the flow cells for 30
seconds at 100 uL/minute.
[0334] The resulting data were analyzed by BiaEvaluation software
(Biacore) using a global fit simultaneous ka/kd assuming Langmuir
binding. The Rmax parameter of the software was set to local to
allow compensation for minor variations in the anti-mouse IgG Fc
capture step. The calculated affinities presented in Table 4, which
are in the 10.sup.-9 to 10.sup.-13 M range, are sufficiently high
to achieve a therapeutic dose in-vivo at less than or equal to 10
mg/kg.
TABLE-US-00012 TABLE 4 Ovr110 MAb AFFINITIES Affinity MAb KD (M) KA
(MS) kd (1/s) ka (1/Ms) A72.1 1.68E-09 8.17E+08 1.35E-05 1.36E+04
A57.1 1.95E-09 5.5E+08 1.61E-05 8.86E+03 A7.1 9.51E-13 1.14E+12
8.00E-09 9.12E+03
Western Blots
[0335] 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 Ovr110-293T transfectants and mammalian
adenocarcinoma 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 (Ovr110 monoclonal
antibodies: A10.2, A13.1, A31.1, A57.1, A72.1, A77.1, A89, A107,
C3.2, C5.1, C5.3, C6.3, C7.1, C9.1, C11.1, C12.1 or C17.1) and then
with horseradish-peroxidase conjugated goat anti-mouse IgG
secondary antibody (Jackson Immunoresearch Laboratories, Inc.) and
finally visualized by chemiluminescence using an ECL advance
western blotting detection kit (Amersham Biosiences, Piscataway,
N.J.).
[0336] Deglycosylation experiments were performed on protein
extracts from Ovr110-293T transfectants, Ovr110 mRNA positive
(QPCR+) and Ovr110 mRNA negative (QPCR-) mammalian adenocarcinoma
cell lines and ovarian tumors using Peptide N-Glycosidase F (New
England Biolabs, Inc., Beverly, Mass.) as per the directions
provided by the manufacturer. The deglycosylated samples were then
analyzed by western blots as described above. Briefly, 100 ug of
protein extract was denatured in Glycoprotein denaturing buffer
(0.5% SDS+reducing agent) at 100.degree. C. for 10 min. This was
followed by the addition of kit reaction buffers at a final
concentration of 1% NP-40 and 50 mM sodium phosphate before the
addition of 100 units of PNGase F and incubated at 37.degree. C.
for 4 hours.
TABLE-US-00013 TABLE 5A RESULTS FROM WESTERN BLOTS USING OVR110
MABS WITH EXTRACTS FROM TRANSFECTED 293T CELLS & BREAST,
OVARIAN & COLON CANCER CELL LINES A10.1 A13.1 A72.1 A31.1 A57.1
A77.1 A89 A107 Ovr110- + + + + + + + + HA-293T multiple multiple
multiple multiple multiple multiple multiple multiple bands bands
bands bands bands bands bands bands major major major major major
major major major band at band at band at band at band at band at
band at band at 49-60 kDa 49-60 kDa 49-60 kDa 49-60 kDa 49-60 kDa
49-60 kDa 49-60 kDa 49-60 kDa & minor & minor & minor
& minor & minor & minor & minor & minor band
band band band band band band band at ~30 kDa at ~30 kDa at ~30 kDa
at ~30 kDa at ~30 kDa at ~30 kDa at ~30 kDa at ~30 kDa Degly- Major
band cosylated at ~30 kDa Ovr110- minor band HA-293T at ~16 kDa
MCF7 Weak ++ ++++ +++ ++++ Weak ++ + & SKBR3 50-60 kDa 50-60
kDa 50-60 kDa 50-60 kDa 50-60 kDa 50-60 kDa 50-60 kDa 50-60 kDa
(QPCR+) Degly- ~30 kDa cosylated MCF7 & SKBR3 (QPCR+) CaOV3 --
-- -- -- -- -- -- -- & HT29 (QPCR-)
[0337] Results of the western blot experiments are summarized in
Tables 5A & 5B. As can be observed, the Ovr110 MAbs A10.1,
A13.1, A31.1, A57.1, A72.1, A77.1, A89, A107, C3.2, C5.1, C5.3,
C6.3, C7.1, C9.1, C11.1, C12.1 and C17.1 identified minor bands of
the predicted size for the non-glycosylated Ovr110 protein (30 kDa)
and major bands at 49-60 kDa, in lysates of Ovr110 transfected
human 293T cells. The larger bands were consistent with the
presence of several glycosylation sites on the Ovr110 protein.
Major bands of 50-60 kDa were also detected by the same Ovr110
MAbs, in lysates from the QPCR+human breast cancer cell lines SKBR3
and MCF.sub.7 (ATCC, Manassas, Va.), but were not detected in
lysates from the QPCR- cell lines CaOV3 and HT29 (ATCC).
[0338] Deglycosylation with PNGase reduced the size of the bands
detected by Ovr110 MAb A57.1 from .about.60 kDa (glycosylated) to
.about.30 kDa (predicted non-glycosylated size), in lysates from
Ovr110 transfected human 293T cells, and in lysates from SKBR3 and
MCF7 (ATCC) breast cancer cell lines. Deglycosylation of lysates
from 3 ovarian tumor samples, with PNGase F, also reduced the size
of the bands detected by Ovr110 MAb 57.1 from .about.60 kDa
(glycosylated) to .about.30 kDa.
TABLE-US-00014 TABLE 5B RESULTS FROM WESTERN BLOTS USING OVR110
MABS WITH EXTRACTS FROM BREAST & COLON CANCER CELL LINES C3.2
C5.1 C5.3 C6.3 C7.1 C9.1 C11.1 C12.1 C17.1 A57.1 SKBR3 + + + + + ++
++ ++ + ++ (QPCR+) 50 kDa 50 kDa 50 kDa 50 & 62 50 kDa 50 kDa
50 kDa 50 kDa 50 kDa 50-60 & weak & weak & weak kDa
& weak & weak & weak & weak & weak kDa 30 &
60 30 & 60 bands at & weak bands at bands at bands at bands
at bands at & minor kDa kDa 30 & 60 band at 30 & 60 30
& 60 30 & 60 30 & 60 30 & 60 band kDa 30 kDa kDa
kDa kDa kDa kDa at ~30 kDa HT29 -- -- -- -- -- -- -- -- -- --
(QPCR-)
Example 2
Cell Surface Binding of Ovr110 MAbs in Live Cancer Cells
Demonstrated by Immunofluorescence
[0339] The following cancer cell lines were used in this study:
Ovarian OvCar-3, ovarian CaOV-3 and breast SKBr-3. OvCAR-3 and
SKBR-3 cells but not the control CaOV-3 cells express Ovr110.
[0340] Cells were seeded on 18 mm glass coverslips and cultured at
37.degree. C. in DMEM containing 10% fetal bovine serum and
penicillin and streptomycin for 48 hr prior to treatment with the
anti-Ovr110 MAbs.
[0341] Eleven Ovr110 MAbs (Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1,
Ovr110.A31.1, Ovr110.A57.1, Ovr110.A77.1, Ovr110.A87.1,
Ovr110.A89.1, Ovr110.A99.1, Ovr110.A102.1 and Ovr110.A107.1) were
tested to determine which antibody binds to the cell surface of
Ovr110 expressing cancer cells. Primary MAbs were added to the
medium at a final concentration of 10 ug/ml and incubated for one
hour at 37.degree. C. Following fixation with 3% formaldehyde in
Phosphate Buffered Saline (PBS), the cells were incubated with a
secondary Cy3-labeled donkey anti-mouse (Jackson Immunoresearch
Laboratories, West Grove, Pa.) at a concentration of 10 ug/ml for
30 min. Following washing, the cells were mounted in a medium
containing DAPI (Vectastain, Vector, Burlingame, Calif.) to
visualize the cell nuclei and provide a counterstain, and observed
in a Zeiss Fluorescence Microscope Axiophot equipped with the
appropriate fluorescent filters. Micrographs were obtained with a
CCD camera.
Results
[0342] Of the eleven MAbs tested (Ovr110.A7.1, Ovr110.A13.1,
Ovr110.A72.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A77.1,
Ovr110.A87.1, Ovr110.A89.1, Ovr110.A99.1, Ovr110.A102.1 and
Ovr110.A107.1), ten antibodies were able to bind at least a portion
of Ovr110 expressing cells. FIG. 2 shows the binding of
Ovr110.A57.1 to the cell membrane of OvCAR-3 ovarian cancer cells
(arrows in A) and SKBR-3 breast cancer cells (arrows in B). The
cell membrane of CaOV-3 cells, a control cell line that does not
express Ovr110, was not labeled when the cells were incubated with
the same antibody (FIG. 2C).
Binding and Internalization in Live Cancer Cells
[0343] This study was performed using fluorescent antibodies. By
labeling antibodies with the fluorescent dye Cy3, antibody binding
and internalization can be visualized by fluorescence microscopy.
The technology is well established. OvCAR-3 cells that do not
express Ovr110 were used as negative controls.
[0344] Cy3 Conjugation
[0345] Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1, and
Ovr110.A87.1 were labeled with Cy-3. Cy3 conjugation was carried
out according to standard procedures and the manufacturer's
guidelines. Briefly, 1 mg of antibody was dialyzed against 0.1M
bicarbonate buffer (pH 9.3) for 60 min, mixed with Cy3 dye and
incubated at RT for 2 hr, and then transferred in a Pierce Slide-A
Lyzer Dialysis cassette for dialysis in 2 liters of PBS for 6 hr at
4 C. The operation was repeated 6 times. The Cy3 conjugated
antibodies were recovered and concentration was measured in a
spectrometer at 280 nm.
[0346] Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1 and
Ovr110.A87.1 MAbs were then incubated at a concentration of 10
ug/ml with the cells at 37.degree. C. in a water chamber for 60
min, washed in PBS and fix with 3% formaldehyde in PBS for 10 min.
Following fixation, the coverslips with the cells were mounted in a
medium containing DAPI (Vectastain) to visualize cell nuclei, and
observed in a Zeiss fluorescence Microscope Axiophot equipped with
the appropriate fluorescent filters. Micrographs were obtained with
a color CCD camera.
[0347] Results
[0348] Immunofluorescence microscopy of cancer cells treated with
Cy3-Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1, and
Ovr110.A87.1 indicated that the cancer cells expressing Ovr110 bind
and internalize the fluorescent antibodies to varying extent FIG. 3
A (arrows) shows that following binding Cy3-Ovr110.A57.1 is
internalized by SKOV-3 cells and to a lesser degree by SKBr-3 cells
(FIG. 3B). No or low binding of Cy3-Ovr110.A57.1 was observed in
the CaOV-3 control cells (FIG. 3C). The internalization pattern
staining in the SKOV-3 cells was characterized by the presence of
perinuclear vesicles likely to correspond to endosomes located in
the proximity of the Golgi apparatus (FIGS. 3A and B).
CONCLUSIONS
[0349] Ovr110 MAbs are internalized in vitro upon binding to Ovr110
on the cell surface of Ovr110 expressing cancer cells.
Ovr110 Distribution in Tumors and Normal Tissues Assessed by
Immunohistochemistry
[0350] Tissues
[0351] Formalin fixed paraffin embedded blocks of breast, ovarian
cancer and normal adjacent tissues were obtained from National
Disease Research Interchange (Philadelphia, Pa.). OCT embedded
blocks of normal organs were obtained from Zoion (Hawthorne,
N.Y.).
[0352] Immunohistochemical Staining for Formalin Fixed Paraffin
Embedded Sections
[0353] Six-.mu.m-thick sections cut from formalin fixed paraffin
embedded blocks were baked at 45.degree. C., deparaffinized in
Histoclear and rehydrated through a series of ethanol until PBS.
Antigen retrieval was performed by boiling the section slides in 10
mM sodium citrate buffer (pH 6.0) at 120.degree. C., 1517 PSI in
decloaking chamber (Biocare, Walnut Creek, Calif.) for 10 min
Endogenous peroxidase activity was quenched by treating with 3%
hydrogen peroxide solution for 15 min. Slides were incubated with
1% BSA to block nonspecific antibody binding and then reacted with
6 different primary Ovr110 MAbs used at a concentration of 1 ug/ml
for 1 hour in room temperature in a DAKO autostainer (Dako Co.,
Carpinteria, Calif.). After washing in Tris-Buffered Saline (TBS)
with 0.5% Tween -20, slides were incubated with anti-mouse IgG as
the secondary antibody conjugated to horse radish peroxidase (HRP).
After washing in TBS with 0.5% Tween -20, sections were visualized
by 3,3'-diaminobenzidine chromagen for 2-5 minutes (Immunovision
Technologies, Co. Daly City, Calif.) and counterstained with
hematoxylin before mounting in Permount medium after dehydration.
Normal mouse IgG at the same concentration as the primary antibody
served as negative controls.
[0354] Immunohistochemical Staining for OCT Embedded Frozen Unfixed
Sections
[0355] Slides were cut in the cryochamber at 5-8 um at an
appropriate temperature, air dried for a minimum of 1/2 hour at
room temperature. IHC was performed using the Immunovision
Powervision Kit (Immunovision Technologies Co. Daly City, Calif.).
Briefly, slides were rinsed in TBS to remove off OCT and incubated
with the primary antibody Ovr110.A13.1 and Ovr110.A57.1 for 1 hour
at room temperature. They were then post-fixed in 4%
paraformaldehyde fixative for 10 minutes and treated as described
above.
[0356] Results
[0357] Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A57.1 and
Ovr110.A87.1 were used to immunolabel sections of clinical samples
of ovarian serous adenocarcinoma. FIG. 4 shows the distribution of
Ovr110 in ovarian tumors as evaluated by IHC using
Ovr110.A57.1.
[0358] Thirteen out of fifteen clinical samples (87%) showed
positive immunolabeling using Ovr110.A57.1. while fourteen out of
fifteen (93%) were positive using Ovr110.A13.1 (Table 6A). Specific
immunostaining was restricted to the epithelial cells in the tumors
and the number of positive cells varied between 50% to almost all
of the tumor cells. FIG. 4A and FIG. 4B show that the epithelial
cells of the tumor displayed a strong membranous staining (arrows)
with less intense cytoplasmic staining and no background staining
in the stroma. FIG. 4C shows lack of specific labeling in a control
experiment in which the primary antibody was replaced with a mouse
IgG fraction.
[0359] Eight out of ten (80%) breast cancer clinical samples were
positive when Ovr110.A57.1 was used while five out of ten (50%)
were positive using Ovr110.A13.1 (Table 6A). FIG. 5 shows the
pattern of expression in clinical samples of breast infiltratrating
ductal carcinoma. The labeling was restricted to the cell surface
of the epithelial cells of the tumors (FIGS. 5A and B, arrows).
FIG. 5C shows the absence of specific labeling in a control
experiment in which the primary antibody was replaced with a mouse
IgG fraction. As judged by the intensity of the immunolabeling, the
level of expression for Ovr110 in the neoplastic ovarian and breast
tissues was high. A limited number of pancreatic cancer samples
were investigated for Ovr110 expression. Two out of four clinical
samples (50%) showed expression of Ovr110 with Ovr110.A57.1 and
three out of five with Ovr110.A13.1 (Table 6A). FIGS. 6 A and B
shows the immunolabeling pattern obtained using Ovr110.A57.1 in
clinical samples of pancreatic adenocarcinoma. The labeling is
restricted to the cell surface of epithelial cells (arrows). No
specific labeling was observed when normal mouse IgG was used
instead of Ovr110.A57.1. Lung cancer tissues were also found to be
positive for immunolabeling with A7.1, A13.1 and A31.1 (2/2, 2/3
& 1/2 cases respectively).
[0360] Ovr110 expression was also analyzed in normal tissues and
generally found to be negative in the following organs: liver,
stomach, bladder, testis, colon, ovary, prostate and lung (1/7
positive only with A13.1). The cells of the normal heart showed
moderate cytoplasmic but no cell surface staining. The kidney
showed moderate membranous staining of some distal convoluted
tubules and the ascending loop. The apical membrane of the normal
breast and pancreatic ducts was also labeled.
TABLE-US-00015 TABLE 6A Summary of immunohistochemistry results
showing the number of positive cases in normal human tissue samples
and ovarian, breast and pancreatic cancer clinical samples. B MAb O
C O N B C NAT* B N P C P N L C L N A7.1 12/15 0/3 2/2 NA 2/5 2/2
2/3 2/2 0/5 A13.1 14/15 0/3 5/10 2/2 3/8 3/5 0/3 2/3 1/7 A31.1 1/2
0/2 1/2 NA 1/5 0/2 0/3 1/2 0/5 A57.1 13/15 0/3 8/10 2/2 2/3 2/4 NA
NA NA *NAT = Normal adjacent tissue O C = ovarian cancer; O N =
ovary normal; B C = breast cancer; B NAT = breast NAT; BN = breast
normal; P C = pancreatric cancer; P N = pancreatic normal; L C =
lung cancer; L N = lung normal
TABLE-US-00016 TABLE 6B Binding of Ovr110 MAbs to normal adult
mouse mammary tissue Mammary gland Lymph node Ductal Smooth in the
Pad Conc. Epithelium Stroma Muscle Lymphocytes MAb A57.1 1 ug/ml 3+
C/M* -- -- Lymphatic vessel 2+ C C3.2 1 ug/ml 3+ C -- -- Some 1+ C
C6.3 1 ug/ml 3+ C -- -- 2+ C C12.1 1 ug/ml 3+ -- -- -- apical M
Controls Pro104 2 ug/ml -- -- -- D133.1 E- 0.25 ug/ml 3+ M -- -- --
cadherin IgG1 10 ug/ml -- -- -- -- *Grading 1-3+ using Carr's
scale, C = cytoplasmic & M = membrane
[0361] Because binding to the rodent homolog of Ovr110 would
facilitate preclinical safety testing for the binding of the
anti-Ovr110 MAbs, several anti-Ovr110 Mabs was tested in normal
mouse mammary tissue that was prepared, sectioned and stained in
the same manner as the normal human tissues. Results of this
testing are presented in Table 6B. Ovr110.A57.1, Ovr110.C3.2,
Ovr110.C6.3 and Ovr110.C12.1 all reacted with the ductal epithelial
cells in mouse mammary glands, in a similar pattern to that in
normal human mammary tissues.
SUMMARY
[0362] The results demonstrate that Ovr110 expression can be used
as a highly sensitive and specific indicator for serous carcinomas
of the ovary and breast infiltrating ductal carcinoma, even though,
Ovr110 was also expressed in some pancreatic and lung cancers and
several anti-Ovr110 MAbs apparently also reacted with a related
molecule in mouse mammary tissue. The cell membrane staining
pattern indicates that Ovr110 should be an ideal therapeutic
target.
Example 3
Killing of Ovr110 Transfected Cho Cells by Incubation with MAbs and
Anti-Mouse MAb Saporin Conjugate
[0363] Experiments were performed by incubating Ovr110 transfected
CHO cells (Ovr110-CHO) with Ovr110 Mabs premixed with Mab-zap goat
anti-mouse Ig saporin conjugate (Advanced Targeting Systems, San
Diego, Calif.) and measuring cell viability at 72 and 96 h, to
detect potential killing effects on these Ovr110 expressing cells.
On day 1, Ovr110-CHO cells were placed into 96 well, flat bottom,
sterile cell culture plates (Corning), in triplicate wells, at 2000
cells/75 uL/well, in F12 medium with 10% FBS, P/S. Plates were
incubated at 37.degree. C., in 5% CO2, overnight. Duplicate plates
were set up to allow readings at 72 h and 96 h. On Day 2 (0 h), 25
uL of 4.times. final MAb concentrations alone, or 25 uL of
4.times.MAb premixed with 25 uL of 4.times.Mab Zap, or 25 uL of
4.times.Mab Zap alone, or 25 uL of medium alone were added to wells
of the 96 well plates, in triplicate, to a final volume of 100 uL.
Final MAb concentrations were 2 ug/mL, 0.4 ug/mL, 0.08 ug/mL and 0
ug/mL and the final concentration of MAb Zap was 1 ug/mL.
Triplicate wells with medium alone, MAb alone (2 ug/mL only) and
MAb Zap alone were used as negative controls. The anti-transferin
receptor MAb 5E9 (ATCC, Manassus, Va.) was used as a positive
control MAb for killing. Plates were shaken gently for five minutes
to mix the reagents and then incubated at 37.degree. C., in 5% CO2.
On day 5 (72 h), 10 uL of a of Alamar Blue stock solution
(Biosource International, Camarillo, Calif.) was added to wells of
the first set of plates and they were incubated at 37.degree. C.,
in 5% CO2 for 2-7 h. Plates were then analyzed on a SpectraMAX
GeminiEM spectraphotometer (Molecular Devices, Sunnyvale, Calif.)
(emission=590 nm, excitation=560 nm and Autocutoff=570 nm) and
viability was expressed as a percentage the control wells with
medium alone.
TABLE-US-00017 TABLE 7 Ovr110-CHO killing by Ovr110 MAb & MAb
Zap Saporin Conjugate Percent Growth Compared to Wells with Medium
Alone % Ovr110- Ovr110- CHO Positive CHO MAb MAb + MAb Zap MAb with
MAb (2 ug/mL) + MAb Mab MAb MAb MAb Clone (IF) * MAb Zap Zap (2
ug/mL) (0.08 ug/mL) (0.4 ug/mL) (2 ug/mL) 5E9 -- 93.5 78.0 96.7
66.1 75.8 87.1 A10.1 70 91.9 61.3 101.6 48.4 50.0 45.2 A31.1 40
91.2 59.6 96.2 43.1 44.2 43.7 A57.1 40 100.0 57.7 101.9 36.6 36.5
42.3 A87.1 70 92.2 58.8 98.0 45.8 39.2 43.1 C3.2 60 97.0 71.7 98.9
50.9 55.5 56.6 C5.1 40 98.1 73.1 100.0 52.0 50.0 46.9 C5.3 40 96.2
75.0 103.8 57.7 53.8 59.6 C6.3 40 96.2 73.1 100.1 51.9 43.1 50.0
C9.1 20 98.1 78.1 102.6 58.5 80.0 67.9 C11.1 1 98.0 78.4 101.9 58.8
70.6 80.4 C12.1 20 100.0 80.4 103.9 66.7 72.5 78.4 *
Immunofluorescence microscopy as detailed in Example 2.
[0364] Results of testing Ovr110.A10.1, Ovr110.A31.1, Ovr110.A57.1,
Ovr110.A87.1, Ovr110.C3.2, Ovr110.C5.1, Ovr110.C5.3, Ovr110.C6.3,
Ovr110.C9.1, Ovr110.C11.1 and Ovr110.C12.1 are presented in Table
8. As can be seen, the MAb Zap alone resulted in a high background
and inhibited growth of the Ovr110-CHO cells from 0-41.4%. This was
not the case for the negative control wells with Pro104-CHO cells
and MAb Zap alone, which resulted in 0-10% growth inhibition (data
not shown). However, none of the Ovr110 MAbs alone, produced more
than 3.8% growth inhibition of Ovr110-CHO cells. Whereas, when
added with MAb Zap saporin conjugate, all of the Ovr110 MAbs tested
produced greater than 10% more growth inhibition than with MAb Zap
alone. Ovr110.A57.1 in particular, at concentrations of 0.08, 0.4
and 2.0 ug/mL together with MAb Zap resulted in 15.4-21.1% greater
Ovr110-CHO cell growth inhibition, than MAb Zap alone and
57.7-63.4% growth inhibition compared to wells with medium alone.
In conclusion, growth inhibition of Ovr110 expressing CHO cells,
was obtained at concentrations of MAb which are easily achievable
in-vivo, for therapeutic purpose. These in-vitro data suggest that
the Ovr110 MAbs above would be suitable for targeting of drug or
isotopes to tumor cells, in-vivo.
Example 4
Binding of Ovr110 MAbs and Soluble BTLA-Fc to Activated T-Cells and
Tumor Cells
[0365] Anti-human B7x/B7H4 and anti-mouse B7S1 MAbs were previously
shown to bind to activated T-cells (Prasad et al., Immunity
18:863-73 (2003); Sica et al., Immunity 18:849-61 (2003); Zang et
al., Proc. Natl. Acad. Sci. USA. 100:10388-92 (2003)). In order to
verify binding of the Ovr110 MAbs of this invention to activated
cells, fresh human T-cells were purified and stimulated with
different compounds, as discussed infra. The binding of the Ovr110
MAbs to activated CD3 positive T-cells expressing CD25 (IL-2R) and
CD71 (TFR) was analyzed by FACS. Because BTLA has been identified
as the putative receptor for Ovr110 (B7x/B7H4) (Watanabe et al.,
Nat. Immunol. 2003 4:670-9; Carreno & Collins Trends Immunol.
2003 24:524-7), we also examined the binding of the human
BTLA-mouse IgG2a Fc fusion disclosed herein to these activated
T-cells and tumor cells.
Preparation of Human Peripheral Blood Leukocytes (PBL)
[0366] Human peripheral blood from normal, male donors was obtained
from volunteer donors at Stanford Blood Center (Palo Alto, Calif.).
Mononuclear cells were isolated using standard Ficoll/Hypaque
single step density gradient centrifugation (1.077 g/mL)
methods.
[0367] Activation of T-Cells
[0368] Mononuclear cells at a final concentration of 10.sup.6/mL
were cultured for 3 days, at 37.degree. C., in RPMI-1640 (CeliGro),
supplemented with 10% FCS (Hyclone, Utah), with phytohemagglutinin
(PHA-M) (Sigma, St. Louis, Mo.) at 10 ug/mL, or lipopolysaccharide
(LPS) (Sigma) at 10 ug/mL, or a combination of phorbol myristic
acetate (PMA) (Sigma) at 10 ng/mL and ionomycin (Sigma) at 1 uM, in
standard 25 cm.sup.2 tissue culture flasks in 10% CO.sub.2.
[0369] Immunofluorescence and Flow Cytometry
[0370] The cells were collected after 3 days of PHA stimulation and
washed extensively.
[0371] The mononuclear cells were distributed into a 96 well
V-bottom plate and incubated in autologous serum to block Fc
receptors. Anti-CD3 FITC antibody (Serotec, Raleigh, N.C.) was
added to each well and either CD80PE, CD86PE, CD25PE, or
biotinylated anti-CD71 (Serotec), Ovr110.A57.1 or Ovr110.C3.2 were
added as a second MAbs, at 20 ug/mL, for dual color analysis. The
cells were washed twice and Phycoerythrin-Streptavidin (PESA) was
added to the wells preincubated with biotinylated MAbs. The cells
were washed twice and resuspended in FACS buffer. Cells were
preincubated in autologous serum and stained with Ovr110-Ig or
BTLA-Ig fusion proteins, at 20 ug/mL. The cells were washed twice,
donkey anti-mouse PE (1 ug/mL) was added to the samples and the
cells were then washed twice and incubated in mouse serum to block
free binding sites on the donkey anti-mouse antibody. Anti-CD3 FITC
antibody was then added as a last step to identify T-cells. After
washing twice, the cells were resuspended in FACS buffer and
analyzed by flow cytometry. The human tumor cell line SKBR3 was
incubated with MAbs or BTLA-Fc as previously described above.
[0372] All samples were analyzed on an EPICS Elite Flow Cytometer.
All histograms were generated using the Winmdi program. CD3
positive T cells were used as a gate to analyze the expression of
the B7 family and activation markers (CD71 and CD25).
TABLE-US-00018 TABLE 8A Binding of anti-Ovr110 MAbs to tumor cells
and activated T-cells Resting PHA 72 h HT29 SKBR3 (0 h) Activated
(QPCR-) (QPCR+) T-Cells T-Cells % % % % Cells MFI* Cells MFI Cells
MFI Cells MFI Neg. 2 0.5 2 0.6 4 6.4 1 8 Control (Pro104 D9.1) CD25
0 7.6 77 19 CD71 100 50 99 217 8 7.6 95 219 A7.1 2 0.5 71 4.2 A57.1
2 0.6 4 1 6 10 82 246 A72.1 21 1.0 6 1.2 C3.2 1 0.5 60 3.6 6 10 2 5
*Mean of fluorescence intensity
TABLE-US-00019 TABLE 8B Binding of Anti-Ovr110 MAbs to PHA
Activated T-cells from Normal Male Donors Day 0% Cells Positive Day
3% Cells Positive N (Average .+-. St Dev) N (Average .+-. St Dev)
Neg. control 5 1.8 .+-. 1.4 6 1.9 .+-. 0.4 Total CD3+ 5 82.6 .+-.
14.1 6 92.4 .+-. 6.4 Percentage of CD3 Gated Cells Positive
Ovr110.A57.1+ 5 2.8 .+-. 1.7 6 57 .+-. 34.0 Ovr110.C3.2+ 3 3.2 .+-.
2.6 5 8.8 .+-. 12.4 CD80+ 5 1.2 .+-. 0.8 6 3.6 .+-. 3.1 CD86+ 5 1.4
.+-. 0.8 6 12.8 .+-. 11.4 CD25+ 5 1.9 .+-. 1.1 6 86.3 .+-. 8.1
CD71+ 5 7.2 .+-. 1.0 6 95.9 .+-. 3.5 Ovr110-Fc 1 0.9 4 82.8 .+-.
15.9 BTLA-Fc 1 1.4 4 20.2 .+-. 32.5
TABLE-US-00020 TABLE 8C Binding of anti-Ovr110 MAbs to Activated B
Cells, Dendritic Cells and Monocytes Percent Cells Positive by FACS
B Cells Dendritic Monocytes (CD19+) Cells (CD1c+) (CD14+) MAb 0 h
72 h 0 h 72 h 0 h 72 h Negative 1 2 1 1 1 1 Control Positive 31 22
11 15 94 96 Control Ovr110.A57.1 12 13 26 72 8 22 CD80 16 16 14 2 4
4 CD86 5 27 50 94 70 20 CD25 3 14 2 1 1 2 CD71 61 64# 89 100* 32 72
#Fluorescence intensity increased ~2-fold over 0 h *Fluorescence
intensity increased 4-fold over 0 h
[0373] As can be observed in FIG. 7, where the filled curves
represent the binding of MAbs to non-stimulated T-cells, and in
Tables 8A, 8B and 8C, an increase in the expression of CD25 and
CD71 (i.e. increase in PE mean fluorescence) was achieved on the
PHA activated T-cells (gated on CD3), compared to non-stimulated
T-cells. An increase in the expression of CD71 (i.e. increase in
positive cells or fluorescence intensity) was achieved on the
activated dendritic cells (gated on CD1c) and activated monocytes.
These data demonstrate positive activation of T-cells, dendritic
cells and monocytes. The fluorescence profiles in FIG. 7 and Tables
8A, 8B and 8C demonstrate that expression of CD86 (B7.2) and Ovr110
(MAb A57.1) were increased in the activated T-cells and activated
dendritic cells and Ovr110 was increased somewhat in activated
monocytes.
[0374] As a further proof that BTLA is the ligand for Ovr110 we
tested the binding of the BTLA-Fc (mouse IgG2a) fusion protein to
Ovr110 transfected 293F cells (Ovr110-293F). From FIGS. 7G, 7H and
7I, it may be observed that the BTLA-Fc fusion protein bound
significantly to the Ovr110-293F cells (17% cells positive, MFI
24.57), but not to the control 293F cells (2% cells positive, MFI
3.44). Furthermore no appreciable binding of the mouse IgG2a to
Ovr110-293F cells via the Fc fragment was observed (3% cells
positive, MFI 4.32). From the data presented in Table 8B, the
binding of BTLA-Fc and Ovr110-Fc to activated cells, and FIGS. 7G,
7H and 7I, the binding of BTLA-Fc to Ovr110-293F cells, it is
apparent that these two recombinant proteins may be useful as
diagnostic or therapeutic agents, by blocking tumor function. In
addition, modified versions of BTLA-Fc and Ovr110-Fc conjugated to,
e.g., a cytotoxic or cytostatic component, or other functionality,
could be also used as a therapeutic agent.
[0375] The data presented in Tables 8A and 8B, demonstrate that the
MAb A57.1 apparently binds preferentially to the activated T-cells,
and MAb C3.2 binds preferentially to the tumor cell line SKBR3.
These data suggest differences between the epitopes that these two
MAbs bind to, which may be important in decreasing the immune
suppressing effects of tumor-expressed or shed Ovr110, but which
also may be important in minimizing any immunosuppressive effect
due to the use of Mab C3.2 as a therapeutic anti-tumor
antibody.
Example 5
Functional Validation of Ovr110
Materials and Methods
[0376] Cells and Cell Culture
[0377] RK3E, 293T, IEC-18, SKOV3, HeLa, CaOV3, HT29, MCF7 and SKBR3
cell lines were purchased from American Type Culture Collection
(Manassas, Va.). Cells were grown in DMEM (Invitrogen) with
L-glutamine plus 4.5 g/L glucose and supplemented with 10% FBS and
100 U/mL Penicillin/Streptomycin (Cellgro). All cells were
maintained in a humidified 37.degree. C. incubator with 5%
CO.sub.2.
[0378] siRNA Oligonucleotide Design and Preparation
[0379] To design siRNA molecules, sequences were selected from the
open reading frame of the Ovr110 mRNA based on methods previously
described (Elbashir et al., 2001). A random "scrambled" siRNA
sequence which should not generate knockdown of any known cellular
mRNA was used as a negative control. As an additional negative
control, a siRNA targeting Emerin was used to demonstrate that
knockdown of a non-essential mRNA did not affect Ovr110 levels nor
any of the biological endpoints studied (data not shown). As a
positive control for knockdown of an mRNA leading to apoptosis
induction, a siRNA targeting DAXX was used, based on published data
(Michaelson et al., J Cell Sci. 2003 Jan. 15; 116(Pt 2):345-52). A
BLAST search against the human genome was performed with each
selected siRNA sequence to ensure that the siRNA was
target-specific and would not function to knockdown other
sequences. All siRNA molecules (HPP purified grade) were chemically
synthesized by Xeragon Inc. (Germantown, Md.). siRNA's were
dissolved in sterile buffer, heated at 90.degree. C. for 1 minute
and then incubated at 37.degree. C. for 1 hour prior to use. siRNA
oligonucleotides with two thymidine residues (dTdT) at the 3' end
of the sequence consisted of the following specific RNA
sequences:
TABLE-US-00021 Ovr110 #37: (SEQ ID NO: 14) sense
5'-GGUGUUUUAGGCUUGGUCC-3' (BEST) Ovr110 #39: (SEQ ID NO: 15) sense
5'-CUCACAGAUGCUGGCACCU-3' Ovr110 #41: (SEQ ID NO: 16) sense
5'-GGUUGUGUCUGUGCUCUAC-3' Emerin: (SEQ ID NO: 17) sense
5'-CCGUGCUCCUGGGGCUGGG-3' Scrambled: (SEQ ID NO: 18) sense
5'-UUCUCCGAACGUGUCACGU-3' DAXX: (SEQ ID NO: 19) sense
5'-GGAGUUGGAUCUCUCAGAA-3'
[0380] Transfection with siRNA Oligonucleotides
[0381] 6.times.10.sup.4 SKBR3 cells were seeded in 12-well plates
for 18-24 hours prior to transfection. Transient transfection was
carried out using Oligofectamine reagent (Invitrogen) according to
the manufacturer's protocol. A final concentration of 100 nM siRNA
(except DAXX siRNA which was 200 nM) and 1.5 ul Oligofectamine were
used per well of cells. siRNA's were transfected in triplicate for
all experiments. Parallel wells of cells were evaluated 72 hours
after transfection for changes in mRNA levels by quantitative
real-time RT-PCR (QPCR), changes in protein levels by Western
immunoblot and changes in apoptosis by two different assay systems
(see below). The results demonstrating down regulation of the
Ovr110 protein are shown in FIG. 11. The siRNA #37 against Ovr110
was also tested with cells that did not express Ovr110 and there
was no effect on apoptosis (data not shown). All findings were
confirmed with at least 2 additional experiments.
[0382] Quantitative Real Time RT-PCR (QPCR)
[0383] A QuantiTech SYBR Green RT-PCR kit from Qiagen Inc. was used
for QPCR evaluation. Between 20 and 40 ng of template RNA was used
per reaction. QPCR was performed using a Taqman 7700 Sequence
Detection system (Applied Biosystem Inc).
[0384] Apoptosis Assays
[0385] Two different assay kits were used to evaluate the effects
of siRNA on apoptosis. With the "Apo-ONE Homogeneous Caspase-3/7
Assay" kit (Promega Inc.) the test cells were solubilized directly
in the culture plate and caspase activity, reflected as a
fluorescent readout, was measured according to supplier's
instructions. With the second kit, "Guava Nexin V-PE Kit" (Guava
Technologies Inc.), treated cells were harvested by trypsinization
and washing and approximately 10.sup.5 cells were resuspended in 40
ul provided buffer and 5 ul each Annexin V (+) and 7-AAD (-) were
added. Following 20 minutes incubation on ice, cells were analyzed
using the Guava PCA Flowcytometer according to manufacturer's
instructions. The results demonstrating that Ovr110 knockdown
induces apoptosis are shown in FIG. 12 and FIG. 13.
[0386] For the anoikis assays IEC-18 and RK3E cells expressing the
genes indicated were trypsinized and re-suspended in FBS free media
at a density of 150,000 and 200,000 cells/ml, respectively. A 1 ml
aliquot of the mix was plated into each well of a 12-well plate and
the samples incubated at 37.degree. C. for 24 hrs. Cells were then
collected and evaluated using the Guave-Nexin V-PE kit as above.
Ras, a potent oncogene, served as a positive control and AP as a
negative control for the anoikis assay. The results are shown in
FIG. 15.
[0387] SDS-PAGE and Western Immunoblot Analysis 72 hrs after
transfection with siRNA, cell extracts were prepared on ice using
solubilization buffer (1% NP40, 10 mM Na.sub.2PO4, 0.15M NaCl) plus
a protease inhibitor cocktail (Roche Inc.). Extracts for other
experiments with virus infected or untransfected cells were
prepared in a similar fashion. Protein extracts from harvested
tumors were prepared by homogenization of snap-frozen, minced tumor
tissue in extraction buffer (50 mM Tris-HCl, pH=7.2, 150 mM NaCl, 5
mM EDTA, 0.5% IG-Pal plus protease inhibitors) followed by
sonication and then centrifugation in a microfuge to clarify the
extracts. Between 20 and 50 ug of protein extract were used for
each gel lane; protein equivalent concentrations were evaluated for
protein level comparisons on the same gel. Clarified extracts were
mixed with an equal volume of 2.times. concentrated Laemmli sample
buffer (Invitrogen), heated to 70.degree. C. for 10 minutes and
then analyzed using pre-cast 4-12% SDS-polyacrylamide minigels
(Nupage, Invitrogen) with MES running buffer (Nupage; Invitrogen).
Gels were transferred to Immobilon-P PVDF membranes (0.45 .mu.m
pore size, Invitrogen) using 1.times. Nupage transfer buffer plus
10% Methanol. The membranes were rinsed and blocked for 1 hour at
room temperature using 5% nonfat dry milk in PBS with 0.05%
Tween-20. Membranes were incubated with primary antibody overnight
in 5% nonfat dry milk in PBS with 0.05% Tween-20. A mouse
monoclonal antibody directed against Ovr110 was produced using
recombinant Ovr110 protein. The monoclonal antibody against Ovr110
was used at a final concentration of 1 ug/ml and a mouse monoclonal
antibody against GAPDH (Chemicon Inc.) at a final concentration of
2 ug/ml. Following primary antibody incubation, membranes were
washed four times at room temperature for 10 min. each in
1.times.PBS with 0.05% Tween-20. Horseradish peroxidase linked goat
anti-mouse immunoglobulin (Jackson Lab Inc.) was used (1:10,000
dilution) in 5% nonfat dry milk in PBS plus 0.05% Tween-20 for 1
hour at room temperature to detect the primary monoclonal antibody.
Membranes were finally washed four times for 10 min. in 1.times.PBS
plus 0.05% Tween-20 followed by detection using enhanced
chemiluminescence (ECL) reagent per manufacturer's directions
(Amersham).
[0388] Expression Vector Construction
[0389] For expression of Ovr110 protein in mammalian cells, Ovr110
cDNA was sub-cloned into the pLXSN vector (BD Bioscience/Clontech)
and sequence verified. The pLXSN retrovirus vector utilizes the MLV
LTR to drive expression of cDNA's cloned into the multiple cloning
site and an SV40 promoter driving expression of a Neo gene encoding
G418 resistance. pLAPSN, a retroviral expression vector encoding
alkaline phosphatase (AP), was purchased from BD
Bioscience/Clontech (pLXSN-AP).
[0390] Virus Production
[0391] Ecotropic virus was used to infect RK3E and IEC-18 cells and
amphotropic virus to infect SKOV3 cells. For ecotropic virus
packaging, one day prior to transfection, 293T cells were seeded at
a density of 8.times.10.sup.5 cells per well of a 6 well dish onto
Biocoat collagen coated plates (BD). Cells were transfected with
purified plasmid DNA's using Lipofectamine with the addition of
PLUS reagent (Invitrogen). Per well of cells 0.8 .mu.g of virus
plasmid DNA: pLXSN-Ovr110, pLXSN-Ovr110HA or pLXSN-AP plus 0.8
.mu.g pVpack-ECO and 0.8 .mu.g pVpackGP (Stratagene) were added to
a stock of 125 .mu.L DMEM without serum and 10 .mu.L of PLUS
reagent followed by incubation for 15 minutes at room temperature.
Subsequently, 8 .mu.L of lipofectamine diluted into 125 .mu.L of
DMEM medium were added to the DNA/PLUS reagent mixture and
incubated for 15 minutes as room temperature. One ml of DMEM was
added to the final lipofectamine/DNA mixture and applied to the
cell monolayer, already containing 1 ml DMEM without serum,
followed by incubation at 37.degree. C. for 3 hours. The
transfection mix was replaced with DMEM containing 20% FBS and
cells grown overnight. Finally, the media was changed to DMEM
supplemented with 10% FBS+100 U/mL Pen/Strep for virus collection.
Virus-containing media were harvested 24 hours later and filtered
through a 0.45 .mu.m polysulfonic filter. For amphotropic virus
packaging the same procedure was followed except that the pVpack
Ampho plasmid (Stratagene) was used instead of the pVpack Eco
plasmid.
[0392] Virus Infection and Selection
[0393] Polybrene (Hexadimethrine Bromide; Sigma) was added to fresh
virus-containing medium at a final concentration of 4 .mu.g/ml.
RK3E, IEC-18 or SKOV3 cells, plated the day before at a density of
3.times.10.sup.5 cells per 100 mm2 dish, were washed once with
phosphate-buffered saline including Ca2+ and Mg2+ (cellgro). The
virus solution (6 ml per 100 mm2 dish) was applied directly to the
cells and then incubated for 3 hours in a humidified 37.degree. C.
incubator with 5% CO.sub.2 with occasional swirling. The
virus-containing medium was replaced by fresh growth medium and the
cells incubated at 37.degree. C. for 60-72 hours at which point a
final concentration of 350 ug/mL of G418 sulfate (Cellgro) was
included in the growth medium to select for virus-infected cells.
Cells were maintained between 70-80% confluence and G418-containing
media was changed every 2 days. Following G418 selection, pools of
cells were used for subsequent experiments including verification
of Ovr110 protein expression by Western immunoblot analysis where
cells were extracted and analyzed as described above. Expression of
AP by infected cell monolayers was monitored by staining whereby
monolayers of cells were fixed for 10 minutes at room temperature
with a solution of 0.5% glutaraldehyde, rinsed with PBS, heated to
65.degree. C. for 30 minutes and AP was visualized by incubation
with BCIP/NBT liquid substrate (Sigma) for 2-3 hours.
Tumor Xenograft Experiments
[0394] Retrovirus-infected, G418-selected pools of SKOV3 cells
expressing either AP or Ovr110 were injected subcutaneously into
nude mice. Parental SKOV3 cells were also used for comparison.
10.sup.7 of each cell type were implanted with matrigel into each
of 6 mice. 100% of mice injected with tumor cells developed tumors
and tumor formation was monitored by palpation and caliper
measurement when possible every 4 days for the duration of the
study. The results are shown in FIG. 14. Data is expressed as mean
group tumor volume over time.
Example 6
Monoclonal Sandwich ELISA Detection of Ovr110
[0395] High binding polystyrene plates (Corning Life Sciences (MA))
were coated overnight at 4.degree. C. with 0.8 .mu.g/well of
anti-Ovr110 MAb. The coating solution was aspirated off and free
binding sites were blocked with 3000/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 mammalian Ovr110 (extracellular portion). For each
sandwich ELISA, standards of 10, 2.5, 0.5, 0.25, 0.1 and 0 ng/ml
Ovr110 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).
[0396] For the checkerboard ELISA, all possible combination of
antibodies, were tested for efficiency as coating or detecting
reagents. The pairs A72.1/A7.1, A77.1/A57.1, A57.1/A7.1 and
A57.1/C3.2 gave the best signal/noise ratio and were further
evaluated in sandwich ELISA assays to analyze the efficiency of
detection of endogenous Ovr110 in lysates from cancer cell lines
and body fluids. The pair A72.1/A7.1 was used to test the 2700
serum samples listed below.
Results
[0397] The results of the checkerboard ELISA on 10 MAb of the
A-series and 8 MAb of the C-series are shown in Tables 9A and 9B.
Each antibody was tested as both a coating and detecting antibody,
in all possible combination. All pairs were tested in duplicates
with 100 ng of recombinant Ovr110B protein in buffer, with buffer
alone as a blank. The results are shown as specific signal/noise
ratio. The MAbs detect two distinct epitopes, based on these
pairing data. The Ovr110 A7, A77, A87 and A10 MAbs react with one
epitope or epitopes which are close enough to sterically hinder the
binding of the other three MAbs. All C-series antibodies detect
this epitope (or overlapping epitopes) as well. The other distinct
epitope or epitopes is detected by Ovr110 A89, A57, A31, A72, A107
MAbs. Several pairs with the highest signal/noise ratio were used
to test sensitivity for recombinant protein, reactivity towards
native protein in cell lines and some initial serum samples.
Epitope Specificities--Binning of MAb & Epitope Mapping
TABLE-US-00022 [0398] TABLE 9A Pairing of Ovr110 A-series MAb by
Sandwich ELISA Coating Detecting MAb MAb A7 A10 A13 A22 A31 A57 A77
A87 A89 A107 A7 1.8 2.9 1.1 9.7 7.24 10 2.4 1.7 7.9 9.7 A10 3.5 3.2
3.5 19.9 14.4 19.9 4.5 3.5 16.6 18.5 A13 1.5 4.8 1.1 7.9 5.9 8.1
2.1 1.4 6.2 8.1 A22 21 25 6.5 5.8 3.6 7.13 12.6 15.5 4.7 5.8 A31
11.6 18.77 4.8 7.9 4.7 8.5 6.8 11.1 6.1 7.7 A57 7.1 26 7 8.5 5.7
9.7 13.3 14.5 7.1 8.7 A77 7.7 12 2.9 17.3 16 19.6 2 7.3 16.6 18.8
A87 1.7 2.7 1.1 7.1 5.6 8 2 1.6 6.2 7.8 A89 18 22.5 6.9 8.2 5.7 8.9
12.2 14.2 6.9 8.7 A107 21.5 25.5 6.7 7.3 4.7 7.9 12.7 15.4 5.7
7.3
TABLE-US-00023 TABLE 9B Pairing of Ovr110 C-series MAb by Sandwich
ELISA Coat Det MAb MAb C3.2 C5.1 C5.3 C7 C9 C11 C12 C17 A72 A7.1
A57.1 A77.1 C3 1 1 1 7 2 3 4 1 8 1 8 2 C5.1 1 1 1 5 1 2 3 1 6 1 6 1
C5.3 1 1 1 6 2 2 3 1 7 1 7 2 C7 12 8 9 1 4 11 14 3 34 14 45 2 C9 1
1 1 2 1 1 2 1 2 1 3 1 C11 4 3 3 14 2 1 7 1 2 5 2 2 C12 2 2 2 4 1 2
1 1 7 3 8 1 C17 1 1 1 3 1 1 2 1 4 1 4 1 A72 11 8 9 33 5 1 24 3 1 18
1 3 A57 12 8 10 33 6 1 24 3 1 18 1 8 A77 6 4 4 2 2 5 5 2 19 8 21 1
Control 1 1 1 1 1 1 1 1 1 1 1 1 MAb
[0399] The epitope map of the Ovr110 MAbs derived from the results
in these tables is shown in FIG. 16.
Human Serum Samples
[0400] The human cancer and benign serum samples were obtained from
IMPATH-BCP, Inc and DSS (Diagnostic Support Service). The serum
samples from healthy women were obtained from ProMedex, LCC. All
samples were aliquoted upon arrival and stored at -80 C until
use.
[0401] Results
[0402] As described above, for the detection of Ovr110 in serum
samples, a sensitive detection system based on the use of horse
radish peroxidase (HRP) and a high sensitivity TMB substrate
(DAKO), was used. The minimal detectable dose (MDD) for Ovr110 in
this ELISA format is 100 pg/ml. For calculation of median values,
samples with values below the MDD were defined as 100 pg/ml Ovr110.
The minimum detectable dose is defined as two standard
abbreviations above the background signal. Most of the serum
samples from healthy patients showed low Ovr110 concentrations in
the sandwich ELISA while sera from ovarian cancer patients have
elevated levels of Ovr110.
[0403] We tested the Ovr110 concentration in more than 2700 serum
samples from patients with lung, breast, colon, prostate or ovarian
cancer or with non-cancerous, benign diseases. For a complete list
of all tested samples, see Table 10 below.
TABLE-US-00024 TABLE 10 Serum Samples Tested by Sandwich ELISA
Sample Type No. of Samples Normal 555 (281-M, 274-F) Breast Cancer
260 Breast Benign 180 Colon Cancer 150 (71-M, 79-F) Colon Benign
296 (151-M, 145-F) Lung Cancer 323 (235-M, 93-F) Lung Benign 250
(130-M, 120-F) Ovarian Cancer 236 Ovarian Benign 150 Prostate
Cancer 138 Prostate Benign 147
[0404] FIG. 17 shows the Ovr110 concentration in serum from 540
healthy donors and more than 1200 patients with cancer. Elevated
levels of Ovr110 are observed in some patients of all cancer types
but patients with ovarian cancer have the highest median Ovr110
concentration.
[0405] We tested the concentration of Ovr110 in sera of one hundred
forty seven women with serous or endometrial ovarian cancer and
sixty seven sera of women with mucinous cancer, using sera which
represent all four stages of tumor progression. As shown in FIG.
18, the first two ovarian cancer types are positive for Ovr110 by
IHC while mucinous cancer is not. In good agreement with these
data, the median Ovr110 concentration in serum of patients with
endometrial and serous cancer is higher than in mucinous cancer
patients.
[0406] When compared with healthy women, the median concentration
of Ovr110 in serous and endometrial cancer is more than 2-fold
higher. Most of the women in this group of 260 healthy women are
above 50 years of age to mirror the age distribution of women with
ovarian cancer. We can not see differences in Ovr110 detection in
healthy women of pre-menopause and post-menopause age. More
important, we also do not detect an elevated level of Ovr110 in
sera of one hundred fifty women with benign ovarian diseases (50
sera of patients with endometriosis, enlarged ovaries and
polycystic ovaries, respectively).
[0407] In agreement with our findings that Ovr110 is expressed as a
cell surface membrane protein, the overall concentration of Ovr110
in serum is very low even in women with serous cancer. Hence, the
Ovr110 concentration detected in sera from women with serous cancer
is below 20 ng/ml.
Example 7
Deposits
Deposit of Cell Lines and DNA
[0408] The following 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. Ovr110.A57.1 (PTA-5180) was deposited
May 8, 2003. Ovr110.A7.1 (PTA-5855) and Ovr110.A72.1 (PTA-5856)
were deposited Mar. 11, 2004. Ovr110.C3.2 (PTA-5884) was deposited
Mar. 23, 2004. The names of the deposited hybridoma cell lines
above may be shortened for convenience of reference. E.g. A57.1
corresponds to Ovr110.A57.1. These hybridomas correspond to the
clones (with their full names) listed in Table 11.
TABLE-US-00025 TABLE 11 ATCC deposits Hybridoma ATCC Accession No.
Deposit Date Ovr110.A57.1 PTA-5180 May 8, 2003 Ovr110.A7.1 PTA-5855
Mar. 11, 2004 Ovr110.A72.1 PTA-5856 Mar. 11, 2004 Ovr110.C3.2
PTA-5884 Mar. 23, 2004
[0409] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations there under (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of deposit. The
organisms will be made available by ATCC under the terms of the
Budapest Treaty, and subject to an agreement between diaDexus, Inc.
and ATCC, which assures permanent and unrestricted availability of
the progeny of the cultures to the public upon issuance of the
pertinent U.S. patent or upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC .sctn.122 and the Commissioner's rules pursuant
thereto (including 3 7 CFR .sctn.1.14 with particular reference to
886 OG 638).
[0410] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strains are not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws. The making of these deposits is by
no means an admission that deposits are required to enable the
invention
Sequence CWU 1
1
191306PRTArtificial sequenceSynthetic 1Met Leu Gln Asn Ser Ala Val
Leu Leu Val Leu Val Ile Ser Ala Ser1 5 10 15Ala Thr Met Ala Ser Leu
Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser 20 25 30Ile Ile Ile Ile Leu
Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly 35 40 45Ile Ser Gly Arg
His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly 50 55 60Asn Ile Gly
Glu Asp Gly Ile Gln Ser Cys Thr Phe Glu Pro Asp Ile65 70 75 80Lys
Leu Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly 85 90
95Leu Val His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp
100 105 110Glu Met Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val
Ile Val 115 120 125Gly Asn Ala Ser Leu Arg Leu Lys Asn Val Gln Leu
Thr Asp Ala Gly 130 135 140Thr Tyr Lys Cys Tyr Ile Ile Thr Ser Lys
Gly Lys Gly Asn Ala Asn145 150 155 160Leu Glu Tyr Lys Thr Gly Ala
Phe Ser Met Pro Glu Val Asn Val Asp 165 170 175Tyr Asn Ala Ser Ser
Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe 180 185 190Pro Gln Pro
Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn 195 200 205Phe
Ser Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val 210 215
220Thr Met Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn
Thr225 230 235 240Tyr Ser Cys Met Ile Glu Asn Asp Ile Ala Lys Ala
Thr Gly Asp Ile 245 250 255Lys Val Thr Glu Ser Glu Ile Lys Arg Arg
Ser His Leu Gln Leu Leu 260 265 270Asn Ser Lys Ala Ser Leu Cys Val
Ser Ser Phe Phe Ala Ile Ser Trp 275 280 285Ala Leu Leu Pro Leu Ser
Pro Tyr Leu Met Leu Lys His His His His 290 295 300His
His3052278PRTArtificial sequenceSynthetic 2Met Leu Gln Asn Ser Ala
Val Leu Leu Val Leu Val Ile Ser Ala Ser1 5 10 15Ala Thr Met Gly Ile
Ser Gly Arg His Ser Ile Thr Val Thr Thr Val 20 25 30Ala Ser Ala Gly
Asn Ile Gly Glu Asp Gly Ile Gln Ser Cys Thr Phe 35 40 45Glu Pro Asp
Ile Lys Leu Ser Asp Ile Val Ile Gln Trp Leu Lys Glu 50 55 60Gly Val
Leu Gly Leu Val His Glu Phe Lys Glu Gly Lys Asp Glu Leu65 70 75
80Ser Glu Gln Asp Glu Met Phe Arg Gly Arg Thr Ala Val Phe Ala Asp
85 90 95Gln Val Ile Val Gly Asn Ala Ser Leu Arg Leu Lys Asn Val Gln
Leu 100 105 110Thr Asp Ala Gly Thr Tyr Lys Cys Tyr Ile Ile Thr Ser
Lys Gly Lys 115 120 125Gly Asn Ala Asn Leu Glu Tyr Lys Thr Gly Ala
Phe Ser Met Pro Glu 130 135 140Val Asn Val Asp Tyr Asn Ala Ser Ser
Glu Thr Leu Arg Cys Glu Ala145 150 155 160Pro Arg Trp Phe Pro Gln
Pro Thr Val Val Trp Ala Ser Gln Val Asp 165 170 175Gln Gly Ala Asn
Phe Ser Glu Val Ser Asn Thr Ser Phe Glu Leu Asn 180 185 190Ser Glu
Asn Val Thr Met Lys Val Val Ser Val Leu Tyr Asn Val Thr 195 200
205Ile Asn Asn Thr Tyr Ser Cys Met Ile Glu Asn Asp Ile Ala Lys Ala
210 215 220Thr Gly Asp Ile Lys Val Thr Glu Ser Glu Ile Lys Arg Arg
Ser His225 230 235 240Leu Gln Leu Leu Asn Ser Lys Ala Ser Leu Cys
Val Ser Ser Phe Phe 245 250 255Ala Ile Ser Trp Ala Leu Leu Pro Leu
Ser Pro Tyr Leu Met Leu Lys 260 265 270His His His His His His
275330DNAArtificial sequenceSynthetic 3ccaatgcatg gtatttcagg
gagacactcc 30430DNAArtificial sequenceSynthetic 4cggctagctt
ttagcatcag gtaagggctg 305292PRTArtificial sequenceSynthetic 5Met
Leu Gln Asn Ser Ala Val Leu Leu Val Leu Val Ile Ser Ala Ser1 5 10
15Ala Thr His Glu Ala Glu Gln Ser Arg Met His Gly Ile Ser Gly Arg
20 25 30His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile Gly
Glu 35 40 45Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu
Ser Asp 50 55 60Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu
Val His Glu65 70 75 80Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln
Asp Glu Met Phe Arg 85 90 95Gly Arg Thr Ala Val Phe Ala Asp Gln Val
Ile Val Gly Asn Ala Ser 100 105 110Leu Arg Leu Lys Asn Val Gln Leu
Thr Asp Ala Gly Thr Tyr Lys Cys 115 120 125Tyr Ile Ile Thr Ser Lys
Gly Lys Gly Asn Ala Asn Leu Glu Tyr Lys 130 135 140Thr Gly Ala Phe
Ser Met Pro Glu Val Asn Val Asp Tyr Asn Ala Ser145 150 155 160Ser
Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln Pro Thr 165 170
175Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser Glu Val
180 185 190Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met
Lys Val 195 200 205Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr
Tyr Ser Cys Met 210 215 220Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly
Asp Ile Lys Val Thr Glu225 230 235 240Ser Glu Ile Lys Arg Arg Ser
His Leu Gln Leu Leu Asn Ser Lys Ala 245 250 255Ser Leu Cys Val Ser
Ser Phe Phe Ala Ile Ser Trp Ala Leu Leu Pro 260 265 270Leu Ser Pro
Tyr Leu Met Leu Lys Ala Ser His His His His His His 275 280 285His
His His His 290633DNAArtificial sequenceSynthetic 6ctttgtttaa
acatgaagac attgcctgcc atg 33729DNAArtificial sequenceSynthetic
7cggctagcac tcctcacaca tatggatgc 29829DNAArtificial
sequenceSynthetic 8cggctagcgg gtctgcttgc cacttcgtc
299289PRTArtificial sequenceSynthetic 9Met Lys Thr Leu Pro Ala Met
Leu Gly Thr Gly Lys Leu Phe Trp Val1 5 10 15Phe Phe Leu Ile Pro Tyr
Leu Asp Ile Trp Asn Ile His Gly Lys Glu 20 25 30Ser Cys Asp Val Gln
Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile 35 40 45Leu Ala Gly Asp
Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr Cys Ala 50 55 60Asn Arg Pro
His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr Cys Val65 70 75 80Lys
Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser 85 90
95Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn Gly Ser
100 105 110Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser
His Ser 115 120 125Thr Thr Leu Tyr Val Thr Asp Val Lys Ser Ala Ser
Glu Arg Pro Ser 130 135 140Lys Asp Glu Met Ala Ser Arg Pro Trp Leu
Leu Tyr Ser Leu Leu Pro145 150 155 160Leu Gly Gly Leu Pro Leu Leu
Ile Thr Thr Cys Phe Cys Leu Phe Cys 165 170 175Cys Leu Arg Arg His
Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala 180 185 190Gly Arg Glu
Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr 195 200 205Glu
Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly 210 215
220Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly
Ser225 230 235 240Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys
Pro Gly Ile Val 245 250 255Tyr Ala Ser Leu Asn His Ser Val Ile Gly
Leu Asn Ser Arg Leu Ala 260 265 270Arg Asn Val Lys Glu Ala Pro Thr
Glu Tyr Ala Ser Ile Cys Val Arg 275 280 285Ser10241PRTArtificial
sequenceSynthetic 10Met Lys Thr Leu Pro Ala Met Leu Gly Thr Gly Lys
Leu Phe Trp Val1 5 10 15Phe Phe Leu Ile Pro Tyr Leu Asp Ile Trp Asn
Ile His Gly Lys Glu 20 25 30Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg
Gln Ser Glu His Ser Ile 35 40 45Leu Ala Gly Asp Pro Phe Glu Leu Glu
Cys Pro Val Lys Tyr Cys Ala 50 55 60Asn Arg Pro His Val Thr Trp Cys
Lys Leu Asn Gly Thr Thr Cys Val65 70 75 80Lys Leu Glu Asp Arg Gln
Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser 85 90 95Phe Phe Ile Leu His
Phe Glu Pro Val Leu Pro Asn Asp Asn Gly Ser 100 105 110Tyr Arg Cys
Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser His Ser 115 120 125Thr
Thr Leu Tyr Val Thr Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala 130 135
140Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln
Thr145 150 155 160Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu
Ser Glu Thr Gly 165 170 175Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe
Arg Met Gln Glu Gly Ser 180 185 190Glu Val Tyr Ser Asn Pro Cys Leu
Glu Glu Asn Lys Pro Gly Ile Val 195 200 205Tyr Ala Ser Leu Asn His
Ser Val Ile Gly Leu Asn Ser Arg Leu Ala 210 215 220Arg Asn Val Lys
Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg225 230 235
240Ser11390PRTArtificial sequenceSynthetic 11Met Lys Thr Leu Pro
Ala Met Leu Gly Thr Gly Lys Leu Phe Trp Val1 5 10 15Phe Phe Leu Ile
Pro Tyr Leu Asp Ile Trp Asn Ile His Gly Lys Glu 20 25 30Ser Cys Asp
Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile 35 40 45Leu Ala
Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr Cys Ala 50 55 60Asn
Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr Cys Val65 70 75
80Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser
85 90 95Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn Gly
Ser 100 105 110Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu
Ser His Ser 115 120 125Thr Thr Leu Tyr Val Thr Asp Val Lys Ser Ala
Ser Glu Arg Pro Ser 130 135 140Lys Asp Glu Met Ala Ser Arg Pro Ala
Ser Glu Asn Leu Tyr Phe Gln145 150 155 160Gly Pro Arg Gly Pro Thr
Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro 165 170 175Ala Pro Asn Leu
Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 180 185 190Ile Lys
Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val 195 200
205Val Val Asp Val Ser Glu Asp Pro Asp Val Gln Ile Ser Trp Phe Val
210 215 220Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
Glu Asp225 230 235 240Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu
Pro Ile Gln His Gln 245 250 255Asp Trp Met Ser Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp 260 265 270Leu Pro Ala Pro Ile Glu Arg
Thr Ile Ser Lys Pro Lys Gly Val Arg 275 280 285Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys 290 295 300Lys Gln Val
Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp305 310 315
320Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys
325 330 335Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
Tyr Ser 340 345 350Lys Leu Arg Val Glu Lys Asn Trp Val Glu Arg Asn
Ser Tyr Ser Cys 355 360 365Ser Val Val His Glu Gly Leu His Asn His
His Thr Thr Lys Ser Phe 370 375 380Ser Arg Thr Pro Gly Lys385
39012614PRTArtificial sequenceSynthetic 12Met Asn Arg Thr Trp Pro
Arg Arg Ile Trp Gly Ser Ser Gln Asp Glu1 5 10 15Ala Glu Leu Ile Arg
Glu Asp Ile Gln Gly Ala Leu His Asn Tyr Arg 20 25 30Ser Gly Arg Gly
Glu Arg Arg Ala Ala Ala Leu Arg Ala Thr Gln Glu 35 40 45Glu Leu Gln
Arg Asp Arg Ser Pro Ala Ala Glu Thr Pro Pro Leu Gln 50 55 60Arg Arg
Pro Ser Val Arg Ala Val Ile Ser Thr Val Glu Arg Gly Ala65 70 75
80Gly Arg Gly Arg Pro Gln Ala Lys Pro Ile Pro Glu Ala Glu Glu Ala
85 90 95Gln Arg Pro Glu Pro Val Gly Thr Ser Ser Asn Ala Asp Ser Ala
Ser 100 105 110Pro Asp Leu Gly Pro Arg Gly Pro Asp Leu Val Val Leu
Gln Ala Glu 115 120 125Arg Glu Val Asp Ile Leu Asn His Val Phe Asp
Asp Val Glu Ser Phe 130 135 140Val Ser Arg Leu Gln Lys Ser Ala Glu
Ala Ala Arg Val Leu Glu His145 150 155 160Arg Glu Arg Gly Arg Arg
Ser Arg Arg Arg Ala Ala Gly Glu Gly Leu 165 170 175Leu Thr Leu Arg
Ala Lys Pro Pro Ser Glu Ala Glu Tyr Thr Asp Val 180 185 190Leu Gln
Lys Ile Lys Tyr Ala Phe Ser Leu Leu Ala Arg Leu Arg Gly 195 200
205Asn Ile Ala Asp Pro Ser Ser Pro Glu Leu Leu His Phe Leu Phe Gly
210 215 220Pro Leu Gln Met Ile Val Asn Thr Ser Gly Gly Pro Glu Phe
Ala Ser225 230 235 240Ser Val Arg Arg Pro His Leu Thr Ser Asp Ala
Val Ala Leu Leu Arg 245 250 255Asp Asn Val Thr Pro Arg Glu Asn Glu
Leu Trp Thr Ser Leu Gly Asp 260 265 270Ser Trp Thr Arg Pro Gly Leu
Glu Leu Ser Pro Glu Glu Gly Pro Pro 275 280 285Tyr Arg Pro Glu Phe
Phe Ser Gly Trp Glu Pro Pro Val Thr Asp Pro 290 295 300Gln Ser Arg
Ala Trp Glu Asp Pro Val Glu Lys Gln Leu Gln His Glu305 310 315
320Arg Arg Arg Arg Gln Gln Ser Ala Pro Gln Val Ala Val Asn Gly His
325 330 335Arg Asp Leu Glu Pro Glu Ser Glu Pro Gln Leu Glu Ser Glu
Thr Ala 340 345 350Gly Lys Trp Val Leu Cys Asn Tyr Asp Phe Gln Ala
Arg Asn Ser Ser 355 360 365Glu Leu Ser Val Lys Gln Arg Asp Val Leu
Glu Val Leu Asp Asp Ser 370 375 380Arg Lys Trp Trp Lys Val Arg Asp
Pro Ala Gly Gln Glu Gly Tyr Val385 390 395 400Pro Tyr Asn Ile Leu
Thr Pro Tyr Pro Gly Pro Arg Leu His His Ser 405 410 415Gln Ser Pro
Ala Arg Ser Leu Asn Ser Thr Pro Pro Pro Pro Pro Ala 420 425 430Pro
Ala Pro Ala Pro Pro Pro Ala Leu Ala Arg Pro Arg Trp Asp Arg 435 440
445Pro Arg Trp Asp Ser Cys Asp Ser Leu Asn Gly Leu Asp Pro Ser Glu
450 455 460Lys Glu Lys Phe Ser Gln Met Leu Ile Val Asn Glu Glu Leu
Gln Ala465 470 475 480Arg Leu Ala Gln Gly Arg Ser Gly Pro Ser Arg
Ala Val Pro Gly Pro 485 490 495Arg Ala Pro Glu Pro Gln Leu Ser Pro
Gly Ser Asp Ala Ser Glu Val 500 505 510Arg Ala Trp Leu Gln Ala Lys
Gly Phe Ser Ser Gly Thr Val Asp Ala 515 520 525Leu Gly Val Leu Thr
Gly Ala Gln Leu Phe Ser Leu Gln Lys Glu Glu 530 535 540Leu Arg Ala
Val Ser Pro Glu Glu Gly Ala Arg Val Tyr Ser Gln Val545 550 555
560Thr Val Gln Arg Ser Leu Leu Glu Asp Lys Glu Lys Val Ser Glu Leu
565 570 575Glu Ala Val
Met Glu Lys Gln Lys Lys Lys Val Glu Gly Glu Val Glu 580 585 590Met
Glu Val Ile Asp Pro Ala Phe Leu Tyr Lys Val Val Arg Trp Ala 595 600
605His His His His His His 61013309PRTArtificial sequenceSynthetic
13Met Leu Gln Asn Ser Ala Val Leu Leu Val Leu Val Ile Ser Ala Ser1
5 10 15Ala Thr Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile
Ser 20 25 30Ile Ile Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly
Phe Gly 35 40 45Ile Ser Gly Arg His Ser Ile Thr Val Thr Thr Val Ala
Ser Ala Gly 50 55 60Asn Ile Gly Glu Asp Gly Ile Gln Ser Cys Thr Phe
Glu Pro Asp Ile65 70 75 80Lys Leu Ser Asp Ile Val Ile Gln Trp Leu
Lys Glu Gly Val Leu Gly 85 90 95Leu Val His Glu Phe Lys Glu Gly Lys
Asp Glu Leu Ser Glu Gln Asp 100 105 110Glu Met Phe Arg Gly Arg Thr
Ala Val Phe Ala Asp Gln Val Ile Val 115 120 125Gly Asn Ala Ser Leu
Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly 130 135 140Thr Tyr Lys
Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn145 150 155
160Leu Glu Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp
165 170 175Tyr Asn Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg
Trp Phe 180 185 190Pro Gln Pro Thr Val Val Trp Ala Ser Gln Val Asp
Gln Gly Ala Asn 195 200 205Phe Ser Glu Val Ser Asn Thr Ser Phe Glu
Leu Asn Ser Glu Asn Val 210 215 220Thr Met Lys Val Val Ser Val Leu
Tyr Asn Val Thr Ile Asn Asn Thr225 230 235 240Tyr Ser Cys Met Ile
Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile 245 250 255Lys Val Thr
Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu 260 265 270Asn
Ser Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp 275 280
285Ala Leu Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys Tyr Pro Tyr Asp
290 295 300Val Pro Asp Tyr Ala3051419RNAArtificial
sequenceSynthetic 14gguguuuuag gcuuggucc 191519RNAArtificial
sequenceSynthetic 15cucacagaug cuggcaccu 191619RNAArtificial
sequenceSynthetic 16gguugugucu gugcucuac 191719RNAArtificial
sequenceSynthetic 17ccgugcuccu ggggcuggg 191819RNAArtificial
sequenceSynthetic 18uucuccgaac gugucacgu 191919RNAArtificial
sequenceSynthetic 19ggaguuggau cucucagaa 19
* * * * *