U.S. patent application number 13/328458 was filed with the patent office on 2012-10-18 for pro104 antibody compositions and methods of use.
This patent application is currently assigned to diaDexus, Inc.. Invention is credited to Laura Corral, Gilbert-Andre Keller, Muriel Kmet, Wenlu Li, Jackie Papkoff, Glenn Pilkington, Iris Simon, Jianwen Tang.
Application Number | 20120263647 13/328458 |
Document ID | / |
Family ID | 34594531 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120263647 |
Kind Code |
A1 |
Papkoff; Jackie ; et
al. |
October 18, 2012 |
Pro104 Antibody Compositions and Methods of Use
Abstract
The invention provides isolated anti-ovarian, pancreatic, lung
or breast cancer antigen (Pro104) antibodies that bind to Pro104 on
a mammalian cell in vivo. The invention also encompasses
compositions comprising an anti-Pro104 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-Pro104 antibody, as well as an expression vector
comprising the isolated nucleic acid. Also provided are cells that
produce the anti-Pro104 antibodies. The invention encompasses a
method of producing the anti-Pro104 antibodies. Other aspects of
the invention are a method of killing a Pro104-expressing cancer
cell, comprising contacting the cancer cell with an anti-Pro104
antibody and a method of alleviating or treating a
Pro104-expressing cancer in a mammal, comprising administering a
therapeutically effective amount of the anti-Pro104 antibody to the
mammal.
Inventors: |
Papkoff; Jackie; (San
Francisco, CA) ; Pilkington; Glenn; (Rye, AU)
; Keller; Gilbert-Andre; (Belmont, CA) ; Li;
Wenlu; (South San Francisco, CA) ; Corral; Laura;
(Belmont, MA) ; Simon; Iris; (Awstodam, NL)
; Kmet; Muriel; (Palo Alto, CA) ; Tang;
Jianwen; (Freemont, CA) |
Assignee: |
diaDexus, Inc.
South San Francisco
CA
|
Family ID: |
34594531 |
Appl. No.: |
13/328458 |
Filed: |
December 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12354047 |
Jan 15, 2009 |
8080650 |
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13328458 |
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10562259 |
Dec 21, 2005 |
7479546 |
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PCT/US04/20741 |
Jun 28, 2004 |
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12354047 |
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60485346 |
Jun 27, 2003 |
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60523271 |
Nov 17, 2003 |
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Current U.S.
Class: |
424/9.1 ;
424/142.1; 424/146.1; 424/178.1; 435/338; 435/7.23; 530/388.15;
530/388.26; 530/391.3; 530/391.7; 536/24.5 |
Current CPC
Class: |
C07K 2317/77 20130101;
G01N 33/57449 20130101; A61K 47/6869 20170801; A61P 35/00 20180101;
A61K 47/6857 20170801; A61K 49/0058 20130101; A61K 47/6855
20170801; A61K 47/6859 20170801; G01N 2500/00 20130101; C07K
16/3069 20130101; A61K 49/0032 20130101; G01N 33/57415 20130101;
C07K 16/3023 20130101; C07K 16/3015 20130101; A61P 43/00 20180101;
C07K 16/303 20130101 |
Class at
Publication: |
424/9.1 ;
530/388.26; 530/388.15; 530/391.7; 424/146.1; 435/7.23; 530/391.3;
536/24.5; 435/338; 424/142.1; 424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/577 20060101 G01N033/577; A61P 35/00 20060101
A61P035/00; C07H 21/02 20060101 C07H021/02; C12N 5/16 20060101
C12N005/16; C07K 16/40 20060101 C07K016/40; A61K 49/00 20060101
A61K049/00 |
Claims
1. An isolated antibody which is produced by a hybridoma selected
from the group consisting of American Type Culture Collection
accession number PTA-5277, 6076, 6077 and 6078 or competes for
binding to the same epitope as the epitope bound by a monoclonal
antibody produced by a hybridoma selected from the group consisting
of ATCC accession number PTA-5277, 6076, 6077 and 6078.
2. The antibody of claim 1 wherein said antibody is a human
antibody.
3. The antibody of claim 1 which binds to Pro104 on a mammalian
cell.
4. The antibody of claim 3 which internalizes upon binding to
Pro104 on a mammalian cell.
5. The antibody of claim 1 which is conjugated to a growth
inhibitory agent or a cytotoxic agent.
6. The antibody of claim 1 wherein said antibody inhibits the
growth of Pro104-expressing cells.
7. An isolated cell that produces the antibody of claim 1.
8. A method of killing a Pro104-expressing cell, comprising
contacting the cell with the antibody of claim 1.
9. The method of claim 8, wherein the antibody is a human
antibody.
10. The method of claim 8, wherein the antibody is conjugated to a
cytotoxic agent.
11. The method of claim 8, wherein the antibody is administered in
conjunction with at least one chemotherapeutic agent.
12. A method for detecting Pro104 overexpression in a subject in
need thereof comprising comparing the level of Pro104 in a sample,
determined by the binding of a Pro104 antibody of claim 1, from
said subject to the level of Pro104 in a control, wherein an
increase in the level of Pro104 in the sample from the subject as
compared to the control is indicative of Pro104 overexpression in
the subject.
13. The antibody of claim 1 which is detectably labeled.
14. A method of imaging Pro104-expressing cells in a subject
comprising detecting the presence of the antibody of claim 13 which
has been administered to said subject.
15. An article of manufacture comprising a container and the
antibody of claim 1.
16. The article of manufacture of claim 15 which further comprises
a carrier.
17. The article of manufacture of claim 15 which further comprises
a label or package insert.
18. An isolated Pro104 specific siRNA which knocks-down Pro104
mRNA.
19. The Pro104 specific siRNA of claim 18 which down regulates
Pro104 protein expression.
20. The Pro104 specific siRNA of claim 18 which induces apoptosis.
Description
[0001] This patent application is a continuation of U.S.
application Ser. No. 12/354,047 filed Jan. 15, 2009, which is a
continuation of U.S. application Ser. No. 10/562,259, filed Dec.
21, 2005, now issued as U.S. Pat. No. 7,479,546, which is the U.S.
National Stage of PCT Application number PCT/US2004/020741, filed
Jun. 28, 2004, which claims the benefit of priority from U.S.
Provisional Patent Application Ser. No. 60/523,271, filed Nov. 17,
2003 and U.S. Provisional Patent Application Ser. No. 60/485,346,
filed Jun. 27, 2003, each of which are herein incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-Pro104 antibody
compositions and methods of detecting Pro104 expressing cancers and
killing Pro104-expressing breast, ovarian pancreatic and lung
cancers cells. In addition, this invention relates to methods of
modulating or killing a Pro104 expressing cell by administering an
effective amount of a compound capable of modulating Pro104
function.
BACKGROUND OF THE INVENTION
Breast Cancer
[0003] 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. In the United States it is
estimated there will be 217,440 new cases of breast cancer and
40,580 deaths due to breast cancer in 2004. (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.
[0004] 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.
[0005] 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).
[0006] 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.
[0007] 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.
[0008] 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%).
[0009] There are four primary classifications of breast cancer
varying by the site of origin and the extent of disease
development. [0010] 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. [0011] 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. [0012] III. Lobular carcinoma in situ
(LCIS): Malignancy arising in a single lobule of the breast that
fails to extend through the lobule wall, it generally remains
localized. [0013] IV. Infiltrating lobular carcinoma (ILC):
Malignancy arising in a single lobule of the breast and invading
directly through the lobule wall into adjacent tissues. By virtue
of its invasion beyond the lobule wall, ILC may penetrate
lymphatics and blood vessels and spread to distant sites.
[0014] 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.
[0015] 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 carcinoma. AJCC Cancer Staging
Handbook pp. 159-70 (Irvin D. Fleming et al. eds., 5.sup.th 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).
[0016] In addition to the staging of the primary tumor, breast
cancer metastases 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.
[0017] 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 which
could differentiate between different stages of invasion. Progress
in this field will allow more rapid and reliable method for
treating breast cancer patients.
[0018] 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.
[0019] 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.
[0020] Patients with stage I and stage II breast cancer requires
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).
[0021] In an effort to provide more treatment options to patients,
efforts are underway to define an earlier stage of breast cancer
with low recurrence which 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).
Ovarian Cancer
[0022] Cancer of the ovaries is the fourth-most common cause of
cancer death in women in the United States, with more than 23,000
new cases and roughly 14,000 deaths predicted for the year 2001.
Shridhar, V. et al., Cancer Res. 61(15): 5895-904 (2001);
Memarzadeh, S. & Berek, J. S., J. Reprod. Med. 46(7): 621-29
(2001). The American Cancer Society estimates that there will be
about 25,580 new cases of ovarian cancer in 2004 in the United
States alone. Ovarian cancer will cause about 16,090 deaths in the
United States. ACS Website: cancer with the extension .org of the
world wide web. The incidence of ovarian cancer is of serious
concern worldwide, with an estimated 191,000 new cases predicted
annually. Runnebaum, I. B. & Stickeler, E., J. Cancer Res.
Clin. Oncol. 127(2): 73-79 (2001). Unfortunately, women with
ovarian cancer are typically asymptomatic until the disease has
metastasized. Because effective screening for ovarian cancer is not
available, roughly 70% of women diagnosed have an advanced stage of
the cancer with a five-year survival rate of approximately 25-30%.
Memarzadeh, S. & Berek, J. S., supra; Nunns, D. et al., Obstet.
Gynecol. Surv. 55(12): 746-51. Conversely, women diagnosed with
early stage ovarian cancer enjoy considerably higher survival
rates. Werness, B. A. & Eltabbakh, G. H., Int'l. J. Gynecol.
Pathol. 20(1): 48-63 (2001). Although our understanding of the
etiology of ovarian cancer is incomplete, the results of extensive
research in this area point to a combination of age, genetics,
reproductive, and dietary/environmental factors. Age is a key risk
factor in the development of ovarian cancer: while the risk for
developing ovarian cancer before the age of 30 is slim, the
incidence of ovarian cancer rises linearly between ages 30 to 50,
increasing at a slower rate thereafter, with the highest incidence
being among septagenarian women. Jeanne M. Schilder et al.,
Hereditary Ovarian Cancer: Clinical Syndromes and Management, in
Ovarian Cancer 182 (Stephen C. Rubin & Gregory P. Sutton eds.,
2d ed. 2001).
[0023] 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.
[0024] 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 that pregnancy, lactation, and
the use of oral contraceptives, all of which suppress ovulation,
confer a protective effect with respect to developing ovarian
cancer. Id.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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 IA,
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.
[0030] 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.
[0031] 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.
[0032] 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. Vartainen, 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.
[0033] 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 approximately 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.
[0034] 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.
[0035] Accordingly, there is a great need for more sensitive and
accurate methods for predicting whether a person is likely to
develop ovarian cancer, for diagnosing ovarian cancer, for
monitoring the progression of the disease, for staging the ovarian
cancer, for determining whether the ovarian cancer has
metastasized, and for imaging the ovarian cancer. There is also a
need for better treatment of ovarian cancer.
Pancreatic Cancer
[0036] Pancreatic cancer is the thirteenth-most common cancer and
eighth-most cause of cancer death worldwide. Donghui Li, Molecular
Epidemiology, in Pancreatic Cancer 3 (Douglas B. Evans et al. eds.,
2002). In the United States, cancer of the pancreas is the
fourth-most common cancer in both males and females, accounting for
five percent of cancer deaths and nearly 30,000 deaths overall. Id.
The rates of pancreatic cancer are higher in men than women and
higher in African-Americans as opposed to Caucasians. Id. at 9. The
most significant predictor of pancreatic cancer is patient age;
among Caucasians, the age-related incidence of pancreatic cancer
increases continuously, even through the 85 and older category. Id.
at 3. Approximately 80% of cases occur in the age range of 60 to
80, with those in their 80s experiencing a risk of acquiring the
disease 40 times that of those in their 40s. Id. Furthermore, the
American Cancer Society estimates that there will be about 31,800
new cases of pancreatic cancer in 2004 in the United States alone.
Pancreatic cancer will cause about 31,200 deaths in the United
States in the same year. ACS Website: cancer with the extension
.org of the world wide web. Despite the efforts of researchers and
physicians in devising treatments for pancreatic cancer, it remains
almost universally fatal. James R. Howe, Molecular Markers as a
Tool for the Early Diagnosis of Pancreatic Cancer, in Pancreatic
Cancer 29 (Douglas B. Evans et al. eds., 2002).
[0037] Aside from age, a number of risk factors for pancreatic
cancer have been identified, including smoking, diet, occupation,
certain medical conditions, heredity, and molecular biologic.
Smoking is the most important risk factor for acquiring the
disease, with the link between smoking and pancreatic cancer being
established in numerous studies. Li, supra at 3. The relative risk
amounts to at least 1.5, increasing with the level of smoking to an
outer risk ratio of 10-fold. Id. The next most important factor
would appear to be diet, with increased risk associated with animal
protein and fat intake, and decreased risk associated with intake
of fruits and vegetables. Id. at 3-4. As for particular
occupations, excessive rates of pancreatic cancer have been
associated with workers in chemistry, coal and gas exploration, the
metal industry, leather tanning, textiles, aluminum milling, and
transportation. Id. at 4. A number of medical conditions have also
been associated with an increased incidence of pancreatic cancer,
including diabetes, chronic pancreatitis, gastrectomy, and
cholecystectomy, although the cause and effect relationship between
these conditions and pancreatic cancer has not been established.
Id.
[0038] Hereditary genetic factors comprise less than 10% of the
pancreatic cancer burden, with associations documented with
hereditary pancreatitis, as well as germline mutations in familial
cancer syndrome genes such as hMSH2 and hMLH1 (hereditary
nonpolyposis colon cancer), p16 (familial atypical multiple
mole-melanoma) and BRCA1/BRCA2 (breast and ovarian cancer). Id. at
3. While no other organ has a higher inherited basis for cancer
than the pancreas, researchers have been unable to pinpoint the
particular genetic defect(s) that contribute to one's
susceptibility to pancreatic cancer. David H. Berger & William
E. Fisher, Inherited Pancreatic Cancer Syndromes, in Pancreatic
Cancer 73 (Douglas B. Evans et al. eds., 2002).
[0039] From the standpoint of molecular biology, research has
revealed an association between pancreatic cancer and a number of
genetic mutations, including the activation of the proto-oncogene
K-ras and the inactivation of the tumor suppressor genes p53, p16,
and DPC4. Marina E. Jean et al., The Molecular Biology of
Pancreatic Cancer, in Pancreatic Cancer 15 (Douglas B. Evans et al.
eds., 2002).
[0040] In one study of pancreatic adenocarcinomas, 83% possessed
K-ras activation along with inactivation of p16 and p53. Id. K-ras
mutations are found in 80 to 95% of pancreatic adenocarcinomas,
with p53, p16, and DPC4 genes being the must frequently deleted
tumor suppressor genes in cancer of the pancreas. Howe, supra at
29. Homozygous deletions, hypermethylation, and mutations of the
p16 gene have been discovered in 85 to 98% of adenocarcinomas of
the pancreas. Id. As might be expected by the role of alterations
in the K-ras, p53, p16, and DPC4 genes, loss of regulation of the
cell cycle would appear to be key to tumorigenesis in the pancreas,
and may explain why this cancer is so aggressive. Jean, supra at
15. Research has also revealed a link between this cancer and
abnormal regulation of certain growth factors and growth factor
receptors, as well as an upregulation of matrix metalloproteinases
and tumor angiogenesis regulators. Id. Epidermal growth factor,
fibroblast growth factor, transforming growth factor-.E-backward.,
insulin-like growth factor, hepatocyte growth factor, and vascular
endothelial growth factor may play various roles in pancreatic
cancer, although such roles have not been elucidated. Id. at
18-22.
[0041] The development of screening techniques to detect the
presence of pancreatic cancer is particularly essential for this
deadly cancer, as most patients fail to present until their
pancreatic tumors obstruct the bile duct or induce pain, at which
point the tumors have invaded the capillary and lymphatic vessels
that surround the pancreas, Howe, supra at 29; unfortunately,
patients with the metastatic form of the disease typically survive
less than one year after diagnosis, Jean et al., supra at 15. While
computed tomography (CT) and endoscopic retrograde
cholangiopancreatography (ERCP) may assist in the diagnosis of
symptomatic patients, there is presently no tool for screening for
pancreatic tumors that would permit their early discovery, at which
point they might be curable. Howe, supra at 29. Markers such as
carcinoembryonic antigen, and antibodies generated against cell
lines of human colonic cancer (CA 19-9 and CA 195), human ovarian
cancer (CA 125), and human pancreatic cancer (SPAN-1 and DUPAN-2)
may be elevated in the serum of patients with pancreatic cancer,
but these markers are not sufficiently reliable to serve as
screening tools due to their lack of specificity and appearance
late in the disease. Walter J. Burdette, Cancer: Etiology,
Diagnosis, and Treatment 99 (1998); Hasholzner, U. et al.,
Anticancer Res. 19(4A): 2477-80 (1999).
[0042] Due to the present lack of adequate screening methods,
physicians are increasingly turning to techniques which employ
methods of molecular biology as the most promising means for early
diagnosis of the disease. Howe, supra at 30. At present, there is
no high sensitivity, high specificity marker that enables the
detection of pancreatic cancer in asymptomatic individuals, but
several biological markers are under investigation. Id.
Considerable efforts are currently focusing on K-ras, with
researchers devising techniques to screen samples of pancreatic
juice, bile, duodenal juice, or ERCP brushings to detect K-ras
mutations. Id. Because the collection of these samples is invasive
and not particularly helpful in screening those who are
asymptomatic, researchers have also turned to serum and stool
analysis for K-ras mutations, with the former being the most
promising, as the latter is hindered by the complexity of the
source material. Id. at 35-38, 42. Moreover, because serum levels
of the transcription factor protein p53 may parallel cancer
progression, p53 is likewise being studied as possible tumor
marker. Id. at 37; Jean et al., supra at 17.
[0043] Once pancreatic cancer has been diagnosed, treatment
decisions are made in reference to the stage of cancer progression.
A number of imaging techniques are employed to stage pancreatic
cancer, with computed tomography (CT) being the present method of
choice, Harmeet Kaur et al., Pancreatic Cancer: Radiologic Staging,
in Pancreatic Cancer 86 (Douglas B. Evans et al. eds., 2002);
Ishiguchi, T. et al., Hepatogastroenterology 48(40): 923-27 (2001),
despite the fact that it frequently underestimates the extent of
the cancer, as small-volume metastases are often beyond the
resolution of CT, H. J. Kim & K. C. Conlon, Laparascopic
Staging, in Pancreatic Cancer 15 (Douglas B. Evans et al. eds.,
2002). MRI may at some point supplant CT in view of, inter alia,
its ability to (1) contrast among various tissue, (2) modify pulse
sequences to improve visualization of lesions and minimize
artifacts, (3) perform imaging while limiting a patient's exposure
to ionizing radiation, and (4) visualize vessels without using IV
iodinated contrast reagents. Kaur et al., supra at 87. At present,
however, MRI has not demonstrated a clear advantage over CT. Kim
& Conlon, supra at 116.
[0044] A variety of ultrasonic techniques are also currently
employed in staging, including transabdominal ultrasound (TUS),
endoscopic ultrasound (EUS), and intraoperative ultrasound (IUS),
with EUS being one of the most promising. Kaur et al., supra at 86;
Richard A. Erickson, Endoscopic Diagnosis and Staging: Endoscopic
Ultrasound, Endoscopic Retrograde Cholangiopancreatography, in
Pancreatic Cancer 97-106 (Douglas B. Evans et al. eds., 2002).
These techniques, however, are each limited by a variety of
factors: TUS is hindered by gas in the gastrointestinal tract and
fat in the peritoneum, EUS requires considerable experience in
ultrasonography and endoscopy and may not be widely available, and
IUS can only be used intraoperatively. Kaur et al., supra at
86.
[0045] Although in its nascent stages, the search for markers that
will assist in staging pancreatic cancer has found some possible
leads. For example, research has revealed that two
metastasis-suppressing genes, nm23-H1 and KAI1, are differentially
expressed depending on the stage of pancreatic cancer, with their
expression being upregulated at early stages and down regulated at
later stages of the disease. Friess, H. et al., J. Clin. Oncol.
19(9): 2422-32 (2001). Researchers have also focused on genetic
lymph node staging, particularly searching for mutations in the
K-ras proto-oncogene. Yamada, T. et al., Int'l J. Oncol. 16(6):
1165-71 (2000). Likewise, research has identified that the presence
of mutated K-ras sequences in plasma/serum is associated with late
stage pancreatic cancer, although the presence of early stage
pancreatic cancer can be detected this way as well. Sorenson, G.
D., Clin. Cancer Res. 6(6): 2129-37 (2000). A promising staging
technique using a multimarker reverse transcriptase-polymerase
chain reaction assay has successfully distinguished pancreatic
cancer stages by assaying blood and tissue samples for mRNA
expression of the following tumor markers: the .E-backward.-human
chorionic gonadotropin gene, the hepatocyte growth factor receptor
gene c-met, and the
.E-backward.-1,4-N-acetyl-galactosaminyl-transferase gene. Bilchik,
A. et al., Cancer 88(5): 1037-44 (2000).
[0046] One classification system commonly used to stage pancreatic
cancer is the TNM system devised by the Union Internationale Contre
le Cancer. AJCC Cancer Staging Handbook 3 (Irvin D. Fleming et al.
eds., 5.sup.th ed. 1998). This system is divided into several
stages, each of which evaluates the extent of cancer growth with
respect to primary tumor (T), regional lymph nodes (N), and distant
metastasis (M). Id.
[0047] Stage 0 is characterized by carcinoma in situ (Tis), with no
regional lymph node metastasis (N0) and no distant metastasis (M0).
Id. at 113. Stages I and II differ from stage 0 only in terms of
tumor category: stage I involves a tumor limited only to the
pancreas that is either (1) 2 cm or less in greatest dimension (T1)
or (2) more than 2 cm in greatest dimension (T2), while stage II
involves a tumor that extends directly into the duodenum, bile
duct, or peripancreatic tissues (T3). Id. Stage III involves tumor
category T1, T2, or T3; regional lymph node metastasis (N1), which
involves either a single lymph node (pN1a) or multiple lymph nodes
(pN1b); and no distant metastasis (M0). Stage IVA is characterized
by tumor extension directly into the stomach, spleen, colon, or
adjacent large vessels (T4); any N category; and no distant
metastasis (M0). Lastly, stage IVB is characterized by any T
category, any N category, and distant metastasis (M1). Id.
[0048] Once the cancer has been staged, the only consistently
effective treatment for the disease is surgery, and with only ten
to fifteen percent of patients being able to undergo potentially
curative resection. Jean et al., supra at 15; Fleming et al. eds.,
supra at 111; William F. Regine, Postoperative Adjuvant Therapy:
Past, Present, and Future Trial Development, in Pancreatic Cancer
235 (Douglas B. Evans et al. eds., 2002). Moreover, the five-year
survival of those patients undergoing resection is below twenty
percent. Regine, supra at 235. While chemotherapeutic agents such
as gemcitabine and 5-fluorouracil have shown some effectiveness
against pancreatic carcinomas, the reality is that chemotherapy has
shown little impact on survival from pancreatic cancer. Burdette,
supra at 101. Radiation therapy has provided conflicting results
with respect to its efficacy, id., although radiation in
combination with 5-fluorouracil has shown some promise, Regine,
supra at 235.
[0049] In view of the failure of conventional techniques at
treating pancreatic cancer, a number of novel approaches employing
the techniques of molecular biology have been investigated.
Considerable research has been performed in the area of gene
therapy, including antisense technology, gene-directed prodrug
activation strategies, promoter gene strategies, and oncolytic
viral therapies. Eugene A. Choi & Francis R. Spitz, Strategies
for Gene Therapy, in Pancreatic Cancer 331 (Douglas B. Evans et al.
eds., 2002); Kasuya, H. et al., Hepatogastroenterology 48(40):
957-61 (2001). Other recent approaches have focused on the
inhibition of matrix metalloproteinases, enzymes which facilitate
the metastasis and invasion of tumor cells through their
degradation of basement membranes, and their role in peritumoral
stromal degradation and angiogenesis. Alexander S. Rosemurgy, II
& Mahmudul Haq, Role of Matrix Metalloproteinase Inhibition in
the Treatment of Pancreatic Cancer, in Pancreatic Cancer 369
(Douglas B. Evans et al. eds., 2002).
[0050] From the foregoing, it is clear that procedures used for
detecting, diagnosing, monitoring, staging, prognosticating, and
preventing the recurrence of pancreatic 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.
Lung Cancer
[0051] Throughout the last hundred years, the incidence of lung
cancer has steadily increased, so much so that now in many
countries, it is the most common cancer. In fact, lung cancer is
the second most prevalent type of cancer for both men and women in
the United States and is the most common cause of cancer death in
both sexes. Lung cancer deaths have increased ten-fold in both men
and women since 1930, primarily due to an increase in cigarette
smoking, but also due to an increased exposure to arsenic,
asbestos, chromates, chloromethyl ethers, nickel, polycyclic
aromatic hydrocarbons and other agents. See Scott, Lung Cancer: A
Guide to Diagnosis and Treatment, Addicus Books (2000) and Alberg
et al., in Kane et al. (eds.) Biology of Lung Cancer, pp. 11-52,
Marcel Dekker, Inc. (1998). The American Cancer Society estimates
there will be over 173,550 new cases of lung cancer in 2004.
Additionally, there will be an estimated 160,440 deaths from lung
cancer in 2004. ACS Website: cancer with the extension .org of the
world wide web.
[0052] Lung cancer may result from a primary tumor originating in
the lung or a secondary tumor which has spread from another organ
such as the bowel or breast. Although there are over a dozen types
of lung cancer, over 90% fall into two categories: small cell lung
cancer (SCLC) and non-small cell lung cancer (NSCLC). See Scott,
supra. About 20-25% of all lung cancers are characterized as SCLC,
while 70-80% are diagnosed as NSCLC. Id. A rare type of lung cancer
is mesothelioma, which is generally caused by exposure to asbestos,
and which affects the pleura of the lung. Lung cancer is usually
diagnosed or screened for by chest x-ray, CAT scans, PET scans, or
by sputum cytology. A diagnosis of lung cancer is usually confirmed
by biopsy of the tissue. Id.
[0053] SCLC tumors are highly metastatic and grow quickly. By the
time a patient has been diagnosed with SCLC, the cancer has usually
already spread to other parts of the body, including lymph nodes,
adrenals, liver, bone, brain and bone marrow. See Scott, supra; Van
Houtte et al. (eds.), Progress and Perspective in the Treatment of
Lung Cancer, Springer-Verlag (1999). Because the disease has
usually spread to such an extent that surgery is not an option, the
current treatment of choice is chemotherapy plus chest irradiation.
See Van Houtte, supra. The stage of disease is a principal
predictor of long-term survival. Less than 5% of patients with
extensive disease that has spread beyond one lung and surrounding
lymph nodes, live longer than two years. Id. However, the
probability of five-year survival is three to four times higher if
the disease is diagnosed and treated when it is still in a limited
stage, i.e., not having spread beyond one lung. Id.
[0054] NSCLC is generally divided into three types: squamous cell
carcinoma, adenocarcinoma and large cell carcinoma. Both squamous
cell cancer and adenocarcinoma develop from the cells that line the
airways; however, adenocarcinoma develops from the goblet cells
that produce mucus. Large cell lung cancer has been thus named
because the cells look large and rounded when viewed
microscopically, and generally are considered relatively
undifferentiated. See Yesner, Atlas of Lung Cancer,
Lippincott-Raven (1998).
[0055] Secondary lung cancer is a cancer initiated elsewhere in the
body that has spread to the lungs. Cancers that metastasize to the
lung include, but are not limited to, breast cancer, melanoma,
colon cancer and Hodgkin's lymphoma. Treatment for secondary lung
cancer may depend upon the source of the original cancer. In other
words, a lung cancer that originated from breast cancer may be more
responsive to breast cancer treatments and a lung cancer that
originated from the colon cancer may be more responsive to colon
cancer treatments.
[0056] The stage of a cancer indicates how far it has spread and is
an important indicator of the prognosis. In addition, staging is
important because treatment is often decided according to the stage
of a cancer. SCLC is divided into two stages: limited disease,
i.e., cancer that can only be seen in one lung and in nearby lymph
nodes; and extensive disease, i.e., cancer that has spread outside
the lung to the chest or to other parts of the body. For most
patients with SCLC, the disease has already progressed to lymph
nodes or elsewhere in the body at the time of diagnosis. See Scott,
supra. Even if spreading is not apparent on the scans, it is likely
that some cancer cells may have spread away and traveled through
the bloodstream or lymph system. In general, chemotherapy with or
without radiotherapy is often the preferred treatment. The initial
scans and tests done at first will be used later to see how well a
patient is responding to treatment.
[0057] In contrast, non-small cell cancer may be divided into four
stages. Stage I is highly localized cancer with no cancer in the
lymph nodes. Stage II cancer has spread to the lymph nodes at the
top of the affected lung. Stage III cancer has spread near to where
the cancer started. This can be to the chest wall, the covering of
the lung (pleura), the middle of the chest (mediastinum) or other
lymph nodes. Stage IV cancer has spread to another part of the
body. Stage I-III cancer is usually treated with surgery, with or
without chemotherapy. Stage IV cancer is usually treated with
chemotherapy and/or palliative care.
[0058] A number of chromosomal and genetic abnormalities have been
observed in lung cancer. In NSCLC, chromosomal aberrations have
been described on 3p, 9p, 11p, 15p and 17p, and chromosomal
deletions have been seen on chromosomes 7, 11, 13 and 19. See
Skarin (ed.), Multimodality Treatment of Lung Cancer, Marcel
Dekker, Inc. (2000); Gemmill et al., pp. 465-502, in Kane, supra;
Bailey-Wilson et al., pp. 53-98, in Kane, supra. Chromosomal
abnormalities have been described on 1p, 3p, 5q, 6q, 8q, 13q and
17p in SCLC. Id. In addition, the loss of the short arm of
chromosome 3p has also been seen in greater than 90% of SCLC tumors
and approximately 50% of NSCLC tumors. Id.
[0059] A number of oncogenes and tumor suppressor genes have been
implicated in lung cancer. See Mabry, pp. 391-412, in Kane, supra
and Sclafani et al., pp. 295-316, in Kane, supra. In both SCLC and
NSCLC, the p53 tumor suppressor gene is mutated in over 50% of lung
cancers. See Yesner, supra. Another tumor suppressor gene, FHIT,
which is found on chromosome 3p, is mutated by tobacco smoke. Id.;
Skarin, supra. In addition, more than 95% of SCLCs and
approximately 20-60% of NSCLCs have an absent or abnormal
retinoblastoma (Rb) protein, another tumor suppressor gene. The ras
oncogene (particularly K-ras) is mutated in 20-30% of NSCLC
specimens and the c-erbB2 oncogene is expressed in 18% of stage 2
NSCLC and 60% of stage 4 NSCLC specimens. See Van Houtte, supra.
Other tumor suppressor genes that are found in a region of
chromosome 9, specifically in the region of 9p21, are deleted in
many cancer cells, including p16.sup.INK4A and p15.sup.INK4B. See
Bailey-Wilson, supra; Sclafani et al., supra. These tumor
suppressor genes may also be implicated in lung cancer
pathogenesis.
[0060] In addition, many lung cancer cells produce growth factors
that may act in an autocrine or paracrine fashion on lung cancer
cells. See Siegfried et al., pp. 317-336, in Kane, supra; Moody,
pp. 337-370, in Kane, supra and Heasley et al., 371-390, in Kane,
supra. In SCLC, many tumor cells produce gastrin-releasing peptide
(GRP), which is a proliferative growth factor for these cells. See
Skarin, supra. Many NSCLC tumors express epidermal growth factor
(EGF) receptors, allowing NSCLC cells to proliferate in response to
EGF. Insulin-like growth factor (IGF-I) is elevated in greater than
95% of SCLC and greater than 80% of NSCLC tumors; it is thought to
function as an autocrine growth factor. Id. Finally, stem cell
factor (SCF, also known as steel factor or kit ligand) and c-Kit (a
proto-oncoprotein tyrosine kinase receptor for SCF) are both
expressed at high levels in SCLC, and thus may form an autocrine
loop that increases proliferation. Id.
[0061] Although the majority of lung cancer cases are attributable
to cigarette smoking, most smokers do not develop lung cancer.
Epidemiological evidence has suggested that susceptibility to lung
cancer may be inherited in a Mendelian fashion, and thus have an
inherited genetic component. Bailey-Wilson, supra. Thus, it is
thought that certain allelic variants at some genetic loci may
affect susceptibility to lung cancer. Id. One way to identify which
allelic variants are likely to be involved in lung cancer
susceptibility, as well as susceptibility to other diseases, is to
look at allelic variants of genes that are highly expressed in
lung.
[0062] The lung is susceptible to a number of other debilitating
diseases as well, including, without limitation, emphysema,
pneumonia, cystic fibrosis and asthma. See Stockley (ed.),
Molecular Biology of the Lung, Volume I: Emphysema and Infection,
Birkhauser Verlag (1999), hereafter Stockley I, and Stockley (ed.),
Molecular Biology of the Lung, Volume II: Asthma and Cancer,
Birkhauser Verlag (1999), hereafter Stockley II. The cause of many
these disorders is still not well understood and there are few, if
any, good treatment options for many of these noncancerous lung
disorders. Thus, there remains a need to understand various
noncancerous lung disorders and to identify treatments for these
diseases.
[0063] The development and differentiation of lung tissue during
embryonic development is also very important. All of the epithelial
cells of the respiratory tract, including those of the lung and
bronchi, are derived from the primitive endodermal cells that line
the embryonic outpouching. See Yesner, supra. During embryonic
development, multipotent endodermal stem cells differentiate into
many different types of specialized cells, which include ciliated
cells for moving inhaled particles, goblet cells for producing
mucus, Kulchitsky's cells for endocrine function, and Clara cells
and type II pneumocytes for secreting surfactant protein. Id.
Improper development and differentiation may cause respiratory
disorders and distress in infants, particularly in premature
infants, whose lungs cannot produce sufficient surfactant when they
are born. Further, some lung cancer cells, particularly small cell
carcinomas, are plastic and can alter their phenotype into a number
of cell types, including large cell carcinoma, adenocarcinoma and
squamous cell carcinoma. Id. Thus, a better understanding of lung
development and differentiation may help facilitate understanding
of lung cancer initiation and progression.
[0064] The most common screening tests for lung cancer are chest
x-ray and sputum cytology. Randomized controlled trials have not
demonstrated a reduction in lung cancer mortality resulting from
screening with chest x-ray and/or sputum cytology. Additionally,
sputum cytology has not been shown to be effective when used as an
adjunct to annual chest x-ray. Screening with chest x-ray plus
sputum cytology appears to detect lung cancer at an earlier stage,
but this would be expected in a screening test whether or not it
was effective at reducing mortality. Since early detection by
current screening methods fails to reduce mortality in lung cancer
patients, current lung cancer screening methods are inadequate.
[0065] There are two important potential hazards associated with
chest radiography screening. First, false positive test results can
lead to an unnecessary invasive procedure, such as percutaneous
needle biopsy or thoracotomy. These procedures are costly and due
to their invasive nature carry risks of their own. The second
hazard with chest radiography screening is overdiagnosis.
Overdiagnosis is the diagnosis of a small or slowly growing tumor
that would not have become clinically significant had it not been
detected by screening. Although overdiagnosis is almost impossible
to document in a living individual, autopsy studies suggest that
many individuals die with lung cancer rather than from it.
[0066] Additionally, the spectrum of lung cancer type has shifted
over the last two decades. Whereas the most common type used to be
squamous cell cancer (usually centrally located), the most common
type now is adenocarcinoma (usually peripherally located). The
latter may be more amenable to early detection by chest x-ray, the
limitations of which are described above. In contrast, sputum
cytology, is more sensitive in the detection of squamous cell
cancer than in detecting adenocarcinoma, and therefore lacks
usefulness in detecting the more common adenocarcinomas. Clearly,
new highly sensitive non-invasive methods of detecting lung cancer
are needed.
[0067] There are intensive efforts to improve lung cancer screening
with newer technologies, including low-dose helical computed
tomography (LDCT) and molecular techniques. LDCT is far more
sensitive than chest radiography. In a recent screening study, CT
detected almost 6 times as many stage I lung cancers as chest
radiography and most of these tumors were 1 cm or less in diameter.
However, the effectiveness of screening with LDCT has not yet been
evaluated in a controlled clinical trial.
[0068] There are two potential hazards that must be considered
against any potential benefit of screening with LDCT. The more
common and familiar hazard is the false positive test result, which
may lead to anxiety and invasive diagnostic procedures. A less
familiar hazard is overdiagnosis, the diagnosis of a condition that
would not have become clinically significant had it not been
detected by screening. In the case of screening with LDCT,
overdiagnosis could lead to unnecessary diagnosis of lung cancer
requiring some combination of surgery, e.g., lobectomy,
chemotherapy and radiation therapy. As stated above, overdiagnosis
is almost impossible to document in a living individual. In one
large study, about one-sixth of all lung cancers found at autopsy
had not been clinically recognized before death. Furthermore,
autopsy probably fails to detect many small lung cancers that are
detectable by CT.
[0069] Current therapies for lung cancer are quite limited.
Generally, patient options comprise surgery, radiation therapy, and
chemotherapy.
[0070] Depending on the type and stage of a lung cancer, surgery
may be used to remove the tumor along with some surrounding lung
tissue. A lobectomy refers to a lobe (section) of the lung being
removed. If the entire lung is removed, the surgery is called a
pneumonectomy. Removing only part of a lobe is known as a
segmentectomy or wedge resection.
[0071] If the cancer has spread to the brain, benefit may be gained
from removal of the brain metastasis. This involves a craniotomy
(surgery through a hole in the skull).
[0072] For radiation therapy several methods exist. External beam
radiation therapy uses radiation delivered from outside the body
that is focused on the cancer. This type of radiation therapy is
most often used to treat a primary lung cancer or its metastases to
other organs.
[0073] Brachytherapy uses a small pellet of radioactive material
placed directly into the cancerous tissue or into the airway next
to the cancer. Radiation therapy is sometimes used as the main
(primary) treatment of lung cancer, especially if the general
health of the patient is too poor to undergo surgery. Brachytherapy
can also be used to help relieve blockage of large airways by
cancer.
[0074] Additionally, radiation therapy can be used as a post
surgical treatment to kill very small deposits of cancer that
cannot be seen or removed during surgery. Radiation therapy can
also be used to palliate (relieve) symptoms of lung cancer such as
pain, bleeding, difficulty swallowing, and problems caused by brain
metastases.
[0075] For chemotherapy, cisplatin or a related drug, carboplatin,
are the chemotherapy agents most often used in treating NSCLC.
Recent studies found that combining either of these with drugs such
as gemcitabine, paclitaxel, docetaxel, etoposide, or vinorelbine
appear to be more effective in treating NSCLC.
[0076] Recently, the National Comprehensive Cancer Network (NCCN;
nccn with the extension .org of the world wide web), an alliance of
nineteen of the world's leading cancer centers, announces a major
update of the NCCN Non-Small Cell Lung Cancer Clinical Practice
Guidelines. The NCCN is widely recognized as a standard for
clinical policy in oncology.
[0077] Recently approved targeted therapy, gefitinib (IRESSA.RTM.,
AstraZeneca Pharmaceuticals LP) is now recommended as third-line
therapy and as second-line only if the platinum/docetaxel
combination was used as first-line therapy.
[0078] The NCCN's Non-Small Cell Lung Cancer (NSCLC) guidelines
contain recommendations for administration of chemotherapy to
patients with this disease including patient selection criteria and
definition of first-, second-, and third-line agents and
combinations.
[0079] Chemotherapeutic agents are specified as two-agent regimens
for first-line therapy, two agent regimens or single agents for
second-line therapy, and one single agent for third-line therapy.
Agents used in first- and second-line therapy are: cisplatin
(PLATINOL.RTM., Bristol-Myers Squibb Company), carboplatin
(PARAPLATIN.RTM., Bristol-Myers Squibb Company), paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb Company), docetaxel
(TAXOTERE.RTM., Aventis Pharmaceuticals Inc.), vinorelbine
(NAVELBINE.RTM., GlaxoSmithKline), gemcitabine (GEMZAR.RTM., Eli
Lilly and Company), etoposide (TOPOSAR.RTM., Pfizer, Inc.;
VEPESID.RTM., Bristol-Myers Squibb Company; ETOPOPHOS.RTM.,
Bristol-Myers Squibb Company), irinotecan (CAMPTOSAR.RTM., Pfizer,
Inc.), vinblastine (VELBAN.RTM., Eli Lilly and Company), mitomycin
(MUTAMYCIN.RTM., Bristol-Myers Squibb Company), and ifosfamide
(IFEX.RTM., Bristol-Myers Squibb Company).
[0080] Some of the usual chemotherapy combinations used for
patients with SCLC include: EP (etoposide and cisplatin); ET
(etoposide and carboplatin); ICE (ifosfamide, carboplatin, and
etoposide); and CAV (cyclophosphamide, doxorubicin, and
vincristine).
[0081] New drugs such as gemcitabine, paclitaxel, vinorelbine,
topotecan, and teniposide have shown promising results in some SCLC
studies. Growth factors may be given in conjunction to chemotherapy
agents if patient health is good. The administration of growth
factors help prevent bone marrow side effects.
[0082] Ongoing or recently completed therapeutic trials for various
compounds to treat lung cancer include alitretinoin (PANRETIN.RTM.,
Ligand Pharmaceuticals), topotecan HCl (HYCAMTIN.RTM.,
GlaxoSmithKline), liposomal ether lipid (Elan Pharmaceutical),
cantuzumab mertansine (ImmunoGen), oncolytic virus therapy
(GAVAX.RTM., Cell Genesys), vincristine (ONCO TCS.RTM., Inex
Pharmaceuticals), a concentrate of shark cartilage (NEOVASTAT.RTM.,
AEterna Laboratories), squalamine (Genaera), mirostipen (Human
Genome Sciences Inc.), p53 tumor suppressor therapy (ADVEXIN.RTM.,
Introgen Therapeutics), biricodar dicitrate (INCEL.RTM., Vertex
Pharmaceuticals), flavopiridol (Aventis), pemetrexed (ALIMTA.RTM.,
Eli Lilly and Company), pivaloyloxymethylbutyrate (PIVANEX.RTM.,
Titan Pharmaceuticals), tirapazamine (TIRAZONE.RTM.,
Sanofi-Synthelabo Pharmaceuticals), irinotecan (CAMPTOSAR.RTM.,
Pharmacia), tezacitabine (Chiron), cisplatin/vinblastine/amifostine
(MedImmune), paclitaxel/carboplatin/amifostine (MedImmune),
antisense agent (ONCOMYC-NG.RTM., AVI BioPharma),
exisulind/vinorelbine (APTOSYN.RTM./NAVELBINE.RTM., Cell
Pathyways), tariquidar (QLT), paclitaxel poliglumex (XYOTAX.RTM.,
Cell Therapeutics), PEG-camptothecin (PROTHECAN.RTM., Enzon),
decitabine (SuperGen), erlotinib (TARCEVA.RTM., OSI
Pharmaceuticals), ABX-EGF (Abgenix), vitamin E-based emulsion
formulation of paclitaxel (TOCOSOL PACLITAXEL.RTM., Sonus
Pharmaceuticals), a fragment of the mouse antibody HMFG-1 labeled
with yttrium 90 (THERAFAB.RTM., Antisoma), minodronate (Yamanouchi
Pharmaceutical), exisulind/docetaxel/carboplatin
(APTOSYN.RTM./TAXOTERE.RTM./PARAPLATIN.RTM., Cell Pathways),
exisulind/gemcitabine HCl (APTOSYN.RTM./GEMZAR.RTM., Cell
Pathways), IMC-C225/carboplatin/paclitaxel
(ERBITUX.RTM./CARBOPLATIN.RTM./PACLITAXEL.RTM., ImClone Systems),
and vinorelbine (NAVELBINE.RTM., GlaxoSmithKline).
[0083] As indicated above, many therapeutics are recommended for
use in combination as a first-line therapy or only if other
therapeutics have failed as second-, and third-line agents. While
there are many compounds in ongoing or recently completed
therapeutic trials, there is great need for additional therapeutic
compounds capable of treating early stage and advanced or
metastasized lung cancer.
[0084] Accordingly, there is a great need for more sensitive and
accurate methods for predicting whether a person is likely to
develop lung cancer, for diagnosing lung cancer, for monitoring the
progression of the disease, for staging the lung cancer, for
determining whether the lung cancer has metastasized and for
imaging the lung cancer. There is also a need for better treatment
of lung cancer. Further, there is a great need for diagnosing and
treating noncancerous lung disorders such as emphysema, pneumonia,
lung infection, pulmonary fibrosis, cystic fibrosis and asthma.
There is also a need for compositions and methods of using these
compositions to identify lung tissue for forensic purposes and for
determining whether a particular cell or tissue exhibits
lung-specific characteristics.
[0085] As discussed above, each of the methods for diagnosing and
staging breast, ovarian pancreatic, and lung cancer is limited by
the technology employed. Accordingly, there is need for sensitive
molecular and cellular markers and reagents for the detection of
breast, ovarian, pancreatic and lung cancer including metastatic
cancer. There is a need for molecular markers and reagents for the
accurate staging, including clinical and pathological staging, of
breast, ovarian, pancreatic and lung cancers to optimize treatment
methods. Finally, there is a need for sensitive molecular and
cellular markers and reagents to monitor the progress of cancer
treatments, including markers that can detect recurrence of breast,
ovarian, pancreatic and lung cancers following remission.
[0086] The present invention provides alternative reagents and
methods for treating breast, ovarian, pancreatic and lung cancer
that overcome the limitations of conventional therapeutic methods
as well as offer additional advantages that will be apparent from
the detailed description below.
Angiogenesis in Cancer
[0087] Growth and metastasis of solid tumors are also dependent on
angiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473;
Folkman, J., 1989, Journal of the National Cancer Institute, 82,
4-6. It has been shown, for example, that tumors which enlarge to
greater than 2 mm must obtain their own blood supply and do so by
inducing the growth of new capillary blood vessels. Once these new
blood vessels become embedded in the tumor, they provide a means
for tumor cells to enter the circulation and metastasize to distant
sites such as liver, lung or bone. Weidner, N., et al., 1991, The
New England Journal of Medicine, 324(1), 1-8.
[0088] Angiogenesis, defined as the growth or sprouting of new
blood vessels from existing vessels, is a complex process that
primarily occurs during embryonic development. The process is
distinct from vasculogenesis, in that the new endothelial cells
lining the vessel arise from proliferation of existing cells,
rather than differentiating from stem cells. The process is
invasive and dependent upon proteolysis of the extracellular matrix
(ECM), migration of new endothelial cells, and synthesis of new
matrix components. Angiogenesis occurs during embryogenic
development of the circulatory system; however, in adult humans,
angiogenesis only occurs as a response to a pathological condition
(except during the reproductive cycle in women).
[0089] Under normal physiological conditions in adults,
angiogenesis takes place only in very restricted situations such as
hair growth and wounding healing. Auerbach, W. and Auerbach, R.,
1994, Pharmacol Ther. 63(3):265-3 11; Ribatti et al., 1991,
Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4.
Angiogenesis progresses by a stimulus which results in the
formation of a migrating column of endothelial cells. Proteolytic
activity is focused at the advancing tip of this "vascular sprout",
which breaks down the ECM sufficiently to permit the column of
cells to infiltrate and migrate. Behind the advancing front, the
endothelial cells differentiate and begin to adhere to each other,
thus forming a new basement membrane. The cells then cease
proliferation and finally define a lumen for the new arteriole or
capillary.
[0090] Unregulated angiogenesis has gradually been recognized to be
responsible for a wide range of disorders, including, but not
limited to, cancer, cardiovascular disease, rheumatoid arthritis,
psoriasis and diabetic retinopathy. Folkman, 1995, Nat Med
1(1):27-31; Isner, 1999, Circulation 99(13): 1653-5; Koch, 1998,
Arthritis Rheum 41(6):951-62; Walsh, 1999, Rheumatology (Oxford)
38(2):103-12; Ware and Simons, 1997, Nat Med 3(2): 158-64.
[0091] Of particular interest is the observation that angiogenesis
is required by solid tumors for their growth and metastases.
Folkman, 1986 supra; Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6;
Folkman, 1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu Rev
Med 49:407-24. A tumor usually begins as a single aberrant cell
which can proliferate only to a size of a few cubic millimeters due
to the distance from available capillary beds, and it can stay
`dormant` without further growth and dissemination for a long
period of time. Some tumor cells then switch to the angiogenic
phenotype to activate endothelial cells, which proliferate and
mature into new capillary blood vessels. These newly formed blood
vessels not only allow for continued growth of the primary tumor,
but also for the dissemination and recolonization of metastatic
tumor cells. The precise mechanisms that control the angiogenic
switch is not well understood, but it is believed that
neovascularization of tumor mass results from the net balance of a
multitude of angiogenesis stimulators and inhibitors Folkman, 1995,
supra.
[0092] One of the most potent angiogenesis inhibitors is endostatin
identified by O'Reilly and Folkman. O'Reilly et al., 1997, Cell
88(2):277-85; O'Reilly et al., 1994, Cell 79(2):3 15-28. Its
discovery was based on the phenomenon that certain primary tumors
can inhibit the growth of distant metastases. O'Reilly and Folkman
hypothesized that a primary tumor initiates angiogenesis by
generating angiogenic stimulators in excess of inhibitors. However,
angiogenic inhibitors, by virtue of their longer half-life in the
circulation, reach the site of a secondary tumor in excess of the
stimulators. The net result is the growth of primary tumor and
inhibition of secondary tumor. Endostatin is one of a growing list
of such angiogenesis inhibitors produced by primary tumors. It is a
proteolytic fragment of a larger protein: endostatin is a 20 kDa
fragment of collagen XVIII (amino acid H1132-K1315 in murine
collagen XVIII). Endostatin has been shown to specifically inhibit
endothelial cell proliferation in vitro and block angiogenesis in
vivo. More importantly, administration of endostatin to
tumor-bearing mice leads to significant tumor regression, and no
toxicity or drug resistance has been observed even after multiple
treatment cycles. Boehm et al., 1997, Nature 390(6658):404-407. The
fact that endostatin targets genetically stable endothelial cells
and inhibits a variety of solid tumors makes it a very attractive
candidate for anticancer therapy. Fidler and Ellis, 1994, Cell
79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84; Hinsbergh
et al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition, angiogenesis
inhibitors have been shown to be more effective when combined with
radiation and chemotherapeutic agents. Klement, 2000, J. Clin
Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,
Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,
Nature 394(6690):287-91.
[0093] The present invention provides alternative methods of
treating breast, ovarian, pancreatic and lung 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
[0094] This invention is directed to an isolated Pro104 antibody
that binds to Pro104 on a mammalian cell in vivo. The invention is
further directed to an isolated Pro104 antibody that internalizes
upon binding to Pro104 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-5277, 6076, 6077 and 6078.
[0095] 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-5277, 6076,
6077 and 6078.
[0096] 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.
[0097] The mammalian cell may be a cancer cell. Preferably, the
anti-Pro104 monoclonal antibody inhibits the growth of
Pro104-expressing cancer cells in vivo.
[0098] The antibody may be produced in bacteria. Alternatively, the
antibody may be a humanized form of an anti-Pro104 antibody
produced by a hybridoma selected from the group of hybridomas
having ATCC accession number PTA-5277, 6076, 6077 and 6078.
[0099] Preferably, the cancer is selected from the group consisting
of breast, ovarian, pancreatic and lung 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.
[0100] 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.
[0101] The invention is also directed to a method of killing a
Pro104-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 breast, ovarian, pancreatic and lung cancer
cells.
[0102] The ovarian cancer may be ovarian serous adenocarcinoma.
[0103] The breast cancer may be breast infiltrating ductal
carcinoma.
[0104] The breast, ovarian, pancreatic or lung cancer may also be
metastatic.
[0105] The invention is also directed to a method of alleviating a
Pro104-expressing cancer in a mammal, comprising administering a
therapeutically effective amount of the antibodies to the
mammal.
[0106] 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 or pancreatic cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0107] FIG. 1 shows that Pro104.D116.1 MAb binds to 293F cells
transiently transfected with Pro104.
[0108] FIG. 2 shows that Pro104.D118.1 MAb Binds to 293F cells
transiently transfected with Pro104.
[0109] FIG. 3 shows that Pro104.C19.1 binds to live HeLa cancer
cells expressing Pro104.
[0110] FIG. 4 shows that Cy3-Pro104.C25.1 binds to live HeLa cancer
cells expressing Pro104.
[0111] FIG. 5 shows that Cy3-Pro104.C25.1 binds to and is
internalized in live HeLa cancer cells expressing Pro104.
[0112] FIG. 6 shows that Cy3-Pro104.C19.1 binds to and is
internalized in pancreatic cancer cells expressing Pro104.
[0113] FIG. 7 shows that Cy3-Pro104.C55.1 binds to and is
internalized in pancreatic cancer cells expressing Pro104.
[0114] FIG. 8 shows that Pro104.C25.1 binds to Pro104 on cancer
cells in ovarian tumors.
[0115] FIG. 9 shows that Pro104.C25.1 binds to Pro104 on the cell
membrane of ovarian cancer cells.
[0116] FIG. 10 shows that Pro104.D9 binds to Pro104 on the cell
membrane of ovarian cancer cells.
[0117] FIG. 11 shows that Pro104.D133 binds to Pro104 on the cell
membrane of serous ovarian cancer cells.
[0118] FIG. 12 shows that Pro104.C25.1 binds to Pro104 on cancer
cells in pancreatic tumors.
[0119] FIG. 13 shows controls demonstrating Pro104 MAb
immunolabeling specificity.
[0120] FIG. 14 shows an epitope map of Pro104 MAbs.
[0121] FIG. 15 shows a western blot showing detection of Pro104
protein in mRNA+ cell lines and ovarian tumor tissue (T) but not
normal adjacent tissue (N).
[0122] FIG. 16 shows that overexpression of Pro104 leads to
phosphorylation of EGF Receptor.
[0123] FIG. 17 shows that the Pro104 protein is glycosylated and
GPI-Linked.
[0124] FIG. 18 shows the surface biotinylation of native Pro104 in
cell lines.
[0125] FIG. 19 shows retroviral-mediated overexpression of Pro104
protein in RK3E cells.
[0126] FIG. 20 shows retroviral-mediated overexpression of Pro104
protein in SKOV3 Cells.
[0127] FIG. 21 shows siRNA mediates specific down-regulation of
Pro104 protein in HeLa cells.
[0128] FIG. 22 shows siRNA mediates down-regulation of Pro104
protein in CaOV3 cells.
[0129] FIG. 23 shows Pro104 siRNA specific knockdown of Pro104 mRNA
in CaOV3 cells.
[0130] FIG. 24 shows Pro104 siRNA specific knockdown of Pro104 mRNA
in HeLa cells.
[0131] FIG. 25 shows Pro104 siRNA specific knockdown of Pro104 mRNA
in HeLa cells, compared to a positive control.
[0132] FIG. 26 shows different Pro104 siRNAs inducing specific mRNA
knockdown and apoptosis in HeLa cells.
[0133] FIG. 27 shows specific knockdown of Pro104 mRNA in HeLa
Cells inducing cell death.
[0134] FIG. 28 shows specific mRNA knockdown by Pro104 siRNA
inducing apoptosis in HeLa cells.
[0135] FIG. 29 shows specific knockdown of Pro104 mRNA in CaOV3
Cells inducing apoptosis.
[0136] FIG. 30 shows Pro104 siRNA having no effect on apoptosis in
cells without Pro104 mRNA.
[0137] FIG. 31 shows overexpression of Pro104 inducing cell growth
in soft agar.
[0138] FIG. 32 shows Pro104 protease activity is required for cell
growth.
[0139] FIG. 33 shows knockdown of Pro104 mRNA by siRNA inhibiting
growth of HeLa cells in soft agar.
[0140] FIG. 34 shows knockdown of Pro104 mRNA by siRNA inhibiting
growth of HeLa cells in soft agar.
[0141] FIG. 35 shows increased growth of human tumor cells
over-expressing Pro104.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
[0142] Human "Pro104" as used herein, refers to a protein of 314
amino acids that is expressed on the cell surface as a
glycoprotein. The nucleotide and amino acid sequences of Pro104
have been disclosed, e.g., WO200016805-A1 PA (DIAD-) DIADEXUS Human
cancer-specific gene, Pro104; WO9836054-A1 PA (AMRA-) AMRAD
Nucleotide sequence of short isoform of HELA2; and J. D. Hooper et
al. Testisin, a new human serine protease expressed by premeiotic
testicular germ cells and lost in testicular germ cell tumors.
Cancer Research 59:3199-3205 (1999)). Pro104 has also been
disclosed in the REFSEQ database as: NM.sub.--006799.2 (GI:
21614534) Homo sapiens protease, serine, 21 (testisin) (PRSS21),
transcript variant 1, mRNA. RefSeq gives the following summary of
PRSS21 (Pro104): [0143] This gene encodes a cell-surface anchored
serine protease, which is a member of the trypsin family of serine
proteases. It is predicted to be active on peptide linkages
involving the carboxyl group of lysine or arginine. The protein
localizes to the cytoplasm and the plasma membrane of premeiotic
testicular germ cells and it may be involved in progression of
testicular tumors of germ cell origin. Alternative splicing of this
gene results in three transcript variants encoding three different
isoforms.
[0144] The amino acids of Pro104 are presumably located on the cell
surface. Pro104 as used herein include allelic variants and
conservative substitution mutants of the protein which have Pro104
biological activity. Additionally, splice variants may have Pro104
biological activity. The RefSeq accessions for the splice variants
referenced above include: NM.sub.--144956.1 (GI: 21614530) Homo
sapiens protease, serine, 21 (testisin) (PRSS21), transcript
variant 2, mRNA; and NM.sub.--144957 (GI: 21614532) Homo sapiens
protease, serine, 21 (testisin) (PRSS21), transcript variant 3,
mRNA.
[0145] Our findings that Pro104 is apparently associated with the
more aggressive breast, ovarian, pancreatic and lung cancers makes
this cell surface antigen an attractive target for immunotherapy of
these and possibly other tumor types.
[0146] 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.
[0147] An "isolated antibody" is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. Preferably, the antibody
will be purified (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0148] 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 units along with an additional polypeptide
called J chain, and therefore contains 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (VH) followed by three constant domains (CH) for each of the
.alpha., .delta. and .gamma. chains and four CH domains for .mu.
and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (VL) followed by a constant domain (CL) at its
other end.
[0149] The VL is aligned with the VH and the CL is aligned with the
first constant domain of the heavy chain (CHI). Particular amino
acid residues are believed to form an interface between the light
chain and heavy chain variable domains. The pairing of a VH and VL
together forms a single antigen-binding site. For the structure and
properties of the different classes of antibodies, see, e.g., Basic
and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I.
Teff and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk,
Conn., 1994, page 71 and Chapter 6.
[0150] 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.
[0151] 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).
[0152] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (LI), 5056 (L2) and 89-97 (L3) in
the VL, and around about 1-35 (HI), 50-65 (H2) and 95-102 (113) in
the VH; Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)) and/or those residues from a
"hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and
91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and 96-101 (1-13)
in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0153] 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.
[0154] 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.
[0155] An "intact" antibody is one which comprises an
antigen-binding site as well as a CL and at least heavy chain
constant domains, CHI, CH2 and CH3. The constant domains may be
native sequence constant domains (e.g. human native sequence
constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0156] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S.
Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):
1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. Papain
digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. The Fab
fragment consists of an entire L chain along with the variable
region domain of the H chain (VH), and the first constant domain of
one heavy chain (CHI). Each Fab fragment is monovalent with respect
to antigen binding, i.e., it has a single antigen-binding site.
Pepsin treatment of an antibody yields a single large F(ab')2
fragment which roughly corresponds to two disulfide linked Fab
fragments having divalent antigen-binding activity and is still
capable of cross-linking antigen. Fab' fragments differ from Fab
fragments by having additional few residues at the carboxy terminus
of the CHI domain including one or more cysteines from the antibody
hinge region. Fab'-SH is the designation herein for Fab' in which
the cysteine residue(s) of the constant domains bear a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs
of 8 Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0157] 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.
[0158] "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.
[0159] "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.
[0160] 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).
[0161] 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.
[0162] 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 Pro104 will possess at least about 70%
homology with the native sequence Pro104, 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.
[0163] 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.
[0164] "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).
[0165] "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).
[0166] As used herein, an anti-Pro104 antibody that "internalizes"
is one that is taken up by (i.e., enters) the cell upon binding to
Pro104 on a mammalian cell (i.e. cell surface Pro104). The
internalizing antibody will of course include antibody fragments,
human or humanized antibody and antibody conjugate. For therapeutic
applications, internalization in vivo is contemplated. The number
of antibody molecules internalized will be sufficient or adequate
to kill a Pro104-expressing cell, especially a Pro104-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.
[0167] Whether an anti-Pro104 antibody internalizes upon binding
Pro104 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 Pro104
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 Pro104-expressing tumor
transplant or xenograft, or a mouse into which cells transfected
with human Pro104 have been introduced, or a transgenic mouse
expressing the human Pro104 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
Pro104-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.
[0168] The faster the rate of internalization of the antibody upon
binding to the Pro104-expressing cell in vivo, the faster the
desired killing or growth inhibitory effect on the target
Pro104-expressing cell can be achieved, e.g., by a cytotoxic
immunoconjugate. Preferably, the kinetics of internalization of the
anti-Pro104 antibodies are such that they favor rapid killing of
the Pro104-expressing target cell. Therefore, it is desirable that
the anti-Pro104 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-Pro104 antibody in vivo. The antibody will
preferably be internalized into the cell within a few hours upon
binding to Pro104 on the cell surface, preferably within 1 hour,
even more preferably within 15-30 minutes.
[0169] To determine if a test antibody can compete for binding to
the same epitope as the epitope bound by the anti-Pro104 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, Pro104-coated wells of a microtiter plate,
or Pro104-coated sepharose beads, are pre-incubated with or without
candidate competing antibody and then a biotin-labeled anti-Pro104
antibody of the invention is added. The amount of labeled
anti-Pro104 antibody bound to the Pro104 antigen in the wells or on
the beads is measured using avidin-peroxidase conjugate and
appropriate substrate.
[0170] Alternatively, the anti-Pro104 antibody can be labeled,
e.g., with a radioactive or fluorescent label or some other
detectable and measurable label. The amount of labeled anti-Pro104
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-Pro104 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-Pro104 antibody
of the invention if the candidate competing antibody can block
binding of the anti-Pro104 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.
[0171] An antibody having a "biological characteristic" of a
designated antibody, such as any of the monoclonal antibodies
Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18,
Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34,
Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50,
Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60,
Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6,
Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,
Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31,
Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56,
Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68,
Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,
Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111,
Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116,
Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121,
Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125,
Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129,
Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134,
Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139,
Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71,
Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78,
Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,
Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,
Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,
Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,
Pro104.K358 or Pro104.K362, 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, Pro104.C1,
Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19,
Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37,
Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53,
Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60, Pro104.C66,
Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9,
Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20,
Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43,
Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58,
Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69,
Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88, Pro104.D91,
Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111, Pro104.D112,
Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116, Pro104.D117,
Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121, Pro104.D122,
Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125, Pro104.D126,
Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129, Pro104.D130,
Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134, Pro104.D135,
Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139, Pro104.K14,
Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71, Pro104.K72,
Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78, Pro104.K81,
Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155, Pro104.K156,
Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160, Pro104.K163,
Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226, Pro104.K227,
Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281, Pro104.K358 or
Pro104.K362 will bind the same epitope as that bound by Pro104.C1,
Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19,
Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37,
Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53,
Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60, Pro104.C66,
Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9,
Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20,
Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43,
Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58,
Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69,
Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88, Pro104.D91,
Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111, Pro104.D112,
Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116, Pro104.D117,
Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121, Pro104.D122,
Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125, Pro104.D126,
Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129, Pro104.D130,
Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134, Pro104.D135,
Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139, Pro104.K14,
Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71, Pro104.K72,
Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78, Pro104.K81,
Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155, Pro104.K156,
Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160, Pro104.K163,
Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226, Pro104.K227,
Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281, Pro104.K358 or
Pro104.K362 (e.g. which competes for binding or blocks binding of
monoclonal antibody Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17,
Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27,
Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49,
Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57,
Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,
Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18,
Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29,
Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55,
Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64,
Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85,
Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106,
Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115,
Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120,
Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124,
Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128,
Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133,
Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138,
Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47,
Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,
Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89,
Pro104.K155, Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159,
Pro104.K160, Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217,
Pro104.K226, Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264,
Pro104.K281, Pro104.K358 or Pro104.K362 to Pro104), be able to
target a Pro104-expressing tumor cell in vivo and will bind to
Pro104 on a mammalian cell in vivo.
[0172] Furthermore, an antibody with the biological characteristic
of the Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18,
Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34,
Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50,
Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60,
Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6,
Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,
Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31,
Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56,
Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68,
Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,
Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111,
Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116,
Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121,
Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125,
Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129,
Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134,
Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139,
Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71,
Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78,
Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,
Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,
Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,
Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,
Pro104.K358 or Pro104.K362 antibody will internalize upon binding
to Pro104 on a mammalian cell in vivo.
[0173] Likewise, an antibody with the biological characteristic of
the Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18,
Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34,
Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50,
Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60,
Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6,
Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,
Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31,
Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56,
Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68,
Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,
Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111,
Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116,
Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121,
Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125,
Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129,
Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134,
Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139,
Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71,
Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78,
Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,
Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,
Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,
Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,
Pro104.K358 or Pro104.K362 antibody will have the same epitope
binding, targeting, internalizing, tumor growth inhibitory and
cytotoxic properties of the antibody.
[0174] 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 Pro104
protein disclosed herein. Methods for identifying antagonists of a
Pro104 polypeptide may comprise contacting a Pro104 polypeptide or
a cell expressing Pro104 on the cell surface, with a candidate
antagonist antibody and measuring a detectable change in one or
more biological activities normally associated with the Pro104
polypeptide.
[0175] An "antibody that inhibits the growth of tumor cells
expressing Pro104" or a "growth inhibitory" antibody is one which
binds to and results in measurable growth inhibition of cancer
cells expressing or overexpressing Pro104. Preferred growth
inhibitory anti-Pro104 antibodies inhibit growth of
Pro104-expressing tumor cells e.g., breast, ovarian, pancreatic and
lung 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-Pro104 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.
[0176] 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 Pro104. Preferably the cell is a tumor cell, e.g. an
breast, ovarian, pancreatic and lung 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.
[0177] 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.
[0178] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0179] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126.330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer, of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0180] "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.
[0181] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may be
performed.
[0182] 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.
[0183] A "Pro104-expressing cell" is a cell which expresses
endogenous or transfected Pro104 on the cell surface. A
"Pro104-expressing cancer" is a cancer comprising cells that have
Pro104 protein present on the cell surface. A "Pro104-expressing
cancer" produces sufficient levels of Pro104 on the surface of
cells thereof, such that an anti-Pro104 antibody can bind thereto
and have a therapeutic effect with respect to the cancer. A cancer
which "overexpresses" Pro104 is one which has significantly higher
levels of Pro104 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. Pro104 overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the Pro104 protein present on the surface of a cell (e.g. via an
immunohistochemistry assay; FACS analysis). Alternatively, or
additionally, one may measure levels of Pro104-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 Pro104 overexpression by measuring shed antigen in a
biological fluid such as serum, e.g., using antibody-based assays
(see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990;
WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued
Mar. 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80
(1990)). Aside from the above assays, various in vivo assays are
available to the skilled practitioner. For example, one may expose
cells within the body of the patient to an antibody which is
optionally labeled with a detectable label, e.g. a radioactive
isotope, and binding of the antibody to cells in the patient can be
evaluated, e.g. by external scanning for radioactivity or by
analyzing a biopsy taken from a patient previously exposed to the
antibody. A Pro104-expressing cancer includes ovarian, pancreatic,
lung or breast cancer.
[0184] 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.
[0185] "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 a
Pro104-expressing cancer if, after receiving a therapeutic amount
of an anti-Pro104 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-Pro104 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.
[0186] 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).
[0187] 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.
[0188] "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.
[0189] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0190] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0191] "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.
[0192] 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..
[0193] 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.
[0194] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a Pro104-expressing cancer cell, either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of Pro104-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 semi synthetic 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.
[0195] "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.
[0196] The term "epitope tagged" used herein refers to a chimeric
polypeptide comprising an anti-Pro104 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).
[0197] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0198] 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.
[0199] 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.
[0200] "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.
[0201] The cell that produces an anti-Pro104 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.
[0202] 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.
[0203] 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).
[0204] 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).
[0205] 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.
[0206] 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.
[0207] 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
[0208] The invention provides anti-Pro104 antibodies. Preferably,
the anti-Pro104 antibodies internalize upon binding to cell surface
Pro104 on a mammalian cell. The anti-Pro104 antibodies may also
destroy or lead to the destruction of tumor cells bearing
Pro104.
[0209] It was not apparent that Pro104 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 Pro104 is
internalization competent upon binding by the anti-Pro 104
antibodies of the invention. Additionally, it was demonstrated that
the anti-Pro104 antibodies of the present invention can
specifically target Pro104-expressing tumor cells in vivo and
inhibit or kill these cells. These in vivo tumor targeting,
internalization and growth inhibitory properties of the anti-Pro104
antibodies make these antibodies very suitable for therapeutic
uses, e.g., in the treatment of various cancers including breast,
ovarian, pancreatic and lung cancer. Internalization of the
anti-Pro104 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.
[0210] The anti-Pro104 antibodies of the invention also have
various non-therapeutic applications. The anti-Pro104 antibodies of
the present invention can be useful for diagnosis and staging of
Pro104-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 Pro104 from cells, for detection and quantitation of Pro104 in
vitro, e.g. in an ELISA or a Western blot, to kill and eliminate
Pro104-expressing cells from a population of mixed cells as a step
in the purification of other cells. The internalizing anti-Pro104
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.
[0211] 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-Pro104 antibodies of the invention are also contemplated,
e.g., an anti-Pro 104 antibody which has the biological
characteristics of a monoclonal antibody produced by the hybridomas
accorded ATCC accession numbers PTA-5277, 6076, 6077 and 6078,
specifically including the in vivo tumor targeting, internalization
and any cell proliferation inhibition or cytotoxic characteristics.
Specifically provided are anti-Pro104 antibodies that bind to an
epitope present in amino acids 1-10, 10-20, 20-30, 30-40, 40-50,
50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130,
130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200,
200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270,
270-280, 280-290, 290-300, 300-310, 310-314 of human Pro104.
[0212] Methods of producing the above antibodies are described in
detail below.
[0213] The present anti-Pro104 antibodies are useful for treating a
Pro104-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal. Such cancers include ovarian and pancreatic
cancer, cancer of the urinary tract, prostate cancer, breast
cancer, colon cancer, and lung cancer. Such a cancer includes more
specifically, ovarian serous adenocarcinoma, breast infiltrating
ductal carcinoma, prostate adenocarcinoma, renal cell carcinomas,
colorectal adenocarcinomas, lung adenocarcinomas, lung squamous
cell carcinomas, and pleural mesothelioma. The breast cancer may be
HER-2 negative or positive breast cancer. The cancers encompass
metastatic cancers of any of the preceding, e.g., breast, ovarian,
pancreatic and lung cancer metastases. The antibody is able to bind
to at least a portion of the cancer cells that express Pro104 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 Pro104-expressing tumor cells or inhibit the growth
of such tumor cells, in vitro or in vivo, upon binding to Pro104 on
the cell. Such an antibody includes a naked anti-Pro104 antibody
(not conjugated to any agent). Naked anti-Pro104 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-Pro104 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.
[0214] The invention provides a composition comprising an
anti-Pro104 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-Pro104 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-Pro 104 antibody of the invention, and a
carrier. The formulation may be a therapeutic formulation
comprising a pharmaceutically acceptable carrier.
[0215] Another aspect of the invention is isolated nucleic acids
encoding the internalizing anti-Pro104 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.
[0216] The invention also provides methods useful for treating a
Pro104-expressing cancer or alleviating one or more symptoms of the
cancer in a mammal, comprising administering a therapeutically
effective amount of an internalizing anti-Pro104 antibody to the
mammal. The antibody therapeutic compositions can be administered
short term (acute) or chronic, or intermittent as directed by
physician. Also provided are methods of inhibiting the growth of,
and killing a Pro104 expressing cell. Finally, the invention also
provides kits and articles of manufacture comprising at least one
antibody of this invention, preferably at least one anti-Pro104
antibody of this invention that binds to Pro104 on a mammalian cell
in vivo or at least one internalizing anti-Pro104 antibody of this
invention. Kits containing anti-Pro104 antibodies find use in
detecting Pro104 expression, or in therapeutic or diagnostic
assays, e.g., for Pro104 cell killing assays or for purification
and/or immunoprecipitation of Pro104 from cells. For example, for
isolation and purification of Pro104, the kit can contain an
anti-Pro104 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
Pro104 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-Pro104 Antibodies
[0217] 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 Pro104
antigen to be used for production of antibodies may be, e.g., the
full length polypeptide or a portion thereat including a soluble
form of Pro104 lacking the membrane spanning sequence, or synthetic
peptides to selected portions of the protein.
[0218] Alternatively, cells expressing Pro104 at their cell surface
(e.g. CHO or NIH-3T3 cells transformed to overexpress Pro104;
ovarian, pancreatic, lung, breast or other Pro104-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 Pro104 are available as provided above. Pro104 can
be produced recombinantly in and isolated from, prokaryotic cells,
e.g., bacterial cells, or eukaryotic cells using standard
recombinant DNA methodology. Pro104 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.
[0219] Antibodies or binding proteins that bind to various tags and
fusion sequences are available as elaborated below. Other forms of
Pro104 useful for generating antibodies will be apparent to those
skilled in the art.
[0220] Tags
[0221] 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)).
[0222] Polyclonal Antibodies
[0223] 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 mmunized. 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.
[0224] 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.
[0225] Monoclonal Antibodies
[0226] 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)).
[0227] 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.
[0228] 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)).
[0229] 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).
[0230] 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.
[0231] 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.
[0232] 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).
[0233] 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. Mal. 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.
[0234] 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.
[0235] Humanized Antibodies
[0236] 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.
[0237] 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)).
[0238] 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.
[0239] 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.
[0240] Various forms of a humanized anti-Pro104 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.
[0241] Human Antibodies
[0242] 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 MI3 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).
[0243] Antibody Fragments
[0244] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors. Various techniques have been
developed for the production of antibody fragments. Traditionally,
these fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods 24:107-117 (1992); and Brennan et al., Science,
229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. Fab, Fv and ScFv antibody
fragments can all be expressed in and secreted from E. coli, thus
allowing the facile production of large amounts of these fragments.
Antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab)2 fragments (Carter et al., Bio/Technology 10: 163-167
(1992)). According to another approach, F(ab)2 fragments can be
isolated directly from recombinant host cell culture. Fab and
F(ab)2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues are described in U.S.
Pat. No. 5,869,046. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. The
antibody of choice may also be a single chain Fv fragment (scFv).
See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. Fv and sFv are the only species with intact combining
sites that are devoid of constant regions; thus, they are suitable
for reduced nonspecific binding during in vivo use. sFv fusion
proteins may be constructed to yield fusion of an effector protein
at either the amino or the carboxy terminus of an sFv. See Antibody
Engineering, ed. Borrebaeck, supra. The antibody fragment may also
be a "linear antibody", e.g., as described in U.S. Pat. No.
5,641,870 for example. Such linear antibody fragments may be
monospecific or bispecific.
[0245] Bispecific Antibodies
[0246] 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
Pro104 protein. Other such antibodies may combine a Pro104 binding
site with a binding site for another protein. Alternatively, an
anti-Pro104.Arm may be combined with an arm which binds to a
triggering molecule on a leukocyte such as a Teen receptor molecule
(e.g. C133), or Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16),
so as to focus and localize cellular defense mechanisms to the
Pro104-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express Pro104. These
antibodies possess a Pro104-binding arm and an arm which binds the
cytotoxic agent (e.g. saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab)2 bispecific
antibodies). WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0247] 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).
[0248] 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.
[0249] 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).
[0250] 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.
[0251] 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.
[0252] 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.
[0253] 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.
[0254] 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.
[0255] 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 Umber et al., J. Immunol.,
152:5368 (1994).
[0256] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0257] Multivalent Antibodies
[0258] 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 VDI(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.
[0259] Other Amino Acid Sequence Modifications
[0260] Amino acid sequence modification(s) of the anti-Pro104
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-Pro104 antibody are prepared by introducing appropriate
nucleotide changes into the anti-Pro104 antibody nucleic acid, or
by peptide synthesis.
[0261] Such modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the anti-Pro104 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-Pro104 antibody,
such as changing the number or position of glycosylation sites.
[0262] A useful method for identification of certain residues or
regions of the anti-Pro104 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-Pro104
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 Pro104 antigen.
[0263] 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-Pro104
antibody variants are screened for the desired activity.
[0264] 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-Pro104 antibody
with an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the
anti-Pro104 antibody molecule include the fusion to the N- or
C-terminus of the anti-Pro104 antibody to an enzyme (e.g. for
ADEPT) or a fusion to a polypeptide which increases the serum
half-life of the antibody.
[0265] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-Pro104 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 Original Exemplary
Substitutions Preferred Substitutions Ala (A) val; leu; ile Val Arg
(R) lys; g1n; asn lys Asn (N) g1n; 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; g1n; lys; arg arg Ile (I) leu;
val; met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ile
Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser Phe Val (V)
ile; leu; met; phe; ala; leu
[0266] 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.
[0267] 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-Pro104 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).
[0268] 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 MI3 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 Pro104. 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.
[0269] 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).
[0270] Nucleic acid molecules encoding amino acid sequence variants
of the anti-Pro104 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-Pro104 antibody.
[0271] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0272] 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
[0273] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0274] The growth inhibitory effects of an anti-Pro104 antibody of
the invention may be assessed by methods known in the art, e.g.,
using cells which express Pro104 either endogenously or following
transfection with the Pro104 gene. For example, the tumor cell
lines and Pro104-transfected cells provided in Example 1 below may
be treated with an anti-Pro104 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-Pro104 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 Pro104. Preferably, the
anti-Pro104 antibody will inhibit cell proliferation of a
Pro104-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-Pro104 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.
[0275] 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. Pro104-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.
[0276] To screen for antibodies which bind to an epitope on Pro104
bound by an antibody of interest, e.g., the Pro104 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-Pro104 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 Pro104 can
be used in competition assays with the test antibodies or with a
test antibody and an antibody with a characterized or known
epitope.
[0277] For example, a method to screen for antibodies that bind to
an epitope which is bound by an antibody this invention may
comprise combining a Pro104-containing sample with a test antibody
and an antibody of this invention to form a mixture, the level of
Pro104 antibody bound to Pro104 in the mixture is then determined
and compared to the level of Pro104 antibody bound in the mixture
to a control mixture, wherein the level of Pro104 antibody binding
to Pro104 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-Pro104 antibody of this invention. The level of Pro104
antibody bound to Pro104 is determined by ELISA. The control may be
a positive or negative control or both. For example, the control
may be a mixture of Pro104, Pro104 antibody of this invention and
an antibody known to bind the epitope bound by the Pro104 antibody
of this invention. The anti-Pro104 antibody labeled with a label
such as those disclosed herein. The Pro104 may be bound to a solid
support, e.g., a tissue culture plate or to beads, e.g., sepharose
beads.
Immunoconjugates
[0278] 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.
[0279] 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.
[0280] Maytansine and Maytansinoids
[0281] Preferably, an anti-Pro104 antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0282] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the cast African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
[0283] Maytansinoid-Antibody Conjugates
[0284] 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.
[0285] Anti-Pro104 Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0286] Anti-Pro104 antibody-maytansinoid conjugates are prepared by
chemically linking an anti-Pro104 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.
[0287] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al. Cancer Research 52: 127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred. Conjugates of the antibody and
maytansinoid may be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl (2-pyridyldithio) propionate
(SPDP), succinimidyl-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as his (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include N-succinimidyl
(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J.
173:723-737 [1978]) and N-succinimidyl (2-pyridylthio)pentanoate
(SPP) to provide for a disulfide linkage.
[0288] 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.
[0289] Calicheamicin
[0290] Another immunoconjugate of interest comprises an anti-Pro104
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
[0291] Other antitumor agents that can be conjugated to the
anti-Pro104 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).
[0292] 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-Pro104 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.
[0293] 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.
[0294] 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 glutaraldehyde), 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.
[0295] Alternatively, a fusion protein comprising the anti-Pro104
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.
[0296] 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)
[0297] 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.
[0298] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form. Enzymes that
are useful in the method of this invention include, but are not
limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as O-galactosidase and neuraminidase useful for converting
glycosylated prodrugs into free drugs; P-lactamase useful for
converting drugs derivatized with P-lactams into free drugs; and
penicillin amidases, such as penicillin V amidase or penicillin G
amidase, useful for converting drugs derivatized at their amine
nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,
into free drugs. Alternatively, antibodies with enzymatic activity,
also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g.,
Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can
be prepared as described herein for delivery of the abzyme to a
tumor cell population. The enzymes of this invention can be
covalently bound to the anti-Pro104 antibodies by techniques well
known in the art such as the use of the heterobifunctional
crosslinking reagents discussed above.
[0299] 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
[0300] 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).
[0301] The anti-Pro104 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
[0302] The invention also provides isolated nucleic acid molecule
encoding the humanized anti-Pro104 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.
[0303] Signal Sequence Component
[0304] The anti-Pro104 antibody of this invention may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the native anti-Pro 104 antibody signal sequence, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group of the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, oc factor leader (including
Saccharomyces and Kluyveromyces .alpha.-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-Pro104 antibody.
[0305] Origin of Replication
[0306] 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).
[0307] Selection Gene Component
[0308] 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.
[0309] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-Pro104 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).
[0310] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-Pro104 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.
[0311] 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.
[0312] 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).
[0313] Promoter Component
[0314] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-Pro104 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-Pro104 antibody.
[0315] 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.
[0316] 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.
[0317] Anti-Pro104 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.
[0318] 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
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.
[0319] Enhancer Element Component
[0320] Transcription of a DNA encoding the anti-Pro104 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-Pro
104 antibody-encoding sequence, but is preferably located at a site
5' from the promoter.
[0321] Transcription Termination Component
[0322] 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-Pro104 antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO 94/11026 and the expression vector disclosed therein.
[0323] Selection and Transformation of Host Cells
[0324] 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.
[0325] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation region
(TIR) and signal sequences for optimizing expression and secretion,
these patents incorporated herein by reference. After expression,
the antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g, in CHO
cells.
[0326] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-Pro104 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. lactic, 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.
[0327] Suitable host cells for the expression of glycosylated
anti-Pro104 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.
[0328] 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.
[0329] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0330] Host cells are transformed with the above-described
expression or cloning vectors for anti-Pro104 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0331] Culturing Host Cells
[0332] The host cells used to produce the anti-Pro104 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. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0333] Purification of Anti-Pro104 Antibody
[0334] 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.
[0335] 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.
[0336] 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
[0337] 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.
[0338] 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-Pro104 antibody which internalizes, it may be desirable to
include in the one formulation, an additional antibody, e.g. a
second anti-Pro104 antibody which binds a different epitope on
Pro104, 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.
[0339] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0340] 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.
[0341] 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-Pro104 Antibodies
[0342] According to the present invention, the anti-Pro104 antibody
that internalizes upon binding Pro104 on a cell surface is used to
treat a subject in need thereof having a cancer characterized by
Pro104-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.
[0343] The cancer will generally comprise Pro104-expressing cells,
such that the anti-Pro104 antibody is able to bind thereto. While
the cancer may be characterized by overexpression of the Pro104
molecule, the present application further provides a method for
treating cancer which is not considered to be a
Pro104-overexpressing cancer.
[0344] This invention also relates to methods for detecting cells
which overexpress Pro104 and to diagnostic kits useful in detecting
cells expressing Pro104 or in detecting Pro104 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 Pro104 overexpressing
cells. A level of Pro104 binding higher than that of such a control
sample would be indicative of the test sample containing cells that
overexpress Pro104. Alternatively the control may be a sample of
cells known to contain cells that overexpress Pro104. In such a
case, a level of Pro104 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
Pro104.
[0345] Pro104 overexpression may be detected with a various
diagnostic assays. For example, over expression of Pro104 may be
assayed by immunohistochemistry (IHC). Parrafin embedded tissue
sections from a tumor biopsy may be subjected to the IHC assay and
accorded a Pro104 protein staining intensity criteria as
follows.
[0346] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0347] 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.
[0348] Score 2+ a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0349] Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0350] Those tumors with 0 or 1+ scores for Pro104 expression may
be characterized as not overexpressing Pro104, whereas those tumors
with 2+ or 3+ scores may be characterized as overexpressing
Pro104.
[0351] 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 Pro104
overexpression in the tumor. Pro104 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 Pro104 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.
[0352] A sample suspected of containing cells expressing or
overexpressing Pro104 is combined with the antibodies of this
invention under conditions suitable for the specific binding of the
antibodies to Pro104. Binding and/or internalizing the Pro104
antibodies of this invention is indicative of the cells expressing
Pro104. The level of binding may be determined and compared to a
suitable control, wherein an elevated level of bound Pro104 as
compared to the control is indicative of Pro104 overexpression. The
sample suspected of containing cells overexpressing Pro104 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 Pro104 by combining a serum
sample from a subject with a Pro104 antibody of this invention,
determining the level of Pro104 bound to the antibody and comparing
the level to a control, wherein an elevated level of Pro104 in the
serum of the patient as compared to a control is indicative of
overexpression of Pro104 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.
[0353] 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-Pro104 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-Pro104 antibodies of the invention are useful to alleviate
Pro104-expressing cancers, e.g., ovarian, pancreatic, lung or
breast cancers upon initial diagnosis of the disease or during
relapse. For therapeutic applications, the anti-Pro104 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-Pro104 antibody treatment can be administered in
conjunction with other forms of conventional therapy, either
consecutively with, pre- or post-conventional therapy,
Chemotherapeutic drugs such as Taxotere.RTM. (docetaxel),
Taxol.RTM. (paclitaxel), estramustine and mitoxantrone are used in
treating metastatic and hormone refractory ovarian, pancreatic,
lung or breast cancer, in particular, in good risk patients. In the
present method of the invention for treating or alleviating cancer,
in particular, androgen independent and/or metastatic ovarian,
pancreatic, lung or breast cancer, the cancer patient can be
administered anti-Pro104 antibody in conjunction with treatment
with the one or more of the preceding chemotherapeutic agents. In
particular, combination therapy with palictaxel and modified
derivatives (see, e.g., EP0600517) is contemplated. The anti-Pro104
antibody will be administered with a therapeutically effective dose
of the chemotherapeutic agent. The anti-Pro104 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.
[0354] Particularly, an immunoconjugate comprising the anti-Pro104
antibody conjugated with a cytotoxic agent may be administered to
the patient. Preferably, the immunoconjugate bound to the Pro104
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.
[0355] The anti-Pro104 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-Pro104 antibody.
[0356] 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.
[0357] It may also be desirable to combine administration of the
anti-Pro104 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 Pro104-expressing tumor cells. The
cocktail may also comprise antibodies that are directed to other
epitopes of Pro104. Preferably the other antibodies do not
interfere with the binding and or internalization of the antibodies
of this invention.
[0358] The antibody therapeutic treatment method of the present
invention may involve the combined administration of an anti-Pro104
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).
[0359] 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-Pro104 antibody (and optionally other agents
as described herein) may be administered to the patient.
[0360] 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-Pro104
antibody.
[0361] 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-Pro104 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.
[0362] 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.
[0363] 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.
[0364] 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
[0365] The invention also relates to an article of manufacture
containing materials useful for the detection for Pro104
overexpressing cells and/or the treatment of Pro104 expressing
cancer, in particular breast, ovarian, pancreatic and lung 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
Pro104 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-Pro 104 antibody of the invention. The label
or package insert indicates that the composition is used for
detecting Pro104 expressing cells and/or for treating breast,
ovarian, pancreatic and lung cancer, or more specifically ovarian
serous adenocarcinoma, breast infiltrating ductal carcinoma,
prostate adenocarcinoma, renal cell carcinomas, colorectal
adenocarcinomas, lung adenocarcinomas, lung squamous cell
carcinomas, and pleural mesothelioma, in a patient in need thereof.
The breast cancer may be HER-2 negative or positive breast cancer.
The cancers encompass metastatic cancers of any of the preceding,
e.g., breast, ovarian, pancreatic and lung cancer metastases. The
label or package insert may further comprise instructions for
administering the antibody composition to a cancer patient.
Additionally, the article of manufacture may further comprise a
second container comprising a substance which detects the antibody
of this invention, e.g., a second antibody which binds to the
antibodies of this invention. The substance may be labeled with a
detectable label such as those disclosed herein. The second
container may contain e.g., a pharmaceutically-acceptable buffer,
such as bacteriostatic water for injection (BWFI),
phosphate-buffered saline, Ringer's solution and dextrose solution.
The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0366] Kits are also provided that are useful for various purposes,
e.g., for Pro104 cell killing assays, for purification or
immunoprecipitation of Pro104 from cells or for detecting the
presence of Pro104 in a serum sample or detecting the presence of
Pro104-expressing cells in a cell sample. For isolation and
purification of Pro104, the kit can contain an anti-Pro104 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 Pro104 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
[0367] The following MAb/hybridomas of the present invention are
described below: Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17,
Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27,
Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49,
Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57,
Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,
Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18,
Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29,
Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55,
Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64,
Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85,
Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106,
Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115,
Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120,
Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124,
Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128,
Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133,
Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138,
Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47,
Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,
Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89,
Pro104.K155, Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159,
Pro104.K160, Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217,
Pro104.K226, Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264,
Pro104.K281, Pro104.K358 or Pro104.K362.
[0368] 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)
[0369] Pro104 (Testisin) Full Length, Fragment E. coli Expressed
Sequence & Protein Production
[0370] For immunization of mice and production of the C series of
MAbs, a Pro104 construct encoding a region of Pro104 from Lys20 to
Trp297 was introduced into a standard E. coli expression vector via
restriction enzyme sites. The construct was cloned in-frame to the
C-terminus of a six-histidine tag so that the Pro104 construct
would be expressed as a six-histidine tagged protein of 288 amino
acids. The recombinant plasmid was used to transform competent E.
coli cells and Pro104 expression was performed in shaker flasks.
The bacterial paste, collected after induction with IPTG, was used
for Pro104 purification via Ni-NTA column chromatography.
TABLE-US-00002 Pro104 (Lys20 (underlined)-Trp297) expressed amino
acid sequence (the bold type represents the hexa histidine tag)
(SEQ ID NO. 1)
MAKPESQEAAPLSGPOGRRVITSRIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWA
LTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYD
IALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVA
IINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVVS
WGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWLEHHHHHH
[0371] Pro104 (Testisin) Insect Cell Expressed Sequence &
Protein Production
[0372] For immunization of mice and production of the D series of
MAbs, a Pro104 construct encoding a honey bee melletin secretion
signal, a region of Pro104 from Ile42 to Trp297 and a six histidine
tag was cloned and expressed using standard techniques. Pro104 was
purified using Ni-NTA resin.
TABLE-US-00003 Pro104 (Ile42-Trp297) expressed amino acid sequence
(underlined portion represents the honey bee melletin secretion
signal and the bold type represents the hexa histidine tag) (SEQ ID
NO. 2) MKFLVNVALVFMVVYISYIYADPMAIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRW
ALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPY
DIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQV
AIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVV
SWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWHHHHHH
[0373] Pro104 (Testisin) 293T & LMTK Cell Expressed Sequences
& Protein Production
[0374] For screening of both C and D series Pro104 MAbs, full
length Pro104 protein (Met1-Val314) was expressed both with and
without an HA tag (bold) and spacer (underlined) located at the
C-terminus, downstream from the recombination site (italics), using
standard mammalian expression techniques.
TABLE-US-00004 Pro104 transfected human 293T and mouse LMTK cell
amino acid sequence (SEQ ID NO. 3)
MGARGALLLALLLARAGLRKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLR
LWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYF
VSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIK
EDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGG
PLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWPLL
FFPLLWALPLLGPVDPAFLYKVVRSRMASYPYDVPDYASL
[0375] Pro104 (Testisin) CHO Cell Expressed Sequences & Protein
Production
[0376] For immunization of mice and production of the K-series
Pro104 MAbs, full length Pro104 protein (Met1-Val314) was expressed
without a tag using standard mammalian expression techniques.
[0377] Hamster CHO cells were stably transfected with Tetracycline
Receptor (TR) using standard recombinant techniques. Prior to
Pro104 transfection, CHO-TR cells were cultured in HAM F12 medium
with 10% fetal bovine serum (FBS). A vector encoding full length
Pro104 protein (Met1-Val314) was transfected into the CHO cells.
Stable transfectants were selected in HAM F12 medium with 10% FBS
with Hydromycin B at 300 ug/ml, for 15 days. Hydromycin B-resistant
cells were checked for expression of Pro104 by western blot using
diaDexus Pro104.C25.1 monoclonal antibody after 16-20 hour
stimulation with 1 ug/ml Tetracycline. Cells were expanded,
scaled-up and cryopreserved in FBS with 10% DMSO and stored in
liquid nitrogen at -196.degree. C. to assure maintenance of viable
clone cultures.
TABLE-US-00005 Pro104 transfected hamster CHO cell amino acid
sequence (SEQ ID NO. 4)
MGARGALLLALLLARAGLRKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLR
LWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYF
VSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIK
EDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGG
PLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWPLL
FFPLLWALPLLGPV
[0378] Ovr115 Serine Protease Domain Sequence & Protein
Production
[0379] Ovr115 (TMPRSS4) was used to screen out cross reactive
hybridoma clones, since this antigen was also upregulated in
ovarian and pancreatic cancers and since it also contained a
potentially cross-reactive serine protease domain.
[0380] An Ovr115 construct encoding a tobacco etch virus protease
(TEV) recognition site and the serine protease domain of Ovr115
from Val203 to Leu435 was cloned in-frame to the C-terminus of
glutathione S-transferase (GST) so that the Ovr115 construct was
expressed as a GST-fusion protein of 486 amino acids using standard
techniques. Purification of Ovr115 was completed via glutathione
sepharose column.
TABLE-US-00006 Ovr115 serine protease domain construct amino acid
sequence (GST sequence is underlined, TEV sequence is in italics
and tag sequence is in bold type) (SEQ ID NO. 5)
MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYID
GDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV
DELSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFK
KRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRHNQTSLYKKAGFENLY
FQGVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLTAAHCFRKHTDVFNWKVRAG
SDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTFSGTVRPICLPFFDEELTPA
TPLWIIGWGFTKQNGGKMSDILLQASVQVIDSTRCNADDAYQGEVTEKMMCAGIPEGGVD
TCQGDSGGPLMYQSDQWHVVGIVSWGYGCGGPSTPGVYTKVSAYLNWIYNVWKAELSNWS
HPQFEK
[0381] Ovr115 Extracellular Fragment Sequence & Protein
Production
[0382] An Ovr115 (TMPRSS4) construct encoding a region of Ovr115
from Lys52 to Leu435, which constituted only the predicted
extracellular portion of the molecule, was cloned with a
six-histidine tag immediately downstream of codon Leu435. Ovr115
was purified using Ni-NTA resin.
TABLE-US-00007 Ovr115 extracellular construct (Ovr115 Lys52
(underlined)-Leu435) amino acid sequence (6 His tag sequence is in
bold type) (SEQ ID NO. 6)
MKVILDKYYFLCGQPLHFIPRKQLCDGELDCPLGEDEEHCVKSFPEGPAV
AVRLSKDRSTLQVLDSATGNWFSACFDNFTEALAETACRQMGYSSKPTFR
AVEIGPDQDLDVVEITENSQELRMRNSSGPCLSGSLVSLHCLACGKSLKT
PRVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLTAAHCFRKHTD
VFNWKVRAGSDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTF
SGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQVID
STRCNADDAYQGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQWHVVG
IVSWGYGCGGPSTPGVYTKVSAYLNWIYNVWKAELHHHHHH
[0383] Generation of Stable Ovr115 LMTK Mouse Cell Lines
[0384] Full length HA-tagged Ovr115 (Met1-Leu435) (underlined) was
transfected into mouse LMTK cells after cloning into a mammalian
expression vector with an HA tag. Individual clones were checked
for expression of Ovr115 by western blot using anti-HA antibody
(Covance, Richmond, Calif.), after 1 week in culture.
TABLE-US-00008 Ovr115 transfected LMTK amino acid sequence (SEQ ID
NO. 7) MDPDSDQPLNSLDVKPLRKPRIPMETFRKVGIPIIIALLSLASIIIVVVLIKVILDKYYF
LCGQPLHFIPRKQLCDGELDCPLGEDEEHCVKSFPEGPAVAVRLSKDRSTLQVLDSATGN
WFSACFDNFTEALAETACRQMGYSSKPTFRAVEIGPDQDLDVVEITENSQELRMRNSSGP
CLSGSLVSLHCLACGKSLKTPRVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLT
AAHCFRKHTDVFNWKVRAGSDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTF
SGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQVIDSTRCNADDAY
QGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQWHVVGIVSWGYGCGGPSTPGVYTKV
SAYLNWIYNVWKAELDPAFLYKVVRSRMASYPYDVPDYASL
Immunizations
[0385] For generation of the C series MAbs mice were immunized with
soluble E. coli expressed Pro104 recombinant protein, encoding a
region of Pro104 from Lys20 to Trp297 of the full length protein.
Groups of 8 BALB/c mice were immunized intradermally in both rear
footpads. All injections were 25 uL per foot. The first injection
(day 1) of 10 ug of 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.
[0386] For generation of the D series MAbs mice were immunized as
above with soluble insect cell expressed Pro104 recombinant
protein, which corresponded to a region from Ile42 to Trp297 of the
full length protein.
[0387] For the K series MAbs mice were immunized with a stably
transfected CHO cell line expressing Pro104 on the cell surface.
The cell surface expression of Pro104 ranged from 13.3 to 97.0%. In
the first two injections, the mice were immunized with
1.25.times.10.sup.6 cells/mouse, in injections 3-9, the mice were
injected with 3.75.times.10.sup.6 cells/mouse. The mice were given
a final injection of 2.5.times.10.sup.6 cells. Whole cells in HBSS
(Hanks Balanced Salt Solution) with no adjuvants were used
throughout the immunization series. The K series immunization
schedule was the same one used for the C and D series above.
[0388] Hybridoma Fusions
[0389] Mice were sacrificed at the completion of the immunization
protocol and draining lymph node (popliteal) tissue was collected
by sterile dissection. Lymph node cells were dispersed using a
Tenbroeck tissue grinder (Wheaton #357426, VWR, Brisbane, Calif.)
followed by pressing through a sterile 40 uM sieve (VWR) into DMEM
and removing T-cells via anti-CD90 (Thy1.2) coated magnetic beads
(Miltenyl Biotech, Baraisch-Gladbach, Germany).
[0390] These primary B-cell enriched lymph node cells were then
immortalized by electro-cell fusion (BTX, San Diego, Calif.) with
the continuous myeloma cell line P3x63Ag8.653 (Kearney, J. F. et
al., J. Immunology 123: 1548-1550, 1979). Successfully fused cells
were selected by culturing in standard Hypoxanthine, Azaserine (HA)
(Sigma, St. Louis, Mo.) containing selection medium (DMEM/15%
FBS/0.5 ng/mL rIL-6 (Sigma, Saint Louis, Miss.)/10% P388D.sub.1
(ATCC, Manassas, Va.) conditioned medium). These fusion cultures
were immediately distributed, 2 million cells per plate, into wells
of 96 well culture plates (Costar Cat. #3585, VWR). Distributing
the culture in 96 well culture plates, immediately following
fusion, facilitated selection of a larger diversity of hybridoma
clones producing single, specific antibodies. Supernatants from
wells were screened by ELISA, for reactivity against Pro104 E. coli
expressed protein, Pro104 insect expressed protein, and for no
cross-reactivity with the serine protease Ovr115 extracellular
domain (insect expressed).
[0391] Monoclonal cultures, consisting of the genetically uniform
progeny from single cells, were established after the screening
procedure above, by limiting dilution (Coller, H and Coller, B.
Hybridoma 2: 91-6, 1983), or cell sorting of single viable cells
into wells of two 96 well plates (VWR), using flow cytometry
(Coulter Elite, Beckman Coulter, Miami, Fla.). The resulting murine
B-cell hybridoma cultures were expanded using standard tissue
culture techniques. Selected hybridomas were cryopreserved in fetal
bovine serum (FBS) with 10% DMSO and stored in Liquid Nitrogen at
-196.degree. C. to assure maintenance of viable clone cultures.
Screening & Selection of Antibody Producing Hybridomas
[0392] Hybridoma cell lines were selected for production of Pro104
specific antibody by enzyme linked solid phase immunoassay (ELISA).
Pro104 or Ovr115 proteins were nonspecifically adsorbed to wells of
96 well polystyrene EIA plates (VWR). One hundred uL volumes of
Pro104 or Ovr115 proteins at approximately 1 ug/mL in (DPBS) were
incubated overnight at 4.degree. C. in wells of 96 well polystyrene
EIA plates. Plates were washed twice with Tris buffered saline with
0.05% Tween 20, pH 7.4 (TBST). The plate wells were then emptied
and nonspecific binding capacity was blocked by completely filling
the assay wells with TBST/0.5% bovine serum albumin (TBST/BSA) and
incubating for 30 minutes at room temperature (RT). The plate wells
were then emptied, 100 uL of hybridoma culture medium samples
diluted 1:1 with TBST/BSA was added to the wells and incubated for
1 hour at RT. The wells were then washed 3 times with (TBST). One
hundred uL of alkaline phosphatase conjugated goat anti-mouse IgG
(Fc) (Pierce Chemical Co., Rockford, Ill.), diluted 1:5000 in
TBST/BSA, was then added to each well and incubated for 1 hour at
RT. The wells were then washed 3 times with TBST. One hundred uL of
alkaline phosphatase substrate para-nitrophenylphosphate (pNPP)
(Sigma, Saint Louis, Miss.) at 1 mg/mL in 1 M Diethanolamine buffer
pH 8.9 (Pierce, Rockford, Ill.) was then added to each well and
incubated for 20 min. at RT. Color development was stopped by
addition of 50 uL of 2N NaOH/well. Bound alkaline phosphatase
activity was indicated by the development of a visible yellow
color. The enzymatic reaction was quantified by measuring the
solution's absorbance at 405 nm wavelength. Cultures producing the
highest absorbance values were chosen for expansion and further
evaluation. Selected ELISA positive cultures from the original 96
well plates were transferred to new 96 well tissue culture plates
(VWR).
ELISA Screening of Pro104 MAbs
[0393] Hybridomas were retested to confirm continued production of
Pro104 specific MAbs. Hybridoma cultures with supernatants
producing ELISA absorbance values greater than 1.0 with Pro104 and
less than 0.2 with Ovr115 were expanded in tissue culture and
cryopreserved, as described above. Selected Pro104 specific
cultures were subcloned by limiting dilution or single cell sorting
(Coulter Elite) to ensure genetically stable and uniform
progeny.
Results from ELISA Screening of Cloned Pro104 MAbs
[0394] Clones Pro104.C13, Pro104.C18, Pro104.C25 and Pro104.C55
from the first immunization with E. coli expressed Pro104 were
positive by ELISA with E. coli expressed Pro104, insect expressed
Pro104, human 293T cell expressed Pro104 with and without the HA
tag (Table 1 below) and did not react with the other human serine
proteases pancreatic trypsin, lung tryptase and kallikrein (Cal
Biochem, San Diego, Calif.) nor plasmin or urokinase (American
Diagnostics, Greenwich, Conn.). Pro104.C13, Pro104.C18, Pro104.C19,
Pro104.C25 and Pro104.C55 were subcloned and scaled up for further
characterization by western blot, immunohistochemistry and
immunofluorescence. MAbs Pro104.C37 and Pro104.C48 cross-reacted
with human urokinase and were not evaluated further by
immunohistochemistry or immunofluorescence.
[0395] Clones Pro104.D4, Pro104.D6, Pro104.D9, Pro104.D14,
Pro104.D18, Pro104.D19, Pro104.D31, Pro104.D43, Pro104.D47,
Pro104.D51, Pro104.D56, Pro104.D58, Pro104.D64, Pro104.D81,
Pro104.D88, Pro104.D91 and Pro104.D94 from immunization with the
insect expressed Pro104, were positive by ELISA with E. coli
expressed Pro104, insect expressed Pro104, human 293T cell
expressed Pro104 with and without the HA tag (Table 3) and did not
react with the other human serine proteases pancreatic trypsin,
lung tryptase, kallikrein, plasmin nor urokinase, see Table 3
below. Clones Pro104.D12, Pro104.D15, Pro104.D55, Pro104.D62,
Pro104.D68 and Pro104.D106 were positive by ELISA with E. coli
expressed Pro104, insect expressed Pro104 and were more weakly
reactive (ELISA OD 405 inn from 0.3-0.8) with the human 293T cell
expressed Pro104 (data not shown), but did not react with
pancreatic trypsin, lung tryptase and kallikrein, plasmin or
urokinase (Table 3). Pro104.D20 and Pro104.D21 were positive only
with mammalian (293T) Pro104 protein. Pro104.D4, Pro104.D6,
Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,
Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31,
Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56,
Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68,
Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,
Pro104.D91, Pro104.D94, Pro104.D102 and Pro104.D106 were subcloned
and scaled up for further characterization by western blot,
immunohistochemistry and immunofluorescence. Pro104 MAbs
cross-reacting with other human serine proteases (Pro104.D29 &
Pro104.D31) were not evaluated further by immunohistochemistry or
immunofluorescence.
FACS Screening for Cell Surface Binding of Pro104 C-Series MAbs
[0396] CAOV3 (RT-PCR positive for Pro104) and SKOV3 (RT-PCR
negative for Pro104) ovarian carcinoma cell lines (ATCC) were grown
in DMEM/10% FBS. Prior to staining, the cells 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 min. 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. The cells were
then centrifuged for 5 minutes at 1300 rpm and resuspended in
DMEM/10% FBS. The cells were then 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, Hayward,
Calif.) and if >90% viable they were distributed into 96-well
v-bottom plates (VWR) for staining with MAbs. 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 a 200 ul volume of DPBS/3% FBS/0.01% Na Azide (FACS buffer)
was added to each well. Centrifugation and aspiration was repeated,
then 25 uL volumes of hybridoma supernatant or purified MAb were
added to the cells. Plates were vortexed to resuspend cells, stored
on ice for 15 min., then washed in 200 uL of FACS buffer and
centrifuged as above. This washing procedure was repeated a twice
and then 25 uL volumes 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 volumes of FACS buffer and 67 uL of 1%
paraformaldehyde/DPBS were added to wells, for fixation, then the
volumes were increased to 250 uL with DPBS. Stained cells were
analyzed on an Elite fluorescent activated cell sorter (FACS)
(Beckman-Coulter, Miami, Fla.).
[0397] Results demonstrating cell surface binding of several of the
C series MAbs by immunofluorescent FACS and microscopic analysis,
are summarized in Table 2. Further immunofluorescence microscopy
data with human tumor cell lines are presented below. The isotypes
of the C series MAbs were determined using commercially available
mouse monoclonal antibody isotyping immunoassay test kits
(IsoStrip, Roche Diagnostic Corp., Indianapolis, Ind.). Results of
the isotyping are listed in Table 2.
TABLE-US-00009 TABLE 2 ISOTYPE, ELISA, IMMUNOFLUORESCENCE FACS
& MICROSCOPY RESULTS OF Pro104 C SERIES MAbs Direct ELISA
Microscopy Pur. E. coli Pur. Insect FL FACS Co-localization Pro104
expressed insect Pro104 Pro104 Pro104- CaOv-3 SkOv-3 with HA on
Pro104- MAb Full Lgth expressed Crude 293T HA 293T (RT- (RT- HA
Transfected Clone Isotype Pro104 Pro104 Lysate Lysate Lysate PCR+)
PCR-) 293T cells C4 IgG1 k + + + + + - - 4* C13 IgG1 k + + - + + +
- 3 C18 IgG1 k + + + + + - - 3 C19 IgG1 k + + + - - - - 4 C25 IgG1
k + + + + + + + 3 C34 IgG1 k + + + - - - - 4 C37 IgG1 k + + + + + +
3 C46 IgG2b k + + - - - - - 2 C48 IgG1 k + + - + + + - 3 C50 IgG2b
k + + - - - - - 3 C53 IgG2b k + + - - - - - 3 C54 IgG2b k + + + - -
- - 4 C55 IgG1 k + + + + - - - 4 C57 IgG2b k + + - - - + - 2 C60
IgG1 k + + + - - + - 4 C66 IgG1 k + + - - - - - 1 C84 IgG1 k + - +
+ + - - 1 C1 IgG2b k + - - - - + + 2 C17 IgG2b k + - - - - + + 2
C24 IgG2a k + - - - - + - 2 C27 IgG3 k + - - - - + - 3 C49 IgG2a k
+ + - - - + - 2 C75 IgG3 k + + - - - + - 3 *4 = Strong
co-localization with HA with no background staining of
non-transfected 293T cells, 3 = Strong co-localization with HA
& background staining of non-transfected 293T cells, 2 =
Partial co-localization with HA & background staining of
non-transfected 293T cells, 1 = Weak HA staining (possibly blocked
by test MAb) & high background staining of non-transfected 293T
cells.
TABLE-US-00010 TABLE 3 RESULTS OF ELISA SCREENING OF THE Pro104 D
SERIES MAbs Pro104 Pro104 MAb Pro104 Pro104 Ovr115 293T Clone
(Insect) (E. coli) LMTK Urokinase Plasmin Tryptase Kallikrein
Trypsin lysate D4 + + - - - - - - + D6 + + - - - - - - + D9 + + - -
- - - - + D12 + + - - - - - - + D14 + + - - - - - - + D15 + + - - -
- - - + D18 + + - - - - - - + D19 + + - - - - - - + D20 - - - - - -
- - + D21 - - - - - - - - + D26 + + - + D29 + - - - + - - - + D31 +
+ - - + - - - + D43 + + - + D47 + + - + D51 + + - + D55 + - - - - -
- - + D56 + + - + D58 + + - - - - - - + D62 + + - - - - - - + D63 +
+ - + D64 + + - + D68 + + - - - - - - + D69 + + - + D75 - - - + D81
+ + - + D85 + + - + D88 + + - - - - - - + D91 + + - + D94 + + - +
D102 + + - + D106 + + - - - - - - +
FACS Screening for Cell Surface Binding of Pro104 D-Series and
K-Series MAbs
[0398] Fifty million 293F cells were transfected by a preparation
of lipid-DNA complexes by performing a dilution of 50 .mu.g of
plasmid DNA in Opti-MEM I reduced serum medium (GIBCO) to a total
volume of 1.5 ml followed by gentle mixing. A dilution of 75 .mu.l
of 293Fectin (Invitrogen) in Opti-MEM I to a total volume of 1.5 ml
was mixed gently and incubated for 5 minutes at room temperature.
After the 5 minute incubation, the diluted DNA was mixed with the
diluted 293fectin. This mixture was allowed to incubate for 20-30
minutes at room temperature to allow the DNA-293fectin complexes to
form. While the DNA-293fectin complexes incubated, an aliquot of 50
ml of cell suspension (1E6 viable cells/ml) was placed into a
sterile, disposable flask. After the DNA-293fectin complex
incubation was completed, they were transferred to each flask of
cells. The flasks were incubated in a 37.degree. C. incubator with
shaking at 120 rpm. The cells were used for staining experiments at
approximately 48 hours post-transfection.
[0399] The DNA sequence used for transfecting the 293F cells was
the full length Pro104 sequence (met.sup.1 to val.sup.314), with no
tags, See SEQ ID NO: 3 above.
[0400] Prior to staining, the viability of the 293F and control
cells was measured using Guava Viacount (Guava Technologies,
Hayward, Calif.) and if >90% were viable they were distributed
into 96-well v-bottom plates (VWR) for staining with MAbs.
[0401] 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 hybridoma supernatant or 1
ug/million cells of 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 twice and then 25 uL of goat anti-mouse Ig (H+L) biotin
conjugated antibody (Caltag Laboratories, Burlingame, Calif.) was
added to the wells for 15 minutes and washed as above. 25 ul of
phycoerythrin Streptavidin (PESA) was 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. Stained cells were analyzed on an Elite
fluorescence activated cell sorter (Beckman-Coulter, Miami,
Fla.).
[0402] Results demonstrating cell surface binding of many of the
D-series and K-series MAbs by FACS analysis, are listed in Tables
4A, 4B, and 4C below. Results of representative experiments
demonstrating cell surface expression by FACS analysis are depicted
in FIG. 1 (A and B) and FIG. 2 (A and B).
[0403] Specifically, FIG. 1A demonstrates cell surface binding of
the Pro104.D116.1 antibody to transiently transfected 293F cells
compared to a control antibody (Ovr110.A57.1). FIG. 1B indicates
the binding observed in FIG. 1A is specific to Pro104. In addition,
FIG. 2A demonstrates cell surface binding of the Pro104.D118.1
antibody to transiently transfected 293F cells compared to a
control antibody (Ovr110.A57.1). FIG. 2B indicates the binding
observed in FIG. 2A is specific to Pro104.
[0404] Binding of the MAb Pro104.D116.1 resulted in 85% of Pro104
transfected human 293F cells being positive, with a MFI (mean
fluorescence intensity) 9-fold higher than cells stained with a
control antibody (Ovr110.A57.1) alone. Binding of Pro104.D118.1
resulted in a bimodal distribution with 70% of the cells being
positive for Pro104 and a mean fluorescence intensity 22-fold
higher than the control antibody.
[0405] The other D-series antibodies that bound significantly to
Pro104-293F transfected cells and not to untransfected 293F cells
were Pro104.D9.1, Pro104.D112.1, Pro104.D113.1, Pro104.D114.1,
Pro104.D115.1, Pro104.D119.1, Pro104.D120.1, Pro104.D121.1,
Pro104.D122.1, Pro104.D123.1, Pro104.D124.1, Pro104.D125.1,
Pro104.D126.1, Pro104.D127.1, Pro104.D129.1, Pro104.D130.1,
Pro104.D131.1, Pro104.D132.1, Pro104.D133.1, Pro104.D134.1,
Pro104.D135.1, Pro104.D136.1, Pro104.D137.1, Pro104.D138.1, and
Pro104.D139.1 (see table 4A below).
TABLE-US-00011 TABLE 4A Cell Surface Binding of Pro104 D-Series
MAbs to Pro104 Transfected 293F Cells Pro104-Transfected
Untransfected 293F 293F Cells Cells % Cells % Cells Sample Positive
MFI Positive MFI No Stain 1.6 0.429 1.5 0.465 GAMBio SAPE 4.5 0.421
1.7 0.538 SAPE alone 3.8 0.411 1.4 0.511 Ovr110.A57.1 3.1 0.372 1.6
0.504 (negative control) 5E9C11 80.7 25 99.2 18.1 (positive
control) Pro104.D9.1 38 2.63 9.2 0.802 Pro104.D111.1 5.2 0.593 1.2
0.479 Pro104.D112.1 28.7 2.38 1.4 0.515 Pro104.D113.1 68.7 6.2 1.8
0.571 Pro104.D114.1 51.5 4.18 1.7 0.509 Pro104.D115.1 28.3 2.25 3.9
0.672 Pro104.D116.1 84.8 3.88 1.9 0.594 Pro104.D117.1 1.5 2.04 1.2
0.524 Pro104.D118.1 69.9 9.01 1.6 0.524 Pro104.D119.1 83.4 3.96 1.2
0.503 Pro104.D120.1 36.4 2.68 1.8 0.531 Pro104.D121.1 76.3 7.58 1.9
0.545 Pro104.D122.1 21.3 2.41 1.3 0.513 Pro104.D123.1 63 3.12 1.3
0.475 Pro104.D124.1 74.5 3.41 2 0.595 Pro104.D125.1 65.1 6.62 3
0.609 Pro104.D126.1 62.5 3.16 1.5 0.483 Pro104.D127.1 66.3 3.39 1.4
0.514 Pro104.D128.1 1.9 2.06 1.3 0.504 Pro104.D129.1 72.5 3.33 2.9
0.59 Pro104.D130.1 56.7 6.26 3.7 0.65 Pro104.D131.1 31.2 2.58 9.7
0.822 Pro104.D132.1 61.2 3.1 2.4 0.614 Pro104.D133.1 55 2.99 2.1
0.511 Pro104.D134.1 14.2 2.19 2.3 0.561 Pro104.D135.1 53.3 5.87 2.7
0.538 Pro104.D136.1 40.2 2.52 2.3 0.52 Pro104.D137.1 56.3 5.86 2.9
0.646 Pro104.D138.1 68.3 6.23 3.1 0.624 Pro104.D139.1 59.4 5.98 2.7
0.633
[0406] Pro104.K81 (from the K series) antibody also bound to 293F
transiently transfected with Pro104. Approximately 54% of the cells
were positive with a mean fluorescence intensity of 1.69 which was
3-fold over the negative control antibody.
[0407] The other K-series antibodies that bound significantly to
Pro104-293F transfected cells and not to untransfected 293F cells
were Pro104.K72, Pro104.K78, Pro104.K81, Pro104.K88, Pro104.K156,
Pro104.K159, Pro104.K164 and Pro104.K176 (see table 4B below).
TABLE-US-00012 TABLE 4B Cell Surface Binding of Pro104 K-Series
MAbs to Pro104 Transfected 293F Cells Pro104-Transfected
Untransfected 293F 293F Cells Cells % Cells % Cells Sample Positive
MFI Positive MFI No Stain 1.1 0.41 0.8 0.456 GAMBio SAPE 1.8 0.428
1.5 0.482 SAPE 1.4 0.397 0.8 0.459 Pro104.D9.1 70.3 3 1.7 0.482
Cln242.B53.1 3.6 0.544 1.4 0.535 (negative control) Pro104.K15 18.1
0.919 3.2 0.557 Pro104.K47 16.2 0.901 11.7 0.824 Pro104.K71 18.1
0.963 1.8 0.479 Pro104.K72 19.7 1.03 1.5 0.475 Pro104.K75 99.2 15.5
13.4 0.816 Pro104.K78 99 17.2 2.7 0.495 Pro104.K81 54.2 1.69 3.1
0.56 Pro104.K88 55.5 1.95 1.9 0.51 Pro104.K156 98.7 18.7 7.7 0.907
Pro104.K159 39.1 1.34 1.9 0.488 Pro104.K164 81.8 3.75 4.2 0.581
Pro104.K176 81.1 3.23 2.4 0.546
[0408] Pro104 D-series and K-series antibodies were also tested on
cell lines that were QPCR positive for Pro104 transcript (HeLa) and
QPCR negative for Pro104 transcript (HCT116). Pro104.K81 bound to
97% of HeLa and to 9% of HCT116 cells, with a 8-fold higher shift
in mean fluorescence intensity. (See table 4C below).
TABLE-US-00013 TABLE 4C Cell Surface Binding of Pro104 D-Series and
K-Series MAbs to Pro104 QPCR Positive Cell Line HeLa Hela Cells
HCT116 Cells % Cells % Cells Sample Positive MFI Positive MFI No
Stain 1.3 0.339 1.6 0.332 GAMBio SAPE 1.4 0.38 1.25 0.48 SAPE 1.3
0.333 1.4 0.423 Ovr110.A57.1 1.3 0.375 1.3 0.522 (negative control)
anti-CD71 99.8 49.6 99.9 38.9 (positive control) Pro104.D4.1 2
0.425 0.7 0.447 Pro104.6.1 4.4 0.692 1.5 0.448 Pro104.D9.1 3.3
0.694 0.5 0.408 Pro104.D14.1 2.2 0.428 1 0.404 Pro104.D18.1 2.3
0.486 1 0.421 Pro104.D19.1 2.1 0.506 1.1 0.421 Pro104.D31.1 6.9
0.634 3.6 0.527 Pro104.D58.1 2 0.441 0.5 0.421 Pro104.D116.1 5.5
0.738 0.6 0.428 Pro104.D118.1 2 0.478 1.1 0.449 Pro104.D119.1 6.3
0.801 0.8 0.425 Pro104.D121.1 4.1 0.656 1 0.432 Pro104.D123.1 3.8
0.696 0.6 0.416 Pro104.D124.1 5.3 0.746 0.7 0.418 Pro104.D126.1 4.6
0.711 0.8 0.428 Pro104.D132.1 4.6 0.689 1.2 0.431 Pro104.D133.1 5.2
0.706 0.8 0.408 Pro104.K14 2.3 0.472 1 0.538 Pro104.K15 92.6 5.84
80.2 3.29 Pro104.K16 2.8 0.455 1.9 0.505 Pro104.K47 51.8 1.6 69.5
2.09 Pro104.K71 10.9 0.744 8.9 0.776 Pro104.K72 10.4 0.769 8.3
0.719 Pro104.K74 1.6 0.442 0.2 0.465 Pro104.K75 98.8 14.5 93 7.62
Pro104.K76 2.2 0.42 1.3 0.485 Pro104.K78 2.7 0.409 1 0.444
Pro104.K81 96.9 6.51 8.9 0.772 Pro104.K87 3.3 0.531 3.9 0.641
Pro104.K88 99.3 10.2 96.7 10.1 Pro104.K89 99.6 19.1 43.7 1.34
Pro104.K155 1.5 0.413 0.4 0.468 Pro104.K156 2.4 0.491 1.1 0.505
Pro104.K157 97.4 10.3 92.9 6.47 Pro104.K158 2.3 0.454 2.4 0.519
Pro104.K159 15.8 0.87 22.3 0.908 Pro104.K160 36.7 1.3 29 1.08
Pro104.K163 4.1 0.49 13.9 0.751 Pro104.K176 31.7 1.18 47 1.29
Pro104.K217 11.9 0.767 25.4 0.949 Pro104.K226 9.6 0.736 11.4 0.75
Pro104.K227 42.4 1.42 65.5 2.19 Pro104.K240 97.5 8 95.8 15.2
Pro104.K274 23 0.923 15.1 0.622 Pro104.K264 3.6 0.497 1.5 0.53
Pro104.K281 55.2 2.08 70.7 3 Pro104.K358 3.8 0.539 4.1 0.625
Pro104.K362 3.9 0.534 3.2 0.61
[0409] These results indicate that the antibodies in Tables 4A, 4B
and 4C and in particular, Pro104.K81 Mab are suitable for
immunotherapy of tumors with or without conjugated drugs, toxins,
enzymes, prodrug activating molecules or isotopes.
Pro104 MAb Affinity Analyses
[0410] 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 Pro104 MAbs.
[0411] Pro104 protein lot#060402 (diaDexus) was immobilized on flow
cell 2 of a CM5 sensor chip (Biacore) by standard amine coupling
(Biacore). Flow cell 1 was used as a blank surface for reference
subtractions, and was activated and then inactivated with
ethanolamine. Pro104 MAbs were diluted in HBS EP buffer (Biacore)
and passed over flow cells 1 and 2 in series. MAbs were injected in
duplicate, sequentially, for each of five concentrations: 200, 100,
50, 25, 12.5 ug/mL. Assuming a molecular weight for the MAbs of
158,000 kDa, the respective concentrations in nM were calculated to
be: 1266, 633, 317, 158 and 79. MAbs were injected for 3 minutes at
30 uL/min followed by a dissociation time of 12 minutes. The
regeneration of the chip surface, or removal of MAb between cycles,
was performed by passing two injections of 100 mM Glycine pH 1.5
through the flow cells firstly for 30 seconds and then for 12
seconds, both at 100 uL/minute.
[0412] The above procedure was performed by using the Biacore's
kinetic analysis wizard included in the Biacore control software.
The resulting sensograms were fitted automatically, assuming a 1:1
Langmuir binding model. The results presented in Table 5 below were
calculated using the wizard data processing function. The
calculated affinities presented in Table 4 were all in the
10.sup.-9M range except for Pro104.C34 and hence were sufficiently
high to achieve a therapeutic dose in-vivo at less than or equal to
10 mg/kg, excepting for Pro104.C34.
TABLE-US-00014 TABLE 5 Pro104 C Series MAb Affinities Pro104
Biacore Affinity Characterization Anti-Pro104 Antibody Clone KA KD
ka Kd C4 3.5E+08 2.9E-09 2.6E+04 7.4E-05 C18 3.4E+08 2.9E-09
2.6E+04 7.5E-05 C25 3.5E+08 2.8E-09 3.0E+04 8.5E-05 C37 5.5E+08
1.8E-09 5.4E+45 9.7E-05 C48 3.6E+08 2.8E-09 4.0E+04 1.1E-04 C60
1.6E+08 6.4E-09 3.0E+04 1.9E-04 C19 1.9E+08 5.2E-09 3.8E+04 2.0E-04
C34 3.2E+08 3.0E-05 2.0E+01 6.2E-04 C54 5.5E+08 1.8E-09 3.7E+04
6.7E-05 C55 1.0E+08 9.8E-09 1.6E+04 1.5E-04
Western Blots
[0413] 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 Pro104-293T transient 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 a
Novex-XCell II Minicell gel apparatus (Invitrogen Life
Technologies, Carlsbad, Calif.) and subsequently transferred to
PVDF membranes using an XCell II Blot Module (Invitrogen Life
Technologies, Carlsbad, Calif.). Following the transfer of
proteins, the membranes were blocked in 1% blocking reagent (Cat.
#1 096 176, Roche Diagnostic Corp., Indianapolis, Ind.) and
incubated overnight at 4.degree. C. with purified primary
antibodies Pro104.C4, Pro104.C13, Pro104.C18, Pro104.C19,
Pro104.C25, Pro104.C34, Pro104.C37, Pro104.C48, Pro104.C55,
Pro104.C60 or Pro104.C66, and then with horseradish-peroxidase
conjugated goat anti-mouse IgG (Cat. #115-036-062, Jackson
Immunoresearch Laboratories, Inc.). Bands were visualized by
chemiluminescence using an ECL advance western blotting detection
kit (Cat. #RPN2135, Amersham Biosciences, Piscataway, N.J.).
[0414] Deglycosylation experiments were performed on protein
extracts from Pro104-293T transfectants, mammalian adenocarcinoma
cell lines and normal human testis by treating with peptide
N-glycosidase F (PNGaseF, Cat. #P0704S, New England Biolabs, Inc,
Beverly, Mass.) as directed by the manufacturer. The deglycosylated
samples were then analyzed by western blotting as described above.
Briefly, 100 ug volumes of protein extracts were denatured in
glycoprotein denaturing buffer (0.5% SDS/1% beta-mercaptoethanol),
at 100.degree. C. for 10 minutes. This was followed by the addition
of kit reaction buffers (New England Biolabs) to a final
concentration of 1% NP-40 and 50 mM sodium phosphate, addition of
100 units of PNGase F and incubation at 37.degree. C. for 4
hours.
TABLE-US-00015 TABLE 6 RESULTS FROM WESTERN BLOTS USING PRO104 MABS
WITH EXTRACTS FROM TRANSFECTED 293T CELLS & HUMAN
ADENOCARCINOMA CELL LINES HeLa & Pro104 Deglycosylated CaOv3
Deglycosylated SkOv3 MAb Pro104-293T Pro104-293T (RT-PCR+) HeLa
& CaOv3 (RT-PCR-) C4 +Multiple bands 35-40 kDa C13 +Multiple
bands 35-40 kDa C18 +Multiple bands 35-40 kDa C19 +Multiple bands
35-40 kDa C25 +Multiple approx. approx. approx. -- bands 35-40 30
kDa 38 kDa 30 kDa kDa C34 +Multiple bands 35-40 kDa C37 +Multiple
approx. -- bands 35-40 38 kDa kDa C48 +Multiple bands 35-40 kDa C55
+Multiple approx. -- bands 35-40 38 kDa kDa C60 +Multiple bands
35-40 kDa C66 --
[0415] Results of the western blot experiments are summarized in
Table 6 above. In whole cell lysates from Pro104-293T
transfectants, the MAbs Pro104.C4, Pro104.C13, Pro104.C18,
Pro104.C19, Pro104.C25, Pro104.C34, Pro104.C37, Pro104.C48,
Pro104.C55 and Pro104.C60 reacted specifically with several protein
bands from approximately 35 kDa to 40 kDa, which were consistent
with glycosylated forms of Pro104 formed after processing of full
length Pro104/testisin. These bands were absent in the
non-transfected 293T cell line sample. Pro104 MAb-C66 was not
reactive by western blot analysis and was therefore eliminated from
further studies. A protein eband at approximately 38 kDa was
detected by MAbs Pro104.C25, Pro104.C55 and Pro104.C37 in lysates
from Pro104 mRNA positive (RT-PCR+) cancer cell lines HeLa and
CaOv3 (ATCC), but was absent as expected, in lysates from the
RT-PCR negative ovarian cancer cell line SkOv3 (ATCC). Similarly,
MAbs Pro104.C25 and Pro104.C55 detected a band of the predicted
molecular weight (38 kDa), in lysate from normal human testis (data
not shown). MAbs Pro104.C25 and Pro104.C55 also reacted with
recombinant and native mouse testisin (data not shown), in western
blots.
[0416] In western blots on deglycosylated lysates from Pro104
transfectants, RT-PCR positive cell lines and normal human testis,
the migration of the Pro104 protein as detected by Pro104.C25,
shifted from approximately 38-40 kDa (glycosylated) to
approximately 30 kDa (non-glycosylated). This reduction in
molecular weight of Pro104 is consistent with the prediction of
three N-glycosylation sites on the catalytic subunit of Pro104
protein.
Example 2
Cell Surface Binding of Pro104 MAbs in Live Cancer Cells
Demonstrated by Immunofluorescence
[0417] The following cancer cell lines were used in this study and
were obtained from the ATCC: Cervical (HeLa), Ovarian (Tov-112D,
Tov-21G, CaOV-3 and SKOV-3), colon (HCT116) as well as the
pancreatic (MIA Paca-2 and AsPC). HeLa, CaOV-3 cell lines express
Pro104 RNA as determined by QPCR. Control HCT116 and SKOV-3 cells
do not express Pro104 RNA.
[0418] The above cell lines were seeded onto sterile 18 mm glass
coverslips and cultured at 37.degree. C. in DMEM/10% FBS with
penicillin and streptomycin for 48 hr prior to treatment with the
primary antibodies (Pro104 MAbs). MAbs Pro104.C19.1, Pro104.C25.1,
Pro104.C55.1 and Pro104.D9 were tested by immunofluorescence
microscopy to determine which of these antibodies bound
specifically to the cell surface of Pro104 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 5 ug/ml for 30 min. Following washing, the
cells were mounted in Vectastain (Vector, Burlingame, Calif.), a
medium containing DAPI to visualize the cell nuclei and observed in
a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Thornwood,
N.Y.) equipped with the appropriate fluorescent filters.
Micrographs were recorded using a CCD camera.
[0419] Pro104.C19.1, Pro104.C25.1, Pro104.C55.1 and Pro104.D9 all
bound to Pro104 expressing cells. FIGS. 3A and 3B demonstrate the
binding of Pro104.C19.1 to HeLa cells (FIG. 3A). Most of the cells
in the field showed labeling for Pro104. Pro104.C19.1 could clearly
be seen decorating the cell membrane of the cells (arrows).
However, Pro104.C19.1 did not bind to the control negative (QPCR)
SKOV-3 cancer cells (FIG. 3B, N indicates the position of the cell
nuclei).
Binding and Internalization in Live Cancer Cells by Cy3 Conjugated
Antibodies
[0420] This study was performed using directly conjugated
fluorescent antibodies (MAbs). Using antibodies directly conjugated
with the fluorescent dye Cy3, antibody binding and internalization
can be visualized by fluorescence microscopy. This technology is
well known in the art. SKOV-3 cells that do not express Pro104
(QPCR negative) were used as negative controls.
[0421] Cy3 Conjugation
[0422] Pro104.C19.1, Pro104.C25.1 and Pro104.A55.1 MAbs were each
conjugated to Cy3. Cy3 conjugation was carried out according to
standard procedures in the manufacturer's guidelines (Pierce).
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, then transferred to a Slide-A Lyzer Dialysis cassette
(Pierce) and dialyzed in 2 liters of PBS for 6 hr at 4.degree. C.
The dialysis buffer was replaced and dialysis was repeated 6 times.
The Cy3 conjugated antibodies were recovered and concentration was
measured in a spectrometer at 280 nm.
[0423] Cell Labeling
[0424] Cy3-Pro104.C19.1, Cy3-Pro104.C25.1 and Cy3-Pro104.A55.1 MAbs
were incubated with the cells at a concentration of 10 ug/ml at
37.degree. C. in water chambers for 60 min and then the coverslips
with cells were washed in PBS and the cells were fixed 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 the cells observed using
a Zeiss fluorescence Microscope Axiophot equipped with the
appropriate fluorescent filters. Micrographs were obtained with a
CCD camera.
[0425] Results
[0426] Immunofluorescence microscopy of cancer cells treated with
Cy3-Pro104.C19.1, Cy3-Pro104.C25.1 and Cy3-Pro104.A55.1 indicated
that ovarian and pancreatic cancer cells expressing Pro104 were
able to bind and internalize the fluorescent antibodies. FIG. 4A
shows the binding of Cy3-Pro104.C25.1 to the cell surface of HeLa
cells (arrows), a cell line that expresses Pro104. The
Cy3-Pro104.C25.1 antibody did not bind to the control cells SKOV-3
which do not express Pro104. See FIG. 4B, N indicates the nuclei of
several unlabeled cells. FIG. 5 demonstrates that, following the
binding to the cell membrane, Cy3-Pro104.C25.1 was internalized in
live HeLa cells and that internalization vesicles could be observed
in the cytoplasm of these cells. In particular, vesicles could be
often visualized in close proximity to the cell nuclei (N) (arrow).
FIG. 6A and FIG. 7A show the binding and internalization of
Cy3-Pro104.19.1 and Cy3-Pro104.C55.1 in MIA-PaCa-2 cells,
respectively. MIA-PaCa-2 cells are a pancreatic cell line that
expresses Pro104. The internalization pattern was characterized by
the presence of perinuclear vesicles likely to correspond to
endosomes located in the proximity of the Golgi apparatus (arrows).
Cy3-Pro104.C19.1 and Cy3-Pro104.C55.1 did not bind to the cells of
the control cell line HCT-116 which does not express Pro104 (FIGS.
6B and 7B).
[0427] Cy3 conjugated MAbs Pro104.C19.1, Pro104.C25.1 and
Pro104.A55.1 were all internalized upon binding to the cell surface
of Pro104 expressing cancer cells, in-vitro. These results indicate
anti-Pro104 antibodies, and in particular, Pro104.C19.1,
Pro104.C25.1 and Pro104.A55.1 MAbs are suitable for immunotherapy
of tumors with or without conjugated drugs, toxins, enzymes,
prodrug activating molecules or isotopes.
Distribution of Pro104 in Tumors and Normal Tissues by
Immunohistochemistry (IHC)
[0428] Formalin fixed paraffin embedded blocks of ovarian and
pancreatic cancer and normal adjacent tissues were obtained from
the National Disease Research Interchange (Philadelphia, Pa.). OCT
embedded blocks of normal organs were obtained from Zoion
(Hawthorne, N.Y.).
[0429] Immunohistochemical Staining for Formalin Fixed Paraffin
Embedded Sections
[0430] Six .mu.m thick sections cut from formalin fixed paraffin
embedded blocks were heated at 45.degree. C. for 15 min,
deparaffinized in Histoclear (National Diagnostics, Atlanta, Ga.),
deparaffinized in Histoclear and rehydrated through a series of
reducing ethanol concentrations to PBS. Antigen retrieval was
performed by boiling the section slides in 10 mM sodium citrate
buffer (pH 6.0), at 120.degree. C., 15-17 PSI in a decloaking
chamber (Biocare, Walnut Creek, Calif.) for 10 min. Endogenous
peroxidase activity was quenched by treating the sections with 3%
hydrogen peroxide solution for 15 min. Slides were incubated with
1% BSA to block nonspecific antibody binding and then reacted with
the primary Pro104 MAbs used at a concentration of 10 ug/ml for 1
hour at room temperature in a DAKO autostainer (Dako Co.,
Carpinteria, Calif.). After washing in Tris-Buffered Saline (TBS)
with 0.5% Tween-20, slides were then incubated with anti-mouse IgG
conjugated to horse radish peroxidase (HRP) (Immunovision
technologies, Daly City, Calif.). After washing in TBS with 0.5%
Tween-20, sections were treated by 3,3'-diaminobenzidine chromagen
for 2-5 minutes (Immunovision Technologies) and counterstained with
hematoxylin before mounting in Permount medium (American Master
Tech Scientific, Inc, Lodi, Calif.) after dehydration. Normal mouse
IgG at the same concentration as the primary antibody, served as
negative a control for immunolabeling specificity. In additional
control experiments, the Pro104 MAbs were incubated with the
antigen Pro104 before being applied to the histological
sections.
[0431] Immunohistochemical Staining for OCT Embedded Frozen Unfixed
Sections
[0432] Slides were cut in the cryochamber at 5-8 um at an
appropriate temperature, air dried for a minimum of thirty minutes
at room temperature. Briefly, slides were rinsed in TBS to remove
off OCT and incubated at room temperature. IHC was performed using
the Immunovision Powervision Kit (Immunovision Technologies, Co.
Daly City, Calif.). Briefly, slides were rinsed in TBS-T to remove
off OCT and incubated with Pro104 primary antibodies for 1 hour at
room temperature. They were then post-fixed in 4% paraformaldehyde
fixative for 10 min at room temperature and treated as described
above.
[0433] Results
[0434] Pro104.C25.1, Pro104.A55.1, Pro104.D9 and Pro104.D133 were
used to immunolabel sections of ovarian and pancreatic cancer.
Epithelial cells in the ovarian and pancreatic tumors but not in
normal ovary and pancreas were labeled. Pro104.C25.1 labeled the
cell surface of 10 out of 17 (58%) serous ovarian cancer and 11/11
(100%) pancreatic cancer clinical samples. Pro104.C55.1 labeled the
cell surface of 6 out of 8 (75%) ovarian cancer and 3/3 (100%)
pancreatic cancer clinical samples. Pro104.D9 labeled the cell
surface in 1/4 ovarian cancer (25%). FIGS. 8A, 8B, 8C and 8D
illustrate the IHC results obtained with Pro104.C25.1 in two
ovarian cancer clinical samples (FIGS. 8A and 8C). Control normal
ovaries were not labeled by the Pro104.C25.1 MAb (FIGS. 8B and 8D).
FIG. 9 shows a higher magnification of an ovarian cancer
histological section labeled with Pro104.C25.1. The labeling
clearly localized to the cell membrane of the tumor epithelial
cells (arrows).
[0435] FIG. 10 demonstrates that Pro104.D9 labeled the cell surface
of ovarian cancer cells (arrow). Additionally, FIG. 11 shows that
Pro104.D133 labeled the cell surface of serous ovarian cancer
cells.
[0436] FIGS. 12A and 12C illustrate the immunolabeling pattern
obtained with Pro104.C25.1 in clinical samples of pancreatic
adenocarcinoma. Pro104 labeling was mostly restricted to the cell
surface of epithelial cells (arrows) with occasional cytoplasmic
labeling (FIG. 12C). Normal pancreatic cells were mostly devoid of
specific labeling (FIGS. 12B and 12D). Additionally, FIG. 13 shows
that no specific labeling was observed when normal mouse IgG was
used instead of Pro104.C25 (FIG. 13A) or when Pro104.C25.1 was
adsorbed with Pro104 antigen prior to processing for IHC (FIG.
13B).
[0437] Pro104 expression was also analyzed in normal organs. IHC on
OCT frozen sections showed no detectable labeling on the cell
surface in the cells of normal heart, liver, kidney, brain, colon,
stomach, lung, prostate, ovary, pancreas and breast. However, the
membrane of the germ cells in the testis showed strong Pro104
immuno labeling. This result was expected from data published in
the scientific literature (J. D. Hooper et al. Testisin, a new
human serine protease expressed by premeiotic testicular germ cells
and lost in testicular germ cell tumors. Cancer Research
59:3199-3205 (1999)). See table 7 below for summary.
TABLE-US-00016 TABLE 7 Summary of IHC results for Pro104 D-Series
MAbs Pro104 D MAb Immunohistochemistry Results Unfixed OCT Formalin
Fixed (FFPE) Testis Normal Ovarian Cancers Ovarian cancers mAb
Dilution Germ Smooth vital (no. positive/ (Ratio positive/ Clone
(ug/ml) cell Stroma muscle Other organs* no. tested) Testis tested)
IgG1 10 ug/ml - - - - Anti- 3+ (5/5) 3+ (2/2) Keratin D9 5 ug/ml 3+
- - - - 3+ (2/3) 3+ D116 10 ug/ml 3+ - - - - 1+ (3/5) D119 10 ug/ml
3+ - - - - 1+ (3/5) D121 10 ug/ml 3+ - - - 3+ 1+ (4/5) 2+ (3/3)
(all 3) D124 10 ug/ml 3+ - - - - 1+ (3/5) D123 10 ug/ml 3+ - - - -
2+ (2/5) 3+ 2+ (2/2) D126 10 ug/ml 3+ - - - +/- 2+ (1/5) 3+ 1+
(2/2) D132 10 ug/ml 3+ - - - - 2+ (1/5) 3+ 2+ (5/5) D133 10 ug/ml
3+ - - - - 2+ (1/5) 3+ 2+ (2/2) *Normal vital organs include heart,
liver and kidney.
[0438] The immunohistochemistry results above demonstrate Pro104 is
expressed in a high percentage of ovarian and pancreatic cancer
cases. The fact that Pro104 is expressed on the cell surface of
cancer cells makes it an ideal target for antibody based therapy.
Additionally, binding of anti-Pro104 antibodies to ovarian and
pancreatic cancer cells demonstrated by IHC indicates anti-Pro104
antibodies, and in particular, Pro104.C25.1, Pro104.D9 and
Pro104.D133 MAbs are suitable for immunotherapy of tumors with or
without conjugated drugs, toxins, enzymes, prodrug activating
molecules or isotopes.
Example 3
Mouse Monoclonal Sandwich ELISA Detection of Pro104
Pro104 Competitive Checkerboard ELISA
[0439] High binding polystyrene plates (Corning Life Sciences (MA))
were coated overnight at 4.degree. C. with 1 .mu.g/well of
anti-Pro104 MAb. The coating solution was aspirated off and free
binding sites were blocked by adding 300 .mu.l/well of
Superblock-TBS (Pierce Biotechnology, Illinois) for 1 hour at RT.
After washing twice with Wash Buffer (1.times.TBS/0.05% Tween20),
100 .mu.l volumes of Pro104 antigen were added to each well. Each
pair was tested with 100 ng/ml and 0 ng/ml of recombinant Pro104 E.
coli expressed protein diluted in Assay Buffer (1.times.TBS, 1%
BSA/1% Mouse Serum/1% Calf Serum/0.1% Tween20). After addition,
plates were incubated for 1 hour at RT with shaking, and washed
4.times. with 360 .mu.l of Wash Buffer. Then 50 .mu.l volumes of
unlabeled coating MAb, at 20 .mu.g/ml in Assay Buffer, were added
and incubated with shaking at RT for 10 min. Afterwards, 50 .mu.l
volumes of biotinylated detecting MAb (2 .mu.g/ml) were added to
each well and plates were incubated for 1 hour at RT, with shaking.
After washing, 100 volumes of alkaline phosphatase conjugated
streptavidin (Jackson ImmunoResearch Laboratories, PA, 1:2000
dilution) were added to wells and plates were incubated for 30 min.
at RT, with shaking. After washing, the plate was then developed
using pNPP substrate in 1.times.DEA buffer (Pierce Biotechnology,
Illinois) for 30 min. at RT. The reaction was stopped by adding 100
.mu.l/well 1N NaOH, and plates were read at 405 nm using a
Spectramax 190 plate reader Molecular Devices, CA). OD readings at
405 nm were used to calculate signal to noise ratio (OD at 100
ng/mL divided by OD at 0 ng/mL) of each Ab pairs.
[0440] The results of the checkerboard ELISA testing 13 MAb are
shown in Table 8 below. Each antibody was used as a coating as well
as a detecting antibody in all possible combinations. All pairs
were tested in duplicate on 100 and 0 ng of Pro104 E. coli protein
in assay buffer (containing mouse serum, calf serum and BSA to be
used as blank). The results are shown as specific signal/noise
(assay buffer alone) ratio. During the incubation with detecting
antibody, a 10-fold higher concentration of coating antibody was
added to the wells to prevent self-pairing. Self-pairing may be
observed when antigens are partly multimerized and may confound MAb
pairing results. Performing the ELISA assay under competitive
conditions ensures that antibodies cannot bind to the same or
proximal epitopes when the antigen is aggregated.
[0441] The data suggest a minimum of five epitopes have been
identified since steric hindrance may also be a contributing factor
to the non-pairing of MAbs. The epitope map of the Pro104 MAbs
derived from the results in Table 8 is shown in FIG. 14. More than
50% of the monoclonal antibodies (Pro104.C4, Pro104.C18,
Pro104.C25, Pro104.C37, Pro104.C48 and Pro104.C60) reacted with one
epitope or epitopes proximal enough to cause steric hindrance and
so block MAb pairing in the assay. The MAbs Pro104.C34 and
Pro104.C13 both reacted with epitopes that were distinct from one
another and distinct from the other epitopes or MAb groups. The
MAbs Pro104.C55 and Pro104.C19 reacted with an epitope or two
proximal epitopes which were sufficiently close to the epitope
identified by Pro104.C66 to cause partial blocking. The MAbs
Pro104.C55 and Pro104.C19 also reacted with an epitope or epitopes
which were sufficiently close to the epitope or epitopes identified
by the MAb group Pro104.C4, Pro104.C18, Pro104.C25, Pro104.C37,
Pro104.C48 and Pro104.C60 to cause partial blocking. However, MAb
Pro104.C66 reacted with an epitope which was sufficiently distant
from the epitope or epitopes identified by Pro104.C4, Pro104.C18,
Pro104.C25, Pro104.C37, Pro104.C48 and Pro104.C60 to allow pairing
with MAbs of this group.
[0442] Several different MAb combinations were tested to establish
a sandwich ELISA assay for the detection of native Pro104 from
medium or lysates of cancer cell lines, transfected cell lines and
cancer tissues. The pairs Pro104.C19/C48 and Pro104.C55/C34
performed best in the Sandwich ELISA with a sensitivity for
recombinant Pro104 at approximately 1 ng/ml. Pro104 was detected by
sandwich ELISA in CHAPS (Pierce) detergent lysates from RT-PCR
positive CaOV3 ovarian cancer cells, RT-PCR positive HeLa cervical
cancer cells and in lysates from Pro104 transfected 293T and LMTK
cells, 48 hours post transfection. Pro104 was not detected in
lysates from the prostate cancer cell line PC3 (ATCC) nor the colon
cancer line HT29 (ATCC), which are Pro104 negative by RT-PCR. These
results were also in agreement with immunofluorescence data.
However, Pro104 protein could not be detected in the tissue culture
medium from any of these cancer cell lines, or in the medium from
Pro104 transfected cells, at 48 hours post transfection.
TABLE-US-00017 TABLE 8 Identification of Pro104 ELISA MAb Pairs by
Competition ELISA Detecting Antibody C4 C13 C18 C25 C37 C48 C60 C66
C19 C34 C55 Coating C4 4.3* 50 4 5.3 4.7 5.1 2.1 12 Antibody C13 33
5.3 40 32 26.7 32 28 8.3 17 9 16 C18 3.2 43 2.7 3.1 3.6 3.9 1.54 10
C25 4 47 4.3 5 5.6 5.1 1.67 1.3 C37 4.2 46 4 4.3 4.4 4.9 2.3 13 C48
3 50 3.7 3.9 3.7 3.8 1.9 14.7 1.3 19.5 1 C60 22 46 21 19 18 25 4.38
7.8 C66 31 30 34 23 19 22 13 2 12 11 10 B8 22.8 22.4 21/40 16 13 17
7.8 2.6 26 15.6 27 B11 17 14 9.7 7.9 11 5.3 1.4 C19 34 33 34 14 1.6
31 11 C34 8.7 7.7 5.6 1.9 4.3 1.4 4.3 C55 38 7.8 13.4 13.6 3 27 2.3
*Signal to noise (OD at 100 ng/mL divided by OD at 0 ng/mL (assay
buffer alone)) ratio.
Example 4
Detection of Pro104 Protein and Phosphorylation of EGF Receptor
Detection of Pro104 Protein in Cell Lines and Ovarian Tumors by
Western Blot
[0443] Rk3E, HeLa, AsPC1 and HT29 cells lines were evaluated for
expression of Pro104. The RK3E and HT29 cell lines are negative for
Pro104 mRNA. As a control RK3E was transfected with Pro104
(RK3E-104) using methods known in the art. As an additional control
RK3E cells were also transfected with Alkaline Phosphatase
(RK3E-AP). HeLa and AsPC1 are positive for Pro104 mRNA. In addition
to the cell lines, ovarian tumor and normal adjacent tissue to the
tumor was evaluated for the presence of Pro104.
[0444] Cell extracts were prepared on ice using modified RIPA
buffer (1% NP40, 10 mM Na.sub.2PO.sub.4, 0.15M NaCl) plus a
protease inhibitor cocktail (Roche Inc.). 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 Life
Technologies, Carlsbad, Calif.), heated to 70.degree. C. for 10
minutes and then analyzed using pre-cast 4-12% SDS-polyacrylamide
minigels (Nupage; Invitrogen Life Technologies, Carlsbad, Calif.)
with MES running buffer (Nupage; Invitrogen Life Technologies,
Carlsbad, Calif.). Gels were transferred to Immobilon-P PVDF
membranes with a 0.45 .mu.m pore size (Invitrogen Life
Technologies, Carlsbad, Calif.) 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 Pro104 was produced in house
using recombinant bacterial Pro104 protein. The Pro104 monoclonal
antibody was diluted 1:1000 for a final concentration of 1 ug/ml
and a mouse monoclonal antibody against GAPDH (Chemicon Inc.,
Temecula, Calif.) was diluted 1:5000 (for a final concentration of
0.2 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., Bar Harbor, Me.) 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, Piscataway, N.J.) and exposure to X-ray film
(Kodak, Rochester, N.Y.). For the Western immunoblot experiment
comparing RK3E cells infected with an AP (alkaline
phosphatase)-expressing retrovirus with the same cells infected
with a Pro104-expressing retrovirus, cells were plated in growth
medium containing either 1% or 10% FBS for 48 hours. Cell extracts
were prepared using modified RIPA buffer including a phosphatase
inhibitor cocktail (Calbiochem) and 25 ug of clarified extract were
evaluated by SDS-PAGE and Western immunoblot with a polyclonal
antibody specific for the phosphorylated EGF receptor (BioSource
International, Camarillo, Calif.).
[0445] FIG. 15A demonstrates by western blot Pro104 protein was
detected in Pro104 transfected cells lines (RK3E-104) and cell
lines natively expressing Pro104 (HeLa and AsPC1). Pro104 protein
was not detected in AP transfected cell lines (Rk3E-AP) and mRNA
negative cell lines (HT29). Additionally, FIG. 15B illustrated
detection of Pro104 protein by western blot in ovarian tumor
tissues but not in normal adjacent tissues.
[0446] The fact that Pro104 is detectable in cancer cell lines and
ovarian tumor tissue makes it an ideal target for antibody based
therapy. Anti-Pro104 antibodies are suitable for immunotherapy of
tumors with or without conjugated drugs, toxins, enzymes, prodrug
activating molecules or isotopes.
Phosphorylation of EGF Receptor
[0447] RK3E transfected cell lines overexpressing Pro104
(RK3E-Pro104) were evaluated for phosphorylation of the Epidermal
Growth Factor (EGF) Receptor. As a control RK3E cells were also
transfected with Alkaline Phosphatase (RK3E-AP). Using methods
known in the art, phosphorylation of the EGF receptor was evaluated
with 10% and 1% serum from RK3E-Pro104 and RK3E-AP cells.
[0448] Over expression of Pro104 was found lead to phosphorylation
of EGF receptor. FIG. 16 is a western immunoblot against
phosphorylated EGF Receptor which demonstrates EGF receptor is
phosphorylated from overexpression of Pro104 compared to AP
controls.
Example 5
Glycosylation, GPI-Linkage and Biotinylation of Pro104 Protein
Pro104 Glycosylation
[0449] Deglycosylation experiments were performed on protein
extracts from HeLa cell lines (Pro104 mRNA positive) and ovarian
cancer tumor samples using Peptide N-Glycosidase F (PNGaseF,
Cat#P0704S, New England Biolabs, Inc, Beverly, Mass.) as per the
directions provided by the manufacturer. The deglycosylated samples
were then analyzed by western blotting as described above. Briefly,
100 ug of protein extract was denatured in glycoprotein denaturing
buffer/0.5% SDS/1% beta-mercaptoethanol, at 100.degree. C. for 10
minutes. This was followed by the addition of kit reaction buffers
(New England Biolabs) 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.
[0450] FIG. 17A illustrates a shift in the migration of Pro104
protein from both the HeLa cell line and ovarian cancer samples
when treated with Pangs. These results demonstrate not only that
Pro104 is glycosylated, but that anti-Pro104 antibodies are capable
of detecting both glycosylated and deglycosylated forms of native
Pro104.
Pro104 GPI-Linkage Characterization by PI-PLC
[0451] HeLa cells were seeded in 6 well plates. 48 hours later, at
90% confluence, the media were replaced with 1 ml fresh growth
media, with and without 0.5 unit phosphatidylinositol-specific
phospholipase C (PI-PLC, Sigma). After one hour incubation at
37.degree. C., the media were harvested and briefly microfuged. 15
.mu.l of unconcentrated media were analyzed by SDS-PAGE. Cells were
solubilized for immunoblot analysis as described above.
[0452] Since human Pro104 was predicted to be a GPI-linked protein,
this was tested by treating live HeLa cells with
phosphatidylinositol-specific phospholipase C (PI-PLC) as described
above. PI-PLC cleaves the membrane anchor from GPI-linked proteins
and releases the protein into the medium. FIG. 17B demonstrated no
Pro104 protein was shed into the medium of untreated HeLa cells,
however, treatment with PI-PLC released Pro104 into the medium
where it could be detected by immunoblot. The PI-PLC treatment did
not release other non-GPI-linked membrane proteins indicating that
the release of Pro104 was due to specific cleavage of the
GPI-anchor by the PI-PLC. This experiment shows that Pro104 is
localized to the surface of tumor cells via a GPI-linkage.
Pro104 Biotinylation
[0453] Attached Cells
[0454] Caco2, CaOV3, or HeLa cells were removed from a 37.degree.
C. incubator, place on ice, and remained on ice for duration of the
experiment. Cells were washed 3 times with ice cold PBS (10 mM
Na-P) at pH 7.4. Biotinylation reagent (Sulfa-NHS-SS-Biotin;
Pierce, Rockford, Ill.) dissolved in ice cold PBS to final
concentration 010.5 mg/ml was added to cover the cells completely
(approximately 200 .mu.l) and incubated on ice for 30 minutes.
Biotinylation reagent was removed and cells were washed with
1.times.PBS+25 mM Tris once followed by three washes with ice cold
PBS. 500 .mu.l of Lysis Buffer (1.times.PBS+1.0% Triton) with
1.times. protease inhibitors was added to cells and incubated on
ice for 10 minutes. Resulting lysate was transferred into a
microcentrifuge tube and spun for 2 minutes at 14,000 rpm at
4.degree. C. 50 .mu.l of supernatant was saved to be run on gels as
total protein extract, while the remaining volume of supernatant
was immunoprecipitated with 20 .mu.l of Streptavidin Agarose beads
(Pierce). After immunoprecipitation, the beads were washed three
times with cell lysis buffer (1.times.PBS+1.0% Triton). 100 .mu.l
of 1.times.LDS Sample Buffer (NuPage; Invitrogen) and 1.times.
Sample Reducing Agent (NuPage; Invitrogen) were added to each
sample and incubated at 70.degree. C. for 10 minutes prior to
running on gel. A standard western blot was then performed as
described above.
[0455] Detached Cells
[0456] Caco2, CaOV3, or HeLa cells were removed from a 37.degree.
C. incubator, place on ice, and remained on ice for duration of the
experiment. Cells were detached and washed 3 times with ice cold
PBS (10 mM Na-P) at pH 7.4 and resuspended in 500 .mu.l PBS.
Biotinylation reagent (Sulfo-NHS-SS-Biotin; Pierce, Rockford, Ill.)
dissolved in ice cold PBS with a concentration of 1.0 mg/ml was
added to the cells for a final concentration of 0.5 mg/ml and
incubated on ice for 30 minutes. Cells were spun at 2000 rpm for 5
minutes. Biotinylation reagent was removed and cells were washed
with 1.times.PBS+25 mM Tris once followed by three washes with ice
cold PBS. Cells were spun at 2000 rpm for 5 minutes in between
washings to remove buffer. 500 .mu.l of Lysis Buffer
(1.times.PBS+1.0% Triton) with 1.times. protease inhibitors was
added to cells and incubated on ice for 10 minutes. Resulting
lysate was transferred into a microcentrifuge tube and spun for 2
minutes at 14,000 rpm at 4.degree. C. Supernatant was transferred
to a new microcentrifuge tube and protein concentration was
determined using the BCA assay (Pierce). 50 .mu.l of supernatant
was saved to be run on gels as total protein extract, while the
remaining volume of supernatant was immunoprecipitated with 20
.mu.l of Streptavidin Agarose beads (Pierce). After
immunoprecipitation, the beads were washed three times with cell
lysis buffer (1.times.PBS+1.0% Triton). 100 .mu.l of 1.times.LDS
Sample Buffer (NuPage; Invitrogen) and 1.times. Sample Reducing
Agent (NuPage; Invitrogen) were added to each sample and incubated
at 70.degree. C. for 10 minutes prior to running on gel. A standard
western blot was then performed as described above.
[0457] FIG. 18 demonstrates that native Pro104 is biotinylated on
the cell surface compared to NaK-ATPase (positive control) and
GAPDH (negative control).
[0458] The fact that Pro104 is located on the cell surface via
GPI-Linkage in cell lines makes it an ideal target for antibody
based therapy. Furthermore, binding of anti-Pro104 antibodies to
glycosylated and deglycosylated Pro104 on cell lines and ovarian
cancer cells indicates anti-Pro104 antibodies are suitable for
immunotherapy of tumors with or without conjugated drugs, toxins,
enzymes, prodrug activating molecules or isotopes.
Example 6
Generation of Pro104 Expressing Cell Lines Cells and Cell
Cultures
[0459] SKOV3, RK3E, 293T, HeLa, CaOV3, NCIH522 and HCT116 cell
lines were purchased from American Type Culture Collection
(Manassas, Va.). Cells were grown in DMEM (Invitrogen Life
Technologies, Carlsbad, Calif.) with L-glutamine plus 4.5 g/L
glucose and supplemented with 10% FBS and 100 U/mL
Penicillin/Streptomycin (Cellgro, Herndon, Va.). All cells were
maintained in a humidified 37.degree. C. incubator with 5%
CO.sub.2.
Expression Vector Construction
[0460] As a source for cloning of Pro104, human ovarian cancer cDNA
was prepared from poly-A+mRNA using a BD SMART PCR cDNA synthesis
kit (BD Bioscience/Clontech, Palo Alto, Calif.). For construction
of a retroviral expression vector encoding untagged Pro104
(pLXSN-Pro104), Pro104 cDNA was synthesized by PCR reaction using
ovarian cancer 5'-RACE-ready cDNA as template and the following
gene specific primers:
TABLE-US-00018 (SEQ ID NO: 8) 5'-end:
5'-ATGGGCGCGCGCGGGGCGCTGCTGCTG-3' (SEQ ID NO: 9) 3'-end:
5'-TTATCAGACCGGCCCCAGGAGTGGGAGAGCCCA-3'
The PCR fragment was cloned into the Hpa I cloning site of the
pLXSN vector (BD Bioscience/Clontech) and sequence verified. The
Genbank accession for pLXSN vector is #M28248.
[0461] For construction of a retroviral expression vector encoding
Pro104 with an in-frame COOH-terminal hemagglutinin tag
(pLXSN-Pro104HA), the same procedure was used except that the 3'
primer used was:
TABLE-US-00019 (SEQ ID NO: 10) 3'-end (HA tag is bold): 5'-
TTATCACGCGTAGTCCGGCACGTCGTACGGGTAGCCGACCGGCCCCAGGAGTGGGAGAGC
CCA-3'
[0462] The pLXSN retrovirus vector utilizes a 5'Mo-MuSV (Moloney
Murine Sarcoma Virus) LTR and 3''Mo-MuLV (Moloney Murine Leukemia
Virus) LTR to drive expression of cDNA's cloned into the multiple
cloning site and an SV40 promoter driving expression of a Neo.sup.r
gene encoding G418 resistance. pLAPSN, a retroviral expression
vector encoding alkaline phosphatase (AP), was purchased from BD
Bioscience/Clontech (referred to as pLXSN-AP).
Virus Production
[0463] Ecotropic virus was used to infect RK3E 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 Life Technologies, Carlsbad, Calif.). Per
well of cells 0.8 mg of virus plasmid DNA: pLXSN-Pro104,
pLXSN-Pro104HA or pLXSN-AP plus 0.8 .mu.g pVpack-ECO and 0.8 .mu.g
pVpackGP (Stratagene, La Jolla, Calif.) were added to a stock of
125 .mu.L DMEM without serum and 104 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. One mL of DMEM containing 20% FBS was
added to the transfection mix after the 3 h incubation and 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.
[0464] For amphotropic virus packaging the same procedure was
followed except that the pVpack Ampho plasmid (Stratagene) was used
instead of the pVpack Eco plasmid.
Virus Infection and Selection
[0465] Polybrene (Hexadimethrine Bromide; Sigma, Saint Louis,
Miss.) was added to fresh virus-containing medium at a final
concentration of 4 .mu.g/ml. RK3E or SKOV3 cells, plated the day
before at a density of 3.times.10.sup.5 cells per 100 mm.sup.2
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 Pro104 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 visualized by incubation with BCIP/NBT liquid
substrate (Sigma, Saint Louis, Miss.) for 2-3 hours.
Results of Cell Line Virus Infection and Selection
[0466] SKOV3, RK3E, 293T, HeLa, CaOV3, NCIH522 and HCT116 cell
lines underwent virus infection and selection to overexpress
Pro104.
[0467] Retroviral-mediated overexpression of Pro104 protein in RK3E
cells was confirmed by Western Immunoblot. FIG. 19 is a western
immunoblot demonstrating Pro104 protein expression in retroviral
packaging cell lines and virus infected RK3E cells.
[0468] Retroviral-mediated overexpression of Pro104 protein in
SKOV3 cells was confirmed by Western Immunoblot. FIG. 20 is a
western immunoblot demonstrating Pro104 protein expression in
retroviral packaging cell lines and virus infected SKOV3 cells.
Example 7
siRNA Generation and Transfection
[0469] siRNA Oligonucleotide Design and Preparation
[0470] To design Pro104 specific siRNA molecules, sequences were
selected from the open reading frame of the Pro104 mRNA based on
methods previously described (Elbashir et al., 2001, Nature
411:494-498A 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 Pro104 levels nor any of the
biological endpoints studied. As a positive control for knockdown
of an mRNA leading to apoptosis induction, a siRNA targeting either
DAXX or OPA1 was used, based on published data. Michaelson et al.,
2002, Journal of Cell Science, 116:345-352; Oichen et al., 2003, J
Biol Chem., 278(10):7743-6. 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. The siRNA sequences used to knockdown Emerin and DAXX
were obtained from published papers. Michaelson et al., 2002;
Harborth et al., 2001, Journal of Cell Science, 114:4557-4565.
[0471] 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-00020 (SEQ ID NO: 11) Pro104 #56: sense
5'-CACAUCCAGCCCAUCUGUC-3' (SEQ ID NO: 12) Pro104 #79: sense
5'-GAGGAUGAGGCACUGCCAU-3' (SEQ ID NO: 13) Pro104 #80: sense
5'-CUCUAUGUGCAACCACCUC-3' (SEQ ID NO: 14) Pro104 #81: sense
5'-GUACAGUUUCCGCAAGGAC-3' (SEQ ID NO: 15) Scrambled: sense
5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO: 16) Emerin: sense
5'-CCGUGCUCCUGGGGCUGGG-3' (SEQ ID NO: 17) DAXX: sense
5'-GGAGUUGGAUCUCUCAGAA-3' (SEQ ID NO: 18) OPAI sense
5'-GUUAUCAGUCUGAGCCAGG-3'
Additional siRNA oligonucleotides specific for Pro104 with two
thymidine residues (dTdT) at the 3' end of the sequence consisted
of the following specific RNA sequences:
TABLE-US-00021 (SEQ ID NO: 19) Pro104_siRNA#1: gccggagucgcaggaggcg
(SEQ ID NO: 20) Pro104_siRNA#2: cucgggcguuggccguggc (SEQ ID NO: 21)
Pro104_siRNA#3: accuauagugaccuuagug (SEQ ID NO: 22) Pro104_siRNA#4:
ccuauagugaccuuaguga (SEQ ID NO: 23) Pro104_siRNA#5:
uucacccuaugacauugcc (SEQ ID NO: 24) Pro104_siRNA#6:
gcugucugcaccugucacc (SEQ ID NO: 25) Pro104_siRNA#7:
ccggacagacugcugggug (SEQ ID NO: 26) Pro104_siRNA#8:
agaggaugaggcacugcca (SEQ ID NO: 27) Pro104_siRNA#9:
guucaggucgccaucauaa (SEQ ID NO: 28) Pro104_siRNA#10:
ggacaucuuuggagacaug (SEQ ID NO: 29) Pro104_siRNA#11:
caagaauggacugugguau (SEQ ID NO: 30) Pro104_siRNA#12:
gaauggacugugguaucag (SEQ ID NO: 31) Pro104_siRNA#13:
uggacugugguaucagauu (SEQ ID NO: 32) Pro104_siRNA#14:
ucggcccggugucuacacc (SEQ ID NO: 33) Pro104_siRNA#15:
uaucagccaccacuuugag (SEQ ID NO: 34) Pro104_siRNA#16:
gucaggcccugguucucuu (SEQ ID NO: 35) Pro104_siRNA#17:
uaaacacauuccaguugau (SEQ ID NO: 36) Pro104_siRNA#18:
uaaacacauuccaguugau (SEQ ID NO: 37) Pro104_siRNA#19:
acacauuccaguugaugcc (SEQ ID NO: 38) Pro104_siRNA#20:
cacauuccaguugaugccu
Transfection with siRNA Oligonucleotides
[0472] HeLa (4.times.10.sup.4 cells) and CaOV3 (6.times.10.sup.4
cells) cells expressing Pro104 were seeded in 12-well plates for
18-24 hours prior to transfection. Transient transfection was
carried out using Oligofectamine reagent (Invitrogen Life
Technologies, Carlsbad, Calif.) 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. Pro104, Scrambled, DAXX and Emerin 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). All findings were confirmed with at least 2
additional experiments.
Example 8
SDS-PAGE and Western Immunoblot Analysis
[0473] 72 hrs after transfection with siRNA, cell extracts were
prepared on ice using modified RIPA buffer (1% NP40, 10 mM
Na.sub.2PO.sub.4, 0.15M NaCl) plus a protease inhibitor cocktail
(Roche Inc.). 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 Life Technologies, Carlsbad, Calif.), heated to
70.degree. C. for 10 minutes and then analyzed using pre-cast 4-12%
SDS-polyacrylamide minigels (Nupage; Invitrogen Life Technologies,
Carlsbad, Calif.) with MES running buffer (Nupage; Invitrogen Life
Technologies, Carlsbad, Calif.). Gels were transferred to
Immobilon-P PVDF membranes with a 0.45 .mu.m pore size (Invitrogen
Life Technologies, Carlsbad, Calif.) 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 Pro104 was produced in house
using recombinant bacterial Pro104 protein. The Pro104 monoclonal
antibody was diluted 1:1000 for a final concentration of 1 ug/ml
and a mouse monoclonal antibody against GAPDH (Chemicon Inc.,
Temecula, Calif.) was diluted 1:5000 (for a final concentration of
0.2 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., Bar Harbor, Me.) 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, Piscataway, N.J.) and exposure to X-ray film
(Kodak, Rochester, N.Y.). For the Western immunoblot experiment
comparing RK3E cells infected with an AP (alkaline
phosphatase)-expressing retrovirus with the same cells infected
with a Pro104-expressing retrovirus, cells were plated in growth
medium containing either 1% or 10% FBS for 48 hours. Cell extracts
were prepared using modified RIPA buffer including a phosphatase
inhibitor cocktail (Calbiochem) and 25 ug of clarified extract were
evaluated by SDS-PAGE and Western immunoblot with a polyclonal
antibody specific for the phosphorylated EGF receptor (Bio Source
International, Camarillo, Calif.).
[0474] Effects of Pro104 specific siRNA on Pro104 protein was
determined by western immunoblot. FIG. 21 shows siRNA Mediates
Specific Down-Regulation of Pro104 Protein in HeLa Cells. A 60%
knockdown (.DELTA.CT=1.3) of Pro104 protein was observed. These
results were not confined to a single cell type. FIG. 22 shows
siRNA Mediates Specific Down-Regulation of Pro104 Protein in CaOV3
Cells. A 55% knockdown (.DELTA.CT=1.2) of Pro104 protein was
observed.
Example 9
Quantitative Real Time RT-PCR (QPCR)
[0475] A QuantiTech SYBR Green RT-PCR kit from Qiagen Inc. was used
for QPCR evaluation. The final reaction volume was 20 ul, including
10 ul RT-PCR Master Mix, 2 ul forward primer (5 uM), 2 ul reverse
primer (5 uM), QuantiTect RT mix 0.2 ul and RNase-free water.
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.) with the following cycle conditions:
50.degree. C. for 30 min., 95.degree. C. for 15 min, 40 cycles at
94.degree. C. for 15 s, 55.degree. C. for 30 s, 72.degree. C. for
30 s, then held at 72.degree. C. for 2 min.
[0476] The QPCR assay was used to determine the effect of Pro104
siRNA on gene transcription levels.
[0477] QPCR assays demonstrated Pro104 siRNA specifically knockdown
Pro104 mRNA in CaOV3 cells. FIG. 23A shows that Pro104 siRNA do not
knockdown non-Pro104 mRNA (GAPDH) compared to negative controls in
CaOV3 cells. FIG. 23B demonstrates Pro104 siRNA knockdown Pro104
mRNA compared to negative controls in CaOV3 cells.
[0478] Specificity of Pro104 siRNA was not limited by cell type.
QPCR assays further demonstrated Pro104 siRNA specifically
knockdown Pro104 mRNA in HeLa cells. FIG. 24A shows that Pro104
siRNA do not knockdown non-Pro104 mRNA (GAPDH) compared to negative
controls in HeLa cells. FIG. 24B demonstrates Pro104 siRNA
knockdown Pro104 mRNA compared to negative controls in HeLa cells.
A 75% knockdown (.DELTA.CT=2) of Pro104 was observed in HeLa
cells.
[0479] Knockdown of essential mRNA such as DAXX leads to apoptosis
induction. Michaelson 2002 supra; Olichen 2002, supra. QPCR
experiments demonstrated that knockdown of Pro104 mRNA may lead to
apoptosis induction as well. FIG. 25 shows Pro104 siRNA knockdown
of Pro104 mRNA in HeLa cells compared to a positive control for
apoptosis induction (DAXX).
[0480] Furthermore, QPCR assays confirmed that different Pro104
siRNA knockdown Pro104 mRNA in HeLa cells. FIG. 26B demonstrates
that Pro104 siRNAs #56 (SEQ ID NO: 10), #79 (SEQ ID NO: 11), #80
(SEQ ID NO: 12) and #81 (SEQ ID NO: 13) knockdown Pro104 mRNA
compared to the negative control (scrambled siRNA). Knockdown of
Pro104 mRNA by four different siRNA specifically designed for
Pro104 mRNA is indicative that siRNA designed to interfere with
Pro104 mRNA may knockdown Pro104 mRNA and protein expression.
Example 10
Apoptosis Assays
[0481] Two different assay kits, Annexin V assay and Caspase assay,
were used to evaluate the effects of siRNA on apoptosis.
[0482] With the "Apo-ONE Homogeneous Caspase-3/7 Assay" kit
(Promega Inc., Madison, Wis.) 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.
[0483] 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 machine according to manufacturer's
instructions.
[0484] Annexin V assay results demonstrates that different Pro104
siRNA which knockdown Pro104 mRNA induces apoptosis. FIG. 26A shows
that Pro104 siRNAs #56 (SEQ ID NO: 10), #79 (SEQ ID NO: 11), #80
(SEQ ID NO: 12) and #81 (SEQ ID NO: 13) or DAXX siRNA induce
apoptosis compared to scrambled siRNA (negative control) in HeLa
cells.
[0485] Annexin V assay results also demonstrate specific knockdown
of Pro104 mRNA with Pro104 siRNA induces cell death. FIG. 27A shows
a greater percentage of HeLa cells are early apoptotic when
transfected with Pro104 siRNA compared to negative controls (no
siRNA, and scrambled siRNA). Additionally, FIG. 27B shows a greater
percentage of HeLa cells are necrotic when transfected with Pro104
siRNA compared to negative controls (no siRNA, and scrambled
siRNA).
[0486] Induction of apoptosis by knockdown of Pro104 mRNA by Pro104
siRNA was demonstrated by the Anexin V assay, and the Caspase
assay. Results of Annexin V assay in FIG. 28A show a greater
percentage HeLa cells are apoptotic when transfected with Pro104
siRNA, or DAXX siRNA (positive control) compared to scrambled siRNA
(negative control). Results of Caspase assay in FIG. 28B show a
greater percentage HeLa cells are apoptotic when transfected with
Pro104 siRNA, or DAXX siRNA (positive control) compared to
scrambled siRNA (negative control).
[0487] Induction of apoptosis by Pro104 mRNA knockdown by Pro104
siRNA was not limited by cell type. Results of Annexin V assay in
FIG. 29 show a greater percentage CaOV3 cells also are apoptotic
when transfected with Pro104 siRNA, or OPAI siRNA (positive
control) compared to scrambled siRNA (negative control) and no
siRNA (negative control).
[0488] Induction of apoptosis by knockdown of Pro104 mRNA is due
loss of Pro104 function. Pro104 siRNA does not induce apoptosis in
cells that do not express Pro104. FIG. 30A demonstrates knockdown
levels of Pro104 mRNA, Emerin mRNA (positive control,
non-essential) and DAXX mRNA (positive control, essential) in SKBR3
cells which do not express Pro104 mRNA. There is no difference
between knockdown levels of Pro104 mRNA due to scrambled siRNA and
Pro104 specific siRNA while Emerin and DAXX mRNA levels are
knocked-down, 50% and 65% respectively, by specific siRNA compared
to scrambled siRNA.
[0489] Results from a Caspase assay in FIG. 30B demonstrate that
SKBR3 cells which do not express Pro104 mRNA do not undergo
apoptosis when transfected with Pro104 siRNA while apoptosis is
induced by transfection by DAXX siRNA (positive control).
Furthermore, SKBR3 cells which do not express Pro104 mRNA do not
undergo apoptosis when transfected with Emerin siRNA
(non-essential) or scrambled siRNA (negative control).
[0490] Results from FIGS. 30A and 30B serve as a negative control
to show Pro104 siRNA transfection induces apoptosis by specifically
knockdown of Pro104 mRNA and down-regulation of Pro104 protein.
[0491] Furthermore, Pro104 is shown to be essential to cell
survival. FIGS. 30A and 30B demonstrate that knockdown of
non-essential mRNA (Emerin) does not induce apoptosis compared to
scrambled siRNA (negative control). Only knockdown of essential
mRNA such as DAXX (positive control) and Pro104 will induce
apoptosis.
Example 11
Soft Agar Assay
[0492] Soft agar assays were conducted using 6-well plates
(Corning, VWR). The 2 ml bottom agar base layer consisted of 0.8%
agar, 10% FBS in Iscove's medium (Invitrogen Life Technologies,
Carlsbad, Calif.). Trypsinized cells were suspended in 0.4% agar,
10% FBS in Iscove's medium and applied in a 5 ml final volume on
top of the solidified base layer. Three different viable cell
numbers, 10.sup.5, 10.sup.4 and 5.times.10.sup.3 cells, were seeded
in agar per 6 cm.sup.2 well in duplicate. A final 2 ml layer
consisting of 0.8% agar, 10% FBS in Iscove's medium was applied on
top of the solidified cell layer. The agar plates were then
incubated in a humidified 37.degree. C. incubator with 5% CO.sub.2
for approximately 2 weeks before colonies appeared. The soft agar
was maintained by weekly feedings with growth medium. Colonies were
counted between 2 and 4 weeks. 24 to 36 hours after siRNA
transfection, HeLa cells were trypsinized and plated in soft agar
at a density of 10.sup.4 cells per well as described above.
[0493] Soft agar assays were conducted to evaluate the effects of
over-expression of Pro104, Pro104 protease activity and knockdown
of Pro104 on cells.
[0494] FIG. 31 demonstrates that over expression of Pro104 induces
cell growth in soft agar. Table 9 below shows the number of
colonies observed in soft agar plates for each cell type in FIG.
31.
TABLE-US-00022 TABLE 9 Number of Colonies in Soft Agar Plates
Number of Figure Cell Type Colonies 31A RK3E-AP 0 31B RK3E-Pro104
60 31C RK3E-Pro104-HA 68 31D NCIH522 (-control) 0 31E HCT116
(+control) >200
Pro104 Protease Activity is Required for Cell Growth
[0495] RK3E cells were infected with retrovirus vectors expressing
wild-type Pro104 protein (Pro104), with and without a C-terminal
hemagglutinin tag (HA). Additionally, RK3E cells were infected with
retrovirus vectors expressing Pro104 protein lacking enzymatic
activity with a point mutation within the catalytic triad
(Pro104-mut) or Alkaline Phosphatase (AP-control). Retroviral
infection was followed by G418 selection for infected cells.
[0496] Expression of Pro104 proteins in the G418-selected cell
pools was verified by immunoblot with a monoclonal antibody
directed against Pro104, FIG. 32A. Expression of AP in the
G418-selected cells was evaluated by staining cell monolayers for
AP activity which showed that essentially all of the cells were
positive (FIG. 32B) and, therefore, most of the G418-selected cells
were expressing the gene of interest. The virus-infected, selected
cells were then plated in soft agar and monitored for colony
formation. The parental RK3E cells did not form any colonies under
the conditions used for the assay nor did the AP-expressing cells
(FIG. 32C, 32D). However, cells expressing either HA-tagged
(Pro104-HA) or untagged Pro104 protein formed colonies
demonstrating that ectopic expression of the protein can promote
transformation (FIG. 32C, 32D). The mutant Pro104 (Pro104-mut)
protein was unable to induce soft agar growth of RK3E cells (FIG.
32C, 32D) indicating that the catalytic function of Pro104 is
required for transformation.
Knockdown of Pro104 mRNA by siRNA Inhibits Cell Growth
[0497] As shown above, specific knockdown of Pro104 mRNA and
protein in Hela cells led to an increase in apoptosis, measured by
two different methods. We next examined whether knockdown of Pro104
could affect the ability of HeLa cells to form colonies in soft
agar. HeLa cells were treated with scrambled, Pro104- or
DAXX-specific siRNA and subsequently plated in soft agar to
evaluate colony formation. Scrambled siRNA served as a negative
control while DAXX-specific siRNA served as positive control for
inducing apoptosis. HeLa cells form numerous large colonies in agar
and this was not affected by the scrambled siRNA as demonstrated in
FIGS. 33C and 33F. In contrast, both Pro104- and DAXX-specific
siRNA's inhibited the number of colonies formed by approximately
88% and 80%, respectively (FIGS. 33A and 33B, respectively).
Furthermore, the size and morphology of colonies formed by cells
treated with Pro104- and DAXX-specific siRNAs was smaller and
restricted (FIGS. 33D and 33E, respectively).
[0498] QPCR performed after transfection with siRNA showed that the
Pro104, DAXX and Emerin mRNA levels were decreased compared to
transfection with scrambled siRNA (FIG. 34A). In this experiment
the Pro104 and DAXX siRNA's were again able to induce caspase
activity whereas the scrambled and Emerin-specific siRNA did not
(FIG. 34B). A siRNA against emerin had no effect on the ability of
the Hela cells to form colonies.
Results
[0499] These assays confirm that Pro104 is essential to cell
survival and over-expression induces cell growth (FIG. 31).
Knockdown of Pro104 mRNA by siRNA (FIGS. 23-26) reduces protein
expression (FIGS. 21, 22) which induces apoptosis (FIGS. 27-30).
This in turn results in fewer colonies and colony size and
morphology indicative of apoptosis in soft agar (FIG. 33). Mutated
Pro104 lacking protease activity does not induce cell growth
confirming Pro104 activity is essential for cell growth (FIG.
32).
Example 12
Tumor Xenograft Experiment
[0500] To further evaluate the transforming ability of Pro104, RK3E
cells expressing Pro104 or AP were implanted subcutaneously into
Nude or SCID/Beige mice and tumor formation was monitored.
Increased Growth of Ovarian Tumor Cells Over-Expressing Pro104 in
Nude Mice
[0501] Retrovirus-infected, G418-selected pools of SKOV3 or RK3E
cells expressing either AP or Pro104 were injected subcutaneously
into nude mice. Parental SKOV3 cells were also used for comparison.
For SKOV3 cells, 10.sup.7 of each type were implanted with matrigel
into each of 6 mice. For RK3E cells, 5.times.10.sup.6 cells were
implanted into each of 8 mice without matrigel. Tumor formation was
monitored by palpation and caliper measurement where possible every
4 days for a period of 4 weeks.
[0502] An animal model demonstrated growth of human ovarian tumor
cells over-expressing Pro104. SKOV3 cells over-expressing Pro104
increased in volume compared to Parental SKOV3 cells (control) or
AP expressing SKOV3 cells (non-growth inducing control).
[0503] Additionally, table 10 below shows over-expression of Pro104
promotes tumor formation in subcutaneous cell xenografts in nude
mice.
TABLE-US-00023 TABLE 10 Tumor formation in subcutaneous cell
xenografts. RK3E Cell Line 5 .times. 10.sup.6 cells implanted #
mice with nodules* or tumor** by 4 weeks P (negative control) 0/8
Pro104 8/8* V-Ras (positive control) 8/8**
These animal models demonstrate that Pro104 overexpressing cells
grow and form nodules.
Increased Growth of Ovarian Tumor Cells Over-Expressing Pro104 in
SCID Mice
[0504] Retrovirus-infected, G418-selected pools of SKOV3 or RK3E
cells expressing either AP or Pro104 were injected subcutaneously
into SCID/Beige mice (Charles River Laboratories). Nine or ten mice
were used per group as indicated. For SKOV3 cells, 10.sup.7 cells
in 100 ul PBS were implanted with matrigel and for RK3E cells,
5.times.10.sup.6 cells in 100 .mu.l PBS were implanted without
using matrigel. Tumor formation was monitored by palpation and
caliper measurement and tumor volume was calculated using the
formula: (length.times.width.sup.2)/2. The graphs shown in FIGS.
36A and 36C plot mean group tumor volume over time. All animal
experiments were performed in complete compliance with
institutional guidelines.
[0505] For the SKOV3 xenograft studies a single factor ANOVA was
performed to test whether on the last day of measurement the tumor
volumes between control and Pro104 groups differed. The results
indicated a >99.0% probability that the two groups do not have
the same tumor volume. Furthermore, Pairwise Two-Sample t-Tests
Assuming Unequal Variances with Bonferroni Correction analysis were
performed comparing the SKOV3-testisin tumors to the SKOV3-control
tumors. Analysis of data from the last day of measurement revealed
that the SKOV3-Pro104 tumors had significantly larger volumes than
SKOV3-control tumors at a 99.0% confidence level.
[0506] KR3E-Pro104 Tumor Cell Growth
[0507] Nine out of nine mice implanted with Pro104 expressing RK3E
cells developed large tumors whereas none of the mice implanted
with AP-expressing cells formed tumors (FIG. 35A). At the
conclusion of the xenograft study tumors were harvested and
evaluated by immunoblot for the presence of Pro104 protein. The
tumors maintained expression of Pro104 protein at a level similar
to that observed in the infected RK3E cells prior to implantation
(FIG. 35B).
[0508] SKOV3-Pro104 Tumor Cell Growth
[0509] We next evaluated the effect of ectopic Pro104 expression on
tumor formation by the human SKOV3 ovarian cancer cell line which
was chosen for this purpose since it does not express endogenous
Pro104 mRNA nor Pro104 protein (evaluated by immunoblot-FIG. 35D).
SKOV3 cells were infected with either a retrovirus expressing
Pro104 or the AP control followed by G418-selection. Expression of
Pro104 protein in the selected cells was verified by immunoblot
(FIG. 35D). AP control and Pro104-expressing SKOV3 cells were
implanted subcutaneously into SCID/Beige mice and monitored for
tumor formation. SKOV3 cancer cells are known to form tumors as
xenografts in mice. As expected, the AP control-expressing SKOV3
cells were also capable of growth as xenografts where 10 out of 10
mice implanted formed tumors (FIG. 35C). However, cells expressing
ectopic Pro104 protein formed larger tumors throughout the time
course when compared to the AP-control cells (FIG. 35C).
Statistical analysis of the data showed that the increased size of
SKOV3-Pro104 tumors compared to AP control-SKOV3 tumors was
significant.
Example 13
Anti-Pro104 Molecules in Combination with Anti-Angiogenesis and
Anti-Vascular Molecules
[0510] Angiogenesis plays a critical role in many physiological
processes, such as embryogenesis, wound healing, and menstruation
and in certain pathological events, such as solid tumor growth and
metastasis, arthritis, psoriasis, and diabetic retinopathy as
described above.
[0511] Additionally, vascular targeting agents, which selectively
destroy tumor blood vessels, may be attractive agents for the
treatment of solid tumors. They differ from anti-angiogenic agents
in that they target the mature, blood-conducting vessels of the
tumors. They are better suited for larger tumors where angiogenesis
can occur less frequently. Vascular targeting agents include
antibodies which bind to specific targets or complexes. For
application in man, target molecules are needed that are
selectively expressed on the vascular endothelium of tumors.
[0512] In addition to targeting Pro104 to modulate growth of Pro104
expressing tumors, targeting of angiogenesis associated molecules
or vascular associated molecules and complexes may be used to
enhance anti-Pro104 therapies. Specifically, anti-Pro104 antibodies
may be used in combination with antibodies which specifically
target angiogenesis associated molecules or vascular associated
molecules and complexes to slow, stop, regress, reverse or inhibit
growth or metastasis of Pro104 expressing tumors.
[0513] See Feng D., et al. J Histochem Cytochem. 2000 April;
48(4):545-56; Brekken R A., et al. Cancer Res. 2000 Sep. 15;
60(18):5117-24; Brekken R A., et al., Anticancer Res. 2001
November-December; 21(6B):4221-9; and Brekken R A., et al., Int J
Cancer. 2002 Jul. 10; 100(2):123-30.
Anti-Pro104 Antibodies in Combination with Anti-VEGF Antibodies
[0514] Vascular permeability factor/vascular endothelial growth
factor (VPF/VEGF) is a potent multifunctional cytokine that
permeabilizes vascular endothelium to plasma proteins and
reprograms endothelial cell gene expression so as to induce
angiogenesis. VPF/VEGF is secreted by many tumors and by activated
macrophages, keratinocytes, synovial cells, various embryonic
cells, and cultured epithelial and mesenchymal cell lines. There
are at least five splice variants of VEGF, encoding proteins of
121, 145, 165, 189, and 206 amino acids. The smaller versions
having 121, 145, or 165 amino acids are secreted from cells.
Secreted VEGF is an obligate dimer of between Mr 38,000 and Mr
46,000 in which the monomers are linked by two disulfide bonds. The
VEGF dimer binds to one of two well-characterized receptors, VEGFR1
(FLT-1) and VEGFR2 (KDR/Flk-1), that are selectively expressed on
endothelial cells. A recently identified third cell surface
protein, neuropilin-1, binds VEGF165 with high affinity. VPF/VEGF
induces its biological effects by binding to these receptors which
are selectively expressed in vascular endothelium.
[0515] Anti-Pro104 antibodies may be used in combination with
anti-VEGF antibodies to slow, stop, regress, reverse or inhibit
growth or metastasis of Pro104 expressing tumors.
Anti-Pro104 Antibodies in Combination with Anti-VEGF Receptor
Antibodies
[0516] VEGFR1 and VEGFR2 are members of the type III receptor
tyrosine kinase family that is characterized by seven extracellular
IgG-like repeats, a single spanning transmembrane domain, and an
intracellular split tyrosine kinase domain. Both receptors are
strikingly upregulated in tumors, wounds, and in certain types of
inflammation (e.g., rheumatoid arthritis, psoriasis) in which
VPF/VEGF is overexpressed. The complex that forms between
tumor-secreted VPF/VEGF and its receptors has been recognized as an
attractive potential target for antiangiogenesis therapy. VEGF
binds to VEGFR1 and VEGFR2 with high affinities having a Kd
(dissociation constant) of 15-100 pM and 400-800 pM, respectively.
VEGFR2 appears to be the dominant signaling receptor in
VEGF-induced mitogenesis and permeability.
[0517] Expression of both VEGFR-1 and VEGFR-2 has been localized by
in situ hybridization to microvascular endothelium of normal
kidneys and to tumors, healing wounds, and inflammatory sites.
VEGFR-2 has also been identified in the blood vessels of human
placentas, breast cancers, and gastric carcinomas by light
microscopic immunohistochemistry. See
[0518] Anti-Pro104 antibodies may be used in combination with
antibodies against VEGFR-1, VEGFR-2 or neuropilin-1, to slow, stop,
regress, reverse or inhibit growth or metastasis of Pro104
expressing tumors.
Anti-Pro104 Antibodies in Combination with Anti-Vascular Targeting
Antibodies
[0519] Vascular targeting antibodies specifically bind to vascular
associated markers. Such markers include the complexes that are
formed when vascular endothelial growth factor (VEGF) binds to its
receptors (VEGFR). VEGF production by tumor cells is induced by
oncogenic gene mutations and by the hypoxic conditions within the
tumor mass. The receptors, VEGFR1 (FLT-1) and VEGFR2 (KDR/Flk-1),
are upregulated on vascular endothelial cells in tumors by hypoxia
and by the increased local concentration of VEGF. Consequently,
there is a high concentration of occupied receptors on tumor
vascular endothelium.
[0520] Vascular targeting with monoclonal antibodies that bind to
VEGF: VEGFR complexes and their use as tumor vascular targeting
agents are known to those of skill in the art. Antibodies which
blocks VEGF from binding to VEGFR2 but not VEGFR1 might have dual
activity as an anti-angiogenic agent by inhibiting VEGFR2 activity
and as a vascular targeting agent for selective drug delivery to
tumor vessels.
[0521] Anti-Pro104 antibodies may be used in combination with
antibodies against VEGF:VEGFR complexes to slow, stop, regress,
reverse or inhibit growth or metastasis of Pro104 expressing
tumors. Examples of antibodies include but are not limited to
anti-Pro104, Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17,
Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27,
Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49,
Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57,
Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,
Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18,
Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29,
Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55,
Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64,
Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85,
Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106,
Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115,
Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120,
Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124,
Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128,
Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133,
Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138,
Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47,
Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,
Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89,
Pro104.K155, Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159,
Pro104.K160, Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217,
Pro104.K226, Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264,
Pro104.K281, Pro104.K358 or Pro104.K362; anti-VEGF, bevacizumab
(Avastin; Genentech Inc., South San Francisco, Calif.; Rini et al.
Clin Cancer Res. 2004 Apr. 15; 10(8):2584-6), infliximab (Canete et
al. Arthritis Rheum. 2004 May; 50(5):1636-41, Klimiuk et al. Arch
Immunol Ther Exp (Warsz). 2004 January-February; 52(1):36-42);
anti-VEGF-R, vatalanib (Manley et al. Biochim Biophys Acta. 2004
Mar. 11; 1697(1-2):17-27); anti-VEGF-R2, DC101 (Tong et al. Cancer
Res. 2004 Jun. 1; 64(11):3731-6, Kiessling et al. Neoplasia. 2004
May-June; 6(3):213-23); anti-VEGF-3, hF4-3C5 (Persaud et al. J Cell
Sci. 2004 Jun. 1; 117(Pt 13):2745-56). In addition, combination
therapy for EGFR may be used e.g. anti-EGFR, cetuximab, C225 (Andre
et al. Bull Cancer. 2004 January; 91(1):75-80), gefitinib, ZD1839
(Ciardiello et al. Clin Cancer Res. 2004 Jan. 15;
10(2):784-93).
Example 14
Deposit of Cell Lines and DNA
[0522] 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.
[0523] The following hybridoma cell lines were deposited with ATCC,
Pro104.C55.1, Pro104.C25.1, Pro104.D9.1 and Pro104.K81.15. The
names of the deposited hybridoma cell lines above may be shortened
for convenience of reference. E.g. A01.1 corresponds to
Pro104.A01.1. Additionally, the names of the deposited hybridoma
cell lines may or may not contain the period punctuation mark
separating "Pro104" from the hybridoma clone. E.g. Pro104 C55.1
corresponds to Pro104.C55.1. These hybridomas correspond to the
clones (with their full names) deposited with the ATCC. Table 11
lists the hybridoma clone deposited with the ATCC, the accorded
ATCC accession number, and the date of deposit.
TABLE-US-00024 TABLE 11 ATCC deposits Hybridoma ATCC Accession No.
Deposit Date Pro104.C55.1 PTA-5277 23 Jun. 2003 Pro104.C25.1
PTA-6076 15 Jun. 2004 Pro104.D9.1 PTA-6077 15 Jun. 2004
Pro104.K81.15 PTA-6078 15 Jun. 2004
[0524] 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).
[0525] 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
381288PRTHomo sapiens 1Met Ala Lys Pro Glu Ser Gln Glu Ala Ala Pro
Leu Ser Gly Pro Cys1 5 10 15Gly Arg Arg Val Ile Thr Ser Arg Ile Val
Gly Gly Glu Asp Ala Glu 20 25 30Leu Gly Arg Trp Pro Trp Gln Gly Ser
Leu Arg Leu Trp Asp Ser His 35 40 45Val Cys Gly Val Ser Leu Leu Ser
His Arg Trp Ala Leu Thr Ala Ala 50 55 60His Cys Phe Glu Thr Tyr Ser
Asp Leu Ser Asp Pro Ser Gly Trp Met65 70 75 80Val Gln Phe Gly Gln
Leu Thr Ser Met Pro Ser Phe Trp Ser Leu Gln 85 90 95Ala Tyr Tyr Thr
Arg Tyr Phe Val Ser Asn Ile Tyr Leu Ser Pro Arg 100 105 110Tyr Leu
Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val Lys Leu Ser Ala 115 120
125Pro Val Thr Tyr Thr Lys His Ile Gln Pro Ile Cys Leu Gln Ala Ser
130 135 140Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val Thr Gly
Trp Gly145 150 155 160Tyr Ile Lys Glu Asp Glu Ala Leu Pro Ser Pro
His Thr Leu Gln Glu 165 170 175Val Gln Val Ala Ile Ile Asn Asn Ser
Met Cys Asn His Leu Phe Leu 180 185 190Lys Tyr Ser Phe Arg Lys Asp
Ile Phe Gly Asp Met Val Cys Ala Gly 195 200 205Asn Ala Gln Gly Gly
Lys Asp Ala Cys Phe Gly Asp Ser Gly Gly Pro 210 215 220Leu Ala Cys
Asn Lys Asn Gly Leu Trp Tyr Gln Ile Gly Val Val Ser225 230 235
240Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr Thr Asn
245 250 255Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met Ala Gln
Ser Gly 260 265 270Met Ser Gln Pro Asp Pro Ser Trp Leu Glu His His
His His His His 275 280 2852287PRTHomo sapiens 2Met Lys Phe Leu Val
Asn Val Ala Leu Val Phe Met Val Val Tyr Ile1 5 10 15Ser Tyr Ile Tyr
Ala Asp Pro Met Ala Ile Val Gly Gly Glu Asp Ala 20 25 30Glu Leu Gly
Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu Trp Asp Ser 35 40 45His Val
Cys Gly Val Ser Leu Leu Ser His Arg Trp Ala Leu Thr Ala 50 55 60Ala
His Cys Phe Glu Thr Tyr Ser Asp Leu Ser Asp Pro Ser Gly Trp65 70 75
80Met Val Gln Phe Gly Gln Leu Thr Ser Met Pro Ser Phe Trp Ser Leu
85 90 95Gln Ala Tyr Tyr Thr Arg Tyr Phe Val Ser Asn Ile Tyr Leu Ser
Pro 100 105 110Arg Tyr Leu Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val
Lys Leu Ser 115 120 125Ala Pro Val Thr Tyr Thr Lys His Ile Gln Pro
Ile Cys Leu Gln Ala 130 135 140Ser Thr Phe Glu Phe Glu Asn Arg Thr
Asp Cys Trp Val Thr Gly Trp145 150 155 160Gly Tyr Ile Lys Glu Asp
Glu Ala Leu Pro Ser Pro His Thr Leu Gln 165 170 175Glu Val Gln Val
Ala Ile Ile Asn Asn Ser Met Cys Asn His Leu Phe 180 185 190Leu Lys
Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met Val Cys Ala 195 200
205Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp Ser Gly Gly
210 215 220Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile Gly
Val Val225 230 235 240Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg
Pro Gly Val Tyr Thr 245 250 255Asn Ile Ser His His Phe Glu Trp Ile
Gln Lys Leu Met Ala Gln Ser 260 265 270Gly Met Ser Gln Pro Asp Pro
Ser Trp His His His His His His 275 280 2853340PRTHomo sapiens 3Met
Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala1 5 10
15Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu Ser Gly Pro
20 25 30Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly Glu Asp
Ala 35 40 45Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu Trp
Asp Ser 50 55 60His Val Cys Gly Val Ser Leu Leu Ser His Arg Trp Ala
Leu Thr Ala65 70 75 80Ala His Cys Phe Glu Thr Tyr Ser Asp Leu Ser
Asp Pro Ser Gly Trp 85 90 95Met Val Gln Phe Gly Gln Leu Thr Ser Met
Pro Ser Phe Trp Ser Leu 100 105 110Gln Ala Tyr Tyr Thr Arg Tyr Phe
Val Ser Asn Ile Tyr Leu Ser Pro 115 120 125Arg Tyr Leu Gly Asn Ser
Pro Tyr Asp Ile Ala Leu Val Lys Leu Ser 130 135 140Ala Pro Val Thr
Tyr Thr Lys His Ile Gln Pro Ile Cys Leu Gln Ala145 150 155 160Ser
Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val Thr Gly Trp 165 170
175Gly Tyr Ile Lys Glu Asp Glu Ala Leu Pro Ser Pro His Thr Leu Gln
180 185 190Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys Asn His
Leu Phe 195 200 205Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp
Met Val Cys Ala 210 215 220Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys
Phe Gly Asp Ser Gly Gly225 230 235 240Pro Leu Ala Cys Asn Lys Asn
Gly Leu Trp Tyr Gln Ile Gly Val Val 245 250 255Ser Trp Gly Val Gly
Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr Thr 260 265 270Asn Ile Ser
His His Phe Glu Trp Ile Gln Lys Leu Met Ala Gln Ser 275 280 285Gly
Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu Phe Phe Pro Leu 290 295
300Leu Trp Ala Leu Pro Leu Leu Gly Pro Val Asp Pro Ala Phe Leu
Tyr305 310 315 320Lys Val Val Arg Ser Arg Met Ala Ser Tyr Pro Tyr
Asp Val Pro Asp 325 330 335Tyr Ala Ser Leu 3404314PRTHomo sapiens
4Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala1 5
10 15Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu Ser Gly
Pro 20 25 30Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly Glu
Asp Ala 35 40 45Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu
Trp Asp Ser 50 55 60His Val Cys Gly Val Ser Leu Leu Ser His Arg Trp
Ala Leu Thr Ala65 70 75 80Ala His Cys Phe Glu Thr Tyr Ser Asp Leu
Ser Asp Pro Ser Gly Trp 85 90 95Met Val Gln Phe Gly Gln Leu Thr Ser
Met Pro Ser Phe Trp Ser Leu 100 105 110Gln Ala Tyr Tyr Thr Arg Tyr
Phe Val Ser Asn Ile Tyr Leu Ser Pro 115 120 125Arg Tyr Leu Gly Asn
Ser Pro Tyr Asp Ile Ala Leu Val Lys Leu Ser 130 135 140Ala Pro Val
Thr Tyr Thr Lys His Ile Gln Pro Ile Cys Leu Gln Ala145 150 155
160Ser Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val Thr Gly Trp
165 170 175Gly Tyr Ile Lys Glu Asp Glu Ala Leu Pro Ser Pro His Thr
Leu Gln 180 185 190Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys
Asn His Leu Phe 195 200 205Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe
Gly Asp Met Val Cys Ala 210 215 220Gly Asn Ala Gln Gly Gly Lys Asp
Ala Cys Phe Gly Asp Ser Gly Gly225 230 235 240Pro Leu Ala Cys Asn
Lys Asn Gly Leu Trp Tyr Gln Ile Gly Val Val 245 250 255Ser Trp Gly
Val Gly Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr Thr 260 265 270Asn
Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met Ala Gln Ser 275 280
285Gly Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu Phe Phe Pro Leu
290 295 300Leu Trp Ala Leu Pro Leu Leu Gly Pro Val305
3105486PRTHomo sapiens 5Met Ala Pro Ile Leu Gly Tyr Trp Lys Ile Lys
Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu
Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp
Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro
Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile
Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys
Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu
Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp
Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120
125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala
Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp
Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala
Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile
Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly
Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220His Asn Gln
Thr Ser Leu Tyr Lys Lys Ala Gly Phe Glu Asn Leu Tyr225 230 235
240Phe Gln Gly Val Val Gly Gly Glu Glu Ala Ser Val Asp Ser Trp Pro
245 250 255Trp Gln Val Ser Ile Gln Tyr Asp Lys Gln His Val Cys Gly
Gly Ser 260 265 270Ile Leu Asp Pro His Trp Val Leu Thr Ala Ala His
Cys Phe Arg Lys 275 280 285His Thr Asp Val Phe Asn Trp Lys Val Arg
Ala Gly Ser Asp Lys Leu 290 295 300Gly Ser Phe Pro Ser Leu Ala Val
Ala Lys Ile Ile Ile Ile Glu Phe305 310 315 320Asn Pro Met Tyr Pro
Lys Asp Asn Asp Ile Ala Leu Met Lys Leu Gln 325 330 335Phe Pro Leu
Thr Phe Ser Gly Thr Val Arg Pro Ile Cys Leu Pro Phe 340 345 350Phe
Asp Glu Glu Leu Thr Pro Ala Thr Pro Leu Trp Ile Ile Gly Trp 355 360
365Gly Phe Thr Lys Gln Asn Gly Gly Lys Met Ser Asp Ile Leu Leu Gln
370 375 380Ala Ser Val Gln Val Ile Asp Ser Thr Arg Cys Asn Ala Asp
Asp Ala385 390 395 400Tyr Gln Gly Glu Val Thr Glu Lys Met Met Cys
Ala Gly Ile Pro Glu 405 410 415Gly Gly Val Asp Thr Cys Gln Gly Asp
Ser Gly Gly Pro Leu Met Tyr 420 425 430Gln Ser Asp Gln Trp His Val
Val Gly Ile Val Ser Trp Gly Tyr Gly 435 440 445Cys Gly Gly Pro Ser
Thr Pro Gly Val Tyr Thr Lys Val Ser Ala Tyr 450 455 460Leu Asn Trp
Ile Tyr Asn Val Trp Lys Ala Glu Leu Ser Asn Trp Ser465 470 475
480His Pro Gln Phe Glu Lys 4856391PRTHomo sapiens 6Met Lys Val Ile
Leu Asp Lys Tyr Tyr Phe Leu Cys Gly Gln Pro Leu1 5 10 15His Phe Ile
Pro Arg Lys Gln Leu Cys Asp Gly Glu Leu Asp Cys Pro 20 25 30Leu Gly
Glu Asp Glu Glu His Cys Val Lys Ser Phe Pro Glu Gly Pro 35 40 45Ala
Val Ala Val Arg Leu Ser Lys Asp Arg Ser Thr Leu Gln Val Leu 50 55
60Asp Ser Ala Thr Gly Asn Trp Phe Ser Ala Cys Phe Asp Asn Phe Thr65
70 75 80Glu Ala Leu Ala Glu Thr Ala Cys Arg Gln Met Gly Tyr Ser Ser
Lys 85 90 95Pro Thr Phe Arg Ala Val Glu Ile Gly Pro Asp Gln Asp Leu
Asp Val 100 105 110Val Glu Ile Thr Glu Asn Ser Gln Glu Leu Arg Met
Arg Asn Ser Ser 115 120 125Gly Pro Cys Leu Ser Gly Ser Leu Val Ser
Leu His Cys Leu Ala Cys 130 135 140Gly Lys Ser Leu Lys Thr Pro Arg
Val Val Gly Gly Glu Glu Ala Ser145 150 155 160Val Asp Ser Trp Pro
Trp Gln Val Ser Ile Gln Tyr Asp Lys Gln His 165 170 175Val Cys Gly
Gly Ser Ile Leu Asp Pro His Trp Val Leu Thr Ala Ala 180 185 190His
Cys Phe Arg Lys His Thr Asp Val Phe Asn Trp Lys Val Arg Ala 195 200
205Gly Ser Asp Lys Leu Gly Ser Phe Pro Ser Leu Ala Val Ala Lys Ile
210 215 220Ile Ile Ile Glu Phe Asn Pro Met Tyr Pro Lys Asp Asn Asp
Ile Ala225 230 235 240Leu Met Lys Leu Gln Phe Pro Leu Thr Phe Ser
Gly Thr Val Arg Pro 245 250 255Ile Cys Leu Pro Phe Phe Asp Glu Glu
Leu Thr Pro Ala Thr Pro Leu 260 265 270Trp Ile Ile Gly Trp Gly Phe
Thr Lys Gln Asn Gly Gly Lys Met Ser 275 280 285Asp Ile Leu Leu Gln
Ala Ser Val Gln Val Ile Asp Ser Thr Arg Cys 290 295 300Asn Ala Asp
Asp Ala Tyr Gln Gly Glu Val Thr Glu Lys Met Met Cys305 310 315
320Ala Gly Ile Pro Glu Gly Gly Val Asp Thr Cys Gln Gly Asp Ser Gly
325 330 335Gly Pro Leu Met Tyr Gln Ser Asp Gln Trp His Val Val Gly
Ile Val 340 345 350Ser Trp Gly Tyr Gly Cys Gly Gly Pro Ser Thr Pro
Gly Val Tyr Thr 355 360 365Lys Val Ser Ala Tyr Leu Asn Trp Ile Tyr
Asn Val Trp Lys Ala Glu 370 375 380Leu His His His His His His385
3907461PRTHomo sapiens 7Met Asp Pro Asp Ser Asp Gln Pro Leu Asn Ser
Leu Asp Val Lys Pro1 5 10 15Leu Arg Lys Pro Arg Ile Pro Met Glu Thr
Phe Arg Lys Val Gly Ile 20 25 30Pro Ile Ile Ile Ala Leu Leu Ser Leu
Ala Ser Ile Ile Ile Val Val 35 40 45Val Leu Ile Lys Val Ile Leu Asp
Lys Tyr Tyr Phe Leu Cys Gly Gln 50 55 60Pro Leu His Phe Ile Pro Arg
Lys Gln Leu Cys Asp Gly Glu Leu Asp65 70 75 80Cys Pro Leu Gly Glu
Asp Glu Glu His Cys Val Lys Ser Phe Pro Glu 85 90 95Gly Pro Ala Val
Ala Val Arg Leu Ser Lys Asp Arg Ser Thr Leu Gln 100 105 110Val Leu
Asp Ser Ala Thr Gly Asn Trp Phe Ser Ala Cys Phe Asp Asn 115 120
125Phe Thr Glu Ala Leu Ala Glu Thr Ala Cys Arg Gln Met Gly Tyr Ser
130 135 140Ser Lys Pro Thr Phe Arg Ala Val Glu Ile Gly Pro Asp Gln
Asp Leu145 150 155 160Asp Val Val Glu Ile Thr Glu Asn Ser Gln Glu
Leu Arg Met Arg Asn 165 170 175Ser Ser Gly Pro Cys Leu Ser Gly Ser
Leu Val Ser Leu His Cys Leu 180 185 190Ala Cys Gly Lys Ser Leu Lys
Thr Pro Arg Val Val Gly Gly Glu Glu 195 200 205Ala Ser Val Asp Ser
Trp Pro Trp Gln Val Ser Ile Gln Tyr Asp Lys 210 215 220Gln His Val
Cys Gly Gly Ser Ile Leu Asp Pro His Trp Val Leu Thr225 230 235
240Ala Ala His Cys Phe Arg Lys His Thr Asp Val Phe Asn Trp Lys Val
245 250 255Arg Ala Gly Ser Asp Lys Leu Gly Ser Phe Pro Ser Leu Ala
Val Ala 260 265 270Lys Ile Ile Ile Ile Glu Phe Asn Pro Met Tyr Pro
Lys Asp Asn Asp 275 280 285Ile Ala Leu Met Lys Leu Gln Phe Pro Leu
Thr Phe Ser Gly Thr Val 290 295 300Arg Pro Ile Cys Leu Pro Phe Phe
Asp Glu Glu Leu Thr Pro Ala Thr305 310 315 320Pro Leu Trp Ile Ile
Gly Trp Gly Phe Thr Lys Gln Asn Gly Gly Lys 325 330 335Met Ser Asp
Ile Leu Leu Gln Ala Ser Val Gln Val Ile Asp Ser Thr 340 345 350Arg
Cys Asn Ala Asp Asp Ala Tyr Gln Gly Glu
Val Thr Glu Lys Met 355 360 365Met Cys Ala Gly Ile Pro Glu Gly Gly
Val Asp Thr Cys Gln Gly Asp 370 375 380Ser Gly Gly Pro Leu Met Tyr
Gln Ser Asp Gln Trp His Val Val Gly385 390 395 400Ile Val Ser Trp
Gly Tyr Gly Cys Gly Gly Pro Ser Thr Pro Gly Val 405 410 415Tyr Thr
Lys Val Ser Ala Tyr Leu Asn Trp Ile Tyr Asn Val Trp Lys 420 425
430Ala Glu Leu Asp Pro Ala Phe Leu Tyr Lys Val Val Arg Ser Arg Met
435 440 445Ala Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu 450
455 460827DNAArtificial sequenceSynthetic 8atgggcgcgc gcggggcgct
gctgctg 27933DNAArtificial sequenceSynthetic 9ttatcagacc ggccccagga
gtgggagagc cca 331063DNAArtificial sequenceSynthetic 10ttatcacgcg
tagtccggca cgtcgtacgg gtagccgacc ggccccagga gtgggagagc 60cca
631119RNAArtificial sequenceSynthetic 11cacauccagc ccaucuguc
191219RNAArtificial sequenceSynthetic 12gaggaugagg cacugccau
191319RNAArtificial sequenceSynthetic 13cucuaugugc aaccaccuc
191419RNAArtificial sequenceSynthetic 14guacaguuuc cgcaaggac
191519RNAArtificial sequenceSynthetic 15uucuccgaac gugucacgu
191619RNAArtificial sequenceSynthetic 16ccgugcuccu ggggcuggg
191719RNAArtificial sequenceSynthetic 17ggaguuggau cucucagaa
191819RNAArtificial sequenceSynthetic 18guuaucaguc ugagccagg
191919RNAArtificial sequenceSynthetic 19gccggagucg caggaggcg
192019RNAArtificial sequenceSynthetic 20cucgggcguu ggccguggc
192119RNAArtificial sequenceSynthetic 21accuauagug accuuagug
192219RNAArtificial sequenceSynthetic 22ccuauaguga ccuuaguga
192319RNAArtificial sequenceSynthetic 23uucacccuau gacauugcc
192419RNAArtificial sequenceSynthetic 24gcugucugca ccugucacc
192519RNAArtificial sequenceSynthetic 25ccggacagac ugcugggug
192619RNAArtificial sequenceSynthetic 26agaggaugag gcacugcca
192719RNAArtificial sequenceSynthetic 27guucaggucg ccaucauaa
192819RNAArtificial sequenceSynthetic 28ggacaucuuu ggagacaug
192919RNAArtificial sequenceSynthetic 29caagaaugga cugugguau
193019RNAArtificial sequenceSynthetic 30gaauggacug ugguaucag
193119RNAArtificial sequenceSynthetic 31uggacugugg uaucagauu
193219RNAArtificial sequenceSynthetic 32ucggcccggu gucuacacc
193319RNAArtificial sequenceSynthetic 33uaucagccac cacuuugag
193419RNAArtificial sequenceSynthetic 34gucaggcccu gguucucuu
193519RNAArtificial sequenceSynthetic 35uaaacacauu ccaguugau
193619RNAArtificial sequenceSynthetic 36uaaacacauu ccaguugau
193719RNAArtificial sequenceSynthetic 37acacauucca guugaugcc
193819RNAArtificial sequenceSynthetic 38cacauuccag uugaugccu 19
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