U.S. patent application number 13/662332 was filed with the patent office on 2014-02-20 for nucleic acid and corresponding protein named 158p1d7 useful in the treatment and detection of bladder and other cancers.
This patent application is currently assigned to Agensys, Inc.. The applicant listed for this patent is AGENSYS, INC.. Invention is credited to Pia M. Challita-Eid, Mary Faris, Wangmao Ge, Jean Gudas, Aya JAKOBOVITS, Steven B. Kanner, Karen Jane Meyrick Morrison, Robert Kendall Morrison, Juan J. Perez-Villar, Arthur B. Raitano.
Application Number | 20140051096 13/662332 |
Document ID | / |
Family ID | 46301840 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140051096 |
Kind Code |
A9 |
JAKOBOVITS; Aya ; et
al. |
February 20, 2014 |
NUCLEIC ACID AND CORRESPONDING PROTEIN NAMED 158P1D7 USEFUL IN THE
TREATMENT AND DETECTION OF BLADDER AND OTHER CANCERS
Abstract
The invention described herein relates to novel nucleic acid
sequences and their encoded proteins, referred to as 158P1D7 and
variants thereof, and to diagnostic and therapeutic methods and
compositions useful in the management of various cancers that
express 158P1D7 and variants thereof.
Inventors: |
JAKOBOVITS; Aya; (Beverly
Hills, CA) ; Morrison; Robert Kendall; (Santa Monica,
CA) ; Raitano; Arthur B.; (Los Alamitos, CA) ;
Challita-Eid; Pia M.; (Encino, CA) ; Perez-Villar;
Juan J.; (Puzol, ES) ; Morrison; Karen Jane
Meyrick; (Santa Monica, CA) ; Faris; Mary;
(Los Angeles, CA) ; Ge; Wangmao; (Tampa, FL)
; Gudas; Jean; (Los Angeles, CA) ; Kanner; Steven
B.; (Santa Monica, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENSYS, INC. |
Santa Monica |
CA |
US |
|
|
Assignee: |
Agensys, Inc.
Santa Monica
CA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130052667 A1 |
February 28, 2013 |
|
|
Family ID: |
46301840 |
Appl. No.: |
13/662332 |
Filed: |
October 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12050895 |
Mar 18, 2008 |
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13662332 |
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10776773 |
Feb 10, 2004 |
7358353 |
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12050895 |
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10280340 |
Oct 25, 2002 |
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10776773 |
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10277292 |
Oct 21, 2002 |
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10280340 |
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09935430 |
Aug 22, 2001 |
6863892 |
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10280340 |
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09935430 |
Aug 22, 2001 |
6863892 |
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10277292 |
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60446633 |
Feb 10, 2003 |
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60227098 |
Aug 22, 2000 |
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60282739 |
Apr 10, 2001 |
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Current U.S.
Class: |
435/7.92 ;
435/7.1; 436/501 |
Current CPC
Class: |
A61K 47/6813 20170801;
C07K 16/30 20130101; C07K 2317/622 20130101; C07K 16/18 20130101;
G01N 33/57484 20130101; A61K 38/00 20130101; C07K 2317/55 20130101;
C07K 14/47 20130101; C07K 16/3038 20130101; A61K 51/1093 20130101;
A61K 47/646 20170801; A61P 35/00 20180101; C07K 2317/54 20130101;
Y10T 436/25 20150115 |
Class at
Publication: |
435/7.92 ;
436/501; 435/7.1 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/577 20060101 G01N033/577; G01N 33/566 20060101
G01N033/566 |
Claims
1. A method for detection, the method comprising: providing a test
sample, and determining an expression level of a protein in the
test sample, wherein the determining is achieved by measuring
specific binding of a binding partner to the protein in the test
sample, wherein the binding partner is a substance that
specifically binds to a protein comprising the amino acid sequence
of SEQ ID NO: 3.
2. The method of claim 1, which further comprises: providing a
normal sample; determining an expression level of the protein in
the normal sample, wherein the determining is achieved by measuring
specific binding of the binding partner to the protein in the
normal sample; and comparing the expression levels of the protein
detected in the test sample and the normal sample.
3. The method of claim 1, wherein the binding partner is an
antibody or antigen binding fragment thereof that specifically
binds to a protein comprising the amino acid sequence of SEQ ID NO:
3.
4. The method of claim 3, wherein the antibody or fragment is
monoclonal.
5. The method of claim 1, wherein the test sample is a bladder test
sample.
6. The method of claim 2, wherein the normal sample is a normal
bladder sample.
7. A method to detect the presence of malignancy in a tissue, which
method comprises: contacting said tissue with an antibody or
antigen binding fragment thereof that binds specifically to a
protein comprising amino acid sequence of SEQ ID NO: 3 under
conditions wherein a complex is formed of said antibody or antigen
binding fragment thereof with a protein present in said tissue,
and, detecting the presence of said complex, wherein the presence
of said complex indicates the presence of malignancy in said
tissue.
8. The method of claim 7, wherein said antibody or fragment is
monoclonal.
9. The method of claim 7, wherein the tissue is a bladder
tissue.
10. The method of claim 7, wherein said detecting step comprises
immunohistochemical analysis.
11. The method of claim 7, wherein said contacting is in situ.
12. The method of claim 7, wherein said contacting is on a Western
blot.
13. The method of claim 7, wherein said contacting is on a tissue
array.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/050,895, filed Mar. 18, 2008, now pending, which is a
divisional application of U.S. application Ser. No. 10/776,773,
filed Feb. 10, 2004, now U.S. Pat. No. 7,358,353, which claims the
benefit of priority of U.S. Provisional Application No. 60/446,633,
filed Feb. 10, 2003, and is a continuation-in-part of U.S. patent
application Ser. No. 10/280,340, filed Oct. 25, 2002, now
abandoned, and a continuation-in-part of U.S. patent application
Ser. No. 10/277,292, filed Oct. 21, 2002, now abandoned, both of
which are continuations of U.S. patent application Ser. No.
09/935,430, filed Aug. 22, 2001, now U.S. Pat. No. 6,863,892, which
claims the benefit of priority of U.S. Provisional Application No.
60/227,098, filed Aug. 22, 2000, and U.S. Provisional Application
No. 60/282,793, filed Apr. 10, 2001. All applications are
incorporated by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
511582005006SeqList.txt, date recorded: Oct. 22, 2012, size:
287,914 bytes).
FIELD OF THE INVENTION
[0003] The invention described herein relates to novel nucleic acid
sequences and their encoded proteins, referred to as 158P1D7 and
variants thereof, and to diagnostic and therapeutic methods and
compositions useful in the management of various cancers that
express 158P1D7 and variants thereof.
BACKGROUND ART
[0004] Cancer is the second leading cause of human death next to
coronary disease. Worldwide, millions of people die from cancer
every year. In the United States alone, as reported by the American
Cancer Society, cancer causes the death of well over a half-million
people annually, with over 1.2 million new cases diagnosed per
year. While deaths from heart disease have been declining
significantly, those resulting from cancer generally are on the
rise. In the early part of the next century, cancer is predicted to
become the leading cause of death.
[0005] Of all new cases of cancer in the United States, bladder
cancer represents approximately 5 percent in men (fifth most common
neoplasm) and 3 percent in women (eighth most common neoplasm). The
incidence is increasing slowly, concurrent with an increasing older
population. In 1998, there was an estimated 54,500 cases, including
39,500 in men and 15,000 in women. The age-adjusted incidence in
the United States is 32 per 100,000 for men and 8 per 100,000 in
women. The historic male/female ratio of 3:1 may be decreasing
related to smoking patterns in women. There were an estimated
11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900
in women). Bladder cancer incidence and mortality strongly increase
with age and will be an increasing problem as the population
becomes more elderly.
[0006] Bladder cancers comprise a heterogeneous group of diseases.
The main determinants of disease control and survival are histology
and extent of disease. The main codes for these factors include
pathology classification, the International Classification of
Diseases-Oncology (ICDO), and staging classification of extent of
disease, the TNM classification (Table XXI). For a general
discussion of bladder and other urogenital cancers, see, e.g.,
Volgelzang, et al, Eds. Comprehensive Textbook of Genitourinary
Oncology, (Williams & Wilkins, Baltimore 1996), in particular
pages 295-556.
[0007] Three primary types of tumors have been reported in the
bladder. The most common type of bladder cancer is Transitional
cell carcinoma (TCC); this accounts for about 90% of all bladder
cancers. The second form of bladder cancer is squamous cell
carcinoma, which accounts for about 8% of all bladder cancers where
schistosomiasis is not endemic, and approximately 75% of bladder
carcinomas where schistosomiasis is endemic. Squamous cell
carcinomas tend to invade deeper layers of the bladder. The third
type of bladder cancer is adenocarcinoma, which account for 1%-2%
of bladder cancers; these are primarily invasive forms of
cancer.
[0008] Bladder cancer is commonly detected and diagnosed using
cytoscopy and urine cytology. However these methods demonstrate
poor sensitivity. Relatively more reliable methods of detection
currently used in the clinic include the bladder tumor antigen
(BTA) stat test, NMP22 protein assay, telomerase expression and
hyaluronic acid and hyaluronidase (HA-HAase) urine test. The
advantage of using such markers in the diagnosis of bladder cancer
is their relative high sensitivity in earlier tumor stages compared
to standard cytology.
[0009] For example, the BTA stat test has 60-80% sensitivity and
50-70% specificity for bladder cancer, while the HA-HAase urine
test shows 90-92% sensitivity and 80-84% specificity for bladder
cancer (J Urol 2001 165:1067). In general, sensitivity for stage Ta
tumors was 81% for nuclear matrix protein (NMP22), 70% for
telomerase, 32% for bladder tumor antigen (BTA) and 26% for
cytology (J Urol 2001 166:470; J Urol 1999, 161:810). Although the
telomeric repeat assay which measures telomerase activity is
relatively sensitive, instability of telomerase in urine presently
renders this detection method unreliable.
[0010] Most bladder cancers recur in the bladder. Generally,
bladder cancer is managed with a combination of transurethral
resection of the bladder (TUR) and intravesical chemotherapy or
immunotherapy. The multifocal and recurrent nature of bladder
cancer points out the limitations of TUR. Most muscle-invasive
cancers are not cured by TUR alone. Radical cystectomy and urinary
diversion is the most effective means to eliminate the cancer but
carry an undeniable impact on urinary and sexual function.
[0011] Intravesical bacilli Calmette-Guerin (BCG) is a common and
efficacious immunotherapeutic agent used in the treatment of
bladder cancer. BCG is also used as a prophylactic agent to prevent
recurrence of bladder cancer. However, 30% of patients fail to
respond to BCG therapy and go on to develop invasive and metastatic
disease (Catalona et al. J Urol 1987, 137:220-224). BCG-related
side effects have been frequently observed such as drug-induced
cystitis, risk of bacterial infection, and hematuria, amongst
others. Other alternative immunotherapies have been used for the
treatment of bladder cancer, such as KLH (Flamm et al. Urologe
1994; 33:138-143) interferons (Bazarbashi et al. J Surg Oncol.
2000; 74:181-4), and MAGE-3 peptide loaded dendritic cells
(Nishiyama et al. Clin Cancer Res 2001; 7:23-31). All these
approaches are still experimental (Zlotta et al. Eur Urol 2000; 37
Suppl 3:10-15). There continues to be a significant need for
diagnostic and treatment modalities that are beneficial for bladder
cancer patients. Furthermore, from a worldwide standpoint, several
cancers stand out as the leading killers. In particular, carcinomas
of the lung, prostate, breast, colon, pancreas, and ovary are
primary causes of cancer death. These and virtually all other
carcinomas share a common lethal feature. With very few exceptions,
metastatic disease from a carcinoma is fatal. Moreover, even for
those cancer patients who initially survive their primary cancers,
their lives are dramatically altered. Many cancer patients
experience strong anxieties driven by the awareness of the
potential for recurrence or treatment failure. Many cancer patients
experience physical debilitations following treatment. Furthermore,
many cancer patients experience a recurrence.
[0012] Prostate cancer is the fourth most prevalent cancer in men
worldwide. In North America and Northern Europe, it is by far the
most common cancer in males and is the second leading cause of
cancer death in men. In the United States alone, well over 30,000
men die annually of this disease, second only to lung cancer.
Despite the magnitude of these figures, there is still no effective
treatment for metastatic prostate cancer. Surgical prostatectomy,
radiation therapy, hormone ablation therapy, surgical castration
and chemotherapy continue to be the main treatment modalities.
Unfortunately, these treatments are ineffective for many and are
often associated with undesirable consequences.
[0013] On the diagnostic front, the lack of a prostate tumor marker
that can accurately detect early-stage, localized tumors remains a
significant limitation in the diagnosis and management of this
disease. Although the serum prostate specific antigen (PSA) assay
has been a very useful tool, however its specificity and general
utility is widely regarded as lacking in several important
respects. While previously identified markers such as PSA, PSM,
PCTA and PSCA have facilitated efforts to diagnose and treat
prostate cancer, there is need for the identification of additional
markers and therapeutic targets for prostate and related cancers in
order to further improve diagnosis and therapy.
[0014] Renal cell carcinoma (RCC) accounts for approximately 3
percent of adult malignancies. Once adenomas reach a diameter of 2
to 3 cm, malignant potential exists. In the adult, the two
principal malignant renal tumors are renal cell adenocarcinoma and
transitional cell carcinoma of the renal pelvis or ureter. The
incidence of renal cell adenocarcinoma is estimated at more than
29,000 cases in the United States, and more than 11,600 patients
died of this disease in 1998. Transitional cell carcinoma is less
frequent, with an incidence of approximately 500 cases per year in
the United States.
[0015] Surgery has been the primary therapy for renal cell
adenocarcinoma for many decades. Until recently, metastatic disease
has been refractory to any systemic therapy. With recent
developments in systemic therapies, particularly immunotherapies,
metastatic renal cell carcinoma may be approached aggressively in
appropriate patients with a possibility of durable responses.
Nevertheless, there is a remaining need for effective therapies for
these patients.
[0016] An estimated 130,200 cases of colorectal cancer occurred in
2000 in the United States, including 93,800 cases of colon cancer
and 36,400 of rectal cancer. Colorectal cancers are the third most
common cancers in men and women. Incidence rates declined
significantly during 1992-1996 (-2.1% per year). Research suggests
that these declines have been due to increased screening and polyp
removal, preventing progression of polyps to invasive cancers.
There were an estimated 56,300 deaths (47,700 from colon cancer,
8,600 from rectal cancer) in 2000, accounting for about 11% of all
U.S. cancer deaths.
[0017] At present, surgery is the most common form of therapy for
colorectal cancer, and for cancers that have not spread, it is
frequently curative. Chemotherapy, or chemotherapy plus radiation
is given before or after surgery to most patients whose cancer has
deeply perforated the bowel wall or has spread to the lymph nodes.
A permanent colostomy (creation of an abdominal opening for
elimination of body wastes) is occasionally needed for colon cancer
and is infrequently required for rectal cancer. There continues to
be a need for effective diagnostic and treatment modalities for
colorectal cancer.
[0018] There were an estimated 164,100 new cases of lung and
bronchial cancer in 2000, accounting for 14% of all U.S. cancer
diagnoses. The incidence rate of lung and bronchial cancer is
declining significantly in men, from a high of 86.5 per 100,000 in
1984 to 70.0 in 1996. In the 1990s, the rate of increase among
women began to slow. In 1996, the incidence rate in women was 42.3
per 100,000.
[0019] Lung and bronchial cancer caused an estimated 156,900 deaths
in 2000, accounting for 28% of all cancer deaths. During 1992-1996,
mortality from lung cancer declined significantly among men (-1.7%
per year) while rates for women were still significantly increasing
(0.9% per year). Since 1987, more women have died each year of lung
cancer than breast cancer, which, for over 40 years, was the major
cause of cancer death in women. Decreasing lung cancer incidence
and mortality rates most likely resulted from decreased smoking
rates over the previous 30 years; however, decreasing smoking
patterns among women lag behind those of men. Of concern, although
the declines in adult tobacco use have slowed, tobacco use in youth
is increasing again.
[0020] Treatment options for lung and bronchial cancer are
determined by the type and stage of the cancer and include surgery,
radiation therapy, and chemotherapy. For many localized cancers,
surgery is usually the treatment of choice. Because the disease has
usually spread by the time it is discovered, radiation therapy and
chemotherapy are often needed in combination with surgery.
Chemotherapy alone or combined with radiation is the treatment of
choice for small cell lung cancer; on this regimen, a large
percentage of patients experience remission, which in some cases is
long lasting. There is however, an ongoing need for effective
treatment and diagnostic approaches for lunch and bronchial
cancers.
[0021] An estimated 182,800 new invasive cases of breast cancer
were expected to have occurred among women in the United States
during 2000. Additionally, about 1,400 new cases of breast cancer
were expected to be diagnosed in men in 2000. After increasing
about 4% per year in the 1980s, breast cancer incidence rates in
women have leveled off in the 1990s to about 110.6 cases per
100,000.
[0022] In the U.S. alone, there were an estimated 41,200 deaths
(40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer
ranks second among cancer deaths in women. According to the most
recent data, mortality rates declined significantly during
1992-1996 with the largest decreases in younger women, both white
and black. These decreases were probably the result of earlier
detection and improved treatment.
[0023] Taking into account the medical circumstances and the
patient's preferences, treatment of breast cancer may involve
lumpectomy (local removal of the tumor) and removal of the lymph
nodes under the arm; mastectomy (surgical removal of the breast)
and removal of the lymph nodes under the arm; radiation therapy;
chemotherapy; or hormone therapy. Often, two or more methods are
used in combination. Numerous studies have shown that, for early
stage disease, long-term survival rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical
mastectomy. Significant advances in reconstruction techniques
provide several options for breast reconstruction after mastectomy.
Recently, such reconstruction has been done at the same time as the
mastectomy.
[0024] Local excision of ductal carcinoma in situ (DCIS) with
adequate amounts of surrounding normal breast tissue may prevent
the local recurrence of the DCIS. Radiation to the breast and/or
tamoxifen may reduce the chance of DCIS occurring in the remaining
breast tissue. This is important because DCIS, if left untreated,
may develop into invasive breast cancer. Nevertheless, there are
serious side effects or sequelae to these treatments. There is,
therefore, a need for efficacious breast cancer treatments.
[0025] There were an estimated 23,100 new cases of ovarian cancer
in the United States in 2000. It accounts for 4% of all cancers
among women and ranks second among gynecologic cancers. During
1992-1996, ovarian cancer incidence rates were significantly
declining. Consequent to ovarian cancer, there were an estimated
14,000 deaths in 2000. Ovarian cancer causes more deaths than any
other cancer of the female reproductive system.
[0026] Surgery, radiation therapy, and chemotherapy are treatment
options for ovarian cancer. Surgery usually includes the removal of
one or both ovaries, the fallopian tubes (salpingo-oophorectomy),
and the uterus (hysterectomy). In some very early tumors, only the
involved ovary will be removed, especially in young women who wish
to have children. In advanced disease, an attempt is made to remove
all intra-abdominal disease to enhance the effect of chemotherapy.
There continues to be an important need for effective treatment
options for ovarian cancer.
[0027] There were an estimated 28,300 new cases of pancreatic
cancer in the United States in 2000. Over the past 20 years, rates
of pancreatic cancer have declined in men. Rates among women have
remained approximately constant but may be beginning to decline.
Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the
United States. Over the past 20 years, there has been a slight but
significant decrease in mortality rates among men (about -0.9% per
year) while rates have increased slightly among women.
[0028] Surgery, radiation therapy, and chemotherapy are treatment
options for pancreatic cancer. These treatment options can extend
survival and/or relieve symptoms in many patients but are not
likely to produce a cure for most. There is a significant need for
additional therapeutic and diagnostic options for pancreatic
cancer.
SUMMARY OF THE INVENTION
[0029] The present invention relates to a novel nucleic acid
sequence and its encoded polypeptide, designated 158P1D7. As used
herein, "158P1D7" may refer to the novel polynucleotides or
polypeptides or variants thereof or both of the disclosed
invention.
[0030] Nucleic acids encoding 158P1D7 are over-expressed in the
cancer(s) listed in Table I. Northern blot expression analysis of
158P1D7 expression in normal tissues shows a restricted expression
pattern in adult tissues. The nucleotide (FIG. 2) and amino acid
(FIG. 2, and FIG. 3) sequences of 158P1D7 are provided. The
tissue-related profile of 158P1D7 in normal adult tissues, combined
with the over-expression observed in bladder tumors, shows that
158P1D7 is aberrantly over-expressed in at least some cancers.
Thus, 158P1D7 nucleic acids and polypeptides serve as a useful
diagnostic agent (or indicator) and/or therapeutic target for
cancers of the tissues, such as those listed in Table I.
[0031] The invention provides polynucleotides corresponding or
complementary to all or part of the 158P1D7 nucleic acids, mRNAs,
and/or coding sequences, preferably in isolated form, including
polynucleotides encoding 158P1D7-related proteins and fragments of
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, or more than 25 contiguous amino acids; at least
about 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or
more than 100 contiguous amino acids of a 158P1D7-related protein,
as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA
hybrids, and related molecules (such as PNAs), polynucleotides or
oligonucleotides complementary or having at least a 90% homology to
158P1D7 nucleic acid sequences or mRNA sequences or parts thereof,
and polynucleotides or oligonucleotides that hybridize to the
158P1D7 genes, mRNAs, or to 158P1D7-encoding polynucleotides. Also
provided are means for isolating cDNAs and the gene(s) encoding
158P1D7. Recombinant DNA molecules containing 158P1D7
polynucleotides, cells transformed or transduced with such
molecules, and host-vector systems for the expression of 158P1D7
gene products are also provided. The invention further provides
antibodies that bind to 158P1D7 proteins and polypeptide fragments
thereof, including polyclonal and monoclonal antibodies, murine and
other mammalian antibodies, chimeric antibodies, humanized and
fully human antibodies, and antibodies labeled with a detectable
marker. The invention also comprises T cell clones that recognize
an epitope of 158P1D7 in the context of a particular HLA
molecule.
[0032] The invention further provides methods for detecting the
presence, amount, and status of 158P1D7 polynucleotides and
proteins in various biological samples, as well as methods for
identifying cells that express 158P1D7 polynucleotides and
polypeptides. A typical embodiment of this invention provides
methods for monitoring 158P1D7 polynucleotides and polypeptides in
a tissue or hematology sample having or suspected of having some
form of growth dysregulation such as cancer.
[0033] Note that to determine the starting position of any peptide
set forth in Tables V-XVIII and XXII to XLIX (collectively HLA
Peptide Tables) respective to its parental protein, e.g., variant
1, variant 2, etc., reference is made to three factors: the
particular variant, the length of the peptide in an HLA Peptide
Table, and the Search Peptides in Table VII. Generally, a unique
Search Peptide is used to obtain HLA peptides of a particular for a
particular variant. The position of each Search Peptide relative to
its respective parent molecule is listed in Table 55. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X-1" to each position in Tables V-XVIII and XXII to XLIX to obtain
the actual position of the HLA peptides in their parental molecule.
For example, if a particular Search Peptide begins at position 150
of its parental molecule, one must add 150-1, i.e., 149 to each HLA
peptide amino acid position to calculate the position of that amino
acid in the parent molecule.
[0034] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 158P1D7 such as bladder cancers, including therapies aimed
at inhibiting the transcription, translation, processing or
function of 158P1D7 as well as cancer vaccines.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1. 158P1D7 SSH nucleic acid sequence. The 158P1D7 SSH
sequence contains 231 bp.
[0036] FIG. 2. A) The cDNA and amino acid sequence of 158P1D7
variant 1 (also called "158P1D7 v.1" or "158P1D7 variant 1") is
shown in FIG. 2A. The start methionine is underlined. The open
reading frame extends from nucleic acid 23-2548 including the stop
codon.
[0037] B) The cDNA and amino acid sequence of 158P1D7 variant 2
(also called "158P1D7 v.2") is shown in FIG. 2B. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 23-2548 including the stop codon.
[0038] C) The cDNA and amino acid sequence of 158P1D7 variant 3
(also called "158P1D7 v.3") is shown in FIG. 2C. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 23-2221 including the stop codon.
[0039] D) The cDNA and amino acid sequence of 158P1D7 variant 4
(also called "158P1D7 v.4") is shown in FIG. 2D. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 23-1210 including the stop codon.
[0040] E) The cDNA and amino acid sequence of 158P1D7 variant 5
(also called "158P1D7 v.5") is shown in FIG. 2E. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 480-3005 including the stop codon.
[0041] F) The cDNA and amino acid sequence of 158P1D7 variant 6
(also called "158P1D7 v.6") is shown in FIG. 2F. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 23-1612 including the stop codon.
[0042] FIG. 3. A) The amino acid sequence of 158P1D7 v.1 is shown
in FIG. 3A; it has 841 amino acids. B) The amino acid sequence of
158P1D7 v.3 is shown in FIG. 3B; it has 732 amino acids. C) The
amino acid sequence of 158P1D7 v.4 is shown in FIG. 3C; it has 395
amino acids. D) The amino acid sequence of 158P1D7 v.6 is shown in
FIG. 3D; it has 529 amino acids. As used herein, a reference to
158P1D7 includes all variants thereof, including those shown in
FIGS. 2, 3, 10, 11, and 12 unless the context clearly indicates
otherwise.
[0043] FIG. 4. Alignment BLAST homology of 158P1D7 v.1 amino acid
to hypothetical protein FLJ22774.
[0044] FIG. 5. FIG. 5A: Amino acid sequence alignment of 158P1D7
with human protein.
[0045] FIG. 5B: Amino acid sequence alignment of 158P1D7 with human
protein similar to IGFALS.
[0046] FIG. 6. Expression of 158P1D7 by RT-PCR. First strand cDNA
was prepared from vital pool 1 (VP1: liver, lung and kidney), vital
pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool
(LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool,
bladder cancer pool, colon cancer pool, lung cancer pool, ovary
cancer pool, breast cancer pool, and metastasis pool. Normalization
was performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to 158P1D7, was performed at
30 cycles of amplification. Strong expression of 158P1D7 is
observed in bladder cancer pool and breast cancer pool. Lower
levels of expression are observed in VP1, VP2, xenograft pool,
prostate cancer pool, colon cancer pool, lung cancer pool, ovary
cancer pool, and metastasis pool.
[0047] FIG. 7. Expression of 158P1D7 in normal human tissues. Two
multiple tissue northern blots, with 2 .mu.g of mRNA/lane, were
probed with the 158P1D7 fragment. Size standards in kilobases (kb)
are indicated on the side. The results show expression of 158P1D7
in prostate, liver, placenta, heart and, to lower levels, in small
intestine and colon.
[0048] FIG. 8. Expression of 158P1D7 in bladder cancer patient
specimens. FIG. 8A. RNA was extracted from the bladder cancer cell
lines (CL), normal bladder (N), bladder tumors (T) and matched
normal adjacent tissue (NAT) isolated from bladder cancer patients.
Northern blots with 10 .mu.g of total RNA/lane were probed with the
158P1D7 fragment. Size standards in kilobases (kb) are indicated on
the side. The results show expression of 158P1D7 in 1 of 3 bladder
cancer cell lines. In patient specimens, 158P1D7 expression is
detected in 4 of 6 tumors tested. FIG. 8B. In another study,
158P1D7 expression is detected in all patient tumors tested (8B).
The expression observed in normal adjacent tissues (isolated from
diseased tissues) but not in normal tissue, isolated from healthy
donors, may indicate that these tissues are not fully normal and
that 158P1D7 may be expressed in early stage tumors.
[0049] FIG. 9. Expression of 158P1D7 in lung cancer patient
specimens. RNA was extracted from lung cancer cell lines (CL), lung
tumors (T), and their normal adjacent tissues (NAT) isolated from
lung cancer patients. Northern blot with 10 .mu.g of total RNA/lane
was probed with the 158P1D7 fragment. Size standards in kilobases
(kb) are indicated on the side. The results show expression of
158P1D7 in 1 of 3 lung cancer cell lines and in all 3 lung tumors
tested, but not in normal lung tissues.
[0050] FIG. 10. Expression of 158P1D7 in breast cancer patient
specimens. RNA was extracted from breast cancer cell lines (CL),
normal breast (N), and breast tumors (T) isolated from breast
cancer patients. Northern blot with 10 .mu.g of total RNA/lane was
probed with the 158P1D7 fragment. Size standards in kilobases (kb)
are indicated on the side. The results show expression of 158P1D7
in 2 of 3 breast cancer cell lines and in 2 breast tumors, but not
in normal breast tissue.
[0051] FIG. 11. FIGS. 11(a)-(d): Hydrophilicity amino acid profile
of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm
sequence analysis using the method of Hopp and Woods (Hopp T. P.,
Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828)
accessed on the Protscale website located on the World Wide Web
through the ExPasy molecular biology server.
[0052] FIG. 12. FIGS. 12(a)-(d): Hydropathicity amino acid profile
of 158P1D7 v.1, v.3,
[0053] v.4, and v.6 determined by computer algorithm sequence
analysis using the method of Kyte and Doolittle (Kyte J., Doolittle
R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale
website located on the World Wide Web at the ExPasy molecular
biology server.
[0054] FIG. 13. FIGS. 13(a)-(d): Percent accessible residues amino
acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by
computer algorithm sequence analysis using the method of Janin
(Janin J., 1979 Nature 277:491-492) accessed on the ProtScale
website located on the World Wide Web at the ExPasy molecular
biology server.
[0055] FIG. 14. FIGS. 14(a)-(d): Average flexibility amino acid
profile of 158P1D7 v.1,
[0056] v.3, v.4, and v.6 determined by computer algorithm sequence
analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran
R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.
32:242-255) accessed on the ProtScale website located on the World
Wide Web at the ExPasy molecular biology server.
[0057] FIG. 15. FIGS. 15(a)-(d): Beta-turn amino acid profile of
158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm
sequence analysis using the method of Deleage and Roux (Deleage,
G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the
ProtScale website located on the World Wide Web at the ExPasy
molecular biology server.
[0058] FIG. 16. FIGS. 16(A)-(D): Secondary structure and
transmembrane domains prediction for 158P1D7 protein variants. The
secondary structures of 158P1D7 protein variants 1 (SEQ ID NO:
104), v.3 (SEQ ID NO: 105), v.4 (SEQ ID NO: 106), and v.6 (SEQ ID
NO: 107), respectively, were predicted using the HNN--Hierarchical
Neural Network method (NPS@: Network Protein Sequence Analysis TIBS
2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C.,
Geourjon C. and Deleage G.), accessed from the ExPasy molecular
biology server located on the World Wide Web at
(.expasy.ch/tools/). This method predicts the presence and location
of alpha helices, extended strands, and random coils from the
primary protein sequence. The percent of the protein variant in a
given secondary structure is also listed. FIGS. 16E, 16G, 16I, and
16K: Schematic representation of the probability of existence of
transmembrane regions of 158P1D7 protein variants 1, 3, 4, and 6,
respectively, based on the TMpred algorithm of Hofmann and Stoffel
which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE--A database
of membrane spanning protein segments Biol. Chem. Hoppe-Seyler
374:166, 1993). FIGS. 16F, 16H, 16J, and 16L: Schematic
representation of the probability of the existence of transmembrane
regions of 158P1D7 protein variants 1, 3, 4, and 6, respectively,
based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh
(Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A
hidden Markov model for predicting transmembrane helices in protein
sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for
Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F.
Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.:
AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed
from the ExPasy molecular biology server located on the World Wide
Web at (.expasy.ch/tools/). Protein variants 1 and 3 are predicted
to contain 1 transmembrane region and protein variants 3 and 4 are
not predicted to have transmembrane regions. All variants contain a
hydrophobic stretch at their amino terminus that may encode a
signal peptide.
[0059] FIG. 17. Schematic alignment of SNP variants of 158P1D7.
Schematic alignment of SNP variants of 158P1D7. Variant 158P1D7 v.2
is a variant with single nucleotide differences at 1546. Though
this SNP variant is shown on transcript variant 158P1D7 v.1, it
could also occur in any other transcript variants that contains the
base pairs. Numbers correspond to those of 158P1D7 v.1. Black box
shows sequence similar to 158P1D7 v.1. SNP is indicated above the
box.
[0060] FIG. 18. Schematic alignment of protein variants of 158P1D7.
Schematic alignment of protein variants of 158P1D7. Protein
variants correspond to nucleotide variants. Nucleotide variant
158P1D7 v.2 and v.5 code for the same protein as v.1. Nucleotide
variants 158P1D7 v.3 and v.4 are transcript variants of v.1, as
shown in FIG. 12. Variant v.6 is a single nucleotide different from
v.4 but codes for a protein that differs in the C-terminal portion
from the protein coded by v.4. Black boxes represent sequence
similar to v.1. Hatched box represents amino acid sequence not
present in v.1. Numbers underneath the box correspond to 158P1D7
v.1.
[0061] FIG. 19. Exon compositions of transcript variants of
158P1D7. Variant 158P1D7 v.3, v.4, v.5 and v.6 are transcript
variants of 158P1D7 v.1. Variant 158P1D7 v.3 spliced 2069-2395 out
of variant 158P1D7 v.1 and variant v.4 spliced out 1162-2096 out of
v.1. Variant v.5 added another exon and 2 bp to the 5' end and
extended 288 bp to the 3' end of variant v.1. Variant v.6 spliced
at the same site as v.4 but spliced out an extra `g` at the
boundary. Numbers in "( )" underneath the boxes correspond to those
of 158P1D7 v.1. Lengths of introns and exons are not
proportional.
[0062] FIG. 20. 158P1D7 Expression in Melanoma Cancer. RNA was
extracted from normal skin cell line Detroit-551, and from the
melanoma cancer cell line A375. Northern blots with 10 ug of total
RNA were probed with the 158P1D7 DNA probe. Size standards in
kilobases are on the side. Results show expression of 158P1D7 in
the melanoma cancer cell line but not in the normal cell line.
[0063] FIG. 21. 158P1D7 Expression in cervical cancer patient
specimens. First strand cDNA was prepared from normal cervix,
cervical cancer cell line Hela, and a panel of cervical cancer
patient specimens. Normalization was performed by PCR using primers
to actin and GAPDH. Semi-quantitative PCR, using primers to
158P1D7, was performed at 26 and 30 cycles of amplification.
Results show expression of 158P1D7 in 5 out of 14 tumor specimens
tested but not in normal cervix nor in the cell line.
[0064] FIG. 22. Detection of 158P1D7 protein in recombinant cells
with monoclonal antibodies. Cell lysates from the indicated cell
lines were separated by SDS-PAGE and then transferred to
nitrocellulose for Western blotting. The blots were probed with 5
ug/ml of the indicated anti-158P1D7 monoclonal antibodies (MAbs) in
PBS+0.2% Tween 20+1% non-fat milk, washed, and then incubated with
goat anti-mouse IgG-HRP secondary Ab. Immunoreactive bands were
then visualized by enhanced chemoluminescence and exposure to
autoradiographic film. Arrows indicate the .about.95 KD and 90 kD
158P1D7 protein doublet band which suggest 158P1D7 is
post-translationally modified to generate 2 different molecular
weight species. These results demonstrate expression of 158P1D7
protein in recombinant cells and specific detection of the protein
with monoclonal antibodies.
[0065] FIG. 23. Surface staining of 158P1D7-expressing 293T and
UMUC cells with anti-158P1D7 monoclonal antibodies. Transiently
transfected 293T cells expressing 158P1D7 and stable
158P1D7-expressing UMUC bladder cancer cells were analyzed for
surface expression of 158P1D7 with monoclonal antibodies (MAbs) by
flow cytometry. Transfected 293T control vector and 158P1D7 vector
cells and stable UMUC-neo and UMUC-158P1D7 cells were stained with
10 ug/ml and 1 ug/ml, respectively, of the indicated MAbs. Surface
bound MAbs were detected by incubation with goat anti-mouse IgG-PE
secondary Ab and then subjected to FACS analysis.
158P1D7-expressing 293T and UMUC cells exhibited an increase in
relative fluorescence comnpared to control cells demonstrating
surface expression and detection of 158P1D7 protein by each of the
MAbs.
[0066] FIG. 24. Surface staining of endogenous 158P1D7-expressing
LAPC9 prostate cancer and UGB1 bladder cancer xenograft cells with
MAb M15-68(2)22.1.1. LAPC9 and UGB1 xenograft cells were subjected
to surface staining with either control mouse IgG antibody or MAb
M15-68(2).1.1 at 1 ug/ml. Surface bound MAbs were detected by
incubation with goat anti-mouse IgG-PE secondary Ab and then
subjected to FACS analysis. Both LAPC9 and UGB 1 cells exhibited an
increase in relative fluorescence with the anti-158P1D7 MAb
demonstrating surface expression and detection of 158P1D7
protein.
[0067] FIG. 25. Monoclonal antibody-mediated internalization of
endogenous surface 158P1D7 in NCI-H146 small cell lung cancer
cells. NCI-H146 cells were stained with 5 ug/ml of the indicated
MAbs at 4.degree. C. for 1.5 hours, washed, and then either left at
4.degree. C. or moved to 37.degree. C. for 10 and 30 minutes.
Residual surface bound MAb was then detected with anti-mouse IgG-PE
secondary antibody. The decrease in the mean fluorescence intensity
(MF) of cells moved to 37.degree. C. compared to cells left at
4.degree. C. demonstrates internalization of surface bound
158P1D7/MAb complexes.
[0068] FIG. 26. Binding of the 158P1D7 extracellular domain to
human umbilical vein endothelial cells. The recombinant
extracellular domain (ECD) of 158P1D7 (amino acids 16-608) was
iodinated to high specific activity using the iodogen
(1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril) method. Human
umbilical vein endothelial cells (HUVEC) at 90% confluency in 6
well plates was incubated with 1 nM of 125I-158P1D7 ECD in the
presence (non-specific binding) or absence (Total binding) of 50
fold excess unlabeled ECD for 2 hours at either 4.degree. C. or
37.degree. C. Cells were washed, solubilized in 0.5M NaOH, and
subjected to gamma counting. The data shows specific binding of
158P1D7 ECD to HUVEC cells suggesting the presence of an 158P1D7
receptor on HUVEC cells. FIG. 26A. Shows that the 158P1D7 ECD bound
directly to the surface of HUVEC cells as detected by the 158P1D7
specific MAb. FIG. 26B. Shows specific binding of 158P1D7 ECD to
HUVEC cells suggesting the presence of an 158P1D7 receptor on HUVEC
cells.
[0069] FIG. 27. 158P1D7 enhances the growth of bladder cancer in
mice. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory,
Wilmington, Mass.) were used and maintained in a strictly
controlled environment in accordance with the NIH Guide for the
Care and Use of Laboratory Animals. 158P1D7 transfected UM-UC-3
cells and parental cells were injected into the subcutaneous space
of SCID mice. Each mouse received 4.times.10.sup.6 cells suspended
in 50% (v/v) of Matrigel. Tumor size was monitored through caliper
measurements twice a week. The longest dimension (L) and the
dimension perpendicular to it (W) were taken to calculate tumor
volume according to the formula W2.times.L/2. The Mann-Whitney U
test was used to evaluate differences of tumor growth. All tests
were two sided with {acute over (.alpha.)}=0.05.
[0070] FIG. 28. Internalization of M15-68(2).31.1.1 in NCI-H146
cells. Endogenous-158P1D7 expressing NCI-H146 cells were incubated
with 5 ug/ml of MAb M15-68(2).31.1.1 at 4.degree. C. for 1 hour,
washed, and then incubated with goat anti-mouse IgG-PE secondary
antibody and washed. Cells were then either left at 4.degree. C. or
moved to 37.degree. C. for 30 minutes. Cells were then subjected to
fluorescent and brightfield microscopy. Cells that remained at
4.degree. C. exhibited a halo of fluorescence on the cells
demonstrative of surface staining. Cells moved to 37.degree. C.
exhibited a loss of the halo of surface fluorescence and the
generation of punctuate internal fluorescence indicative of
internalization of the 158P1D7/MAb complexes.
[0071] FIG. 29. Effect of 158P1D7 RNAi on cell survival. As
control, 3T3 cells, a cell line with no detectable expression of
158P1D7 mRNA, was also treated with the panel of siRNAs (including
oligo 158P1D7.b) and no phenotype was observed. This result
reflects the fact that the specific protein knockdown in the LNCaP
and PC3 cells is not a function of general toxicity, since the 3T3
cells did not respond to the 158P1D7.b oligo. The differential
response of the three cell lines to the Eg5 control is a reflection
of differences in levels of cell transfection and responsiveness of
the cell lines to oligo treatment.
[0072] FIG. 30. 158P1D7 MAbs Retard Growth of Human Prostate Cancer
Xenografts in Mice. Male ICR-SCID mice, 5-6 weeks old (Charles
River Laboratory, Wilmington, Mass.) were used and were maintained
in a strictly-controlled environment in accordance with the NIH
Guide for the Care and Use of Laboratory Animals. LAPC-9AD, an
androgen-dependent human prostate cancer, was used to establish
xenograft models. Stock tumors were regularly maintained in SCID
mice. At the day of implantation, stock tumors were harvested and
trimmed of necrotic tissues and minced to 1 mm3 pieces. Each mouse
received 4 pieces of tissues at the subcutaneous site of right
flank. Murine monoclonal antibodies to 158P1D7 and PSCA were tested
at a dose of 1000 .mu.g/mouse and 500 .mu.g/mouse respectively. PBS
and anti-KLH monoclonal antibody were used as controls. The study
cohort consisted of 4 groups with 6 mice in each group. MAbs were
dosed intra-peritoneally twice a week for a total of 8 doses.
Treatment was started when tumor volume reached 45 mm3. Tumor size
was monitored through caliper measurements twice a week. The
longest dimension (L) and the dimension perpendicular to it (W)
were taken to calculate tumor volume according to the formula:
W2.times.L/2. The Student's t test and the Mann-Whitney U test,
where applicable, were used to evaluate differences of tumor
growth. All tests were two-sided with .alpha.=0.05.
[0073] FIG. 31. Anti-PSCA and 158P1D7 MAbs Retard the Growth of
Human Bladder Cancer Xenografts in Mice. Male ICR-SCID mice, 5-6
weeks old (Charles River Laboratory, Wilmington, Mass.) were used
and were maintained in a strictly-controlled environment in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals.
[0074] UG-B1, a patient bladder cancer, was used to establish
xenograft models. Stock tumors regularly maintained in SCID mice
were sterilely dissected, minced, and digested using Pronase
(Calbiochem, San Diego, Calif.). Cell suspensions generated were
incubated overnight at 37.degree. C. to obtain a homogeneous
single-cell suspension. Each mouse received 2.5.times.10.sup.6
cells at the subcutaneous site of right flank. Murine monoclonal
antibodies to 158P1D7 and PSCA were tested at a dose of 500
.mu.g/mouse in the study. PBS was used as control. MAbs were dosed
intra-peritoneally twice a week for a total of 12 doses, starting
on the same day of tumor cell injection. Tumor size was monitored
through caliper measurements twice a week. The longest dimension
(L) and the dimension perpendicular to it (W) were taken to
calculate tumor volume according to the formula: W2.times.L/2. The
results show that Anti-158P1D7 mAbs are capable of inhibiting the
growth of human bladder carcinoma in mice.
[0075] FIG. 32. Effect of 158P1D7 on Proliferation of Rat1 cells.
Cells were grown overnight in 0.5% FBS and then compared to cells
treated with 10% FBS. The cells were evaluated for proliferation at
18-96 hr post-treatment by a 3H-thymidine incorporation assay and
for cell cycle analysis by a BrdU incorporation/propidium iodide
staining assay. The results show that the Rat-1 cells expressing
the 158P1D7 antigen grew effectively in low serum concentrations
(0.1%) compared to the Rat-1-Neo cells.
[0076] FIG. 33. 158P1D7 Enhances Entry Into the S Phase. Cells were
labeled with 10 .mu.M BrdU, washed, trypsinized and fixed in 0.4%
paraformaldehyde and 70% ethanol. Anti-BrdU-FITC (Pharmigen) was
added to the cells, the cells were washed and then incubated with
10 .mu.g/ml propidium iodide for 20 min prior to washing and
analysis for fluorescence at 488 nm. The results show that there
was increased labeling of cells in S-phase (DNA synthesis phase of
the cell cycle) in 3T3 cells that expressed the 158P1D7 antigen
relative to control cells.
[0077] FIG. 34. FIG. 34A. The cDNA (SEQ ID NO: 108) and amino acid
sequence (SEQ ID NO: 109) of M15/X68(2)18 VH clone #1. FIG. 34B.
The cDNA (SEQ ID NO: 110) and amino acid sequence (SEQ ID NO: 111)
of M15/X68(2)18 VL clone #2.
[0078] FIG. 35. FIG. 35A. The amino acid sequence (SEQ ID NO: 112)
of M15/X68(2)18 VH clone #1. FIG. 35B. The amino acid sequence (SEQ
ID NO: 113) of M15/X68(2)18 VL clone #2.
[0079] FIG. 36. Detection of 158P1D7 protein by
immunohistochemistry in various cancer patient specimens. Tissue
was obtained from patients with bladder transitional cell
carcinoma, breast ductal carcinoma and lung carcinoma. The results
showed expression of 158P1D7 in the tumor cells of the cancer
patients' tissue panel (A) bladder transitional cell carcinoma,
invasive Grade III (B) bladder transitional cell carcinoma,
papillary Grade II. (C) breast infiltrating ductal carcinoma,
moderately differentiated, (D) breast infiltrating ductal
carcinoma, moderate to poorly differentiated, (E) lung squamous
cell carcinoma, (F) lung adenocarcinoma, well differentiated. The
expression of 158P1D7 in bladder transitional cell carcinoma
tissues was detected mostly around the cell membrane indicating
that 158P1D7 is membrane associated.
DETAILED DESCRIPTION OF THE INVENTION
I.) Definitions
[0080] Unless otherwise defined, all terms of art, notations and
other scientific terms or terminology used herein are intended to
have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial
difference over what is generally understood in the art. Many of
the techniques and procedures described or referenced herein are
well understood and commonly employed using conventional
methodology by those skilled in the art, such as, for example, the
widely utilized molecular cloning methodologies described in
Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd.
edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. As appropriate, procedures involving the use of
commercially available kits and reagents are generally carried out
in accordance with manufacturer defined protocols and/or parameters
unless otherwise noted.
[0081] The terms "invasive bladder cancer" means bladder cancers
that have extended into the bladder muscle wall, and are meant to
include stage stage T2-T4 and disease under the TNM (tumor, node,
metastasis) system. In general, these patients have substantially
less favorable outcomes compared to patients having non-invasive
cancer. Following cystectomy, 50% or more of the patients with
invasive cancer will develop metastasis (Whittmore. Semin Urol
1983; 1:4-10).
[0082] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 158P1D7 (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence 158P1D7. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0083] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 158P1D7-related protein). For example an analog of
the 158P1D7 protein can be specifically bound by an antibody or T
cell that specifically binds to 158P1D7 protein.
[0084] The term "antibody" is used in the broadest sense. Therefore
an "antibody" can be naturally occurring or man-made such as
monoclonal antibodies produced by conventional hybridoma
technology. Anti-158P1D7 antibodies bind 158P1D7 proteins, or a
fragment thereof, and comprise monoclonal and polyclonal antibodies
as well as fragments containing the antigen-binding domain and/or
one or more complementarity determining regions of these
antibodies.
[0085] An "antibody fragment" is defined as at least a portion of
the variable region of the immunoglobulin molecule that binds to
its target, i.e., the antigen-binding region. In one embodiment it
specifically covers single anti-158P1D7 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-158P1D7 antibody compositions with polyepitopic
specificity.
[0086] The term "codon optimized sequences" refers to nucleotide
sequences that have been optimized for a particular host species by
replacing any one or more than one codon having a usage frequency
of less than about 20%, more preferably less than about 30% or 40%.
A sequence may be "completely optimized" to contain no codon having
a usage frequency of less than about 20%, more preferably less than
about 30% or 40%. Nucleotide sequences that have been optimized for
expression in a given host species by elimination of spurious
polyadenylation sequences, elimination of exon/intron splicing
signals, elimination of transposon-like repeats and/or optimization
of GC content in addition to codon optimization are referred to
herein as an "expression enhanced sequences."
[0087] The term "cytotoxic agent" refers to a substance that
inhibits or prevents one or more than one function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes chemotherapeutic agents, and toxins such as
small molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants
thereof. Examples of cytotoxic agents include, but are not limited
to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,
doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicine,
dihydroxy anthracin dione, actinomycin, diphtheria toxin,
Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A
chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis
inhibitor, and glucocorticoid and other chemotherapeutic agents, as
well as radioisotopes such as 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. Antibodies may also be conjugated
to an anti-cancer pro-drug activating enzyme capable of converting
the pro-drug to its active form.
[0088] The term "homolog" refers to a molecule which exhibits
homology to another molecule, by for example, having sequences of
chemical residues that are the same or similar at corresponding
positions.
[0089] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see, e.g.,
Stites, et al., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing, Los
Altos, Calif. (1994).
[0090] The terms "hybridize", "hybridizing", "hybridizes" and the
like, used in the context of polynucleotides, are meant to refer to
conventional hybridization conditions, preferably such as
hybridization in 50% formamide/6.times.SSC/0.1% SDS/100 .mu.g/ml
ssDNA, in which temperatures for hybridization are above 37 degrees
C. and temperatures for washing in 0.1.times.SSC/0.1% SDS are above
55 degrees C.
[0091] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated, or
present, with the peptides in their in situ environment. For
example, a polynucleotide is said to be "isolated" when it is
substantially separated from contaminant polynucleotides that
correspond or are complementary to nucleic acids other than those
of 158P1D7 or that encode polypeptides other than 158P1D7 gene
product or fragments thereof. A skilled artisan can readily employ
nucleic acid isolation procedures to obtain an isolated 158P1D7
polynucleotide. A protein is said to be "isolated," for example,
when physical, mechanical and/or chemical methods are employed to
remove the 158P1D7 protein from cellular constituents that are
normally associated, or present, with the protein. A skilled
artisan can readily employ standard purification methods to obtain
an isolated 158P1D7 protein. Alternatively, an isolated protein can
be prepared by synthetic or chemical means.
[0092] The term "mammal" refers to any organism classified as a
mammal, including mice, rats, rabbits, dogs, cats, cows, horses and
humans. In one embodiment of the invention, the mammal is a mouse.
In another embodiment of the invention, the mammal is a human.
[0093] The terms "metastatic bladder cancer" and "metastatic
disease" mean bladder cancers that have spread to regional lymph
nodes or to distant sites, and are meant to stage
T.times.N.times.M+ under the TNM system. The most common site for
bladder cancer metastasis is lymph node. Other common sites for
metastasis include lung, bone and liver.
[0094] The term "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the antibodies comprising the population are identical except
for possible naturally occurring mutations that are present in
minor amounts.
[0095] A "motif", as in biological motif of an 158P1D7-related
protein, refers to any pattern of amino acids forming part of the
primary sequence of a protein, that is associated with a particular
function (e.g. protein-protein interaction, protein-DNA
interaction, etc) or modification (e.g. that is phosphorylated,
glycosylated or amidated), or localization (e.g. secretory
sequence, nuclear localization sequence, etc.) or a sequence that
is correlated with being immunogenic, either humorally or
cellularly. A motif can be either contiguous or capable of being
aligned to certain positions that are generally correlated with a
certain function or property. In the context of HLA motifs, "motif"
refers to the pattern of residues in a peptide of defined length,
usually a peptide of from about 8 to about 13 amino acids for a
class I HLA motif and from about 6 to about 25 amino acids for a
class II HLA motif, which is recognized by a particular HLA
molecule. Peptide motifs for HLA binding are typically different
for each protein encoded by each human HLA allele and differ in the
pattern of the primary and secondary anchor residues.
[0096] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
[0097] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with mammals,
such as humans.
[0098] The term "polynucleotide" means a polymeric form of
nucleotides of at least 3, 4, 5, 6, 7, 8, 9, or 10 bases or base
pairs in length, either ribonucleotides or deoxynucleotides or a
modified form of either type of nucleotide, and is meant to include
single and double stranded forms of DNA and/or RNA. In the art,
this term is often used interchangeably with "oligonucleotide",
although "oligonucleotide" may be used to refer to the subset of
polynucleotides less than about 50 nucleotides in length. A
polynucleotide can comprise a nucleotide sequence disclosed herein
wherein thymidine (T) (as shown for example in can also be uracil
(U); this definition pertains to the differences between the
chemical structures of DNA and RNA, in particular the observation
that one of the four major bases in RNA is uracil (U) instead of
thymidine (T).
[0099] The term "polypeptide" means a polymer of at least about 4,
5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter or single letter designations for amino acids are
used. In the art, this term is often used interchangeably with
"peptide" or "protein", thus "peptide" may be used to refer to the
subset of polypeptides less than about 50 amino acids in
length.
[0100] An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the HLA
molecule. One to three, usually two, primary anchor residues within
a peptide of defined length generally defines a "motif" for an
immunogenic peptide. These residues are understood to fit in close
contact with peptide binding groove of an HLA molecule, with their
side chains buried in specific pockets of the binding groove. In
one embodiment, for example, the primary anchor residues for an HLA
class I molecule are located at position 2 (from the amino terminal
position) and at the carboxyl terminal position of a 8, 9, 10, 11,
or 12 residue peptide epitope in accordance with the invention. In
another embodiment, for example, the primary anchor residues of a
peptide that will bind an HLA class II molecule are spaced relative
to each other, rather than to the termini of a peptide, where the
peptide is generally of at least 9 amino acids in length. The
primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the
primary and/or secondary anchor positions shown in Table IV. Such
analogs are used to modulate the binding affinity and/or population
coverage of a peptide comprising a particular HLA motif or
supermotif.
[0101] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0102] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured nucleic acid sequences to reanneal when
complementary strands are present in an environment below their
melting temperature. The higher the degree of desired homology
between the probe and hybridizable sequence, the higher the
relative temperature that can be used. As a result, it follows that
higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For
additional details and explanation of stringency of hybridization
reactions, see Ausubel et al., Current Protocols in Molecular
Biology, Wiley Interscience Publishers, (1995).
[0103] "Stringent conditions" or "high stringency conditions", as
defined herein, are identified by, but not limited to, those that:
(1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium
dodecyl sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium. citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C. "Moderately stringent conditions"
are described by, but not limited to, those in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, New York: Cold Spring
Harbor Press, 1989, and include the use of washing solution and
hybridization conditions (e.g., temperature, ionic strength and %
SDS) less stringent than those described above. An example of
moderately stringent conditions is overnight incubation at
37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times.Denhardt's solution, 10% dextran sulfate, and 20
mg/mL denatured sheared salmon sperm DNA, followed by washing the
filters in 1.times.SSC at about 37-50.degree. C. The skilled
artisan will recognize how to adjust the temperature, ionic
strength, etc. as necessary to accommodate factors such as probe
length and the like.
[0104] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles.
[0105] A "transgenic animal" (e.g., a mouse or rat) is an animal
having cells that contain a transgene, which transgene was
introduced into the animal or an ancestor of the animal at a
prenatal, e.g., an embryonic stage. A "transgene" is a DNA that is
integrated into the genome of a cell from which a transgenic animal
develops.
[0106] As used herein, an HLA or cellular immune response "vaccine"
is a composition that contains or encodes one or more peptides of
the invention. There are numerous embodiments of such vaccines,
such as a cocktail of one or more individual peptides; one or more
peptides of the invention comprised by a polyepitopic peptide; or
nucleic acids that encode such individual peptides or polypeptides,
e.g., a minigene that encodes a polyepitopic peptide. The "one or
more peptides" can include any whole unit integer from 1-150 or
more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, or 150 or more peptides of the
invention. The peptides or polypeptides can optionally be modified,
such as by lipidation, addition of targeting or other sequences.
HLA class I peptides of the invention can be admixed with, or
linked to, HLA class II peptides, to facilitate activation of both
cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can
also comprise peptide-pulsed antigen presenting cells, e.g.,
dendritic cells.
[0107] The term "variant" refers to a molecule that exhibits a
variation from a described type or norm, such as a protein that has
one or more different amino acid residues in the corresponding
position(s) of a specifically described protein (e.g. the 158P1D7
protein shown in FIG. 2 or FIG. 3). An analog is an example of a
variant protein.
[0108] The 158P1D7-related proteins of the invention include those
specifically identified herein, as well as allelic variants,
conservative substitution variants, analogs and homologs that can
be isolated/generated and characterized without undue
experimentation following the methods outlined herein or readily
available in the art. Fusion proteins that combine parts of
different 158P1D7 proteins or fragments thereof, as well as fusion
proteins of a 158P1D7 protein and a heterologous polypeptide are
also included. Such 158P1D7 proteins are collectively referred to
as the 158P1D7-related proteins, the proteins of the invention, or
158P1D7. The term "158P1D7-related protein" refers to a polypeptide
fragment or an 158P1D7 protein sequence of 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more
than 25 amino acids; or, at least about 30, 35, 40, 45, 50, 55, 60,
65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.
II.) 158P1D7 Polynucleotides
[0109] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of an 158P1D7 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding an 158P1D7-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to an 158P1D7
gene or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to an 158P1D7 gene, mRNA, or to an
158P1D7 encoding polynucleotide (collectively, "158P1D7
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0110] Embodiments of a 158P1D7 polynucleotide include: a 158P1D7
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 158P1D7 as shown in FIG. 2, wherein T is U; at least 10
contiguous nucleotides of a polynucleotide having the sequence as
shown in FIG. 2; or, at least 10 contiguous nucleotides of a
polynucleotide having the sequence as shown in FIG. 2 where T is U.
For example, embodiments of 158P1D7 nucleotides comprise, without
limitation: [0111] (a) a polynucleotide comprising or consisting of
the sequence as shown in FIG. 2, wherein T can also be U; [0112]
(b) a polynucleotide comprising or consisting of the sequence as
shown in FIG. 2, from nucleotide residue number 23 through
nucleotide residue number 2548, wherein T can also be U; [0113] (c)
a polynucleotide that encodes a 158P1D7-related protein whose
sequence is encoded by the cDNAs contained in the plasmid
designated p158P1D7-Turbo/3PX deposited with American Type Culture
Collection as Accession No. PTA-3662 on 22 Aug. 2001 (sent via
Federal Express on 20 Aug. 2001); [0114] (d) a polynucleotide that
encodes an 158P1D7-related protein that is at least 90% homologous
to the entire amino acid sequence shown in FIG. 2; [0115] (e) a
polynucleotide that encodes an 158P1D7-related protein that is at
least 90% identical to the entire amino acid sequence shown in FIG.
2; [0116] (f) a polynucleotide that encodes at least one peptide
set forth in Tables V-XVIII; [0117] (g) a polynucleotide that
encodes a peptide region of at least 5 amino acids of FIG. 3 in any
whole number increment up to 841 that includes an amino acid
position having a value greater than 0.5 in the Hydrophilicity
profile of FIG. 11; [0118] (h) a polynucleotide that encodes a
peptide region of at least 5 amino acids of FIG. 3 in any whole
number increment up to 841 that includes an amino acid position
having a value less than 0.5 in the Hydropathicity profile of FIG.
12; [0119] (i) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
841 that includes an amino acid position having a value greater
than 0.5 in the Percent Accessible Residues profile of FIG. 13;
[0120] (j) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
841 that includes an amino acid position having a value greater
than 0.5 in the Average Flexibility profile on FIG. 14; [0121] (k)
a polynucleotide that encodes a peptide region of at least 5 amino
acids of FIG. 3 in any whole number increment up to 841 that
includes an amino acid position having a value greater than 0.5 in
the Beta-turn profile of FIG. 15; [0122] (l) a polynucleotide that
is fully complementary to a polynucleotide of any one of (a)-(k);
[0123] (m) a polynucleotide that selectively hybridizes under
stringent conditions to a polynucleotide of (a)-(l); [0124] (n) a
peptide that is encoded by any of (a)-(k); and, [0125] (o) a
polynucleotide of any of (a)-(m) or peptide of (n) together with a
pharmaceutical excipient and/or in a human unit dose form.
[0126] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0127] Typical embodiments of the invention disclosed herein
include 158P1D7 polynucleotides that encode specific portions of
the 158P1D7 mRNA sequence (and those which are complementary to
such sequences) such as those that encode the protein and fragments
thereof, for example of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,
625, 650, 675, 700, 725, 750, 775, 800, 825 or 841 contiguous amino
acids.
[0128] For example, representative embodiments of the invention
disclosed herein include: polynucleotides and their encoded
peptides themselves encoding about amino acid 1 to about amino acid
10 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 158P1D7 protein shown in FIG. 2, or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 158P1D7
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 158P1D7 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 158P1D7 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 158P1D7
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 158P1D7 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 158P1D7 protein shown in FIG. 2 or
FIG. 3, in increments of about 10 amino acids, ending at the
carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3.
Accordingly polynucleotides encoding portions of the amino acid
sequence (of about 10 amino acids), of amino acids 100 through the
carboxyl terminal amino acid of the 158P1D7 protein are embodiments
of the invention. Wherein it is understood that each particular
amino acid position discloses that position plus or minus five
amino acid residues.
[0129] Polynucleotides encoding relatively long portions of the
158P1D7 protein are also within the scope of the invention. For
example, polynucleotides encoding from about amino acid 1 (or 20 or
30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of
the 158P1D7 protein shown in FIG. 2 or FIG. 3 can be generated by a
variety of techniques well known in the art. These polynucleotide
fragments can include any portion of the 158P1D7 sequence as shown
in FIG. 2 or FIG. 3.
[0130] Additional illustrative embodiments of the invention
disclosed herein include 158P1D7 polynucleotide fragments encoding
one or more of the biological motifs contained within the 158P1D7
protein sequence, including one or more of the motif-bearing
subsequences of the 158P1D7 protein set forth in Tables V-XVIII. In
another embodiment, typical polynucleotide fragments of the
invention encode one or more of the regions of 158P1D7 that exhibit
homology to a known molecule. In another embodiment of the
invention, typical polynucleotide fragments can encode one or more
of the 158P1D7 N-glycosylation sites, cAMP and cGMP-dependent
protein kinase phosphorylation sites, casein kinase II
phosphorylation sites or N-myristoylation site and amidation
sites.
II.A.) Uses of 158P1D7 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
[0131] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 158P1D7 gene maps to
the chromosomal location set forth in Example 3. For example,
because the 158P1D7 gene maps to this chromosome, polynucleotides
that encode different regions of the 158P1D7 protein are used to
characterize cytogenetic abnormalities of this chromosomal locale,
such as abnormalities that are identified as being associated with
various cancers. In certain genes, a variety of chromosomal
abnormalities including rearrangements have been identified as
frequent cytogenetic abnormalities in a number of different cancers
(see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);
Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,
P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding
specific regions of the 158P1D7 protein provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 158P1D7 that may contribute to the malignant phenotype. In
this context, these polynucleotides satisfy a need in the art for
expanding the sensitivity of chromosomal screening in order to
identify more subtle and less common chromosomal abnormalities (see
e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057
(1994)).
[0132] Furthermore, as 158P1D7 was shown to be highly expressed in
bladder and other cancers, 158P1D7 polynucleotides are used in
methods assessing the status of 158P1D7 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 158P1D7 protein are used to assess the
presence of perturbations (such as deletions, insertions, point
mutations, or alterations resulting in a loss of an antigen etc.)
in specific regions of the 158P1D7 gene, such as such regions
containing one or more motifs. Exemplary assays include both RT-PCR
assays as well as single-strand conformation polymorphism (SSCP)
analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8):
369-378 (1999), both of which utilize polynucleotides encoding
specific regions of a protein to examine these regions within the
protein.
II.A.2.) Antisense Embodiments
[0133] Other specifically contemplated nucleic acid related
embodiments of the invention disclosed herein are genomic DNA,
cDNAs, ribozymes, and antisense molecules, as well as nucleic acid
molecules based on an alternative backbone, or including
alternative bases, whether derived from natural sources or
synthesized, and include molecules capable of inhibiting the RNA or
protein expression of 158P1D7. For example, antisense molecules can
be RNAs or other molecules, including peptide nucleic acids (PNAs)
or non-nucleic acid molecules such as phosphorothioate derivatives,
that specifically bind DNA or RNA in a base pair-dependent manner.
A skilled artisan can readily obtain these classes of nucleic acid
molecules using the 158P1D7 polynucleotides and polynucleotide
sequences disclosed herein.
[0134] Antisense technology entails the administration of exogenous
oligonucleotides that bind to a target polynucleotide located
within the cells. The term "antisense" refers to the fact that such
oligonucleotides are complementary to their intracellular targets,
e.g., 158P1D7. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 158P1D7 antisense oligonucleotides of
the present invention include derivatives such as
S-oligonucleotides (phosphorothioate derivatives or S-oligos, see,
Jack Cohen, supra), which exhibit enhanced cancer cell growth
inhibitory action. S-oligos (nucleoside phosphorothioates) are
isoelectronic analogs of an oligonucleotide (O-oligo) in which a
nonbridging oxygen atom of the phosphate group is replaced by a
sulfur atom. The S-oligos of the present invention can be prepared
by treatment of the corresponding O-oligos with
3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer
reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990);
and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).
Additional 158P1D7 antisense oligonucleotides of the present
invention include morpholino antisense oligonucleotides known in
the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic
Acid Drug Development 6: 169-175).
[0135] The 158P1D7 antisense oligonucleotides of the present
invention typically can be RNA or DNA that is complementary to and
stably hybridizes with the first 100 5' codons or last 100 3'
codons of the 158P1D7 genomic sequence or the corresponding mRNA.
Absolute complementarity is not required, although high degrees of
complementarity are preferred. Use of an oligonucleotide
complementary to this region allows for the selective hybridization
to 158P1D7 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 158P1D7 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 158P1D7 mRNA. Optionally, 158P1D7 antisense
oligonucleotide is a 30-mer oligonucleotide that is complementary
to a region in the first 10 5' codons or last 10 3' codons of
158P1D7. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 158P1D7 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12:
510-515 (1996).
II.A.3.) Primers and Primer Pairs
[0136] Further specific embodiments of the nucleotides of the
invention include primers and primer pairs, which allow the
specific amplification of polynucleotides of the invention or of
any specific parts thereof, and probes that selectively or
specifically hybridize to nucleic acid molecules of the invention
or to any part thereof. Primers may also be used as probes and can
be labeled with a detectable marker, such as, for example, a
radioisotope, fluorescent compound, bioluminescent compound, a
chemiluminescent compound, metal chelator or enzyme. Such probes
and primers are used to detect the presence of a 158P1D7
polynucleotide in a sample and as a means for detecting a cell
expressing a 158P1D7 protein.
[0137] Examples of such probes include polypeptides comprising all
or part of the human 158P1D7 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 158P1D7
mRNAs are also described in the Examples. As will be understood by
the skilled artisan, a great many different primers and probes can
be prepared based on the sequences provided herein and used
effectively to amplify and/or detect a 158P1D7 mRNA. Preferred
probes of the invention are polynucleotides of more than about 9,
about 12, about 15, about 18, about 20, about 23, about 25, about
30, about 35, about 40, about 45, and about 50 consecutive
nucleotides found in 158P1D7 nucleic acids disclosed herein.
[0138] The 158P1D7 polynucleotides of the invention are useful for
a variety of purposes, including but not limited to their use as
probes and primers for the amplification and/or detection of the
158P1D7 gene(s), mRNA(s), or fragments thereof; as reagents for the
diagnosis and/or prognosis of bladder cancer and other cancers; as
coding sequences capable of directing the expression of 158P1D7
polypeptides; as tools for modulating or inhibiting the expression
of the 158P1D7 gene(s) and/or translation of the 158P1D7
transcript(s); and as therapeutic agents.
II.A.4.) Isolation of 158P1D7-Encoding Nucleic Acid Molecules
[0139] The 158P1D7 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 158P1D7 gene
product(s), as well as the isolation of polynucleotides encoding
158P1D7 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of the 158P1D7 gene product as
well as polynucleotides that encode analogs of 158P1D7-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding an 158P1D7 gene are well known
(see, for example, Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York,
1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,
Wiley and Sons, 1995). For example, lambda phage cloning
methodologies can be conveniently employed, using commercially
available cloning systems (e.g., Lambda ZAP Express, Stratagene).
Phage clones containing 158P1D7 gene cDNAs can be identified by
probing with a labeled 158P1D7 cDNA or a fragment thereof. For
example, in one embodiment, the 158P1D7 cDNA (FIG. 2) or a portion
thereof can be synthesized and used as a probe to retrieve
overlapping and full-length cDNAs corresponding to a 158P1D7 gene.
The 158P1D7 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 158P1D7
DNA probes or primers.
[0140] The present invention includes the use of any probe as
described herein to identify and isolate a 158P1D7 or 158P1D7
related nucleic acid sequence from a naturally occurring source,
such as humans or other mammals, as well as the isolated nucleic
acid sequence per se, which would comprise all or most of the
sequences found in the probe used.
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0141] The invention also provides recombinant DNA or RNA molecules
containing an 158P1D7 polynucleotide, a fragment, analog or
homologue thereof, including but not limited to phages, plasmids,
phagemids, cosmids, YACs, BACs, as well as various viral and
non-viral vectors well known in the art, and cells transformed or
transfected with such recombinant DNA or RNA molecules. Methods for
generating such molecules are well known (see, for example,
Sambrook et al, 1989, supra). The invention further provides a
host-vector system comprising a recombinant DNA molecule containing
a 158P1D7 polynucleotide, fragment, analog or homologue thereof
within a suitable prokaryotic or eukaryotic host cell. Examples of
suitable eukaryotic host cells include a yeast cell, a plant cell,
or an animal cell, such as a mammalian cell or an insect cell
(e.g., a baculovirus-infectible cell such as an Sf9 or HighFive
cell). Examples of suitable mammalian cells include various bladder
cancer cell lines such as SCaBER, UM-UC3, HT1376, RT4, T24,
TCC-SUP, J82 and SW780, other transfectable or transducible bladder
cancer cell lines, as well as a number of mammalian cells routinely
used for the expression of recombinant proteins (e.g., COS, CHO,
293, 293T cells). More particularly, a polynucleotide comprising
the coding sequence of 158P1D7 or a fragment, analog or homolog
thereof can be used to generate 158P1D7 proteins or fragments
thereof using any number of host-vector systems routinely used and
widely known in the art.
[0142] A wide range of host-vector systems suitable for the
expression of 158P1D7 proteins or fragments thereof are available,
see for example, Sambrook et al., 1989, supra; Current Protocols in
Molecular Biology, 1995, supra). Preferred vectors for mammalian
expression include but are not limited to pcDNA 3.1 myc-His-tag
(Invitrogen) and the retroviral vector pSR.alpha.tkneo (Muller et
al., 1991, MCB 11:1785). Using these expression vectors, 158P1D7
can be expressed in several bladder cancer and non-bladder cell
lines, including for example SCaBER, UM-UC3, HT1376, RT4, T24,
TCC-SUP, J82 and SW780. The host-vector systems of the invention
are useful for the production of a 158P1D7 protein or fragment
thereof. Such host-vector systems can be employed to study the
functional properties of 158P1D7 and 158P1D7 mutations or
analogs.
[0143] Recombinant human 158P1D7 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 158P1D7-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 158P1D7 or fragment, analog or homolog thereof, the
158P1D7 or related protein is expressed in the 293T cells, and the
recombinant 158P1D7 protein is isolated using standard purification
methods (e.g., affinity purification using anti-158P1D7
antibodies). In another embodiment, a 158P1D7 coding sequence is
subcloned into the retroviral vector pSR.alpha.MSVtkneo and used to
infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293
and rat-1 in order to establish 158P1D7 expressing cell lines.
Various other expression systems well known in the art can also be
employed. Expression constructs encoding a leader peptide joined in
frame to the 158P1D7 coding sequence can be used for the generation
of a secreted form of recombinant 158P1D7 protein.
[0144] As discussed herein, redundancy in the genetic code permits
variation in 158P1D7 gene sequences. In particular, it is known in
the art that specific host species often have specific codon
preferences, and thus one can adapt the disclosed sequence as
preferred for a desired host. For example, preferred analog codon
sequences typically have rare codons (i.e., codons having a usage
frequency of less than about 20% in known sequences of the desired
host) replaced with higher frequency codons. Codon preferences for
a specific species are calculated, for example, by utilizing codon
usage tables available on the INTERNET.
[0145] Additional sequence modifications are known to enhance
protein expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon/intron
splice site signals, transposon-like repeats, and/or other such
well-characterized sequences that are deleterious to gene
expression. The GC content of the sequence is adjusted to levels
average for a given cellular host, as calculated by reference to
known genes expressed in the host cell. Where possible, the
sequence is modified to avoid predicted hairpin secondary mRNA
structures. Other useful modifications include the addition of a
translational initiation consensus sequence at the start of the
open reading frame, as described in Kozak, Mol. Cell Biol.,
9:5073-5080 (1989) Skilled artisans understand that the general
rule that eukaryotic ribosomes initiate translation exclusively at
the 5' proximal AUG codon is abrogated only under rare conditions
(see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR
15(20): 8125-8148 (1987)).
III.) 158P1D7-Related Proteins
[0146] Another aspect of the present invention provides
158P1D7-related proteins. Specific embodiments of 158P1D7 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 158P1D7 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 158P1D7 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 158P1D7 shown in FIG. 2 or FIG. 3.
[0147] In general, naturally occurring allelic variants of human
158P1D7 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of the
158P1D7 protein contain conservative amino acid substitutions
within the 158P1D7 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 158P1D7. One class of 158P1D7 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 158P1D7 amino acid sequence, but further
contain a radical departure from the sequence, such as a
non-conservative substitution, truncation, insertion or frame
shift. In comparisons of protein sequences, the terms, similarity,
identity, and homology each have a distinct meaning as appreciated
in the field of genetics. Moreover, orthology and paralogy can be
important concepts describing the relationship of members of a
given protein family in one organism to the members of the same
family in other organisms.
[0148] Amino acid abbreviations are provided in Table II.
Conservative amino acid substitutions can frequently be made in a
protein without altering either the conformation or the function of
the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conservative
substitutions. Such changes include substituting any of isoleucine
(I), valine (V), and leucine (L) for any other of these hydrophobic
amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine (N) and vice versa; and serine
(S) for threonine (T) and vice versa. Other substitutions can also
be considered conservative, depending on the environment of the
particular amino acid and its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can alanine (A) and valine
(V). Methionine (M), which is relatively hydrophobic, can
frequently be interchanged with leucine and isoleucine, and
sometimes with valine. Lysine (K) and arginine (R) are frequently
interchangeable in locations in which the significant feature of
the amino acid residue is its charge and the differing pK's of
these two amino acid residues are not significant. Still other
changes can be considered "conservative" in particular environments
(see, e.g. Table III herein; pages 13-15 "Biochemistry" 2nd ED.
Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992
Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;
270(20):11882-6).
[0149] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 158P1D7 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 158P1D7 variants can be made using methods known in
the art such as site-directed mutagenesis, alanine scanning, and
PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.,
10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315
(1985)), restriction selection mutagenesis (Wells et al., Philos.
Trans. R. Soc. London SerA, 317:415 (1986)) or other known
techniques can be performed on the cloned DNA to produce the
158P1D7 variant DNA.
[0150] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence that
is involved in a specific biological activity such as a
protein-protein interaction. Among the preferred scanning amino
acids are relatively small, neutral amino acids. Such amino acids
include alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid among this group because
it eliminates the side-chain beyond the beta-carbon and is less
likely to alter the main-chain conformation of the variant. Alanine
is also typically preferred because it is the most common amino
acid. Further, it is frequently found in both buried and exposed
positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does
not yield adequate amounts of variant, an isosteric amino acid can
be used.
[0151] As defined herein, 158P1D7 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 158P1D7 protein having the amino
acid sequence of FIG. 2. As used in this sentence, "cross reactive"
means that an antibody or T cell that specifically binds to an
158P1D7 variant also specifically binds to the 158P1D7 protein
having the amino acid sequence of FIG. 2. A polypeptide ceases to
be a variant of the protein shown in FIG. 2 when it no longer
contains any epitope capable of being recognized by an antibody or
T cell that specifically binds to the 158P1D7 protein. Those
skilled in the art understand that antibodies that recognize
proteins bind to epitopes of varying size, and a grouping of the
order of about four or five amino acids, contiguous or not, is
regarded as a typical number of amino acids in a minimal epitope.
See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes
et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol
(1985) 135(4):2598-608.
[0152] Another class of 158P1D7-related protein variants share 70%,
75%, 80%, 85% or 90% or more similarity with the amino acid
sequence of FIG. 2 or a fragment thereof. Another specific class of
158P1D7 protein variants or analogs comprise one or more of the
158P1D7 biological motifs described herein or presently known in
the art. Thus, encompassed by the present invention are analogs of
158P1D7 fragments (nucleic or amino acid) that have altered
functional (e.g. immunogenic) properties relative to the starting
fragment. It is to be appreciated that motifs now or which become
part of the art are to be applied to the nucleic or amino acid
sequences of FIG. 2 or FIG. 3.
[0153] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of the 158P1D7 protein shown in FIG. 2 or FIG. 3. For
example, representative embodiments of the invention comprise
peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or more contiguous amino acids of the 158P1D7 protein shown in
FIG. 2 or FIG. 3.
[0154] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of the 158P1D7 protein shown in FIG.
2 or FIG. 3, polypeptides consisting of about amino acid 10 to
about amino acid 20 of the 158P1D7 protein shown in FIG. 2 or FIG.
3, polypeptides consisting of about amino acid 20 to about amino
acid 30 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 30 to about amino acid
40 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 40 to about amino acid 50 of the
158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 50 to about amino acid 60 of the 158P1D7
protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about
amino acid 60 to about amino acid 70 of the 158P1D7 protein shown
in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70
to about amino acid 80 of the 158P1D7 protein shown in FIG. 2 or
FIG. 3, polypeptides consisting of about amino acid 80 to about
amino acid 90 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 90 to about amino acid
100 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, etc.
throughout the entirety of the 158P1D7 amino acid sequence.
Moreover, polypeptides consisting of about amino acid 1 (or 20 or
30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.)
of the 158P1D7 protein shown in FIG. 2 or FIG. 3 are embodiments of
the invention. It is to be appreciated that the starting and
stopping positions in this paragraph refer to the specified
position as well as that position plus or minus 5 residues.
[0155] 158P1D7-related proteins are generated using standard
peptide synthesis technology or using chemical cleavage methods
well known in the art. Alternatively, recombinant methods can be
used to generate nucleic acid molecules that encode a
158P1D7-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of the 158P1D7
protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
[0156] Additional illustrative embodiments of the invention
disclosed herein include 158P1D7 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within the 158P1D7 polypeptide sequence set forth in FIG. 2 or FIG.
3. Various motifs are known in the art, and a protein can be
evaluated for the presence of such motifs by a number of publicly
available Internet sites (see, e.g., Epimatrix.TM. and Epimer.TM.,
Brown University, and BIMAS.).
[0157] Motif bearing subsequences of the 158P1D7 protein are set
forth and identified in Table XIX.
[0158] Table XX sets forth several frequently occurring motifs
based on pfam searches (see URL address pfam.wustl.edu/). The
columns of Table XX list (1) motif name abbreviation, (2) percent
identity found amongst the different member of the motif family,
(3) motif name or description and (4) most common function;
location information is included if the motif is relevant for
location.
[0159] Polypeptides comprising one or more of the 158P1D7 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 158P1D7 motifs discussed above are associated with growth
dysregulation and because 158P1D7 is overexpressed in certain
cancers (See, e.g., Table I). Casein kinase II, cAMP and
camp-dependent protein kinase, and Protein Kinase C, for example,
are enzymes known to be associated with the development of the
malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):
165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338
(1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126
(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and
O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both
glycosylation and myristoylation are protein modifications also
associated with cancer and cancer progression (see e.g. Dennis et
al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp.
Cell Res. 235(1): 145-154 (1997)). Amidation is another protein
modification also associated with cancer and cancer progression
(see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13):
169-175 (1992)).
[0160] In another embodiment, proteins of the invention comprise
one or more of the immunoreactive epitopes identified in accordance
with art-accepted methods, such as the peptides set forth in Tables
V-XVIII. CTL epitopes can be determined using specific algorithms
to identify peptides within an 158P1D7 protein that are capable of
optimally binding to specified HLA alleles (e.g., Table IV;
Epimatrix.TM. and Epimer.TM., Brown University; and BIMAS.)
Moreover, processes for identifying peptides that have sufficient
binding affinity for HLA molecules and which are correlated with
being immunogenic epitopes, are well known in the art, and are
carried out without undue experimentation. In addition, processes
for identifying peptides that are immunogenic epitopes, are well
known in the art, and are carried out without undue experimentation
either in vitro or in vivo.
[0161] Also known in the art are principles for creating analogs of
such epitopes in order to modulate immunogenicity. For example, one
begins with an epitope that bears a CTL or HTL motif (see, e.g.,
the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
The epitope is analoged by substituting out an amino acid at one of
the specified positions, and replacing it with another amino acid
specified for that position. For example, one can substitute out a
deleterious residue in favor of any other residue, such as a
preferred residue as defined in Table IV; substitute a
less-preferred residue with a preferred residue as defined in Table
IV; or substitute an originally-occurring preferred residue with
another preferred residue as defined in Table IV. Substitutions can
occur at primary anchor positions or at other positions in a
peptide; see, e.g., Table IV.
[0162] A variety of references reflect the art regarding the
identification and generation of epitopes in a protein of interest
as well as analogs thereof. See, for example, WO 9733602 to Chesnut
et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al.,
J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol.
1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4):
249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk
et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3
(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et
al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8):
3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278;
Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;
Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al.,
J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994
1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2):
79-92.
[0163] Related embodiments of the inventions include polypeptides
comprising combinations of the different motifs set forth in Table
XIX, and/or, one or more of the predicted CTL epitopes of Table V
through Table XVIII, and/or, one or more of the T cell binding
motifs known in the art. Preferred embodiments contain no
insertions, deletions or substitutions either within the motifs or
the intervening sequences of the polypeptides. In addition,
embodiments which include a number of either N-terminal and/or
C-terminal amino acid residues on either side of these motifs may
be desirable (to, for example, include a greater portion of the
polypeptide architecture in which the motif is located). Typically
the number of N-terminal and/or C-terminal amino acid residues on
either side of a motif is between about 1 to about 100 amino acid
residues, preferably 5 to about 50 amino acid residues.
[0164] 158P1D7-related proteins are embodied in many forms,
preferably in isolated form. A purified 158P1D7 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 158P1D7 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 158P1D7-related protein include
purified 158P1D7-related proteins and functional, soluble
158P1D7-related proteins. In one embodiment, a functional, soluble
158P1D7 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0165] The invention also provides 158P1D7 proteins comprising
biologically active fragments of the 158P1D7 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
158P1D7 protein, such as the ability to elicit the generation of
antibodies that specifically bind an epitope associated with the
158P1D7 protein; to be bound by such antibodies; to elicit the
activation of HTL or CTL; and/or, to be recognized by HTL or
CTL.
[0166] 158P1D7-related polypeptides that contain particularly
interesting structures can be predicted and/or identified using
various analytical techniques well known in the art, including, for
example, the methods of Chou-Fasman, Garnier-Robson,
Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf
analysis, or on the basis of immunogenicity. Fragments that contain
such structures are particularly useful in generating
subunit-specific anti-158P1D7 antibodies, or T cells or in
identifying cellular factors that bind to 158P1D7.
[0167] CTL epitopes can be determined using specific algorithms to
identify peptides within an 158P1D7 protein that are capable of
optimally binding to specified HLA alleles (e.g., by using the
SYFPEITHI site at World Wide Web; the listings in Table IV(A)-(E);
Epimatrix.TM. and Epimer.TM. Brown University; and BIMAS).
Illustrating this, peptide epitopes from 158P1D7 that are presented
in the context of human MHC class 1 molecules HLA-A1, A2, A3, All,
A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the
complete amino acid sequence of the 158P1D7 protein was entered
into the HLA Peptide Motif Search algorithm found in the
Bioinformatics and Molecular Analysis Section (BIMAS) web site
listed above. The HLA peptide motif search algorithm was developed
by Dr. Ken Parker based on binding of specific peptide sequences in
the groove of HLA Class 1 molecules, in particular HLA-A2 (see,
e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science
255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992);
Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm
allows location and ranking of 8-mer, 9-mer, and 10-mer peptides
from a complete protein sequence for predicted binding to HLA-A2 as
well as numerous other HLA Class I molecules. Many HLA class I
binding peptides are 8-, 9-, 10 or 11-mers. For example, for class
I HLA-A2, the epitopes preferably contain a leucine (L) or
methionine (M) at position 2 and a valine (V) or leucine (L) at the
C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7
(1992)). Selected results of 158P1D7 predicted binding peptides are
shown in Tables V-XVIII herein. In Tables V-XVIII, the top 50
ranking candidates, 9-mers and 10-mers, for each family member are
shown along with their location, the amino acid sequence of each
specific peptide, and an estimated binding score. The binding score
corresponds to the estimated half time of dissociation of complexes
containing the peptide at 37.degree. C. at pH 6.5. Peptides with
the highest binding score are predicted to be the most tightly
bound to HLA Class I on the cell surface for the greatest period of
time and thus represent the best immunogenic targets for T-cell
recognition.
[0168] Actual binding of peptides to an HLA allele can be evaluated
by stabilization of HLA expression on the antigen-processing
defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8
(1997) and Peshwa et al., Prostate 36:129-38 (1998)).
Immunogenicity of specific peptides can be evaluated in vitro by
stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence
of antigen presenting cells such as dendritic cells.
[0169] It is to be appreciated that every epitope predicted by the
BIMAS site, Epimer.TM. and Epimatrix.TM. sites, or specified by the
HLA class I or class II motifs available in the art or which become
part of the art such as set forth in Table IV (or determined using
World Wide Web) are to be "applied" to the 158P1D7 protein. As used
in this context "applied" means that the 158P1D7 protein is
evaluated, e.g., visually or by computer-based patterns finding
methods, as appreciated by those of skill in the relevant art.
Every subsequence of the 158P1D7 of 8, 9, 10, or 11 amino acid
residues that bears an HLA Class I motif, or a subsequence of 9 or
more amino acid residues that bear an HLA Class II motif are within
the scope of the invention.
III.B.) Expression of 158P1D7-Related Proteins
[0170] In an embodiment described in the examples that follow,
158P1D7 can be conveniently expressed in cells (such as 293T cells)
transfected with a commercially available expression vector such as
a CMV-driven expression vector encoding 158P1D7 with a C-terminal
6.times.His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5,
GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides
an IgGK secretion signal that can be used to facilitate the
production of a secreted 158P1D7 protein in transfected cells. The
secreted HIS-tagged 158P1D7 in the culture media can be purified,
e.g., using a nickel column using standard techniques.
III. C.) Modifications of 158P1D7-Related Proteins
[0171] Modifications of 158P1D7-related proteins such as covalent
modifications are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of a 158P1D7 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of the 158P1D7. Another type of covalent
modification of the 158P1D7 polypeptide included within the scope
of this invention comprises altering the native glycosylation
pattern of a protein of the invention. Another type of covalent
modification of 158P1D7 comprises linking the 158P1D7 polypeptide
to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0172] The 158P1D7-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 158P1D7
fused to another, heterologous polypeptide or amino acid sequence.
Such a chimeric molecule can be synthesized chemically or
recombinantly. A chimeric molecule can have a protein of the
invention fused to another tumor-associated antigen or fragment
thereof. Alternatively, a protein in accordance with the invention
can comprise a fusion of fragments of the 158P1D7 sequence (amino
or nucleic acid) such that a molecule is created that is not,
through its length, directly homologous to the amino or nucleic
acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule
can comprise multiples of the same subsequence of 158P1D7. A
chimeric molecule can comprise a fusion of a 158P1D7-related
protein with a polyhistidine epitope tag, which provides an epitope
to which immobilized nickel can selectively bind, with cytokines or
with growth factors. The epitope tag is generally placed at the
amino- or carboxyl-terminus of the 158P1D7. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
158P1D7-related protein with an immunoglobulin or a particular
region of an immunoglobulin. For a bivalent form of the chimeric
molecule (also referred to as an "immunoadhesin"), such a fusion
could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a 158P1D7 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 158P1D7-Related Proteins
[0173] The proteins of the invention have a number of different
uses. As 158P1D7 is highly expressed in bladder and other cancers,
158P1D7-related proteins are used in methods that assess the status
of 158P1D7 gene products in normal versus cancerous tissues,
thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of the 158P1D7 protein are used
to assess the presence of perturbations (such as deletions,
insertions, point mutations etc.) in those regions (such as regions
containing one or more motifs). Exemplary assays utilize antibodies
or T cells targeting 158P1D7-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within the 158P1D7 polypeptide sequence in order to evaluate the
characteristics of this region in normal versus cancerous tissues
or to elicit an immune response to the epitope. Alternatively,
158P1D7-related proteins that contain the amino acid residues of
one or more of the biological motifs in the 158P1D7 protein are
used to screen for factors that interact with that region of
158P1D7.
[0174] 158P1D7 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of an 158P1D7 protein), for identifying agents or cellular
factors that bind to 158P1D7 or a particular structural domain
thereof, and in various therapeutic and diagnostic contexts,
including but not limited to diagnostic assays, cancer vaccines and
methods of preparing such vaccines.
[0175] Proteins encoded by the 158P1D7 genes, or by analogs,
homologs or fragments thereof, have a variety of uses, including
but not limited to generating antibodies and in methods for
identifying ligands and other agents and cellular constituents that
bind to an 158P1D7 gene product. Antibodies raised against an
158P1D7 protein or fragment thereof are useful in diagnostic and
prognostic assays, and imaging methodologies in the management of
human cancers characterized by expression of 158P1D7 protein, such
as those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 158P1D7-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0176] Various immunological assays useful for the detection of
158P1D7 proteins are used, including but not limited to various
types of radioimmunoassays, enzyme-linked immunosorbent assays
(ELISA), enzyme-linked immunofluorescent assays (ELIFA),
immunocytochemical methods, and the like. Antibodies can be labeled
and used as immunological imaging reagents capable of detecting
158P1D7-expressing cells (e.g., in radioscintigraphic imaging
methods). 158P1D7 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
IV.) 158P1D7 Antibodies
[0177] Another aspect of the invention provides antibodies that
bind to 158P1D7-related proteins. Preferred antibodies specifically
bind to a 158P1D7-related protein and do not bind (or bind weakly)
to peptides or proteins that are not 158P1D7-related proteins. For
example, antibodies bind 158P1D7 can bind 158P1D7-related proteins
such as the homologs or analogs thereof.
[0178] 158P1D7 antibodies of the invention are particularly useful
in bladder cancer diagnostic and prognostic assays, and imaging
methodologies. Similarly, such antibodies are useful in the
treatment, diagnosis, and/or prognosis of other cancers, to the
extent 158P1D7 is also expressed or overexpressed in these other
cancers. Moreover, intracellularly expressed antibodies (e.g.,
single chain antibodies) are therapeutically useful in treating
cancers in which the expression of 158P1D7 is involved, such as
advanced or metastatic bladder cancers.
[0179] The invention also provides various immunological assays
useful for the detection and quantification of 158P1D7 and mutant
158P1D7-related proteins. Such assays can comprise one or more
158P1D7 antibodies capable of recognizing and binding a
158P1D7-related protein, as appropriate. These assays are performed
within various immunological assay formats well known in the art,
including but not limited to various types of radioimmunoassays,
enzyme-linked immunosorbent assays (ELISA), enzyme-linked
immunofluorescent assays (ELIFA), and the like.
[0180] Immunological non-antibody assays of the invention also
comprise T cell immunogenicity assays (inhibitory or stimulatory)
as well as major histocompatibility complex (MHC) binding
assays.
[0181] In addition, immunological imaging methods capable of
detecting bladder cancer and other cancers expressing 158P1D7 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 158P1D7
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 158P1D7 expressing cancers such as
bladder cancer.
[0182] 158P1D7 antibodies are also used in methods for purifying a
158P1D7-related protein and for isolating 158P1D7 homologues and
related molecules. For example, a method of purifying a
158P1D7-related protein comprises incubating an 158P1D7 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 158P1D7-related protein under conditions that
permit the 158P1D7 antibody to bind to the 158P1D7-related protein;
washing the solid matrix to eliminate impurities; and eluting the
158P1D7-related protein from the coupled antibody. Other uses of
the 158P1D7 antibodies of the invention include generating
anti-idiotypic antibodies that mimic the 158P1D7 protein.
[0183] Various methods for the preparation of antibodies are well
known in the art. For example, antibodies can be prepared by
immunizing a suitable mammalian host using a 158P1D7-related
protein, peptide, or fragment, in isolated or immunoconjugated form
(Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane
(1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
In addition, fusion proteins of 158P1D7 can also be used, such as a
158P1D7 GST-fusion protein. In a particular embodiment, a GST
fusion protein comprising all or most of the amino acid sequence of
FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate
appropriate antibodies. In another embodiment, a 158P1D7-related
protein is synthesized and used as an immunogen.
[0184] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 158P1D7-related protein or
158P1D7 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0185] The amino acid sequence of 158P1D7 as shown in FIG. 2 or
FIG. 3 can be analyzed to select specific regions of the 158P1D7
protein for generating antibodies. For example, hydrophobicity and
hydrophilicity analyses of the 158P1D7 amino acid sequence are used
to identify hydrophilic regions in the 158P1D7 structure (see,
e.g., the Example entitled "Antigenicity profiles"). Regions of the
158P1D7 protein that show immunogenic structure, as well as other
regions and domains, can readily be identified using various other
methods known in the art, such as Chou-Fasman, Hopp and Woods,
Kyte-Doolittle, Janin, Bhaskaran and Ponnuswamy, Deleage and Roux,
Garnier-Robson, Eisenberg, Karplus-Schultz, or Jameson-Wolf
analysis. Thus, each region identified by any of these programs or
methods is within the scope of the present invention. Methods for
the generation of 158P1D7 antibodies are further illustrated by way
of the examples provided herein. Methods for preparing a protein or
polypeptide for use as an immunogen are well known in the art. Also
well known in the art are methods for preparing immunogenic
conjugates of a protein with a carrier, such as BSA, KLH or other
carrier protein. In some circumstances, direct conjugation using,
for example, carbodiimide reagents are used; in other instances
linking reagents such as those supplied by Pierce Chemical Co.,
Rockford, Ill., are effective. Administration of a 158P1D7
immunogen is often conducted by injection over a suitable time
period and with use of a suitable adjuvant, as is understood in the
art. During the immunization schedule, titers of antibodies can be
taken to determine adequacy of antibody formation.
[0186] 158P1D7 monoclonal antibodies can be produced by various
means well known in the art. For example, immortalized cell lines
that secrete a desired monoclonal antibody are prepared using the
standard hybridoma technology of Kohler and Milstein or
modifications that immortalize antibody-producing B cells, as is
generally known. Immortalized cell lines that secrete the desired
antibodies are screened by immunoassay in which the antigen is a
158P1D7-related protein. When the appropriate immortalized cell
culture is identified, the cells can be expanded and antibodies
produced either from in vitro cultures or from ascites fluid.
[0187] One embodiment of the invention is a mouse hybridoma that
produces murine monoclonal antibodies designated X68(2)18 (a.k.a.
M15-68(2)18.1.1) deposited with American Type Culture Collection
(ATCC), P.O. Box 1549, Manassas, Va. 20108 on 6 Feb. 2004 and
assigned Accession No. PTA-5801.
[0188] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of the 158P1D7 protein can also be produced in
the context of chimeric or complementarity determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
158P1D7 antibodies can also be produced, and are preferred for use
in therapeutic contexts. Methods for humanizing murine and other
non-human antibodies, by substituting one or more of the non-human
antibody CDRs for corresponding human antibody sequences, are well
known (see for example, Jones et al., 1986, Nature 321: 522-525;
Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al.,
1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.
Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol.
151: 2296.
[0189] Methods for producing fully human monoclonal antibodies
include phage display and transgenic methods (for review, see
Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully
human 158P1D7 monoclonal antibodies can be generated using cloning
technologies employing large human Ig gene combinatorial libraries
(i.e., phage display) (Griffiths and Hoogenboom, Building an in
vitro immune system: human antibodies from phage display libraries.
In: Protein Engineering of Antibody Molecules for Prophylactic and
Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham
Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from
combinatorial libraries. Id., pp 65-82). Fully human 158P1D7
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued
19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and, 6,114,598 issued
5 Sep. 2000). This method avoids the in vitro manipulation required
with phage display technology and efficiently produces high
affinity authentic human antibodies.
[0190] Reactivity of 158P1D7 antibodies with an 158P1D7-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 158P1D7-related proteins,
158P1D7-expressing cells or extracts thereof. A 158P1D7 antibody or
fragment thereof can be labeled with a detectable marker or
conjugated to a second molecule. Suitable detectable markers
include, but are not limited to, a radioisotope, a fluorescent
compound, a bioluminescent compound, chemiluminescent compound, a
metal chelator or an enzyme. Further, bi-specific antibodies
specific for two or more 158P1D7 epitopes are generated using
methods generally known in the art. Homodimeric antibodies can also
be generated by cross-linking techniques known in the art (e.g.,
Wolff et al., Cancer Res. 53: 2560-2565).
V.) 158P1D7 Cellular Immune Responses
[0191] The mechanism by which T cells recognize antigens has been
delineated. Efficacious peptide epitope vaccine compositions of the
invention induce a therapeutic or prophylactic immune responses in
very broad segments of the world-wide population. For an
understanding of the value and efficacy of compositions of the
invention that induce cellular immune responses, a brief review of
immunology-related technology is provided.
[0192] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the
study of single amino acid substituted antigen analogs and the
sequencing of endogenously bound, naturally processed peptides,
critical residues that correspond to motifs required for specific
binding to HLA antigen molecules have been identified and are set
forth in Table IV (see also, e.g., Southwood, et al., J. Immunol.
160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al., SYFPEITHI, access via World Wide Web at URL
syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.
Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,
1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;
Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et
al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.
155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490,
1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and
Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12,
Review).
[0193] Furthermore, x-ray crystallographic analyses of HLA-peptide
complexes have revealed pockets within the peptide binding
cleft/groove of HLA molecules which accommodate, in an
allele-specific mode, residues borne by peptide ligands; these
residues in turn determine the HLA binding capacity of the peptides
in which they are present. (See, e.g., Madden, D. R. Annu. Rev.
Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont
et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994;
Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al.,
Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA
90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.
L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science
257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et
al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and
Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
[0194] Accordingly, the definition of class I and class II
allele-specific HLA binding motifs, or class I or class II
supermotifs allows identification of regions within a protein that
are correlated with binding to particular HLA antigen(s).
[0195] Thus, by a process of HLA motif identification, candidates
for epitope-based vaccines have been identified; such candidates
can be further evaluated by HLA-peptide binding assays to determine
binding affinity and/or the time period of association of the
epitope and its corresponding HLA molecule. Additional confirmatory
work can be performed to select, amongst these vaccine candidates,
epitopes with preferred characteristics in terms of population
coverage, and/or immunogenicity.
[0196] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0197] 1) Evaluation of primary T cell cultures from normal
individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol.
32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105,
1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et
al., Human Immunol. 59:1, 1998). This procedure involves the
stimulation of peripheral blood lymphocytes (PBL) from normal
subjects with a test peptide in the presence of antigen presenting
cells in vitro over a period of several weeks. T cells specific for
the peptide become activated during this time and are detected
using, e.g., a lymphokine- or 51Cr-release assay involving peptide
sensitized target cells.
[0198] 2) Immunization of HLA transgenic mice (see, e.g.,
Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A.
et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J.
Immunol. 159:4753, 1997). For example, in such methods peptides in
incomplete Freund's adjuvant are administered subcutaneously to HLA
transgenic mice. Several weeks following immunization, splenocytes
are removed and cultured in vitro in the presence of test peptide
for approximately one week. Peptide-specific T cells are detected
using, e.g., a 51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0199] 3) Demonstration of recall T cell responses from immune
individuals who have been either effectively vaccinated and/or from
chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp.
Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997;
Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S.
C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J.
Virol. 71:6011, 1997). Accordingly, recall responses are detected
by culturing PBL from subjects that have been exposed to the
antigen due to disease and thus have generated an immune response
"naturally", or from patients who were vaccinated against the
antigen. PBL from subjects are cultured in vitro for 1-2 weeks in
the presence of test peptide plus antigen presenting cells (APC) to
allow activation of "memory" T cells, as compared to "naive" T
cells. At the end of the culture period, T cell activity is
detected using assays including 51Cr release involving
peptide-sensitized targets, T cell proliferation, or lymphokine
release.
VI.) 158P1D7 Transgenic Animals
[0200] Nucleic acids that encode a 158P1D7-related protein can also
be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. In accordance with established
techniques, cDNA encoding 158P1D7 can be used to clone genomic DNA
that encodes 158P1D7. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 158P1D7. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. Nos.
4,736,866 issued 12 Apr. 1988, and 4,870,009 issued 26 Sep. 1989.
Typically, particular cells would be targeted for 158P1D7 transgene
incorporation with tissue-specific enhancers.
[0201] Transgenic animals that include a copy of a transgene
encoding 158P1D7 can be used to examine the effect of increased
expression of DNA that encodes 158P1D7. Such animals can be used as
tester animals for reagents thought to confer protection from, for
example, pathological conditions associated with its
overexpression. In accordance with this aspect of the invention, an
animal is treated with a reagent and a reduced incidence of a
pathological condition, compared to untreated animals that bear the
transgene, would indicate a potential therapeutic intervention for
the pathological condition.
[0202] Alternatively, non-human homologues of 158P1D7 can be used
to construct a 158P1D7 "knock out" animal that has a defective or
altered gene encoding 158P1D7 as a result of homologous
recombination between the endogenous gene encoding 158P1D7 and
altered genomic DNA encoding 158P1D7 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 158P1D7 can be
used to clone genomic DNA encoding 158P1D7 in accordance with
established techniques. A portion of the genomic DNA encoding
158P1D7 can be deleted or replaced with another gene, such as a
gene encoding a selectable marker that can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA
(both at the 5' and 3' ends) are included in the vector (see, e.g.,
Thomas and Capecchi, Cell, 51:503 (1987) for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced DNA has homologously recombined with the
endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915
(1992)). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse or rat) to form aggregation chimeras (see,
e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.
113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal, and the embryo brought to term
to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knock out animals
can be characterized, for example, for their ability to defend
against certain pathological conditions or for their development of
pathological conditions due to absence of the 158P1D7
polypeptide.
VII.) Methods for the Detection of 158P1D7
[0203] Another aspect of the present invention relates to methods
for detecting 158P1D7 polynucleotides and polypeptides and
158P1D7-related proteins, as well as methods for identifying a cell
that expresses 158P1D7. The expression profile of 158P1D7 makes it
a diagnostic marker for metastasized disease. Accordingly, the
status of 158P1D7 gene products provides information useful for
predicting a variety of factors including susceptibility to
advanced stage disease, rate of progression, and/or tumor
aggressiveness. As discussed in detail herein, the status of
158P1D7 gene products in patient samples can be analyzed by a
variety protocols that are well known in the art including
immunohistochemical analysis, the variety of Northern blotting
techniques including in situ hybridization, RT-PCR analysis (for
example on laser capture micro-dissected samples), Western blot
analysis and tissue array analysis.
[0204] More particularly, the invention provides assays for the
detection of 158P1D7 polynucleotides in a biological sample, such
as urine, serum, bone, prostatic fluid, tissues, semen, cell
preparations, and the like. Detectable 158P1D7 polynucleotides
include, for example, a 158P1D7 gene or fragment thereof, 158P1D7
mRNA, alternative splice variant 158P1D7 mRNAs, and recombinant DNA
or RNA molecules that contain a 158P1D7 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 158P1D7
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0205] In one embodiment, a method for detecting an 158P1D7 mRNA in
a biological sample comprises producing cDNA from the sample by
reverse transcription using at least one primer; amplifying the
cDNA so produced using an 158P1D7 polynucleotides as sense and
antisense primers to amplify 158P1D7 cDNAs therein; and detecting
the presence of the amplified 158P1D7 cDNA. Optionally, the
sequence of the amplified 158P1D7 cDNA can be determined.
[0206] In another embodiment, a method of detecting a 158P1D7 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 158P1D7
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 158P1D7 gene. Any number of appropriate
sense and antisense probe combinations can be designed from the
nucleotide sequence provided for the 158P1D7 (FIG. 2) and used for
this purpose.
[0207] The invention also provides assays for detecting the
presence of an 158P1D7 protein in a tissue or other biological
sample such as urine, serum, semen, bone, prostate, cell
preparations, and the like. Methods for detecting a 158P1D7-related
protein are also well known and include, for example,
immunoprecipitation, immunohistochemical analysis, Western blot
analysis, molecular binding assays, ELISA, ELIFA and the like. For
example, a method of detecting the presence of a 158P1D7-related
protein in a biological sample comprises first contacting the
sample with a 158P1D7 antibody, a 158P1D7-reactive fragment
thereof, or a recombinant protein containing an antigen binding
region of a 158P1D7 antibody; and then detecting the binding of
158P1D7-related protein in the sample.
[0208] Methods for identifying a cell that expresses 158P1D7 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 158P1D7 gene comprises
detecting the presence of 158P1D7 mRNA in the cell. Methods for the
detection of particular mRNAs in cells are well known and include,
for example, hybridization assays using complementary DNA probes
(such as in situ hybridization using labeled 158P1D7 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 158P1D7, and other amplification type detection
methods, such as, for example, branched DNA, SISBA, TMA and the
like). Alternatively, an assay for identifying a cell that
expresses a 158P1D7 gene comprises detecting the presence of
158P1D7-related protein in the cell or secreted by the cell.
Various methods for the detection of proteins are well known in the
art and are employed for the detection of 158P1D7-related proteins
and cells that express 158P1D7-related proteins.
[0209] 158P1D7 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 158P1D7 gene
expression. For example, 158P1D7 expression is significantly
upregulated in bladder cancer, and is expressed in cancers of the
tissues listed in Table I. Identification of a molecule or
biological agent that inhibits 158P1D7 expression or
over-expression in cancer cells is of therapeutic value. For
example, such an agent can be identified by using a screen that
quantifies 158P1D7 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
VIII.) Methods for Monitoring the Status of 158P1D7-Related Genes
and Their Products
[0210] Oncogenesis is known to be a multistep process where
cellular growth becomes progressively dysregulated and cells
progress from a normal physiological state to precancerous and then
cancerous states (see, e.g., Alers et al., Lab Invest. 77(5):
437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)).
In this context, examining a biological sample for evidence of
dysregulated cell growth (such as aberrant 158P1D7 expression in
cancers) allows for early detection of such aberrant physiology,
before a pathologic state such as cancer has progressed to a stage
that therapeutic options are more limited and or the prognosis is
worse. In such examinations, the status of 158P1D7 in a biological
sample of interest can be compared, for example, to the status of
158P1D7 in a corresponding normal sample (e.g. a sample from that
individual or alternatively another individual that is not affected
by a pathology). An alteration in the status of 158P1D7 in the
biological sample (as compared to the normal sample) provides
evidence of dysregulated cellular growth. In addition to using a
biological sample that is not affected by a pathology as a normal
sample, one can also use a predetermined normative value such as a
predetermined normal level of mRNA expression (see, e.g., Greyer et
al., J. Comp. Neurol. 1996 Dec. 9; 376(2):306-14 and U.S. Pat. No.
5,837,501) to compare 158P1D7 status in a sample.
[0211] The term "status" in this context is used according to its
art accepted meaning and refers to the condition or state of a gene
and its products. Typically, skilled artisans use a number of
parameters to evaluate the condition or state of a gene and its
products. These include, but are not limited to the location of
expressed gene products (including the location of 158P1D7
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 158P1D7 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 158P1D7
comprises a change in the location of 158P1D7 and/or 158P1D7
expressing cells and/or an increase in 158P1D7 mRNA and/or protein
expression.
[0212] 158P1D7 status in a sample can be analyzed by a number of
means well known in the art, including without limitation,
immunohistochemical analysis, in situ hybridization, RT-PCR
analysis on laser capture micro-dissected samples, Western blot
analysis, and tissue array analysis. Typical protocols for
evaluating the status of the 158P1D7 gene and gene products are
found, for example in Ausubel et al. eds., 1995, Current Protocols
In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the
status of 158P1D7 in a biological sample is evaluated by various
methods utilized by skilled artisans including, but not limited to
genomic Southern analysis (to examine, for example perturbations in
the 158P1D7 gene), Northern analysis and/or PCR analysis of 158P1D7
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 158P1D7 mRNAs), and, Western
and/or immunohistochemical analysis (to examine, for example
alterations in polypeptide sequences, alterations in polypeptide
localization within a sample, alterations in expression levels of
158P1D7 proteins and/or associations of 158P1D7 proteins with
polypeptide binding partners). Detectable 158P1D7 polynucleotides
include, for example, a 158P1D7 gene or fragment thereof, 158P1D7
mRNA, alternative splice variants, 158P1D7 mRNAs, and recombinant
DNA or RNA molecules containing a 158P1D7 polynucleotide.
[0213] The expression profile of 158P1D7 makes it a diagnostic
marker for local and/or metastasized disease, and provides
information on the growth or oncogenic potential of a biological
sample. In particular, the status of 158P1D7 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 158P1D7 status and diagnosing
cancers that express 158P1D7, such as cancers of the tissues listed
in Table I. For example, because 158P1D7 mRNA is so highly
expressed in bladder and other cancers relative to normal bladder
tissue, assays that evaluate the levels of 158P1D7 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 158P1D7 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0214] The expression status of 158P1D7 provides information
including the presence, stage and location of dysplastic,
precancerous and cancerous cells, predicting susceptibility to
various stages of disease, and/or for gauging tumor aggressiveness.
Moreover, the expression profile makes it useful as an imaging
reagent for metastasized disease. Consequently, an aspect of the
invention is directed to the various molecular prognostic and
diagnostic methods for examining the status of 158P1D7 in
biological samples such as those from individuals suffering from,
or suspected of suffering from a pathology characterized by
dysregulated cellular growth, such as cancer.
[0215] As described above, the status of 158P1D7 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 158P1D7 in a biological sample
taken from a specific location in the body can be examined by
evaluating the sample for the presence or absence of 158P1D7
expressing cells (e.g. those that express 158P1D7 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 158P1D7-expressing cells are
found in a biological sample that does not normally contain such
cells (such as a lymph node), because such alterations in the
status of 158P1D7 in a biological sample are often associated with
dysregulated cellular growth. Specifically, one indicator of
dysregulated cellular growth is the metastases of cancer cells from
an organ of origin (such as the bladder) to a different area of the
body (such as a lymph node). By example, evidence of dysregulated
cellular growth is important because occult lymph node metastases
can be detected in a substantial proportion of patients with
prostate cancer, and such metastases are associated with known
predictors of disease progression (see, e.g., Murphy et al.,
Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol.
18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt
1):474-8).
[0216] In one aspect, the invention provides methods for monitoring
158P1D7 gene products by determining the status of 158P1D7 gene
products expressed by cells from an individual suspected of having
a disease associated with dysregulated cell growth (such as
hyperplasia or cancer) and then comparing the status so determined
to the status of 158P1D7 gene products in a corresponding normal
sample. The presence of aberrant 158P1D7 gene products in the test
sample relative to the normal sample provides an indication of the
presence of dysregulated cell growth within the cells of the
individual.
[0217] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 158P1D7 mRNA or protein
expression in a test cell or tissue sample relative to expression
levels in the corresponding normal cell or tissue. The presence of
158P1D7 mRNA can, for example, be evaluated in tissue samples
including but not limited to those listed in Table I. The presence
of significant 158P1D7 expression in any of these tissues is useful
to indicate the emergence, presence and/or severity of a cancer,
since the corresponding normal tissues do not express 158P1D7 mRNA
or express it at lower levels.
[0218] In a related embodiment, 158P1D7 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 158P1D7 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 158P1D7 expressed in a corresponding
normal sample. In one embodiment, the presence of 158P1D7 protein
is evaluated, for example, using immunohistochemical methods.
158P1D7 antibodies or binding partners capable of detecting 158P1D7
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0219] In a further embodiment, one can evaluate the status of
158P1D7 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules. These perturbations can include insertions, deletions,
substitutions and the like. Such evaluations are useful because
perturbations in the nucleotide and amino acid sequences are
observed in a large number of proteins associated with a growth
dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.
Pathol. 26(8):369-378). For example, a mutation in the sequence of
158P1D7 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 158P1D7 indicates a potential loss of function or
increase in tumor growth.
[0220] A wide variety of assays for observing perturbations in
nucleotide and amino acid sequences are well known in the art. For
example, the size and structure of nucleic acid or amino acid
sequences of 158P1D7 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and 5,952,170
issued 17 Jan. 1995).
[0221] Additionally, one can examine the methylation status of the
158P1D7 gene in a biological sample. Aberrant demethylation and/or
hypermethylation of CpG islands in gene 5' regulatory regions
frequently occurs in immortalized and transformed cells, and can
result in altered expression of various genes. For example,
promoter hypermethylation of the DBCCR1, PAX6 and APC genes have
been detected in bladder cancers leading to aberrant expression of
the genes (Esteller et al., Cancer Res 2001; 61:3225-3229) A
variety of assays for examining methylation status of a gene are
well known in the art. For example, one can utilize, in Southern
hybridization approaches, methylation-sensitive restriction enzymes
which cannot cleave sequences that contain methylated CpG sites to
assess the methylation status of CpG islands. In addition, MSP
(methylation specific PCR) can rapidly profile the methylation
status of all the CpG sites present in a CpG island of a given
gene. This procedure involves initial modification of DNA by sodium
bisulfite (which will convert all unmethylated cytosines to uracil)
followed by amplification using primers specific for methylated
versus unmethylated DNA. Protocols involving methylation
interference can also be found for example in Current Protocols In
Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds.,
1995.
[0222] Gene amplification is an additional method for assessing the
status of 158P1D7. Gene amplification is measured in a sample
directly, for example, by conventional Southern blotting or
Northern blotting to quantitate the transcription of mRNA (Thomas,
1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies are employed that recognize specific duplexes, including
DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or
DNA-protein duplexes. The antibodies in turn are labeled and the
assay carried out where the duplex is bound to a surface, so that
upon the formation of duplex on the surface, the presence of
antibody bound to the duplex can be detected.
[0223] Biopsied tissue or peripheral blood can be conveniently
assayed for the presence of cancer cells using for example,
Northern, dot blot or RT-PCR analysis to detect 158P1D7 expression.
The presence of RT-PCR amplifiable 158P1D7 mRNA provides an
indication of the presence of cancer. RT-PCR assays are well known
in the art. RT-PCR detection assays for tumor cells in peripheral
blood are currently being evaluated for use in the diagnosis and
management of a number of human solid tumors.
[0224] A further aspect of the invention is an assessment of the
susceptibility that an individual has for developing cancer. In one
embodiment, a method for predicting susceptibility to cancer
comprises detecting 158P1D7 mRNA or 158P1D7 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 158P1D7 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 158P1D7
in bladder or other tissue is examined, with the presence of
158P1D7 in the sample providing an indication of bladder cancer
susceptibility (or the emergence or existence of a bladder tumor).
Similarly, one can evaluate the integrity 158P1D7 nucleotide and
amino acid sequences in a biological sample, in order to identify
perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The presence of
one or more perturbations in 158P1D7 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0225] The invention also comprises methods for gauging tumor
aggressiveness. In one embodiment, a method for gauging
aggressiveness of a tumor comprises determining the level of
158P1D7 mRNA or 158P1D7 protein expressed by tumor cells, comparing
the level so determined to the level of 158P1D7 mRNA or 158P1D7
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 158P1D7 mRNA or 158P1D7 protein expression in the tumor
sample relative to the normal sample indicates the degree of
aggressiveness. In a specific embodiment, aggressiveness of a tumor
is evaluated by determining the extent to which 158P1D7 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 158P1D7 nucleotide and amino acid
sequences in a biological sample, in order to identify
perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The presence of
one or more perturbations indicates more aggressive tumors.
[0226] Another embodiment of the invention is directed to methods
for observing the progression of a malignancy in an individual over
time. In one embodiment, methods for observing the progression of a
malignancy in an individual over time comprise determining the
level of 158P1D7 mRNA or 158P1D7 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 158P1D7 mRNA or 158P1D7 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 158P1D7 mRNA or 158P1D7 protein expression in
the tumor sample over time provides information on the progression
of the cancer. In a specific embodiment, the progression of a
cancer is evaluated by determining 158P1D7 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
158P1D7 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules such as insertions, deletions, substitutions and the
like, where the presence of one or more perturbations indicates a
progression of the cancer.
[0227] The above diagnostic approaches can be combined with any one
of a wide variety of prognostic and diagnostic protocols known in
the art. For example, another embodiment of the invention is
directed to methods for observing a coincidence between the
expression of 158P1D7 gene and 158P1D7 gene products (or
perturbations in 158P1D7 gene and 158P1D7 gene products) and a
factor that is associated with malignancy, as a means for
diagnosing and prognosticating the status of a tissue sample. A
wide variety of factors associated with malignancy can be utilized,
such as the expression of genes associated with malignancy (e.g.
PSCA, H-ras and p53 expression etc.) as well as gross cytological
observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.
6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et
al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J.
Surg. Pathol. 23(8):918-24). Methods for observing a coincidence
between the expression of 158P1D7 gene and 158P1D7 gene products
(or perturbations in 158P1D7 gene and 158P1D7 gene products) and
another factor that is associated with malignancy are useful, for
example, because the presence of a set of specific factors that
coincide with disease provides information crucial for diagnosing
and prognosticating the status of a tissue sample.
[0228] In one embodiment, methods for observing a coincidence
between the expression of 158P1D7 gene and 158P1D7 gene products
(or perturbations in 158P1D7 gene and 158P1D7 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 158P1D7 mRNA or protein in a tissue sample,
detecting the overexpression of BLCA-4A mRNA or protein in a tissue
sample (or PSCA expression), and observing a coincidence of 158P1D7
mRNA or protein and BLCA-4 mRNA or protein overexpression (or PSCA
expression) (Amara et al., 2001, Cancer Res 61:4660-4665; Konety et
al., Clin Cancer Res, 2000, 6(7):2618-2625). In a specific
embodiment, the expression of 158P1D7 and BLCA-4 mRNA in bladder
tissue is examined, where the coincidence of 158P1D7 and BLCA-4
mRNA overexpression in the sample indicates the existence of
bladder cancer, bladder cancer susceptibility or the emergence or
status of a bladder tumor.
[0229] Methods for detecting and quantifying the expression of
158P1D7 mRNA or protein are described herein, and standard nucleic
acid and protein detection and quantification technologies are well
known in the art. Standard methods for the detection and
quantification of 158P1D7 mRNA include in situ hybridization using
labeled 158P1D7 riboprobes, Northern blot and related techniques
using 158P1D7 polynucleotide probes, RT-PCR analysis using primers
specific for 158P1D7, and other amplification type detection
methods, such as, for example, branched DNA, SISBA, TMA and the
like. In a specific embodiment, semi-quantitative RT-PCR is used to
detect and quantify 158P1D7 mRNA expression. Any number of primers
capable of amplifying 158P1D7 can be used for this purpose,
including but not limited to the various primer sets specifically
described herein. In a specific embodiment, polyclonal or
monoclonal antibodies specifically reactive with the wild-type
158P1D7 protein can be used in an immunohistochemical assay of
biopsied tissue.
IX.) Identification of Molecules that Interact with 158P1D7
[0230] The 158P1D7 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 158P1D7, as well as
pathways activated by 158P1D7 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999,
5,925,523 issued 20 Jul. 1999, 5,846,722 issued 8 Dec. 1998 and
6,004,746 issued 21 Dec. 1999. Algorithms are also available in the
art for genome-based predictions of protein function (see, e.g.,
Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
[0231] Alternatively one can screen peptide libraries to identify
molecules that interact with 158P1D7 protein sequences. In such
methods, peptides that bind to 158P1D7 are identified by screening
libraries that encode a random or controlled collection of amino
acids. Peptides encoded by the libraries are expressed as fusion
proteins of bacteriophage coat proteins, the bacteriophage
particles are then screened against the 158P1D7 protein.
[0232] Accordingly, peptides having a wide variety of uses, such as
therapeutic, prognostic or diagnostic reagents, are thus identified
without any prior information on the structure of the expected
ligand or receptor molecule. Typical peptide libraries and
screening methods that can be used to identify molecules that
interact with 158P1D7 protein sequences are disclosed for example
in U.S. Pat. Nos. 5,723,286 issued 3 Mar. 1998 and 5,733,731 issued
31 Mar. 1998.
[0233] Alternatively, cell lines that express 158P1D7 are used to
identify protein-protein interactions mediated by 158P1D7. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B J, et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 158P1D7 protein can be immunoprecipitated from
158P1D7-expressing cell lines using anti-158P1D7 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 158P1D7 and a His-tag
(vectors mentioned above). The immunoprecipitated complex can be
examined for protein association by procedures such as Western
blotting, 35S-methionine labeling of proteins, protein
microsequencing, silver staining and two-dimensional gel
electrophoresis.
[0234] Small molecules and ligands that interact with 158P1D7 can
be identified through related embodiments of such screening assays.
For example, small molecules can be identified that interfere with
protein function, including molecules that interfere with 158P1D7's
ability to mediate phosphorylation and de-phosphorylation,
interaction with DNA or RNA molecules as an indication of
regulation of cell cycles, second messenger signaling or
tumorigenesis. Similarly, small molecules that modulate 158P1D7
related ion channel, protein pump, or cell communication functions
158P1D7 are identified and used to treat patients that have a
cancer that expresses 158P1D7 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass.,
1992). Moreover, ligands that regulate 158P1D7 function can be
identified based on their ability to bind 158P1D7 and activate a
reporter construct. Typical methods are discussed for example in
U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods
for forming hybrid ligands in which at least one ligand is a small
molecule. In an illustrative embodiment, cells engineered to
express a fusion protein of 158P1D7 and a DNA-binding protein are
used to co-express a fusion protein of a hybrid ligand/small
molecule and a cDNA library transcriptional activator protein. The
cells further contain a reporter gene, the expression of which is
conditioned on the proximity of the first and second fusion
proteins to each other, an event that occurs only if the hybrid
ligand binds to target sites on both hybrid proteins. Those cells
that express the reporter gene are selected and the unknown small
molecule or the unknown ligand is identified. This method provides
a means of identifying modulators which activate or inhibit
158P1D7.
[0235] An embodiment of this invention comprises a method of
screening for a molecule that interacts with an 158P1D7 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with the 158P1D7 amino acid
sequence, allowing the population of molecules and the 158P1D7
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 158P1D7 amino acid sequence, and then separating molecules
that do not interact with the 158P1D7 amino acid sequence from
molecules that do. In a specific embodiment, the method further
comprises purifying, characterizing and identifying a molecule that
interacts with the 158P1D7 amino acid sequence. The identified
molecule can be used to modulate a function performed by 158P1D7.
In a preferred embodiment, the 158P1D7 amino acid sequence is
contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
[0236] The identification of 158P1D7 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in bladder and other cancers, opens a number of
therapeutic approaches to the treatment of such cancers. As
contemplated herein, 158P1D7 functions as a transcription factor
involved in activating tumor-promoting genes or repressing genes
that block tumorigenesis.
[0237] Accordingly, therapeutic approaches that inhibit the
activity of the 158P1D7 protein are useful for patients suffering
from a cancer that expresses 158P1D7. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of the 158P1D7
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of the 158P1D7 gene or translation of 158P1D7
mRNA.
[0238] X.A.) Anti-Cancer Vaccines
[0239] The invention provides cancer vaccines comprising a
158P1D7-related protein or 158P1D7-related nucleic acid. In view of
the expression of 158P1D7, cancer vaccines prevent and/or treat
158P1D7-expressing cancers with minimal or no effects on non-target
tissues. The use of a tumor antigen in a vaccine that generates
humoral and/or cell-mediated immune responses as anti-cancer
therapy is well known in the art (see, e.g., Hodge et al., 1995,
Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol.
159:3113-3117).
[0240] Such methods can be readily practiced by employing a
158P1D7-related protein, or a 158P1D7-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 158P1D7 immunogen (which typically comprises a
number of antibody or T cell epitopes). Skilled artisans understand
that a wide variety of vaccine systems for delivery of
immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer
Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of
generating an immune response (e.g. humoral and/or cell-mediated)
in a mammal, comprise the steps of: exposing the mammal's immune
system to an immunoreactive epitope (e.g. an epitope present in the
158P1D7 protein shown in FIG. 2 or analog or homolog thereof) so
that the mammal generates an immune response that is specific for
that epitope (e.g. generates antibodies that specifically recognize
that epitope). In a preferred method, the 158P1D7 immunogen
contains a biological motif, see e.g., Tables V-XVIII, or a peptide
of a size range from 158P1D7 indicated in FIG. 11, FIG. 12, FIG.
13, FIG. 14, and FIG. 15.
[0241] The entire 158P1D7 protein, immunogenic regions or epitopes
thereof can be combined and delivered by various means. Such
vaccine compositions can include, for example, lipopeptides (e.g.,
Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide
compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG")
microspheres (see, e.g., Eldridge, et al., Molec. Immunol.
28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et
al., Vaccine 13:675-681, 1995), peptide compositions contained in
immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al.,
Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243,
1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J.
P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P.,
J. Immunol. Methods 196:17-32, 1996), peptides formulated as
multivalent peptides; peptides for use in ballistic delivery
systems, typically crystallized peptides, viral delivery vectors
(Perkus, M. E. et al., In: Concepts in vaccine development,
Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al.,
Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;
Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et
al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology
175:535, 1990), particles of viral or synthetic origin (e.g.,
Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J.
H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al.,
Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,
and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et
al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J.
Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996),
or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science
259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G.,
Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine
development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B.,
and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and
Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted
delivery technologies, also known as receptor mediated targeting,
such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.)
may also be used.
[0242] In patients with 158P1D7-associated cancer, the vaccine
compositions of the invention can also be used in conjunction with
other treatments used for cancer, e.g., surgery, chemotherapy, drug
therapies, radiation therapies, etc. including use in combination
with immune adjuvants such as IL-2, IL-12, GM-CSF, and the
like.
[0243] Cellular Vaccines:
[0244] CTL epitopes can be determined using specific algorithms to
identify peptides within 158P1D7 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University, BIMAS, and SYFPEITHI). In a preferred embodiment,
the 158P1D7 immunogen contains one or more amino acid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables V-XVIII or a peptide of 8, 9, 10 or 11
amino acids specified by an HLA Class I motif/supermotif (e.g.,
Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at
least 9 amino acids that comprises an HLA Class II motif/supermotif
(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art,
the HLA Class I binding groove is essentially closed ended so that
peptides of only a particular size range can fit into the groove
and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11
amino acids long. In contrast, the HLA Class II binding groove is
essentially open ended; therefore a peptide of about 9 or more
amino acids can be bound by an HLA Class II molecule. Due to the
binding groove differences between HLA Class I and II, HLA Class I
motifs are length specific, i.e., position two of a Class I motif
is the second amino acid in an amino to carboxyl direction of the
peptide. The amino acid positions in a Class II motif are relative
only to each other, not the overall peptide, i.e., additional amino
acids can be attached to the amino and/or carboxyl termini of a
motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino
acids long, or longer than 25 amino acids.
[0245] Antibody-Based Vaccines
[0246] A wide variety of methods for generating an immune response
in a mammal are known in the art (for example as the first step in
the generation of hybridomas). Methods of generating an immune
response in a mammal comprise exposing the mammal's immune system
to an immunogenic epitope on a protein (e.g. the 158P1D7 protein)
so that an immune response is generated. A typical embodiment
consists of a method for generating an immune response to 158P1D7
in a host, by contacting the host with a sufficient amount of at
least one 158P1D7 B cell or cytotoxic T-cell epitope or analog
thereof; and at least one periodic interval thereafter
re-contacting the host with the 158P1D7 B cell or cytotoxic T-cell
epitope or analog thereof. A specific embodiment consists of a
method of generating an immune response against a 158P1D7-related
protein or a man-made multiepitopic peptide comprising:
administering 158P1D7 immunogen (e.g. the 158P1D7 protein or a
peptide fragment thereof, an 158P1D7 fusion protein or analog etc.)
in a vaccine preparation to a human or another mammal. Typically,
such vaccine preparations further contain a suitable adjuvant (see,
e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such
as a PADRE.TM. peptide (Epimmune Inc., San Diego, Calif.; see,
e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;
Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al.,
Immunol. Res. 1998 18(2): 79-92). An alternative method comprises
generating an immune response in an individual against a 158P1D7
immunogen by: administering in vivo to muscle or skin of the
individual's body a DNA molecule that comprises a DNA sequence that
encodes an 158P1D7 immunogen, the DNA sequence operatively linked
to regulatory sequences which control the expression of the DNA
sequence; wherein the DNA molecule is taken up by cells, the DNA
sequence is expressed in the cells and an immune response is
generated against the immunogen (see, e.g., U.S. Pat. No.
5,962,428). Optionally a genetic vaccine facilitator such as
anionic lipids; saponins; lectins; estrogenic compounds;
hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also
administered.
[0247] Nucleic Acid Vaccines:
[0248] Vaccine compositions of the invention include nucleic
acid-mediated modalities. DNA or RNA that encode protein(s) of the
invention can be administered to a patient. Genetic immunization
methods can be employed to generate prophylactic or therapeutic
humoral and cellular immune responses directed against cancer cells
expressing 158P1D7. Constructs comprising DNA encoding a
158P1D7-related protein/immunogen and appropriate regulatory
sequences can be injected directly into muscle or skin of an
individual, such that the cells of the muscle or skin take-up the
construct and express the encoded 158P1D7 protein/immunogen.
Alternatively, a vaccine comprises a 158P1D7-related protein.
Expression of the 158P1D7-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear 158P1D7 protein. Various
prophylactic and therapeutic genetic immunization techniques known
in the art can be used (for review, see information and references
published at Internet address URL: genweb.com). Nucleic acid-based
delivery is described, for instance, in Wolff et. al., Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples
of DNA-based delivery technologies include "naked DNA", facilitated
(bupivicaine, polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0249] For therapeutic or prophylactic immunization purposes,
proteins of the invention can be expressed via viral or bacterial
vectors. Various viral gene delivery systems that can be used in
the practice of the invention include, but are not limited to,
vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus,
adeno-associated virus, lentivirus, and sindbis virus (see, e.g.,
Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J.
Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems
can also be employed by introducing naked DNA encoding a
158P1D7-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0250] Vaccinia virus is used, for example, as a vector to express
nucleotide sequences that encode the peptides of the invention.
Upon introduction into a host, the recombinant vaccinia virus
expresses the protein immunogenic peptide, and thereby elicits a
host immune response. Vaccinia vectors and methods useful in
immunization protocols are described in, e.g., U.S. Pat. No.
4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG
vectors are described in Stover et al., Nature 351:456-460 (1991).
A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention,
e.g. adeno and adeno-associated virus vectors, retroviral vectors,
Salmonella typhi vectors, detoxified anthrax toxin vectors, and the
like, will be apparent to those skilled in the art from the
description herein.
[0251] Thus, gene delivery systems are used to deliver a
158P1D7-related nucleic acid molecule. In one embodiment, the
full-length human 158P1D7 cDNA is employed. In another embodiment,
158P1D7 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0252] Ex Vivo Vaccines
[0253] Various ex vivo strategies can also be employed to generate
an immune response. One approach involves the use of antigen
presenting cells (APCs) such as dendritic cells (DC) to present
158P1D7 antigen to a patient's immune system. Dendritic cells
express MHC class I and II molecules, B7 co-stimulator, and IL-12,
and are thus highly specialized antigen presenting cells. In
bladder cancer, autologous dendritic cells pulsed with peptides of
the MAGE-3 antigen are being used in a Phase I clinical trial to
stimulate bladder cancer patients' immune systems (Nishiyama et
al., 2001, Clin Cancer Res, 7(1):23-31). Thus, dendritic cells can
be used to present 158P1D7 peptides to T cells in the context of
MHC class I or II molecules. In one embodiment, autologous
dendritic cells are pulsed with 158P1D7 peptides capable of binding
to MHC class I and/or class II molecules. In another embodiment,
dendritic cells are pulsed with the complete 158P1D7 protein. Yet
another embodiment involves engineering the overexpression of the
158P1D7 gene in dendritic cells using various implementing vectors
known in the art, such as adenovirus (Arthur et al., 1997, Cancer
Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer
Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA
transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or
tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.
186:1177-1182). Cells that express 158P1D7 can also be engineered
to express immune modulators, such as GM-CSF, and used as
immunizing agents.
[0254] X.B.) 158P1D7 as a Target for Antibody-Based Therapy
[0255] 158P1D7 is an attractive target for antibody-based
therapeutic strategies. A number of antibody strategies are known
in the art for targeting both extracellular and intracellular
molecules (see, e.g., complement and ADCC mediated killing as well
as the use of intrabodies). Because 158P1D7 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 158P1D7-immunoreactive compositions are
prepared that exhibit excellent sensitivity without toxic,
non-specific and/or non-target effects caused by binding of the
immunoreactive composition to non-target organs and tissues.
Antibodies specifically reactive with domains of 158P1D7 are useful
to treat 158P1D7-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0256] 158P1D7 antibodies can be introduced into a patient such
that the antibody binds to 158P1D7 and modulates a function, such
as an interaction with a binding partner, and consequently mediates
destruction of the tumor cells and/or inhibits the growth of the
tumor cells. Mechanisms by which such antibodies exert a
therapeutic effect can include complement-mediated cytolysis,
antibody-dependent cellular cytotoxicity, modulation of the
physiological function of 158P1D7, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0257] Those skilled in the art understand that antibodies can be
used to specifically target and bind immunogenic molecules such as
an immunogenic region of the 158P1D7 sequence shown in FIG. 2 or
FIG. 3. In addition, skilled artisans understand that it is routine
to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et
al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or
therapeutic agents are delivered directly to cells, such as by
conjugating them to antibodies specific for a molecule expressed by
that cell (e.g. 158P1D7), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0258] A wide variety of compositions and methods for using
antibody-cytotoxic agent conjugates to kill cells are known in the
art. In the context of cancers, typical methods entail
administering to an animal having a tumor a biologically effective
amount of a conjugate comprising a selected cytotoxic and/or
therapeutic agent linked to a targeting agent (e.g. an anti-158P1D7
antibody) that binds to a marker (e.g. 158P1D7) expressed,
accessible to binding or localized on the cell surfaces. A typical
embodiment is a method of delivering a cytotoxic and/or therapeutic
agent to a cell expressing 158P1D7, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
158P1D7 epitope, and, exposing the cell to the antibody-agent
conjugate. Another illustrative embodiment is a method of treating
an individual suspected of suffering from metastasized cancer,
comprising a step of administering parenterally to said individual
a pharmaceutical composition comprising a therapeutically effective
amount of an antibody conjugated to a cytotoxic and/or therapeutic
agent.
[0259] Cancer immunotherapy using anti-158P1D7 antibodies can be
done in accordance with various approaches that have been
successfully employed in the treatment of other types of cancer,
including but not limited to colon cancer (Arlen et al., 1998,
Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al.,
1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood
90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.
52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.
Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et
al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,
1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.
55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.
Immunol. 11:117-127). Some therapeutic approaches involve
conjugation of naked antibody to a toxin, such as the conjugation
of Y91 or 1131 to anti-CD20 antibodies (e.g., Zevalin.TM., IDEC
Pharmaceuticals Corp. or Bexxar.TM., Coulter Pharmaceuticals),
while others involve co-administration of antibodies and other
therapeutic agents, such as Herceptin.TM. (trastuzumab) with
paclitaxel (Genentech, Inc.). To treat bladder cancer, for example,
158P1D7 antibodies can be administered in conjunction with
radiation, chemotherapy or hormone ablation.
[0260] Although 158P1D7 antibody therapy is useful for all stages
of cancer, antibody therapy can be particularly appropriate in
advanced or metastatic cancers. Treatment with the antibody therapy
of the invention is indicated for patients who have received one or
more rounds of chemotherapy. Alternatively, antibody therapy of the
invention is combined with a chemotherapeutic or radiation regimen
for patients who have not received chemotherapeutic treatment.
Additionally, antibody therapy can enable the use of reduced
dosages of concomitant chemotherapy, particularly for patients who
do not tolerate the toxicity of the chemotherapeutic agent very
well.
[0261] Cancer patients can be evaluated for the presence and level
of 158P1D7 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 158P1D7 imaging, or other
techniques that reliably indicate the presence and degree of
158P1D7 expression. Immunohistochemical analysis of tumor biopsies
or surgical specimens is preferred for this purpose. Methods for
immunohistochemical analysis of tumor tissues are well known in the
art.
[0262] Anti-158P1D7 monoclonal antibodies that treat bladder and
other cancers include those that initiate a potent immune response
against the tumor or those that are directly cytotoxic. In this
regard, anti-158P1D7 monoclonal antibodies (mAbs) can elicit tumor
cell lysis by either complement-mediated or antibody-dependent cell
cytotoxicity (ADCC) mechanisms, both of which require an intact Fc
portion of the immunoglobulin molecule for interaction with
effector cell Fc receptor sites on complement proteins. In
addition, anti-158P1D7 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 158P1D7.
Mechanisms by which directly cytotoxic mAbs act include: inhibition
of cell growth, modulation of cellular differentiation, modulation
of tumor angiogenesis factor profiles, and the induction of
apoptosis. The mechanism(s) by which a particular anti-158P1D7 mAb
exerts an anti-tumor effect is evaluated using any number of in
vitro assays that evaluate cell death such as ADCC, ADMMC,
complement-mediated cell lysis, and so forth, as is generally known
in the art.
[0263] In some patients, the use of murine or other non-human
monoclonal antibodies, or human/mouse chimeric mAbs can induce
moderate to strong immune responses against the non-human antibody.
This can result in clearance of the antibody from circulation and
reduced efficacy. In the most severe cases, such an immune response
can lead to the extensive formation of immune complexes which,
potentially, can cause renal failure. Accordingly, preferred
monoclonal antibodies used in the therapeutic methods of the
invention are those that are either fully human or humanized and
that bind specifically to the target 158P1D7 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0264] Therapeutic methods of the invention contemplate the
administration of single anti-158P1D7 mAbs as well as combinations,
or cocktails, of different mAbs. Such mAb cocktails can have
certain advantages inasmuch as they contain mAbs that target
different epitopes, exploit different effector mechanisms or
combine directly cytotoxic mAbs with mAbs that rely on immune
effector functionality. Such mAbs in combination can exhibit
synergistic therapeutic effects. In addition, anti-158P1D7 mAbs can
be administered concomitantly with other therapeutic modalities,
including but not limited to various chemotherapeutic agents,
androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery
or radiation. The anti-158P1D7 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0265] Anti-158P1D7 antibody formulations are administered via any
route capable of delivering the antibodies to a tumor cell. Routes
of administration include, but are not limited to, intravenous,
intraperitoneal, intramuscular, intratumor, intradermal, and the
like. Treatment generally involves repeated administration of the
anti-158P1D7 antibody preparation, via an acceptable route of
administration such as intravenous injection (IV), typically at a
dose in the range of about 0.1 to about 10 mg/kg body weight. In
general, doses in the range of 10-500 mg mAb per week are effective
and well tolerated.
[0266] Based on clinical experience with the Herceptin mAb in the
treatment of metastatic breast cancer, an initial loading dose of
approximately 4 mg/kg patient body weight IV, followed by weekly
doses of about 2 mg/kg IV of the anti-158P1D7 mAb preparation
represents an acceptable dosing regimen. Preferably, the initial
loading dose is administered as a 90 minute or longer infusion. The
periodic maintenance dose is administered as a 30 minute or longer
infusion, provided the initial dose was well tolerated. As
appreciated by those of skill in the art, various factors can
influence the ideal dose regimen in a particular case. Such factors
include, for example, the binding affinity and half life of the Ab
or mAbs used, the degree of 158P1D7 expression in the patient, the
extent of circulating shed 158P1D7 antigen, the desired
steady-state antibody concentration level, frequency of treatment,
and the influence of chemotherapeutic or other agents used in
combination with the treatment method of the invention, as well as
the health status of a particular patient.
[0267] Optionally, patients should be evaluated for the levels of
158P1D7 in a given sample (e.g. the levels of circulating 158P1D7
antigen and/or 158P1D7 expressing cells) in order to assist in the
determination of the most effective dosing regimen, etc. Such
evaluations are also used for monitoring purposes throughout
therapy, and are useful to gauge therapeutic success in combination
with the evaluation of other parameters (for example, urine
cytology and/or ImmunoCyt levels in bladder cancer therapy, or by
analogy, serum PSA levels in prostate cancer therapy).
[0268] Anti-idiotypic anti-158P1D7 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 158P1D7-related protein. In particular, the
generation of anti-idiotypic antibodies is well known in the art;
this methodology can readily be adapted to generate anti-idiotypic
anti-158P1D7 antibodies that mimic an epitope on a 158P1D7-related
protein (see, for example, Wagner et al., 1997, Hybridoma 16:
33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et
al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an
anti-idiotypic antibody can be used in cancer vaccine
strategies.
[0269] X.C.) 158P1D7 as a Target for Cellular Immune Responses
[0270] Vaccines and methods of preparing vaccines that contain an
immunogenically effective amount of one or more HLA-binding
peptides as described herein are further embodiments of the
invention. Furthermore, vaccines in accordance with the invention
encompass compositions of one or more of the claimed peptides. A
peptide can be present in a vaccine individually. Alternatively,
the peptide can exist as a homopolymer comprising multiple copies
of the same peptide, or as a heteropolymer of various peptides.
Polymers have the advantage of increased immunological reaction
and, where different peptide epitopes are used to make up the
polymer, the additional ability to induce antibodies and/or CTLs
that react with different antigenic determinants of the pathogenic
organism or tumor-related peptide targeted for an immune response.
The composition can be a naturally occurring region of an antigen
or can be prepared, e.g., recombinantly or by chemical
synthesis.
[0271] Carriers that can be used with vaccines of the invention are
well known in the art, and include, e.g., thyroglobulin, albumins
such as human serum albumin, tetanus toxoid, polyamino acids such
as poly 1-lysine, poly 1-glutamic acid, influenza, hepatitis B
virus core protein, and the like. The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water,
or saline, preferably phosphate buffered saline. The vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, as
disclosed herein, CTL responses can be primed by conjugating
peptides of the invention to lipids, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, an
adjuvant such as a synthetic
cytosine-phosphorothiolated-guanine-containing (CpG)
oligonucleotides has been found to increase CTL responses 10- to
100-fold. (see, e.g. Davila and Celis J. Immunol. 165:539-547
(2000))
[0272] Upon immunization with a peptide composition in accordance
with the invention, via injection, aerosol, oral, transdermal,
transmucosal, intrapleural, intrathecal, or other suitable routes,
the immune system of the host responds to the vaccine by producing
large amounts of CTLs and/or HTLs specific for the desired antigen.
Consequently, the host becomes at least partially immune to later
development of cells that express or overexpress 158P1D7 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0273] In some embodiments, it may be desirable to combine the
class I peptide components with components that induce or
facilitate neutralizing antibody and or helper T cell responses
directed to the target antigen. A preferred embodiment of such a
composition comprises class I and class II epitopes in accordance
with the invention. An alternative embodiment of such a composition
comprises a class I and/or class II epitope in accordance with the
invention, along with a cross reactive HTL epitope such as
PADRE.TM. (Epimmune, San Diego, Calif.) molecule (described e.g.,
in U.S. Pat. No. 5,736,142).
[0274] A vaccine of the invention can also include
antigen-presenting cells (APC), such as dendritic cells (DC), as a
vehicle to present peptides of the invention. Vaccine compositions
can be created in vitro, following dendritic cell mobilization and
harvesting, whereby loading of dendritic cells occurs in vitro. For
example, dendritic cells are transfected, e.g., with a minigene in
accordance with the invention, or are pulsed with peptides. The
dendritic cell can then be administered to a patient to elicit
immune responses in vivo. Vaccine compositions, either DNA- or
peptide-based, can also be administered in vivo in combination with
dendritic cell mobilization whereby loading of dendritic cells
occurs in vivo.
[0275] Preferably, the following principles are utilized when
selecting an array of epitopes for inclusion in a polyepitopic
composition for use in a vaccine, or for selecting discrete
epitopes to be included in a vaccine and/or to be encoded by
nucleic acids such as a minigene. It is preferred that each of the
following principles be balanced in order to make the selection.
The multiple epitopes to be incorporated in a given vaccine
composition may be, but need not be, contiguous in sequence in the
native antigen from which the epitopes are derived.
[0276] 1.) Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with
tumor clearance. For HLA Class I this includes 3-4 epitopes that
come from at least one tumor associated antigen (TAA). For HLA
Class II a similar rationale is employed; again 3-4 epitopes are
selected from at least one TAA (see, e.g., Rosenberg et al.,
Science 278:1447-1450). Epitopes from one TAA may be used in
combination with epitopes from one or more additional TAAs to
produce a vaccine that targets tumors with varying expression
patterns of frequently-expressed TAAs.
[0277] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC50 of 500 nM or less, often 200 nM or less; and for
Class II an IC50 of 1000 nM or less.
[0278] 3.) Sufficient supermotif bearing-peptides, or a sufficient
array of allele-specific motif-bearing peptides, are selected to
give broad population coverage. For example, it is preferable to
have at least 80% population coverage. A Monte Carlo analysis, a
statistical evaluation known in the art, can be employed to assess
the breadth, or redundancy of, population coverage.
[0279] 4.) When selecting epitopes from cancer-related antigens it
is often useful to select analogs because the patient may have
developed tolerance to the native epitope.
[0280] 5.) Of particular relevance are epitopes referred to as
"nested epitopes." Nested epitopes occur where at least two
epitopes overlap in a given peptide sequence. A nested peptide
sequence can comprise B cell, HLA class I and/or HLA class II
epitopes. When providing nested epitopes, a general objective is to
provide the greatest number of epitopes per sequence. Thus, an
aspect is to avoid providing a peptide that is any longer than the
amino terminus of the amino terminal epitope and the carboxyl
terminus of the carboxyl terminal epitope in the peptide. When
providing a multi-epitopic sequence, such as a sequence comprising
nested epitopes, it is generally important to screen the sequence
in order to insure that it does not have pathological or other
deleterious biological properties.
[0281] 6.) If a polyepitopic protein is created, or when creating a
minigene, an objective is to generate the smallest peptide that
encompasses the epitopes of interest. This principle is similar, if
not the same as that employed when selecting a peptide comprising
nested epitopes. However, with an artificial polyepitopic peptide,
the size minimization objective is balanced against the need to
integrate any spacer sequences between epitopes in the polyepitopic
protein. Spacer amino acid residues can, for example, be introduced
to avoid junctional epitopes (an epitope recognized by the immune
system, not present in the target antigen, and only created by the
man-made juxtaposition of epitopes), or to facilitate cleavage
between epitopes and thereby enhance epitope presentation.
Junctional epitopes are generally to be avoided because the
recipient may generate an immune response to that non-native
epitope. Of particular concern is a junctional epitope that is a
"dominant epitope." A dominant epitope may lead to such a zealous
response that immune responses to other epitopes are diminished or
suppressed.
[0282] 7.) Where the sequences of multiple variants of the same
target protein are present, potential peptide epitopes can also be
selected on the basis of their conservancy. For example, a
criterion for conservancy may define that the entire sequence of an
HLA class I binding peptide or the entire 9-mer core of a class II
binding peptide be conserved in a designated percentage of the
sequences evaluated for a specific protein antigen.
[0283] X.C.1. Minigene Vaccines
[0284] A number of different approaches are available which allow
simultaneous delivery of multiple epitopes. Nucleic acids encoding
the peptides of the invention are a particularly useful embodiment
of the invention. Epitopes for inclusion in a minigene are
preferably selected according to the guidelines set forth in the
previous section. A preferred means of administering nucleic acids
encoding the peptides of the invention uses minigene constructs
encoding a peptide comprising one or multiple epitopes of the
invention.
[0285] The use of multi-epitope minigenes is described below and
in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and
Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J.
Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348,
1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a
multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing
epitopes derived 158P1D7, the PADRE.RTM. universal helper T cell
epitope (or multiple HTL epitopes from 158P1D7), and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0286] The immunogenicity of a multi-epitopic minigene can be
confirmed in transgenic mice to evaluate the magnitude of CTL
induction responses against the epitopes tested. Further, the
immunogenicity of DNA-encoded epitopes in vivo can be correlated
with the in vitro responses of specific CTL lines against target
cells transfected with the DNA plasmid. Thus, these experiments can
show that the minigene serves to both: 1.) generate a CTL response
and 2.) that the induced CTLs recognized cells expressing the
encoded epitopes.
[0287] For example, to create a DNA sequence encoding the selected
epitopes (minigene) for expression in human cells, the amino acid
sequences of the epitopes may be reverse translated. A human codon
usage table can be used to guide the codon choice for each amino
acid. These epitope-encoding DNA sequences may be directly
adjoined, so that when translated, a continuous polypeptide
sequence is created. To optimize expression and/or immunogenicity,
additional elements can be incorporated into the minigene design.
Examples of amino acid sequences that can be reverse translated and
included in the minigene sequence include: HLA class I epitopes,
HLA class II epitopes, antibody epitopes, a ubiquitination signal
sequence, and/or an endoplasmic reticulum targeting signal. In
addition, HLA presentation of CTL and HTL epitopes may be improved
by including synthetic (e.g. poly-alanine) or naturally-occurring
flanking sequences adjacent to the CTL or HTL epitopes; these
larger peptides comprising the epitope(s) are within the scope of
the invention.
[0288] The minigene sequence may be converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the
minigene. Overlapping oligonucleotides (30-100 bases long) may be
synthesized, phosphorylated, purified and annealed under
appropriate conditions using well known techniques. The ends of the
oligonucleotides can be joined, for example, using T4 DNA ligase.
This synthetic minigene, encoding the epitope polypeptide, can then
be cloned into a desired expression vector.
[0289] Standard regulatory sequences well known to those of skill
in the art are preferably included in the vector to ensure
expression in the target cells. Several vector elements are
desirable: a promoter with a down-stream cloning site for minigene
insertion; a polyadenylation signal for efficient transcription
termination; an E. coli origin of replication; and an E. coli
selectable marker (e.g. ampicillin or kanamycin resistance).
Numerous promoters can be used for this purpose, e.g., the human
cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos.
5,580,859 and 5,589,466 for other suitable promoter sequences.
[0290] Additional vector modifications may be desired to optimize
minigene expression and immunogenicity. In some cases, introns are
required for efficient gene expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the
transcribed region of the minigene. The inclusion of mRNA
stabilization sequences and sequences for replication in mammalian
cells may also be considered for increasing minigene
expression.
[0291] Once an expression vector is selected, the minigene is
cloned into the polylinker region downstream of the promoter. This
plasmid is transformed into an appropriate E. coli strain, and DNA
is prepared using standard techniques. The orientation and DNA
sequence of the minigene, as well as all other elements included in
the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells harboring the correct plasmid
can be stored as a master cell bank and a working cell bank.
[0292] In addition, immunostimulatory sequences (ISSs or CpGs)
appear to play a role in the immunogenicity of DNA vaccines. These
sequences may be included in the vector, outside the minigene
coding sequence, if desired to enhance immunogenicity.
[0293] In some embodiments, a bi-cistronic expression vector which
allows production of both the minigene-encoded epitopes and a
second protein (included to enhance or decrease immunogenicity) can
be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include
cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules
(e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR
binding proteins (PADRE.TM., Epimmune, San Diego, Calif.). Helper
(HTL) epitopes can be joined to intracellular targeting signals and
expressed separately from expressed CTL epitopes; this allows
direction of the HTL epitopes to a cell compartment different than
that of the CTL epitopes. If required, this could facilitate more
efficient entry of HTL epitopes into the HLA class II pathway,
thereby improving HTL induction. In contrast to HTL or CTL
induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0294] Therapeutic quantities of plasmid DNA can be produced for
example, by fermentation in E. coli, followed by purification.
Aliquots from the working cell bank are used to inoculate growth
medium, and grown to saturation in shaker flasks or a bioreactor
according to well-known techniques. Plasmid DNA can be purified
using standard bioseparation technologies such as solid phase
anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.).
If required, supercoiled DNA can be isolated from the open circular
and linear forms using gel electrophoresis or other methods.
[0295] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0296] Target cell sensitization can be used as a functional assay
for expression and HLA class I presentation of minigene-encoded CTL
epitopes. For example, the plasmid DNA is introduced into a
mammalian cell line that is suitable as a target for standard CTL
chromium release assays. The transfection method used will be
dependent on the final formulation. Electroporation can be used for
"naked" DNA, whereas cationic lipids allow direct in vitro
transfection. A plasmid expressing green fluorescent protein (GFP)
can be co-transfected to allow enrichment of transfected cells
using fluorescence activated cell sorting (FACS). These cells are
then chromium-51 (51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by 51Cr release,
indicates both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be
evaluated in an analogous manner using assays to assess HTL
activity.
[0297] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human HLA proteins are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g., IM for DNA in PBS, intraperitoneal (i.p.) for
lipid-complexed DNA). Twenty-one days after immunization,
splenocytes are harvested and restimulated for one week in the
presence of peptides encoding each epitope being tested.
Thereafter, for CTL effector cells, assays are conducted for
cytolysis of peptide-loaded, 51Cr-labeled target cells using
standard techniques. Lysis of target cells that were sensitized by
HLA loaded with peptide epitopes, corresponding to minigene-encoded
epitopes, demonstrates DNA vaccine function for in vivo induction
of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic
mice in an analogous manner.
[0298] Alternatively, the nucleic acids can be administered using
ballistic delivery as described, for instance, in U.S. Pat. No.
5,204,253. Using this technique, particles comprised solely of DNA
are administered. In a further alternative embodiment, DNA can be
adhered to particles, such as gold particles.
[0299] Minigenes can also be delivered using other bacterial or
viral delivery systems well known in the art, e.g., an expression
construct encoding epitopes of the invention can be incorporated
into a viral vector such as vaccinia.
[0300] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0301] Vaccine compositions comprising CTL peptides of the
invention can be modified, e.g., analoged, to provide desired
attributes, such as improved serum half life, broadened population
coverage or enhanced immunogenicity.
[0302] For instance, the ability of a peptide to induce CTL
activity can be enhanced by linking the peptide to a sequence which
contains at least one epitope that is capable of inducing a T
helper cell response. Although a CTL peptide can be directly linked
to a T helper peptide, often CTL epitope/HTL epitope conjugates are
linked by a spacer molecule. The spacer is typically comprised of
relatively small, neutral molecules, such as amino acids or amino
acid mimetics, which are substantially uncharged under
physiological conditions. The spacers are typically selected from,
e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or
neutral polar amino acids. It will be understood that the
optionally present spacer need not be comprised of the same
residues and thus may be a hetero- or homo-oligomer. When present,
the spacer will usually be at least one or two residues, more
usually three to six residues and sometimes 10 or more residues.
The CTL peptide epitope can be linked to the T helper peptide
epitope either directly or via a spacer either at the amino or
carboxy terminus of the CTL peptide. The amino terminus of either
the immunogenic peptide or the T helper peptide may be
acylated.
[0303] In certain embodiments, the T helper peptide is one that is
recognized by T helper cells present in a majority of a genetically
diverse population. This can be accomplished by selecting peptides
that bind to many, most, or all of the HLA class II molecules.
Examples of such amino acid bind many HLA Class II molecules
include sequences from antigens such as tetanus toxoid at positions
830-843 (QYIKANSKFIGITE; SEQ ID NO: 24), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 25), and Streptococcus 18 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 26).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0304] Alternatively, it is possible to prepare synthetic peptides
capable of stimulating T helper lymphocytes, in a loosely
HLA-restricted fashion, using amino acid sequences not found in
nature (see, e.g., PCT publication WO 95/07707). These synthetic
compounds called Pan-DR-binding epitopes (e.g., PADRE.TM.,
Epimmune, Inc., San Diego, Calif.) are designed to most preferably
bind most HLA-DR (human HLA class II) molecules. For instance, a
pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa
(SEQ ID NO: 27), where "X" is either cyclohexylalanine,
phenylalanine, or tyrosine, and a is either d-alanine or 1-alanine,
has been found to bind to most HLA-DR alleles, and to stimulate the
response of T helper lymphocytes from most individuals, regardless
of their HLA type. An alternative of a pan-DR binding epitope
comprises all "L" natural amino acids and can be provided in the
form of nucleic acids that encode the epitope.
[0305] HTL peptide epitopes can also be modified to alter their
biological properties. For example, they can be modified to include
d-amino acids to increase their resistance to proteases and thus
extend their serum half life, or they can be conjugated to other
molecules such as lipids, proteins, carbohydrates, and the like to
increase their biological activity. For example, a T helper peptide
can be conjugated to one or more palmitic acid chains at either the
amino or carboxyl termini.
[0306] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0307] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes B lymphocytes or T lymphocytes. Lipids have been
identified as agents capable of priming CTL in vivo. For example,
palmitic acid residues can be attached to the .epsilon.- and
.alpha.-amino groups of a lysine residue and then linked, e.g., via
one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser,
or the like, to an immunogenic peptide. The lipidated peptide can
then be administered either directly in a micelle or particle,
incorporated into a liposome, or emulsified in an adjuvant, e.g.,
incomplete Freund's adjuvant. In a preferred embodiment, a
particularly effective immunogenic composition comprises palmitic
acid attached to .epsilon.- and .alpha.-amino groups of Lys, which
is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the immunogenic peptide.
[0308] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to
prime virus specific CTL when covalently attached to an appropriate
peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides
of the invention can be coupled to P3CSS, for example, and the
lipopeptide administered to an individual to specifically prime an
immune response to the target antigen. Moreover, because the
induction of neutralizing antibodies can also be primed with
P3CSS-conjugated epitopes, two such compositions can be combined to
more effectively elicit both humoral and cell-mediated
responses.
[0309] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0310] An embodiment of a vaccine composition in accordance with
the invention comprises ex vivo administration of a cocktail of
epitope-bearing peptides to PBMC, or isolated DC therefrom, from
the patient's blood. A pharmaceutical to facilitate harvesting of
DC can be used, such as Progenipoietin.TM. (Pharmacia-Monsanto, St.
Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and
prior to reinfusion into patients, the DC are washed to remove
unbound peptides. In this embodiment, a vaccine comprises
peptide-pulsed DCs which present the pulsed peptide epitopes
complexed with HLA molecules on their surfaces.
[0311] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 158P1D7. Optionally, a
helper T cell (HTL) peptide, such as a natural or artificial
loosely restricted HLA Class II peptide, can be included to
facilitate the CTL response. Thus, a vaccine in accordance with the
invention is used to treat a cancer which expresses or
overexpresses 158P1D7.
[0312] X.D. Adoptive Immunotherapy
[0313] Antigenic 158P1D7-related peptides are used to elicit a CTL
and/or HTL response ex vivo, as well. The resulting CTL or HTL
cells, can be used to treat tumors in patients that do not respond
to other conventional forms of therapy, or will not respond to a
therapeutic vaccine peptide or nucleic acid in accordance with the
invention. Ex vivo CTL or HTL responses to a particular antigen are
induced by incubating in tissue culture the patient's, or
genetically compatible, CTL or HTL precursor cells together with a
source of antigen-presenting cells (APC), such as dendritic cells,
and the appropriate immunogenic peptide. After an appropriate
incubation time (typically about 7-28 days), in which the precursor
cells are activated and expanded into effector cells, the cells are
infused back into the patient, where they will destroy (CTL) or
facilitate destruction (HTL) of their specific target cell (e.g., a
tumor cell). Transfected dendritic cells may also be used as
antigen presenting cells.
[0314] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0315] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 158P1D7. In therapeutic applications, peptide and/or
nucleic acid compositions are administered to a patient in an
amount sufficient to elicit an effective B cell, CTL and/or HTL
response to the antigen and to cure or at least partially arrest or
slow symptoms and/or complications. An amount adequate to
accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on, e.g., the particular
composition administered, the manner of administration, the stage
and severity of the disease being treated, the weight and general
state of health of the patient, and the judgment of the prescribing
physician.
[0316] For pharmaceutical compositions, the immunogenic peptides of
the invention, or DNA encoding them, are generally administered to
an individual already bearing a tumor that expresses 158P1D7. The
peptides or DNA encoding them can be administered individually or
as fusions of one or more peptide sequences. Patients can be
treated with the immunogenic peptides separately or in conjunction
with other treatments, such as surgery, as appropriate.
[0317] For therapeutic use, administration should generally begin
at the first diagnosis of 158P1D7-associated cancer. This is
followed by boosting doses until at least symptoms are
substantially abated and for a period thereafter. The embodiment of
the vaccine composition (i.e., including, but not limited to
embodiments such as peptide cocktails, polyepitopic polypeptides,
minigenes, or TAA-specific CTLs or pulsed dendritic cells)
delivered to the patient may vary according to the stage of the
disease or the patient's health status. For example, in a patient
with a tumor that expresses 158P1D7, a vaccine comprising
158P1D7-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0318] It is generally important to provide an amount of the
peptide epitope delivered by a mode of administration sufficient to
effectively stimulate a cytotoxic T cell response; compositions
which stimulate helper T cell responses can also be given in
accordance with this embodiment of the invention.
[0319] The dosage for an initial therapeutic immunization generally
occurs in a unit dosage range where the lower value is about 1, 5,
50, 500, or 1,000 .mu.g and the higher value is about 10,000;
20,000; 30,000; or 50,000 .mu.g. Dosage values for a human
typically range from about 500 .mu.g to about 50,000 .mu.g per 70
kilogram patient. Boosting dosages of between about 1.0 .mu.g to
about 50,000 .mu.g of peptide pursuant to a boosting regimen over
weeks to months may be administered depending upon the patient's
response and condition as determined by measuring the specific
activity of CTL and HTL obtained from the patient's blood.
Administration should continue until at least clinical symptoms or
laboratory tests indicate that the neoplasia, has been eliminated
or reduced and for a period thereafter. The dosages, routes of
administration, and dose schedules are adjusted in accordance with
methodologies known in the art.
[0320] In certain embodiments, the peptides and compositions of the
present invention are employed in serious disease states, that is,
life-threatening or potentially life threatening situations. In
such cases, as a result of the minimal amounts of extraneous
substances and the relative nontoxic nature of the peptides in
preferred compositions of the invention, it is possible and may be
felt desirable by the treating physician to administer substantial
excesses of these peptide compositions relative to these stated
dosage amounts.
[0321] The vaccine compositions of the invention can also be used
purely as prophylactic agents. Generally the dosage for an initial
prophylactic immunization generally occurs in a unit dosage range
where the lower value is about 1, 5, 50, 500, or 1000 .mu.g and the
higher value is about 10,000; 20,000; 30,000; or 50,000 .mu.g.
Dosage values for a human typically range from about 500 .mu.g to
about 50,000 .mu.g per 70 kilogram patient. This is followed by
boosting dosages of between about 1.0 .mu.g to about 50,000 .mu.g
of peptide administered at defined intervals from about four weeks
to six months after the initial administration of vaccine. The
immunogenicity of the vaccine can be assessed by measuring the
specific activity of CTL and HTL obtained from a sample of the
patient's blood.
[0322] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral, nasal, intrathecal, or
local (e.g. as a cream or topical ointment) administration.
Preferably, the pharmaceutical compositions are administered
parentally, e.g., intravenously, subcutaneously, intradermally, or
intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier.
[0323] A variety of aqueous carriers may be used, e.g., water,
buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the
like. These compositions may be sterilized by conventional,
well-known sterilization techniques, or may be sterile filtered.
The resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration.
[0324] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservatives, and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
[0325] The concentration of peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0326] A human unit dose form of the peptide composition is
typically included in a pharmaceutical composition that comprises a
human unit dose of an acceptable carrier, preferably an aqueous
carrier, and is administered in a volume of fluid that is known by
those of skill in the art to be used for administration of such
compositions to humans (see, e.g., Remington's Pharmaceutical
Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co.,
Easton, Pa., 1985).
[0327] Proteins(s) of the invention, and/or nucleic acids encoding
the protein(s), can also be administered via liposomes, which may
also serve to: 1) target the proteins(s) to a particular tissue,
such as lymphoid tissue; 2) to target selectively to diseases
cells; or, 3) to increase the half-life of the peptide composition.
Liposomes include emulsions, foams, micelles, insoluble monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the
like. In these preparations, the peptide to be delivered is
incorporated as part of a liposome, alone or in conjunction with a
molecule which binds to a receptor prevalent among lymphoid cells,
such as monoclonal antibodies which bind to the CD45 antigen, or
with other therapeutic or immunogenic compositions. Thus, liposomes
either filled or decorated with a desired peptide of the invention
can be directed to the site of lymphoid cells, where the liposomes
then deliver the peptide compositions. Liposomes for use in
accordance with the invention are formed from standard
vesicle-forming lipids, which generally include neutral and
negatively charged phospholipids and a sterol, such as cholesterol.
The selection of lipids is generally guided by consideration of,
e.g., liposome size, acid lability and stability of the liposomes
in the blood stream. A variety of methods are available for
preparing liposomes, as described in, e.g., Szoka, et al., Ann.
Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871,
4,501,728, 4,837,028, and 5,019,369.
[0328] For targeting cells of the immune system, a ligand to be
incorporated into the liposome can include, e.g., antibodies or
fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome suspension containing a
peptide may be administered intravenously, locally, topically, etc.
in a dose which varies according to, inter alia, the manner of
administration, the peptide being delivered, and the stage of the
disease being treated.
[0329] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0330] For aerosol administration, immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are about 0.01%-20%
by weight, preferably about 1%-10%. The surfactant must, of course,
be nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters of
fatty acids containing from about 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids with an aliphatic polyhydric alcohol or
its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be employed. The surfactant may constitute about
0.1%-20% by weight of the composition, preferably about 0.25-5%.
The balance of the composition is ordinarily propellant. A carrier
can also be included, as desired, as with, e.g., lecithin for
intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 158P1D7
[0331] As disclosed herein, 158P1D7 polynucleotides, polypeptides,
reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and
anti-polypeptide antibodies are used in well known diagnostic,
prognostic and therapeutic assays that examine conditions
associated with dysregulated cell growth such as cancer, in
particular the cancers listed in Table I (see, e.g., both its
specific pattern of tissue expression as well as its overexpression
in certain cancers as described for example in Example 4).
[0332] 158P1D7 can be used in a manner analogous to, or as
complementary to, the bladder associated antigen combination,
mucins and CEA, represented in a diagnostic kit called
ImmunoCyt.TM.. ImmunoCyt a is a commercially available assay to
identify and monitor the presence of bladder cancer (see Fradet et
al., 1997, Can J Urol, 4(3):400-405). A variety of other diagnostic
markers are also used in similar contexts including p53 and H-ras
(see, e.g., Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102
and Minimoto et al., Cancer Detect Prey 2000; 24(1):1-12).
Therefore, this disclosure of the 158P1D7 polynucleotides and
polypeptides (as well as the 158P1D7 polynucleotide probes and
anti-158P1D7 antibodies used to identify the presence of these
molecules) and their properties allows skilled artisans to utilize
these molecules in methods that are analogous to those used, for
example, in a variety of diagnostic assays directed to examining
conditions associated with cancer.
[0333] Typical embodiments of diagnostic methods which utilize the
158P1D7 polynucleotides, polypeptides, reactive T cells and
antibodies are analogous to those methods from well-established
diagnostic assays which employ, e.g., PSA polynucleotides,
polypeptides, reactive T cells and antibodies. For example, just as
PSA polynucleotides are used as probes (for example in Northern
analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int.
33(3):567-74(1994)) and primers (for example in PCR analysis, see,
e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe
the presence and/or the level of PSA mRNAs in methods of monitoring
PSA overexpression or the metastasis of prostate cancers, the
158P1D7 polynucleotides described herein can be utilized to detect
158P1D7 overexpression or the metastasis of bladder and other
cancers expressing this gene. Alternatively, just as PSA
polypeptides are used to generate antibodies specific for PSA which
can then be used to observe the presence and/or the level of PSA
proteins in methods to monitor PSA protein overexpression (see,
e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis
of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract.
192(3):233-7 (1996)), the 158P1D7 polypeptides described herein can
be utilized to generate antibodies for use in detecting 158P1D7
overexpression or the metastasis of bladder cells and cells of
other cancers expressing this gene.
[0334] Specifically, because metastases involves the movement of
cancer cells from an organ of origin (such as the lung or bladder
etc.) to a different area of the body (such as a lymph node),
assays which examine a biological sample for the presence of cells
expressing 158P1D7 polynucleotides and/or polypeptides can be used
to provide evidence of metastasis. For example, when a biological
sample from tissue that does not normally contain
158P1D7-expressing cells (lymph node) is found to contain
158P1D7-expressing cells such as the 158P1D7 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0335] Alternatively 158P1D7 polynucleotides and/or polypeptides
can be used to provide evidence of cancer, for example, when cells
in a biological sample that do not normally express 158P1D7 or
express 158P1D7 at a different level are found to express 158P1D7
or have an increased expression of 158P1D7 (see, e.g., the 158P1D7
expression in the cancers listed in Table I and in patient samples
etc. shown in the accompanying Figures). In such assays, artisans
may further wish to generate supplementary evidence of metastasis
by testing the biological sample for the presence of a second
tissue restricted marker (in addition to 158P1D7) such as
ImmunoCyt.TM. PSCA etc. (see, e.g., Fradet et al., 1997, Can J
Urol, 4(3):400-405; Amara et al., 2001, Cancer Res 61:4660-4665).
Just as PSA polynucleotide fragments and polynucleotide variants
are employed by skilled artisans for use in methods of monitoring
PSA, 158P1D7 polynucleotide fragments and polynucleotide variants
are used in an analogous manner. In particular, typical PSA
polynucleotides used in methods of monitoring PSA are probes or
primers which consist of fragments of the PSA cDNA sequence.
Illustrating this, primers used to PCR amplify a PSA polynucleotide
must include less than the whole PSA sequence to function in the
polymerase chain reaction. In the context of such PCR reactions,
skilled artisans generally create a variety of different
polynucleotide fragments that can be used as primers in order to
amplify different portions of a polynucleotide of interest or to
optimize amplification reactions (see, e.g., Caetano-Anolles, G.
Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al.,
Methods Mol. Biol. 98:121-154 (1998)). An additional illustration
of the use of such fragments is provided in Example 4, where a
158P1D7 polynucleotide fragment is used as a probe to show the
expression of 158P1D7 RNAs in cancer cells. In addition, variant
polynucleotide sequences are typically used as primers and probes
for the corresponding mRNAs in PCR and Northern analyses (see,
e.g., Sawai et al., Fetal Diagn. Ther. 1996 November-December
11(6):407-13 and Current Protocols In Molecular Biology, Volume 2,
Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide
fragments and variants are useful in this context where they are
capable of binding to a target polynucleotide sequence (e.g. the
158P1D7 polynucleotide shown in FIG. 2) under conditions of high
stringency.
[0336] Furthermore, PSA polypeptides which contain an epitope that
can be recognized by an antibody or T cell that specifically binds
to that epitope are used in methods of monitoring PSA. 158P1D7
polypeptide fragments and polypeptide analogs or variants can also
be used in an analogous manner. This practice of using polypeptide
fragments or polypeptide variants to generate antibodies (such as
anti-PSA antibodies or T cells) is typical in the art with a wide
variety of systems such as fusion proteins being used by
practitioners (see, e.g., Current Protocols In Molecular Biology,
Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this
context, each epitope(s) functions to provide the architecture with
which an antibody or T cell is reactive. Typically, skilled
artisans create a variety of different polypeptide fragments that
can be used in order to generate immune responses specific for
different portions of a polypeptide of interest (see, e.g., U.S.
Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may
be preferable to utilize a polypeptide comprising one of the
158P1D7 biological motifs discussed herein or a motif-bearing
subsequence which is readily identified by one of skill in the art
based on motifs available in the art. Polypeptide fragments,
variants or analogs are typically useful in this context as long as
they comprise an epitope capable of generating an antibody or T
cell specific for a target polypeptide sequence (e.g. the 158P1D7
polypeptide shown in FIG. 2).
[0337] As shown herein, the 158P1D7 polynucleotides and
polypeptides (as well as the 158P1D7 polynucleotide probes and
anti-158P1D7 antibodies or T cells used to identify the presence of
these molecules) exhibit specific properties that make them useful
in diagnosing cancers such as those listed in Table I. Diagnostic
assays that measure the presence of 158P1D7 gene products, in order
to evaluate the presence or onset of a disease condition described
herein, such as bladder cancer, are used to identify patients for
preventive measures or further monitoring, as has been done so
successfully with PSA for monitoring prostate cancer. Materials
such as 158P1D7 polynucleotides and polypeptides (as well as the
158P1D7 polynucleotide probes and anti-158P1D7 antibodies used to
identify the presence of these molecules) satisfy a need in the art
for molecules having similar or complementary characteristics to
PSA in situations of bladder cancer. Finally, in addition to their
use in diagnostic assays, the 158P1D7 polynucleotides disclosed
herein have a number of other utilities such as their use in the
identification of oncogenetic associated chromosomal abnormalities
in the chromosomal region to which the 158P1D7 gene maps (see
Example 3 below). Moreover, in addition to their use in diagnostic
assays, the 158P1D7-related proteins and polynucleotides disclosed
herein have other utilities such as their use in the forensic
analysis of tissues of unknown origin (see, e.g., Takahama K
Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).
[0338] Additionally, 158P1D7-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 158P1D7. For example, the
amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or
fragments of either, can be used to generate an immune response to
the 158P1D7 antigen. Antibodies or other molecules that react with
158P1D7 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
XII.) Inhibition of 158P1D7 Protein Function
[0339] The invention includes various methods and compositions for
inhibiting the binding of 158P1D7 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
158P1D7 function.
[0340] XII.A.) Inhibition of 158P1D7 with Intracellular
Antibodies
[0341] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 158P1D7 are introduced
into 158P1D7 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-158P1D7 antibody is
expressed intracellularly, binds to 158P1D7 protein, and thereby
inhibits its function. Methods for engineering such intracellular
single chain antibodies are well known. Such intracellular
antibodies, also known as "intrabodies", are specifically targeted
to a particular compartment within the cell, providing control over
where the inhibitory activity of the treatment is focused. This
technology has been successfully applied in the art (for review,
see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies
have been shown to virtually eliminate the expression of otherwise
abundant cell surface receptors (see, e.g., Richardson et al.,
1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,
1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene
Ther. 1: 332-337).
[0342] Single chain antibodies comprise the variable domains of the
heavy and light chain joined by a flexible linker polypeptide, and
are expressed as a single polypeptide. Optionally, single chain
antibodies are expressed as a single chain variable region fragment
joined to the light chain constant region. Well-known intracellular
trafficking signals are engineered into recombinant polynucleotide
vectors encoding such single chain antibodies in order to precisely
target the intrabody to the desired intracellular compartment. For
example, intrabodies targeted to the endoplasmic reticulum (ER) are
engineered to incorporate a leader peptide and, optionally, a
C-terminal ER retention signal, such as the KDEL amino acid motif.
Intrabodies intended to exert activity in the nucleus are
engineered to include a nuclear localization signal. Lipid moieties
are joined to intrabodies in order to tether the intrabody to the
cytosolic side of the plasma membrane. Intrabodies can also be
targeted to exert function in the cytosol. For example, cytosolic
intrabodies are used to sequester factors within the cytosol,
thereby preventing them from being transported to their natural
cellular destination.
[0343] In one embodiment, intrabodies are used to capture 158P1D7
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 158P1D7
intrabodies in order to achieve the desired targeting. Such 158P1D7
intrabodies are designed to bind specifically to a particular
158P1D7 domain. In another embodiment, cytosolic intrabodies that
specifically bind to the 158P1D7 protein are used to prevent
158P1D7 from gaining access to the nucleus, thereby preventing it
from exerting any biological activity within the nucleus (e.g.,
preventing 158P1D7 from forming transcription complexes with other
factors).
[0344] In order to specifically direct the expression of such
intrabodies to particular cells, the transcription of the intrabody
is placed under the regulatory control of an appropriate
tumor-specific promoter and/or enhancer. In order to target
intrabody expression specifically to bladder, for example, the PSCA
promoter and/or promoter/enhancer can be utilized (See, for
example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999 and Lin et al.
PNAS, USA 92(3):679-683 (1995)).
[0345] XII.B.) Inhibition of 158P1D7 with Recombinant Proteins
[0346] In another approach, recombinant molecules bind to 158P1D7
and thereby inhibit 158P1D7 function. For example, these
recombinant molecules prevent or inhibit 158P1D7 from
accessing/binding to its binding partner(s) or associating with
other protein(s). Such recombinant molecules can, for example,
contain the reactive part(s) of a 158P1D7 specific antibody
molecule. In a particular embodiment, the 158P1D7 binding domain of
a 158P1D7 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 158P1D7 ligand
binding domains linked to the Fc portion of a human IgG, such as
human IgG1. Such IgG portion can contain, for example, the C.sub.H2
and C.sub.H3 domains and the hinge region, but not the C.sub.H1
domain. Such dimeric fusion proteins are administered in soluble
form to patients suffering from a cancer associated with the
expression of 158P1D7, whereby the dimeric fusion protein
specifically binds to 158P1D7 and blocks 158P1D7 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0347] XII.C.) Inhibition of 158P1D7 Transcription or
Translation
[0348] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 158P1D7 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 158P1D7 mRNA into protein.
[0349] In one approach, a method of inhibiting the transcription of
the 158P1D7 gene comprises contacting the 158P1D7 gene with a
158P1D7 antisense polynucleotide. In another approach, a method of
inhibiting 158P1D7 mRNA translation comprises contacting the
158P1D7 mRNA with an antisense polynucleotide. In another approach,
a 158P1D7 specific ribozyme is used to cleave the 158P1D7 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
158P1D7 gene, such as the 158P1D7 promoter and/or enhancer
elements. Similarly, proteins capable of inhibiting a 158P1D7 gene
transcription factor are used to inhibit 158P1D7 mRNA
transcription. The various polynucleotides and compositions useful
in the aforementioned methods have been described above. The use of
antisense and ribozyme molecules to inhibit transcription and
translation is well known in the art.
[0350] Other factors that inhibit the transcription of 158P1D7 by
interfering with 158P1D7 transcriptional activation are also useful
to treat cancers expressing 158P1D7. Similarly, factors that
interfere with 158P1D7 processing are useful to treat cancers that
express 158P1D7. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0351] XII.D.) General Considerations for Therapeutic
Strategies
[0352] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 158P1D7 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 158P1D7 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 158P1D7 antisense polynucleotides, ribozymes,
factors capable of interfering with 158P1D7 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0353] The above therapeutic approaches can be combined with any
one of a wide variety of surgical, chemotherapy or radiation
therapy regimens. The therapeutic approaches of the invention can
enable the use of reduced dosages of chemotherapy (or other
therapies) and/or less frequent administration, an advantage for
all patients and particularly for those that do not tolerate the
toxicity of the chemotherapeutic agent well.
[0354] The anti-tumor activity of a particular composition (e.g.,
antisense, ribozyme, intrabody), or a combination of such
compositions, can be evaluated using various in vitro and in vivo
assay systems. In vitro assays that evaluate therapeutic activity
include cell growth assays, soft agar assays and other assays
indicative of tumor promoting activity, binding assays capable of
determining the extent to which a therapeutic composition will
inhibit the binding of 158P1D7 to a binding partner, etc.
[0355] In vivo, the effect of a 158P1D7 therapeutic composition can
be evaluated in a suitable animal model. For example, xenogenic
bladder cancer models can be used, wherein human bladder cancer
explants or passaged xenograft tissues are introduced into immune
compromised animals, such as nude or SCID mice (Shibayama et al.,
1991, J Urol., 146(4):1136-7; Beecken et al., 2000, Urology,
56(3):521-526). Efficacy can be predicted using assays that measure
inhibition of tumor formation, tumor regression or metastasis, and
the like.
[0356] In vivo assays that evaluate the promotion of apoptosis are
useful in evaluating therapeutic compositions. In one embodiment,
xenografts from tumor bearing mice treated with the therapeutic
composition can be examined for the presence of apoptotic foci and
compared to untreated control xenograft-bearing mice. The extent to
which apoptotic foci are found in the tumors of the treated mice
provides an indication of the therapeutic efficacy of the
composition.
[0357] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16th
Edition, A. Osal., Ed., 1980).
[0358] Therapeutic formulations can be solubilized and administered
via any route capable of delivering the therapeutic composition to
the tumor site. Potentially effective routes of administration
include, but are not limited to, intravenous, parenteral,
intraperitoneal, intramuscular, intratumor, intradermal,
intraorgan, orthotopic, and the like. A preferred formulation for
intravenous injection comprises the therapeutic composition in a
solution of preserved bacteriostatic water, sterile unpreserved
water, and/or diluted in polyvinylchloride or polyethylene bags
containing 0.9% sterile Sodium Chloride for Injection, USP.
Therapeutic protein preparations can be lyophilized and stored as
sterile powders, preferably under vacuum, and then reconstituted in
bacteriostatic water (containing for example, benzyl alcohol
preservative) or in sterile water prior to injection.
[0359] Dosages and administration protocols for the treatment of
cancers using the foregoing methods will vary with the method and
the target cancer, and will generally depend on a number of other
factors appreciated in the art.
XIII.) Identification, Characterization and Use of Modulators of
158P1D7
[0360] Methods to Identify and Use Modulators
[0361] In one embodiment, screening is performed to identify
modulators that induce or suppress a particular expression profile,
suppress or induce specific pathways, preferably generating the
associated phenotype thereby. In another embodiment, having
identified differentially expressed genes important in a particular
state; screens are performed to identify modulators that alter
expression of individual genes, either increase or decrease. In
another embodiment, screening is performed to identify modulators
that alter a biological function of the expression product of a
differentially expressed gene. Again, having identified the
importance of a gene in a particular state, screens are performed
to identify agents that bind and/or modulate the biological
activity of the gene product.
[0362] In addition, screens are done for genes that are induced in
response to a candidate agent. After identifying a modulator (one
that suppresses a cancer expression pattern leading to a normal
expression pattern, or a modulator of a cancer gene that leads to
expression of the gene as in normal tissue) a screen is performed
to identify genes that are specifically modulated in response to
the agent. Comparing expression profiles between normal tissue and
agent-treated cancer tissue reveals genes that are not expressed in
normal tissue or cancer tissue, but are expressed in agent treated
tissue, and vice versa. These agent-specific sequences are
identified and used by methods described herein for cancer genes or
proteins. In particular these sequences and the proteins they
encode are used in marking or identifying agent-treated cells. In
addition, antibodies are raised against the agent-induced proteins
and used to target novel therapeutics to the treated cancer tissue
sample.
[0363] Modulator-Related Identification and Screening Assays:
[0364] Gene Expression-Related Assays
[0365] Proteins, nucleic acids, and antibodies of the invention are
used in screening assays. The cancer-associated proteins,
antibodies, nucleic acids, modified proteins and cells containing
these sequences are used in screening assays, such as evaluating
the effect of drug candidates on a "gene expression profile,"
expression profile of polypeptides or alteration of biological
function. In one embodiment, the expression profiles are used,
preferably in conjunction with high throughput screening techniques
to allow monitoring for expression profile genes after treatment
with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69
(2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome
Res 6:986-94, 1996).
[0366] The cancer proteins, antibodies, nucleic acids, modified
proteins and cells containing the native or modified cancer
proteins or genes are used in screening assays. That is, the
present invention comprises methods for screening for compositions
which modulate the cancer phenotype or a physiological function of
a cancer protein of the invention. This is done on a gene itself or
by evaluating the effect of drug candidates on a "gene expression
profile" or biological function. In one embodiment, expression
profiles are used, preferably in conjunction with high throughput
screening techniques to allow monitoring after treatment with a
candidate agent, see Zlokarnik, supra.
[0367] A variety of assays are executed directed to the genes and
proteins of the invention. Assays are run on an individual nucleic
acid or protein level. That is, having identified a particular gene
as up regulated in cancer, test compounds are screened for the
ability to modulate gene expression or for binding to the cancer
protein of the invention. "Modulation" in this context includes an
increase or a decrease in gene expression. The preferred amount of
modulation will depend on the original change of the gene
expression in normal versus tissue undergoing cancer, with changes
of at least 10%, preferably 50%, more preferably 100-300%, and in
some embodiments 300-1000% or greater. Thus, if a gene exhibits a
4-fold increase in cancer tissue compared to normal tissue, a
decrease of about four-fold is often desired; similarly, a 10-fold
decrease in cancer tissue compared to normal tissue a target value
of a 10-fold increase in expression by the test compound is often
desired. Modulators that exacerbate the type of gene expression
seen in cancer are also useful, e.g., as an upregulated target in
further analyses.
[0368] The amount of gene expression is monitored using nucleic
acid probes and the quantification of gene expression levels, or,
alternatively, a gene product itself is monitored, e.g., through
the use of antibodies to the cancer protein and standard
immunoassays. Proteomics and separation techniques also allow for
quantification of expression.
[0369] Expression Monitoring to Identify Compounds that Modify Gene
Expression
[0370] In one embodiment, gene expression monitoring, i.e., an
expression profile, is monitored simultaneously for a number of
entities. Such profiles will typically involve one or more of the
genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid
probes are attached to biochips to detect and quantify cancer
sequences in a particular cell. Alternatively, PCR can be used.
Thus, a series, e.g., wells of a microtiter plate, can be used with
dispensed primers in desired wells. A PCR reaction can then be
performed and analyzed for each well.
[0371] Expression monitoring is performed to identify compounds
that modify the expression of one or more cancer-associated
sequences, e.g., a polynucleotide sequence set out in FIG. 2.
Generally, a test modulator is added to the cells prior to
analysis. Moreover, screens are also provided to identify agents
that modulate cancer, modulate cancer proteins of the invention,
bind to a cancer protein of the invention, or interfere with the
binding of a cancer protein of the invention and an antibody or
other binding partner.
[0372] In one embodiment, high throughput screening methods involve
providing a library containing a large number of potential
therapeutic compounds (candidate compounds). Such "combinatorial
chemical libraries" are then screened in one or more assays to
identify those library members (particular chemical species or
subclasses) that display a desired characteristic activity. The
compounds thus identified can serve as conventional "lead
compounds," as compounds for screening, or as therapeutics.
[0373] In certain embodiments, combinatorial libraries of potential
modulators are screened for an ability to bind to a cancer
polypeptide or to modulate activity. Conventionally, new chemical
entities with useful properties are generated by identifying a
chemical compound (called a "lead compound") with some desirable
property or activity, e.g., inhibiting activity, creating variants
of the lead compound, and evaluating the property and activity of
those variant compounds. Often, high throughput screening (HTS)
methods are employed for such an analysis.
[0374] As noted above, gene expression monitoring is conveniently
used to test candidate modulators (e.g., protein, nucleic acid or
small molecule). After the candidate agent has been added and the
cells allowed to incubate for a period, the sample containing a
target sequence to be analyzed is, e.g., added to a biochip.
[0375] If required, the target sequence is prepared using known
techniques. For example, a sample is treated to lyse the cells,
using known lysis buffers, electroporation, etc., with purification
and/or amplification such as PCR performed as appropriate. For
example, an in vitro transcription with labels covalently attached
to the nucleotides is performed. Generally, the nucleic acids are
labeled with biotin-FITC or PE, or with cy3 or cy5.
[0376] The target sequence can be labeled with, e.g., a
fluorescent, a chemiluminescent, a chemical, or a radioactive
signal, to provide a means of detecting the target sequence's
specific binding to a probe. The label also can be an enzyme, such
as alkaline phosphatase or horseradish peroxidase, which when
provided with an appropriate substrate produces a product that is
detected. Alternatively, the label is a labeled compound or small
molecule, such as an enzyme inhibitor, that binds but is not
catalyzed or altered by the enzyme. The label also can be a moiety
or compound, such as, an epitope tag or biotin which specifically
binds to streptavidin. For the example of biotin, the streptavidin
is labeled as described above, thereby, providing a detectable
signal for the bound target sequence. Unbound labeled streptavidin
is typically removed prior to analysis.
[0377] As will be appreciated by those in the art, these assays can
be direct hybridization assays or can comprise "sandwich assays",
which include the use of multiple probes, as is generally outlined
in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117;
5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802;
5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this
embodiment, in general, the target nucleic acid is prepared as
outlined above, and then added to the biochip comprising a
plurality of nucleic acid probes, under conditions that allow the
formation of a hybridization complex.
[0378] A variety of hybridization conditions are used in the
present invention, including high, moderate and low stringency
conditions as outlined above. The assays are generally run under
stringency conditions which allow formation of the label probe
hybridization complex only in the presence of target. Stringency
can be controlled by altering a step parameter that is a
thermodynamic variable, including, but not limited to, temperature,
formamide concentration, salt concentration, chaotropic salt
concentration pH, organic solvent concentration, etc. These
parameters may also be used to control non-specific binding, as is
generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be
desirable to perform certain steps at higher stringency conditions
to reduce non-specific binding.
[0379] The reactions outlined herein can be accomplished in a
variety of ways. Components of the reaction can be added
simultaneously, or sequentially, in different orders, with
preferred embodiments outlined below. In addition, the reaction may
include a variety of other reagents. These include salts, buffers,
neutral proteins, e.g. albumin, detergents, etc. which can be used
to facilitate optimal hybridization and detection, and/or reduce
nonspecific or background interactions. Reagents that otherwise
improve the efficiency of the assay, such as protease inhibitors,
nuclease inhibitors, anti-microbial agents, etc., may also be used
as appropriate, depending on the sample preparation methods and
purity of the target. The assay data are analyzed to determine the
expression levels of individual genes, and changes in expression
levels as between states, forming a gene expression profile.
[0380] Biological Activity-Related Assays
[0381] The invention provides methods identify or screen for a
compound that modulates the activity of a cancer-related gene or
protein of the invention. The methods comprise adding a test
compound, as defined above, to a cell comprising a cancer protein
of the invention. The cells contain a recombinant nucleic acid that
encodes a cancer protein of the invention. In another embodiment, a
library of candidate agents is tested on a plurality of cells.
[0382] In one aspect, the assays are evaluated in the presence or
absence or previous or subsequent exposure of physiological
signals, e.g. hormones, antibodies, peptides, antigens, cytokines,
growth factors, action potentials, pharmacological agents including
chemotherapeutics, radiation, carcinogenics, or other cells (i.e.,
cell-cell contacts). In another example, the determinations are
made at different stages of the cell cycle process. In this way,
compounds that modulate genes or proteins of the invention are
identified. Compounds with pharmacological activity are able to
enhance or interfere with the activity of the cancer protein of the
invention. Once identified, similar structures are evaluated to
identify critical structural features of the compound.
[0383] In one embodiment, a method of modulating (e.g., inhibiting)
cancer cell division is provided; the method comprises
administration of a cancer modulator. In another embodiment, a
method of modulating (e.g., inhibiting) cancer is provided; the
method comprises administration of a cancer modulator. In a further
embodiment, methods of treating cells or individuals with cancer
are provided; the method comprises administration of a cancer
modulator.
[0384] In one embodiment, a method for modulating the status of a
cell that expresses a gene of the invention is provided. As used
herein status comprises such art-accepted parameters such as
growth, proliferation, survival, function, apoptosis, senescence,
location, enzymatic activity, signal transduction, etc. of a cell.
In one embodiment, a cancer inhibitor is an antibody as discussed
above. In another embodiment, the cancer inhibitor is an antisense
molecule. A variety of cell growth, proliferation, and metastasis
assays are known to those of skill in the art, as described
herein.
[0385] High Throughput Screening to Identify Modulators
[0386] The assays to identify suitable modulators are amenable to
high throughput screening. Preferred assays thus detect enhancement
or inhibition of cancer gene transcription, inhibition or
enhancement of polypeptide expression, and inhibition or
enhancement of polypeptide activity.
[0387] In one embodiment, modulators evaluated in high throughput
screening methods are proteins, often naturally occurring proteins
or fragments of naturally occurring proteins. Thus, e.g., cellular
extracts containing proteins, or random or directed digests of
proteinaceous cellular extracts, are used. In this way, libraries
of proteins are made for screening in the methods of the invention.
Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred.
Particularly useful test compound will be directed to the class of
proteins to which the target belongs, e.g., substrates for enzymes,
or ligands and receptors.
[0388] Use of Soft Agar Growth and Colony Formation to Identify and
Characterize Modulators
[0389] Normal cells require a solid substrate to attach and grow.
When cells are transformed, they lose this phenotype and grow
detached from the substrate. For example, transformed cells can
grow in stirred suspension culture or suspended in semi-solid
media, such as semi-solid or soft agar. The transformed cells, when
transfected with tumor suppressor genes, can regenerate normal
phenotype and once again require a solid substrate to attach to and
grow. Soft agar growth or colony formation in assays are used to
identify modulators of cancer sequences, which when expressed in
host cells, inhibit abnormal cellular proliferation and
transformation. A modulator reduces or eliminates the host cells'
ability to grow suspended in solid or semisolid media, such as
agar.
[0390] Techniques for soft agar growth or colony formation in
suspension assays are described in Freshney, Culture of Animal
Cells a Manual of Basic Technique (3rd ed., 1994). See also, the
methods section of Garkavtsev et al. (1996), supra.
[0391] Evaluation of Contact Inhibition and Growth Density
Limitation to Identify and Characterize Modulators
[0392] Normal cells typically grow in a flat and organized pattern
in cell culture until they touch other cells. When the cells touch
one another, they are contact inhibited and stop growing.
Transformed cells, however, are not contact inhibited and continue
to grow to high densities in disorganized foci. Thus, transformed
cells grow to a higher saturation density than corresponding normal
cells. This is detected morphologically by the formation of a
disoriented monolayer of cells or cells in foci. Alternatively,
labeling index with (3H)-thymidine at saturation density is used to
measure density limitation of growth, similarly an MTT or Alamar
blue assay will reveal proliferation capacity of cells and the
ability of modulators to affect same. See Freshney (1994), supra.
Transformed cells, when transfected with tumor suppressor genes,
can regenerate a normal phenotype and become contact inhibited and
would grow to a lower density.
[0393] In this assay, labeling index with 3H)-thymidine at
saturation density is a preferred method of measuring density
limitation of growth. Transformed host cells are transfected with a
cancer-associated sequence and are grown for 24 hours at saturation
density in non-limiting medium conditions. The percentage of cells
labeling with (3H)-thymidine is determined by incorporated cpm.
[0394] Contact independent growth is used to identify modulators of
cancer sequences, which had led to abnormal cellular proliferation
and transformation. A modulator reduces or eliminates contact
independent growth, and returns the cells to a normal
phenotype.
[0395] Evaluation of Growth Factor or Serum Dependence to Identify
and Characterize Modulators
[0396] Transformed cells have lower serum dependence than their
normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst.
37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970));
Freshney, supra. This is in part due to release of various growth
factors by the transformed cells. The degree of growth factor or
serum dependence of transformed host cells can be compared with
that of control. For example, growth factor or serum dependence of
a cell is monitored in methods to identify and characterize
compounds that modulate cancer-associated sequences of the
invention.
[0397] Use of Tumor-Specific Marker Levels to Identify and
Characterize Modulators
[0398] Tumor cells release an increased amount of certain factors
(hereinafter "tumor specific markers") than their normal
counterparts. For example, plasminogen activator (PA) is released
from human glioma at a higher level than from normal brain cells
(see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and
Potential Interference with Tumor Growth, in Biological Responses
in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor
Angiogenesis Factor (TAF) is released at a higher level in tumor
cells than their normal counterparts. See, e.g., Folkman,
Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is
released from endothelial tumors (Ensoli, B et al).
[0399] Various techniques which measure the release of these
factors are described in Freshney (1994), supra. Also, see, Unkless
et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland &
Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J.
Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor
Vascularization, and Potential Interference with Tumor Growth, in
Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985);
Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor
specific marker levels are monitored in methods to identify and
characterize compounds that modulate cancer-associated sequences of
the invention.
[0400] Invasiveness into Matrigel to Identify and Characterize
Modulators
[0401] The degree of invasiveness into Matrigel or an extracellular
matrix constituent can be used as an assay to identify and
characterize compounds that modulate cancer associated sequences.
Tumor cells exhibit a positive correlation between malignancy and
invasiveness of cells into Matrigel or some other extracellular
matrix constituent. In this assay, tumorigenic cells are typically
used as host cells. Expression of a tumor suppressor gene in these
host cells would decrease invasiveness of the host cells.
Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994),
supra, can be used. Briefly, the level of invasion of host cells is
measured by using filters coated with Matrigel or some other
extracellular matrix constituent. Penetration into the gel, or
through to the distal side of the filter, is rated as invasiveness,
and rated histologically by number of cells and distance moved, or
by prelabeling the cells with .sup.125I and counting the
radioactivity on the distal side of the filter or bottom of the
dish. See, e.g., Freshney (1984), supra.
[0402] Evaluation of Tumor Growth In Vivo to Identify and
Characterize Modulators
[0403] Effects of cancer-associated sequences on cell growth are
tested in transgenic or immune-suppressed organisms. Transgenic
organisms are prepared in a variety of art-accepted ways. For
example, knock-out transgenic organisms, e.g., mammals such as
mice, are made, in which a cancer gene is disrupted or in which a
cancer gene is inserted. Knock-out transgenic mice are made by
insertion of a marker gene or other heterologous gene into the
endogenous cancer gene site in the mouse genome via homologous
recombination. Such mice can also be made by substituting the
endogenous cancer gene with a mutated version of the cancer gene,
or by mutating the endogenous cancer gene, e.g., by exposure to
carcinogens.
[0404] To prepare transgenic chimeric animals, e.g., mice, a DNA
construct is introduced into the nuclei of embryonic stem cells.
Cells containing the newly engineered genetic lesion are injected
into a host mouse embryo, which is re-implanted into a recipient
female. Some of these embryos develop into chimeric mice that
possess germ cells some of which are derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric
mice can be derived according to U.S. Pat. No. 6,365,797, issued 2
Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et
al., Manipulating the Mouse Embryo: A laboratory Manual, Cold
Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, Robertson, ed., IRL Press,
Washington, D.C., (1987).
[0405] Alternatively, various immune-suppressed or immune-deficient
host animals can be used. For example, a genetically athymic "nude"
mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921
(1974)), a SCID mouse, a thymectornized mouse, or an irradiated
mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978);
Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host.
Transplantable tumor cells (typically about 106 cells) injected
into isogenic hosts produce invasive tumors in a high proportion of
cases, while normal cells of similar origin will not. In hosts
which developed invasive tumors, cells expressing cancer-associated
sequences are injected subcutaneously or orthotopically. Mice are
then separated into groups, including control groups and treated
experimental groups) e.g. treated with a modulator). After a
suitable length of time, preferably 4-8 weeks, tumor growth is
measured (e.g., by volume or by its two largest dimensions, or
weight) and compared to the control. Tumors that have statistically
significant reduction (using, e.g., Student's T test) are said to
have inhibited growth.
[0406] In Vitro Assays to Identify and Characterize Modulators
[0407] Assays to identify compounds with modulating activity can be
performed in vitro. For example, a cancer polypeptide is first
contacted with a potential modulator and incubated for a suitable
amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the
cancer polypeptide levels are determined in vitro by measuring the
level of protein or mRNA. The level of protein is measured using
immunoassays such as Western blotting, ELISA and the like with an
antibody that selectively binds to the cancer polypeptide or a
fragment thereof. For measurement of mRNA, amplification, e.g.,
using PCR, LCR, or hybridization assays, e.g., Northern
hybridization, RNAse protection, dot blotting, are preferred. The
level of protein or mRNA is detected using directly or indirectly
labeled detection agents, e.g., fluorescently or radioactively
labeled nucleic acids, radioactively or enzymatically labeled
antibodies, and the like, as described herein.
[0408] Alternatively, a reporter gene system can be devised using a
cancer protein promoter operably linked to a reporter gene such as
luciferase, green fluorescent protein, CAT, or P-gal. The reporter
construct is typically transfected into a cell. After treatment
with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured according to
standard techniques known to those of skill in the art (Davis GF,
supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol.
1998: 9:624).
[0409] As outlined above, in vitro screens are done on individual
genes and gene products. That is, having identified a particular
differentially expressed gene as important in a particular state,
screening of modulators of the expression of the gene or the gene
product itself is performed.
[0410] In one embodiment, screening for modulators of expression of
specific gene(s) is performed. Typically, the expression of only
one or a few genes is evaluated. In another embodiment, screens are
designed to first find compounds that bind to differentially
expressed proteins. These compounds are then evaluated for the
ability to modulate differentially expressed activity. Moreover,
once initial candidate compounds are identified, variants can be
further screened to better evaluate structure activity
relationships.
[0411] Binding Assays to Identify and Characterize Modulators
[0412] In binding assays in accordance with the invention, a
purified or isolated gene product of the invention is generally
used. For example, antibodies are generated to a protein of the
invention, and immunoassays are run to determine the amount and/or
location of protein. Alternatively, cells comprising the cancer
proteins are used in the assays.
[0413] Thus, the methods comprise combining a cancer protein of the
invention and a candidate compound such as a ligand, and
determining the binding of the compound to the cancer protein of
the invention. Preferred embodiments utilize the human cancer
protein; animal models of human disease of can also be developed
and used. Also, other analogous mammalian proteins also can be used
as appreciated by those of skill in the art. Moreover, in some
embodiments variant or derivative cancer proteins are used.
[0414] Generally, the cancer protein of the invention, or the
ligand, is non-diffusibly bound to an insoluble support. The
support can, e.g., be one having isolated sample receiving areas (a
microtiter plate, an array, etc.). The insoluble supports can be
made of any composition to which the compositions can be bound, is
readily separated from soluble material, and is otherwise
compatible with the overall method of screening. The surface of
such supports can be solid or porous and of any convenient
shape.
[0415] Examples of suitable insoluble supports include microtiter
plates, arrays, membranes and beads. These are typically made of
glass, plastic (e.g., polystyrene), polysaccharide, nylon,
nitrocellulose, or Teflon.TM., etc. Microtiter plates and arrays
are especially convenient because a large number of assays can be
carried out simultaneously, using small amounts of reagents and
samples. The particular manner of binding of the composition to the
support is not crucial so long as it is compatible with the
reagents and overall methods of the invention, maintains the
activity of the composition and is nondiffusable. Preferred methods
of binding include the use of antibodies which do not sterically
block either the ligand binding site or activation sequence when
attaching the protein to the support, direct binding to "sticky" or
ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface, etc. Following binding of the protein or
ligand/binding agent to the support, excess unbound material is
removed by washing. The sample receiving areas may then be blocked
through incubation with bovine serum albumin (BSA), casein or other
innocuous protein or other moiety.
[0416] Once a cancer protein of the invention is bound to the
support, and a test compound is added to the assay. Alternatively,
the candidate binding agent is bound to the support and the cancer
protein of the invention is then added. Binding agents include
specific antibodies, non-natural binding agents identified in
screens of chemical libraries, peptide analogs, etc.
[0417] Of particular interest are assays to identify agents that
have a low toxicity for human cells. A wide variety of assays can
be used for this purpose, including proliferation assays, cAMP
assays, labeled in vitro protein-protein binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, functional assays (phosphorylation assays, etc.) and the
like.
[0418] A determination of binding of the test compound (ligand,
binding agent, modulator, etc.) to a cancer protein of the
invention can be done in a number of ways. The test compound can be
labeled, and binding determined directly, e.g., by attaching all or
a portion of the cancer protein of the invention to a solid
support, adding a labeled candidate compound (e.g., a fluorescent
label), washing off excess reagent, and determining whether the
label is present on the solid support. Various blocking and washing
steps can be utilized as appropriate.
[0419] In certain embodiments, only one of the components is
labeled, e.g., a protein of the invention or ligands labeled.
Alternatively, more than one component is labeled with different
labels, e.g., I125, for the proteins and a fluorophor for the
compound. Proximity reagents, e.g., quenching or energy transfer
reagents are also useful.
[0420] Competitive Binding to Identify and Characterize
Modulators
[0421] In one embodiment, the binding of the "test compound" is
determined by competitive binding assay with a "competitor." The
competitor is a binding moiety that binds to the target molecule
(e.g., a cancer protein of the invention). Competitors include
compounds such as antibodies, peptides, binding partners, ligands,
etc. Under certain circumstances, the competitive binding between
the test compound and the competitor displaces the test compound.
In one embodiment, the test compound is labeled. Either the test
compound, the competitor, or both, is added to the protein for a
time sufficient to allow binding. Incubations are performed at a
temperature that facilitates optimal activity, typically between
four and 40.degree. C. Incubation periods are typically optimized,
e.g., to facilitate rapid high throughput screening; typically
between zero and one hour will be sufficient. Excess reagent is
generally removed or washed away. The second component is then
added, and the presence or absence of the labeled component is
followed, to indicate binding.
[0422] In one embodiment, the competitor is added first, followed
by the test compound. Displacement of the competitor is an
indication that the test compound is binding to the cancer protein
and thus is capable of binding to, and potentially modulating, the
activity of the cancer protein. In this embodiment, either
component can be labeled. Thus, e.g., if the competitor is labeled,
the presence of label in the post-test compound wash solution
indicates displacement by the test compound. Alternatively, if the
test compound is labeled, the presence of the label on the support
indicates displacement.
[0423] In an alternative embodiment, the test compound is added
first, with incubation and washing, followed by the competitor. The
absence of binding by the competitor indicates that the test
compound binds to the cancer protein with higher affinity than the
competitor. Thus, if the test compound is labeled, the presence of
the label on the support, coupled with a lack of competitor
binding, indicates that the test compound binds to and thus
potentially modulates the cancer protein of the invention.
[0424] Accordingly, the competitive binding methods comprise
differential screening to identity agents that are capable of
modulating the activity of the cancer proteins of the invention. In
this embodiment, the methods comprise combining a cancer protein
and a competitor in a first sample. A second sample comprises a
test compound, the cancer protein, and a competitor. The binding of
the competitor is determined for both samples, and a change, or
difference in binding between the two samples indicates the
presence of an agent capable of binding to the cancer protein and
potentially modulating its activity. That is, if the binding of the
competitor is different in the second sample relative to the first
sample, the agent is capable of binding to the cancer protein.
[0425] Alternatively, differential screening is used to identify
drug candidates that bind to the native cancer protein, but cannot
bind to modified cancer proteins. For example the structure of the
cancer protein is modeled and used in rational drug design to
synthesize agents that interact with that site, agents which
generally do not bind to site-modified proteins. Moreover, such
drug candidates that affect the activity of a native cancer protein
are also identified by screening drugs for the ability to either
enhance or reduce the activity of such proteins.
[0426] Positive controls and negative controls can be used in the
assays. Preferably control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples occurs for a time sufficient to allow for
the binding of the agent to the protein. Following incubation,
samples are washed free of non-specifically bound material and the
amount of bound, generally labeled agent determined. For example,
where a radiolabel is employed, the samples can be counted in a
scintillation counter to determine the amount of bound
compound.
[0427] A variety of other reagents can be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc. which are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., can be used. The mixture of components
is added in an order that provides for the requisite binding.
[0428] Use of Polynucleotides to Down-Regulate or Inhibit a Protein
of the Invention.
[0429] Polynucleotide modulators of cancer can be introduced into a
cell containing the target nucleotide sequence by formation of a
conjugate with a ligand-binding molecule, as described in WO
91/04753. Suitable ligand-binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell. Alternatively, a polynucleotide
modulator of cancer can be introduced into a cell containing the
target nucleic acid sequence, e.g., by formation of a
polynucleotide-lipid complex, as described in WO 90/10448. It is
understood that the use of antisense molecules or knock out and
knock in models may also be used in screening assays as discussed
above, in addition to methods of treatment.
[0430] Inhibitory and Antisense Nucleotides
[0431] In certain embodiments, the activity of a cancer-associated
protein is down-regulated, or entirely inhibited, by the use of
antisense polynucleotide or inhibitory small nuclear RNA (snRNA),
i.e., a nucleic acid complementary to, and which can preferably
hybridize specifically to, a coding mRNA nucleic acid sequence,
e.g., a cancer protein of the invention, mRNA, or a subsequence
thereof. Binding of the antisense polynucleotide to the mRNA
reduces the translation and/or stability of the mRNA.
[0432] In the context of this invention, antisense polynucleotides
can comprise naturally occurring nucleotides, or synthetic species
formed from naturally occurring subunits or their close homologs.
Antisense polynucleotides may also have altered sugar moieties or
inter-sugar linkages. Exemplary among these are the
phosphorothioate and other sulfur containing species which are
known for use in the art. Analogs are comprised by this invention
so long as they function effectively to hybridize with nucleotides
of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad,
Calif.; Sequitor, Inc., Natick, Mass.
[0433] Such antisense polynucleotides can readily be synthesized
using recombinant means, or can be synthesized in vitro. Equipment
for such synthesis is sold by several vendors, including Applied
Biosystems. The preparation of other oligonucleotides such as
phosphorothioates and alkylated derivatives is also well known to
those of skill in the art.
[0434] Antisense molecules as used herein include antisense or
sense oligonucleotides. Sense oligonucleotides can, e.g., be
employed to block transcription by binding to the anti-sense
strand. The antisense and sense oligonucleotide comprise a single
stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target mRNA (sense) or DNA (antisense) sequences for
cancer molecules. Antisense or sense oligonucleotides, according to
the present invention, comprise a fragment generally at least about
12 nucleotides, preferably from about 12 to 30 nucleotides. The
ability to derive an antisense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in,
e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol
et al. (BioTechniques 6:958 (1988)).
[0435] Ribozymes
[0436] In addition to antisense polynucleotides, ribozymes can be
used to target and inhibit transcription of cancer-associated
nucleotide sequences. A ribozyme is an RNA molecule that
catalytically cleaves other RNA molecules. Different kinds of
ribozymes have been described, including group I ribozymes,
hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead
ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25:
289-317 (1994) for a general review of the properties of different
ribozymes).
[0437] The general features of hairpin ribozymes are described,
e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990);
European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678.
Methods of preparing are well known to those of skill in the art
(see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA
90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45
(1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699-703
(1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and
Yamada et al., Virology 205: 121-126 (1994)).
[0438] Use of Modulators in Phenotypic Screening
[0439] In one embodiment, a test compound is administered to a
population of cancer cells, which have an associated cancer
expression profile. By "administration" or "contacting" herein is
meant that the modulator is added to the cells in such a manner as
to allow the modulator to act upon the cell, whether by uptake and
intracellular action, or by action at the cell surface. In some
embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a
peptide) is put into a viral construct such as an adenoviral or
retroviral construct, and added to the cell, such that expression
of the peptide agent is accomplished, e.g., PCT US97/01019.
Regulatable gene therapy systems can also be used. Once the
modulator has been administered to the cells, the cells are washed
if desired and are allowed to incubate under preferably
physiological conditions for some period. The cells are then
harvested and a new gene expression profile is generated. Thus,
e.g., cancer tissue is screened for agents that modulate, e.g.,
induce or suppress, the cancer phenotype. A change in at least one
gene, preferably many, of the expression profile indicates that the
agent has an effect on cancer activity. Similarly, altering a
biological function or a signaling pathway is indicative of
modulator activity. By defining such a signature for the cancer
phenotype, screens for new drugs that alter the phenotype are
devised. With this approach, the drug target need not be known and
need not be represented in the original gene/protein expression
screening platform, nor does the level of transcript for the target
protein need to change. The modulator inhibiting function will
serve as a surrogate marker
[0440] As outlined above, screens are done to assess genes or gene
products. That is, having identified a particular differentially
expressed gene as important in a particular state, screening of
modulators of either the expression of the gene or the gene product
itself is performed.
[0441] Use of Modulators to Affect Peptides of the Invention
[0442] Measurements of cancer polypeptide activity, or of the
cancer phenotype are performed using a variety of assays. For
example, the effects of modulators upon the function of a cancer
polypeptide(s) are measured by examining parameters described
above. A physiological change that affects activity is used to
assess the influence of a test compound on the polypeptides of this
invention. When the functional outcomes are determined using intact
cells or animals, a variety of effects can be assesses such as, in
the case of a cancer associated with solid tumors, tumor growth,
tumor metastasis, neovascularization, hormone release,
transcriptional changes to both known and uncharacterized genetic
markers (e.g., by Northern blots), changes in cell metabolism such
as cell growth or pH changes, and changes in intracellular second
messengers such as cGNIP.
[0443] Methods of Identifying Characterizing Cancer-Associated
Sequences
[0444] Expression of various gene sequences is correlated with
cancer. Accordingly, disorders based on mutant or variant cancer
genes are determined. In one embodiment, the invention provides
methods for identifying cells containing variant cancer genes,
e.g., determining the presence of, all or part, the sequence of at
least one endogenous cancer gene in a cell. This is accomplished
using any number of sequencing techniques. The invention comprises
methods of identifying the cancer genotype of an individual, e.g.,
determining all or part of the sequence of at least one gene of the
invention in the individual. This is generally done in at least one
tissue of the individual, e.g., a tissue set forth in Table I, and
may include the evaluation of a number of tissues or different
samples of the same tissue. The method may include comparing the
sequence of the sequenced gene to a known cancer gene, i.e., a
wild-type gene to determine the presence of family members,
homologies, mutations or variants. The sequence of all or part of
the gene can then be compared to the sequence of a known cancer
gene to determine if any differences exist. This is done using any
number of known homology programs, such as BLAST, Bestfit, etc. The
presence of a difference in the sequence between the cancer gene of
the patient and the known cancer gene correlates with a disease
state or a propensity for a disease state, as outlined herein.
[0445] In a preferred embodiment, the cancer genes are used as
probes to determine the number of copies of the cancer gene in the
genome. The cancer genes are used as probes to determine the
chromosomal localization of the cancer genes. Information such as
chromosomal localization finds use in providing a diagnosis or
prognosis in particular when chromosomal abnormalities such as
translocations, and the like are identified in the cancer gene
locus.
XIV.) RNAi and Therapeutic Use of Small Interfering RNA
(siRNAs)
[0446] The present invention is also directed towards siRNA
oligonucleotides, particularly double stranded RNAs encompassing at
least a fragment of the 158P1D7 coding region or 5'' UTR regions,
or complement, or any antisense oligonucleotide specific to the
158P1D7 sequence. In one embodiment such oligonucleotides are used
to elucidate a function of 158P1D7, or are used to screen for or
evaluate modulators of 158P1D7 function or expression. In another
embodiment, gene expression of 158P1D7 is reduced by using siRNA
transfection and results in significantly diminished proliferative
capacity of transformed cancer cells that endogenously express the
antigen; cells treated with specific 158P1D7 siRNAs show reduced
survival as measured, e.g., by a metabolic readout of cell
viability, correlating to the reduced proliferative capacity. Thus,
158P1D7 siRNA compositions comprise siRNA (double stranded RNA)
that correspond to the nucleic acid ORF sequence of the 158P1D7
protein or subsequences thereof; these subsequences are generally
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35
contiguous RNA nucleotides in length and contain sequences that are
complementary and non-complementary to at least a portion of the
mRNA coding sequence In a preferred embodiment, the subsequences
are 19-25 nucleotides in length, most preferably 21-23 nucleotides
in length.
[0447] RNA interference is a novel approach to silencing genes in
vitro and in vivo, thus small double stranded RNAs (siRNAs) are
valuable therapeutic agents. The power of siRNAs to silence
specific gene activities has now been brought to animal models of
disease and is used in humans as well. For example, hydrodynamic
infusion of a solution of siRNA into a mouse with a siRNA against a
particular target has been proven to be therapeutically
effective.
[0448] The pioneering work by Song et al. indicates that one type
of entirely natural nucleic acid, small interfering RNAs (siRNAs),
served as therapeutic agents even without further chemical
modification (Song, E., et al. "RNA interference targeting Fas
protects mice from fulminant hepatitis" Nat. Med. 9(3): 347-51
(2003)). This work provided the first in vivo evidence that
infusion of siRNAs into an animal could alleviate disease. In that
case, the authors gave mice injections of siRNA designed to silence
the FAS protein (a cell death receptor that when over-activated
during inflammatory response induces hepatocytes and other cells to
die). The next day, the animals were given an antibody specific to
Fas. Control mice died of acute liver failure within a few days,
while over 80% of the siRNA-treated mice remained free from serious
disease and survived. About 80% to 90% of their liver cells
incorporated the naked siRNA oligonucleotides. Furthermore, the RNA
molecules functioned for 10 days before losing effect after 3
weeks.
[0449] For use in human therapy, siRNA is delivered by efficient
systems that induce long-lasting RNAi activity. A major caveat for
clinical use is delivering siRNAs to the appropriate cells.
Hepatocytes seem to be particularly receptive to exogenous RNA.
Today, targets located in the liver are attractive because liver is
an organ that can be readily targeted by nucleic acid molecules and
viral vectors. However, other tissue and organs targets are
preferred as well.
[0450] Formulations of siRNAs with compounds that promote transit
across cell membranes are used to improve administration of siRNAs
in therapy. Chemically modified synthetic siRNA, that are resistant
to nucleases and have serum stability have concomitant enhanced
duration of RNAi effects, are an additional embodiment.
[0451] Thus, siRNA technology is a therapeutic for human malignancy
by delivery of siRNA molecules directed to 158P1D7 to individuals
with the cancers, such as those listed in Table 1. Such
administration of siRNAs leads to reduced growth of cancer cells
expressing 158P1D7, and provides an anti-tumor therapy, lessening
the morbidity and/or mortality associated with malignancy.
[0452] The effectiveness of this modality of gene product knockdown
is significant when measured in vitro or in vivo. Effectiveness in
vitro is readily demonstrable through application of siRNAs to
cells in culture (as described above) or to aliquots of cancer
patient biopsies when in vitro methods are used to detect the
reduced expression of 158P1D7 protein.
XV.) Kits/Articles of Manufacture
[0453] For use in the laboratory, prognostic, prophylactic,
diagnostic and therapeutic applications described herein, kits are
within the scope of the invention. Such kits can comprise a
carrier, package, or container that is compartmentalized to receive
one or more containers such as vials, tubes, and the like, each of
the container(s) comprising one of the separate elements to be used
in the method, along with a label or insert comprising instructions
for use, such as a use described herein. For example, the
container(s) can comprise a probe that is or can be detectably
labeled. Such probe can be an antibody or polynucleotide specific
for a protein or a gene or message of the invention, respectively.
Where the method utilizes nucleic acid hybridization to detect the
target nucleic acid, the kit can also have containers containing
nucleotide(s) for amplification of the target nucleic acid
sequence. Kits can comprise a container comprising a reporter, such
as a biotin-binding protein, such as avidin or streptavidin, bound
to a reporter molecule, such as an enzymatic, fluorescent, or
radioisotope label; such a reporter can be used with, e.g., a
nucleic acid or antibody. The kit can include all or part of the
amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a
nucleic acid molecule that encodes such amino acid sequences.
[0454] The kit of the invention will typically comprise the
container described above and one or more other containers
associated therewith that comprise materials desirable from a
commercial and user standpoint, including buffers, diluents,
filters, needles, syringes; carrier, package, container, vial
and/or tube labels listing contents and/or instructions for use,
and package inserts with instructions for use.
[0455] A label can be present on or with the container to indicate
that the composition is used for a specific therapy or
non-therapeutic application, such as a prognostic, prophylactic,
diagnostic or laboratory application, and can also indicate
directions for either in vivo or in vitro use, such as those
described herein. Directions and or other information can also be
included on an insert(s) or label(s) which is included with or on
the kit. The label can be on or associated with the container. A
label a can be on a container when letters, numbers or other
characters forming the label are molded or etched into the
container itself; a label can be associated with a container when
it is present within a receptacle or carrier that also holds the
container, e.g., as a package insert. The label can indicate that
the composition is used for diagnosing, treating, prophylaxing or
prognosing a condition, such as a neoplasia of a tissue set forth
in Table I.
[0456] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0457] In another embodiment of the invention, an article(s) of
manufacture containing compositions, such as amino acid
sequence(s), small molecule(s), nucleic acid sequence(s), and/or
antibody(s), e.g., materials useful for the diagnosis, prognosis,
prophylaxis and/or treatment of neoplasias of tissues such as those
set forth in Table I is provided. The article of manufacture
typically comprises at least one container and at least one label.
Suitable containers include, for example, bottles, vials, syringes,
and test tubes. The containers can be formed from a variety of
materials such as glass, metal or plastic. The container can hold
amino acid sequence(s), small molecule(s), nucleic acid
sequence(s), cell population(s) and/or antibody(s). In one
embodiment, the container holds a polynucleotide for use in
examining the mRNA expression profile of a cell, together with
reagents used for this purpose. In another embodiment a container
comprises an antibody, binding fragment thereof or specific binding
protein for use in evaluating protein expression of 158P1D7 in
cells and tissues, or for relevant laboratory, prognostic,
diagnostic, prophylactic and therapeutic purposes; indications
and/or directions for such uses can be included on or with such
container, as can reagents and other compositions or tools used for
these purposes. In another embodiment, a container comprises
materials for eliciting a cellular or humoral immune response,
together with associated indications and/or directions. In another
embodiment, a container comprises materials for adoptive
immunotherapy, such as cytotoxic T cells (CTL) or helper T cells
(HTL), together with associated indications and/or directions;
reagents and other compositions or tools used for such purpose can
also be included.
[0458] The container can alternatively hold a composition that is
effective for treating, diagnosis, prognosing or prophylaxing a
condition and can have a sterile access port (for example the
container can be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agents in the composition can be an antibody capable of
specifically binding 158P1D7 and modulating the function of
158P1D7.
[0459] The article of manufacture can further comprise a second
container comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and/or dextrose
solution. It can further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, stirrers, needles, syringes, and/or package inserts with
indications and/or instructions for use.
EXAMPLES
[0460] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of a cDNA Fragment of the 158P1D7 Gene
[0461] To isolate genes that are over-expressed in bladder cancer
we used the Suppression Subtractive Hybridization (SSH) procedure
using cDNA derived from bladder cancer tissues, including invasive
transitional cell carcinoma. The 158P1D7 SSH cDNA sequence was
derived from a bladder cancer pool minus normal bladder cDNA
subtraction. Included in the driver were also cDNAs derived from 9
other normal tissues. The 158P1D7 cDNA was identified as highly
expressed in the bladder cancer tissue pool, with lower expression
seen in a restricted set of normal tissues.
[0462] The SSH DNA sequence of 231 bp (FIG. 1) has high homology
(230/231 identity) to a hypothetical protein FLJ22774 (GenBank
accession XM.sub.--033183) derived from a chromosome 13 genomic
clone. A 158P1D7 cDNA clone (TurboScript3PX) of 2,555 bp was
isolated from bladder cancer cDNA, revealing an ORF of 841 amino
acids (FIG. 2 and FIG. 3).
[0463] The 158P1D7 protein has a signal sequence and a
transmembrane domain and is predicted to be localized to the cell
surface using the PSORT-I program (URL
psort.nibb.ac.jp:8800/form.html). Amino acid sequence analysis of
158P1D7 reveals 100% identity over 798 amino acid region to a human
hypothetical protein FLJ22774 (GenBank Accession XP.sub.--033182)
(FIG. 4).
[0464] Materials and Methods
[0465] Human Tissues:
[0466] The bladder cancer patient tissues were purchased from
several sources such as from the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues were purchased from Clontech, Palo Alto,
Calif.
[0467] RNA Isolation:
[0468] Tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA.
Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA
Mini and Midi kits. Total and mRNA were quantified by
spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel
electrophoresis.
[0469] Oligonucleotides:
[0470] The following HPLC purified oligonucleotides were used:
TABLE-US-00001 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 28)
5'TTTTGATCAAGCTT.sub.303' Adaptor 1: (SEQ ID NO: 29)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 30)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 31)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 32)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 33)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO: 34)
5'TCGAGCGGCCGCCCGGGCAGGA3' Nested primer (NP)2: (SEQ ID NO: 35)
5'AGCGTGGTCGCGGCCGAGGA3'
[0471] Suppression Subtractive Hybridization:
[0472] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in bladder cancer. The SSH reaction utilized cDNA from
bladder cancer and normal tissues.
[0473] The gene 158P1D7 sequence was derived from a bladder cancer
pool minus normal bladder cDNA subtraction. The SSH DNA sequence
(FIG. 1) was identified.
[0474] The cDNA derived from of pool of normal bladder tissues was
used as the source of the "driver" cDNA, while the cDNA from a pool
of bladder cancer tissues was used as the source of the "tester"
cDNA. Double stranded cDNAs corresponding to tester and driver
cDNAs were synthesized from 2 .mu.g of poly(A)+ RNA isolated from
the relevant xenograft tissue, as described above, using CLONTECH's
PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN
as primer. First- and second-strand synthesis were carried out as
described in the Kit's user manual protocol (CLONTECH Protocol No.
PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested
with Dpn II for 3 hrs at 37.degree. C. Digested cDNA was extracted
with phenol/chloroform (1:1) and ethanol precipitated.
[0475] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from the relevant tissue source (see above) with a
mix of digested cDNAs derived from the nine normal tissues:
stomach, skeletal muscle, lung, brain, liver, kidney, pancreas,
small intestine, and heart.
[0476] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l, 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH).
Ligation was terminated with 1 .mu.l of 0.2 M EDTA and heating at
72.degree. C. for 5 min.
[0477] The first hybridization was performed by adding 1.5 .mu.l
(600 ng) of driver cDNA to each of two tubes containing 1.5 .mu.l
(20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final
volume of 4 .mu.l, the samples were overlaid with mineral oil,
denatured in an MJ Research thermal cycler at 98.degree. C. for 1.5
minutes, and then were allowed to hybridize for 8 hrs at 68.degree.
C. The two hybridizations were then mixed together with an
additional 1 .mu.l of fresh denatured driver cDNA and were allowed
to hybridize overnight at 68.degree. C. The second hybridization
was then diluted in 200 .mu.l of 20 mM Hepes, pH 8.3, 50 mM NaCl,
0.2 mM EDTA, heated at 70.degree. C. for 7 min. and stored at
-20.degree. C.
[0478] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0479] To amplify gene fragments resulting from SSH reactions, two
PCR amplifications were performed. In the primary PCR reaction 1
.mu.l of the diluted final hybridization mix was added to 1 of PCR
primer 1 (10 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5 .mu.l
10.times. reaction buffer (CLONTECH) and 0.5 .mu.l 50.times.
Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25
.mu.l. PCR 1 was conducted using the following conditions:
75.degree. C. for 5 min., 94.degree. C. for 25 sec., then 27 cycles
of 94.degree. C. for 10 sec, 66.degree. C. for 30 sec, 72.degree.
C. for 1.5 min. Five separate primary PCR reactions were performed
for each experiment. The products were pooled and diluted 1:10 with
water. For the secondary PCR reaction, 1 .mu.l from the pooled and
diluted primary PCR reaction was added to the same reaction mix as
used for PCR 1, except that primers NP1 and NP2 (10 .mu.M) were
used instead of PCR primer 1. PCR 2 was performed using 10-12
cycles of 94.degree. C. for 10 sec, 68.degree. C. for 30 sec, and
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0480] The PCR products were inserted into pCR2.1 using the T/A
vector cloning kit (Invitrogen). Transformed E. coli were subjected
to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in liquid culture
overnight. To identify inserts, PCR amplification was performed on
1 ml of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0481] Bacterial clones were stored in 20% glycerol in a 96 well
format. Plasmid DNA was prepared, sequenced, and subjected to
nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP
databases.
[0482] RT-PCR Expression Analysis:
[0483] First strand cDNAs can be generated from 1 .mu.g of mRNA
with oligo (dT)12-18 priming using the Gibco-BRL Superscript
Preamplification system. The manufacturer's protocol was used which
included an incubation for 50 min at 42.degree. C. with reverse
transcriptase followed by RNAse H treatment at 37.degree. C. for 20
min. After completing the reaction, the volume can be increased to
200 .mu.l with water prior to normalization. First strand cDNAs
from 16 different normal human tissues can be obtained from
Clontech.
[0484] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers 5'
atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 36) and 5'
agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 37) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1.times.PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM
MgCl.sub.2, 50 mM KCl, pH8.3) and 1.times. Klentaq DNA polymerase
(Clontech). Five .mu.l of the PCR reaction can be removed at 18,
20, and 22 cycles and used for agarose gel electrophoresis. PCR was
performed using an MJ Research thermal cycler under the following
conditions: Initial denaturation can be at 94.degree. C. for 15
sec, followed by a 18, 20, and 22 cycles of 94.degree. C. for 15,
65.degree. C. for 2 min, 72.degree. C. for 5 sec. A final extension
at 72.degree. C. was carried out for 2 min. After agarose gel
electrophoresis, the band intensities of the 283 b.p. .beta.-actin
bands from multiple tissues were compared by visual inspection.
Dilution factors for the first strand cDNAs were calculated to
result in equal .beta.-actin band intensities in all tissues after
22 cycles of PCR. Three rounds of normalization can be required to
achieve equal band intensities in all tissues after 22 cycles of
PCR.
[0485] To determine expression levels of the 158P1D7 gene, 5 .mu.l
of normalized first strand cDNA were analyzed by PCR using 26, and
30 cycles of amplification. Semi-quantitative expression analysis
can be achieved by comparing the PCR products at cycle numbers that
give light band intensities. The primers used for RT-PCR were
designed using the 158P1D7 SSH sequence and are listed below:
TABLE-US-00002 158P1D7.1 (SEQ ID NO: 38) 5'
ATAAGCTTTCAATGTTGCGCTCCT 3' 158P1D7.2 (SEQ ID NO: 39) 5'
TGTCAACTAAGACCACGTCCATTC3'
[0486] A typical RT-PCR expression analysis is shown in FIG. 6.
RT-PCR expression analysis was performed on first strand cDNAs
generated using pools of tissues from multiple samples. The cDNAs
were shown to be normalized using beta-actin PCR. Expression of
158P1D7 was observed in bladder cancer pool.
Example 2
Full Length Cloning of 158P1D7
[0487] The 158P1D7 SSH cDNA sequence was derived from a bladder
cancer pool minus normal bladder cDNA subtraction. The SSH cDNA
sequence (FIG. 1) was designated 158P1D7. The full-length cDNA
clone 158P1D7-clone TurboScript3PX (FIG. 2) was cloned from bladder
cancer pool cDNA.
[0488] 158P1D7 clone cDNA was deposited under the terms of the
Budapest Treaty on 22 Aug. 2001, with the American Type Culture
Collection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209
USA) as plasmid p158P1D7-Turbo/3PX, and has been assigned Accession
No. PTA-3662.
Example 3
Chromosomal Mapping of 158P1D7
[0489] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are available
including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics
7:22; Research Genetics, Huntsville Ala.), human-rodent somatic
cell hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0490] 158P1D7 maps to chromosome 13, using 158P1D7 sequence and
the NCBI BLAST tool. This is a region of frequent amplification in
bladder cancer (Prat et al., Urology 2001 May; 57(5):986-92;
Muscheck et al., Carcinogenesis 2000 September; 21(9):1721-26) and
is associated with rapid tumor cell proliferation in advanced
bladder cancer (Tomovska et al., Int J Oncol 2001 June;
18(6):1239-44).
Example 4
Expression Analysis of 158P1D7 in Normal Tissues and Patient
Specimens
[0491] Analysis of 158P1D7 by RT-PCR is shown in FIG. 6. Strong
expression of 158P1D7 is observed in bladder cancer pool and breast
cancer pool. Lower levels of expression are observed in VP1, VP2,
xenograft pool, prostate cancer pool, colon cancer pool, lung
cancer pool, ovary cancer pool, and metastasis pool.
[0492] Extensive northern blot analysis of 158P1D7 in 16 human
normal tissues confirms the expression observed by RT-PCR (FIG. 7).
Two transcripts of approximately 4.6 and 4.2 kb are detected in
prostate and, to lower levels, in heart, placenta, liver, small
intestine and colon.
[0493] Northern blot analysis on patient tumor specimens shows
expression of 158P1D7 in most bladder tumor tissues tested and in
the bladder cancer cell line SCaBER (FIGS. 8A and 8B). The
expression detected in normal adjacent tissues (isolated from
patients) but not in normal tissues (isolated from a healthy donor)
may indicate that these tissues are not fully normal and that
158P1D7 may be expressed in early stage tumors. Expression of
158P1D7 is also detected in 2 of 4 lung cancer cell lines, and in
all 3 lung cancer tissues tested (FIG. 9). In breast cancer
samples, 158P1D7 expression is observed in the MCF7 and CAMA-1
breast cancer cell lines, in breast tumor tissues isolated from
breast cancer patients, but not in normal breast tissues (FIG. 10).
158P1D7 shows expression in melanoma cancer. RNA was extracted from
normal skin cell line Detroit-551, and from the melanoma cancer
cell line A375. Northern blots with 10 ug of total RNA were probed
with the 158P1D7 DNA probe. Results show expression of 158P1D7 in
the melanoma cancer cell line but not in the normal cell line (FIG.
20). 158P1D7 shows expression in cervical cancer patient specimens.
First strand cDNA was prepared from normal cervix, cervical cancer
cell line Hela, and a panel of cervical cancer patient specimens.
Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using primers to 158P1D7, was
performed at 26 and 30 cycles of amplification. Results show
expression of 158P1D7 in 5 out of 14 tumor specimens tested but not
in normal cervix nor in the cell line (FIG. 21).
[0494] The restricted expression of 158P1D7 in normal tissues and
the expression detected in prostate cancer, bladder cancer, colon
cancer, lung cancer, ovarian cancer, breast cancer, melanoma
cancer, and cervical cancer suggest that 158P1D7 is a potential
therapeutic target and a diagnostic marker for human cancers.
Example 5
Production of Recombinant 158P1D7 in Prokaryotic Systems
[0495] To express recombinant 158P1D7 and 158P1D7 variants in
prokaryotic cells, the full or partial length 158P1D7 and 158P1D7
variant cDNA sequences are cloned into any one of a variety of
expression vectors known in the art. One or more of the following
regions of 158P1D7 variants are expressed: the full length sequence
presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
contiguous amino acids from 158P1D7, variants, or analogs
thereof.
[0496] A. In Vitro Transcription and Translation Constructs:
[0497] pCRII:
[0498] To generate 158P1D7 sense and anti-sense RNA probes for RNA
in situ investigations, pCRII constructs (Invitrogen, Carlsbad
Calif.) are generated encoding either all or fragments of the
158P1D7 cDNA. The pCRII vector has Sp6 and T7 promoters flanking
the insert to drive the transcription of 158P1D7 RNA for use as
probes in RNA in situ hybridization experiments. These probes are
used to analyze the cell and tissue expression of 158P1D7 at the
RNA level. Transcribed 158P1D7 RNA representing the cDNA amino acid
coding region of the 158P1D7 gene is used in in vitro translation
systems such as the TnT.TM. Coupled Reticulolysate System (Promega,
Corp., Madison, Wis.) to synthesize 158P1D7 protein.
[0499] B. Bacterial Constructs:
[0500] pGEX Constructs:
[0501] To generate recombinant 158P1D7 proteins in bacteria that
are fused to the Glutathione S-transferase (GST) protein, all or
parts of the 158P1D7 cDNA protein coding sequence are cloned into
the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech,
Piscataway, N.J.). These constructs allow controlled expression of
recombinant 158P1D7 protein sequences with GST fused at the
amino-terminus and a six histidine epitope (6.times.His) at the
carboxyl-terminus. The GST and 6.times.His tags permit purification
of the recombinant fusion protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-GST and anti-His antibodies. The 6.times.His tag
is generated by adding 6 histidine codons to the cloning primer at
the 3' end, e.g., of the open reading frame (ORF). A proteolytic
cleavage site, such as the PreScission.TM. recognition site in
pGEX-6P-1, may be employed such that it permits cleavage of the GST
tag from 158P1D7-related protein. The ampicillin resistance gene
and pBR322 origin permits selection and maintenance of the pGEX
plasmids in E. coli.
[0502] pMAL Constructs:
[0503] To generate, in bacteria, recombinant 158P1D7 proteins that
are fused to maltose-binding protein (MBP), all or parts of the
158P1D7 cDNA protein coding sequence are fused to the MBP gene by
cloning into the pMAL-c2X and pMAL-p2X vectors (New England
Biolabs, Beverly, Mass.). These constructs allow controlled
expression of recombinant 158P1D7 protein sequences with MBP fused
at the amino-terminus and a 6.times.His epitope tag at the
carboxyl-terminus. The MBP and 6.times.His tags permit purification
of the recombinant protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-MBP and anti-His antibodies. The 6.times.His
epitope tag is generated by adding 6 histidine codons to the 3'
cloning primer. A Factor Xa recognition site permits cleavage of
the pMAL tag from 158P1D7. The pMAL-c2X and pMAL-p2X vectors are
optimized to express the recombinant protein in the cytoplasm or
periplasm respectively. Periplasm expression enhances folding of
proteins with disulfide bonds. Amino acids 356-608 of 158P1D7
variant 1 have been cloned into the pMALc2X vector.
[0504] pET Constructs:
[0505] To express 158P1D7 in bacterial cells, all or parts of the
158P1D7 cDNA protein coding sequence are cloned into the pET family
of vectors (Novagen, Madison, Wis.). These vectors allow tightly
controlled expression of recombinant 158P1D7 protein in bacteria
with and without fusion to proteins that enhance solubility, such
as NusA and thioredoxin (Trx), and epitope tags, such as
6.times.His and S-Tag.TM. that aid purification and detection of
the recombinant protein. For example, constructs are made utilizing
pET NusA fusion system 43.1 such that regions of the 158P1D7
protein are expressed as amino-terminal fusions to NusA.
[0506] C. Yeast Constructs:
[0507] pESC Constructs:
[0508] To express 158P1D7 in the yeast species Saccharomyces
cerevisiae for generation of recombinant protein and functional
studies, all or parts of the 158P1D7 cDNA protein coding sequence
are cloned into the pESC family of vectors each of which contain 1
of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La
Jolla, Calif.). These vectors allow controlled expression from the
same plasmid of up to 2 different genes or cloned sequences
containing either Flag.TM. or Myc epitope tags in the same yeast
cell. This system is useful to confirm protein-protein interactions
of 158P1D7. In addition, expression in yeast yields similar
post-translational modifications, such as glycosylations and
phosphorylations, that are found when expressed in eukaryotic
cells.
[0509] pESP Constructs:
[0510] To express 158P1D7 in the yeast species Saccharomyces pombe,
all or parts of the 158P1D7 cDNA protein coding sequence are cloned
into the pESP family of vectors. These vectors allow controlled
high level of expression of a 158P1D7 protein sequence that is
fused at either the amino terminus or at the carboxyl terminus to
GST which aids purification of the recombinant protein. A Flag.TM.
epitope tag allows detection of the recombinant protein with
anti-Flag.TM. antibody.
Example 6
Production of Recombinant 158P1D7 in Eukaryotic Systems
[0511] A. Mammalian Constructs:
[0512] To express recombinant 158P1D7 in eukaryotic cells, the full
or partial length 158P1D7 cDNA sequences were cloned into any one
of a variety of expression vectors known in the art. One or more of
the following regions of 158P1D7 were expressed in these
constructs, amino acids 1 to 841, or any 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or
more contiguous amino acids from 158P1D7 v.1; amino acids 1 to 732
of v.3; amino acids 1 to 395 of v.4; amino acids 1 to 529 of v.6;
or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from
158P1D7 variants, or analogs thereof.
[0513] The constructs can be transfected into any one of a wide
variety of mammalian cells such as 293T cells. Transfected 293T
cell lysates can be probed with the anti-158P1D7 polyclonal serum,
described herein.
[0514] pcDNA4/HisMax Constructs:
[0515] To express 158P1D7 in mammalian cells, a 158P1D7 ORF, or
portions thereof, of 158P1D7 are cloned into pcDNA4/H isMax Version
A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from
the cytomegalovirus (CMV) promoter and the SP16 translational
enhancer. The recombinant protein has Xpress.TM. and six histidine
(6.times.His) epitopes fused to the amino-terminus. The pcDNA4/H
isMax vector also contains the bovine growth hormone (BGH)
polyadenylation signal and transcription termination sequence to
enhance mRNA stability along with the SV40 origin for episomal
replication and simple vector rescue in cell lines expressing the
large T antigen. The Zeocin resistance gene allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli.
[0516] pcDNA3.1/MycHis Constructs:
[0517] To express 158P1D7 in mammalian cells, a 158P1D7 ORF, or
portions thereof, of 158P1D7 with a consensus Kozak translation
initiation site was cloned into pcDNA3.1/MycHis Version A
(Invitrogen, Carlsbad, Calif.). Protein expression was driven from
the cytomegalovirus (CMV) promoter. The recombinant proteins have
the myc epitope and 6.times.His epitope fused to the
carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the
bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli.
[0518] The complete ORF of 158P1D7 v.1 was cloned into the
pcDNA3.1/MycHis construct to generate 158P1D7.pcDNA3.1/MycHis. FIG.
23 shows expression of 158P1D7.pcDNA3.1/MycHis following
transfection into 293T cells. 293T cells were transfected with
either 158P1D7.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control.
Forty hours later, cells were collected and analyzed by flow
cytometry using anti-158P1D7 monoclonal antibodies. Results show
expression of 158P1D7 from the 158P1D7.pcDNA3.1/MycHis construct on
the surface of transfected cells.
[0519] pcDNA3.1/CT-GFP-TOPO Construct:
[0520] To express 158P1D7 in mammalian cells and to allow detection
of the recombinant proteins using fluorescence, a 158P1D7 ORF, or
portions thereof, with a consensus Kozak translation initiation
site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein
expression is driven from the cytomegalovirus (CMV) promoter. The
recombinant proteins have the Green Fluorescent Protein (GFP) fused
to the carboxyl-terminus facilitating non-invasive, in vivo
detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector
also contains the bovine growth hormone (BGH) polyadenylation
signal and transcription termination sequence to enhance mRNA
stability along with the SV40 origin for episomal replication and
simple vector rescue in cell lines expressing the large T antigen.
The Neomycin resistance gene allows for selection of mammalian
cells that express the protein, and the ampicillin resistance gene
and ColE1 origin permits selection and maintenance of the plasmid
in E. coli. Additional constructs with an amino-terminal GFP fusion
are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a
158P1D7 protein.
[0521] PAPtag:
[0522] A 158P1D7 ORF, or portions thereof, is cloned into pAPtag-5
(GenHunter Corp. Nashville, Tenn.). This construct generates an
alkaline phosphatase fusion at the carboxyl-terminus of a 158P1D7
protein while fusing the IgG.kappa. signal sequence to the
amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgG.kappa. signal sequence is
fused to the amino-terminus of a 158P1D7 protein. The resulting
recombinant 158P1D7 proteins are optimized for secretion into the
media of transfected mammalian cells and can be used to identify
proteins such as ligands or receptors that interact with 158P1D7
proteins. Protein expression is driven from the CMV promoter and
the recombinant proteins also contain myc and 6.times.His epitopes
fused at the carboxyl-terminus that facilitates detection and
purification. The Zeocin resistance gene present in the vector
allows for selection of mammalian cells expressing the recombinant
protein and the ampicillin resistance gene permits selection of the
plasmid in E. coli.
[0523] pTag5:
[0524] A 158P1D7 ORF, or portions thereof, were cloned into pTag-5.
This vector is similar to pAPtag but without the alkaline
phosphatase fusion. This construct generated a 158P1D7 protein with
an amino-terminal IgG.kappa. signal sequence and myc and
6.times.His epitope tags at the carboxyl-terminus that facilitate
detection and affinity purification. The resulting recombinant
158P1D7 protein was optimized for secretion into the media of
transfected mammalian cells, and was used as immunogen or ligand to
identify proteins such as ligands or receptors that interact with
the 158P1D7 proteins. Protein expression is driven from the CMV
promoter. The Zeocin resistance gene present in the vector allows
for selection of mammalian cells expressing the protein, and the
ampicillin resistance gene permits selection of the plasmid in E.
coli.
[0525] The extracellular domain, amino acids 16-608, 27-300, and
301-608, of 158P1D7 v.1 were cloned into the pTag5 construct to
generate 158P1D7 (16-608).pTag5, 158P1D7(27-300).pTag5, and
158P1D7(301-608).pTag5 respectively. Expression and secretion of
the various segments of the extracellular domain of 158P1D7
following vector transfection into 293T cells was confirmed.
[0526] PsecFc:
[0527] A 158P1D7 ORF, or portions thereof, was also cloned into
psecFc. The psecFc vector was assembled by cloning the human
immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2
(Invitrogen, California). This construct generates an IgG1 Fc
fusion at the carboxyl-terminus of the 158P1D7 proteins, while
fusing the IgGK signal sequence to N-terminus. 158P1D7 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant 158P1D7 proteins are optimized for secretion into the
media of transfected mammalian cells, and can be used as immunogens
or to identify proteins such as ligands or receptors that interact
with 158P1D7 protein. Protein expression is driven from the CMV
promoter. The hygromycin resistance gene present in the vector
allows for selection of mammalian cells that express the
recombinant protein, and the ampicillin resistance gene permits
selection of the plasmid in E. coli.
[0528] The extracellular domain amino acids 16-608 of 158P1D7 v.1
was cloned into the psecFc construct to generate
158P1D7(16-608).psecFc.
[0529] pSR.alpha. Constructs:
[0530] To generate mammalian cell lines that express 158P1D7
constitutively, 158P1D7 ORF, or portions thereof, of 158P1D7 were
cloned into pSR.alpha. constructs. Amphotropic and ecotropic
retroviruses were generated by transfection of pSR.alpha.
constructs into the 293T-10A1 packaging line or co-transfection of
pSR.alpha. and a helper plasmid (containing deleted packaging
sequences) into the 293 cells, respectively. The retrovirus is used
to infect a variety of mammalian cell lines, resulting in the
integration of the cloned gene, 158P1D7, into the host cell-lines.
Protein expression is driven from a long terminal repeat (LTR). The
Neomycin resistance gene present in the vector allows for selection
of mammalian cells that express the protein, and the ampicillin
resistance gene and ColE1 origin permit selection and maintenance
of the plasmid in E. coli. The retroviral vectors can thereafter be
used for infection and generation of various cell lines using, for
example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
[0531] The complete ORF of 158P1D7 v.1 was cloned into the
pSR.alpha. construct to generate 158P1D7.pSR.alpha.. FIG. 23 shows
expression of 158P1D7.pSR.alpha. following transduction into UMUC3
cells. UMUC-3 cells were transduced with either 158P1D7.pSR.alpha.
or vector control. Forty hours later, cells were collected and
analyzed by flow cytometry using anti-158P1D7 monoclonal
antibodies. Results show expression of 158P1D7 from the
158P1D7.pSR.alpha. construct on the surface of the cells.
[0532] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
158P1D7 sequences to allow detection using anti-Flag antibodies.
For example, the FLAG.TM. sequence 5' gat tac aag gat gac gac gat
aag 3' (SEQ ID NO: 40) is added to cloning primer at the 3' end of
the ORF. Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length 158P1D7 proteins.
[0533] Additional Viral Vectors:
[0534] Additional constructs are made for viral-mediated delivery
and expression of 158P1D7. High virus titer leading to high level
expression of 158P1D7 is achieved in viral delivery systems such as
adenoviral vectors and herpes amplicon vectors. A 158P1D7 coding
sequences or fragments thereof are amplified by PCR and subcloned
into the AdEasy shuttle vector (Stratagene). Recombination and
virus packaging are performed according to the manufacturer's
instructions to generate adenoviral vectors. Alternatively, 158P1D7
coding sequences or fragments thereof are cloned into the HSV-1
vector (Imgenex) to generate herpes viral vectors. The viral
vectors are thereafter used for infection of various cell lines
such as PC3, NIH 3T3, 293 or rat-1 cells.
[0535] Regulated Expression Systems:
[0536] To control expression of 158P1D7 in mammalian cells, coding
sequences of 158P1D7, or portions thereof, are cloned into
regulated mammalian expression systems such as the T-Rex System
(Invitrogen), the GeneSwitch System (Invitrogen) and the
tightly-regulated Ecdysone System (Sratagene). These systems allow
the study of the temporal and concentration dependent effects of
recombinant 158P1D7. These vectors are thereafter used to control
expression of 158P1D7 in various cell lines such as PC3, NIH 3T3,
293 or rat-1 cells.
[0537] B. Baculovirus Expression Systems
[0538] To generate recombinant 158P1D7 proteins in a baculovirus
expression system, 158P1D7 ORF, or portions thereof, are cloned
into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen),
which provides a His-tag at the N-terminus. Specifically,
pBlueBac-158P1D7 is co-transfected with helper plasmid pBac-N-Blue
(Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to
generate recombinant baculovirus (see Invitrogen instruction manual
for details). Baculovirus is then collected from cell supernatant
and purified by plaque assay.
[0539] Recombinant 158P1D7 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 158P1D7 protein can be detected using anti-158P1D7 or
anti-His-tag antibody. 158P1D7 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 158P1D7.
Example 7
Antigenicity Profiles and Secondary Structure
[0540] FIG. 11(a)-(d), FIG. 12(a)-(d), FIG. 13(a)-(d), FIG.
14(a)-(d), and FIG. 15(a)-(d) depict graphically five amino acid
profiles each of 158P1D7 protein variants 1, 3, 4, and 6, each
assessment available by accessing the ProtScale website located on
the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the
ExPasy molecular biology server.
[0541] These profiles: FIG. 11, Hydrophilicity, (Hopp T. P., Woods
K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 12,
Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.
157:105-132); FIG. 13, Percentage Accessible Residues (Janin J.,
1979 Nature 277:491-492); FIG. 14, Average Flexibility, (Bhaskaran
R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.
32:242-255); FIG. 15, Beta-turn (Deleage, G., Roux B. 1987 Protein
Engineering 1:289-294); and optionally others available in the art,
such as on the ProtScale website, were used to identify antigenic
regions of each of the 158P1D7 variant proteins. Each of the above
amino acid profiles of 158P1D7 variants were generated using the
following ProtScale parameters for analysis: 1) A window size of 9;
2) 100% weight of the window edges compared to the window center;
and, 3) amino acid profile values normalized to lie between 0 and
1.
[0542] Hydrophilicity (FIG. 11), Hydropathicity (FIG. 12) and
Percentage Accessible Residues (FIG. 13) profiles were used to
determine stretches of hydrophilic amino acids (i.e., values
greater than 0.5 on the Hydrophilicity and Percentage Accessible
Residues profile, and values less than 0.5 on the Hydropathicity
profile). Such regions are likely to be exposed to the aqueous
environment, be present on the surface of the protein, and thus
available for immune recognition, such as by antibodies.
[0543] Average Flexibility (FIG. 14) and Beta-turn (FIG. 15)
profiles determine stretches of amino acids (i.e., values greater
than 0.5 on the Beta-turn profile and the Average Flexibility
profile) that are not constrained in secondary structures such as
beta sheets and alpha helices. Such regions are also more likely to
be exposed on the protein and thus accessible to immune
recognition, such as by antibodies.
[0544] Antigenic sequences of the 158P1D7 variant proteins
indicated, e.g., by the profiles set forth in FIGS. 11(a)-(d), FIG.
12(a)-(d), FIG. 13(a)-(d), FIG. 14(a)-(d), and FIG. 15(a)-(d) are
used to prepare immunogens, either peptides or nucleic acids that
encode them, to generate therapeutic and diagnostic anti-158P1D7
antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50
or more than 50 contiguous amino acids, or the corresponding
nucleic acids that encode them, from the 158P1D7 protein variants
listed in FIGS. 2 and 3. In particular, peptide immunogens of the
invention can comprise, a peptide region of at least 5 amino acids
of FIGS. 2 and 3 in any whole number increment that includes an
amino acid position having a value greater than 0.5 in the
Hydrophilicity profiles of FIG. 11; a peptide region of at least 5
amino acids of FIGS. 2 and 3 in any whole number increment that
includes an amino acid position having a value less than 0.5 in the
Hydropathicity profile of FIG. 12; a peptide region of at least 5
amino acids of FIGS. 2 and 3 in any whole number increment that
includes an amino acid position having a value greater than 0.5 in
the Percent Accessible Residues profiles of FIG. 13; a peptide
region of at least 5 amino acids of FIGS. 2 and 3 in any whole
number increment that includes an amino acid position having a
value greater than 0.5 in the Average Flexibility profiles on FIG.
14; and, a peptide region of at least 5 amino acids of FIGS. 2 and
3 in any whole number increment that includes an amino acid
position having a value greater than 0.5 in the Beta-turn profile
of FIG. 15. Peptide immunogens of the invention can also comprise
nucleic acids that encode any of the forgoing.
[0545] All immunogens of the invention, peptide or nucleic acid,
can be embodied in human unit dose form, or comprised by a
composition that includes a pharmaceutical excipient compatible
with human physiology.
[0546] The secondary structure of 158P1D7 protein variants 1, 3, 4,
and 6, namely the predicted presence and location of alpha helices,
extended strands, and random coils, are predicted from the primary
amino acid sequence using the HNN--Hierarchical Neural Network
method (NPS@: Network Protein Sequence Analysis TIBS 2000 March
Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourj on C.
and Deleage G.,
http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html),
accessed from the ExPasy molecular biology server
(http://www.expasy.ch/tools/). The analysis indicates that 158P1D7
variant 1 is composed of 35.32% alpha helix, 15.93% extended
strand, and 48.75% random coil (FIG. 16A). Variant 3 is composed of
34.97% alpha helix, 16.94% extended strand, and 48.09% random coil
(FIG. 16B). Variant 4 is composed of 24.56% alpha helix, 20.76%
extended strand, and 54.68% random coil (FIG. 16C). Variant 6 is
composed of 28.92% alpha helix, 17.96% extended strand, and 53.12%
random coil (FIG. 16D).
[0547] Analysis for the potential presence of transmembrane domains
in the 158P1D7 variant proteins was carried out using a variety of
transmembrane prediction algorithms accessed from the ExPasy
molecular biology server (http://www.expasy.ch/tools/). Shown
graphically in FIG. 16E, 16G, 16I, 16K, are the results of analysis
of variants 1, 3, 4, and 6, respectively, using the TMpred program.
In FIG. 16F, 16H, 16I, 16L are the results of variants 1, 3, 4, and
6, respectively, using the TMHMM program. Both the TMpred program
and the TMHMM program predict the presence of 1 transmembrane
domain in variant 1 and 3. Variants 4 and 6 are not predicted to
contain transmembrane domains. All variants contain a stretch of
hydrophobic amino acid sequence at their amino terminus that may
encode a signal peptide. Analyses of 158P1D7 and 158P1D7 variants
using other structural prediction programs are summarized in Table
LVI.
Example 8
Generation of 158P1D7 Polyclonal Antibodies
[0548] Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. In addition to immunizing with a full
length 158P1D7 protein variant, computer algorithms are employed in
design of immunogens that, based on amino acid sequence analysis
contain characteristics of being antigenic and available for
recognition by the immune system of the immunized host (see the
Example entitled "Antigenicity Profiles and Secondary Structure").
Such regions would be predicted to be hydrophilic, flexible, in
beta-turn conformations, and be exposed on the surface of the
protein (see, e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15
for amino acid profiles that indicate such regions of 158P1D7
protein variants 1, 3, 4, and 6).
[0549] For example, recombinant bacterial fusion proteins or
peptides containing hydrophilic, flexible, beta-turn regions of
158P1D7 protein variants are used as antigens to generate
polyclonal antibodies in New Zealand White rabbits or monoclonal
antibodies as described in Example 9. For example, in 158P1D7
variant 1, such regions include, but are not limited to, amino
acids 25-45, amino acids 250-385, and amino acids 694-730. It is
useful to conjugate the immunizing agent to a protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include, but are not limited to, keyhole
limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and
soybean trypsin inhibitor. In one embodiment, a peptide encoding
amino acids 274-285 of 158P1D7 variant 1 was synthesized and
conjugated to KLH. This peptide is then used as immunogen.
Alternatively the immunizing agent may include all or portions of
the 158P1D7 variant proteins, analogs or fusion proteins thereof.
For example, the 158P1D7 variant 1 amino acid sequence can be fused
using recombinant DNA techniques to any one of a variety of fusion
protein partners that are well known in the art, such as
glutathione-S-transferase (GST) and HIS tagged fusion proteins. In
another embodiment, amino acids 27-300 of 158P1D7 variant 1 is
fused to GST using recombinant techniques and the pGEX expression
vector, expressed, purified and used to immunize a rabbit. Such
fusion proteins are purified from induced bacteria using the
appropriate affinity matrix.
[0550] Other recombinant bacterial fusion proteins that may be
employed include maltose binding protein, LacZ, thioredoxin, NusA,
or an immunoglobulin constant region (see the section entitled
"Production of 158P1D7 in Prokaryotic Systems" and Current
Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M.
Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Times, M.,
Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med.
174, 561-566).
[0551] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the section entitled "Production of
Recombinant 158P1D7 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, amino acids 16-608 of 158P1D7
variant 1 was cloned into the Tag5 mammalian secretion vector, and
expressed in 293T cells. The recombinant protein was purified by
metal chelate chromatography from tissue culture supernatants of
293T cells stably expressing the recombinant vector. The purified
Tag5 158P1D7 variant 1 protein is then used as immunogen.
[0552] During the immunization protocol, it is useful to mix or
emulsify the antigen in adjuvants that enhance the immune response
of the host animal. Examples of adjuvants include, but are not
limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
[0553] In a typical protocol, rabbits are initially immunized
subcutaneously with up to 200 .mu.g, typically 100-200 .mu.g, of
fusion protein or peptide conjugated to KLH mixed in complete
Freund's adjuvant (CFA). Rabbits are then injected subcutaneously
every two weeks with up to 200 .mu.g, typically 100-200 .mu.g, of
the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds
are taken approximately 7-10 days following each immunization and
used to monitor the titer of the antiserum by ELISA.
[0554] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with the GST-fusion of
158P1D7 variant 1 protein, the full-length 158P1D7 variant 1 cDNA
is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see
the Example entitled "Production of Recombinant 158P1D7 in
Eukaryotic Systems"). After transfection of the constructs into
293T cells, cell lysates are probed with the anti-158P1D7 serum and
with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz,
Calif.) to determine specific reactivity to denatured 158P1D7
protein using the Western blot technique. In addition, the immune
serum is tested by fluorescence microscopy, flow cytometry and
immunoprecipitation against 293T and other recombinant
158P1D7-expressing cells to determine specific recognition of
native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and flow cytometric techniques using cells that
endogenously express 158P1D7 are also carried out to test
reactivity and specificity.
[0555] Anti-serum from rabbits immunized with 158P1D7 variant
fusion proteins, such as GST and MBP fusion proteins, are purified
by depletion of antibodies reactive to the fusion partner sequence
by passage over an affinity column containing the fusion partner
either alone or in the context of an irrelevant fusion protein. For
example, antiserum derived from a GST-158P1D7 variant 1 fusion
protein is first purified by passage over a column of GST protein
covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.).
The antiserum is then affinity purified by passage over a column
composed of a MBP-158P1D7 fusion protein covalently coupled to
Affigel matrix. The serum is then further purified by protein G
affinity chromatography to isolate the IgG fraction. Sera from
other His-tagged antigens and peptide immunized rabbits as well as
fusion partner depleted sera are affinity purified by passage over
a column matrix composed of the original protein immunogen or free
peptide.
Example 9
Generation of 158P1D7 Monoclonal Antibodies (mAbs)
[0556] In one embodiment, therapeutic mAbs to 158P1D7 variants
comprise those that react with epitopes specific for each variant
protein or specific to sequences in common between the variants
that would bind, internalize, disrupt or modulate the biological
function of the 158P1D7 variants, for example those that would
disrupt the interaction with ligands and binding partners.
Immunogens for generation of such mAbs include those designed to
encode or contain the extracellular domain or the entire 158P1D7
protein variant sequence, regions predicted to contain functional
motifs, and regions of the 158P1D7 protein variants predicted to be
antigenic from computer analysis of the amino acid sequence (see,
e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15, and the
Example entitled "Antigenicity Profiles and Secondary Structure").
Immunogens include peptides, recombinant bacterial proteins, and
mammalian expressed Tag 5 proteins and human and murine IgG FC
fusion proteins. In addition, pTAG5 protein, DNA vectors encoding
the pTAG5 cells engineered to express high levels of a respective
158P1D7 variant, such as 293T-158P1D7 variant 1 or 3T3, RAT, or
300.19-158P1D7 variant 1 murine Pre-B cells, are used to immunize
mice.
[0557] To generate mAbs to a 158P1D7 variant, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen or 10.sup.7 158P1D7-expressing cells mixed in
complete Freund's adjuvant. Mice are then subsequently immunized IP
every 2-4 weeks with, typically, 10-50 .mu.g of protein immunogen
or 10.sup.7 cells mixed in incomplete Freund's adjuvant.
Alternatively, MPL-TDM adjuvant is used in immunizations. In
addition to the above protein and cell-based immunization
strategies, a DNA-based immunization protocol is employed in which
a mammalian expression vector encoding a 158P1D7 variant sequence
is used to immunize mice by direct injection of the plasmid DNA.
For example, amino acids 16-608 of 158P1D7 of variant 1 was cloned
into the Tag5 mammalian secretion vector and the recombinant vector
was used as immunogen. In another example, the same amino acids
were cloned into an Fc-fusion secretion vector in which the 158P1D7
variant 1 sequence is fused at the amino-terminus to an IgK leader
sequence and at the carboxyl-terminus to the coding sequence of the
human or murine IgG Fc region. This recombinant vector was then
used as immunogen. The plasmid immunization protocols were used in
combination with purified proteins expressed from the same vector
and with cells expressing the respective 158P1D7 variant.
[0558] During the immunization protocol, test bleeds are taken 7-10
days following an injection to monitor titer and specificity of the
immune response. Once appropriate reactivity and specificity is
obtained as determined by ELISA, Western blotting,
immunoprecipitation, fluorescence microscopy, and flow cytometric
analyses, fusion and hybridoma generation is then carried out with
established procedures well known in the art (see, e.g., Harlow and
Lane, 1988).
[0559] In one embodiment for generating 158P1D7 variant 1
monoclonal antibodies, a peptide encoding amino acids 274-285 was
synthesized, conjugated to KLH and used as immunogen. ELISA on free
peptide was used to identify immunoreactive clones. Reactivity and
specificity of the monoclonal antibodies to full length 158P1D7
variant 1 protein was monitored by Western blotting,
immunoprecipitation, and flow cytometry using both recombinant and
endogenous-expressing 158P1D7 variant 1 cells (See FIGS. 22, 23,
24, 25, and 28).
[0560] The binding affinity of 158P1D7 variant 1 specific
monoclonal antibodies was determined using standard technologies.
Affinity measurements quantify the strength of antibody to epitope
binding and are used to help define which 158P1D7 variant
monoclonal antibodies preferred for diagnostic or therapeutic use,
as appreciated by one of skill in the art. The BIAcore system
(Uppsala, Sweden) is a preferred method for determining binding
affinity. The BIAcore system uses surface plasmon resonance (SPR,
Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,
Methods in Enzymology 295: 268) to monitor biomolecular
interactions in real time. BIAcore analysis conveniently generates
association rate constants, dissociation rate constants,
equilibrium dissociation constants, and affinity constants. Results
of BIAcore analysis of 158P1D7 variant 1 monoclonal antibodies is
shown in Table LVII.
[0561] To generate monoclonal antibodies specific for other 158P1D7
variants, immunogens are designed to encode amino acid sequences
unique to the variants. In one embodiment, a peptide encoding amino
acids 382-395 unique to 158P1D7 variant 4 is synthesized, coupled
to KLH and used as immunogen. In another embodiment, peptides or
bacterial fusion proteins are made that encompass the unique
sequence generated by alternative splicing in the variants. In one
example, a peptide encoding a consecutive sequence containing amino
acids 682 and 683 in 158P1D7 variant 3 is used, such as amino acids
673-693. In another example, a peptide encoding a consecutive
sequence containing amino acids 379-381 in 158P1D7 variant 6 is
used, such as amino acids 369-391. Hybridomas are then selected
that recognize the respective variant specific antigen and also
recognize the full length variant protein expressed in cells. Such
selection utilizes immunoassays described above such as Western
blotting, immunoprecipitation, and flow cytometry.
[0562] To generate 158P1D7 monoclonal antibodies the following
protocols were used. 5 Balb/c mice were immunized subcutaneously
with 2 .mu.g of peptide in Quiagen ImmuneEasy.TM. adjuvant.
Immunizations were given 2 weeks apart. The peptide used was a 12
amino acid peptide consisting of amino acids 274-285 with the
sequence EEHEDPSGSLHL (SEQ ID NO: 41) conjugated to KLH at the C'
terminal (Keyhole Limpet Hemocyanin).
[0563] B-cells from spleens of immunized mice were fused with the
fusion partner Sp2/0 under the influence of polyethylene glycol.
Antibody producing hybridomas were selected by screening on peptide
coated ELISA plates indicating specific binding to the peptide and
then by FACS on cells expressing 158P1D7. This produced and
identified four 158P1D7 extra cellular domain (ECD) specific
antibodies designated: M15-68(2)18.1.1; M15-68(2)22.1.1;
M15-68(2)31.1.1 and M15-68(2)102.1.1.
[0564] The antibody designated M15-68(2)18.1.1 was sent (via
Federal Express) to the American Type Culture Collection (ATCC),
P.O. Box 1549, Manassas, Va. 20108 on 6 Feb. 2004 and assigned
Accession number PTA-5801.
The characteristics of these four antibodies are set forth in Table
LVII.
[0565] To clone the M15-68(2)18.1.1 antibody the following
protocols were used. M15-68(2)18.1.1 hybridoma cells were lysed
with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was
purified and quantified. First strand cDNAs was generated from
total RNA with oligo (dT)12-18 priming using the Gibco-BRL
Superscript Preamplification system. First strand cDNA was
amplified using mouse Ig variable heavy chain primers, and mouse Ig
variable light chain primers. PCR products were cloned into the
pCRScript vector (Stratagene, La Jolla). Several clones were
sequenced and the variable heavy (VH) and variable light (VL) chain
regions determined. The nucleic acid and amino acid sequences of
M15-68(2)18 variable heavy and light chain regions are set forth in
FIGS. 34A and 34B and FIGS. 35A and 35B.
Example 10
HLA Class I and Class II Binding Assays
[0566] HLA class I and class II binding assays using purified HLA
molecules are performed in accordance with disclosed protocols
(e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J.
Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813
(1994)). Briefly, purified MHC molecules (5 to 500 nM) are
incubated with various unlabeled peptide inhibitors and 1-10 nM
.sup.125I-radiolabeled probe peptides as described. Following
incubation, MHC-peptide complexes are separated from free peptide
by gel filtration and the fraction of peptide bound is determined.
Typically, in preliminary experiments, each MHC preparation is
titered in the presence of fixed amounts of radiolabeled peptides
to determine the concentration of HLA molecules necessary to bind
10-20% of the total radioactivity. All subsequent inhibition and
direct binding assays are performed using these HLA
concentrations.
[0567] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq.[HLA], the measured IC.sub.50 values are
reasonable approximations of the true K.sub.D values. Peptide
inhibitors are typically tested at concentrations ranging from 120
.mu.g/ml to 1.2 ng/ml, and are tested in two to four completely
independent experiments. To allow comparison of the data obtained
in different experiments, a relative binding figure is calculated
for each peptide by dividing the IC.sub.50 of a positive control
for inhibition by the IC.sub.50 for each tested peptide (typically
unlabeled versions of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values
are compiled. These values can subsequently be converted back into
IC.sub.50 nM values by dividing the IC.sub.50 nM of the positive
controls for inhibition by the relative binding of the peptide of
interest. This method of data compilation is accurate and
consistent for comparing peptides that have been tested on
different days, or with different lots of purified MHC.
[0568] Binding assays as outlined above may be used to analyze HLA
supermotif and/or HLA motif-bearing peptides.
Example 11
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate
Epitopes
[0569] HLA vaccine compositions of the invention can include
multiple epitopes. The multiple epitopes can comprise multiple HLA
supermotifs or motifs to achieve broad population coverage. This
example illustrates the identification and confirmation of
supermotif- and motif-bearing epitopes for the inclusion in such a
vaccine composition. Calculation of population coverage is
performed using the strategy described below.
[0570] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0571] The searches performed to identify the motif-bearing peptide
sequences in the Example entitled "Antigenicity Profiles" and
Tables V-XVIII and XXII-XLIX employ the protein sequence data from
the gene product of 158P1D7 set forth in FIGS. 2 and 3.
[0572] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
158P1D7 protein sequences are analyzed using a text string search
software program to identify potential peptide sequences containing
appropriate HLA binding motifs; such programs are readily produced
in accordance with information in the art in view of known
motif/supermotif disclosures. Furthermore, such calculations can be
made mentally.
[0573] Identified A2-, A3-, and DR-supermotif sequences are scored
using polynomial algorithms to predict their capacity to bind to
specific HLA-Class I or Class II molecules. These polynomial
algorithms account for the impact of different amino acids at
different positions, and are essentially based on the premise that
the overall affinity (or .DELTA.G) of peptide-HLA molecule
interactions can be approximated as a linear polynomial function of
the type:
".DELTA.G"=a.sub.1i.times.a.sub.2i.times.a.sub.3i . . .
.times.a.sub.ni
where a.sub.ji is a coefficient which represents the effect of the
presence of a given amino acid (j) at a given position (i) along
the sequence of a peptide of n amino acids. The crucial assumption
of this method is that the effects at each position are essentially
independent of each other (i.e., independent binding of individual
side-chains). When residue j occurs at position i in the peptide,
it is assumed to contribute a constant amount j.sub.i to the free
energy of binding of the peptide irrespective of the sequence of
the rest of the peptide.
[0574] The method of derivation of specific algorithm coefficients
has been described in Gulukota et al., J. Mol. Biol. 267:1258-126,
1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and
Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for
all i positions, anchor and non-anchor alike, the geometric mean of
the average relative binding (ARB) of all peptides carrying j is
calculated relative to the remainder of the group, and used as the
estimate of j.sub.i. For Class II peptides, if multiple alignments
are possible, only the highest scoring alignment is utilized,
following an iterative procedure. To calculate an algorithm score
of a given peptide in a test set, the ARB values corresponding to
the sequence of the peptide are multiplied. If this product exceeds
a chosen threshold, the peptide is predicted to bind. Appropriate
thresholds are chosen as a function of the degree of stringency of
prediction desired.
[0575] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0576] Complete protein sequences from 158P1D7 are scanned
utilizing motif identification software, to identify 8-, 9-10- and
11-mer sequences containing the HLA-A2-supermotif main anchor
specificity. Typically, these sequences are then scored using the
protocol described above and the peptides corresponding to the
positive-scoring sequences are synthesized and tested for their
capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201
is considered a prototype A2 supertype molecule).
[0577] These peptides are then tested for the capacity to bind to
additional A2-supertype molecules (A*0202, A*0203, A*0206, and
A*6802). Peptides that bind to at least three of the five
A2-supertype alleles tested are typically deemed A2-supertype
cross-reactive binders. Preferred peptides bind at an affinity
equal to or less than 500 nM to three or more HLA-A2 supertype
molecules.
[0578] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0579] The 158P1D7 protein sequence scanned above is also examined
for the presence of peptides with the HLA-A3-supermotif primary
anchors. Peptides corresponding to the HLA A3 supermotif-bearing
sequences are then synthesized and tested for binding to HLA-A*0301
and HLA-A*1101 molecules, the molecules encoded by the two most
prevalent A3-supertype alleles. The peptides that bind at least one
of the two alleles with binding affinities of .ltoreq.500 nM, often
.ltoreq.200 nM, are then tested for binding cross-reactivity to the
other common A3-supertype alleles (e.g., A*3101, A*3301, and
A*6801) to identify those that can bind at least three of the five
HLA-A3-supertype molecules tested.
[0580] Selection of HLA-B7 Supermotif Bearing Epitopes
[0581] The 158P1D7 protein is also analyzed for the presence of 8-,
9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding
peptides are synthesized and tested for binding to HLA-B*0702, the
molecule encoded by the most common B7-supertype allele (i.e., the
prototype B7 supertype allele). Peptides binding B*0702 with
IC.sub.50 of .ltoreq.500 nM are identified using standard methods.
These peptides are then tested for binding to other common
B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401).
Peptides capable of binding to three or more of the five
B7-supertype alleles tested are thereby identified.
[0582] Selection of A1 and A24 Motif-Bearing Epitopes
[0583] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 158P1D7 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0584] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs are identified using analogous
methodology.
Example 12
Confirmation of Immunogenicity
[0585] Cross-reactive candidate CTL A2-supermotif-bearing peptides
that are identified as described herein are selected to confirm in
vitro immunogenicity. Confirmation is performed using the following
methodology:
[0586] Target Cell Lines for Cellular Screening:
[0587] The 0.221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of HLA-A2.1-restricted CTL. This cell
line is grown in RPMI-1640 medium supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (v/v) heat
inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the antigen of interest,
can be used as target cells to confirm the ability of
peptide-specific CTLs to recognize endogenous antigen.
[0588] Primary CTL Induction Cultures:
[0589] Generation of Dendritic Cells (DC):
[0590] PBMCs are thawed in RPMI with 30 .mu.g/ml DNAse, washed
twice and resuspended in complete medium (RPMI-1640 plus 5% AB
human serum, non-essential amino acids, sodium pyruvate,
L-glutamine and penicillin/streptomycin). The monocytes are
purified by plating 10.times.10.sup.6 PBMC/well in a 6-well plate.
After 2 hours at 37.degree. C., the non-adherent cells are removed
by gently shaking the plates and aspirating the supernatants. The
wells are washed a total of three times with 3 ml RPMI to remove
most of the non-adherent and loosely adherent cells. Three ml of
complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of
IL-4 are then added to each well. TNF.alpha. is added to the DCs on
day 6 at 75 ng/ml and the cells are used for CTL induction cultures
on day 7.
[0591] Induction of CTL with DC and Peptide:
[0592] CD8+ T-cells are isolated by positive selection with Dynal
immunomagnetic beads (Dynabeads.RTM. M-450) and the
Detacha-Bead.RTM. reagent. Typically about 200-250.times.10.sup.6
PBMC are processed to obtain 24.times.10.sup.6 CD8.sup.+ T-cells
(enough for a 48-well plate culture). Briefly, the PBMCs are thawed
in RPMI with 30 .mu.g/ml DNAse, washed once with PBS containing 1%
human AB serum and resuspended in PBS/1% AB serum at a
concentration of 20.times.10.sup.6 cells/ml. The magnetic beads are
washed 3 times with PBS/AB serum, added to the cells (1400
beads/20.times.10.sup.6 cells) and incubated for 1 hour at
4.degree. C. with continuous mixing. The beads and cells are washed
4.times. with PBS/AB serum to remove the nonadherent cells and
resuspended at 100.times.10.sup.6 cells/ml (based on the original
cell number) in PBS/AB serum containing 100 .mu.l/ml
Detacha-Bead.RTM. reagent and 30 .mu.g/ml DNAse. The mixture is
incubated for 1 hour at room temperature with continuous mixing.
The beads are washed again with PBS/AB/DNAse to collect the CD8+
T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7
minutes, washed once with PBS with 1% BSA, counted and pulsed with
40 .mu.g/ml of peptide at a cell concentration of
1-2.times.10.sup.6/ml in the presence of 3 .mu.g/ml
.beta..sub.2-microglobulin for 4 hours at 20.degree. C. The DC are
then irradiated (4,200 rads), washed 1 time with medium and counted
again.
[0593] Setting Up Induction Cultures:
[0594] 0.25 ml cytokine-generated DC (at 1.times.10.sup.5 cells/ml)
are co-cultured with 0.25 ml of CD8+ T-cells (at 2.times.10.sup.6
cell/ml) in each well of a 48-well plate in the presence of 10
ng/ml of IL-7. Recombinant human IL-10 is added the next day at a
final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours
later at 10 IU/ml.
[0595] Restimulation of the Induction Cultures with Peptide-Pulsed
Adherent Cells:
[0596] Seven and fourteen days after the primary induction, the
cells are restimulated with peptide-pulsed adherent cells. The
PBMCs are thawed and washed twice with RPMI and DNAse. The cells
are resuspended at 5.times.10.sup.6 cells/ml and irradiated at
.about.4200 rads. The PBMCs are plated at 2.times.10.sup.6 in 0.5
ml complete medium per well and incubated for 2 hours at 37.degree.
C. The plates are washed twice with RPMI by tapping the plate
gently to remove the nonadherent cells and the adherent cells
pulsed with 10 .mu.g/ml of peptide in the presence of 3 .mu.g/ml
.beta..sub.2 microglobulin in 0.25 ml RPMI/5% AB per well for 2
hours at 37.degree. C. Peptide solution from each well is aspirated
and the wells are washed once with RPMI. Most of the media is
aspirated from the induction cultures (CD8+ cells) and brought to
0.5 ml with fresh media. The cells are then transferred to the
wells containing the peptide-pulsed adherent cells. Twenty four
hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant human IL2 is added the
next day and again 2-3 days later at 50 IU/ml (Tsai et al.,
Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days
later, the cultures are assayed for CTL activity in a .sup.51Cr
release assay. In some experiments the cultures are assayed for
peptide-specific recognition in the in situ IFN.gamma. ELISA at the
time of the second restimulation followed by assay of endogenous
recognition 7 days later. After expansion, activity is measured in
both assays for a side-by-side comparison.
Measurement of CTL Lytic Activity by .sup.51Cr Release
[0597] Seven days after the second restimulation, cytotoxicity is
determined in a standard (5 hr) .sup.51Cr release assay by assaying
individual wells at a single E:T. Peptide-pulsed targets are
prepared by incubating the cells with 10 .mu.g/ml peptide overnight
at 37.degree. C.
[0598] Adherent target cells are removed from culture flasks with
trypsin-EDTA. Target cells are labeled with 200 .mu.Ci of .sup.51Cr
sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37.degree.
C. Labeled target cells are resuspended at 10.sup.6 per ml and
diluted 1:10 with K562 cells at a concentration of
3.3.times.10.sup.6/ml (an NK-sensitive erythroblastoma cell line
used to reduce non-specific lysis). Target cells (100 .mu.l) and
effectors (100 .mu.l) are plated in 96 well round-bottom plates and
incubated for 5 hours at 37.degree. C. At that time, 100 .mu.l of
supernatant are collected from each well and percent lysis is
determined according to the formula:
[(cpm of the test sample-cpm of the spontaneous .sup.51Cr release
sample)/(cpm of the maximal .sup.51Cr release sample-cpm of the
spontaneous .sup.51Cr release sample)].times.100.
[0599] Maximum and spontaneous release are determined by incubating
the labeled targets with 1% Trition X-100 and media alone,
respectively. A positive culture is defined as one in which the
specific lysis (sample-background) is 10% or higher in the case of
individual wells and is 15% or more at the two highest E:T ratios
when expanded cultures are assayed.
[0600] In Situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-Specific and Endogenous Recognition
[0601] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH8.2) overnight
at 4.degree. C. The plates are washed with Ca.sup.2+,
Mg.sup.2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for
two hours, after which the CTLs (100 .mu.l/well) and targets (100
.mu.l/well) are added to each well, leaving empty wells for the
standards and blanks (which received media only). The target cells,
either peptide-pulsed or endogenous targets, are used at a
concentration of 1.times.10.sup.6 cells/ml. The plates are
incubated for 48 hours at 37.degree. C. with 5% CO.sub.2.
[0602] Recombinant human IFN-gamma is added to the standard wells
starting at 400 pg or 1200 pg/100 microliter/well and the plate
incubated for two hours at 37.degree. C. The plates are washed and
100 .mu.l of biotinylated mouse anti-human IFN-gamma monoclonal
antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added
and incubated for 2 hours at room temperature. After washing again,
100 microliter HRP-streptavidin (1:4000) are added and the plates
incubated for one hour at room temperature. The plates are then
washed 6.times. with wash buffer, 100 microliter/well developing
solution (TMB 1:1) are added, and the plates allowed to develop for
5-15 minutes. The reaction is stopped with 50 microliter/well 1M
H.sub.3PO.sub.4 and read at OD450. A culture is considered positive
if it measured at least 50 pg of IFN-gamma/well above background
and is twice the background level of expression.
[0603] CTL Expansion.
[0604] Those cultures that demonstrate specific lytic activity
against peptide-pulsed targets and/or tumor targets are expanded
over a two week period with anti-CD3. Briefly, 5.times.10.sup.4
CD8+ cells are added to a T25 flask containing the following:
1.times.10.sup.6 irradiated (4,200 rad) PBMC (autologous or
allogeneic) per ml, 2.times.10.sup.5 irradiated (8,000 rad)
EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml
in RPMI-1640 containing 10% (v/v) human AB serum, non-essential
amino acids, sodium pyruvate, 25 .mu.M 2-mercaptoethanol,
L-glutamine and penicillin/streptomycin. Recombinant human IL2 is
added 24 hours later at a final concentration of 200 IU/ml and
every three days thereafter with fresh media at 50 IU/ml. The cells
are split if the cell concentration exceeds 1.times.10.sup.6/ml and
the cultures are assayed between days 13 and 15 at E:T ratios of
30, 10, 3 and 1:1 in the .sup.51Cr release assay or at
1.times.10.sup.6/ml in the in situ IFN.gamma. assay using the same
targets as before the expansion.
[0605] Cultures are expanded in the absence of anti-CD3.sup.+ as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8.sup.+ cells are added to a T25 flask
containing the following: 1.times.10.sup.6 autologous PBMC per ml
which have been peptide-pulsed with 10 .mu.g/ml peptide for two
hours at 37.degree. C. and irradiated (4,200 rad); 2.times.10.sup.5
irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640
containing 10% (v/v) human AB serum, non-essential AA, sodium
pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0606] Immunogenicity of A2 Supermotif-Bearing Peptides
[0607] A2-supermotif cross-reactive binding peptides are tested in
the cellular assay for the ability to induce peptide-specific CTL
in normal individuals. In this analysis, a peptide is typically
considered to be an epitope if it induces peptide-specific CTLs in
at least individuals, and preferably, also recognizes the
endogenously expressed peptide.
[0608] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 158P1D7. Briefly,
PBMCs are isolated from patients, re-stimulated with peptide-pulsed
monocytes and assayed for the ability to recognize peptide-pulsed
target cells as well as transfected cells endogenously expressing
the antigen.
[0609] Evaluation of A*03/A11 Immunogenicity
[0610] HLA-A3 supermotif-bearing cross-reactive binding peptides
are also evaluated for immunogenicity using methodology analogous
for that used to evaluate the immunogenicity of the HLA-A2
supermotif peptides.
[0611] Evaluation of B7 Immunogenicity
[0612] Immunogenicity screening of the B7-supertype cross-reactive
binding peptides identified as set forth herein are confirmed in a
manner analogous to the confirmation of A2- and
A3-supermotif-bearing peptides.
[0613] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also confirmed using similar methodology
Example 13
Implementation of the Extended Supermotif to Improve the Binding
Capacity of Native Epitopes by Creating Analogs
[0614] HLA motifs and supermotifs (comprising primary and/or
secondary residues) are useful in the identification and
preparation of highly cross-reactive native peptides, as
demonstrated herein. Moreover, the definition of HLA motifs and
supermotifs also allows one to engineer highly cross-reactive
epitopes by identifying residues within a native peptide sequence
which can be analoged to confer upon the peptide certain
characteristics, e.g. greater cross-reactivity within the group of
HLA molecules that comprise a supertype, and/or greater binding
affinity for some or all of those HLA molecules. Examples of
analoging peptides to exhibit modulated binding affinity are set
forth in this example.
[0615] Analoging at Primary Anchor Residues
[0616] Peptide engineering strategies are implemented to further
increase the cross-reactivity of the epitopes. For example, the
main anchors of A2-supermotif-bearing peptides are altered, for
example, to introduce a preferred L, I, V, or M at position 2, and
I or V at the C-terminus.
[0617] To analyze the cross-reactivity of the analog peptides, each
engineered analog is initially tested for binding to the prototype
A2 supertype allele A*0201, then, if A*0201 binding capacity is
maintained, for A2-supertype cross-reactivity.
[0618] Alternatively, a peptide is confirmed as binding one or all
supertype members and then analogued to modulate binding affinity
to any one (or more) of the supertype members to add population
coverage.
[0619] The selection of analogs for immunogenicity in a cellular
screening analysis is typically further restricted by the capacity
of the parent wild type (WT) peptide to bind at least weakly, i.e.,
bind at an IC.sub.50 of 5000 nM or less, to three of more A2
supertype alleles. The rationale for this requirement is that the
WT peptides must be present endogenously in sufficient quantity to
be biologically relevant. Analoged peptides have been shown to have
increased immunogenicity and cross-reactivity by T cells specific
for the parent epitope (see, e.g., Parkhurst et al., J. Immunol.
157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA
92:8166, 1995).
[0620] In the cellular screening of these peptide analogs, it is
important to confirm that analog-specific CTLs are also able to
recognize the wild-type peptide and, when possible, target cells
that endogenously express the epitope.
[0621] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0622] Analogs of HLA-A3 supermotif-bearing epitopes are generated
using strategies similar to those employed in analoging HLA-A2
supermotif-bearing peptides. For example, peptides binding to 3/5
of the A3-supertype molecules are engineered at primary anchor
residues to possess a preferred residue (V, S, M, or A) at position
2.
[0623] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate 500 nM binding capacity are then confirmed as having
A3-supertype cross-reactivity.
[0624] Similarly to the A2- and A3-motif bearing peptides, peptides
binding 3 or more B7-supertype alleles can be improved, where
possible, to achieve increased cross-reactive binding or greater
binding affinity or binding half life. B7 supermotif-bearing
peptides are, for example, engineered to possess a preferred
residue (V, I, L, or F) at the C-terminal primary anchor position,
as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490,
1996).
[0625] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0626] The analog peptides are then be confirmed for
immunogenicity, typically in a cellular screening assay. Again, it
is generally important to demonstrate that analog-specific CTLs are
also able to recognize the wild-type peptide and, when possible,
targets that endogenously express the epitope.
[0627] Analoging at Secondary Anchor Residues
[0628] Moreover, HLA supermotifs are of value in engineering highly
cross-reactive peptides and/or peptides that bind HLA molecules
with increased affinity by identifying particular residues at
secondary anchor positions that are associated with such
properties. For example, the binding capacity of a B7
supermotif-bearing peptide with an F residue at position 1 is
analyzed. The peptide is then analoged to, for example, substitute
L for F at position 1. The analoged peptide is evaluated for
increased binding affinity, binding half life and/or increased
cross-reactivity. Such a procedure identifies analoged peptides
with enhanced properties.
[0629] Engineered analogs with sufficiently improved binding
capacity or cross-reactivity can also be tested for immunogenicity
in HLA-B7-transgenic mice, following for example, IFA immunization
or lipopeptide immunization. Analogued peptides are additionally
tested for the ability to stimulate a recall response using PBMC
from patients with 158P1D7-expressing tumors.
[0630] Other Analoguing Strategies
[0631] Another form of peptide analoguing, unrelated to anchor
positions, involves the substitution of a cysteine with
.alpha.-amino butyric acid. Due to its chemical nature, cysteine
has the propensity to form disulfide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substitution of .alpha.-amino butyric acid for cysteine not only
alleviates this problem, but has been shown to improve binding and
crossbinding capabilities in some instances (see, e.g., the review
by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and
I. Chen, John Wiley & Sons, England, 1999).
[0632] Thus, by the use of single amino acid substitutions, the
binding properties and/or cross-reactivity of peptide ligands for
HLA supertype molecules can be modulated.
Example 14
Identification and Confirmation of 158P1D7-Derived Sequences with
HLA-DR Binding Motifs
[0633] Peptide epitopes bearing an HLA class II supermotif or motif
are identified and confirmed as outlined below using methodology
similar to that described for HLA Class I peptides.
[0634] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0635] To identify 158P1D7-derived, HLA class II HTL epitopes, the
158P1D7 antigen is analyzed for the presence of sequences bearing
an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are
selected comprising a DR-supermotif, comprising a 9-mer core, and
three-residue N- and C-terminal flanking regions (15 amino acids
total).
[0636] Protocols for predicting peptide binding to DR molecules
have been developed (Southwood et al., J. Immunol. 160:3363-3373,
1998). These protocols, specific for individual DR molecules, allow
the scoring, and ranking, of 9-mer core regions. Each protocol not
only scores peptide sequences for the presence of DR-supermotif
primary anchors (i.e., at position 1 and position 6) within a 9-mer
core, but additionally evaluates sequences for the presence of
secondary anchors. Using allele-specific selection tables (see,
e.g., Southwood et al., ibid.), it has been found that these
protocols efficiently select peptide sequences with a high
probability of binding a particular DR molecule. Additionally, it
has been found that performing these protocols in tandem,
specifically those for DR1, DR4w4, and DR7, can efficiently select
DR cross-reactive peptides.
[0637] The 158P1D7-derived peptides identified above are tested for
their binding capacity for various common HLA-DR molecules. All
peptides are initially tested for binding to the DR molecules in
the primary panel: DR1, DR4w4, and DR7. Peptides binding at least
two of these three DR molecules are then tested for binding to
DR2w2 .beta.1, DR2w2 .beta.2, DR6w19, and DR9 molecules in
secondary assays. Finally, peptides binding at least two of the
four secondary panel DR molecules, and thus cumulatively at least
four of seven different DR molecules, are screened for binding to
DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides
binding at least seven of the ten DR molecules comprising the
primary, secondary, and tertiary screening assays are considered
cross-reactive DR binders. 158P1D7-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0638] Selection of DR3 Motif Peptides
[0639] Because HLA-DR3 is an allele that is prevalent in Caucasian,
Black, and Hispanic populations, DR3 binding capacity is a relevant
criterion in the selection of HTL epitopes. Thus, peptides shown to
be candidates may also be assayed for their DR3 binding capacity.
However, in view of the binding specificity of the DR3 motif,
peptides binding only to DR3 can also be considered as candidates
for inclusion in a vaccine formulation.
[0640] To efficiently identify peptides that bind DR3, target
158P1D7 antigens are analyzed for sequences carrying one of the two
DR3-specific binding motifs reported by Geluk et al. (J. Immunol.
152:5742-5748, 1994). The corresponding peptides are then
synthesized and confirmed as having the ability to bind DR3 with an
affinity of 1 .mu.M or better, i.e., less than 1 .mu.M. Peptides
are found that meet this binding criterion and qualify as HLA class
II high affinity binders.
[0641] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0642] Similarly to the case of HLA class I motif-bearing peptides,
the class II motif-bearing peptides are analoged to improve
affinity or cross-reactivity. For example, aspartic acid at
position 4 of the 9-mer core sequence is an optimal residue for DR3
binding, and substitution for that residue often improves DR 3
binding.
Example 15
Immunogenicity of 158P1D7-Derived HTL Epitopes
[0643] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0644] Immunogenicity of HTL epitopes are confirmed in a manner
analogous to the determination of immunogenicity of CTL epitopes,
by assessing the ability to stimulate HTL responses and/or by using
appropriate transgenic mouse models. Immunogenicity is determined
by screening for: 1.) in vitro primary induction using normal PBMC
or 2.) recall responses from patients who have 158P1D7-expressing
tumors.
Example 16
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0645] This example illustrates the assessment of the breadth of
population coverage of a vaccine composition comprised of multiple
epitopes comprising multiple supermotifs and/or motifs.
[0646] In order to analyze population coverage, gene frequencies of
HLA alleles are determined. Gene frequencies for each HLA allele
are calculated from antigen or allele frequencies utilizing the
binomial distribution formulae gf=1-(SQRT(1-af)) (see, e.g., Sidney
et al., Human Immunol. 45:79-93, 1996). To obtain overall
phenotypic frequencies, cumulative gene frequencies are calculated,
and the cumulative antigen frequencies derived by the use of the
inverse formula [af=1-(1-Cgf).sup.2].
[0647] Where frequency data is not available at the level of DNA
typing, correspondence to the serologically defined antigen
frequencies is assumed. To obtain total potential supertype
population coverage no linkage disequilibrium is assumed, and only
alleles confirmed to belong to each of the supertypes are included
(minimal estimates). Estimates of total potential coverage achieved
by inter-loci combinations are made by adding to the A coverage the
proportion of the non-A covered population that could be expected
to be covered by the B alleles considered (e.g., total=A+B*(1-A)).
Confirmed members of the A3-like supertype are A3, A11, A31,
A*3301, and A*6801. Although the A3-like supertype may also include
A34, A66, and A*7401, these alleles were not included in overall
frequency calculations. Likewise, confirmed members of the A2-like
supertype family are A*0201, A*0202, A*0203, A*0204, A*0205,
A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like
supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301,
B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also
B*1401, B*3504-06, B*4201, and B*5602).
[0648] Population coverage achieved by combining the A2-, A3- and
B7-supertypes is approximately 86% in five major ethnic groups.
Coverage may be extended by including peptides bearing the A1 and
A24 motifs. On average, A1 is present in 12% and A24 in 29% of the
population across five different major ethnic groups (Caucasian,
North American Black, Chinese, Japanese, and Hispanic). Together,
these alleles are represented with an average frequency of 39% in
these same ethnic populations. The total coverage across the major
ethnicities when A1 and A24 are combined with the coverage of the
A2-, A3- and B7-supertype alleles is >95%. An analogous approach
can be used to estimate population coverage achieved with
combinations of class II motif-bearing epitopes.
[0649] Immunogenicity studies in humans (e.g., Bertoni et al., J.
Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997;
and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that
highly cross-reactive binding peptides are almost always recognized
as epitopes. The use of highly cross-reactive binding peptides is
an important selection criterion in identifying candidate epitopes
for inclusion in a vaccine that is immunogenic in a diverse
population.
[0650] With a sufficient number of epitopes (as disclosed herein
and from the art), an average population coverage is predicted to
be greater than 95% in each of five major ethnic populations. The
game theory Monte Carlo simulation analysis, which is known in the
art (see e.g., Osborne, M. J. and Rubinstein, A. "A course in game
theory" MIT Press, 1994), can be used to estimate what percentage
of the individuals in a population comprised of the Caucasian,
North American Black, Japanese, Chinese, and Hispanic ethnic groups
would recognize the vaccine epitopes described herein. A preferred
percentage is 90%. A more preferred percentage is 95%.
Example 17
CTL Recognition of Endogenously Processed Antigens after
Priming
[0651] This example confirms that CTL induced by native or analoged
peptide epitopes identified and selected as described herein
recognize endogenously synthesized, i.e., native antigens.
[0652] Effector cells isolated from transgenic mice that are
immunized with peptide epitopes, for example HLA-A2
supermotif-bearing epitopes, are re-stimulated in vitro using
peptide-coated stimulator cells. Six days later, effector cells are
assayed for cytotoxicity and the cell lines that contain
peptide-specific cytotoxic activity are further re-stimulated. An
additional six days later, these cell lines are tested for
cytotoxic activity on .sup.51Cr labeled Jurkat-A2.1/K.sup.b target
cells in the absence or presence of peptide, and also tested on
.sup.51Cr labeled target cells bearing the endogenously synthesized
antigen, i.e. cells that are stably transfected with 158P1D7
expression vectors.
[0653] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
158P1D7 antigen. The choice of transgenic mouse model to be used
for such an analysis depends upon the epitope(s) that are being
evaluated. In addition to HLA-A*0201/K.sup.b transgenic mice,
several other transgenic mouse models including mice with human
A11, which may also be used to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for
HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed, which may be used to evaluate HTL
epitopes.
Example 18
Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
[0654] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 158P1D7-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 158P1D7-expressing
tumor. The peptide composition can comprise multiple CTL and/or HTL
epitopes. The epitopes are identified using methodology as
described herein. This example also illustrates that enhanced
immunogenicity can be achieved by inclusion of one or more HTL
epitopes in a CTL vaccine composition; such a peptide composition
can comprise an HTL epitope conjugated to a CTL epitope. The CTL
epitope can be one that binds to multiple HLA family members at an
affinity of 500 nM or less, or analogs of that epitope. The
peptides may be lipidated, if desired.
[0655] Immunization Procedures:
[0656] Immunization of transgenic mice is performed as described
(Alexander et al., J. Immunol. 159:4753-4761, 1997). For example,
A2/K.sup.b mice, which are transgenic for the human HLA A2.1 allele
and are used to confirm the immunogenicity of HLA-A*0201 motif- or
HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously
(base of the tail) with a 0.1 ml of peptide in Incomplete Freund's
Adjuvant, or if the peptide composition is a lipidated CTL/HTL
conjugate, in DMSO/saline, or if the peptide composition is a
polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days
after priming, splenocytes obtained from these animals are
restimulated with syngenic irradiated LPS-activated lymphoblasts
coated with peptide.
[0657] Cell Lines:
[0658] Target cells for peptide-specific cytotoxicity assays are
Jurkat cells transfected with the HLA-A2.1/K.sup.b chimeric gene
(e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
[0659] In Vitro CTL Activation:
[0660] One week after priming, spleen cells (30.times.10.sup.6
cells/flask) are co-cultured at 37.degree. C. with syngeneic,
irradiated (3000 rads), peptide coated lymphoblasts
(10.times.10.sup.6 cells/flask) in 10 ml of culture medium/T25
flask. After six days, effector cells are harvested and assayed for
cytotoxic activity.
[0661] Assay for Cytotoxic Activity:
[0662] Target cells (1.0 to 1.5.times.10.sup.6) are incubated at
37.degree. C. in the presence of 200 .mu.l of .sup.51Cr. After 60
minutes, cells are washed three times and resuspended in R10
medium. Peptide is added where required at a concentration of 1
.mu.g/ml. For the assay, 10.sup.4 51Cr-labeled target cells are
added to different concentrations of effector cells (final volume
of 200 .mu.l) in U-bottom 96-well plates. After a six hour
incubation period at 37.degree. C., a 0.1 ml aliquot of supernatant
is removed from each well and radioactivity is determined in a
Micromedic automatic gamma counter. The percent specific lysis is
determined by the formula: percent specific release=100.times.
(experimental release-spontaneous release)/(maximum
release-spontaneous release). To facilitate comparison between
separate CTL assays run under the same conditions, % .sup.51Cr
release data is expressed as lytic units/10.sup.6 cells. One lytic
unit is arbitrarily defined as the number of effector cells
required to achieve 30% lysis of 10,000 target cells in a six hour
.sup.51Cr release assay. To obtain specific lytic units/10.sup.6,
the lytic units/10.sup.6 obtained in the absence of peptide is
subtracted from the lytic units/10.sup.6 obtained in the presence
of peptide. For example, if 30% .sup.51Cr release is obtained at
the effector (E): target (T) ratio of 50:1 (i.e., 5.times.10.sup.5
effector cells for 10,000 targets) in the absence of peptide and
5:1 (i.e., 5.times.10.sup.4 effector cells for 10,000 targets) in
the presence of peptide, the specific lytic units would be: [(
1/50,000)-( 1/500,000)].times.10.sup.6=18 LU.
[0663] The results are analyzed to assess the magnitude of the CTL
responses of animals injected with the immunogenic CTL/HTL
conjugate vaccine preparation and are compared to the magnitude of
the CTL response achieved using, for example, CTL epitopes as
outlined above in the Example entitled "Confirmation of
Immunogenicity". Analyses similar to this may be performed to
confirm the immunogenicity of peptide conjugates containing
multiple CTL epitopes and/or multiple HTL epitopes. In accordance
with these procedures, it is found that a CTL response is induced,
and concomitantly that an HTL response is induced upon
administration of such compositions.
Example 19
Selection of CTL and HTL Epitopes for Inclusion in an
158P1D7-Specific Vaccine
[0664] This example illustrates a procedure for selecting peptide
epitopes for vaccine compositions of the invention. The peptides in
the composition can be in the form of a nucleic acid sequence,
either single or one or more sequences (i.e., minigene) that
encodes peptide(s), or can be single and/or polyepitopic
peptides.
[0665] The following principles are utilized when selecting a
plurality of epitopes for inclusion in a vaccine composition. Each
of the following principles is balanced in order to make the
selection.
[0666] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 158P1D7 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 158P1D7. For example, if it has been observed
that patients who spontaneously clear 158P1D7 generate an immune
response to at least three (3) from 158P1D7 antigen, then three or
four (3-4) epitopes should be included for HLA class I. A similar
rationale is used to determine HLA class II epitopes.
[0667] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 nM or less for an HLA class I molecule, or for
class II, an IC.sub.50 of 1000 nM or less; or HLA Class I peptides
with high binding scores from the BIMAS web site, at URL
bimas.dcrt.nih.gov/.
[0668] In order to achieve broad coverage of the vaccine through
out a diverse population, sufficient supermotif bearing peptides,
or a sufficient array of allele-specific motif bearing peptides,
are selected to give broad population coverage. In one embodiment,
epitopes are selected to provide at least 80% population coverage.
A Monte Carlo analysis, a statistical evaluation known in the art,
can be employed to assess breadth, or redundancy, of population
coverage.
[0669] When creating polyepitopic compositions, or a minigene that
encodes same, it is typically desirable to generate the smallest
peptide possible that encompasses the epitopes of interest. The
principles employed are similar, if not the same, as those employed
when selecting a peptide comprising nested epitopes. For example, a
protein sequence for the vaccine composition is selected because it
has maximal number of epitopes contained within the sequence, i.e.,
it has a high concentration of epitopes. Epitopes may be nested or
overlapping (i.e., frame shifted relative to one another). For
example, with overlapping epitopes, two 9-mer epitopes and one
10-mer epitope can be present in a 10 amino acid peptide. Each
epitope can be exposed and bound by an HLA molecule upon
administration of such a peptide. A multi-epitopic, peptide can be
generated synthetically, recombinantly, or via cleavage from the
native source. Alternatively, an analog can be made of this native
sequence, whereby one or more of the epitopes comprise
substitutions that alter the cross-reactivity and/or binding
affinity properties of the polyepitopic peptide. Such a vaccine
composition is administered for therapeutic or prophylactic
purposes. This embodiment provides for the possibility that an as
yet undiscovered aspect of immune system processing will apply to
the native nested sequence and thereby facilitate the production of
therapeutic or prophylactic immune response-inducing vaccine
compositions. Additionally such an embodiment provides for the
possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment (absent the
creating of any analogs) directs the immune response to multiple
peptide sequences that are actually present in 158P1D7, thus
avoiding the need to evaluate any junctional epitopes. Lastly, the
embodiment provides an economy of scale when producing nucleic acid
vaccine compositions. Related to this embodiment, computer programs
can be derived in accordance with principles in the art, which
identify in a target sequence, the greatest number of epitopes per
sequence length.
[0670] A vaccine composition comprised of selected peptides, when
administered, is safe, efficacious, and elicits an immune response
similar in magnitude to an immune response that controls or clears
cells that bear or overexpress 158P1D7.
Example 20
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0671] This example discusses the construction of a minigene
expression plasmid. Minigene plasmids may, of course, contain
various configurations of B cell, CTL and/or HTL epitopes or
epitope analogs as described herein.
[0672] A minigene expression plasmid typically includes multiple
CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3,
-B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24
motif-bearing peptide epitopes are used in conjunction with DR
supermotif-bearing epitopes and/or DR3 epitopes. HLA class I
supermotif or motif-bearing peptide epitopes derived 158P1D7, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 158P1D7 to provide broad population coverage,
i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3
motif-bearing epitopes are selected for inclusion in the minigene
construct. The selected CTL and HTL epitopes are then incorporated
into a minigene for expression in an expression vector.
[0673] Such a construct may additionally include sequences that
direct the HTL epitopes to the endoplasmic reticulum. For example,
the Ii protein may be fused to one or more HTL epitopes as
described in the art, wherein the CLIP sequence of the Ii protein
is removed and replaced with an HLA class II epitope sequence so
that HLA class II epitope is directed to the endoplasmic reticulum,
where the epitope binds to an HLA class II molecules.
[0674] This example illustrates the methods to be used for
construction of a minigene-bearing expression plasmid. Other
expression vectors that may be used for minigene compositions are
available and known to those of skill in the art.
[0675] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa Ig-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disclosed herein. The sequence
encodes an open reading frame fused to the Myc and His antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0676] Overlapping oligonucleotides that can, for example, average
about 70 nucleotides in length with 15 nucleotide overlaps, are
synthesized and HPLC-purified. The oligonucleotides encode the
selected peptide epitopes as well as appropriate linker
nucleotides, Kozak sequence, and signal sequence. The final
multiepitope minigene is assembled by extending the overlapping
oligonucleotides in three sets of reactions using PCR. A
Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are
performed using the following conditions: 95.degree. C. for 15 sec,
annealing temperature (5.degree. below the lowest calculated Tm of
each primer pair) for 30 sec, and 72.degree. C. for 1 min.
[0677] For example, a minigene is prepared as follows. For a first
PCR reaction, 5 .mu.g of each of two oligonucleotides are annealed
and extended: In an example using eight oligonucleotides, i.e.,
four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are
combined in 100 .mu.l reactions containing Pfu polymerase buffer
(1.times.=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75,
2 mM MgSO4, 0.1% Triton X-100, 100 .mu.g/ml BSA), 0.25 mM each
dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products
are gel-purified, and two reactions containing the product of 1+2
and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and
extended for 10 cycles. Half of the two reactions are then mixed,
and 5 cycles of annealing and extension carried out before flanking
primers are added to amplify the full length product. The
full-length product is gel-purified and cloned into pCR-blunt
(Invitrogen) and individual clones are screened by sequencing.
Example 21
The Plasmid Construct and the Degree to which it Induces
Immunogenicity
[0678] The degree to which a plasmid construct, for example a
plasmid constructed in accordance with the previous Example, is
able to induce immunogenicity is confirmed in vitro by determining
epitope presentation by APC following transduction or transfection
of the APC with an epitope-expressing nucleic acid construct. Such
a study determines "antigenicity" and allows the use of human APC.
The assay determines the ability of the epitope to be presented by
the APC in a context that is recognized by a T cell by quantifying
the density of epitope-HLA class I complexes on the cell surface.
Quantitation can be performed by directly measuring the amount of
peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol.
156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the
number of peptide-HLA class I complexes can be estimated by
measuring the amount of lysis or lymphokine release induced by
diseased or transfected target cells, and then determining the
concentration of peptide necessary to obtain equivalent levels of
lysis or lymphokine release (see, e.g., Kageyama et al., J.
Immunol. 154:567-576, 1995).
[0679] Alternatively, immunogenicity is confirmed through in vivo
injections into mice and subsequent in vitro assessment of CTL and
HTL activity, which are analyzed using cytotoxicity and
proliferation assays, respectively, as detailed e.g., in Alexander
et al., Immunity 1:751-761, 1994.
[0680] For example, to confirm the capacity of a DNA minigene
construct containing at least one HLA-A2 supermotif peptide to
induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are
immunized intramuscularly with 100 .mu.g of naked cDNA. As a means
of comparing the level of CTLs induced by cDNA immunization, a
control group of animals is also immunized with an actual peptide
composition that comprises multiple epitopes synthesized as a
single polypeptide as they would be encoded by the minigene.
[0681] Splenocytes from immunized animals are stimulated twice with
each of the respective compositions (peptide epitopes encoded in
the minigene or the polyepitopic peptide), then assayed for
peptide-specific cytotoxic activity in a 51Cr release assay. The
results indicate the magnitude of the CTL response directed against
the A2-restricted epitope, thus indicating the in vivo
immunogenicity of the minigene vaccine and polyepitopic
vaccine.
[0682] It is, therefore, found that the minigene elicits immune
responses directed toward the HLA-A2 supermotif peptide epitopes as
does the polyepitopic peptide vaccine. A similar analysis is also
performed using other HLA-A3 and HLA-B7 transgenic mouse models to
assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif
epitopes, whereby it is also found that the minigene elicits
appropriate immune responses directed toward the provided
epitopes.
[0683] To confirm the capacity of a class II epitope-encoding
minigene to induce HTLs in vivo, DR transgenic mice, or for those
epitopes that cross react with the appropriate mouse MHC molecule,
I-Ab-restricted mice, for example, are immunized intramuscularly
with 100 .mu.g of plasmid DNA. As a means of comparing the level of
HTLs induced by DNA immunization, a group of control animals is
also immunized with an actual peptide composition emulsified in
complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified
from splenocytes of immunized animals and stimulated with each of
the respective compositions (peptides encoded in the minigene). The
HTL response is measured using a 3H-thymidine incorporation
proliferation assay, (see, e.g., Alexander et al. Immunity
1:751-761, 1994). The results indicate the magnitude of the HTL
response, thus demonstrating the in vivo immunogenicity of the
minigene.
[0684] DNA minigenes, constructed as described in the previous
Example, can also be confirmed as a vaccine in combination with a
boosting agent using a prime boost protocol. The boosting agent can
consist of recombinant protein (e.g., Barnett et al., Aids Res. and
Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant
vaccinia, for example, expressing a minigene or DNA encoding the
complete protein of interest (see, e.g., Hanke et al., Vaccine
16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA
95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181,
1999; and Robinson et al., Nature Med. 5:526-34, 1999).
[0685] For example, the efficacy of the DNA minigene used in a
prime boost protocol is initially evaluated in transgenic mice. In
this example, A2.1/Kb transgenic mice are immunized IM with 100
.mu.g of a DNA minigene encoding the immunogenic peptides including
at least one HLA-A2 supermotif-bearing peptide. After an incubation
period (ranging from 3-9 weeks), the mice are boosted IP with 107
pfu/mouse of a recombinant vaccinia virus expressing the same
sequence encoded by the DNA minigene. Control mice are immunized
with 100 .mu.g of DNA or recombinant vaccinia without the minigene
sequence, or with DNA encoding the minigene, but without the
vaccinia boost. After an additional incubation period of two weeks,
splenocytes from the mice are immediately assayed for
peptide-specific activity in an ELISPOT assay. Additionally,
splenocytes are stimulated in vitro with the A2-restricted peptide
epitopes encoded in the minigene and recombinant vaccinia, then
assayed for peptide-specific activity in an alpha, beta and/or
gamma IFN ELISA.
[0686] It is found that the minigene utilized in a prime-boost
protocol elicits greater immune responses toward the HLA-A2
supermotif peptides than with DNA alone. Such an analysis can also
be performed using HLA-A11 or HLA-B7 transgenic mouse models to
assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif
epitopes. The use of prime boost protocols in humans is described
below in the Example entitled "Induction of CTL Responses Using a
Prime Boost Protocol."
Example 22
Peptide Composition for Prophylactic Uses
[0687] Vaccine compositions of the present invention can be used to
prevent 158P1D7 expression in persons who are at risk for tumors
that bear this antigen. For example, a polyepitopic peptide epitope
composition (or a nucleic acid comprising the same) containing
multiple CTL and HTL epitopes such as those selected in the above
Examples, which are also selected to target greater than 80% of the
population, is administered to individuals at risk for a
158P1D7-associated tumor.
[0688] For example, a peptide-based composition is provided as a
single polypeptide that encompasses multiple epitopes. The vaccine
is typically administered in a physiological solution that
comprises an adjuvant, such as Incomplete Freunds Adjuvant. The
dose of peptide for the initial immunization is from about 1 to
about 50,000 .mu.g, generally 100-5,000 .mu.g, for a 70 kg patient.
The initial administration of vaccine is followed by booster
dosages at 4 weeks followed by evaluation of the magnitude of the
immune response in the patient, by techniques that determine the
presence of epitope-specific CTL populations in a PBMC sample.
Additional booster doses are administered as required. The
composition is found to be both safe and efficacious as a
prophylaxis against 158P1D7-associated disease.
[0689] Alternatively, a composition typically comprising
transfecting agents is used for the administration of a nucleic
acid-based vaccine in accordance with methodologies known in the
art and disclosed herein.
Example 23
Polyepitopic Vaccine Compositions Derived from Native 158P1D7
Sequences
[0690] A native 158P1D7 polyprotein sequence is analyzed,
preferably using computer algorithms defined for each class I
and/or class II supermotif or motif, to identify "relatively short"
regions of the polyprotein that comprise multiple epitopes. The
"relatively short" regions are preferably less in length than an
entire native antigen. This relatively short sequence that contains
multiple distinct or overlapping, "nested" epitopes is selected; it
can be used to generate a minigene construct. The construct is
engineered to express the peptide, which corresponds to the native
protein sequence. The "relatively short" peptide is generally less
than 250 amino acids in length, often less than 100 amino acids in
length, preferably less than 75 amino acids in length, and more
preferably less than 50 amino acids in length. The protein sequence
of the vaccine composition is selected because it has maximal
number of epitopes contained within the sequence, i.e., it has a
high concentration of epitopes. As noted herein, epitope motifs may
be nested or overlapping (i.e., frame shifted relative to one
another). For example, with overlapping epitopes, two 9-mer
epitopes and one 10-mer epitope can be present in a 10 amino acid
peptide. Such a vaccine composition is administered for therapeutic
or prophylactic purposes.
[0691] The vaccine composition will include, for example, multiple
CTL epitopes from 158P1D7 antigen and at least one HTL epitope.
This polyepitopic native sequence is administered either as a
peptide or as a nucleic acid sequence which encodes the peptide.
Alternatively, an analog can be made of this native sequence,
whereby one or more of the epitopes comprise substitutions that
alter the cross-reactivity and/or binding affinity properties of
the polyepitopic peptide.
[0692] The embodiment of this example provides for the possibility
that an as yet undiscovered aspect of immune system processing will
apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic immune response-inducing
vaccine compositions. Additionally such an embodiment provides for
the possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment (excluding an
analoged embodiment) directs the immune response to multiple
peptide sequences that are actually present in native 158P1D7, thus
avoiding the need to evaluate any junctional epitopes. Lastly, the
embodiment provides an economy of scale when producing peptide or
nucleic acid vaccine compositions.
[0693] Related to this embodiment, computer programs are available
in the art which can be used to identify in a target sequence, the
greatest number of epitopes per sequence length.
Example 24
Polyepitopic Vaccine Compositions from Multiple Antigens
[0694] The 158P1D7 peptide epitopes of the present invention are
used in conjunction with epitopes from other target
tumor-associated antigens, to create a vaccine composition that is
useful for the prevention or treatment of cancer that expresses
158P1D7 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 158P1D7 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 158P1D7
expression, or can be administered as a composition comprising a
cocktail of one or more discrete epitopes. Alternatively, the
vaccine can be administered as a minigene construct or as dendritic
cells which have been loaded with the peptide epitopes in
vitro.
Example 25
Use of Peptides to Evaluate an Immune Response
[0695] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 158P1D7. Such an analysis can be performed in a manner
described by Ogg et al., Science 279:2103-2106, 1998. In this
Example, peptides in accordance with the invention are used as a
reagent for diagnostic or prognostic purposes, not as an
immunogen.
[0696] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 158P1D7 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising an 158P1D7
peptide containing an A*0201 motif. Tetrameric complexes are
synthesized as described (Musey et al., N. Engl. J. Med. 337:1267,
1997). Briefly, purified HLA heavy chain (A*0201 in this example)
and .beta.2-microglobulin are synthesized by means of a prokaryotic
expression system. The heavy chain is modified by deletion of the
transmembrane-cytosolic tail and COOH-terminal addition of a
sequence containing a BirA enzymatic biotinylation site. The heavy
chain, .beta.2-microglobulin, and peptide are refolded by dilution.
The 45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0697] For the analysis of patient blood samples, approximately one
million PBMCs are centrifuged at 300 g for 5 minutes and
resuspended in 50 .mu.l of cold phosphate-buffered saline.
Tri-color analysis is performed with the tetramer-phycoerythrin,
along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are
incubated with tetramer and antibodies on ice for 30 to 60 min and
then washed twice before formaldehyde fixation. Gates are applied
to contain >99.98% of control samples. Controls for the
tetramers include both A*0201-negative individuals and
A*0201-positive non-diseased donors. The percentage of cells
stained with the tetramer is then determined by flow cytometry. The
results indicate the number of cells in the PBMC sample that
contain epitope-restricted CTLs, thereby readily indicating the
extent of immune response to the 158P1D7 epitope, and thus the
status of exposure to 158P1D7, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 26
Use of Peptide Epitopes to Evaluate Recall Responses
[0698] The peptide epitopes of the invention are used as reagents
to evaluate T cell responses, such as acute or recall responses, in
patients. Such an analysis may be performed on patients who have
recovered from 158P1D7-associated disease or who have been
vaccinated with an 158P1D7 vaccine.
[0699] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
158P1D7 vaccine. PBMC are collected from vaccinated individuals and
HLA typed. Appropriate peptide epitopes of the invention that,
optimally, bear supermotifs to provide cross-reactivity with
multiple HLA supertype family members, are then used for analysis
of samples derived from individuals who bear that HLA type.
[0700] PBMC from vaccinated individuals are separated on
Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,
Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended
in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2
mM), penicillin (50 U/ml), streptomycin (50 .mu.g/ml), and Hepes
(10 mM) containing 10% heat-inactivated human AB serum (complete
RPMI) and plated using microculture formats. A synthetic peptide
comprising an epitope of the invention is added at 10 .mu.g/ml to
each well and HBV core 128-140 epitope is added at 1 .mu.g/ml to
each well as a source of T cell help during the first week of
stimulation.
[0701] In the microculture format, 4.times.10.sup.5 PBMC are
stimulated with peptide in 8 replicate cultures in 96-well round
bottom plate in 100 .mu.l/well of complete RPMI. On days 3 and 10,
100 ul of complete RPMI and 20 U/ml final concentration of rIL-2
are added to each well. On day 7 the cultures are transferred into
a 96-well flat-bottom plate and restimulated with peptide, rIL-2
and 10.sup.5 irradiated (3,000 rad) autologous feeder cells. The
cultures are tested for cytotoxic activity on day 14. A positive
CTL response requires two or more of the eight replicate cultures
to display greater than 10% specific .sup.51Cr release, based on
comparison with non-diseased control subjects as previously
described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996;
Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and
Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
[0702] Target cell lines are autologous and allogeneic
EBV-transformed B-LCL that are either purchased from the American
Society for Histocompatibility and Immunogenetics (ASHI, Boston,
Mass.) or established from the pool of patients as described
(Guilhot, et al. J. Virol. 66:2670-2678, 1992).
[0703] Cytotoxicity assays are performed in the following manner.
Target cells consist of either allogeneic HLA-matched or autologous
EBV-transformed B lymphoblastoid cell line that are incubated
overnight with the synthetic peptide epitope of the invention at 10
.mu.M, and labeled with 10 .mu.Ci of .sup.51Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0704] Cytolytic activity is determined in a standard 4-h, split
well .sup.51Cr release assay using U-bottomed 96 well plates
containing 3,000 targets/well. Stimulated PBMC are tested at
effector/target (E/T) ratios of 20-50:1 on day 14. Percent
cytotoxicity is determined from the formula:
100.times.[(experimental release-spontaneous release)/maximum
release-spontaneous release)]. Maximum release is determined by
lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co.,
St. Louis, Mo.). Spontaneous release is <25% of maximum release
for all experiments.
[0705] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 158P1D7 or an 158P1D7 vaccine.
[0706] Similarly, Class II restricted HTL responses may also be
analyzed. Purified PBMC are cultured in a 96-well flat bottom plate
at a density of 1.5.times.10.sup.5 cells/well and are stimulated
with 10 .mu.g/ml synthetic peptide of the invention, whole 158P1D7
antigen, or PHA. Cells are routinely plated in replicates of 4-6
wells for each condition. After seven days of culture, the medium
is removed and replaced with fresh medium containing 10 U/ml IL-2.
Two days later, 1 .mu.Ci .sup.3H-thymidine is added to each well
and incubation is continued for an additional 18 hours. Cellular
DNA is then harvested on glass fiber mats and analyzed for
.sup.3H-thymidine incorporation. Antigen-specific T cell
proliferation is calculated as the ratio of .sup.3H-thymidine
incorporation in the presence of antigen divided by the
.sup.3H-thymidine incorporation in the absence of antigen.
Example 27
Induction of Specific CTL Response in Humans
[0707] A human clinical trial for an immunogenic composition
comprising CTL and HTL epitopes of the invention is set up as an
IND Phase I, dose escalation study and carried out as a randomized,
double-blind, placebo-controlled trial. Such a trial is designed,
for example, as follows:
[0708] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0709] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0710] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0711] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0712] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0713] The endpoints measured in this study relate to the safety
and tolerability of the peptide composition as well as its
immunogenicity. Cellular immune responses to the peptide
composition are an index of the intrinsic activity of this the
peptide composition, and can therefore be viewed as a measure of
biological efficacy. The following summarize the clinical and
laboratory data that relate to safety and efficacy endpoints.
[0714] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0715] Evaluation of Vaccine Efficacy: For evaluation of vaccine
efficacy, subjects are bled before and after injection. Peripheral
blood mononuclear cells are isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient centrifugation, aliquoted in
freezing media and stored frozen. Samples are assayed for CTL and
HTL activity.
[0716] The vaccine is found to be both safe and efficacious.
Example 28
Phase II Trials in Patients Expressing 158P1D7
[0717] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 158P1D7. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 158P1D7, to establish the safety of
inducing a CTL and HTL response in these patients, and to see to
what extent activation of CTLs improves the clinical picture of
these patients, as manifested, e.g., by the reduction and/or
shrinking of lesions. Such a study is designed, for example, as
follows:
[0718] The studies are performed in multiple centers. The trial
design is an open-label, uncontrolled, dose escalation protocol
wherein the peptide composition is administered as a single dose
followed six weeks later by a single booster shot of the same dose.
The dosages are 50, 500 and 5,000 micrograms per injection.
Drug-associated adverse effects (severity and reversibility) are
recorded.
[0719] There are three patient groupings. The first group is
injected with 50 micrograms of the peptide composition and the
second and third groups with 500 and 5,000 micrograms of peptide
composition, respectively. The patients within each group range in
age from 21-65 and represent diverse ethnic backgrounds. All of
them have a tumor that expresses 158P1D7.
[0720] Clinical manifestations or antigen-specific T-cell responses
are monitored to assess the effects of administering the peptide
compositions. The vaccine composition is found to be both safe and
efficacious in the treatment of 158P1D7-associated disease.
Example 29
Induction of CTL Responses Using a Prime Boost Protocol
[0721] A prime boost protocol similar in its underlying principle
to that used to confirm the efficacy of a DNA vaccine in transgenic
mice, such as described above in the Example entitled "The Plasmid
Construct and the Degree to Which It Induces Immunogenicity," can
also be used for the administration of the vaccine to humans. Such
a vaccine regimen can include an initial administration of, for
example, naked DNA followed by a boost using recombinant virus
encoding the vaccine, or recombinant protein/polypeptide or a
peptide mixture administered in an adjuvant.
[0722] For example, the initial immunization may be performed using
an expression vector, such as that constructed in the Example
entitled "Construction of `Minigene` Multi-Epitope DNA Plasmids" in
the form of naked nucleic acid administered IM (or SC or ID) in the
amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to
1000 .mu.g) can also be administered using a gene gun. Following an
incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-10.sup.7 to 5.times.10.sup.9 pfu. An
alternative recombinant virus, such as an MVA, canarypox,
adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient
blood samples are obtained before immunization as well as at
intervals following administration of the initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells
are isolated from fresh heparinized blood by Ficoll-Hypaque density
gradient centrifugation, aliquoted in freezing media and stored
frozen. Samples are assayed for CTL and HTL activity.
[0723] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 158P1D7 is generated.
Example 30
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0724] Vaccines comprising peptide epitopes of the invention can be
administered using APCs, or "professional" APCs such as DC. In this
example, peptide-pulsed DC are administered to a patient to
stimulate a CTL response in vivo. In this method, dendritic cells
are isolated, expanded, and pulsed with a vaccine comprising
peptide CTL and HTL epitopes of the invention. The dendritic cells
are infused back into the patient to elicit CTL and HTL responses
in vivo. The induced CTL and HTL then destroy or facilitate
destruction, respectively, of the target cells that bear the
158P1D7 protein from which the epitopes in the vaccine are
derived.
[0725] For example, a cocktail of epitope-comprising peptides is
administered ex vivo to PBMC, or isolated DC therefrom. A
pharmaceutical to facilitate harvesting of DC can be used, such as
Progenipoietin.TM. (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After
pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
[0726] As appreciated clinically, and readily determined by one of
skill based on clinical outcomes, the number of DC reinfused into
the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature
Med. 2:52, 1996 and Prostate 32:272, 1997). Although
2-50.times.10.sup.6 DC per patient are typically administered,
larger number of DC, such as 10.sup.7 or 10.sup.8 can also be
provided. Such cell populations typically contain between 50-90%
DC.
[0727] In some embodiments, peptide-loaded PBMC are injected into
patients without purification of the DC. For example, PBMC
generated after treatment with an agent such as Progenipoietin.TM.
are injected into patients without purification of the DC. The
total number of PBMC that are administered often ranges from
10.sup.8 to 10.sup.10. Generally, the cell doses injected into
patients is based on the percentage of DC in the blood of each
patient, as determined, for example, by immunofluorescence analysis
with specific anti-DC antibodies. Thus, for example, if
Progenipoietin.TM. mobilizes 2% DC in the peripheral blood of a
given patient, and that patient is to receive 5.times.10.sup.6 DC,
then the patient will be injected with a total of
2.5.times.10.sup.8 peptide-loaded PBMC. The percent DC mobilized by
an agent such as Progenipoietin.TM. is typically estimated to be
between 2-10%, but can vary as appreciated by one of skill in the
art.
[0728] Ex Vivo Activation of CTL/HTL Responses
[0729] Alternatively, ex vivo CTL or HTL responses to 158P1D7
antigens can be induced by incubating, in tissue culture, the
patient's, or genetically compatible, CTL or HTL precursor cells
together with a source of APC, such as DC, and immunogenic
peptides. After an appropriate incubation time (typically about
7-28 days), in which the precursor cells are activated and expanded
into effector cells, the cells are infused into the patient, where
they will destroy (CTL) or facilitate destruction (HTL) of their
specific target cells, i.e., tumor cells.
Example 31
An Alternative Method of Identifying and Confirming Motif-Bearing
Peptides
[0730] Another method of identifying and confirming motif-bearing
peptides is to elute them from cells bearing defined MHC molecules.
For example, EBV transformed B cell lines used for tissue typing
have been extensively characterized to determine which HLA
molecules they express. In certain cases these cells express only a
single type of HLA molecule. These cells can be transfected with
nucleic acids that express the antigen of interest, e.g. 158P1D7.
Peptides produced by endogenous antigen processing of peptides
produced as a result of transfection will then bind to HLA
molecules within the cell and be transported and displayed on the
cell's surface. Peptides are then eluted from the HLA molecules by
exposure to mild acid conditions and their amino acid sequence
determined, e.g., by mass spectral analysis (e.g., Kubo et al., J.
Immunol. 152:3913, 1994). Because the majority of peptides that
bind a particular HLA molecule are motif-bearing, this is an
alternative modality for obtaining the motif-bearing peptides
correlated with the particular HLA molecule expressed on the
cell.
[0731] Alternatively, cell lines that do not express endogenous HLA
molecules can be transfected with an expression construct encoding
a single HLA allele. These cells can then be used as described,
i.e., they can then be transfected with nucleic acids that encode
158P1D7 to isolate peptides corresponding to 158P1D7 that have been
presented on the cell surface. Peptides obtained from such an
analysis will bear motif(s) that correspond to binding to the
single HLA allele that is expressed in the cell.
[0732] As appreciated by one in the art, one can perform a similar
analysis on a cell bearing more than one HLA allele and
subsequently determine peptides specific for each HLA allele
expressed. Moreover, one of skill would also recognize that means
other than transfection, such as loading with a protein antigen,
can be used to provide a source of antigen to the cell.
Example 32
Complementary Polynucleotides
[0733] Sequences complementary to the 158P1D7-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 158P1D7. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using, e.g., OLIGO 4.06 software (National Biosciences)
and the coding sequence of 158P1D7. To inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5'
sequence and used to prevent promoter binding to the coding
sequence. To inhibit translation, a complementary oligonucleotide
is designed to prevent ribosomal binding to the 158P1D7-encoding
transcript.
Example 33
Purification of Naturally-Occurring or Recombinant 158P1D7 Using
158P1D7 Specific Antibodies
[0734] Naturally occurring or recombinant 158P1D7 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 158P1D7. An immunoaffinity column is constructed by covalently
coupling anti-158P1D7 antibody to an activated chromatographic
resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia
Biotech). After the coupling, the resin is blocked and washed
according to the manufacturer's instructions.
[0735] Media containing 158P1D7 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 158P1D7 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/158P1D7 binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and GCR.P is collected.
Example 34
Identification of Molecules which Interact with 158P1D7
[0736] 158P1D7, or biologically active fragments thereof, are
labeled with 121 1 Bolton-Hunter reagent.
[0737] (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.)
Candidate molecules previously arrayed in the wells of a multi-well
plate are incubated with the labeled 158P1D7, washed, and any wells
with labeled 158P1D7 complex are assayed. Data obtained using
different concentrations of 158P1D7 are used to calculate values
for the number, affinity, and association of 158P1D7 with the
candidate molecules. Throughout this application, various website
data content, publications, applications and patents are
referenced. (Websites are referenced by their Uniform Resource
Locator, or URL, addresses on the World Wide Web.) The disclosures
of each of these items of information are hereby incorporated by
reference herein in their entireties.
Example 35
In Vivo Assay for 158P1D7 Tumor Growth Promotion
[0738] The effect of the 158P1D7 protein on tumor cell growth can
be confirmed in vivo by gene overexpression in bladder cancer
cells. For example, SCID mice can be injected SQ on each flank with
1.times.10.sup.6 bladder cancer cells (such as SCaBER, UM-UC-3,
HT1376, RT4, T24, TCC-SUP, J82 and SW780 cells) containing tkNeo
empty vector or 158P1D7.
[0739] At least two strategies may be used: (1) Constitutive
158P1D7 expression under regulation of a promoter such as a
constitutive promoter obtained from the genomes of viruses such as
polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), or from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, provided
such promoters are compatible with the host cell systems. (2)
Regulated expression under control of an inducible vector system,
such as ecdysone, tet, etc., can be used provided such promoters
are compatible with the host cell systems. Tumor volume is then
monitored at the appearance of palpable tumors and is followed over
time to determine if 158P1D7-expressing cells grow at a faster rate
and whether tumors produced by 158P1D7-expressing cells demonstrate
characteristics of altered aggressiveness (e.g. enhanced
metastasis, vascularization, reduced responsiveness to
chemotherapeutic drugs). Additionally, mice can be implanted with
the same cells orthotopically to determine if 158P1D7 has an effect
on local growth in the bladder or on the ability of the cells to
metastasize, specifically to lungs or lymph nodes (Fu, X., et al.,
Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer
Res., 1997. 17: p. 3239-3242; Peralta, E. A., et al., J. Urol.,
1999. 162: p. 1806-1811). Furthermore, this assay is useful to
confirm the 158P1D7 inhibitory effect of candidate therapeutic
compositions, such as for example, 158P1D7 antibodies or
intrabodies, and 158P1D7 antisense molecules or ribozymes.
[0740] The assay was performed using the following protocols. Male
ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington,
Mass.) were used and maintained in a strictly controlled
environment in accordance with the NIH Guide for the Care and Use
of Laboratory Animals. 158P1D7 transfected UM-UC-3 cells and
parental cells were injected into the subcutaneous space of SCID
mice. Each mouse received 4.times.10.sup.6 cells suspended in 50%
(v/v) of Matrigel. Tumor size was monitored through caliper
measurements twice a week. The longest dimension (L) and the
dimension perpendicular to it (W) were taken to calculate tumor
volume according to the formula W.sup.2.times.L/2. The Mann-Whitney
U test was used to evaluate differences of tumor growth. All tests
were two sided with {acute over (.alpha.)}=0.05. The results show
that 158P1D7 enhances the growth of bladder cancer in mice (FIG.
27).
Example 36
158P1D7 Monoclonal Antibody-Mediated Inhibition of Bladder and
Prostate Tumors In Vivo
[0741] The significant expression of 158P1D7 in cancer tissues,
together with its restricted expression in normal tissues, makes
158P1D7 an excellent target for antibody therapy. In cases where
the monoclonal antibody target is a cell surface protein,
antibodies have been shown to be efficacious at inhibiting tumor
growth (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or URL:
pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target
is not on the cell surface, such as PSA and PAP in prostate cancer,
antibodies have still been shown to recognize and inhibit growth of
cells expressing those proteins (Saffran, D. C., et al., Cancer and
Metastasis Reviews, 1999. 18: p. 437-449). As with any cellular
protein with a restricted expression profile, 158P1D7 is a target
for T cell-based immunotherapy.
[0742] Accordingly, the therapeutic efficacy of anti-158P1D7 mAbs
in human bladder cancer mouse models is modeled in
158P1D7-expressing bladder cancer xenografts or bladder cancer cell
lines, such as those described in Example (the Example entitled "In
Vivo Assay for 158P1D7 Tumor Growth Promotion", that have been
engineered to express 158P1D7.
[0743] Antibody efficacy on tumor growth and metastasis formation
is confirmed, e.g., in a mouse orthotopic bladder cancer xenograft
model. The antibodies can be unconjugated, as discussed in this
Example, or can be conjugated to a therapeutic modality, as
appreciated in the art. It is confirmed that anti-158P1D7 mAbs
inhibit formation of 158P1D7-expressing bladder tumors.
Anti-158P1D7 mAbs also retard the growth of established orthotopic
tumors and prolong survival of tumor-bearing mice. These results
indicate the utility of anti-158P1D7 mAbs in the treatment of local
and advanced stages of bladder cancer. (See, e.g., Saffran, D., et
al., PNAS 10:1073-1078 or URL:
pnas.org/cgi/doi/10.1073/pnas.051624698)
[0744] Administration of anti-158P1D7 mAbs retard established
orthotopic tumor growth and inhibit metastasis to distant sites,
resulting in a significant prolongation in the survival of
tumor-bearing mice. These studies indicate that 158P1D7 is an
attractive target for immunotherapy and demonstrate the therapeutic
potential of anti-158P1D7 mAbs for the treatment of local and
metastatic bladder cancer.
[0745] This example demonstrates that unconjugated 158P1D7
monoclonal antibodies effectively to inhibit the growth of human
bladder tumors grown in SCID mice; accordingly a combination of
such efficacious monoclonal antibodies is also effective.
[0746] Tumor Inhibition Using Multiple Unconjugated 158P1D7
mAbs
[0747] Materials and Methods
[0748] 158P1D7 Monoclonal Antibodies:
[0749] Monoclonal antibodies are raised against 158P1D7 as
described in the Example entitled "Generation of 158P1D7 Monoclonal
Antibodies (mAbs)." The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation, in accordance with
techniques known in the art, for their capacity to bind 158P1D7.
Epitope mapping data for the anti-158P1D7 mAbs, as determined by
ELISA and Western analysis, recognize epitopes on the 158P1D7
protein. Immunohistochemical analysis of bladder cancer tissues and
cells with these antibodies is performed.
[0750] The monoclonal antibodies are purified from ascites or
hybridoma tissue culture supernatants by Protein-G Sepharose
chromatography, dialyzed against PBS, filter sterilized, and stored
at -20.degree. C. Protein determinations are performed by a
Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic
monoclonal antibody or a cocktail comprising a mixture of
individual monoclonal antibodies is prepared and used for the
treatment of mice receiving subcutaneous or orthotopic injections
of bladder tumor xenografts.
[0751] Bladder Cancer Cell Lines
[0752] Bladder cancer cell lines (Scaber, J82, UM-UC-3, HT1376,
RT4, T24, TCC-SUP, J82 and SW780) expressing 158P1D7 are generated
by retroviral gene transfer as described in Hubert, R. S., et al.,
STEAP: a prostate-specific cell-surface antigen highly expressed in
human prostate tumors. Proc Natl Acad Sci USA, 1999.
96(25):14523-8. Anti-158P1D7 staining is detected by using an
FITC-conjugated goat anti-mouse antibody (Southern Biotechnology
Associates) followed by analysis on a Coulter Epics-XL f low
cytometer.
[0753] In Vivo Mouse Models.
[0754] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10.sup.6 158P1D7-expressing bladder cancer cells mixed at a
1:1 dilution with Matrigel (Collaborative Research) in the right
flank of male SCID mice. To test antibody efficacy on tumor
formation, i.p. antibody injections are started on the same day as
tumor-cell injections. As a control, mice are injected with either
purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody
that recognizes an irrelevant antigen not expressed in human cells.
In preliminary studies, no difference is found between mouse IgG or
PBS on tumor growth. Tumor sizes are determined by vernier caliper
measurements, and the tumor volume is calculated as
length.times.width.times.height. Mice with s.c. tumors greater than
1.5 cm in diameter are sacrificed. Circulating levels of
anti-158P1D7 mAbs are determined by a capture ELISA kit (Bethyl
Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al.,
PNAS 10:1073-1078)
[0755] Orthotopic injections are performed, for example, in two
alternative embodiments, under anesthesia by, for example, use of
ketamine/xylazine. In a first embodiment, an intravesicular
injection of bladder cancer cells is administered directly through
the urethra and into the bladder (Peralta, E. A., et al., J. Urol.,
1999. 162:1806-1811). In a second embodiment, an incision is made
through the abdominal wall, the bladder is exposed, and bladder
tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are
surgically glued onto the exterior wall of the bladder, termed
"onplantation" (Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939;
Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242).
Antibodies can be administered to groups of mice at the time of
tumor injection or onplantation, or after 1-2 weeks to allow tumor
establishment.
[0756] Anti-158P1D7 mAbs Inhibit Growth of 158P1D7-Expressing
Bladder Cancer Tumors
[0757] In one embodiment, the effect of anti-158P1D7 mAbs on tumor
formation is tested by using the bladder onplantation orthotopic
model. As compared with the s.c. tumor model, the orthotopic model,
which requires surgical attachment of tumor tissue directly on the
bladder, results in a local tumor growth, development of metastasis
in distal sites, and subsequent death (Fu, X., et al., Int. J.
Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer Res.,
1997. 17: p. 3239-3242). This features make the orthotopic model
more representative of human disease progression and allows one to
follow the therapeutic effect of mAbs, as well as other therapeutic
modalities, on clinically relevant end points.
[0758] Accordingly, 158P1D7-expressing tumor cells are onplanted
orthotopically, and 2 days later, the mice are segregated into two
groups and treated with either: a) 50-2000 .mu.g, usually 200-500
.mu.g, of anti-158P1D7 Ab, or b) PBS, three times per week for two
to five weeks. Mice are monitored weekly for indications of tumor
growth.
[0759] As noted, a major advantage of the orthotopic bladder cancer
model is the ability to study the development of metastases.
Formation of metastasis in mice bearing established orthotopic
tumors is studied by histological analysis of tissue sections,
including lung and lymph nodes (Fu, X., et al., Int. J. Cancer,
1991. 49:938-939; Chang, S., et al., Anticancer Res., 1997.
17:3239-3242). Additionally, IHC analysis using anti-158P1D7
antibodies can be performed on the tissue sections.
[0760] Mice bearing established orthotopic 158P1D7-expressing
bladder tumors are administered 1000 .mu.g injections of either
anti-158P1D7 mAb or PBS over a 4-week period. Mice in both groups
are allowed to establish a high tumor burden (1-2 weeks growth), to
ensure a high frequency of metastasis formation in mouse lungs and
lymph nodes. Mice are then sacrificed and their local bladder tumor
and lung and lymph node tissue are analyzed for the presence of
tumor cells by histology and IHC analysis.
[0761] In another embodiment, the effect of anti-158P1D7 mAbs on
tumor growth was tested using the following protocols. Male
ICR--SCID mice, 5-6 weeks old (Charles River Laboratory,
Wilmington, Mass.) were used and were maintained in a
strictly-controlled environment in accordance with the NIH Guide
for the Care and Use of Laboratory Animals. UG-B1, a patient
bladder cancer, was used to establish xenograft models. Stock
tumors regularly maintained in SCID mice were sterilely dissected,
minced, and digested using Pronase (Calbiochem, San Diego, Calif.).
Cell suspensions generated were incubated overnight at 37.degree.
C. to obtain a homogeneous single-cell suspension. Each mouse
received 2.5.times.10.sup.6 cells at the subcutaneous site of right
flank. Murine monoclonal antibodies to 158P1D7 and PSCA were tested
at a dose of 500 .mu.g/mouse in the study. PBS was used as control.
MAbs were dosed intra-peritoneally twice a week for a total of 12
doses, starting on the same day of tumor cell injection. Tumor size
was monitored through caliper measurements twice a week. The
longest dimension (L) and the dimension perpendicular to it (W)
were taken to calculate tumor volume according to the formula:
W.sup.2.times.L/2. The results show that Anti-158P1D7 mAbs are
capable of inhibiting the growth of human bladder carcinoma in mice
(FIG. 31).
[0762] Anti-158P1D7 mAbs Inhibit Growth of 158P1D7-Expressing
Prostate Cancer Tumors
[0763] In another embodiment, the effect of anti-158P1D7 mAbs on
tumor growth was tested using the following protocols. Male
ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington,
Mass.) were used and were maintained in a strictly-controlled
environment in accordance with the NIH Guide for the Care and Use
of Laboratory Animals. LAPC-9AD, an androgen-dependent human
prostate cancer, was used to establish xenograft models. Stock
tumors were regularly maintained in SCID mice. At the day of
implantation, stock tumors were harvested and trimmed of necrotic
tissues and minced to 1 mm.sup.3 pieces. Each mouse received 4
pieces of tissues at the subcutaneous site of right flank. Murine
monoclonal antibodies to 158P1D7 and PSCA were tested at a dose of
1000 .mu.g/mouse and 500 .mu.g/mouse respectively. PBS and anti-KLH
monoclonal antibody were used as controls. The study cohort
consisted of 4 groups with 6 mice in each group. MAbs were dosed
intra-peritoneally twice a week for a total of 8 doses. Treatment
was started when tumor volume reached 45 mm.sup.3. Tumor size was
monitored through caliper measurements twice a week. The longest
dimension (L) and the dimension perpendicular to it (W) were taken
to calculate tumor volume according to the formula:
W.sup.2.times.L/2. The Student's t test and the Mann-Whitney U
test, where applicable, were used to evaluate differences of tumor
growth. All tests were two-sided with .alpha.=0.05. The results
show that Anti-158P1D7 mAbs are capable of inhibiting the growth of
human prostate carcinoma in mice (FIG. 30).
[0764] These studies demonstrate a broad anti-tumor efficacy of
anti-158P1D7 antibodies on initiation and progression of bladder
cancer and prostate cancer in mouse models. Anti-158P1D7 antibodies
inhibit tumor formation and retard the growth of already
established tumors and prolong the survival of treated mice.
Moreover, anti-158P1D7 mAbs demonstrate a dramatic inhibitory
effect on the spread of local bladder tumor to distal sites, even
in the presence of a large tumor burden. Thus, anti-158P1D7 mAbs
are efficacious on major clinically relevant end points including
lessened tumor growth, lessened metastasis, and prolongation of
survival.
Example 37
Homology Comparison of 158P1D7 to Known Sequences
[0765] The 158P1D7 protein has 841 amino acids with calculated
molecular weight of 95.1 kDa, and pI of 6.07. 158P1D7 is predicted
to be a plasma membrane protein (0.46 PSORT
http://psort.nibb.ac.jp/form.html) with a possibility of it being a
nuclear protein (65% by PSORT http://psort.nibb.ac.jp/form2.html).
158P1D7 has a potential cleavage site between aa 626 and 627 and a
potential signal site at aa 3-25.
[0766] 158P1D7 contains a single transmembrane region from amino
acids 611-633 with high probability that the amino-terminus resides
outside, consistent with the topology of a Type 1 transmembrane
protein (located on the World Wide Web at
.cbs.dtu.dk/services/TMHMM). Also visualized is a short hydrophobic
stretch from amino acids 3-25, consistent with the existence of an
amino-terminal signal peptide. Based on the TMpred algorithm of
Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel,
TMBASE--A database of membrane spanning protein segments Biol.
Chem. Hoppe-Seyler 374:166, 1993), 158P1D7 contains a primary
transmembrane region from amino acids 609-633 and a secondary
transmembrane region from amino acids 3-25 (contiguous amino acids
with values greater than 0 on the plot have high probability of
being transmembrane regions) with an orientation in which the amino
terminus resides inside and the carboxyl terminus outside. An
alternative model is also predicted that 158P1D7 is a Type 1
transmembrane protein in which the amino-terminus resides outside
and the protein contains a secondary transmembrane domain signal
peptide from amino acids 3-25 and a primary transmembrane domain
from aa615-633. The transmembrane prediction algorithms are
accessed through the ExPasy molecular biology server located on the
World Wide Web at (.expasy.ch/tools/).
[0767] By use of the PubMed website of the N.C.B.I. located on the
World Wide Web at (.ncbi.nlm.nih.gov/entrez), it was found at the
protein level that 158P1D7 shows best homology to the hypothetical
protein FLJ22774 (PubMed record: gi 14149932) of unknown function,
with 97% identity and 97% homology (FIG. 4 and FIG. 5A). The
158P1D7 protein demonstrates homology to a human protein similar to
IGFALS (insulin-like growth factor binding protein, acid labile
subunit) (PubMed record: gi 6691962) with 36% identity and 52%
homology (FIG. 5B), to Slit proteins with 25% identity and 39%
homology and to the leucine-rich repeat transmembrane family of
proteins FLRT (Fibronectin-like domain-containing leucine-rich
transmembrane protein), including FLRT2 with 26% identity and 43%
homology, and FLRT3 with 34% identity and 53% homology.
[0768] Insulin-like growth factors (IGF) have been shown to play an
important role in tumor growth including prostate, breast, brain
and ovarian cancer (O'Brian et al, Urology. 2001, 58:1; Wang J et
al Oncogene. 2001, 20:3857; Helle S et al, Br J Cancer. 2001,
85:74). IGFs produce their oncogenic effect by binding to specific
cell surface receptors and activating survival as well as mitogenic
pathways (Babajko S et al, Med Pediatr Oncol. 2001, 36:154; Scalia
P et al, J Cell Biochem. 2001, 82:610). The activity of
insulin-like growth factors is regulated by IGF binding proteins
(IGF-BP) and the acid labile subunit (ALS) of IGF-BP (Zeslawski W
et al, EMBO J. 2001, 20:3638; Jones J I. and Clemmons D R. Endocr.
Rev. 1995, 16: 3). In the plasma, most IGFs exist as a ternary
complex containing IGF-BP and ALS (Jones J I. and Clemmons D R.
Endocr. Rev. 1995, 16: 3). Association with ALS allows the
retention of the ternary complex in the vasculature and extends its
lifespan (Ueki I et al, Proc Natl Acad Sci USA 2000, 97:6868).
Studies in mice demonstrate the contribution of ALS to cell growth
by showing that mice carrying mutant ALS exhibit a growth deficit
(Ueki I et al, Proc Natl Acad Sci USA 2000, 97:6868), indicating
that ALS plays a critical role in the growth of tumor cells. The
158P1D7 protein serves as an IGF-ALS-like protein in that it
facilitates the formation of the IGF ternary complex. The
158P1D7-induced IGF complex formation leads to increased growth of
tumor cells expressing 158P1D7 which facilitates the growth of this
malignancy in vivo. The induction of the IGF complex allows one to
assay for monoclonal antibodies with neutralizing ability to
disrupt, or enhancing capacity to help form, the ternary
interaction.
[0769] Slit proteins were first identified in Drosophila as
secreted proteins that regulate axon guidance and orientation
(Rajagopalan S et al, Cell. 2000, 103:1033; Chen J et al, J
Neurosci. 2001, 21:1548). Mammalian homologs were cloned in mice
and humans, where they are shown to regulate migration and
chemotaxis (Wu J et al, Nature. 2001, 410:948; Brose K and Tessier
M, Curr Opin Neurobiol. 2001, 10:95). Slit proteins localize at two
distinct subcellular sites within epithelial cells depending on
cell stage, with Slit 3 predominantly localizing in the
mitochondria and targeting to the cell surface in more confluent
cells (Little M H et al, Am J Physiol Cell Physiol. 2001,
281:C486). The differential Slit localization suggests that Slit
may function differently whether it is secreted, associated with
the cell surface or retained in the mitochondria. The 158P1D7
protein functions as a Slit-like protein in that it binds to
Roundabout receptors (Robos) on the surface of cells. 158P1D7 has
homology (83% identity along entire length) with the murine Slitrk6
gene, a member of a new family of Leucine Rich Receptors (LRRs).
The Slit family of LRRs is involved in neurite outgrowth and axonal
guidance during development. These proteins also play a role in
organ development by providing cues for branching morphogenesis in
lung, kidney and other organs. The crystal structure for several
LRRs has been determined. These proteins are shaped like a
horseshoe with LRRs on both sides of a central flexible region.
This horseshoe shape likely forms a central pocket where other
proteins (binding partners) can interact. The term binding partner
includes ligands, receptors, substrates, antibodies, and other
molecules that interact with the 158P1D7 polypeptide through
contact or proximity between particular portions of the binding
partner and the 158P1D7 polypeptide. Binding partners for 158P1D7
polypeptides are expressed on both epithelial and mesenchymal cells
within an organ. Known binding partners for the Slit family of LRRs
include both the Robo family of genes and glypicans. Both of these
potential protein interacting partners are aberrantly expressed in
human cancers. Robos are Ig-like proteins that act as adhesion
molecules. Interaction of specific Robo and Slit proteins results
in cell migration with the ultimate outcome being either repulsion
or attraction depending on intracellular signaling cascades.
Mutations that disrupt interaction of Slit with Robo result in
failure to repel migrating neurons during development. Moreover,
mutations that disrupt functional interactions lead to organ
failure and hyperproliferation in the developing lung. Mutational
analysis has further shown that the LRR region is required for
biologic activity of these receptors. 158P1D7 is overexpressed in a
variety of human cancers including those derived from bladder and
lung. Aberrant expression of this protein leads to enhanced cell
growth, survival, increased metastasis and angiogenesis by
disrupting or promoting protein interactions between 158P1D7 and
specific binding partners on the surface of adjacent cells. Binding
of 158P1D7 to Robo receptors (Robo-1, -2, -3 and -4) is observed in
vitro, both as recombinant proteins and as cell surface molecules.
Biological effects are induced when the Robo-1, -2, -3 or -4
receptors or glypican-binding partners binds to 158P1D7 on the cell
surface. These activities are detected by adhesion, enhanced
migration or repulsion in cell based assays. The interaction
between 158P1D7 and Robo receptors leads to increased adhesion
between 158P1D7-expressing tumor cells and endothelium or other
cell types expressing Robo receptors, leading to spreading and
metastasis of tumor cells as well as enhanced angiogenesis.
Further, the association between 158P1D7 and Robo receptors allows
one to screen for monoclonal antibodies with the ability to block
(or enhance) the interaction in an in vitro assay. Such antibodies
have a modulating effect on growth of 158P1D7 expressing
tumors.
[0770] The FLRT (Fibronectin-like domain-containing leucine-rich
transmembrane protein) family of transmembrane proteins has three
members, FLRT1, FLRT2 and FLRT3, which contain 10 leucine-rich
repeats flanked by cysteine-rich domains, a
fibronectin/collagen-like motif and an intracellular tail (Lacy S E
et al, Genomics 1999, 62:417). Based on overall structure of the
three proteins, a role in cell adhesion and receptor signaling is
predicted. A Xenopus laevis ortholog of FLRT3 (XFLRT3) was
identified that shows co-expression with FGFs (fibroblast growth
factors) and is induced after activation and reduced following
inhibition of signal transduction through the FGFs (Bottcher R T et
al, Nature Cell Biol 2004, 6:38). The interaction between FGFRs
(FGF receptors) and XFLRT3 indicates that XFLRT3 modulates
FGF-induced signal transduction through the MAP kinase pathway. The
158P1D7 protein forms a complex with FGFRs that induces modulation
of FGF-induced signal transduction through the MAP kinase (ERK-1
and ERK-2) pathway. FGF-induced signals are potentiated by
expression of 158P1D7, which leads to an increase in the
proliferative capacity of the cells. This significantly promotes
unregulated growth of cancer cells expressing 158P1D7, contributing
to their growth advantage in vivo. The interaction between 158P1D7
protein and FGFR allows one to screen for monoclonal antibodies
with the ability to disrupt (or enhance) the association of these
two molecules. Such antibodies have a modulating effect on growth
of 158P1D7 expressing tumors.
Example 38
Identification and Confirmation of Signal Transduction Pathways
[0771] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J Neurochem. 2001; 76:217-223). In particular, IGF and
IGF-BP have been shown to regulate mitogenic and survival pathways
(Babajko S et al, Med Pediatr Oncol. 2001, 36:154; Scalia P et al,
J Cell Biochem. 2001, 82:610). Using immunoprecipitation and
Western blotting techniques, proteins are identified that associate
with 158P1D7 and mediate signaling events. Several pathways known
to play a role in cancer biology are regulated by 158P1D7,
including phospholipid pathways such as PI3K, AKT, etc, adhesion
and migration pathways, including FAK, Rho, Rac-1, etc, as well as
mitogenic/survival cascades such as ERK, p38, etc. (Cell Growth
Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,
19:3003, J. Cell Biol. 1997, 138:913.). Bioinformatic analysis
revealed that 158P1D7 can become phosphorylated by serine/threonine
as well as tyrosine kinases. Thus, the phosphorylation of 158P1D7
is provided by the present invention to lead to activation of the
above listed pathways.
[0772] Using, e.g., Western blotting techniques, the ability of
158P1D7 to regulate these pathways is confirmed. Cells expressing
or lacking 158P1D7 are either left untreated or stimulated with
cytokines, hormones and anti-integrin antibodies. Cell lysates are
analyzed using anti-phospho-specific antibodies (Cell Signaling,
Santa Cruz Biotechnology) in order to detect phosphorylation and
regulation of ERK, p38, AKT, PI3K, PLC and other signaling
molecules. When 158P1D7 plays a role in the regulation of signaling
pathways, whether individually or communally, it is used as a
target for diagnostic, prognostic, preventative and therapeutic
purposes.
[0773] To confirm that 158P1D7 directly or indirectly activates
known signal transduction pathways in cells, luciferase (luc) based
transcriptional reporter assays are carried out in cells expressing
individual genes. These transcriptional reporters contain
consensus-binding sites for known transcription factors that lie
downstream of well-characterized signal transduction pathways. The
reporters and examples of these associated transcription factors,
signal transduction pathways, and activation stimuli are listed
below:
[0774] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0775] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0776] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0777] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0778] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0779] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0780] Gene-mediated effects are assayed in cells showing mRNA
expression. Luciferase reporter plasmids are introduced by
lipid-mediated transfection (TFX-50, Promega). Luciferase activity,
an indicator of relative transcriptional activity, is measured by
incubation of cell extracts with luciferin substrate and
luminescence of the reaction is monitored in a luminometer.
[0781] Signaling pathways activated by 158P1D7 are mapped and used
for the identification and validation of therapeutic targets. When
158P1D7 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and therapeutic purposes.
Example 39
Involvement in Tumor Progression
[0782] The 158P1D7 gene can contribute to the growth of cancer
cells. The role of 158P1D7 in tumor growth is confirmed in a
variety of primary and transfected cell lines including prostate,
colon, bladder and kidney cell lines as well as NIH 3T3 cells
engineered to stably express 158P1D7. Parental cells lacking
158P1D7 and cells expressing 158P1D7 are evaluated for cell growth
using a well-documented proliferation assay (see, e.g., Fraser S P,
Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E,
Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).
[0783] To confirm the role of 158P1D7 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH3T3 cells lacking 158P1D7 are compared to NHI-3T3 cells
expressing 158P1D7, using a soft agar assay under stringent and
more permissive conditions (Song Z. et al. Cancer Res. 2000,
60:6730).
[0784] To confirm the role of 158P1D7 in invasion and metastasis of
cancer cells, a well-established assay is used, e.g., a Transwell
Insert System assay (Becton Dickinson) (Cancer Res. 1999, 59:6010).
Control cells, including prostate, colon, bladder and kidney cell
lines lacking 158P1D7 are compared to cells expressing 158P1D7,
respectively. Cells are loaded with the fluorescent dye, calcein,
and plated in the top well of the Transwell insert coated with a
basement membrane analog. Invasion is determined by fluorescence of
cells in the lower chamber relative to the fluorescence of the
entire cell population.
[0785] 158P1D7 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 158P1D7 are compared for
differences in cell cycle regulation using a well-established BrdU
assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short,
cells are grown under both optimal (full serum) and limiting (low
serum) conditions are labeled with BrdU and stained with anti-BrdU
Ab and propidium iodide. Cells are analyzed for entry into the G1,
S, and G2M phases of the cell cycle. Alternatively, the effect of
stress on apoptosis is evaluated in control parental cells and
cells expressing 158P1D7, including normal and tumor bladder cells.
Engineered and parental cells are treated with various
chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and
protein synthesis inhibitors, such as cycloheximide. Cells are
stained with annexin V-FITC and cell death is measured by FACS
analysis. The modulation of cell death by 158P1D7 can play a
critical role in regulating tumor progression and tumor load.
[0786] When 158P1D7 plays a role in cell growth, transformation,
invasion or apoptosis, it is used as a target for diagnostic,
prognostic, preventative and therapeutic purposes.
Example 40
Involvement in Angiogenesis
[0787] Angiogenesis or new capillary blood vessel formation is
necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996,
86:353; Folkman J. Endocrinology. 1998 139:441). Several assays
have been developed to measure angiogenesis in vitro and in vivo,
such as the tissue culture assays, endothelial cell tube formation,
and endothelial cell proliferation. Using these assays as well as
in vitro neo-vascularization, the effect of 158P1D7 on angiogenesis
is confirmed. For example, endothelial cells engineered to express
158P1D7 are evaluated using tube formation and proliferation
assays. The effect of 158P1D7 is also confirmed in animal models in
vivo. For example, cells either expressing or lacking 158P1D7 are
implanted subcutaneously in immunocompromised mice. Endothelial
cell migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. When 158P1D7 affects angiogenesis,
it is used as a target for diagnostic, prognostic, preventative and
therapeutic purposes
Example 41
Regulation of Transcription
[0788] The above-indicated localization of 158P1D7 to the nucleus
and its similarity to IGF-BP which has been found to activate
signaling pathways and to regulate essential cellular functions,
support the present invention use of 158P1D7 based on its role in
the transcriptional regulation of eukaryotic genes. Regulation of
gene expression is confirmed, e.g., by studying gene expression in
cells expressing or lacking 158P1D7. For this purpose, two types of
experiments are performed.
[0789] In the first set of experiments, RNA from parental and
158P1D7-expressing cells are extracted and hybridized to
commercially available gene arrays (Clontech) (Smid-Koopman E et
al. Br J Cancer. 2000. 83:246). Resting cells as well as cells
treated with FBS or androgen are compared. Differentially expressed
genes are identified in accordance with procedures known in the
art. The differentially expressed genes are then mapped to
biological pathways (Chen K et al., Thyroid. 2001. 11:41.).
[0790] In the second set of experiments, specific transcriptional
pathway activation is evaluated using commercially available (e.g.,
Stratagene) luciferase reporter constructs including: NFkB-luc,
SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These
transcriptional reporters contain consensus binding sites for known
transcription factors that lie downstream of well-characterized
signal transduction pathways, and represent a good tool to
ascertain pathway activation and screen for positive and negative
modulators of pathway activation.
[0791] When 158P1D7 plays a role in gene regulation, it is used as
a target for diagnostic, prognostic, preventative and therapeutic
purposes.
Example 42
Subcellular Localization of 158P1D7
[0792] The cellular location of 158P1D7 is assessed using
subcellular fractionation techniques widely used in cellular
biology (Storrie B, et al. Methods Enzymol. 1990; 182:203-25). A
variety of cell lines, including prostate, kidney and bladder cell
lines as well as cell lines engineered to express 158P1D7 are
separated into nuclear, cytosolic and membrane fractions. Gene
expression and location in nuclei, heavy membranes (lysosomes,
peroxisomes, and mitochondria), light membranes (plasma membrane
and endoplasmic reticulum), and soluble protein fractions are
tested using Western blotting techniques.
[0793] Alternatively, 293T cells are transfected with an expression
vector encoding individual genes, HIS-tagged (PCDNA 3.1 MYC/HIS,
Invitrogen) and the subcellular localization of these genes is
determined as described above. In short, the transfected cells are
harvested and subjected to a differential subcellular fractionation
protocol (Pemberton, P. A. et al, 1997, J of Histochemistry and
Cytochemistry, 45:1697-1706). Location of the HIS-tagged genes is
followed by Western blotting.
[0794] Using 158P1D7 antibodies, it is possible to demonstrate
cellular localization by immunofluorescence and
immunohistochemistry. For example, cells expressing or lacking
158P1D7 are adhered to a microscope slide and stained with
anti-158P1D7 specific Ab. Cells are incubated with an FITC-coupled
secondary anti-species Ab, and analyzed by fluorescent microscopy.
Alternatively, cells and tissues lacking or expressing 158P1D7 are
analyzed by IHC as described herein.
[0795] When 158P1D7 is localized to specific cell compartments, it
is used as a target for diagnostic, preventative and therapeutic
purposes.
Example 43
Involvement of 158P1D7 in Protein Trafficking
[0796] Due to its similarity to Slit proteins, 158P1D7 can regulate
intracellular trafficking and retention into mitochondrial and/or
nuclear compartments. Its role in the trafficking of proteins can
be confirmed using well-established methods (Valetti C. et al. Mol
Biol Cell. 1999, 10:4107). For example, FITC-conjugated
.alpha.2-macroglobulin is incubated with 158P1D7-expressing and
158P1D7-negative cells. The location and uptake of
FITC-.alpha.2-macroglobulin is visualized using a fluorescent
microscope. In another approach, the co-localization of 158P1D7
with vesicular proteins is confirmed by co-precipitation and
Western blotting techniques and fluorescent microscopy.
[0797] Alternatively, 158P1D7-expressing and 158P1D7-lacking cells
are compared using bodipy-ceramide labeled bovine serum albumine
(Huber L et al. Mol. Cell. Biol. 1995, 15:918). Briefly, cells are
allowed to take up the labeled BSA and are placed intermittently at
4.degree. C. and 18.degree. C. to allow for trafficking to take
place. Cells are examined under fluorescent microscopy, at
different time points, for the presence of labeled BSA in specific
vesicular compartments, including Golgi, endoplasmic reticulum,
etc.
[0798] In another embodiment, the effect of 158P1D7 on membrane
transport is examined using biotin-avidin complexes. Cells either
expressing or lacking 158P1D7 are transiently incubated with
biotin. The cells are placed at 4.degree. C. or transiently warmed
to 37.degree. C. for various periods of time. The cells are
fractionated and examined by avidin affinity precipitation for the
presence of biotin in specific cellular compartments. Using such
assay systems, proteins, antibodies and small molecules are
identified that modify the effect of 158P1D7 on vesicular
transport. When 158P1D7 plays a role in intracellular trafficking,
158P1D7 is a target for diagnostic, prognostic, preventative and
therapeutic purposes
Example 44
Protein-Protein Association
[0799] IGF and IGF-BP proteins have been shown to interact with
other proteins, thereby forming protein complexes that can regulate
protein localization, biological activity, gene transcription, and
cell transformation (Zeslawski W et al, EMBO J. 2001, 20:3638; Yu
H, Rohan T, J Natl Cancer Inst. 2000, 92:1472). Using
immunoprecipitation techniques as well as two yeast hybrid systems,
proteins are identified that associate with 158P1D7.
Immunoprecipitates from cells expressing 158P1D7 and cells lacking
158P1D7 are compared for specific protein-protein associations.
[0800] Studies are performed to determine the extent of the
association of 158P1D7 with receptors, such as the EGF and IGF
receptors, and with intracellular proteins, such as IGF-BP,
cytoskeletal proteins etc. Studies comparing 158P1D7 positive and
158P1D7 negative cells, as well as studies comparing
unstimulated/resting cells and cells treated with epithelial cell
activators, such as cytokines, growth factors and anti-integrin Ab
reveal unique protein-protein interactions.
[0801] In addition, protein-protein interactions are confirmed
using two yeast hybrid methodology (Curr Opin Chem Biol. 1999,
3:64). A vector carrying a library of proteins fused to the
activation domain of a transcription factor is introduced into
yeast expressing a 158P1D7-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
colorimetric reporter activity. Specific association with surface
receptors and effector molecules directs one of skill to the mode
of action of 158P1D7, and thus identifies therapeutic, prognostic,
preventative and/or diagnostic targets for cancer. This and similar
assays are also used to identify and screen for small molecules
that interact with 158P1D7.
[0802] When 158P1D7 associates with proteins or small molecules it
is used as a target for diagnostic, prognostic, preventative and
therapeutic purposes.
Example 45
Transcript Variants of 158P1D7
[0803] Transcript variants are variants of mature mRNA from the
same gene which arise by alternative transcription or alternative
splicing. Alternative transcripts are transcripts from the same
gene but start transcription at different points. Splice variants
are mRNA variants spliced differently from the same transcript. In
eukaryotes, when a multi-exon gene is transcribed from genomic DNA,
the initial RNA is spliced to produce functional mRNA, which has
only exons and is used for translation into an amino acid sequence.
Accordingly, a given gene can have zero to many alternative
transcripts and each transcript can have zero to many splice
variants. Each transcript variant has a unique exon makeup, and can
have different coding and/or non-coding (5' or 3' end) portions,
from the original transcript. Transcript variants can code for
similar or different proteins with the same or a similar function
or can encode proteins with different functions, and can be
expressed in the same tissue at the same time, or in different
tissues at the same time, or in the same tissue at different times,
or in different tissues at different times. Proteins encoded by
transcript variants can have similar or different cellular or
extracellular localizations, e.g., secreted versus
intracellular.
[0804] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified by full-length cloning experiment,
or by use of full-length transcript and EST sequences. First, all
human ESTs were grouped into clusters which show direct or indirect
identity with each other. Second, ESTs in the same cluster were
further grouped into sub-clusters and assembled into a consensus
sequence. The original gene sequence is compared to the consensus
sequence(s) or other full-length sequences. Each consensus sequence
is a potential splice variant for that gene (see, e.g., URL
www.doubletwist.com/products/c11_agentsOverview.jhtml). Even when a
variant is identified that is not a full-length clone, that portion
of the variant is very useful for antigen generation and for
further cloning of the full-length splice variant, using techniques
known in the art.
[0805] Moreover, computer programs are available in the art that
identify transcript variants based on genomic sequences.
Genomic-based transcript variant identification programs include
FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in
Drosophila genomic DNA," Genome Research. 2000 April; 10(4):
516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and
GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion
of splice variant identification protocols see., e.g., Southan, C.,
A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8;
498(2-3):214-8; de Souza, S. J., et al., Identification of human
chromosome 22 transcribed sequences with ORF expressed sequence
tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.
[0806] To further confirm the parameters of a transcript variant, a
variety of techniques are available in the art, such as full-length
cloning, proteomic validation, PCR-based validation, and 5' RACE
validation, etc. (see e.g., Proteomic Validation: Brennan, S. O.,
et al., Albumin banks peninsula: a new termination variant
characterized by electrospray mass spectrometry, Biochem Biophys
Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al.,
Differential splicing of pre-messenger RNA produces multiple forms
of mature caprine alpha(s1)-casein, Eur J Biochem. 1997 Oct. 1;
249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific
reverse transcription-PCR quantification of vascular endothelial
growth factor (VEGF) splice variants by LightCycler technology,
Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery
of new human beta-defensins using a genomics-based approach, Gene.
2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation:
Brigle, K. E., et al., Organization of the murine reduced folate
carrier gene and identification of variant splice forms, Biochem
Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).
[0807] It is known in the art that genomic regions are modulated in
cancers. When the genomic region to which a gene maps is modulated
in a particular cancer, the alternative transcripts or splice
variants of the gene are modulated as well. Disclosed herein is
that 158P1D7 has a particular expression profile related to cancer.
Alternative transcripts and splice variants of 158P1D7 may also be
involved in cancers in the same or different tissues, thus serving
as tumor-associated markers/antigens.
[0808] Using the full-length gene and EST sequences, four
transcript variants were identified, designated as 158P1D7 v.3,
v.4, v.5 and v.6. The boundaries of the exon in the original
transcript, 158P1D7 v.1 were shown in Table BILL-I. Compared with
158P1D7 v.1, transcript variant 158P1D7 v.3 has spliced out
2069-2395 from variant 158P1D7 v.1, as shown in FIG. 12. Variant
158P1D7 v.4 spliced out 1162-2096 of variant 158P1D7 v.1. Variant
158P1D7 v.5 added one exon to the 5' and extended 2 bp to the 5'
end and 288 bp to the 3' end of variant 158P1D7 v.1. Theoretically,
each different combination of exons in spatial order, e.g. exon 1
of v.5 and exons 1 and 2 of v.3 or v.4, is a potential splice
variant.
[0809] The variants of 158P1D7 include those that lack a
transmembrane motif, but include a signal peptide indicating that
they are secreted proteins (v.4 and v.6). Secreted proteins such as
v.4 and v.6 serve as biomarkers of cancer existence and
progression. The levels of such variant proteins in the serum of
cancer patients serves as a prognostic marker of cancer disease or
its progression, particularly of cancers such as those listed in
Table I. Moreover, such secreted proteins are targets of monoclonal
antibodies and related binding molecules. Accordingly, secreted
proteins such as these serve as targets for diagnostics,
prognostics, prophylactics and therapeutics for human malignancies.
Targeting of secreted variants of 158P1D7 is particularly preferred
when they have pathology-related or cancer-related effects on
cells/tissues.
[0810] Tables LI (a)-(d) through LIV(a)-(d) are set forth on a
variant-by-variant bases. Tables LI(a)-(d) shows nucleotide
sequence of the transcript variant. Tables LII(a)-(d) shows the
alignment of the transcript variant with nucleic acid sequence of
158P1D7 v.1. Tables LIII (a)-(d) lays out amino acid translation of
the transcript variant for the identified reading frame
orientation. Tables LIV(a)-(d) displays alignments of the amino
acid sequence encoded by the splice variant with that of 158P1D7
v.1.
Example 46
Single Nucleotide Polymorphisms of 158P1D7
[0811] A Single Nucleotide Polymorphism (SNP) is a single base pair
variation in a nucleotide sequence at a specific location. At any
given point of the genome, there are four possible nucleotide base
pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base
pair sequence of one or more locations in the genome of an
individual. Haplotype refers to the base pair sequence of more than
one location on the same DNA molecule (or the same chromosome in
higher organisms), often in the context of one gene or in the
context of several tightly linked genes. SNP that occurs on a cDNA
is called cSNP. This cSNP may change amino acids of the protein
encoded by the gene and thus change the functions of the protein.
Some SNP cause inherited diseases; others contribute to
quantitative variations in phenotype and reactions to environmental
factors including diet and drugs among individuals. Therefore, SNP
and/or combinations of alleles (called haplotypes) have many
applications, including diagnosis of inherited diseases,
determination of drug reactions and dosage, identification of genes
responsible for diseases, and analysis of the genetic relationship
between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, "SNP
analysis to dissect human traits," Curr. Opin. Neurobiol. 2001
October; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic
susceptibility to adverse drug reactions," Trends Pharmacol. Sci.
2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A.
Roses, "The use of single nucleotide polymorphisms in the isolation
of common disease genes," Pharmacogenomics. 2000 February;
1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The
predictive power of haplotypes in clinical response,"
Pharmacogenomics. 2000 feb; 1(1):15-26).
[0812] SNP are identified by a variety of art-accepted methods (P.
Bean, "The promising voyage of SNP target discovery," Am. Clin.
Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, "In search of
human variation," Genome Res. 1998 July; 8(7):691-697; M. M. She,
"Enabling large-scale pharmacogenetic studies by high-throughput
mutation detection and genotyping technologies," Clin. Chem. 2001
February; 47(2):164-172). For example, SNP can be identified by
sequencing DNA fragments that show polymorphism by gel-based
methods such as restriction fragment length polymorphism (RFLP) and
denaturing gradient gel electrophoresis (DGGE). They can also be
discovered by direct sequencing of DNA samples pooled from
different individuals or by comparing sequences from different DNA
samples. With the rapid accumulation of sequence data in public and
private databases, one can discover SNP by comparing sequences
using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, "Single
nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998;
12(4):221-225). SNP can be verified and genotype or haplotype of an
individual can be determined by a variety of methods including
direct sequencing and high throughput microarrays (P. Y. Kwok,
"Methods for genotyping single nucleotide polymorphisms," Annu.
Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.
Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A.
Duesterhoeft, "High-throughput SNP genotyping with the Masscode
system," Mol. Diagn. 2000 December; 5(4):329-340).
[0813] Using the methods described above, one SNP was identified in
the original transcript, 158P1D7 v.1, at positions 1546 (A/G). The
transcripts or proteins with alternative allele was designated as
variant 158P1D7 v.2. FIG. 17 shows the schematic alignment of the
SNP variants. FIG. 18 shows the schematic alignment of protein
variants, corresponding to nucleotide variants. Nucleotide variants
that code for the same amino acid sequence as v.1 are not shown in
FIG. 18. These alleles of the SNP, though shown separately here,
can occur in different combinations (haplotypes) and in any one of
the transcript variants (such as 158P1D7 v.5) that contains the
site of the SNP.
Example 47
Therapeutic and Diagnostic Use of Anti-158P1D7 Antibodies in
Humans
[0814] Anti-158P1D7 monoclonal antibodies are safely and
effectively used for diagnostic, prophylactic, prognostic and/or
therapeutic purposes in humans. Western blot and
immunohistochemical analysis of cancer tissues and cancer
xenografts with anti-158P1D7 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 158P1D7 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-158P1D7 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0815] As determined by flow cytometry, anti-158P1D7 mAb
specifically binds to carcinoma cells. Thus, anti-158P1D7
antibodies are used in diagnostic whole body imaging applications,
such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g.,
Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for
the detection of localized and metastatic cancers that exhibit
expression of 158P1D7. Shedding or release of an extracellular
domain of 158P1D7 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 158P1D7 by
anti-158P1D7 antibodies in serum and/or urine samples from suspect
patients.
[0816] Anti-158P1D7 antibodies that specifically bind 158P1D7 are
used in therapeutic applications for the treatment of cancers that
express 158P1D7. Anti-158P1D7 antibodies are used as an
unconjugated modality and as conjugated form in which the
antibodies are attached to one of various therapeutic or imaging
modalities well known in the art, such as a prodrugs, enzymes or
radioisotopes. In preclinical studies, unconjugated and conjugated
anti-158P1D7 antibodies are tested for efficacy of tumor prevention
and growth inhibition in the SCID mouse cancer xenograft models,
e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the
Example entitled "158P1D7 Monoclonal Antibody-mediated Inhibition
of Bladder and Lung Tumors In Vivo"). Either conjugated and
unconjugated anti-158P1D7 antibodies are used as a therapeutic
modality in human clinical trials either alone or in combination
with other treatments as described in following Examples.
Example 48
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of Human Anti-158P1D7 Antibodies In Vivo
[0817] Antibodies are used in accordance with the present invention
which recognize an epitope on 158P1D7, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 158P1D7 expression levels,
tumors such as those listed in Table I are presently preferred
indications. In connection with each of these indications, three
clinical approaches are successfully pursued.
[0818] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-158P1D7 antibodies in combination with a
chemotherapeutic or antineoplastic agent and/or radiation therapy.
Primary cancer targets, such as those listed in Table I, are
treated under standard protocols by the addition anti-158P1D7
antibodies to standard first and second line therapy. Protocol
designs address effectiveness as assessed by reduction in tumor
mass as well as the ability to reduce usual doses of standard
chemotherapy. These dosage reductions allow additional and/or
prolonged therapy by reducing dose-related toxicity of the
chemotherapeutic agent. Anti-158P1D7 antibodies are utilized in
several adjunctive clinical trials in combination with the
chemotherapeutic or antineoplastic agents adriamycin (advanced
prostrate carcinoma), cisplatin (advanced head and neck and lung
carcinomas), taxol (breast cancer), and doxorubicin
(preclinical).
[0819] II.) Monotherapy: In connection with the use of the
anti-158P1D7 antibodies in monotherapy of tumors, the antibodies
are administered to patients without a chemotherapeutic or
antineoplastic agent. In one embodiment, monotherapy is conducted
clinically in end stage cancer patients with extensive metastatic
disease. Patients show some disease stabilization. Trials
demonstrate an effect in refractory patients with cancerous
tumors.
[0820] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-158P1D7 antibodies,
the radiolabeled antibodies are utilized as a diagnostic and/or
imaging agent. In such a role, the labeled antibodies localize to
both solid tumors, as well as, metastatic lesions of cells
expressing 158P1D7. In connection with the use of the anti-158P1D7
antibodies as imaging agents, the antibodies are used as an adjunct
to surgical treatment of solid tumors, as both a pre-surgical
screen as well as a post-operative follow-up to determine what
tumor remains and/or returns. In one embodiment, a
(.sup.111In)-158P1D7 antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses 158P1D7 (by analogy see, e.g., Divgi et al. J. Natl.
Cancer Inst. 83:97-104 (1991)). Patients are followed with standard
anterior and posterior gamma camera. The results indicate that
primary lesions and metastatic lesions are identified.
[0821] Dose and Route of Administration
[0822] As appreciated by those of ordinary skill in the art, dosing
considerations can be determined through comparison with the
analogous products that are in the clinic. Thus, anti-158P1D7
antibodies can be administered with doses in the range of 5 to 400
mg/m.sup.2, with the lower doses used, e.g., in connection with
safety studies. The affinity of anti-158P1D7 antibodies relative to
the affinity of a known antibody for its target is one parameter
used by those of skill in the art for determining analogous dose
regimens. Further, anti-158P1D7 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-158P1D7 antibodies can be lower, perhaps in the range of 50 to
300 mg/m.sup.2, and still remain efficacious. Dosing in mg/m.sup.2,
as opposed to the conventional measurement of dose in mg/kg, is a
measurement based on surface area and is a convenient dosing
measurement that is designed to include patients of all sizes from
infants to adults.
[0823] Three distinct delivery approaches are useful for delivery
of anti-158P1D7 antibodies. Conventional intravenous delivery is
one standard delivery technique for many tumors. However, in
connection with tumors in the peritoneal cavity, such as tumors of
the ovaries, biliary duct, other ducts, and the like,
intraperitoneal administration may prove favorable for obtaining
high dose of antibody at the tumor and to also minimize antibody
clearance. In a similar manner, certain solid tumors possess
vasculature that is appropriate for regional perfusion. Regional
perfusion allows for a high dose of antibody at the site of a tumor
and minimizes short term clearance of the antibody.
[0824] Clinical Development Plan (CDP)
[0825] Overview: The CDP follows and develops treatments of
anti-158P1D7 antibodies in connection with adjunctive therapy,
monotherapy, and as an imaging agent. Trials initially demonstrate
safety and thereafter confirm efficacy in repeat doses. Trails are
open label comparing standard chemotherapy with standard therapy
plus anti-158P1D7 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
158P1D7 expression levels in their tumors as determined by
biopsy.
[0826] As with any protein or antibody infusion-based therapeutic,
safety concerns are related primarily to (i) cytokine release
syndrome, i.e., hypotension, fever, shaking, chills; (ii) the
development of an immunogenic response to the material (i.e.,
development of human antibodies by the patient to the antibody
therapeutic, or HAHA response); and, (iii) toxicity to normal cells
that express 158P1D7. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-158P1D7 antibodies are
found to be safe upon human administration.
Example 49
Human Clinical Trial Adjunctive Therapy with Human Anti-158P1D7
Antibody and Chemotherapeutic Agent
[0827] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-158P1D7 antibody in
connection with the treatment of a solid tumor, e.g., a cancer of a
tissue listed in Table I. In the study, the safety of single doses
of anti-158P1D7 antibodies when utilized as an adjunctive therapy
to an antineoplastic or chemotherapeutic agent as defined herein,
such as, without limitation: cisplatin, topotecan, doxorubicin,
adriamycin, taxol, or the like, is assessed. The trial design
includes delivery of six single doses of an anti-158P1D7 antibody
with dosage of antibody escalating from approximately about 25
mg/m.sup.2 to about 275 mg/m.sup.2 over the course of the treatment
in accordance with the following schedule:
TABLE-US-00003 Day Day Day Day Day 0 Day 7 14 21 28 35 mAb Dose 25
mg/m.sup.2 75 mg/m.sup.2 125 mg/m.sup.2 175 mg/m.sup.2 225
mg/m.sup.2 275 mg/m.sup.2 Chemotherapy + + + + + + (standard
dose)
[0828] Patients are closely followed for one-week following each
administration of antibody and chemotherapy. In particular,
patients are assessed for the safety concerns mentioned above: (i)
cytokine release syndrome, i.e., hypotension, fever, shaking,
chills; (ii) the development of an immunogenic response to the
material (i.e., development of human antibodies by the patient to
the human antibody therapeutic, or HAHA response); and, (iii)
toxicity to normal cells that express 158P1D7. Standard tests and
follow-up are utilized to monitor each of these safety concerns.
Patients are also assessed for clinical outcome, and particularly
reduction in tumor mass as evidenced by MRI or other imaging.
[0829] The anti-158P1D7 antibodies are demonstrated to be safe and
efficacious, Phase II trials confirm the efficacy and refine
optimum dosing.
Example 50
Human Clinical Trial
Monotherapy with Human Anti-158P1D7 Antibody
[0830] Anti-158P1D7 antibodies are safe in connection with the
above-discussed adjunctive trial, a Phase II human clinical trial
confirms the efficacy and optimum dosing for monotherapy. Such
trial is accomplished, and entails the same safety and outcome
analyses, to the above-described adjunctive trial with the
exception being that patients do not receive chemotherapy
concurrently with the receipt of doses of anti-158P1D7
antibodies.
Example 51
Human Clinical Trial
Diagnostic Imaging with Anti-158P1D7 Antibody
[0831] Once again, as the adjunctive therapy discussed above is
safe within the safety criteria discussed above, a human clinical
trial is conducted concerning the use of anti-158P1D7 antibodies as
a diagnostic imaging agent. The protocol is designed in a
substantially similar manner to those described in the art, such as
in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The
antibodies are found to be both safe and efficacious when used as a
diagnostic modality.
Example 52
RNA Interference (RNAi)
[0832] RNA interference (RNAi) technology is implemented to a
variety of cell assays relevant to oncology. RNAi is a
post-transcriptional gene silencing mechanism activated by
double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation
leading to changes in protein expression and subsequently in gene
function. In mammalian cells, these dsRNAs called short interfering
RNA (siRNA) have the correct composition to activate the RNAi
pathway targeting for degradation, specifically some mRNAs. See,
Elbashir S. M., et. al., Duplexes of 21-nucleotide RNAs Mediate RNA
interference in Cultured Mammalian Cells, Nature 411(6836):494-8
(2001). Thus, RNAi technology is used successfully in mammalian
cells to silence targeted genes.
[0833] Loss of cell proliferation control is a hallmark of
cancerous cells; thus, assessing the role of 158P1D7 in cell
survival/proliferation assays is relevant. Accordingly, RNAi was
used to investigate the function of the 158P1D7 antigen. To
generate siRNA for 158P1D7, algorithms were used that predict
oligonucleotides that exhibit the critical molecular parameters
(G:C content, melting temperature, etc.) and have the ability to
significantly reduce the expression levels of the 158P1D7 protein
when introduced into cells. Accordingly, one targeted sequence for
the 158P1D7 siRNA is: 5' AAGCTCATTCTAGCGGGAAAT 3' (SEQ ID NO: 42)
(oligo 158P1D7.b). In accordance with this Example, 158P1D7 siRNA
compositions are used that comprise siRNA (double stranded, short
interfering RNA) that correspond to the nucleic acid ORF sequence
of the 158P1D7 protein or subsequences thereof. Thus, siRNA
subsequences are used in this manner are generally 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA
nucleotides in length. These siRNA sequences are complementary and
non-complementary to at least a portion of the mRNA coding
sequence. In a preferred embodiment, the subsequences are 19-25
nucleotides in length, most preferably 21-23 nucleotides in length.
In preferred embodiments, these siRNA achieve knockdown of 158P1D7
antigen in cells expressing the protein and have functional effects
as described below.
[0834] The selected siRNA (158P1D7.b oligo) was tested in numerous
cell lines in the survival/proliferation MTS assay (measures
cellular metabolic activity). Tetrazolium-based colorimetric assays
(i.e., MTS) detect viable cells exclusively, since living cells are
metabolically active and therefore can reduce tetrazolium salts to
colored formazan compounds; dead cells, however do not. Moreover,
this 158P1D7.b oligo achieved knockdown of 158P1D7 antigen in cells
expressing the protein and had functional effects as described
below using the following protocols.
[0835] Mammalian siRNA Transfections:
[0836] The day before siRNA transfection, the different cell lines
were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at
2.times.10.sup.3 cells/well in 80 .mu.l (96 well plate format) for
the survival/MTS assay. In parallel with the 158P1D7 specific siRNA
oligo, the following sequences were included in every experiment as
controls: a) Mock transfected cells with Lipofectamine 2000
(Invitrogen, Carlsbad, Calif.) and annealing buffer (no siRNA); b)
Luciferase-4 specific siRNA (targeted sequence:
5'-AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO: 43); and, c) Eg5
specific siRNA (targeted sequence: 5'-AACTGAAGACCTGAAGACAATAA-3')
(SEQ ID NO: 44). SiRNAs were used at 10 nM and 1 .mu.g/ml
Lipofectamine 2000 final concentration.
[0837] The procedure was as follows: The siRNAs were first diluted
in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 uM
.mu.M (10-fold concentrated) and incubated 5-10 min RT.
Lipofectamine 2000 was diluted at 10 .mu.g/ml (10-fold
concentrated) for the total number transfections and incubated 5-10
minutes at room temperature (RT). Appropriate amounts of diluted
10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted
10-fold concentrated siRNA and incubated at RT for 20-30'' (5-fold
concentrated transfection solution). 20 .mu.ls of the 5-fold
concentrated transfection solutions were added to the respective
samples and incubated at 37.degree. C. for 96 hours before
analysis.
[0838] MTS Assay:
[0839] The MTS assay is a colorimetric method for determining the
number of viable cells in proliferation, cytotoxicity or
chemosensitivity assays based on a tetrazolium compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium, inner salt; MTS(b)] and an electron coupling
reagent (phenazine ethosulfate; PES). Assays were performed by
adding a small amount of the Solution Reagent directly to culture
wells, incubating for 1-4 hours and then recording absorbance at
490 nm with a 96-well plate reader. The quantity of colored
formazan product as measured by the amount of 490 nm absorbance is
directly proportional to the mitochondrial activity and/or the
number of living cells in culture.
[0840] In order to address the function of 158P1D7 in cells,
158P1D7 was silenced by transfecting the endogenously expressing
158P1D7 cell lines (LNCaP and PC3) with the 158P1D7 specific siRNA
(158P1D7.b) along with negative siRNA controls (Luc4, targeted
sequence not represented in the human genome) and a positive siRNA
control (targeting Eg5) (FIG. 29). The results indicated that when
these cells are treated with siRNA specifically targeting the
158P1D7 mRNA, the resulting "158P1D7 deficient cells" showed
diminished cell viability or proliferation as measured by this
assay (see oligo 158P1D7.b treated cells). This effect is likely
caused by an active induction of apoptosis. The reduced viability
is measured by the increased release (and activity) of a
mitochondrial enzyme that occurs predominantly in apoptotic
cells.
[0841] As control, 3T3 cells, a cell line with no detectable
expression of 158P1D7 mRNA, was also treated with the panel of
siRNAs (including oligo 158P1D7.b) and no phenotype was observed.
This result reflects the fact that the specific protein knockdown
in the LNCaP and PC3 cells is not a function of general toxicity,
since the 3T3 cells did not respond to the 158P1D7.b oligo. The
differential response of the three cell lines to the Eg5 control is
a reflection of differences in levels of cell transfection and
responsiveness of the cell lines to oligo treatment (FIG. 29).
[0842] Together, these data indicate that 158P1D7 plays an
important role in the proliferation of cancer cells and that the
lack of 158P1D7 clearly decreases the survival potential of these
cells. It is to be noted that 158P1D7 is constitutively expressed
in many tumor cell lines. 158P1D7 serves a role in malignancy; it
expression is a primary indicator of disease, where such disease is
often characterized by high rates of uncontrolled cell
proliferation and diminished apoptosis. Correlating cellular
phenotype with gene knockdown following RNAi treatments is
important, and allows one to draw valid conclusions and rule out
toxicity or other non-specific effects of these reagents. To this
end, assays to measure the levels of expression of both protein and
mRNA for the target after RNAi treatments are important, including
Western blotting, FACS staining with antibody, immunoprecipitation,
Northern blotting or RT-PCR (Taqman or standard methods). Any
phenotypic effect of the siRNAs in these assays should be
correlated with the protein and/or mRNA knockdown levels in the
same cell lines. Knockdown of 158P1D7 is achieved using the
158P1D7.b oligo as measured by Western blotting and RT-PCR
analysis.
[0843] A method to analyze 158P1D7 related cell proliferation is
the measurement of DNA synthesis as a marker for proliferation.
Labeled DNA precursors (i.e. .sup.3H-Thymidine) are used and their
incorporation to DNA is quantified. Incorporation of the labeled
precursor into DNA is directly proportional to the amount of cell
division occurring in the culture. Another method used to measure
cell proliferation is performing clonogenic assays. In these
assays, a defined number of cells are plated onto the appropriate
matrix and the number of colonies formed after a period of growth
following siRNA treatment is counted.
[0844] In 158P1D7 cancer target validation, complementing the cell
survival/proliferation analysis with apoptosis and cell cycle
profiling studies are considered. The biochemical hallmark of the
apoptotic process is genomic DNA fragmentation, an irreversible
event that commits the cell to die. A method to observe fragmented
DNA in cells is the immunological detection of histone-complexed
DNA fragments by an immunoassay (i.e. cell death detection ELISA)
which measures the enrichment of histone-complexed DNA fragments
(mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells.
This assay does not require pre-labeling of the cells and can
detect DNA degradation in cells that do not proliferate in vitro
(i.e. freshly isolated tumor cells).
[0845] The most important effector molecules for triggering
apoptotic cell death are caspases. Caspases are proteases that when
activated cleave numerous substrates at the carboxy-terminal site
of an aspartate residue mediating very early stages of apoptosis
upon activation. All caspases are synthesized as pro-enzymes and
activation involves cleavage at aspartate residues. In particular,
caspase 3 seems to play a central role in the initiation of
cellular events of apoptosis. Assays for determination of caspase 3
activation detect early events of apoptosis. Following RNAi
treatments, Western blot detection of active caspase 3 presence or
proteolytic cleavage of products (i.e. PARP) found in apoptotic
cells further support an active induction of apoptosis. Because the
cellular mechanisms that result in apoptosis are complex, each has
its advantages and limitations. Consideration of other
criteria/endpoints such as cellular morphology, chromatin
condensation, membrane blebbing, apoptotic bodies help to further
support cell death as apoptotic. Since not all the gene targets
that regulate cell growth are anti-apoptotic, the DNA content of
permeabilized cells is measured to obtain the profile of DNA
content or cell cycle profile. Nuclei of apoptotic cells contain
less DNA due to the leaking out to the cytoplasm (sub-G1
population). In addition, the use of DNA stains (i.e., propidium
iodide) also differentiate between the different phases of the cell
cycle in the cell population due to the presence of different
quantities of DNA in G0/G1, S and G2/M. In these studies the
subpopulations can be quantified.
[0846] For the 158P1D7 gene, RNAi studies facilitate the
understanding of the contribution of the gene product in cancer
pathways. Such active RNAi molecules have use in identifying assays
to screen for mAbs that are active anti-tumor therapeutics.
Further, siRNA are administered as therapeutics to cancer patients
for reducing the malignant growth of several cancer types,
including those listed in Table 1. When 158P1D7 plays a role in
cell survival, cell proliferation, tumorigenesis, or apoptosis, it
is used as a target for diagnostic, prognostic, preventative and/or
therapeutic purposes
Example 53
158P1D7 Functional Assays
[0847] I. Enhanced Proliferation and Cell Cycle Modulation in
158P1D7 Expressing Cells.
[0848] Enhanced proliferation and entry into S-phase of tumor cells
relative to normal cells is a hallmark of the cancer cell
phenotype. To address the effect of expression of 158P1D7 on the
proliferation rate of normal cells, two rodent cell lines (3T3 and
Rat-1) were infected with virus containing the 158P1D7 gene and
stable cells expressing 158P1D7 antigen were derived, as well as
empty vector control cells expressing the selection marker neomycin
(Neo). The cells were grown overnight in 0.5% FBS and then compared
to cells treated with 10% FBS. The cells were evaluated for
proliferation at 18-96 hr post-treatment by a .sup.3H-thymidine
incorporation assay and for cell cycle analysis by a BrdU
incorporation/propidium iodide staining assay. The results in FIG.
32 show that the Rat-1 cells expressing the 158P1D7 antigen grew
effectively in low serum concentrations (0.1%) compared to the
Rat-1-Neo cells. Similar results were obtained for the 3T3 cells
expressing 158P1D7 versus Neo only. To assess cell proliferation by
another methodology, the cells were stained with BrdU and propidium
iodide. Briefly, cells were labeled with 10 .mu.M BrdU, washed,
trypsinized and fixed in 0.4% paraformaldehyde and 70% ethanol.
Anti-BrdU-FITC (Pharmigen) was added to the cells, the cells were
washed and then incubated with 10 .mu.g/ml propidium iodide for 20
min prior to washing and analysis for fluorescence at 488 nm. The
results in FIG. 33 show that there was increased labeling of cells
in S-phase (DNA synthesis phase of the cell cycle) in 3T3 cells
that expressed the 158P1D7 antigen relative to control cells. These
results confirm those measured by .sup.3H-thymidine incorporation,
and indicate that cells that express 158P1D7 antigen have an
enhanced proliferative capacity and survive in low serum
conditions. Accordingly, 158P1D7 expressing cells have increased
potential for growth as tumor cells in vivo.
[0849] II. Recombinant Extracellular Domain (ECD) Binding to Cell
Surface.
[0850] Cell-cell interactions are essential in maintaining
tissue/organ integrity and homeostasis, both of which become
deregulated during tumor formation and progression. Additionally,
cell-cell interactions facilitate tumor cell attachment during
metastasis and activation of endothelium for increased
angiogenesis. To address interaction between the gene product of
158P1D7 and a putative ligand, an assay was established to identify
the interaction between the extracellular domain (ECD) (amino acids
16-608) of 158P1D7 antigen and primary endothelium. Human umbilical
vein endothelial cells (HUVEC) were grown in 0.1% FBS in media for
3 hr. Cells were washed, detached in 10 mM EDTA and resuspended in
10% FBS. Recombinant 158P1D7 ECD (described in Example entitled
"Production of Recombinant 158P1D7 in Eukaryotic Systems") was
added to cells, and the cells were washed prior to the addition of
MAb M15/X68.2.22 at 1 ug/ml. After washing, secondary Ab
(anti-mouse-PE, 1:400) was added to cells for 1 hr on ice. Cells
were washed and fixed in 1% formalin for 3 hr on ice, then
resuspended in PBS and analyzed by flow cytometry. FIG. 26A shows
that the 158P1D7 ECD bound directly to the surface of HUVEC cells
as detected by the 158P1D7 specific MAb. In a similar embodiment,
recombinant ECD of 158P1D7 was iodinated to high specific activity
using the iodogen (1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril)
method. HUVEC cells at 90% confluency in 6 well plates were
incubated with 1 nM of .sup.125I-158P1D7 ECD in the presence
(non-specific binding) or absence (Total binding) of 50 fold excess
unlabeled ECD for 2 hours at either 4.degree. C. or 37.degree. C.
Cells were washed, solubilized in 0.5M NaOH, and subjected to gamma
counting. The data in FIG. 26B shows specific binding of 158P1D7
ECD to HUVEC cells suggesting the presence of a 158P1D7 receptor on
HUVEC cells. These results indicate that 158P1D7 antigen is
involved in cell-cell interactions that facilitate tumor growth,
activation of endothelium for tumor vascularization or tumor cell
metastasis. The data also indicate that 158P1D7 antigen shed from
the cell surface of expressing cells may bind to cells in an
autocrine or paracrine fashion to induce cell effector
functions.
Example 54
Detection of 158P1D7 Protein in Cancer Patient Specimens Using
Immunohistochemistry
[0851] To determine the expression of 158P1D7 protein, specimens
were obtained from various cancer patients and stained using an
affinity purified monoclonal antibody raised against the peptide
encoding amino acids 274-285 of 158P1D7 (See the Example Entitled
"Generation of 158P1D7 Monoclonal Antibodies (mAbs)"), formalin
fixed, paraffin embedded tissues were cut into 4 micron sections
and mounted on glass slides. The sections were dewaxed, rehydrated
and treated with antigen retrieval solution (Antigen Retrieval
Citra Solution; BioGenex, 4600 Norris Canyon Road, San Ramon,
Calif., 94583) at high temperature. Sections were then incubated in
mouse monoclonal anti-158P1D7 antibody, M15-68(2)22, for 3 hours.
The slides were washed three times in buffer and further incubated
with DAKO EnVision+.TM. peroxidase-conjugated goat anti-mouse
immunoglobulin secondary antibody (DAKO Corporation, Carpenteria,
Calif.) for 1 hour. The sections were then washed in buffer,
developed using the DAB kit (SIGMA Chemicals), counterstained using
hematoxylin, and analyzed by bright field microscopy. The results
showed expression of 158P1D7 in cancer patients' tissue (FIG. 36).
Generally, in bladder transitional cell carcinoma expression of
158P1D7 was mainly around the cell membrane indicating that 158P1D7
is membrane associated in these tissues. 49.3% of bladder
transitional cell carcinoma samples tested were positive for
158P1D7 (Table LVIII).
[0852] These results indicate that 158P1D7 is a target for
diagnostic, prophylactic, prognostic and therapeutic applications
in cancer.
[0853] The present invention is not to be limited in scope by the
embodiments disclosed herein, which are intended as single
illustrations of individual aspects of the invention, and any that
are functionally equivalent are within the scope of the invention.
Various modifications to the models and methods of the invention,
in addition to those described herein, will become apparent to
those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall within the scope of
the invention. Such modifications or other embodiments can be
practiced without departing from the true scope and spirit of the
invention.
[0854] All documents and publications recited herein are hereby
incorporated in their entirety as if fully set forth.
Sequence CWU 1
1
1131231DNAHomo sapiens 1gatctgataa gctttcaatg ttgcgctcct gacaatgtat
tagaagtcct gatggggata 60ggactttgca gttacaagga atagggcaga aaggtcctgg
aagttgagtg gatggctttg 120taatataagg tatcaaacct ggtgctttgg
tgggtagttt tagaatggac gtggtcttag 180ttgacatgcg actatcattt
attgaagatg ttgctgccag atgtaatgat c 23122555DNAHomo
sapiensCDS(23)...(2548) 2tcggatttca tcacatgaca ac atg aag ctg tgg
att cat ctc ttt tat tca 52 Met Lys Leu Trp Ile His Leu Phe Tyr Ser
1 5 10tct ctc ctt gcc tgt ata tct tta cac tcc caa act cca gtg ctc
tca 100Ser Leu Leu Ala Cys Ile Ser Leu His Ser Gln Thr Pro Val Leu
Ser 15 20 25tcc aga ggc tct tgt gat tct ctt tgc aat tgt gag gaa aaa
gat ggc 148Ser Arg Gly Ser Cys Asp Ser Leu Cys Asn Cys Glu Glu Lys
Asp Gly 30 35 40aca atg cta ata aat tgt gaa gca aaa ggt atc aag atg
gta tct gaa 196Thr Met Leu Ile Asn Cys Glu Ala Lys Gly Ile Lys Met
Val Ser Glu45 50 55ata agt gtg cca cca tca cga cct ttc caa cta agc
tta tta aat aac 244Ile Ser Val Pro Pro Ser Arg Pro Phe Gln Leu Ser
Leu Leu Asn Asn60 65 70ggc ttg acg atg ctt cac aca aat gac ttt tct
ggg ctt acc aat gct 292Gly Leu Thr Met Leu His Thr Asn Asp Phe Ser
Gly Leu Thr Asn Ala75 80 85 90att tca ata cac ctt gga ttt aac aat
att gca gat att gag ata ggt 340Ile Ser Ile His Leu Gly Phe Asn Asn
Ile Ala Asp Ile Glu Ile Gly 95 100 105gca ttt aat ggc ctt ggc ctc
ctg aaa caa ctt cat atc aat cac aat 388Ala Phe Asn Gly Leu Gly Leu
Leu Lys Gln Leu His Ile Asn His Asn 110 115 120tct tta gaa att ctt
aaa gag gat act ttc cat gga ctg gaa aac ctg 436Ser Leu Glu Ile Leu
Lys Glu Asp Thr Phe His Gly Leu Glu Asn Leu 125 130 135gaa ttc ctg
caa gca gat aac aat ttt atc aca gtg att gaa cca agt 484Glu Phe Leu
Gln Ala Asp Asn Asn Phe Ile Thr Val Ile Glu Pro Ser 140 145 150gcc
ttt agc aag ctc aac aga ctc aaa gtg tta att tta aat gac aat 532Ala
Phe Ser Lys Leu Asn Arg Leu Lys Val Leu Ile Leu Asn Asp Asn155 160
165 170gct att gag agt ctt cct cca aac atc ttc cga ttt gtt cct tta
acc 580Ala Ile Glu Ser Leu Pro Pro Asn Ile Phe Arg Phe Val Pro Leu
Thr 175 180 185cat cta gat ctt cgt gga aat caa tta caa aca ttg cct
tat gtt ggt 628His Leu Asp Leu Arg Gly Asn Gln Leu Gln Thr Leu Pro
Tyr Val Gly 190 195 200ttt ctc gaa cac att ggc cga ata ttg gat ctt
cag ttg gag gac aac 676Phe Leu Glu His Ile Gly Arg Ile Leu Asp Leu
Gln Leu Glu Asp Asn 205 210 215aaa tgg gcc tgc aat tgt gac tta ttg
cag tta aaa act tgg ttg gag 724Lys Trp Ala Cys Asn Cys Asp Leu Leu
Gln Leu Lys Thr Trp Leu Glu 220 225 230aac atg cct cca cag tct ata
att ggt gat gtt gtc tgc aac agc cct 772Asn Met Pro Pro Gln Ser Ile
Ile Gly Asp Val Val Cys Asn Ser Pro235 240 245 250cca ttt ttt aaa
gga agt ata ctc agt aga cta aag aag gaa tct att 820Pro Phe Phe Lys
Gly Ser Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile 255 260 265tgc cct
act cca cca gtg tat gaa gaa cat gag gat cct tca gga tca 868Cys Pro
Thr Pro Pro Val Tyr Glu Glu His Glu Asp Pro Ser Gly Ser 270 275
280tta cat ctg gca gca aca tct tca ata aat gat agt cgc atg tca act
916Leu His Leu Ala Ala Thr Ser Ser Ile Asn Asp Ser Arg Met Ser Thr
285 290 295aag acc acg tcc att cta aaa cta ccc acc aaa gca cca ggt
ttg ata 964Lys Thr Thr Ser Ile Leu Lys Leu Pro Thr Lys Ala Pro Gly
Leu Ile 300 305 310cct tat att aca aag cca tcc act caa ctt cca gga
cct tac tgc cct 1012Pro Tyr Ile Thr Lys Pro Ser Thr Gln Leu Pro Gly
Pro Tyr Cys Pro315 320 325 330att cct tgt aac tgc aaa gtc cta tcc
cca tca gga ctt cta ata cat 1060Ile Pro Cys Asn Cys Lys Val Leu Ser
Pro Ser Gly Leu Leu Ile His 335 340 345tgt cag gag cgc aac att gaa
agc tta tca gat ctg aga cct cct ccg 1108Cys Gln Glu Arg Asn Ile Glu
Ser Leu Ser Asp Leu Arg Pro Pro Pro 350 355 360caa aat cct aga aag
ctc att cta gcg gga aat att att cac agt tta 1156Gln Asn Pro Arg Lys
Leu Ile Leu Ala Gly Asn Ile Ile His Ser Leu 365 370 375atg aag tct
gat cta gtg gaa tat ttc act ttg gaa atg ctt cac ttg 1204Met Lys Ser
Asp Leu Val Glu Tyr Phe Thr Leu Glu Met Leu His Leu 380 385 390gga
aac aat cgt att gaa gtt ctt gaa gaa gga tcg ttt atg aac cta 1252Gly
Asn Asn Arg Ile Glu Val Leu Glu Glu Gly Ser Phe Met Asn Leu395 400
405 410acg aga tta caa aaa ctc tat cta aat ggt aac cac ctg acc aaa
tta 1300Thr Arg Leu Gln Lys Leu Tyr Leu Asn Gly Asn His Leu Thr Lys
Leu 415 420 425agt aaa ggc atg ttc ctt ggt ctc cat aat ctt gaa tac
tta tat ctt 1348Ser Lys Gly Met Phe Leu Gly Leu His Asn Leu Glu Tyr
Leu Tyr Leu 430 435 440gaa tac aat gcc att aag gaa ata ctg cca gga
acc ttt aat cca atg 1396Glu Tyr Asn Ala Ile Lys Glu Ile Leu Pro Gly
Thr Phe Asn Pro Met 445 450 455cct aaa ctt aaa gtc ctg tat tta aat
aac aac ctc ctc caa gtt tta 1444Pro Lys Leu Lys Val Leu Tyr Leu Asn
Asn Asn Leu Leu Gln Val Leu 460 465 470cca cca cat att ttt tca ggg
gtt cct cta act aag gta aat ctt aaa 1492Pro Pro His Ile Phe Ser Gly
Val Pro Leu Thr Lys Val Asn Leu Lys475 480 485 490aca aac cag ttt
acc cat cta cct gta agt aat att ttg gat gat ctt 1540Thr Asn Gln Phe
Thr His Leu Pro Val Ser Asn Ile Leu Asp Asp Leu 495 500 505gat tta
cta acc cag att gac ctt gag gat aac ccc tgg gac tgc tcc 1588Asp Leu
Leu Thr Gln Ile Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser 510 515
520tgt gac ctg gtt gga ctg cag caa tgg ata caa aag tta agc aag aac
1636Cys Asp Leu Val Gly Leu Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn
525 530 535aca gtg aca gat gac atc ctc tgc act tcc ccc ggg cat ctc
gac aaa 1684Thr Val Thr Asp Asp Ile Leu Cys Thr Ser Pro Gly His Leu
Asp Lys 540 545 550aag gaa ttg aaa gcc cta aat agt gaa att ctc tgt
cca ggt tta gta 1732Lys Glu Leu Lys Ala Leu Asn Ser Glu Ile Leu Cys
Pro Gly Leu Val555 560 565 570aat aac cca tcc atg cca aca cag act
agt tac ctt atg gtc acc act 1780Asn Asn Pro Ser Met Pro Thr Gln Thr
Ser Tyr Leu Met Val Thr Thr 575 580 585cct gca aca aca aca aat acg
gct gat act att tta cga tct ctt acg 1828Pro Ala Thr Thr Thr Asn Thr
Ala Asp Thr Ile Leu Arg Ser Leu Thr 590 595 600gac gct gtg cca ctg
tct gtt cta ata ttg gga ctt ctg att atg ttc 1876Asp Ala Val Pro Leu
Ser Val Leu Ile Leu Gly Leu Leu Ile Met Phe 605 610 615atc act att
gtt ttc tgt gct gca ggg ata gtg gtt ctt gtt ctt cac 1924Ile Thr Ile
Val Phe Cys Ala Ala Gly Ile Val Val Leu Val Leu His 620 625 630cgc
agg aga aga tac aaa aag aaa caa gta gat gag caa atg aga gac 1972Arg
Arg Arg Arg Tyr Lys Lys Lys Gln Val Asp Glu Gln Met Arg Asp635 640
645 650aac agt cct gtg cat ctt cag tac agc atg tat ggc cat aaa acc
act 2020Asn Ser Pro Val His Leu Gln Tyr Ser Met Tyr Gly His Lys Thr
Thr 655 660 665cat cac act act gaa aga ccc tct gcc tca ctc tat gaa
cag cac atg 2068His His Thr Thr Glu Arg Pro Ser Ala Ser Leu Tyr Glu
Gln His Met 670 675 680gtg agc ccc atg gtt cat gtc tat aga agt cca
tcc ttt ggt cca aag 2116Val Ser Pro Met Val His Val Tyr Arg Ser Pro
Ser Phe Gly Pro Lys 685 690 695cat ctg gaa gag gaa gaa gag agg aat
gag aaa gaa gga agt gat gca 2164His Leu Glu Glu Glu Glu Glu Arg Asn
Glu Lys Glu Gly Ser Asp Ala 700 705 710aaa cat ctc caa aga agt ctt
ttg gaa cag gaa aat cat tca cca ctc 2212Lys His Leu Gln Arg Ser Leu
Leu Glu Gln Glu Asn His Ser Pro Leu715 720 725 730aca ggg tca aat
atg aaa tac aaa acc acg aac caa tca aca gaa ttt 2260Thr Gly Ser Asn
Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe 735 740 745tta tcc
ttc caa gat gcc agc tca ttg tac aga aac att tta gaa aaa 2308Leu Ser
Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn Ile Leu Glu Lys 750 755
760gaa agg gaa ctt cag caa ctg gga atc aca gaa tac cta agg aaa aac
2356Glu Arg Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn
765 770 775att gct cag ctc cag cct gat atg gag gca cat tat cct gga
gcc cac 2404Ile Ala Gln Leu Gln Pro Asp Met Glu Ala His Tyr Pro Gly
Ala His 780 785 790gaa gag ctg aag tta atg gaa aca tta atg tac tca
cgt cca agg aag 2452Glu Glu Leu Lys Leu Met Glu Thr Leu Met Tyr Ser
Arg Pro Arg Lys795 800 805 810gta tta gtg gaa cag aca aaa aat gag
tat ttt gaa ctt aaa gct aat 2500Val Leu Val Glu Gln Thr Lys Asn Glu
Tyr Phe Glu Leu Lys Ala Asn 815 820 825tta cat gct gaa cct gac tat
tta gaa gtc ctg gag cag caa aca tag 2548Leu His Ala Glu Pro Asp Tyr
Leu Glu Val Leu Glu Gln Gln Thr * 830 835 840atggaga
25553841PRTHomo sapiens 3Met Lys Leu Trp Ile His Leu Phe Tyr Ser
Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val
Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu
Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly
Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro
Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80
Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85
90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu
Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu
Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu
Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro
Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile
Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe
Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln
Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205
Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210
215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
42555DNAHomo sapiensCDS(23)...(2548) 4tcggatttca tcacatgaca ac atg
aag ctg tgg att cat ctc ttt tat tca 52 Met Lys Leu Trp Ile His Leu
Phe Tyr Ser 1 5 10tct ctc ctt gcc tgt ata tct tta cac tcc caa act
cca gtg ctc tca 100Ser Leu Leu Ala Cys Ile Ser Leu His Ser Gln Thr
Pro Val Leu Ser 15 20 25tcc aga ggc tct tgt gat tct ctt tgc aat tgt
gag gaa aaa gat ggc 148Ser Arg Gly Ser Cys Asp Ser Leu Cys Asn Cys
Glu Glu Lys Asp Gly 30 35 40aca atg cta ata aat tgt gaa gca aaa ggt
atc aag atg gta tct gaa 196Thr Met Leu Ile Asn Cys Glu Ala Lys Gly
Ile Lys Met Val Ser Glu 45 50 55ata agt gtg cca cca tca cga cct ttc
caa cta agc tta tta aat aac 244Ile Ser Val Pro Pro Ser Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn 60 65 70ggc ttg acg atg ctt cac aca aat
gac ttt tct ggg ctt acc aat gct 292Gly Leu Thr Met Leu His Thr Asn
Asp Phe Ser Gly Leu Thr Asn Ala75
80 85 90att tca ata cac ctt gga ttt aac aat att gca gat att gag ata
ggt 340Ile Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile
Gly 95 100 105gca ttt aat ggc ctt ggc ctc ctg aaa caa ctt cat atc
aat cac aat 388Ala Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile
Asn His Asn 110 115 120tct tta gaa att ctt aaa gag gat act ttc cat
gga ctg gaa aac ctg 436Ser Leu Glu Ile Leu Lys Glu Asp Thr Phe His
Gly Leu Glu Asn Leu 125 130 135gaa ttc ctg caa gca gat aac aat ttt
atc aca gtg att gaa cca agt 484Glu Phe Leu Gln Ala Asp Asn Asn Phe
Ile Thr Val Ile Glu Pro Ser 140 145 150gcc ttt agc aag ctc aac aga
ctc aaa gtg tta att tta aat gac aat 532Ala Phe Ser Lys Leu Asn Arg
Leu Lys Val Leu Ile Leu Asn Asp Asn155 160 165 170gct att gag agt
ctt cct cca aac atc ttc cga ttt gtt cct tta acc 580Ala Ile Glu Ser
Leu Pro Pro Asn Ile Phe Arg Phe Val Pro Leu Thr 175 180 185cat cta
gat ctt cgt gga aat caa tta caa aca ttg cct tat gtt ggt 628His Leu
Asp Leu Arg Gly Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly 190 195
200ttt ctc gaa cac att ggc cga ata ttg gat ctt cag ttg gag gac aac
676Phe Leu Glu His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
205 210 215aaa tgg gcc tgc aat tgt gac tta ttg cag tta aaa act tgg
ttg gag 724Lys Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu 220 225 230aac atg cct cca cag tct ata att ggt gat gtt gtc
tgc aac agc cct 772Asn Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val
Cys Asn Ser Pro235 240 245 250cca ttt ttt aaa gga agt ata ctc agt
aga cta aag aag gaa tct att 820Pro Phe Phe Lys Gly Ser Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile 255 260 265tgc cct act cca cca gtg tat
gaa gaa cat gag gat cct tca gga tca 868Cys Pro Thr Pro Pro Val Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser 270 275 280tta cat ctg gca gca
aca tct tca ata aat gat agt cgc atg tca act 916Leu His Leu Ala Ala
Thr Ser Ser Ile Asn Asp Ser Arg Met Ser Thr 285 290 295aag acc acg
tcc att cta aaa cta ccc acc aaa gca cca ggt ttg ata 964Lys Thr Thr
Ser Ile Leu Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile 300 305 310cct
tat att aca aag cca tcc act caa ctt cca gga cct tac tgc cct 1012Pro
Tyr Ile Thr Lys Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro315 320
325 330att cct tgt aac tgc aaa gtc cta tcc cca tca gga ctt cta ata
cat 1060Ile Pro Cys Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile
His 335 340 345tgt cag gag cgc aac att gaa agc tta tca gat ctg aga
cct cct ccg 1108Cys Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg
Pro Pro Pro 350 355 360caa aat cct aga aag ctc att cta gcg gga aat
att att cac agt tta 1156Gln Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn
Ile Ile His Ser Leu 365 370 375atg aag tct gat cta gtg gaa tat ttc
act ttg gaa atg ctt cac ttg 1204Met Lys Ser Asp Leu Val Glu Tyr Phe
Thr Leu Glu Met Leu His Leu 380 385 390gga aac aat cgt att gaa gtt
ctt gaa gaa gga tcg ttt atg aac cta 1252Gly Asn Asn Arg Ile Glu Val
Leu Glu Glu Gly Ser Phe Met Asn Leu395 400 405 410acg aga tta caa
aaa ctc tat cta aat ggt aac cac ctg acc aaa tta 1300Thr Arg Leu Gln
Lys Leu Tyr Leu Asn Gly Asn His Leu Thr Lys Leu 415 420 425agt aaa
ggc atg ttc ctt ggt ctc cat aat ctt gaa tac tta tat ctt 1348Ser Lys
Gly Met Phe Leu Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu 430 435
440gaa tac aat gcc att aag gaa ata ctg cca gga acc ttt aat cca atg
1396Glu Tyr Asn Ala Ile Lys Glu Ile Leu Pro Gly Thr Phe Asn Pro Met
445 450 455cct aaa ctt aaa gtc ctg tat tta aat aac aac ctc ctc caa
gtt tta 1444Pro Lys Leu Lys Val Leu Tyr Leu Asn Asn Asn Leu Leu Gln
Val Leu 460 465 470cca cca cat att ttt tca ggg gtt cct cta act aag
gta aat ctt aaa 1492Pro Pro His Ile Phe Ser Gly Val Pro Leu Thr Lys
Val Asn Leu Lys475 480 485 490aca aac cag ttt acc cat cta cct gta
agt aat att ttg gat gat ctt 1540Thr Asn Gln Phe Thr His Leu Pro Val
Ser Asn Ile Leu Asp Asp Leu 495 500 505gat ttg cta acc cag att gac
ctt gag gat aac ccc tgg gac tgc tcc 1588Asp Leu Leu Thr Gln Ile Asp
Leu Glu Asp Asn Pro Trp Asp Cys Ser 510 515 520tgt gac ctg gtt gga
ctg cag caa tgg ata caa aag tta agc aag aac 1636Cys Asp Leu Val Gly
Leu Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn 525 530 535aca gtg aca
gat gac atc ctc tgc act tcc ccc ggg cat ctc gac aaa 1684Thr Val Thr
Asp Asp Ile Leu Cys Thr Ser Pro Gly His Leu Asp Lys 540 545 550aag
gaa ttg aaa gcc cta aat agt gaa att ctc tgt cca ggt tta gta 1732Lys
Glu Leu Lys Ala Leu Asn Ser Glu Ile Leu Cys Pro Gly Leu Val555 560
565 570aat aac cca tcc atg cca aca cag act agt tac ctt atg gtc acc
act 1780Asn Asn Pro Ser Met Pro Thr Gln Thr Ser Tyr Leu Met Val Thr
Thr 575 580 585cct gca aca aca aca aat acg gct gat act att tta cga
tct ctt acg 1828Pro Ala Thr Thr Thr Asn Thr Ala Asp Thr Ile Leu Arg
Ser Leu Thr 590 595 600gac gct gtg cca ctg tct gtt cta ata ttg gga
ctt ctg att atg ttc 1876Asp Ala Val Pro Leu Ser Val Leu Ile Leu Gly
Leu Leu Ile Met Phe 605 610 615atc act att gtt ttc tgt gct gca ggg
ata gtg gtt ctt gtt ctt cac 1924Ile Thr Ile Val Phe Cys Ala Ala Gly
Ile Val Val Leu Val Leu His 620 625 630cgc agg aga aga tac aaa aag
aaa caa gta gat gag caa atg aga gac 1972Arg Arg Arg Arg Tyr Lys Lys
Lys Gln Val Asp Glu Gln Met Arg Asp635 640 645 650aac agt cct gtg
cat ctt cag tac agc atg tat ggc cat aaa acc act 2020Asn Ser Pro Val
His Leu Gln Tyr Ser Met Tyr Gly His Lys Thr Thr 655 660 665cat cac
act act gaa aga ccc tct gcc tca ctc tat gaa cag cac atg 2068His His
Thr Thr Glu Arg Pro Ser Ala Ser Leu Tyr Glu Gln His Met 670 675
680gtg agc ccc atg gtt cat gtc tat aga agt cca tcc ttt ggt cca aag
2116Val Ser Pro Met Val His Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys
685 690 695cat ctg gaa gag gaa gaa gag agg aat gag aaa gaa gga agt
gat gca 2164His Leu Glu Glu Glu Glu Glu Arg Asn Glu Lys Glu Gly Ser
Asp Ala 700 705 710aaa cat ctc caa aga agt ctt ttg gaa cag gaa aat
cat tca cca ctc 2212Lys His Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn
His Ser Pro Leu715 720 725 730aca ggg tca aat atg aaa tac aaa acc
acg aac caa tca aca gaa ttt 2260Thr Gly Ser Asn Met Lys Tyr Lys Thr
Thr Asn Gln Ser Thr Glu Phe 735 740 745tta tcc ttc caa gat gcc agc
tca ttg tac aga aac att tta gaa aaa 2308Leu Ser Phe Gln Asp Ala Ser
Ser Leu Tyr Arg Asn Ile Leu Glu Lys 750 755 760gaa agg gaa ctt cag
caa ctg gga atc aca gaa tac cta agg aaa aac 2356Glu Arg Glu Leu Gln
Gln Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn 765 770 775att gct cag
ctc cag cct gat atg gag gca cat tat cct gga gcc cac 2404Ile Ala Gln
Leu Gln Pro Asp Met Glu Ala His Tyr Pro Gly Ala His 780 785 790gaa
gag ctg aag tta atg gaa aca tta atg tac tca cgt cca agg aag 2452Glu
Glu Leu Lys Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys795 800
805 810gta tta gtg gaa cag aca aaa aat gag tat ttt gaa ctt aaa gct
aat 2500Val Leu Val Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala
Asn 815 820 825tta cat gct gaa cct gac tat tta gaa gtc ctg gag cag
caa aca tag 2548Leu His Ala Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln
Gln Thr * 830 835 840atggaga 25555841PRTHomo sapiens 5Met Lys Leu
Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser
Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25
30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys
35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro
Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu
Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala
Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu
Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His
Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe
His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn
Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155
160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro
165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu
Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu
Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val
Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280
285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu
290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr
Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile
Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile
His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg
Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn
Ile Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe
Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400
Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405
410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe
Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn
Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro
Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val
Leu Pro Pro His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys
Val Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser
Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu
Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525
Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530
535 540 Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala
Leu545 550 555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn
Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr
Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser
Leu Thr Asp Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu
Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile
Val Val Leu Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys
Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650
655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg
660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser Pro Met
Val His 675 680 685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu
Glu Glu Glu Glu 690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala
Lys His Leu Gln Arg Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His
Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn
Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu
Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu
Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775
780 Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu
Met785 790 795 800 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu
Val Glu Gln Thr 805 810 815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn
Leu His Ala Glu Pro Asp 820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln
Thr 835 840 62228DNAHomo sapiensCDS(23)...(2221) 6tcggatttca
tcacatgaca ac atg aag ctg tgg att cat ctc ttt tat tca 52 Met Lys
Leu Trp Ile His Leu Phe Tyr Ser 1 5 10tct ctc ctt gcc tgt ata tct
tta cac tcc caa act cca gtg ctc tca 100Ser Leu Leu Ala Cys Ile Ser
Leu His Ser Gln Thr Pro Val Leu Ser 15 20 25tcc aga ggc tct tgt gat
tct ctt tgc aat tgt gag gaa aaa gat ggc 148Ser Arg Gly Ser Cys Asp
Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly 30 35 40aca atg cta ata aat
tgt gaa gca aaa ggt atc aag atg gta tct gaa 196Thr Met Leu Ile Asn
Cys Glu Ala Lys Gly Ile Lys Met Val Ser Glu 45 50 55ata agt gtg cca
cca tca cga cct ttc caa cta agc tta tta aat aac 244Ile Ser Val Pro
Pro Ser Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn 60 65 70ggc ttg acg
atg ctt cac aca aat gac ttt tct ggg ctt acc aat gct 292Gly Leu Thr
Met Leu His Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala75 80 85 90att
tca ata cac ctt gga ttt aac aat att gca gat att gag ata ggt 340Ile
Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly 95 100
105gca ttt aat ggc ctt ggc ctc ctg aaa caa ctt cat atc aat cac aat
388Ala Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn
110 115 120tct tta gaa att ctt aaa gag gat act ttc cat gga ctg gaa
aac ctg 436Ser Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu
Asn Leu 125 130 135gaa ttc ctg caa gca gat aac aat ttt atc aca gtg
att gaa cca agt 484Glu Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser 140 145 150gcc ttt agc aag ctc aac aga ctc aaa gtg
tta att tta aat gac aat 532Ala Phe Ser Lys Leu Asn Arg Leu Lys Val
Leu Ile Leu Asn Asp Asn155 160 165 170gct att gag agt ctt cct cca
aac atc ttc cga ttt gtt cct tta acc 580Ala Ile Glu Ser Leu Pro Pro
Asn Ile Phe Arg Phe Val Pro Leu Thr 175 180 185cat cta gat ctt cgt
gga aat caa tta caa aca ttg cct tat gtt ggt 628His Leu Asp Leu Arg
Gly Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly 190 195 200ttt ctc gaa
cac att ggc cga ata ttg gat ctt cag ttg gag gac aac
676Phe Leu Glu His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
205 210 215aaa tgg gcc tgc aat tgt gac tta ttg cag tta aaa act tgg
ttg gag 724Lys Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu 220 225 230aac atg cct cca cag tct ata att ggt gat gtt gtc
tgc aac agc cct 772Asn Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val
Cys Asn Ser Pro235 240 245 250cca ttt ttt aaa gga agt ata ctc agt
aga cta aag aag gaa tct att 820Pro Phe Phe Lys Gly Ser Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile 255 260 265tgc cct act cca cca gtg tat
gaa gaa cat gag gat cct tca gga tca 868Cys Pro Thr Pro Pro Val Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser 270 275 280tta cat ctg gca gca
aca tct tca ata aat gat agt cgc atg tca act 916Leu His Leu Ala Ala
Thr Ser Ser Ile Asn Asp Ser Arg Met Ser Thr 285 290 295aag acc acg
tcc att cta aaa cta ccc acc aaa gca cca ggt ttg ata 964Lys Thr Thr
Ser Ile Leu Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile 300 305 310cct
tat att aca aag cca tcc act caa ctt cca gga cct tac tgc cct 1012Pro
Tyr Ile Thr Lys Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro315 320
325 330att cct tgt aac tgc aaa gtc cta tcc cca tca gga ctt cta ata
cat 1060Ile Pro Cys Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile
His 335 340 345tgt cag gag cgc aac att gaa agc tta tca gat ctg aga
cct cct ccg 1108Cys Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg
Pro Pro Pro 350 355 360caa aat cct aga aag ctc att cta gcg gga aat
att att cac agt tta 1156Gln Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn
Ile Ile His Ser Leu 365 370 375atg aag tct gat cta gtg gaa tat ttc
act ttg gaa atg ctt cac ttg 1204Met Lys Ser Asp Leu Val Glu Tyr Phe
Thr Leu Glu Met Leu His Leu 380 385 390gga aac aat cgt att gaa gtt
ctt gaa gaa gga tcg ttt atg aac cta 1252Gly Asn Asn Arg Ile Glu Val
Leu Glu Glu Gly Ser Phe Met Asn Leu395 400 405 410acg aga tta caa
aaa ctc tat cta aat ggt aac cac ctg acc aaa tta 1300Thr Arg Leu Gln
Lys Leu Tyr Leu Asn Gly Asn His Leu Thr Lys Leu 415 420 425agt aaa
ggc atg ttc ctt ggt ctc cat aat ctt gaa tac tta tat ctt 1348Ser Lys
Gly Met Phe Leu Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu 430 435
440gaa tac aat gcc att aag gaa ata ctg cca gga acc ttt aat cca atg
1396Glu Tyr Asn Ala Ile Lys Glu Ile Leu Pro Gly Thr Phe Asn Pro Met
445 450 455cct aaa ctt aaa gtc ctg tat tta aat aac aac ctc ctc caa
gtt tta 1444Pro Lys Leu Lys Val Leu Tyr Leu Asn Asn Asn Leu Leu Gln
Val Leu 460 465 470cca cca cat att ttt tca ggg gtt cct cta act aag
gta aat ctt aaa 1492Pro Pro His Ile Phe Ser Gly Val Pro Leu Thr Lys
Val Asn Leu Lys475 480 485 490aca aac cag ttt acc cat cta cct gta
agt aat att ttg gat gat ctt 1540Thr Asn Gln Phe Thr His Leu Pro Val
Ser Asn Ile Leu Asp Asp Leu 495 500 505gat tta cta acc cag att gac
ctt gag gat aac ccc tgg gac tgc tcc 1588Asp Leu Leu Thr Gln Ile Asp
Leu Glu Asp Asn Pro Trp Asp Cys Ser 510 515 520tgt gac ctg gtt gga
ctg cag caa tgg ata caa aag tta agc aag aac 1636Cys Asp Leu Val Gly
Leu Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn 525 530 535aca gtg aca
gat gac atc ctc tgc act tcc ccc ggg cat ctc gac aaa 1684Thr Val Thr
Asp Asp Ile Leu Cys Thr Ser Pro Gly His Leu Asp Lys 540 545 550aag
gaa ttg aaa gcc cta aat agt gaa att ctc tgt cca ggt tta gta 1732Lys
Glu Leu Lys Ala Leu Asn Ser Glu Ile Leu Cys Pro Gly Leu Val555 560
565 570aat aac cca tcc atg cca aca cag act agt tac ctt atg gtc acc
act 1780Asn Asn Pro Ser Met Pro Thr Gln Thr Ser Tyr Leu Met Val Thr
Thr 575 580 585cct gca aca aca aca aat acg gct gat act att tta cga
tct ctt acg 1828Pro Ala Thr Thr Thr Asn Thr Ala Asp Thr Ile Leu Arg
Ser Leu Thr 590 595 600gac gct gtg cca ctg tct gtt cta ata ttg gga
ctt ctg att atg ttc 1876Asp Ala Val Pro Leu Ser Val Leu Ile Leu Gly
Leu Leu Ile Met Phe 605 610 615atc act att gtt ttc tgt gct gca ggg
ata gtg gtt ctt gtt ctt cac 1924Ile Thr Ile Val Phe Cys Ala Ala Gly
Ile Val Val Leu Val Leu His 620 625 630cgc agg aga aga tac aaa aag
aaa caa gta gat gag caa atg aga gac 1972Arg Arg Arg Arg Tyr Lys Lys
Lys Gln Val Asp Glu Gln Met Arg Asp635 640 645 650aac agt cct gtg
cat ctt cag tac agc atg tat ggc cat aaa acc act 2020Asn Ser Pro Val
His Leu Gln Tyr Ser Met Tyr Gly His Lys Thr Thr 655 660 665cat cac
act act gaa aga ccc tct gcc tca ctc tat gaa cag cac atg 2068His His
Thr Thr Glu Arg Pro Ser Ala Ser Leu Tyr Glu Gln His Met 670 675
680gga gcc cac gaa gag ctg aag tta atg gaa aca tta atg tac tca cgt
2116Gly Ala His Glu Glu Leu Lys Leu Met Glu Thr Leu Met Tyr Ser Arg
685 690 695cca agg aag gta tta gtg gaa cag aca aaa aat gag tat ttt
gaa ctt 2164Pro Arg Lys Val Leu Val Glu Gln Thr Lys Asn Glu Tyr Phe
Glu Leu 700 705 710aaa gct aat tta cat gct gaa cct gac tat tta gaa
gtc ctg gag cag 2212Lys Ala Asn Leu His Ala Glu Pro Asp Tyr Leu Glu
Val Leu Glu Gln715 720 725 730caa aca tag atggaga 2228Gln Thr
*7732PRTHomo sapiens 7Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Gly Ala His Glu Glu Leu 675 680 685 Lys
Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val 690 695
700 Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His
Ala705 710 715 720 Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr
725 730 81620DNAHomo sapiensCDS(23)...(1210) 8tcggatttca tcacatgaca
ac atg aag ctg tgg att cat ctc ttt tat tca 52 Met Lys Leu Trp Ile
His Leu Phe Tyr Ser 1 5 10tct ctc ctt gcc tgt ata tct tta cac tcc
caa act cca gtg ctc tca 100Ser Leu Leu Ala Cys Ile Ser Leu His Ser
Gln Thr Pro Val Leu Ser 15 20 25tcc aga ggc tct tgt gat tct ctt tgc
aat tgt gag gaa aaa gat ggc 148Ser Arg Gly Ser Cys Asp Ser Leu Cys
Asn Cys Glu Glu Lys Asp Gly 30 35 40aca atg cta ata aat tgt gaa gca
aaa ggt atc aag atg gta tct gaa 196Thr Met Leu Ile Asn Cys Glu Ala
Lys Gly Ile Lys Met Val Ser Glu 45 50 55ata agt gtg cca cca tca cga
cct ttc caa cta agc tta tta aat aac 244Ile Ser Val Pro Pro Ser Arg
Pro Phe Gln Leu Ser Leu Leu Asn Asn 60 65 70ggc ttg acg atg ctt cac
aca aat gac ttt tct ggg ctt acc aat gct 292Gly Leu Thr Met Leu His
Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala75 80 85 90att tca ata cac
ctt gga ttt aac aat att gca gat att gag ata ggt 340Ile Ser Ile His
Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly 95 100 105gca ttt
aat ggc ctt ggc ctc ctg aaa caa ctt cat atc aat cac aat 388Ala Phe
Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn 110 115
120tct tta gaa att ctt aaa gag gat act ttc cat gga ctg gaa aac ctg
436Ser Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu Asn Leu
125 130 135gaa ttc ctg caa gca gat aac aat ttt atc aca gtg att gaa
cca agt 484Glu Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val Ile Glu
Pro Ser 140 145 150gcc ttt agc aag ctc aac aga ctc aaa gtg tta att
tta aat gac aat 532Ala Phe Ser Lys Leu Asn Arg Leu Lys Val Leu Ile
Leu Asn Asp Asn155 160 165 170gct att gag agt ctt cct cca aac atc
ttc cga ttt gtt cct tta acc 580Ala Ile Glu Ser Leu Pro Pro Asn Ile
Phe Arg Phe Val Pro Leu Thr 175 180 185cat cta gat ctt cgt gga aat
caa tta caa aca ttg cct tat gtt ggt 628His Leu Asp Leu Arg Gly Asn
Gln Leu Gln Thr Leu Pro Tyr Val Gly 190 195 200ttt ctc gaa cac att
ggc cga ata ttg gat ctt cag ttg gag gac aac 676Phe Leu Glu His Ile
Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn 205 210 215aaa tgg gcc
tgc aat tgt gac tta ttg cag tta aaa act tgg ttg gag 724Lys Trp Ala
Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu 220 225 230aac
atg cct cca cag tct ata att ggt gat gtt gtc tgc aac agc cct 772Asn
Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val Cys Asn Ser Pro235 240
245 250cca ttt ttt aaa gga agt ata ctc agt aga cta aag aag gaa tct
att 820Pro Phe Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys Lys Glu Ser
Ile 255 260 265tgc cct act cca cca gtg tat gaa gaa cat gag gat cct
tca gga tca 868Cys Pro Thr Pro Pro Val Tyr Glu Glu His Glu Asp Pro
Ser Gly Ser 270 275 280tta cat ctg gca gca aca tct tca ata aat gat
agt cgc atg tca act 916Leu His Leu Ala Ala Thr Ser Ser Ile Asn Asp
Ser Arg Met Ser Thr 285 290 295aag acc acg tcc att cta aaa cta ccc
acc aaa gca cca ggt ttg ata 964Lys Thr Thr Ser Ile Leu Lys Leu Pro
Thr Lys Ala Pro Gly Leu Ile 300 305 310cct tat att aca aag cca tcc
act caa ctt cca gga cct tac tgc cct 1012Pro Tyr Ile Thr Lys Pro Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro315 320 325 330att cct tgt aac
tgc aaa gtc cta tcc cca tca gga ctt cta ata cat 1060Ile Pro Cys Asn
Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile His 335 340 345tgt cag
gag cgc aac att gaa agc tta tca gat ctg aga cct cct ccg 1108Cys Gln
Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro 350 355
360caa aat cct aga aag ctc att cta gcg gga aat att att cac agt tta
1156Gln Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile His Ser Leu
365 370 375atg aag tcc atc ctt tgg tcc aaa gca tct gga aga gga aga
aga gag 1204Met Lys Ser Ile Leu Trp Ser Lys Ala Ser Gly Arg Gly Arg
Arg Glu 380 385 390gaa tga gaaagaagga agtgatgcaa aacatctcca
aagaagtctt ttggaacagg 1260Glu *395aaaatcattc accactcaca gggtcaaata
tgaaatacaa aaccacgaac caatcaacag 1320aatttttatc cttccaagat
gccagctcat tgtacagaaa cattttagaa aaagaaaggg 1380aacttcagca
actgggaatc acagaatacc taaggaaaaa cattgctcag ctccagcctg
1440atatggaggc acattatcct ggagcccacg aagagctgaa gttaatggaa
acattaatgt 1500actcacgtcc aaggaaggta ttagtggaac agacaaaaaa
tgagtatttt gaacttaaag 1560ctaatttaca tgctgaacct gactatttag
aagtcctgga gcagcaaaca tagatggaga 16209395PRTHomo sapiens 9Met Lys
Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15
Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20
25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn
Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val
Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly
Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn
Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile
Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu
Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120
125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp
130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys
Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala
Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu
Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro
Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu
Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu
Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240
Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245
250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro
Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu
Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys
Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu
Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly
Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro
Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser
Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365
Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys Ser Ile Leu Trp 370
375 380 Ser Lys Ala Ser Gly Arg Gly Arg Arg Glu Glu385 390 395
103300DNAHomo sapiensCDS(480)...(3005) 10gcgtcgacaa caagaaatac
tagaaaagga ggaaggagaa cattgctgca gcttggatct 60acaacctaag aaagcaagag
tgatcaatct cagctctgtt aaacatcttg tttacttact 120gcattcagca
gcttgcaaat ggttaactat atgcaaaaaa gtcagcatag ctgtgaagta
180tgccgtgaat tttaattgag ggaaaaagga caattgcttc aggatgctct
agtatgcact 240ctgcttgaaa tattttcaat gaaatgctca gtattctatc
tttgaccaga ggttttaact 300ttatgaagct atgggacttg acaaaaagtg
atatttgaga agaaagtacg cagtggttgg 360tgttttcttt tttttaataa
aggaattgaa ttactttgaa cacctcttcc agctgtgcat 420tacagataac
gtcaggaaga gtctctgctt tacagaatcg gatttcatca catgacaac 479atg aag
ctg tgg att cat ctc ttt tat tca tct ctc ctt gcc tgt ata 527Met Lys
Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15tct
tta cac tcc caa act cca gtg ctc tca tcc aga ggc tct tgt gat 575Ser
Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25
30tct ctt tgc aat tgt gag gaa aaa gat ggc aca atg cta ata aat tgt
623Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys
35 40 45gaa gca aaa ggt atc aag atg gta tct gaa ata agt gtg cca cca
tca 671Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro
Ser 50 55 60cga cct ttc caa cta agc tta tta aat aac ggc ttg acg atg
ctt cac 719Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met
Leu His65 70 75 80aca aat gac ttt tct ggg ctt acc aat gct att tca
ata cac ctt gga 767Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser
Ile His Leu Gly 85 90 95ttt aac aat att gca gat att gag ata ggt gca
ttt aat ggc ctt ggc 815Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110ctc ctg aaa caa ctt cat atc aat cac
aat tct tta gaa att ctt aaa 863Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125gag gat act ttc cat gga ctg
gaa aac ctg gaa ttc ctg caa gca gat 911Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140aac aat ttt atc aca
gtg att gaa cca agt gcc ttt agc aag ctc aac 959Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160aga ctc
aaa gtg tta att tta aat gac aat gct att gag agt ctt cct 1007Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170
175cca aac atc ttc cga ttt gtt cct tta acc cat cta gat ctt cgt gga
1055Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly
180 185 190aat caa tta caa aca ttg cct tat gtt ggt ttt ctc gaa cac
att ggc 1103Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His
Ile Gly 195 200 205cga ata ttg gat ctt cag ttg gag gac aac aaa tgg
gcc tgc aat tgt 1151Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp
Ala Cys Asn Cys 210 215 220gac tta ttg cag tta aaa act tgg ttg gag
aac atg cct cca cag tct 1199Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu
Asn Met Pro Pro Gln Ser225 230 235 240ata att ggt gat gtt gtc tgc
aac agc cct cca ttt ttt aaa gga agt 1247Ile Ile Gly Asp Val Val Cys
Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255ata ctc agt aga cta
aag aag gaa tct att tgc cct act cca cca gtg 1295Ile Leu Ser Arg Leu
Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270tat gaa gaa
cat gag gat cct tca gga tca tta cat ctg gca gca aca 1343Tyr Glu Glu
His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285tct
tca ata aat gat agt cgc atg tca act aag acc acg tcc att cta 1391Ser
Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295
300aaa cta ccc acc aaa gca cca ggt ttg ata cct tat att aca aag cca
1439Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys
Pro305 310 315 320tcc act caa ctt cca gga cct tac tgc cct att cct
tgt aac tgc aaa 1487Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro
Cys Asn Cys Lys 325 330 335gtc cta tcc cca tca gga ctt cta ata cat
tgt cag gag cgc aac att 1535Val Leu Ser Pro Ser Gly Leu Leu Ile His
Cys Gln Glu Arg Asn Ile 340 345 350gaa agc tta tca gat ctg aga cct
cct ccg caa aat cct aga aag ctc 1583Glu Ser Leu Ser Asp Leu Arg Pro
Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365att cta gcg gga aat att
att cac agt tta atg aag tct gat cta gtg 1631Ile Leu Ala Gly Asn Ile
Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380gaa tat ttc act
ttg gaa atg ctt cac ttg gga aac aat cgt att gaa 1679Glu Tyr Phe Thr
Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400gtt
ctt gaa gaa gga tcg ttt atg aac cta acg aga tta caa aaa ctc 1727Val
Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410
415tat cta aat ggt aac cac ctg acc aaa tta agt aaa ggc atg ttc ctt
1775Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu
420 425 430ggt ctc cat aat ctt gaa tac tta tat ctt gaa tac aat gcc
att aag 1823Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala
Ile Lys 435 440 445gaa ata ctg cca gga acc ttt aat cca atg cct aaa
ctt aaa gtc ctg 1871Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys
Leu Lys Val Leu 450 455 460tat tta aat aac aac ctc ctc caa gtt tta
cca cca cat att ttt tca 1919Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu
Pro Pro His Ile Phe Ser465 470 475 480ggg gtt cct cta act aag gta
aat ctt aaa aca aac cag ttt acc cat 1967Gly Val Pro Leu Thr Lys Val
Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495cta cct gta agt aat
att ttg gat gat ctt gat tta cta acc cag att 2015Leu Pro Val Ser Asn
Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510gac ctt gag
gat aac ccc tgg gac tgc tcc tgt gac ctg gtt gga ctg 2063Asp Leu Glu
Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525cag
caa tgg ata caa aag tta agc aag aac aca gtg aca gat gac atc 2111Gln
Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535
540ctc tgc act tcc ccc ggg cat ctc gac aaa aag gaa ttg aaa gcc cta
2159Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala
Leu545 550 555 560aat agt gaa att ctc tgt cca ggt tta gta aat aac
cca tcc atg cca 2207Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn
Pro Ser Met Pro 565 570 575aca cag act agt tac ctt atg gtc acc act
cct gca aca aca aca aat 2255Thr Gln Thr Ser Tyr Leu Met Val Thr Thr
Pro Ala Thr Thr Thr Asn 580 585 590acg gct gat act att tta cga tct
ctt acg gac gct gtg cca ctg tct 2303Thr Ala Asp Thr Ile Leu Arg Ser
Leu Thr Asp Ala Val Pro Leu Ser 595 600 605gtt cta ata ttg gga ctt
ctg att atg ttc atc act att gtt ttc tgt 2351Val Leu Ile Leu Gly Leu
Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610 615 620gct gca ggg ata
gtg gtt ctt gtt ctt cac cgc agg aga aga tac aaa 2399Ala Ala Gly Ile
Val Val Leu Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640aag
aaa caa gta gat gag caa atg aga gac aac agt cct gtg cat ctt 2447Lys
Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650
655cag tac agc atg tat ggc cat aaa acc act cat cac act act gaa aga
2495Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg
660 665 670ccc tct gcc tca ctc tat gaa cag cac atg gtg agc ccc atg
gtt cat 2543Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser Pro Met
Val His 675 680 685gtc tat aga agt cca tcc ttt ggt cca aag cat ctg
gaa gag gaa gaa 2591Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu
Glu Glu Glu Glu 690 695 700gag agg aat gag aaa gaa gga agt gat gca
aaa cat ctc caa aga agt 2639Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala
Lys His Leu Gln Arg Ser705 710 715 720ctt ttg gaa cag gaa aat cat
tca cca ctc aca ggg tca aat atg aaa 2687Leu Leu Glu Gln Glu Asn His
Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730 735tac aaa acc acg aac
caa tca aca gaa ttt tta tcc ttc caa gat gcc 2735Tyr Lys Thr Thr Asn
Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750agc tca ttg
tac aga aac att tta gaa aaa gaa agg gaa ctt cag caa 2783Ser Ser Leu
Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765ctg
gga atc aca gaa tac cta agg aaa aac att gct cag ctc cag cct 2831Leu
Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775
780gat atg gag gca cat tat cct gga gcc cac gaa gag ctg aag tta atg
2879Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu
Met785 790 795 800gaa aca tta atg tac tca cgt cca agg aag gta tta
gtg gaa cag aca 2927Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu
Val Glu Gln Thr 805 810 815aaa aat gag tat ttt gaa ctt aaa gct aat
tta cat gct gaa cct gac 2975Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn
Leu His Ala Glu Pro Asp 820 825 830tat tta gaa gtc ctg gag cag caa
aca tag atggagagtt gagggctttc 3025Tyr Leu Glu Val Leu Glu Gln Gln
Thr * 835 840gccagaaatg ctgtgattct gttattaagt ccataccttg taaataagtg
ccttacgtga 3085gtgtgtcatc aatcagaacc taagcacaga gtaaactatg
gggaaaaaaa aagaagacga 3145aacagaaact cagggatcac tgggagaagc
catggcataa tcttcaggca atttagtctg 3205tcccaaataa acatacatcc
ttggcatgta aatcatcaag ggtaatagta atattcatat 3265acctgaaacg
tgtctcatag gagtcctctc tgcac 330011841PRTHomo sapiens 11Met Lys Leu
Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser
Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25
30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys
35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro
Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu
Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala
Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu
Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His
Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe
His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn
Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155
160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro
165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu
Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu
Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val
Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280
285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu
290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr
Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile
Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile
His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg
Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn
Ile Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe
Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400
Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405
410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe
Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn
Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro
Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val
Leu Pro Pro His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys
Val Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser
Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu
Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525
Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530
535 540 Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala
Leu545 550 555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn
Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr
Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser
Leu Thr Asp Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu
Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile
Val Val Leu Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635
640 Lys Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu
645 650 655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr
Glu Arg 660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser
Pro Met Val His 675 680 685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys
His Leu Glu Glu Glu Glu 690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser
Asp Ala Lys His Leu Gln Arg Ser705 710 715 720 Leu Leu Glu Gln Glu
Asn His Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr
Thr Asn Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser
Ser Leu Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760
765 Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro
770 775 780 Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys
Leu Met785 790 795 800 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val
Leu Val Glu Gln Thr 805 810 815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala
Asn Leu His Ala Glu Pro Asp 820 825 830 Tyr Leu Glu Val Leu Glu Gln
Gln Thr 835 840 121619DNAHomo sapiensCDS(23)...(1612) 12tcggatttca
tcacatgaca ac atg aag ctg tgg att cat ctc ttt tat tca 52 Met Lys
Leu Trp Ile His Leu Phe Tyr Ser 1 5 10tct ctc ctt gcc tgt ata tct
tta cac tcc caa act cca gtg ctc tca 100Ser Leu Leu Ala Cys Ile Ser
Leu His Ser Gln Thr Pro Val Leu Ser 15 20 25tcc aga ggc tct tgt gat
tct ctt tgc aat tgt gag gaa aaa gat ggc 148Ser Arg Gly Ser Cys Asp
Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly 30 35 40aca atg cta ata aat
tgt gaa gca aaa ggt atc aag atg gta tct gaa 196Thr Met Leu Ile Asn
Cys Glu Ala Lys Gly Ile Lys Met Val Ser Glu 45 50 55ata agt gtg cca
cca tca cga cct ttc caa cta agc tta tta aat aac 244Ile Ser Val Pro
Pro Ser Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn 60 65 70ggc ttg acg
atg ctt cac aca aat gac ttt tct ggg ctt acc aat gct 292Gly Leu Thr
Met Leu His Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala75 80 85 90att
tca ata cac ctt gga ttt aac aat att gca gat att gag ata ggt 340Ile
Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly 95 100
105gca ttt aat ggc ctt ggc ctc ctg aaa caa ctt cat atc aat cac aat
388Ala Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn
110 115 120tct tta gaa att ctt aaa gag gat act ttc cat gga ctg gaa
aac ctg 436Ser Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu
Asn Leu 125 130 135gaa ttc ctg caa gca gat aac aat ttt atc aca gtg
att gaa cca agt 484Glu Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser 140 145 150gcc ttt agc aag ctc aac aga ctc aaa gtg
tta att tta aat gac aat 532Ala Phe Ser Lys Leu Asn Arg Leu Lys Val
Leu Ile Leu Asn Asp Asn155 160 165 170gct att gag agt ctt cct cca
aac atc ttc cga ttt gtt cct tta acc 580Ala Ile Glu Ser Leu Pro Pro
Asn Ile Phe Arg Phe Val Pro Leu Thr 175 180 185cat cta gat ctt cgt
gga aat caa tta caa aca ttg cct tat gtt ggt 628His Leu Asp Leu Arg
Gly Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly 190 195 200ttt ctc gaa
cac att ggc cga ata ttg gat ctt cag ttg gag gac aac 676Phe Leu Glu
His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn 205 210 215aaa
tgg gcc tgc aat tgt gac tta ttg cag tta aaa act tgg ttg gag 724Lys
Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu 220 225
230aac atg cct cca cag tct ata att ggt gat gtt gtc tgc aac agc cct
772Asn Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val Cys Asn Ser
Pro235 240 245 250cca ttt ttt aaa gga agt ata ctc agt aga cta aag
aag gaa tct att 820Pro Phe Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile 255 260 265tgc cct act cca cca gtg tat gaa gaa cat
gag gat cct tca gga tca 868Cys Pro Thr Pro Pro Val Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser 270 275 280tta cat ctg gca gca aca tct tca
ata aat gat agt cgc atg tca act 916Leu His Leu Ala Ala Thr Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr 285 290 295aag acc acg tcc att cta
aaa cta ccc acc aaa gca cca ggt ttg ata 964Lys Thr Thr Ser Ile Leu
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile 300 305 310cct tat att aca
aag cca tcc act caa ctt cca gga cct tac tgc cct 1012Pro Tyr Ile Thr
Lys Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro315 320 325 330att
cct tgt aac tgc aaa gtc cta tcc cca tca gga ctt cta ata cat 1060Ile
Pro Cys Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile His 335 340
345tgt cag gag cgc aac att gaa agc tta tca gat ctg aga cct cct ccg
1108Cys Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
350 355 360caa aat cct aga aag ctc att cta gcg gga aat att att cac
agt tta 1156Gln Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile His
Ser Leu 365 370 375atg aat cca tcc ttt ggt cca aag cat ctg gaa gag
gaa gaa gag agg 1204Met Asn Pro Ser Phe Gly Pro Lys His Leu Glu Glu
Glu Glu Glu Arg 380 385 390aat gag aaa gaa gga agt gat gca aaa cat
ctc caa aga agt ctt ttg 1252Asn Glu Lys Glu Gly Ser Asp Ala Lys His
Leu Gln Arg Ser Leu Leu395 400 405 410gaa cag gaa aat cat tca cca
ctc aca ggg tca aat atg aaa tac aaa 1300Glu Gln Glu Asn His Ser Pro
Leu Thr Gly Ser Asn Met Lys Tyr Lys 415 420 425acc acg aac caa tca
aca gaa ttt tta tcc ttc caa gat gcc agc tca 1348Thr Thr Asn Gln Ser
Thr Glu Phe Leu Ser Phe Gln Asp Ala Ser Ser 430 435 440ttg tac aga
aac att tta gaa aaa gaa agg gaa ctt cag caa ctg gga 1396Leu Tyr Arg
Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln Leu Gly 445 450 455atc
aca gaa tac cta agg aaa aac att gct cag ctc cag cct gat atg 1444Ile
Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro Asp Met 460 465
470gag gca cat tat cct gga gcc cac gaa gag ctg aag tta atg gaa aca
1492Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met Glu
Thr475 480 485 490tta atg tac tca cgt cca agg aag gta tta gtg gaa
cag aca aaa aat 1540Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu
Gln Thr Lys Asn 495 500 505gag tat ttt gaa ctt aaa gct aat tta cat
gct gaa cct gac tat tta 1588Glu Tyr Phe Glu Leu Lys Ala Asn Leu His
Ala Glu Pro Asp Tyr Leu 510 515 520gaa gtc ctg gag cag caa aca tag
atggaga 1619Glu Val Leu Glu Gln Gln Thr * 52513529PRTHomo sapiens
13Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1
5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys
Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu
Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile
Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn
Asn Gly Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu
Thr Asn Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala
Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys
Gln Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu
Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135
140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu
Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile
Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr
His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr
Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln
Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln
Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile
Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250
255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val
260 265 270 Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala
Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr
Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile
Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro
Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser
Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu
Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile
Leu Ala Gly Asn Ile Ile His Ser Leu Met Asn Pro Ser Phe Gly 370 375
380 Pro Lys His Leu Glu Glu Glu Glu Glu Arg Asn Glu Lys Glu Gly
Ser385 390 395 400 Asp Ala Lys His Leu Gln Arg Ser Leu Leu Glu Gln
Glu Asn His Ser 405 410 415 Pro Leu Thr Gly Ser Asn Met Lys Tyr Lys
Thr Thr Asn Gln Ser Thr 420 425 430 Glu Phe Leu Ser Phe Gln Asp Ala
Ser Ser Leu Tyr Arg Asn Ile Leu 435 440 445 Glu Lys Glu Arg Glu Leu
Gln Gln Leu Gly Ile Thr Glu Tyr Leu Arg 450 455 460 Lys Asn Ile Ala
Gln Leu Gln Pro Asp Met Glu Ala His Tyr Pro Gly465 470 475 480 Ala
His Glu Glu Leu Lys Leu Met Glu Thr Leu Met Tyr Ser Arg Pro 485 490
495 Arg Lys Val Leu Val Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys
500 505 510 Ala Asn Leu His Ala Glu Pro Asp Tyr Leu Glu Val Leu Glu
Gln Gln 515 520 525 Thr 14841PRTHomo sapiens 14Met Lys Leu Trp Ile
His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His
Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser
Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40
45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser
50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met
Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser
Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly
Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn
His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly
Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile
Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg
Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170
175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly
180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His
Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp
Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu
Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys
Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu
Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu
His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser
Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295
300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys
Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro
Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His
Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro
Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile
Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr
Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400 Val
Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410
415 Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu
420 425 430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala
Ile Lys 435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys
Leu Lys Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu
Pro Pro His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val
Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn
Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu
Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln
Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535
540 Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala
Leu545 550 555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn
Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr
Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser
Leu Thr Asp Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu
Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile
Val Val Leu Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys
Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650
655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg
660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser Pro Met
Val His 675 680 685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu
Glu Glu Glu Glu 690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala
Lys His Leu Gln Arg Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His
Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn
Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu
Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755
760 765 Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln
Pro 770 775 780 Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu
Lys Leu Met785 790 795 800 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys
Val Leu Val Glu Gln Thr 805 810 815 Lys Asn Glu Tyr Phe Glu Leu Lys
Ala Asn Leu His Ala Glu Pro Asp 820 825 830 Tyr Leu Glu Val Leu Glu
Gln Gln Thr 835 840 15732PRTHomo sapiens 15Met Lys Leu Trp Ile His
Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser
Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu
Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45
Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50
55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu
His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile
His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180
185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile
Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala
Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn
Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305
310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn
Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln
Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His
Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu
Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu
Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr
Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425
430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys
435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys
Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro
His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu
Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu
Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn
Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp
Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu
Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550
555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met
Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr
Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp
Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met
Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu
Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val
Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr
Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670
Pro Ser Ala Ser Leu Tyr Glu Gln His Met Gly Ala His Glu Glu Leu 675
680 685 Lys Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu
Val 690 695 700 Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn
Leu His Ala705 710 715 720 Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln
Gln Thr 725 730 16390PRTHomo sapiens 16Met Lys Leu Trp Ile His Leu
Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln
Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys
Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu
Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55
60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu
His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile
His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180
185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile
Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala
Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn
Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305
310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn
Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln
Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His
Ser Leu Met Lys Ser Ile Leu Trp 370 375 380 Ser Lys Ala Ser Gly
Arg385 390 17529PRTHomo sapiens 17Met Lys Leu Trp Ile His Leu Phe
Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr
Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn
Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala
Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60
Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65
70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His
Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe
Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn
Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu
Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys
Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro
Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185
190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly
195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys
Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met
Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser
Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys
Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu
Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile
Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys
Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310
315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys
Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu
Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln
Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser
Leu Met Asn Pro Ser Phe Gly 370 375 380 Pro Lys His Leu Glu Glu Glu
Glu Glu Arg Asn Glu Lys Glu Gly Ser385 390 395 400 Asp Ala Lys His
Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn His Ser 405 410 415 Pro Leu
Thr Gly Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr 420 425 430
Glu Phe Leu Ser Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn Ile Leu 435
440 445 Glu Lys Glu Arg Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr Leu
Arg 450 455 460 Lys Asn Ile Ala Gln Leu Gln Pro Asp Met Glu Ala His
Tyr Pro Gly465 470 475 480 Ala His Glu Glu Leu Lys Leu Met Glu Thr
Leu Met Tyr Ser Arg Pro 485 490 495 Arg Lys Val Leu Val Glu Gln Thr
Lys Asn Glu Tyr Phe Glu Leu Lys 500 505 510 Ala Asn Leu His Ala Glu
Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln 515 520 525 Thr
18798PRTHomo sapiens 18Met Leu Ile Asn Cys Glu Ala Lys Gly Ile Lys
Met Val Ser Glu Ile1 5 10 15 Ser Val Pro Pro Ser Arg Pro Phe Gln
Leu Ser Leu Leu Asn Asn Gly 20 25 30 Leu Thr Met Leu His Thr Asn
Asp Phe Ser Gly Leu Thr Asn Ala Ile 35 40 45 Ser Ile His Leu Gly
Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala 50 55 60 Phe Asn Gly
Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn Ser65 70 75 80 Leu
Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu 85 90
95 Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala
100 105 110 Phe Ser Lys Leu Asn Arg Leu Lys Val Leu Ile Leu Asn Asp
Asn Ala 115 120 125 Ile Glu Ser Leu Pro Pro Asn Ile Phe Arg Phe Val
Pro Leu Thr His 130 135 140 Leu Asp Leu Arg Gly Asn Gln Leu Gln Thr
Leu Pro Tyr Val Gly Phe145 150 155 160 Leu Glu His Ile Gly Arg Ile
Leu Asp Leu Gln Leu Glu Asp Asn Lys 165 170 175 Trp Ala Cys Asn Cys
Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn 180 185 190 Met Pro Pro
Gln Ser Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro 195 200 205 Phe
Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys 210 215
220 Pro Thr Pro Pro Val Tyr Glu Glu His Glu Asp Pro Ser Gly Ser
Leu225 230 235 240 His Leu Ala Ala Thr Ser Ser Ile Asn Asp Ser Arg
Met Ser Thr Lys 245 250 255 Thr Thr Ser Ile Leu Lys Leu Pro Thr Lys
Ala Pro Gly Leu Ile Pro 260 265 270 Tyr Ile Thr Lys Pro Ser Thr Gln
Leu Pro Gly Pro Tyr Cys Pro Ile 275 280 285 Pro Cys Asn Cys Lys Val
Leu Ser Pro Ser Gly Leu Leu Ile His Cys 290 295 300 Gln Glu Arg Asn
Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln305 310 315 320 Asn
Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met 325 330
335 Lys Ser Asp Leu Val Glu Tyr Phe Thr Leu Glu Met Leu His Leu Gly
340 345 350 Asn Asn Arg Ile Glu Val Leu Glu Glu Gly Ser Phe Met Asn
Leu Thr 355 360 365 Arg Leu Gln Lys Leu Tyr Leu Asn Gly Asn His Leu
Thr Lys Leu Ser 370 375 380 Lys Gly Met Phe Leu Gly Leu His Asn Leu
Glu Tyr Leu Tyr Leu Glu385 390 395 400 Tyr Asn Ala Ile Lys Glu Ile
Leu Pro Gly Thr Phe Asn Pro Met Pro 405 410 415 Lys Leu Lys Val Leu
Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro 420 425 430 Pro His Ile
Phe Ser Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr 435 440 445 Asn
Gln Phe Thr His Leu Pro Val Ser Asn Ile Leu Asp Asp Leu Asp 450 455
460 Leu Leu Thr Gln Ile Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser
Cys465 470 475 480 Asp Leu Val Gly Leu Gln Gln Trp Ile Gln Lys Leu
Ser Lys Asn Thr 485 490 495 Val Thr Asp Asp Ile Leu Cys Thr Ser Pro
Gly His Leu Asp Lys Lys 500 505 510 Glu Leu Lys Ala Leu Asn Ser Glu
Ile Leu Cys Pro Gly Leu Val Asn 515 520 525 Asn Pro Ser Met Pro Thr
Gln Thr Ser Tyr Leu Met Val Thr Thr Pro 530 535 540 Ala Thr Thr Thr
Asn Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp545 550 555 560 Ala
Val Pro Leu Ser Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile 565
570
575 Thr Ile Val Phe Cys Ala Ala Gly Ile Val Val Leu Val Leu His Arg
580 585 590 Arg Arg Arg Tyr Lys Lys Lys Gln Val Asp Glu Gln Met Arg
Asp Asn 595 600 605 Ser Pro Val His Leu Gln Tyr Ser Met Tyr Gly His
Lys Thr Thr His 610 615 620 His Thr Thr Glu Arg Pro Ser Ala Ser Leu
Tyr Glu Gln His Met Val625 630 635 640 Ser Pro Met Val His Val Tyr
Arg Ser Pro Ser Phe Gly Pro Lys His 645 650 655 Leu Glu Glu Glu Glu
Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys 660 665 670 His Leu Gln
Arg Ser Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr 675 680 685 Gly
Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe Leu 690 695
700 Ser Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn Ile Leu Glu Lys
Glu705 710 715 720 Arg Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr Leu
Arg Lys Asn Ile 725 730 735 Ala Gln Leu Gln Pro Asp Met Glu Ala His
Tyr Pro Gly Ala His Glu 740 745 750 Glu Leu Lys Leu Met Glu Thr Leu
Met Tyr Ser Arg Pro Arg Lys Val 755 760 765 Leu Val Glu Gln Thr Lys
Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu 770 775 780 His Ala Glu Pro
Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr785 790 795 19798PRTHomo
sapiens 19Met Leu Ile Asn Cys Glu Ala Lys Gly Ile Lys Met Val Ser
Glu Ile1 5 10 15 Ser Val Pro Pro Ser Arg Pro Phe Gln Leu Ser Leu
Leu Asn Asn Gly 20 25 30 Leu Thr Met Leu His Thr Asn Asp Phe Ser
Gly Leu Thr Asn Ala Ile 35 40 45 Ser Ile His Leu Gly Phe Asn Asn
Ile Ala Asp Ile Glu Ile Gly Ala 50 55 60 Phe Asn Gly Leu Gly Leu
Leu Lys Gln Leu His Ile Asn His Asn Ser65 70 75 80 Leu Glu Ile Leu
Lys Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu 85 90 95 Phe Leu
Gln Ala Asp Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala 100 105 110
Phe Ser Lys Leu Asn Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala 115
120 125 Ile Glu Ser Leu Pro Pro Asn Ile Phe Arg Phe Val Pro Leu Thr
His 130 135 140 Leu Asp Leu Arg Gly Asn Gln Leu Gln Thr Leu Pro Tyr
Val Gly Phe145 150 155 160 Leu Glu His Ile Gly Arg Ile Leu Asp Leu
Gln Leu Glu Asp Asn Lys 165 170 175 Trp Ala Cys Asn Cys Asp Leu Leu
Gln Leu Lys Thr Trp Leu Glu Asn 180 185 190 Met Pro Pro Gln Ser Ile
Ile Gly Asp Val Val Cys Asn Ser Pro Pro 195 200 205 Phe Phe Lys Gly
Ser Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys 210 215 220 Pro Thr
Pro Pro Val Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu225 230 235
240 His Leu Ala Ala Thr Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys
245 250 255 Thr Thr Ser Ile Leu Lys Leu Pro Thr Lys Ala Pro Gly Leu
Ile Pro 260 265 270 Tyr Ile Thr Lys Pro Ser Thr Gln Leu Pro Gly Pro
Tyr Cys Pro Ile 275 280 285 Pro Cys Asn Cys Lys Val Leu Ser Pro Ser
Gly Leu Leu Ile His Cys 290 295 300 Gln Glu Arg Asn Ile Glu Ser Leu
Ser Asp Leu Arg Pro Pro Pro Gln305 310 315 320 Asn Pro Arg Lys Leu
Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met 325 330 335 Lys Ser Asp
Leu Val Glu Tyr Phe Thr Leu Glu Met Leu His Leu Gly 340 345 350 Asn
Asn Arg Ile Glu Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr 355 360
365 Arg Leu Gln Lys Leu Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser
370 375 380 Lys Gly Met Phe Leu Gly Leu His Asn Leu Glu Tyr Leu Tyr
Leu Glu385 390 395 400 Tyr Asn Ala Ile Lys Glu Ile Leu Pro Gly Thr
Phe Asn Pro Met Pro 405 410 415 Lys Leu Lys Val Leu Tyr Leu Asn Asn
Asn Leu Leu Gln Val Leu Pro 420 425 430 Pro His Ile Phe Ser Gly Val
Pro Leu Thr Lys Val Asn Leu Lys Thr 435 440 445 Asn Gln Phe Thr His
Leu Pro Val Ser Asn Ile Leu Asp Asp Leu Asp 450 455 460 Leu Leu Thr
Gln Ile Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser Cys465 470 475 480
Asp Leu Val Gly Leu Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr 485
490 495 Val Thr Asp Asp Ile Leu Cys Thr Ser Pro Gly His Leu Asp Lys
Lys 500 505 510 Glu Leu Lys Ala Leu Asn Ser Glu Ile Leu Cys Pro Gly
Leu Val Asn 515 520 525 Asn Pro Ser Met Pro Thr Gln Thr Ser Tyr Leu
Met Val Thr Thr Pro 530 535 540 Ala Thr Thr Thr Asn Thr Ala Asp Thr
Ile Leu Arg Ser Leu Thr Asp545 550 555 560 Ala Val Pro Leu Ser Val
Leu Ile Leu Gly Leu Leu Ile Met Phe Ile 565 570 575 Thr Ile Val Phe
Cys Ala Ala Gly Ile Val Val Leu Val Leu His Arg 580 585 590 Arg Arg
Arg Tyr Lys Lys Lys Gln Val Asp Glu Gln Met Arg Asp Asn 595 600 605
Ser Pro Val His Leu Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His 610
615 620 His Thr Thr Glu Arg Pro Ser Ala Ser Leu Tyr Glu Gln His Met
Val625 630 635 640 Ser Pro Met Val His Val Tyr Arg Ser Pro Ser Phe
Gly Pro Lys His 645 650 655 Leu Glu Glu Glu Glu Glu Arg Asn Glu Lys
Glu Gly Ser Asp Ala Lys 660 665 670 His Leu Gln Arg Ser Leu Leu Glu
Gln Glu Asn His Ser Pro Leu Thr 675 680 685 Gly Ser Asn Met Lys Tyr
Lys Thr Thr Asn Gln Ser Thr Glu Phe Leu 690 695 700 Ser Phe Gln Asp
Ala Ser Ser Leu Tyr Arg Asn Ile Leu Glu Lys Glu705 710 715 720 Arg
Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile 725 730
735 Ala Gln Leu Gln Pro Asp Met Glu Ala His Tyr Pro Gly Ala His Glu
740 745 750 Glu Leu Lys Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg
Lys Val 755 760 765 Leu Val Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu
Lys Ala Asn Leu 770 775 780 His Ala Glu Pro Asp Tyr Leu Glu Val Leu
Glu Gln Gln Thr785 790 795 20405PRTHomo sapiens 20Met Leu Ile Asn
Cys Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile1 5 10 15 Ser Val
Pro Pro Ser Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly 20 25 30
Leu Thr Met Leu His Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile 35
40 45 Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly
Ala 50 55 60 Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn
His Asn Ser65 70 75 80 Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly
Leu Glu Asn Leu Glu 85 90 95 Phe Leu Gln Ala Asp Asn Asn Phe Ile
Thr Val Ile Glu Pro Ser Ala 100 105 110 Phe Ser Lys Leu Asn Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala 115 120 125 Ile Glu Ser Leu Pro
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His 130 135 140 Leu Asp Leu
Arg Gly Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe145 150 155 160
Leu Glu His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys 165
170 175 Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu
Asn 180 185 190 Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro 195 200 205 Phe Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys 210 215 220 Pro Thr Pro Pro Val Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu225 230 235 240 His Leu Ala Ala Thr Ser
Ser Ile Asn Asp Ser Arg Met Ser Thr Lys 245 250 255 Thr Thr Ser Ile
Leu Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro 260 265 270 Tyr Ile
Thr Lys Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile 275 280 285
Pro Cys Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys 290
295 300 Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln305 310 315 320 Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile
His Ser Leu Met 325 330 335 Lys Ser Asp Leu Val Glu Tyr Phe Thr Leu
Glu Met Leu His Leu Gly 340 345 350 Asn Asn Arg Ile Glu Val Leu Glu
Glu Gly Ser Phe Met Asn Leu Thr 355 360 365 Arg Leu Gln Lys Leu Tyr
Leu Asn Gly Asn His Leu Thr Lys Leu Ser 370 375 380 Lys Gly Met Phe
Leu Gly Leu His Ala Ile Lys Glu Ile Leu Pro Gly385 390 395 400 Thr
Phe Asn Pro Met 405 21415PRTHomo sapiens 21Met Leu Ile Asn Cys Glu
Ala Lys Gly Ile Lys Met Val Ser Glu Ile1 5 10 15 Ser Val Pro Pro
Ser Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly 20 25 30 Leu Thr
Met Leu His Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile 35 40 45
Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala 50
55 60 Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn
Ser65 70 75 80 Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu
Asn Leu Glu 85 90 95 Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser Ala 100 105 110 Phe Ser Lys Leu Asn Arg Leu Lys Val
Leu Ile Leu Asn Asp Asn Ala 115 120 125 Ile Glu Ser Leu Pro Pro Asn
Ile Phe Arg Phe Val Pro Leu Thr His 130 135 140 Leu Asp Leu Arg Gly
Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe145 150 155 160 Leu Glu
His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys 165 170 175
Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn 180
185 190 Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val Cys Asn Ser Pro
Pro 195 200 205 Phe Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys Lys Glu
Ser Ile Cys 210 215 220 Pro Thr Pro Pro Val Tyr Glu Glu His Glu Asp
Pro Ser Gly Ser Leu225 230 235 240 His Leu Ala Ala Thr Ser Ser Ile
Asn Asp Ser Arg Met Ser Thr Lys 245 250 255 Thr Thr Ser Ile Leu Lys
Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro 260 265 270 Tyr Ile Thr Lys
Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile 275 280 285 Pro Cys
Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys 290 295 300
Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln305
310 315 320 Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile His Ser
Leu Met 325 330 335 Lys Ser Asp Leu Val Glu Tyr Phe Thr Leu Glu Met
Leu His Leu Gly 340 345 350 Asn Asn Arg Ile Glu Val Leu Glu Glu Gly
Ser Phe Met Asn Leu Thr 355 360 365 Arg Leu Gln Lys Leu Tyr Leu Asn
Gly Asn His Leu Thr Lys Leu Ser 370 375 380 Lys Gly Met Phe Leu Gly
Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu385 390 395 400 Tyr Asn Ala
Ile Lys Glu Ile Leu Pro Gly Thr Phe Asn Pro Met 405 410 415
22777PRTHomo sapiens 22Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Ala Ile Lys Glu Ile Leu Pro Gly Thr Phe Asn Pro Met 435 440 445 His
Ile Phe Ser Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn 450
455 460 Gln Phe Thr His Leu Pro Val Ser Asn Ile Asn Pro Trp Asp Cys
Ser465 470 475 480 Cys Asp Leu Val Gly Leu Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn 485 490 495 Thr Val Thr Asp Asp Ile Leu Cys Thr Ser
Pro Gly His Leu Asp Lys 500 505 510 Lys Glu Leu Lys Ala Leu Asn Ser
Glu Ile Leu Cys Pro Gly Leu Val 515 520 525 Asn Asn Pro Ser Met Pro
Thr Gln Thr Ser Tyr Leu Met Val Ile Leu 530 535 540 Arg Ser Leu Thr
Asp Ala Val Pro Leu Ser Val Leu Ile Leu Gly Leu545 550 555 560 Leu
Ile Met Phe Ile Thr Ile Val Phe Cys Ala Ala Gly Ile Val Val 565 570
575 Leu Val Leu His Arg Arg Arg Arg Tyr Lys Lys Lys Gln Val Asp Glu
580 585 590 Gln Met Arg Asp Asn Ser Pro Val His Leu Gln Tyr Ser Met
Tyr Gly 595 600 605 His Lys Thr Thr His His Thr Thr Glu Arg Pro Ser
Ala Ser Leu Tyr 610 615 620 Glu Gln His Met Val Ser Pro Met Val His
Val Tyr Arg Ser Pro Ser625 630 635 640 Phe Gly Pro Lys His Leu Gly
Ser Asp Ala Lys His Leu Gln Arg Ser 645 650 655 Leu Leu Glu Gln Glu
Asn His Ser Pro Leu Thr Gly Ser Asn Met Lys 660 665 670 Tyr Lys Thr
Thr Asn Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 675 680 685 Ser
Ser Leu Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 690 695
700 Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln
Pro705 710 715 720 Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu
Leu Lys Leu Met 725 730 735 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys
Val Leu Val Glu Gln Thr 740 745 750 Lys Asn Glu Tyr Phe Glu Leu Lys
Ala Asn Leu His Ala Glu Pro Asp 755 760 765 Tyr Leu Glu Val Leu Glu
Gln Gln Thr 770 775 23832PRTHomo sapiens 23Met Phe Leu Trp Leu Phe
Leu Ile Leu Ser Ala Leu Ile Ser Ser Thr1 5 10 15 Asn Ala Asp Ser
Asp Ile Ser Val Glu Ile Cys Asn Val Cys Ser Cys 20 25 30 Val Ser
Val Glu Asn Val Leu Tyr Val Asn Cys Glu Lys Val Ser Val 35 40 45
Tyr Arg Pro Asn Gln Leu Lys Pro Pro Trp Ser Asn Phe Tyr His Leu 50
55 60 Asn Phe Gln Asn Asn Phe Leu Asn Ile Leu Tyr Pro Asn Thr Phe
Leu65 70 75 80 Asn Phe Ser His Ala Val Ser Leu His Leu Gly Asn Asn
Lys Leu Gln 85 90 95 Asn Ile Glu Gly Gly Ala Phe Leu Gly Leu Ser
Ala Leu Lys Gln Leu 100 105 110 His Leu Asn Asn Asn Glu Leu Lys Ile
Leu Arg Ala Asp Thr Phe Leu 115 120 125 Gly Ile Glu Asn Leu Glu Tyr
Leu Gln Ala Asp Tyr Asn Leu Ile Lys 130 135 140 Tyr Ile Glu Arg Gly
Ala Phe Asn Lys Leu His Lys Leu Lys Val Leu145 150 155 160 Ile Leu
Asn Asp Asn Leu Ile Ser Phe Leu Pro Asp Asn Ile Phe Arg 165 170 175
Phe Ala Ser Leu Thr His Leu Asp Ile Arg Gly Asn Arg Ile Gln Lys 180
185 190 Leu Pro Tyr Ile Gly Val Leu Glu His Ile Gly Arg Val Val Glu
Leu 195 200 205 Gln Leu Glu Asp Asn Pro Trp Asn Cys Ser Cys Asp Leu
Leu Pro Leu 210 215 220 Lys Ala Trp Leu Glu Asn Met Pro Tyr Asn Ile
Tyr Ile Gly Glu Ala225 230 235 240 Ile Cys Glu Thr Pro Ser Asp Leu
Tyr Gly Arg Leu Leu Lys Glu Thr 245 250 255 Asn Lys Gln Glu Leu Cys
Pro Met Gly Thr Gly Ser Asp Phe Asp Val 260 265 270 Arg Ile Leu Pro
Pro Ser Gln Leu Glu Asn Gly Tyr Thr Thr Pro Asn 275 280 285 Gly His
Thr Thr Gln Thr Ser Leu His Arg Leu Val Thr Lys Pro Pro 290 295 300
Lys Thr Thr Asn Pro Ser Lys Ile Ser Gly Ile Val Ala Gly Lys Ala305
310 315 320 Leu Ser Asn Arg Asn Leu Ser Gln Ile Val Ser Tyr Gln Thr
Arg Val 325 330 335 Pro Pro Leu Thr Pro Cys Pro Ala Pro Cys Phe Cys
Lys Thr His Pro 340 345 350 Ser Asp Leu Gly Leu Ser Val Asn Cys Gln
Glu Lys Asn Ile Gln Ser 355 360 365 Met Ser Glu Leu Ile Pro Lys Pro
Leu Asn Ala Lys Lys Leu His Val 370 375 380 Asn Gly Asn Ser Ile Lys
Asp Val Asp Val Ser Asp Phe Thr Asp Phe385 390 395 400 Glu Gly Leu
Asp Leu Leu His Leu Gly Ser Asn Gln Ile Thr Val Ile 405 410 415 Lys
Gly Asp Val Phe His Asn Leu Thr Asn Leu Arg Arg Leu Tyr Leu 420 425
430 Asn Gly Asn Gln Ile Glu Arg Leu Tyr Pro Glu Ile Phe Ser Gly Leu
435 440 445 His Asn Leu Gln Tyr Leu Tyr Leu Glu Tyr Asn Leu Ile Lys
Glu Ile 450 455 460 Ser Ala Gly Thr Phe Asp Ser Met Pro Asn Leu Gln
Leu Leu Tyr Leu465 470 475 480 Asn Asn Asn Leu Leu Lys Ser Leu Pro
Val Tyr Ile Phe Ser Gly Ala 485 490 495 Pro Leu Ala Arg Leu Asn Leu
Arg Asn Asn Lys Phe Met Tyr Leu Pro 500 505 510 Val Ser Gly Val Leu
Asp Gln Leu Gln Ser Leu Thr Gln Ile Asp Leu 515 520 525 Glu Gly Asn
Pro Trp Asp Cys Thr Cys Asp Leu Val Ala Leu Lys Leu 530 535 540 Trp
Val Glu Lys Leu Ser Asp Gly Ile Val Val Lys Glu Leu Lys Cys545 550
555 560 Glu Thr Pro Val Gln Phe Ala Asn Ile Glu Leu Lys Ser Leu Lys
Asn 565 570 575 Glu Ile Leu Cys Pro Lys Leu Leu Asn Lys Pro Ser Ala
Pro Phe Thr 580 585 590 Ser Pro Ala Pro Ala Ile Thr Phe Thr Thr Pro
Leu Gly Pro Ile Arg 595 600 605 Ser Pro Pro Gly Gly Pro Val Pro Leu
Ser Ile Leu Ile Leu Ser Ile 610 615 620 Leu Val Val Leu Ile Leu Thr
Val Phe Val Ala Phe Cys Leu Leu Val625 630 635 640 Phe Val Leu Arg
Arg Asn Lys Lys Pro Thr Val Lys His Glu Gly Leu 645 650 655 Gly Asn
Pro Asp Cys Gly Ser Met Gln Leu Gln Leu Arg Lys His Asp 660 665 670
His Lys Thr Asn Lys Lys Asp Gly Leu Ser Thr Glu Ala Phe Ile Pro 675
680 685 Gln Thr Ile Glu Gln Met Ser Lys Ser His Thr Cys Gly Leu Lys
Glu 690 695 700 Ser Glu Thr Gly Phe Met Phe Ser Asp Pro Pro Gly Gln
Lys Val Val705 710 715 720 Met Arg Asn Val Ala Asp Lys Glu Lys Asp
Leu Leu His Val Asp Thr 725 730 735 Arg Lys Arg Leu Ser Thr Ile Asp
Glu Leu Asp Glu Leu Phe Pro Ser 740 745 750 Arg Asp Ser Asn Val Phe
Ile Gln Asn Phe Leu Glu Ser Lys Lys Glu 755 760 765 Tyr Asn Ser Ile
Gly Val Ser Gly Phe Glu Ile Arg Tyr Pro Glu Lys 770 775 780 Gln Pro
Asp Lys Lys Ser Lys Lys Ser Leu Ile Gly Gly Asn His Ser785 790 795
800 Lys Ile Val Val Glu Gln Arg Lys Ser Glu Tyr Phe Glu Leu Lys Ala
805 810 815 Lys Leu Gln Ser Ser Pro Asp Tyr Leu Gln Val Leu Glu Glu
Gln Thr 820 825 830 2414PRTTetanus toxoid 24Gln Tyr Ile Lys Ala Asn
Ser Lys Phe Ile Gly Ile Thr Glu1 5 10 2521PRTPlasmodium falciparum
25Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe1
5 10 15 Asn Val Val Asn Ser 20 2616PRTStreptococcus 26Gly Ala Val
Asp Ser Ile Leu Gly Gly Val Ala Thr Tyr Gly Ala Ala1 5 10 15
2713PRTArtificial Sequencepan-DR binding epitope 27Xaa Lys Xaa Val
Ala Ala Trp Thr Leu Lys Ala Ala Xaa1 5 10 2814DNAArtificial
SequencePrimer 28ttttgatcaa gctt 142942DNAArtificial SequencePrimer
29ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag
423012DNAArtificial SequencePrimer 30gatcctgccc gg
123140DNAArtificial SequencePrimer 31gtaatacgac tcactatagg
gcagcgtggt cgcggccgag 403210DNAArtificial SequencePrimer
32gatcctcggc 103322DNAArtificial SequencePrimer 33ctaatacgac
tcactatagg gc 223422DNAArtificial SequencePrimer 34tcgagcggcc
gcccgggcag ga 223520DNAArtificial SequencePrimer 35agcgtggtcg
cggccgagga 203625DNAArtificial SequencePrimer 36atatcgccgc
gctcgtcgtc gacaa 253726DNAArtificial SequencePrimer 37agccacacgc
agctcattgt agaagg 263824DNAArtificial SequencePrimer 38ataagctttc
aatgttgcgc tcct 243924DNAArtificial SequencePrimer 39tgtcaactaa
gaccacgtcc attc 244024DNAArtificial SequenceFlag Tag 40gattacaagg
atgacgacga taag 244112PRTHomo sapiens 41Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu1 5 10 4221DNAHomo sapiens 42aagctcattc
tagcgggaaa t 214324DNAHomo sapiens 43aagggacgaa gacgaacacu uctt
244423DNAHomo sapiens 44aactgaagac ctgaagacaa taa 23454PRTHomo
sapiens 45Asn Asp Ser Arg1 464PRTHomo sapiens 46Asn Leu Thr Arg1
474PRTHomo sapiens 47Asn Gln Ser Thr1 484PRTHomo sapiens 48Lys Lys
Glu Ser1 494PRTHomo sapiens 49Thr Val Ile Glu1 504PRTHomo sapiens
50Thr His Leu Asp1 514PRTHomo sapiens 51Thr Trp Leu Glu1 524PRTHomo
sapiens 52Ser Ile Asn Asp1 534PRTHomo sapiens 53Ser Leu Ser Asp1
544PRTHomo sapiens 54Thr Gln Ile Asp1 554PRTHomo sapiens 55Thr Val
Thr Asp1 564PRTHomo sapiens 56Ser Leu Thr Asp1 574PRTHomo sapiens
57Ser Leu Tyr Glu1 584PRTHomo sapiens 58Ser Leu Leu Glu1 594PRTHomo
sapiens 59Ser Phe Gln Asp1 604PRTHomo sapiens 60Thr Lys Asn Glu1
618PRTHomo sapiens 61Lys Leu Met Glu Thr Leu Met Tyr1 5 626PRTHomo
sapiens 62Gly Ser Cys Asp Ser Leu1 5 636PRTHomo sapiens 63Gly Leu
Thr Asn Ala Ile1 5 646PRTHomo sapiens 64Gly Ala Phe Asn Gly Leu1 5
656PRTHomo sapiens 65Gly Ser Ile Leu Ser Arg1 5 666PRTHomo sapiens
66Gly Ser Phe Met Asn Leu1 5 676PRTHomo sapiens 67Gly Asn His Leu
Thr Lys1 5 686PRTHomo sapiens 68Gly Met Phe Leu Gly Leu1 5
696PRTHomo sapiens 69Gly Val Pro Leu Thr Lys1 5 702228DNAHomo
sapiens 70tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttatt
catctctcct 60tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct
cttgtgattc 120tctttgcaat tgtgaggaaa aagatggcac aatgctaata
aattgtgaag caaaaggtat 180caagatggta tctgaaataa gtgtgccacc
atcacgacct ttccaactaa gcttattaaa 240taacggcttg acgatgcttc
acacaaatga cttttctggg cttaccaatg ctatttcaat 300acaccttgga
tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg
atactttcca 420tggactggaa aacctggaat tcctgcaagc agataacaat
tttatcacag tgattgaacc 480aagtgccttt agcaagctca acagactcaa
agtgttaatt ttaaatgaca atgctattga 540gagtcttcct ccaaacatct
tccgatttgt tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa
acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa
aaacttggtt 720ggagaacatg cctccacagt ctataattgg tgatgttgtc
tgcaacagcc ctccattttt 780taaaggaagt atactcagta gactaaagaa
ggaatctatt tgccctactc caccagtgta 840tgaagaacat gaggatcctt
caggatcatt acatctggca gcaacatctt caataaatga 900tagtcgcatg
tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960gataccttat attacaaagc catccactca acttccagga ccttactgcc
ctattccttg 1020taactgcaaa gtcctatccc catcaggact tctaatacat
tgtcaggagc gcaacattga 1080aagcttatca gatctgagac ctcctccgca
aaatcctaga aagctcattc tagcgggaaa 1140tattattcac agtttaatga
agtctgatct agtggaatat ttcactttgg aaatgcttca 1200cttgggaaac
aatcgtattg aagttcttga agaaggatcg tttatgaacc taacgagatt
1260acaaaaactc tatctaaatg gtaaccacct gaccaaatta agtaaaggca
tgttccttgg 1320tctccataat cttgaatact tatatcttga atacaatgcc
attaaggaaa tactgccagg 1380aacctttaat ccaatgccta aacttaaagt
cctgtattta aataacaacc tcctccaagt 1440tttaccacca catatttttt
caggggttcc tctaactaag gtaaatctta aaacaaacca 1500gtttacccat
ctacctgtaa gtaatatttt ggatgatctt gatttactaa cccagattga
1560ccttgaggat aacccctggg actgctcctg tgacctggtt ggactgcagc
aatggataca 1620aaagttaagc aagaacacag tgacagatga catcctctgc
acttcccccg ggcatctcga 1680caaaaaggaa ttgaaagccc taaatagtga
aattctctgt ccaggtttag taaataaccc 1740atccatgcca acacagacta
gttaccttat ggtcaccact cctgcaacaa caacaaatac 1800ggctgatact
attttacgat ctcttacgga cgctgtgcca ctgtctgttc taatattggg
1860acttctgatt atgttcatca ctattgtttt ctgtgctgca gggatagtgg
ttcttgttct 1920tcaccgcagg agaagataca aaaagaaaca agtagatgag
caaatgagag acaacagtcc 1980tgtgcatctt cagtacagca tgtatggcca
taaaaccact catcacacta ctgaaagacc 2040ctctgcctca ctctatgaac
agcacatggg agcccacgaa gagctgaagt taatggaaac 2100attaatgtac
tcacgtccaa ggaaggtatt agtggaacag acaaaaaatg agtattttga
2160acttaaagct aatttacatg ctgaacctga ctatttagaa gtcctggagc
agcaaacata 2220gatggaga 2228712555DNAHomo sapiens 71tcggatttca
tcacatgaca acatgaagct gtggattcat ctcttttatt catctctcct 60tgcctgtata
tctttacact cccaaactcc agtgctctca tccagaggct cttgtgattc
120tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaag
caaaaggtat 180caagatggta tctgaaataa gtgtgccacc atcacgacct
ttccaactaa gcttattaaa 240taacggcttg acgatgcttc acacaaatga
cttttctggg cttaccaatg ctatttcaat 300acaccttgga tttaacaata
ttgcagatat tgagataggt gcatttaatg gccttggcct 360cctgaaacaa
cttcatatca atcacaattc tttagaaatt cttaaagagg atactttcca
420tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacag
tgattgaacc 480aagtgccttt agcaagctca acagactcaa agtgttaatt
ttaaatgaca atgctattga 540gagtcttcct ccaaacatct tccgatttgt
tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa acattgcctt
atgttggttt tctcgaacac attggccgaa tattggatct 660tcagttggag
gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt
720ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagcc
ctccattttt 780taaaggaagt atactcagta gactaaagaa ggaatctatt
tgccctactc caccagtgta 840tgaagaacat gaggatcctt caggatcatt
acatctggca gcaacatctt caataaatga 900tagtcgcatg tcaactaaga
ccacgtccat tctaaaacta cccaccaaag caccaggttt 960gataccttat
attacaaagc catccactca acttccagga ccttactgcc ctattccttg
1020taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagc
gcaacattga 1080aagcttatca gatctgagac ctcctccgca aaatcctaga
aagctcattc tagcgggaaa 1140tattattcac agtttaatga agtctgatct
agtggaatat ttcactttgg aaatgcttca 1200cttgggaaac aatcgtattg
aagttcttga agaaggatcg tttatgaacc taacgagatt 1260acaaaaactc
tatctaaatg gtaaccacct gaccaaatta agtaaaggca tgttccttgg
1320tctccataat cttgaatact tatatcttga atacaatgcc attaaggaaa
tactgccagg 1380aacctttaat ccaatgccta aacttaaagt cctgtattta
aataacaacc tcctccaagt 1440tttaccacca catatttttt caggggttcc
tctaactaag gtaaatctta aaacaaacca 1500gtttacccat ctacctgtaa
gtaatatttt ggatgatctt gatttactaa cccagattga 1560ccttgaggat
aacccctggg actgctcctg tgacctggtt ggactgcagc aatggataca
1620aaagttaagc aagaacacag tgacagatga catcctctgc acttcccccg
ggcatctcga 1680caaaaaggaa ttgaaagccc taaatagtga aattctctgt
ccaggtttag taaataaccc 1740atccatgcca acacagacta gttaccttat
ggtcaccact cctgcaacaa caacaaatac 1800ggctgatact attttacgat
ctcttacgga cgctgtgcca ctgtctgttc taatattggg 1860acttctgatt
atgttcatca ctattgtttt ctgtgctgca gggatagtgg ttcttgttct
1920tcaccgcagg agaagataca aaaagaaaca agtagatgag caaatgagag
acaacagtcc 1980tgtgcatctt cagtacagca
tgtatggcca taaaaccact catcacacta ctgaaagacc 2040ctctgcctca
ctctatgaac agcacatggt gagccccatg gttcatgtct atagaagtcc
2100atcctttggt ccaaagcatc tggaagagga agaagagagg aatgagaaag
aaggaagtga 2160tgcaaaacat ctccaaagaa gtcttttgga acaggaaaat
cattcaccac tcacagggtc 2220aaatatgaaa tacaaaacca cgaaccaatc
aacagaattt ttatccttcc aagatgccag 2280ctcattgtac agaaacattt
tagaaaaaga aagggaactt cagcaactgg gaatcacaga 2340atacctaagg
aaaaacattg ctcagctcca gcctgatatg gaggcacatt atcctggagc
2400ccacgaagag ctgaagttaa tggaaacatt aatgtactca cgtccaagga
aggtattagt 2460ggaacagaca aaaaatgagt attttgaact taaagctaat
ttacatgctg aacctgacta 2520tttagaagtc ctggagcagc aaacatagat ggaga
2555722228DNAHomo sapiens 72tcggatttca tcacatgaca acatgaagct
gtggattcat ctcttttatt catctctcct 60tgcctgtata tctttacact cccaaactcc
agtgctctca tccagaggct cttgtgattc 120tctttgcaat tgtgaggaaa
aagatggcac aatgctaata aattgtgaag caaaaggtat 180caagatggta
tctgaaataa gtgtgccacc atcacgacct ttccaactaa gcttattaaa
240taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatg
ctatttcaat 300acaccttgga tttaacaata ttgcagatat tgagataggt
gcatttaatg gccttggcct 360cctgaaacaa cttcatatca atcacaattc
tttagaaatt cttaaagagg atactttcca 420tggactggaa aacctggaat
tcctgcaagc agataacaat tttatcacag tgattgaacc 480aagtgccttt
agcaagctca acagactcaa agtgttaatt ttaaatgaca atgctattga
540gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatc
ttcgtggaaa 600tcaattacaa acattgcctt atgttggttt tctcgaacac
attggccgaa tattggatct 660tcagttggag gacaacaaat gggcctgcaa
ttgtgactta ttgcagttaa aaacttggtt 720ggagaacatg cctccacagt
ctataattgg tgatgttgtc tgcaacagcc ctccattttt 780taaaggaagt
atactcagta gactaaagaa ggaatctatt tgccctactc caccagtgta
840tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatctt
caataaatga 900tagtcgcatg tcaactaaga ccacgtccat tctaaaacta
cccaccaaag caccaggttt 960gataccttat attacaaagc catccactca
acttccagga ccttactgcc ctattccttg 1020taactgcaaa gtcctatccc
catcaggact tctaatacat tgtcaggagc gcaacattga 1080aagcttatca
gatctgagac ctcctccgca aaatcctaga aagctcattc tagcgggaaa
1140tattattcac agtttaatga agtctgatct agtggaatat ttcactttgg
aaatgcttca 1200cttgggaaac aatcgtattg aagttcttga agaaggatcg
tttatgaacc taacgagatt 1260acaaaaactc tatctaaatg gtaaccacct
gaccaaatta agtaaaggca tgttccttgg 1320tctccataat cttgaatact
tatatcttga atacaatgcc attaaggaaa tactgccagg 1380aacctttaat
ccaatgccta aacttaaagt cctgtattta aataacaacc tcctccaagt
1440tttaccacca catatttttt caggggttcc tctaactaag gtaaatctta
aaacaaacca 1500gtttacccat ctacctgtaa gtaatatttt ggatgatctt
gatttactaa cccagattga 1560ccttgaggat aacccctggg actgctcctg
tgacctggtt ggactgcagc aatggataca 1620aaagttaagc aagaacacag
tgacagatga catcctctgc acttcccccg ggcatctcga 1680caaaaaggaa
ttgaaagccc taaatagtga aattctctgt ccaggtttag taaataaccc
1740atccatgcca acacagacta gttaccttat ggtcaccact cctgcaacaa
caacaaatac 1800ggctgatact attttacgat ctcttacgga cgctgtgcca
ctgtctgttc taatattggg 1860acttctgatt atgttcatca ctattgtttt
ctgtgctgca gggatagtgg ttcttgttct 1920tcaccgcagg agaagataca
aaaagaaaca agtagatgag caaatgagag acaacagtcc 1980tgtgcatctt
cagtacagca tgtatggcca taaaaccact catcacacta ctgaaagacc
2040ctctgcctca ctctatgaac agcacatggg agcccacgaa gagctgaagt
taatggaaac 2100attaatgtac tcacgtccaa ggaaggtatt agtggaacag
acaaaaaatg agtattttga 2160acttaaagct aatttacatg ctgaacctga
ctatttagaa gtcctggagc agcaaacata 2220gatggaga 222873732PRTHomo
sapiens 73Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala
Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg
Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly
Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val
Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser
Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe
Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn
Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110
Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115
120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala
Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser
Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn
Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro
Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu
Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp
Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu
Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln Ser225 230 235
240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser
245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro
Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu His
Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr
Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly
Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser Thr Gln Leu Pro
Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser
Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile 340 345 350 Glu
Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360
365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys Ser Asp Leu Val
370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg
Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr
Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys
Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr
Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly
Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn
Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe Ser465 470 475 480
Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn Gln Phe Thr His 485
490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln
Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu
Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr
Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser Pro Gly His Leu Asp
Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn Ser Glu Ile Leu Cys
Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser
Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala
Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro Leu Ser 595 600 605
Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610
615 620 Ala Ala Gly Ile Val Val Leu Val Leu His Arg Arg Arg Arg Tyr
Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser
Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr
His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln
His Met Gly Ala His Glu Glu Leu 675 680 685 Lys Leu Met Glu Thr Leu
Met Tyr Ser Arg Pro Arg Lys Val Leu Val 690 695 700 Glu Gln Thr Lys
Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala705 710 715 720 Glu
Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr 725 730 74841PRTHomo
sapiens 74Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala
Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg
Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly
Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val
Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser
Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe
Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn
Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110
Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115
120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala
Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser
Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn
Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro
Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu
Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp
Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu
Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln Ser225 230 235
240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser
245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro
Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu His
Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr
Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly
Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser Thr Gln Leu Pro
Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser
Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile 340 345 350 Glu
Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360
365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys Ser Asp Leu Val
370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg
Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr
Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys
Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr
Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly
Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn
Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe Ser465 470 475 480
Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn Gln Phe Thr His 485
490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln
Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu
Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr
Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser Pro Gly His Leu Asp
Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn Ser Glu Ile Leu Cys
Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser
Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala
Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro Leu Ser 595 600 605
Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610
615 620 Ala Ala Gly Ile Val Val Leu Val Leu His Arg Arg Arg Arg Tyr
Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser
Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr
His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln
His Met Val Ser Pro Met Val His 675 680 685 Val Tyr Arg Ser Pro Ser
Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695 700 Glu Arg Asn Glu
Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg Ser705 710 715 720 Leu
Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730
735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala
740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu
Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala
Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala His Tyr Pro Gly Ala His
Glu Glu Leu Lys Leu Met785 790 795 800 Glu Thr Leu Met Tyr Ser Arg
Pro Arg Lys Val Leu Val Glu Gln Thr 805 810 815 Lys Asn Glu Tyr Phe
Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp 820 825 830 Tyr Leu Glu
Val Leu Glu Gln Gln Thr 835 840 75732PRTHomo sapiens 75Met Lys Leu
Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser
Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25
30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys
35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro
Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu
Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala
Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu
Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His
Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe
His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn
Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155
160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro
165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu
Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu
Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val
Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280
285 Ser Ser Ile Asn Asp Ser Arg Met
Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala
Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser Thr Gln
Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val
Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile 340 345
350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu
355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys Ser Asp
Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu Gly Asn
Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe Met Asn
Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn His Leu
Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His Asn Leu
Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu Ile Leu
Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455 460 Tyr
Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe Ser465 470
475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn Gln Phe Thr
His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu Asp Leu Leu
Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser Cys
Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys Leu Ser Lys
Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser Pro Gly His
Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn Ser Glu Ile
Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570 575 Thr Gln
Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn 580 585 590
Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro Leu Ser 595
600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr Ile Val Phe
Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His Arg Arg Arg
Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln Met Arg Asp
Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr Gly His Lys
Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala Ser Leu Tyr
Glu Gln His Met Gly Ala His Glu Glu Leu 675 680 685 Lys Leu Met Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val 690 695 700 Glu Gln
Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala705 710 715
720 Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr 725 730
761620DNAHomo sapiens 76tcggatttca tcacatgaca acatgaagct gtggattcat
ctcttttatt catctctcct 60tgcctgtata tctttacact cccaaactcc agtgctctca
tccagaggct cttgtgattc 120tctttgcaat tgtgaggaaa aagatggcac
aatgctaata aattgtgaag caaaaggtat 180caagatggta tctgaaataa
gtgtgccacc atcacgacct ttccaactaa gcttattaaa 240taacggcttg
acgatgcttc acacaaatga cttttctggg cttaccaatg ctatttcaat
300acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatg
gccttggcct 360cctgaaacaa cttcatatca atcacaattc tttagaaatt
cttaaagagg atactttcca 420tggactggaa aacctggaat tcctgcaagc
agataacaat tttatcacag tgattgaacc 480aagtgccttt agcaagctca
acagactcaa agtgttaatt ttaaatgaca atgctattga 540gagtcttcct
ccaaacatct tccgatttgt tcctttaacc catctagatc ttcgtggaaa
600tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaa
tattggatct 660tcagttggag gacaacaaat gggcctgcaa ttgtgactta
ttgcagttaa aaacttggtt 720ggagaacatg cctccacagt ctataattgg
tgatgttgtc tgcaacagcc ctccattttt 780taaaggaagt atactcagta
gactaaagaa ggaatctatt tgccctactc caccagtgta 840tgaagaacat
gaggatcctt caggatcatt acatctggca gcaacatctt caataaatga
900tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaag
caccaggttt 960gataccttat attacaaagc catccactca acttccagga
ccttactgcc ctattccttg 1020taactgcaaa gtcctatccc catcaggact
tctaatacat tgtcaggagc gcaacattga 1080aagcttatca gatctgagac
ctcctccgca aaatcctaga aagctcattc tagcgggaaa 1140tattattcac
agtttaatga agtccatcct ttggtccaaa gcatctggaa gaggaagaag
1200agaggaatga gaaagaagga agtgatgcaa aacatctcca aagaagtctt
ttggaacagg 1260aaaatcattc accactcaca gggtcaaata tgaaatacaa
aaccacgaac caatcaacag 1320aatttttatc cttccaagat gccagctcat
tgtacagaaa cattttagaa aaagaaaggg 1380aacttcagca actgggaatc
acagaatacc taaggaaaaa cattgctcag ctccagcctg 1440atatggaggc
acattatcct ggagcccacg aagagctgaa gttaatggaa acattaatgt
1500actcacgtcc aaggaaggta ttagtggaac agacaaaaaa tgagtatttt
gaacttaaag 1560ctaatttaca tgctgaacct gactatttag aagtcctgga
gcagcaaaca tagatggaga 1620772555DNAHomo sapiens 77tcggatttca
tcacatgaca acatgaagct gtggattcat ctcttttatt catctctcct 60tgcctgtata
tctttacact cccaaactcc agtgctctca tccagaggct cttgtgattc
120tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaag
caaaaggtat 180caagatggta tctgaaataa gtgtgccacc atcacgacct
ttccaactaa gcttattaaa 240taacggcttg acgatgcttc acacaaatga
cttttctggg cttaccaatg ctatttcaat 300acaccttgga tttaacaata
ttgcagatat tgagataggt gcatttaatg gccttggcct 360cctgaaacaa
cttcatatca atcacaattc tttagaaatt cttaaagagg atactttcca
420tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacag
tgattgaacc 480aagtgccttt agcaagctca acagactcaa agtgttaatt
ttaaatgaca atgctattga 540gagtcttcct ccaaacatct tccgatttgt
tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa acattgcctt
atgttggttt tctcgaacac attggccgaa tattggatct 660tcagttggag
gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt
720ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagcc
ctccattttt 780taaaggaagt atactcagta gactaaagaa ggaatctatt
tgccctactc caccagtgta 840tgaagaacat gaggatcctt caggatcatt
acatctggca gcaacatctt caataaatga 900tagtcgcatg tcaactaaga
ccacgtccat tctaaaacta cccaccaaag caccaggttt 960gataccttat
attacaaagc catccactca acttccagga ccttactgcc ctattccttg
1020taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagc
gcaacattga 1080aagcttatca gatctgagac ctcctccgca aaatcctaga
aagctcattc tagcgggaaa 1140tattattcac agtttaatga agtctgatct
agtggaatat ttcactttgg aaatgcttca 1200cttgggaaac aatcgtattg
aagttcttga agaaggatcg tttatgaacc taacgagatt 1260acaaaaactc
tatctaaatg gtaaccacct gaccaaatta agtaaaggca tgttccttgg
1320tctccataat cttgaatact tatatcttga atacaatgcc attaaggaaa
tactgccagg 1380aacctttaat ccaatgccta aacttaaagt cctgtattta
aataacaacc tcctccaagt 1440tttaccacca catatttttt caggggttcc
tctaactaag gtaaatctta aaacaaacca 1500gtttacccat ctacctgtaa
gtaatatttt ggatgatctt gatttactaa cccagattga 1560ccttgaggat
aacccctggg actgctcctg tgacctggtt ggactgcagc aatggataca
1620aaagttaagc aagaacacag tgacagatga catcctctgc acttcccccg
ggcatctcga 1680caaaaaggaa ttgaaagccc taaatagtga aattctctgt
ccaggtttag taaataaccc 1740atccatgcca acacagacta gttaccttat
ggtcaccact cctgcaacaa caacaaatac 1800ggctgatact attttacgat
ctcttacgga cgctgtgcca ctgtctgttc taatattggg 1860acttctgatt
atgttcatca ctattgtttt ctgtgctgca gggatagtgg ttcttgttct
1920tcaccgcagg agaagataca aaaagaaaca agtagatgag caaatgagag
acaacagtcc 1980tgtgcatctt cagtacagca tgtatggcca taaaaccact
catcacacta ctgaaagacc 2040ctctgcctca ctctatgaac agcacatggt
gagccccatg gttcatgtct atagaagtcc 2100atcctttggt ccaaagcatc
tggaagagga agaagagagg aatgagaaag aaggaagtga 2160tgcaaaacat
ctccaaagaa gtcttttgga acaggaaaat cattcaccac tcacagggtc
2220aaatatgaaa tacaaaacca cgaaccaatc aacagaattt ttatccttcc
aagatgccag 2280ctcattgtac agaaacattt tagaaaaaga aagggaactt
cagcaactgg gaatcacaga 2340atacctaagg aaaaacattg ctcagctcca
gcctgatatg gaggcacatt atcctggagc 2400ccacgaagag ctgaagttaa
tggaaacatt aatgtactca cgtccaagga aggtattagt 2460ggaacagaca
aaaaatgagt attttgaact taaagctaat ttacatgctg aacctgacta
2520tttagaagtc ctggagcagc aaacatagat ggaga 2555781620DNAHomo
sapiens 78tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttatt
catctctcct 60tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct
cttgtgattc 120tctttgcaat tgtgaggaaa aagatggcac aatgctaata
aattgtgaag caaaaggtat 180caagatggta tctgaaataa gtgtgccacc
atcacgacct ttccaactaa gcttattaaa 240taacggcttg acgatgcttc
acacaaatga cttttctggg cttaccaatg ctatttcaat 300acaccttgga
tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg
atactttcca 420tggactggaa aacctggaat tcctgcaagc agataacaat
tttatcacag tgattgaacc 480aagtgccttt agcaagctca acagactcaa
agtgttaatt ttaaatgaca atgctattga 540gagtcttcct ccaaacatct
tccgatttgt tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa
acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa
aaacttggtt 720ggagaacatg cctccacagt ctataattgg tgatgttgtc
tgcaacagcc ctccattttt 780taaaggaagt atactcagta gactaaagaa
ggaatctatt tgccctactc caccagtgta 840tgaagaacat gaggatcctt
caggatcatt acatctggca gcaacatctt caataaatga 900tagtcgcatg
tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960gataccttat attacaaagc catccactca acttccagga ccttactgcc
ctattccttg 1020taactgcaaa gtcctatccc catcaggact tctaatacat
tgtcaggagc gcaacattga 1080aagcttatca gatctgagac ctcctccgca
aaatcctaga aagctcattc tagcgggaaa 1140tattattcac agtttaatga
agtccatcct ttggtccaaa gcatctggaa gaggaagaag 1200agaggaatga
gaaagaagga agtgatgcaa aacatctcca aagaagtctt ttggaacagg
1260aaaatcattc accactcaca gggtcaaata tgaaatacaa aaccacgaac
caatcaacag 1320aatttttatc cttccaagat gccagctcat tgtacagaaa
cattttagaa aaagaaaggg 1380aacttcagca actgggaatc acagaatacc
taaggaaaaa cattgctcag ctccagcctg 1440atatggaggc acattatcct
ggagcccacg aagagctgaa gttaatggaa acattaatgt 1500actcacgtcc
aaggaaggta ttagtggaac agacaaaaaa tgagtatttt gaacttaaag
1560ctaatttaca tgctgaacct gactatttag aagtcctgga gcagcaaaca
tagatggaga 162079395PRTHomo sapiens 79Met Lys Leu Trp Ile His Leu
Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln
Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys
Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu
Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55
60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu
His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile
His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180
185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile
Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala
Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn
Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305
310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn
Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln
Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His
Ser Leu Met Lys Ser Ile Leu Trp 370 375 380 Ser Lys Ala Ser Gly Arg
Gly Arg Arg Glu Glu385 390 395 80841PRTHomo sapiens 80Met Lys Leu
Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser
Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25
30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys
35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro
Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu
Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala
Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu
Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His
Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe
His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn
Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155
160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro
165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu
Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu
Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn
Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp
Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val
Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser
Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr
Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280
285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu
290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr
Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile
Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile
His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg
Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn
Ile Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe
Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400
Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405
410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe
Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn
Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro
Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val
Leu Pro Pro His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys
Val Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser
Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu
Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525
Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530
535 540 Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala
Leu545 550 555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn
Pro Ser Met Pro
565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr
Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala
Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe
Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val
Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp
Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser
Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro
Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680
685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu
690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln
Arg Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr
Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu
Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile
Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu
Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu
Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800
Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805
810 815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro
Asp 820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
81395PRTHomo sapiens 81Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Ile Leu Trp 370 375 380 Ser Lys Ala Ser Gly Arg Gly Arg Arg Glu
Glu385 390 395 823300DNAHomo sapiens 82gcgtcgacaa caagaaatac
tagaaaagga ggaaggagaa cattgctgca gcttggatct 60acaacctaag aaagcaagag
tgatcaatct cagctctgtt aaacatcttg tttacttact 120gcattcagca
gcttgcaaat ggttaactat atgcaaaaaa gtcagcatag ctgtgaagta
180tgccgtgaat tttaattgag ggaaaaagga caattgcttc aggatgctct
agtatgcact 240ctgcttgaaa tattttcaat gaaatgctca gtattctatc
tttgaccaga ggttttaact 300ttatgaagct atgggacttg acaaaaagtg
atatttgaga agaaagtacg cagtggttgg 360tgttttcttt tttttaataa
aggaattgaa ttactttgaa cacctcttcc agctgtgcat 420tacagataac
gtcaggaaga gtctctgctt tacagaatcg gatttcatca catgacaaca
480tgaagctgtg gattcatctc ttttattcat ctctccttgc ctgtatatct
ttacactccc 540aaactccagt gctctcatcc agaggctctt gtgattctct
ttgcaattgt gaggaaaaag 600atggcacaat gctaataaat tgtgaagcaa
aaggtatcaa gatggtatct gaaataagtg 660tgccaccatc acgacctttc
caactaagct tattaaataa cggcttgacg atgcttcaca 720caaatgactt
ttctgggctt accaatgcta tttcaataca ccttggattt aacaatattg
780cagatattga gataggtgca tttaatggcc ttggcctcct gaaacaactt
catatcaatc 840acaattcttt agaaattctt aaagaggata ctttccatgg
actggaaaac ctggaattcc 900tgcaagcaga taacaatttt atcacagtga
ttgaaccaag tgcctttagc aagctcaaca 960gactcaaagt gttaatttta
aatgacaatg ctattgagag tcttcctcca aacatcttcc 1020gatttgttcc
tttaacccat ctagatcttc gtggaaatca attacaaaca ttgccttatg
1080ttggttttct cgaacacatt ggccgaatat tggatcttca gttggaggac
aacaaatggg 1140cctgcaattg tgacttattg cagttaaaaa cttggttgga
gaacatgcct ccacagtcta 1200taattggtga tgttgtctgc aacagccctc
cattttttaa aggaagtata ctcagtagac 1260taaagaagga atctatttgc
cctactccac cagtgtatga agaacatgag gatccttcag 1320gatcattaca
tctggcagca acatcttcaa taaatgatag tcgcatgtca actaagacca
1380cgtccattct aaaactaccc accaaagcac caggtttgat accttatatt
acaaagccat 1440ccactcaact tccaggacct tactgcccta ttccttgtaa
ctgcaaagtc ctatccccat 1500caggacttct aatacattgt caggagcgca
acattgaaag cttatcagat ctgagacctc 1560ctccgcaaaa tcctagaaag
ctcattctag cgggaaatat tattcacagt ttaatgaagt 1620ctgatctagt
ggaatatttc actttggaaa tgcttcactt gggaaacaat cgtattgaag
1680ttcttgaaga aggatcgttt atgaacctaa cgagattaca aaaactctat
ctaaatggta 1740accacctgac caaattaagt aaaggcatgt tccttggtct
ccataatctt gaatacttat 1800atcttgaata caatgccatt aaggaaatac
tgccaggaac ctttaatcca atgcctaaac 1860ttaaagtcct gtatttaaat
aacaacctcc tccaagtttt accaccacat attttttcag 1920gggttcctct
aactaaggta aatcttaaaa caaaccagtt tacccatcta cctgtaagta
1980atattttgga tgatcttgat ttactaaccc agattgacct tgaggataac
ccctgggact 2040gctcctgtga cctggttgga ctgcagcaat ggatacaaaa
gttaagcaag aacacagtga 2100cagatgacat cctctgcact tcccccgggc
atctcgacaa aaaggaattg aaagccctaa 2160atagtgaaat tctctgtcca
ggtttagtaa ataacccatc catgccaaca cagactagtt 2220accttatggt
caccactcct gcaacaacaa caaatacggc tgatactatt ttacgatctc
2280ttacggacgc tgtgccactg tctgttctaa tattgggact tctgattatg
ttcatcacta 2340ttgttttctg tgctgcaggg atagtggttc ttgttcttca
ccgcaggaga agatacaaaa 2400agaaacaagt agatgagcaa atgagagaca
acagtcctgt gcatcttcag tacagcatgt 2460atggccataa aaccactcat
cacactactg aaagaccctc tgcctcactc tatgaacagc 2520acatggtgag
ccccatggtt catgtctata gaagtccatc ctttggtcca aagcatctgg
2580aagaggaaga agagaggaat gagaaagaag gaagtgatgc aaaacatctc
caaagaagtc 2640ttttggaaca ggaaaatcat tcaccactca cagggtcaaa
tatgaaatac aaaaccacga 2700accaatcaac agaattttta tccttccaag
atgccagctc attgtacaga aacattttag 2760aaaaagaaag ggaacttcag
caactgggaa tcacagaata cctaaggaaa aacattgctc 2820agctccagcc
tgatatggag gcacattatc ctggagccca cgaagagctg aagttaatgg
2880aaacattaat gtactcacgt ccaaggaagg tattagtgga acagacaaaa
aatgagtatt 2940ttgaacttaa agctaattta catgctgaac ctgactattt
agaagtcctg gagcagcaaa 3000catagatgga gagttgaggg ctttcgccag
aaatgctgtg attctgttat taagtccata 3060ccttgtaaat aagtgcctta
cgtgagtgtg tcatcaatca gaacctaagc acagagtaaa 3120ctatggggaa
aaaaaaagaa gacgaaacag aaactcaggg atcactggga gaagccatgg
3180cataatcttc aggcaattta gtctgtccca aataaacata catccttggc
atgtaaatca 3240tcaagggtaa tagtaatatt catatacctg aaacgtgtct
cataggagtc ctctctgcac 3300832555DNAHomo sapiens 83tcggatttca
tcacatgaca acatgaagct gtggattcat ctcttttatt catctctcct 60tgcctgtata
tctttacact cccaaactcc agtgctctca tccagaggct cttgtgattc
120tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaag
caaaaggtat 180caagatggta tctgaaataa gtgtgccacc atcacgacct
ttccaactaa gcttattaaa 240taacggcttg acgatgcttc acacaaatga
cttttctggg cttaccaatg ctatttcaat 300acaccttgga tttaacaata
ttgcagatat tgagataggt gcatttaatg gccttggcct 360cctgaaacaa
cttcatatca atcacaattc tttagaaatt cttaaagagg atactttcca
420tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacag
tgattgaacc 480aagtgccttt agcaagctca acagactcaa agtgttaatt
ttaaatgaca atgctattga 540gagtcttcct ccaaacatct tccgatttgt
tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa acattgcctt
atgttggttt tctcgaacac attggccgaa tattggatct 660tcagttggag
gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt
720ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagcc
ctccattttt 780taaaggaagt atactcagta gactaaagaa ggaatctatt
tgccctactc caccagtgta 840tgaagaacat gaggatcctt caggatcatt
acatctggca gcaacatctt caataaatga 900tagtcgcatg tcaactaaga
ccacgtccat tctaaaacta cccaccaaag caccaggttt 960gataccttat
attacaaagc catccactca acttccagga ccttactgcc ctattccttg
1020taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagc
gcaacattga 1080aagcttatca gatctgagac ctcctccgca aaatcctaga
aagctcattc tagcgggaaa 1140tattattcac agtttaatga agtctgatct
agtggaatat ttcactttgg aaatgcttca 1200cttgggaaac aatcgtattg
aagttcttga agaaggatcg tttatgaacc taacgagatt 1260acaaaaactc
tatctaaatg gtaaccacct gaccaaatta agtaaaggca tgttccttgg
1320tctccataat cttgaatact tatatcttga atacaatgcc attaaggaaa
tactgccagg 1380aacctttaat ccaatgccta aacttaaagt cctgtattta
aataacaacc tcctccaagt 1440tttaccacca catatttttt caggggttcc
tctaactaag gtaaatctta aaacaaacca 1500gtttacccat ctacctgtaa
gtaatatttt ggatgatctt gatttactaa cccagattga 1560ccttgaggat
aacccctggg actgctcctg tgacctggtt ggactgcagc aatggataca
1620aaagttaagc aagaacacag tgacagatga catcctctgc acttcccccg
ggcatctcga 1680caaaaaggaa ttgaaagccc taaatagtga aattctctgt
ccaggtttag taaataaccc 1740atccatgcca acacagacta gttaccttat
ggtcaccact cctgcaacaa caacaaatac 1800ggctgatact attttacgat
ctcttacgga cgctgtgcca ctgtctgttc taatattggg 1860acttctgatt
atgttcatca ctattgtttt ctgtgctgca gggatagtgg ttcttgttct
1920tcaccgcagg agaagataca aaaagaaaca agtagatgag caaatgagag
acaacagtcc 1980tgtgcatctt cagtacagca tgtatggcca taaaaccact
catcacacta ctgaaagacc 2040ctctgcctca ctctatgaac agcacatggt
gagccccatg gttcatgtct atagaagtcc 2100atcctttggt ccaaagcatc
tggaagagga agaagagagg aatgagaaag aaggaagtga 2160tgcaaaacat
ctccaaagaa gtcttttgga acaggaaaat cattcaccac tcacagggtc
2220aaatatgaaa tacaaaacca cgaaccaatc aacagaattt ttatccttcc
aagatgccag 2280ctcattgtac agaaacattt tagaaaaaga aagggaactt
cagcaactgg gaatcacaga 2340atacctaagg aaaaacattg ctcagctcca
gcctgatatg gaggcacatt atcctggagc 2400ccacgaagag ctgaagttaa
tggaaacatt aatgtactca cgtccaagga aggtattagt 2460ggaacagaca
aaaaatgagt attttgaact taaagctaat ttacatgctg aacctgacta
2520tttagaagtc ctggagcagc aaacatagat ggaga 2555843300DNAHomo
sapiens 84gcgtcgacaa caagaaatac tagaaaagga ggaaggagaa cattgctgca
gcttggatct 60acaacctaag aaagcaagag tgatcaatct cagctctgtt aaacatcttg
tttacttact 120gcattcagca gcttgcaaat ggttaactat atgcaaaaaa
gtcagcatag ctgtgaagta 180tgccgtgaat tttaattgag ggaaaaagga
caattgcttc aggatgctct agtatgcact 240ctgcttgaaa tattttcaat
gaaatgctca gtattctatc tttgaccaga ggttttaact 300ttatgaagct
atgggacttg acaaaaagtg atatttgaga agaaagtacg cagtggttgg
360tgttttcttt tttttaataa aggaattgaa ttactttgaa cacctcttcc
agctgtgcat 420tacagataac gtcaggaaga gtctctgctt tacagaatcg
gatttcatca catgacaaca 480tgaagctgtg gattcatctc ttttattcat
ctctccttgc ctgtatatct ttacactccc 540aaactccagt gctctcatcc
agaggctctt gtgattctct ttgcaattgt gaggaaaaag 600atggcacaat
gctaataaat tgtgaagcaa aaggtatcaa gatggtatct gaaataagtg
660tgccaccatc acgacctttc caactaagct tattaaataa cggcttgacg
atgcttcaca 720caaatgactt ttctgggctt accaatgcta tttcaataca
ccttggattt aacaatattg 780cagatattga gataggtgca tttaatggcc
ttggcctcct gaaacaactt catatcaatc 840acaattcttt agaaattctt
aaagaggata ctttccatgg actggaaaac ctggaattcc 900tgcaagcaga
taacaatttt atcacagtga ttgaaccaag tgcctttagc aagctcaaca
960gactcaaagt gttaatttta aatgacaatg ctattgagag tcttcctcca
aacatcttcc 1020gatttgttcc tttaacccat ctagatcttc gtggaaatca
attacaaaca ttgccttatg 1080ttggttttct cgaacacatt ggccgaatat
tggatcttca gttggaggac aacaaatggg 1140cctgcaattg tgacttattg
cagttaaaaa cttggttgga gaacatgcct ccacagtcta 1200taattggtga
tgttgtctgc aacagccctc cattttttaa aggaagtata ctcagtagac
1260taaagaagga atctatttgc cctactccac cagtgtatga agaacatgag
gatccttcag 1320gatcattaca tctggcagca acatcttcaa taaatgatag
tcgcatgtca actaagacca 1380cgtccattct aaaactaccc accaaagcac
caggtttgat accttatatt acaaagccat 1440ccactcaact tccaggacct
tactgcccta ttccttgtaa ctgcaaagtc ctatccccat 1500caggacttct
aatacattgt caggagcgca acattgaaag cttatcagat ctgagacctc
1560ctccgcaaaa tcctagaaag ctcattctag cgggaaatat tattcacagt
ttaatgaagt 1620ctgatctagt ggaatatttc actttggaaa tgcttcactt
gggaaacaat cgtattgaag 1680ttcttgaaga aggatcgttt atgaacctaa
cgagattaca aaaactctat ctaaatggta 1740accacctgac caaattaagt
aaaggcatgt tccttggtct ccataatctt gaatacttat 1800atcttgaata
caatgccatt aaggaaatac tgccaggaac ctttaatcca atgcctaaac
1860ttaaagtcct gtatttaaat aacaacctcc tccaagtttt accaccacat
attttttcag 1920gggttcctct aactaaggta aatcttaaaa caaaccagtt
tacccatcta cctgtaagta 1980atattttgga tgatcttgat ttactaaccc
agattgacct tgaggataac ccctgggact 2040gctcctgtga cctggttgga
ctgcagcaat ggatacaaaa gttaagcaag aacacagtga 2100cagatgacat
cctctgcact tcccccgggc atctcgacaa aaaggaattg aaagccctaa
2160atagtgaaat tctctgtcca ggtttagtaa ataacccatc catgccaaca
cagactagtt 2220accttatggt caccactcct gcaacaacaa caaatacggc
tgatactatt ttacgatctc 2280ttacggacgc tgtgccactg tctgttctaa
tattgggact tctgattatg ttcatcacta 2340ttgttttctg tgctgcaggg
atagtggttc ttgttcttca ccgcaggaga agatacaaaa 2400agaaacaagt
agatgagcaa atgagagaca acagtcctgt gcatcttcag tacagcatgt
2460atggccataa aaccactcat cacactactg aaagaccctc tgcctcactc
tatgaacagc 2520acatggtgag ccccatggtt catgtctata gaagtccatc
ctttggtcca aagcatctgg 2580aagaggaaga agagaggaat gagaaagaag
gaagtgatgc aaaacatctc caaagaagtc 2640ttttggaaca ggaaaatcat
tcaccactca cagggtcaaa tatgaaatac aaaaccacga 2700accaatcaac
agaattttta tccttccaag atgccagctc attgtacaga aacattttag
2760aaaaagaaag ggaacttcag caactgggaa tcacagaata cctaaggaaa
aacattgctc 2820agctccagcc tgatatggag gcacattatc ctggagccca
cgaagagctg aagttaatgg 2880aaacattaat gtactcacgt ccaaggaagg
tattagtgga acagacaaaa aatgagtatt 2940ttgaacttaa agctaattta
catgctgaac ctgactattt agaagtcctg gagcagcaaa 3000catagatgga
gagttgaggg ctttcgccag aaatgctgtg attctgttat taagtccata
3060ccttgtaaat aagtgcctta cgtgagtgtg tcatcaatca gaacctaagc
acagagtaaa 3120ctatggggaa aaaaaaagaa gacgaaacag aaactcaggg
atcactggga gaagccatgg 3180cataatcttc aggcaattta gtctgtccca
aataaacata catccttggc atgtaaatca 3240tcaagggtaa tagtaatatt
catatacctg aaacgtgtct cataggagtc ctctctgcac 330085841PRTHomo
sapiens 85Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala
Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg
Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly
Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val
Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser
Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe
Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn
Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110
Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115
120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala
Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser
Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn
Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro
Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu
Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp
Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
86841PRTHomo sapiens 86Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
87841PRTHomo sapiens 87Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
881619DNAHomo sapiens 88tcggatttca tcacatgaca acatgaagct gtggattcat
ctcttttatt
catctctcct 60tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct
cttgtgattc 120tctttgcaat tgtgaggaaa aagatggcac aatgctaata
aattgtgaag caaaaggtat 180caagatggta tctgaaataa gtgtgccacc
atcacgacct ttccaactaa gcttattaaa 240taacggcttg acgatgcttc
acacaaatga cttttctggg cttaccaatg ctatttcaat 300acaccttgga
tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg
atactttcca 420tggactggaa aacctggaat tcctgcaagc agataacaat
tttatcacag tgattgaacc 480aagtgccttt agcaagctca acagactcaa
agtgttaatt ttaaatgaca atgctattga 540gagtcttcct ccaaacatct
tccgatttgt tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa
acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa
aaacttggtt 720ggagaacatg cctccacagt ctataattgg tgatgttgtc
tgcaacagcc ctccattttt 780taaaggaagt atactcagta gactaaagaa
ggaatctatt tgccctactc caccagtgta 840tgaagaacat gaggatcctt
caggatcatt acatctggca gcaacatctt caataaatga 900tagtcgcatg
tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960gataccttat attacaaagc catccactca acttccagga ccttactgcc
ctattccttg 1020taactgcaaa gtcctatccc catcaggact tctaatacat
tgtcaggagc gcaacattga 1080aagcttatca gatctgagac ctcctccgca
aaatcctaga aagctcattc tagcgggaaa 1140tattattcac agtttaatga
atccatcctt tggtccaaag catctggaag aggaagaaga 1200gaggaatgag
aaagaaggaa gtgatgcaaa acatctccaa agaagtcttt tggaacagga
1260aaatcattca ccactcacag ggtcaaatat gaaatacaaa accacgaacc
aatcaacaga 1320atttttatcc ttccaagatg ccagctcatt gtacagaaac
attttagaaa aagaaaggga 1380acttcagcaa ctgggaatca cagaatacct
aaggaaaaac attgctcagc tccagcctga 1440tatggaggca cattatcctg
gagcccacga agagctgaag ttaatggaaa cattaatgta 1500ctcacgtcca
aggaaggtat tagtggaaca gacaaaaaat gagtattttg aacttaaagc
1560taatttacat gctgaacctg actatttaga agtcctggag cagcaaacat
agatggaga 1619891619DNAHomo sapiens 89tcggatttca tcacatgaca
acatgaagct gtggattcat ctcttttatt catctctcct 60tgcctgtata tctttacact
cccaaactcc agtgctctca tccagaggct cttgtgattc 120tctttgcaat
tgtgaggaaa aagatggcac aatgctaata aattgtgaag caaaaggtat
180caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaa
gcttattaaa 240taacggcttg acgatgcttc acacaaatga cttttctggg
cttaccaatg ctatttcaat 300acaccttgga tttaacaata ttgcagatat
tgagataggt gcatttaatg gccttggcct 360cctgaaacaa cttcatatca
atcacaattc tttagaaatt cttaaagagg atactttcca 420tggactggaa
aacctggaat tcctgcaagc agataacaat tttatcacag tgattgaacc
480aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgaca
atgctattga 540gagtcttcct ccaaacatct tccgatttgt tcctttaacc
catctagatc ttcgtggaaa 600tcaattacaa acattgcctt atgttggttt
tctcgaacac attggccgaa tattggatct 660tcagttggag gacaacaaat
gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt 720ggagaacatg
cctccacagt ctataattgg tgatgttgtc tgcaacagcc ctccattttt
780taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactc
caccagtgta 840tgaagaacat gaggatcctt caggatcatt acatctggca
gcaacatctt caataaatga 900tagtcgcatg tcaactaaga ccacgtccat
tctaaaacta cccaccaaag caccaggttt 960gataccttat attacaaagc
catccactca acttccagga ccttactgcc ctattccttg 1020taactgcaaa
gtcctatccc catcaggact tctaatacat tgtcaggagc gcaacattga
1080aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattc
tagcgggaaa 1140tattattcac agtttaatga atccatcctt tggtccaaag
catctggaag aggaagaaga 1200gaggaatgag aaagaaggaa gtgatgcaaa
acatctccaa agaagtcttt tggaacagga 1260aaatcattca ccactcacag
ggtcaaatat gaaatacaaa accacgaacc aatcaacaga 1320atttttatcc
ttccaagatg ccagctcatt gtacagaaac attttagaaa aagaaaggga
1380acttcagcaa ctgggaatca cagaatacct aaggaaaaac attgctcagc
tccagcctga 1440tatggaggca cattatcctg gagcccacga agagctgaag
ttaatggaaa cattaatgta 1500ctcacgtcca aggaaggtat tagtggaaca
gacaaaaaat gagtattttg aacttaaagc 1560taatttacat gctgaacctg
actatttaga agtcctggag cagcaaacat agatggaga 1619901619DNAHomo
sapiens 90tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttatt
catctctcct 60tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct
cttgtgattc 120tctttgcaat tgtgaggaaa aagatggcac aatgctaata
aattgtgaag caaaaggtat 180caagatggta tctgaaataa gtgtgccacc
atcacgacct ttccaactaa gcttattaaa 240taacggcttg acgatgcttc
acacaaatga cttttctggg cttaccaatg ctatttcaat 300acaccttgga
tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg
atactttcca 420tggactggaa aacctggaat tcctgcaagc agataacaat
tttatcacag tgattgaacc 480aagtgccttt agcaagctca acagactcaa
agtgttaatt ttaaatgaca atgctattga 540gagtcttcct ccaaacatct
tccgatttgt tcctttaacc catctagatc ttcgtggaaa 600tcaattacaa
acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa
aaacttggtt 720ggagaacatg cctccacagt ctataattgg tgatgttgtc
tgcaacagcc ctccattttt 780taaaggaagt atactcagta gactaaagaa
ggaatctatt tgccctactc caccagtgta 840tgaagaacat gaggatcctt
caggatcatt acatctggca gcaacatctt caataaatga 900tagtcgcatg
tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960gataccttat attacaaagc catccactca acttccagga ccttactgcc
ctattccttg 1020taactgcaaa gtcctatccc catcaggact tctaatacat
tgtcaggagc gcaacattga 1080aagcttatca gatctgagac ctcctccgca
aaatcctaga aagctcattc tagcgggaaa 1140tattattcac agtttaatga
atccatcctt tggtccaaag catctggaag aggaagaaga 1200gaggaatgag
aaagaaggaa gtgatgcaaa acatctccaa agaagtcttt tggaacagga
1260aaatcattca ccactcacag ggtcaaatat gaaatacaaa accacgaacc
aatcaacaga 1320atttttatcc ttccaagatg ccagctcatt gtacagaaac
attttagaaa aagaaaggga 1380acttcagcaa ctgggaatca cagaatacct
aaggaaaaac attgctcagc tccagcctga 1440tatggaggca cattatcctg
gagcccacga agagctgaag ttaatggaaa cattaatgta 1500ctcacgtcca
aggaaggtat tagtggaaca gacaaaaaat gagtattttg aacttaaagc
1560taatttacat gctgaacctg actatttaga agtcctggag cagcaaacat
agatggaga 161991529PRTHomo sapiens 91Met Lys Leu Trp Ile His Leu
Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln
Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys
Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu
Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55
60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu
His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile
His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180
185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile
Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala
Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn
Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305
310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn
Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln
Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His
Ser Leu Met Asn Pro Ser Phe Gly 370 375 380 Pro Lys His Leu Glu Glu
Glu Glu Glu Arg Asn Glu Lys Glu Gly Ser385 390 395 400 Asp Ala Lys
His Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn His Ser 405 410 415 Pro
Leu Thr Gly Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr 420 425
430 Glu Phe Leu Ser Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn Ile Leu
435 440 445 Glu Lys Glu Arg Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr
Leu Arg 450 455 460 Lys Asn Ile Ala Gln Leu Gln Pro Asp Met Glu Ala
His Tyr Pro Gly465 470 475 480 Ala His Glu Glu Leu Lys Leu Met Glu
Thr Leu Met Tyr Ser Arg Pro 485 490 495 Arg Lys Val Leu Val Glu Gln
Thr Lys Asn Glu Tyr Phe Glu Leu Lys 500 505 510 Ala Asn Leu His Ala
Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln 515 520 525 Thr
92841PRTHomo sapiens 92Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
93529PRTHomo sapiens 93Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His 65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185
190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly
195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys
Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met
Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser
Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys
Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu
Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile
Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys
Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310
315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys
Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu
Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln
Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser
Leu Met Asn Pro Ser Phe Gly 370 375 380 Pro Lys His Leu Glu Glu Glu
Glu Glu Arg Asn Glu Lys Glu Gly Ser385 390 395 400 Asp Ala Lys His
Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn His Ser 405 410 415 Pro Leu
Thr Gly Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr 420 425 430
Glu Phe Leu Ser Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn Ile Leu 435
440 445 Glu Lys Glu Arg Glu Leu Gln Gln Leu Gly Ile Thr Glu Tyr Leu
Arg 450 455 460 Lys Asn Ile Ala Gln Leu Gln Pro Asp Met Glu Ala His
Tyr Pro Gly465 470 475 480 Ala His Glu Glu Leu Lys Leu Met Glu Thr
Leu Met Tyr Ser Arg Pro 485 490 495 Arg Lys Val Leu Val Glu Gln Thr
Lys Asn Glu Tyr Phe Glu Leu Lys 500 505 510 Ala Asn Leu His Ala Glu
Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln 515 520 525 Thr
94841PRTHomo sapiens 94Met Lys Leu Trp Ile His Leu Phe Tyr Ser Ser
Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr Pro Val Leu
Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn Cys Glu Glu
Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675 680 685 Val
Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu Glu 690 695
700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu Gln Arg
Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu Thr Gly
Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr Glu Phe
Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn Ile Leu
Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr Glu Tyr
Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met Glu Ala
His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795 800 Glu
Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr 805 810
815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu Pro Asp
820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840 9516PRTHomo
sapiens 95Ala Ser Leu Tyr Glu Gln His Met Gly Ala His Glu Glu Leu
Lys Leu1 5 10 15 9618PRTHomo sapiens 96Ser Ala Ser Leu Tyr Glu Gln
His Met Gly Ala His Glu Glu Leu Lys1 5 10 15 Leu Met9728PRTHomo
sapiens 97Thr Thr Glu Arg Pro Ser Ala Ser Leu Tyr Glu Gln His Met
Gly Ala1 5 10 15 His Glu Glu Leu Lys Leu Met Glu Thr Leu Met Tyr 20
25 9822PRTHomo sapiens 98Ile Ile His Ser Leu Met Lys Ser Ile Leu
Trp Ser Lys Ala Ser Gly1 5 10 15 Arg Gly Arg Arg Glu Glu 20
9923PRTHomo sapiens 99Asn Ile Ile His Ser Leu Met Lys Ser Ile Leu
Trp Ser Lys Ala Ser1 5 10 15 Gly Arg Gly Arg Arg Glu Glu 20
10028PRTHomo sapiens 100Leu Ile Leu Ala Gly Asn Ile Ile His Ser Leu
Met Lys Ser Ile Leu1 5 10 15 Trp Ser Lys Ala Ser Gly Arg Gly Arg
Arg Glu Glu 20 25 10123PRTHomo sapiens 101Gly Asn Ile Ile His Ser
Leu Met Asn Pro Ser Phe Gly Pro Lys His1 5 10 15 Leu Glu Glu Glu
Glu Glu Arg 20 10224PRTHomo sapiens 102Ala Gly Asn Ile Ile His Ser
Leu Met Asn Pro Ser Phe Gly Pro Lys1 5 10 15 His Leu Glu Glu Glu
Glu Glu Arg 20 10329PRTHomo sapiens 103Arg Lys Leu Ile Leu Ala Gly
Asn Ile Ile His Ser Leu Met Asn Pro1 5 10 15 Ser Phe Gly Pro Lys
His Leu Glu Glu Glu Glu Glu Arg 20 25 104841PRTHomo sapiens 104Met
Lys Leu Trp Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10
15 Ser Leu His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp
20 25 30 Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile
Asn Cys 35 40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser
Val Pro Pro Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn
Gly Leu Thr Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr
Asn Ala Ile Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp
Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln
Leu His Ile Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp
Thr Phe His Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140
Asn Asn Phe Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145
150 155 160 Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser
Leu Pro 165 170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu
Asp Leu Arg Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly
Phe Leu Glu His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu
Asp Asn Lys Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys
Thr Trp Leu Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly
Asp Val Val Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile
Leu Ser Arg Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265
270 Tyr Glu Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr
275 280 285 Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser
Ile Leu 290 295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr
Ile Thr Lys Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys
Pro Ile Pro Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu
Leu Ile His Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp
Leu Arg Pro Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala
Gly Asn Ile Ile His Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu
Tyr Phe Thr Leu Glu Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390
395 400 Val Leu Glu Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys
Leu 405 410 415 Tyr Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly
Met Phe Leu 420 425 430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu
Tyr Asn Ala Ile Lys 435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro
Met Pro Lys Leu Lys Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu
Gln Val Leu Pro Pro His Ile Phe Ser465 470 475 480 Gly Val Pro Leu
Thr Lys Val Asn Leu Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro
Val Ser Asn Ile Leu Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510
Asp Leu Glu Asp Asn Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515
520 525 Gln Gln Trp Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp
Ile 530 535 540 Leu Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu
Lys Ala Leu545 550 555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val
Asn Asn Pro Ser Met Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val
Thr Thr Pro Ala Thr Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu
Arg Ser Leu Thr Asp Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu
Gly Leu Leu Ile Met Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala
Gly Ile Val Val Leu Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635
640 Lys Lys Gln Val Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu
645 650 655 Gln Tyr Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr
Glu Arg 660 665 670 Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser
Pro Met Val His 675 680 685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys
His Leu Glu Glu Glu Glu 690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser
Asp Ala Lys His Leu Gln Arg Ser705 710 715 720 Leu Leu Glu Gln Glu
Asn His Ser Pro Leu Thr Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr
Thr Asn Gln Ser Thr Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser
Ser Leu Tyr Arg Asn Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760
765 Leu Gly Ile Thr Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro
770 775 780 Asp Met Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys
Leu Met785 790 795 800 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val
Leu Val Glu Gln Thr 805 810 815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala
Asn Leu His Ala Glu Pro Asp 820 825 830 Tyr Leu Glu Val Leu Glu Gln
Gln Thr 835 840 105732PRTHomo sapiens 105Met Lys Leu Trp Ile His
Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser
Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu
Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45
Glu Ala Lys Gly Ile
Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60 Arg Pro Phe
Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65 70 75 80 Thr
Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His Leu Gly 85 90
95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe Asn Gly Leu Gly
100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn Ser Leu Glu Ile
Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu Asn Leu Glu Phe
Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val Ile Glu Pro Ser
Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys Val Leu Ile Leu
Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro Asn Ile Phe Arg
Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185 190 Asn Gln Leu
Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly 195 200 205 Arg
Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys Asn Cys 210 215
220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met Pro Pro Gln
Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser Pro Pro Phe
Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys Glu Ser Ile
Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu Asp Pro Ser
Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile Asn Asp Ser
Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys Leu Pro Thr
Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310 315 320 Ser
Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys Lys 325 330
335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu Arg Asn Ile
340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln Asn Pro Arg
Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser Leu Met Lys
Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu Met Leu His Leu
Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu Glu Gly Ser Phe
Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr Leu Asn Gly Asn
His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425 430 Gly Leu His
Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys 435 440 445 Glu
Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys Val Leu 450 455
460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro His Ile Phe
Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu Lys Thr Asn
Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu Asp Asp Leu
Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn Pro Trp Asp
Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp Ile Gln Lys
Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu Cys Thr Ser
Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550 555 560 Asn
Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met Pro 565 570
575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr Thr Thr Asn
580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp Ala Val Pro
Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met Phe Ile Thr
Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu Val Leu His
Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val Asp Glu Gln
Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr Ser Met Tyr
Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670 Pro Ser Ala
Ser Leu Tyr Glu Gln His Met Gly Ala His Glu Glu Leu 675 680 685 Lys
Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val 690 695
700 Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His
Ala705 710 715 720 Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr
725 730 106395PRTHomo sapiens 106Met Lys Leu Trp Ile His Leu Phe
Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln Thr
Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys Asn
Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu Ala
Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55 60
Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu His65
70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile His
Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala Phe
Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His Asn
Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu Glu
Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu Lys
Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175 Pro
Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180 185
190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile Gly
195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala Cys
Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn Met
Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn Ser
Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys Lys
Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His Glu
Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser Ile
Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300 Lys
Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305 310
315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn Cys
Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln Glu
Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro Gln
Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His Ser
Leu Met Lys Ser Ile Leu Trp 370 375 380 Ser Lys Ala Ser Gly Arg Gly
Arg Arg Glu Glu385 390 395 107529PRTHomo sapiens 107Met Lys Leu Trp
Ile His Leu Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu
His Ser Gln Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30
Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35
40 45 Glu Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro
Ser 50 55 60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr
Met Leu His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile
Ser Ile His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile
Gly Ala Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile
Asn His Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His
Gly Leu Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe
Ile Thr Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160
Arg Leu Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165
170 175 Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg
Gly 180 185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu
His Ile Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys
Trp Ala Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu
Glu Asn Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val
Cys Asn Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg
Leu Lys Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu
Glu His Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285
Ser Ser Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290
295 300 Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys
Pro305 310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro
Cys Asn Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His
Cys Gln Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro
Pro Pro Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile
Ile His Ser Leu Met Asn Pro Ser Phe Gly 370 375 380 Pro Lys His Leu
Glu Glu Glu Glu Glu Arg Asn Glu Lys Glu Gly Ser385 390 395 400 Asp
Ala Lys His Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn His Ser 405 410
415 Pro Leu Thr Gly Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln Ser Thr
420 425 430 Glu Phe Leu Ser Phe Gln Asp Ala Ser Ser Leu Tyr Arg Asn
Ile Leu 435 440 445 Glu Lys Glu Arg Glu Leu Gln Gln Leu Gly Ile Thr
Glu Tyr Leu Arg 450 455 460 Lys Asn Ile Ala Gln Leu Gln Pro Asp Met
Glu Ala His Tyr Pro Gly465 470 475 480 Ala His Glu Glu Leu Lys Leu
Met Glu Thr Leu Met Tyr Ser Arg Pro 485 490 495 Arg Lys Val Leu Val
Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys 500 505 510 Ala Asn Leu
His Ala Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln 515 520 525 Thr
108347DNAHomo sapiens 108caaactgcag gagtcaggag ttggcctggt
ggcgccctca cagagcctgt ccatcacatg 60caccgtctca ggattctcat tgaccggcta
tggtgtaaac tgggttcgcc agcctccagg 120aaagggtctg gggtggctgg
gaatgatttg gggcgatgga agcacagatt atacttcagc 180tctccaatcc
agactgagca tcaggaagga caattcaaga gccaaacttt cttaaaaaat
240aacagtctgc aaactgatga cacagccagg tattactgtg ccagagatga
agggagggga 300ctctgtttga ttgctggggc caagggacca cggtcaccgt ctcctca
347109115PRTHomo sapiens 109Gln Thr Ala Gly Val Arg Ser Trp Pro Gly
Gly Ala Leu Thr Glu Pro1 5 10 15 Val His His Met His Arg Leu Arg
Ile Leu Ile Asp Arg Leu Trp Cys 20 25 30 Lys Leu Gly Ser Pro Ala
Ser Arg Lys Gly Ser Gly Val Ala Gly Asn 35 40 45 Asp Leu Gly Arg
Trp Lys His Arg Leu Tyr Phe Ser Ser Pro Ile Gln 50 55 60 Thr Glu
His Gln Glu Gly Gln Phe Lys Ser Gln Thr Phe Leu Lys Asn65 70 75 80
Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp 85
90 95 Glu Gly Arg Gly Leu Cys Leu Ile Ala Gly Ala Lys Gly Pro Arg
Ser 100 105 110 Pro Ser Pro 115 110330DNAHomo sapiens 110gacattcagc
tgacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60atctcataca
gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac
120caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa
cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag
acttcaccct caacatccat 240cctgtggagg aggaggatgc tgcaacctat
tactgtcagc acattaggga gcttacacgt 300tcggaggggg gaccaagctg
gagatctaac 330111110PRTHomo sapiens 111Asp Ile Gln Leu Thr Gln Ser
Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15 Gln Arg Ala Thr Ile
Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser
Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg
Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp
Arg Ser Asn 100 105 110 112115PRTHomo sapiens 112Gln Thr Ala Gly
Val Arg Ser Trp Pro Gly Gly Ala Leu Thr Glu Pro1 5 10 15 Val His
His Met His Arg Leu Arg Ile Leu Ile Asp Arg Leu Trp Cys 20 25 30
Lys Leu Gly Ser Pro Ala Ser Arg Lys Gly Ser Gly Val Ala Gly Asn 35
40 45 Asp Leu Gly Arg Trp Lys His Arg Leu Tyr Phe Ser Ser Pro Ile
Gln 50 55 60 Thr Glu His Gln Glu Gly Gln Phe Lys Ser Gln Thr Phe
Leu Lys Asn65 70 75 80 Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr
Tyr Cys Ala Arg Asp 85 90 95 Glu Gly Arg Gly Leu Cys Leu Ile Ala
Gly Ala Lys Gly Pro Arg Ser 100 105 110 Pro Ser Pro 115
113110PRTHomo sapiens 113Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg
Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His
Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile
Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85
90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Arg Ser Asn 100
105 110
* * * * *
References