U.S. patent application number 11/708975 was filed with the patent office on 2007-12-27 for nucleic acids and corresponding proteins entitled 273p4b7 useful in treatment and detection of cancer.
This patent application is currently assigned to AGENSYS, INC.. Invention is credited to Pia M. Challita-Eid, Mary Faris, Wangmao Ge, Aya Jakobovits, Arthur B. Raitano.
Application Number | 20070298424 11/708975 |
Document ID | / |
Family ID | 31891426 |
Filed Date | 2007-12-27 |
United States Patent
Application |
20070298424 |
Kind Code |
A1 |
Challita-Eid; Pia M. ; et
al. |
December 27, 2007 |
Nucleic acids and corresponding proteins entitled 273P4B7 useful in
treatment and detection of cancer
Abstract
A novel gene 273P4B7 and its encoded protein, and variants
thereof, are described wherein 273P4B7 exhibits tissue specific
expression in normal adult tissue, and is aberrantly expressed in
the cancers listed in Table 1. Consequently, 273P4B7 provides a
diagnostic, prognostic, prophylactic and/or therapeutic target for
cancer. The 273P4B7 gene or fragment thereof, or its encoded
protein, or variants thereof, or a fragment thereof, can be used to
elicit a humoral or cellular immune response; antibodies or T cells
reactive with 273P4B7 can be used in active or passive
immunization.
Inventors: |
Challita-Eid; Pia M.;
(Encino, CA) ; Faris; Mary; (Los Angeles, CA)
; Raitano; Arthur B.; (Los Angeles, CA) ;
Jakobovits; Aya; (Beverly Hills, CA) ; Ge;
Wangmao; (Culver City, CA) |
Correspondence
Address: |
AGENSYS C/O MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Assignee: |
AGENSYS, INC.
Santa Monica
CA
|
Family ID: |
31891426 |
Appl. No.: |
11/708975 |
Filed: |
February 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10641633 |
Aug 15, 2003 |
7250498 |
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11708975 |
Feb 20, 2007 |
|
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60404306 |
Aug 16, 2002 |
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60423290 |
Nov 1, 2002 |
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Current U.S.
Class: |
435/6.14 ;
435/7.92; 436/501; 436/503 |
Current CPC
Class: |
Y02A 50/30 20180101;
G01N 33/57484 20130101; A61P 43/00 20180101; C12N 5/0693 20130101;
A61K 2039/505 20130101; Y02A 50/466 20180101; A61K 47/6851
20170801; C07K 14/47 20130101; A61P 37/04 20180101; A01K 2217/075
20130101; C07K 14/435 20130101; A61P 35/00 20180101; A61K 38/00
20130101; C07K 2319/00 20130101; A01K 2217/05 20130101 |
Class at
Publication: |
435/006 ;
435/007.92; 436/501; 436/503 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; G01N 33/566 20060101
G01N033/566; G01N 33/567 20060101 G01N033/567 |
Claims
1. A method for detecting a 273P4B7 gene product in a sample,
comprising: providing the sample and a probe that specifically
binds to the 273P4B7 gene product, wherein the gene product is
encoded directly or indirectly by the nucleotide sequence of SEQ ID
NO:2, and wherein the gene product is selected from the group
consisting of a protein or an mRNA; contacting the sample with the
probe; and determining if the probe is bound specifically to the
273P4B7 gene product, wherein specific probe binding is evidence of
the gene product in the sample.
2. The method of claim 1, wherein the protein comprising the amino
acid sequence of SEQ ID NO:3.
3. The method of claim 2, wherein the probe is an antibody or
antigen-binding fragment thereof that specifically binds to the
protein.
4. The method of claim 1, wherein the mRNA complimentary to SEQ ID
NO:2.
5. The method of claim 4, wherein the probe is a nucleotide
sequence derived from SEQ ID NO:2, which specifically binds to the
mRNA.
6. The method of claim 1, wherein the probe is labeled with a
detectable marker.
7. The method of claim 6, wherein the detectable marker is selected
from the group consisting of a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent compound, a
metal chelator and an enzyme.
8. The method of claim 1, wherein the sample is selected from the
group consisting of serum, blood, urine and tissue.
9. The method of claim 1, wherein the sample is a tissue
sample.
10. The method of claim 9, wherein the tissue sample is obtained
from a source selected from the group consisting of prostate,
testis, kidney, brain, bone, skin, ovary, breast, pancreas, colon,
and lung.
11. A method for determining relative levels of a 273P4B7 gene
product in a test sample and a normal sample from a corresponding
source, comprising: providing the test sample and the normal
sample; contacting the test sample and the normal sample with a
probe that specifically binds to the 273P4B7 gene product, wherein
the gene product is encoded directly or indirectly by the
nucleotide sequence of SEQ ID NO:2, and wherein the gene product is
selected from the group consisting of a protein or an mRNA;
measuring binding of the probe bound specifically to the 273P4B7
gene product in the test sample and the normal sample; and
comparing the measurements of bind in the test sample and the
normal sample to, determining relative levels of the 273P4B7 gene
product in each sample.
12. The method of claim 11, wherein the protein comprising the
amino acid sequence of SEQ ID NO:3.
13. The method of claim 12, wherein the probe is an antibody or
antigen-binding fragment thereof that specifically binds to the
protein.
14. The method of claim 13, wherein the mRNA complimentary to SEQ
ID NO:2.
15. The method of claim 13, wherein the probe is a nucleotide
sequence derived from SEQ ID NO:2, which specifically binds to the
mRNA.
16. The method of claim 11, wherein the probe is labeled with a
detectable marker.
17. The method of claim 16, wherein the detectable marker is
selected from the group consisting of a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent compound, a
metal chelator and an enzyme.
18. The method of claim 11, wherein the sample is selected from the
group consisting of serum, blood, urine and tissue.
19. The method of claim 11, wherein the test sample and the normal
sample is a tissue sample.
20. The method of claim 19, wherein the tissue sample is obtained
from a source selected from the group consisting of prostate,
testis, kidney, brain, bone, skin, ovary, breast, pancreas, colon,
and lung.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/641,633, filed Aug. 15, 2003, and now U.S. Pat. No.
______, which claims the benefit of priority of U.S. Provisional
Application No. 60/404,306, filed Aug. 16, 2002 (expired), and U.S.
Provisional Application No. 60/423,290 Nov. 1, 2002 (expired), all
of which are hereby incorporated by reference in their
entirety.
REFERENCE TO LIST OF TABLES (APPENDIX) SUBMITTED ON COMPACT
DISC
[0002] The Compact Disc Appendix, which is a part of the present
disclosure, is provided in duplicate on compact discs (CD-Rs). Each
contains the following file: 511582008710 Tables, having a date of
creation of Feb. 14, 2007 and a file size of 253,952 bytes. All the
material on the compact discs is hereby expressly incorporated by
reference into the present application.
TECHNICAL FIELD
[0003] The invention described herein relates to genes and their
encoded proteins, termed 273P4B7 and variants thereof, expressed in
certain cancers, and to diagnostic and therapeutic methods and
compositions useful in the management of cancers that express
273P4B7.
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] Worldwide, several cancers stand out as the leading killers.
In particular, carcinomas of the lung, prostate, breast, colon,
pancreas, and ovary represent the 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, common experience has
shown that 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.
[0006] Worldwide, prostate cancer is the fourth most prevalent
cancer in men. 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.
[0007] 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.
[0008] Progress in identifying additional specific markers for
prostate cancer has been improved by the generation of prostate
cancer xenografts that can recapitulate different stages of the
disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts
are prostate cancer xenografts that have survived passage in severe
combined immune deficient (SCID) mice and have exhibited the
capacity to mimic the transition from androgen dependence to
androgen independence (Klein et al., 1997, Nat. Med. 3:402). More
recently identified prostate cancer markers include PCTA-1 (Su et
al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific
membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2
(9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999
Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)
(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
[0009] 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. 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.
[0010] 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.
[0011] 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 eight per 100,000
in women. The historic male to 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.
[0012] Most bladder cancers recur in the bladder. 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. There continues to be a
significant need for treatment modalities that are beneficial for
bladder cancer patients.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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 lung and bronchial
cancers.
[0018] An estimated 182,800 new invasive cases of breast cancer
were expected to occur 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.
[0019] 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.
[0020] Taking into account the medical circumstances and the
patients 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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
[0026] The present invention relates to a gene, designated 273P4B7,
that has now been found to be over-expressed in the cancer(s)
listed in Table I. Northern blot expression analysis of 273P4B7
gene 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 273P4B7 are provided. The
tissue-related profile of 273P4B7 in normal adult tissues, combined
with the over-expression observed in the tissues listed in Table I,
shows that 273P4B7 is aberrantly over-expressed in at least some
cancers, and thus serves as a useful diagnostic, prophylactic,
prognostic, and/or therapeutic target for cancers of the tissue(s)
such as those listed in Table I.
[0027] The invention provides polynucleotides corresponding or
complementary to all or part of the 273P4B7 genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding 273P4B7-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
30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more
than 100 contiguous amino acids of a 273P4B7-related protein, as
well as the peptides/proteins themselves; DNA, RNA, DNA/RNA
hybrids, and related molecules, polynucleotides or oligonucleotides
complementary or having at least a 90% homology to the 273P4B7
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the 273P4B7 genes, mRNAs, or to
273P4B7-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding 273P4B7. Recombinant DNA
molecules containing 273P4B7 polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of 273P4B7 gene products are also provided. The
invention further provides antibodies that bind to 273P4B7 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 or therapeutic agent.
In certain embodiments, there is a proviso that the entire nucleic
acid sequence of FIG. 2 is not encoded and/or the entire amino acid
sequence of FIG. 2 is not prepared. In certain embodiments, the
entire nucleic acid sequence of FIG. 2 is encoded and/or the entire
amino acid sequence of FIG. 2 is prepared, either of which are in
respective human unit dose forms.
[0028] The invention further provides methods for detecting the
presence and status of 273P4B7 polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express 273P4B7. A typical embodiment of this invention
provides methods for monitoring 273P4B7 gene products in a tissue
or hematology sample having or suspected of having some form of
growth dysregulation such as cancer.
[0029] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 273P4B7 such as cancers of tissues listed in Table 1,
including therapies aimed at inhibiting the transcription,
translation, processing or function of 273P4B7 as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
273P4B7 in a human subject wherein the composition comprises a
carrier suitable for human use and a human unit dose of one or more
than one agent that inhibits the production or function of 273P4B7.
Preferably, the carrier is a uniquely human carrier. In another
aspect of the invention, the agent is a moiety that is
immunoreactive with 273P4B7 protein. Non-limiting examples of such
moieties include, but are not limited to, antibodies (such as
single chain, monoclonal, polyclonal, humanized, chimeric, or human
antibodies), functional equivalents thereof (whether naturally
occurring or synthetic), and combinations thereof. The antibodies
can be conjugated to a diagnostic or therapeutic moiety. In another
aspect, the agent is a small molecule as defined herein.
[0030] In another aspect, the agent comprises one or more than one
peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that
binds an HLA class I molecule in a human to elicit a CTL response
to 273P4B7 and/or one or more than one peptide which comprises a
helper T lymphocyte (HTL) epitope which binds an HLA class II
molecule in a human to elicit an HTL response. The peptides of the
invention may be on the same or on one or more separate polypeptide
molecules. In a further aspect of the invention, the agent
comprises one or more than one nucleic acid molecule that expresses
one or more than one of the CTL or HTL response stimulating
peptides as described above. In yet another aspect of the
invention, the one or more than one nucleic acid molecule may
express a moiety that is immunologically reactive with 273P4B7 as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
273P4B7. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of 273P4B7 (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for 273P4B7 production) or a ribozyme effective to lyse
273P4B7 mRNA.
[0031] Note that to determine the starting position of any peptide
set forth in Tables VIII-XXI 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 VII. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X-1" to each position in Tables VIII-XXI 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.
[0032] One embodiment of the invention comprises an HLA peptide,
that occurs at least twice in Tables VIII-XXI and XXII to XLIX
collectively, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention comprises an HLA peptide that
occurs at least once in Tables VIII-XXI and at least once in tables
XXII to XLIX, or an oligonucleotide that encodes the HLA
peptide.
[0033] Another embodiment of the invention is antibody epitopes,
which comprise a peptide regions, or an oligonucleotide encoding
the peptide region, that has one two, three, four, or five of the
following characteristics:
[0034] i) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Hydrophilicity profile of FIG. 5;
[0035] ii) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or less than 0.5, 0.4, 0.3,
0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity
profile of FIG. 6;
[0036] iii) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent
Accessible Residues profile of FIG. 7;
[0037] iv) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average
Flexibility profile of FIG. 8; or
[0038] v) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn
profile of FIG. 9.
[0039] Moreover, the invention comprises 273P4B7 nucleic acid and
amino acid sequences. Further, the invention comprises variants of
273P4B7, and fragments thereof. In an embodiment of the invention a
protein fragment is: a subsequence of at least 158, or 262, or 420
contiguous amino acids of a protein of 273P4B7 v. 1; is an amino
acid subsequence of a protein of 273P4B7 v. 1 with a proviso that
273P4B7 v. 1 protein is such that it does not include an valine (V)
or methionine (M) at position 145; arginine (R) or glycine (G) at
position 172; isoleucine (I) or valine (V) at position 889; or,
lysine (K) or arginine (R) at position 989. An embodiment of an
amino acid sequence of the invention is a fragment of a protein of
273P4B7 v. 1 with a proviso that it is not a protein of 273P4B7 v.
9, v. 10 or v.11. In an embodiment, an amino acid fragment of the
invention is 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, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 170,
175, 180, 185, 190, 195, 200, 225, 250, 260, 261, 262, 263, 264,
265, 270, 275, 300, 325, 350, 375, 400, 418, 419, 420, 421, 422,
423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 422, 434, 435,
450, 475, 500, 525, 550, 575, 600, 650, 675, 700, 705, 710, 715,
716, 717, 718, 719, 720, 725, 750, 775, 800, 825, 850, 875, 900,
925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1127, 1150,
1175, 1200, 1025, or 1250 contiguous amino acids of a protein of
FIG. 2; in certain embodiments the fragment subsequence comprises a
functional or structural motif, e.g., as set forth herein, or
comprises an immune system (antibody or T cell) epitope.
Embodiments of a nucleic acid sequence of the invention comprise a
sequence that encodes an amino acid sequence as set forth
herein.
DESCRIPTION OF THE FIGURES
[0040] FIG. 1. The 273P4B7 SSH sequence of 170 nucleotides.
[0041] FIG. 2. A) The cDNA and amino acid sequence of 273P4B7
variant 1 (also called "273P4B7 v.1" or "273P4B7 variant 1") is
shown in FIG. 2A. The start methionine is underlined. The open
reading frame extends from nucleic acid 95-3847 including the stop
codon. B) The cDNA and amino acid sequence of 273P4B7 variant 2
(also called "273P4B7 v.2") is shown in FIG. 2B. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 604-3987 including the stop codon. C) 273P4B7 v.3
through v.8, SNP variants of 273P4B7 v.1. The 273P4B7 v.3 through
v.8 are variants with single nucleotide difference from 273P4B7
v.1.273P4B7 v.3, v.7, and v.8 code for the same protein as v.1.
273P4B7 v.4, v.5, and v.6 proteins differ from 273P4B7 v.1 by one
amino acid. Though these SNP variants are shown separately, they
can also occur in any combinations and in any of the transcript
variants listed above in FIGS. 2A and 2B. D) The cDNA and amino
acid sequence of 273P4B7 variant 9 (also called "273P4B7 v.9") is
shown in FIG. 2D. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 4-3324 including
the stop codon. E) The cDNA and amino acid sequence of 273P4B7
variant 10 (also called "273P4B7 v.10") is shown in FIG. 2E. The
codon for the start methionine is underlined. The open reading
frame extends from nucleic acid 688-1947 including the stop codon.
F) The cDNA and amino acid sequence of 273P4B7 variant 11 (also
called "273P4B7 v.11") is shown in FIG. 2F. The codon for the start
methionine is underlined. The open reading frame extends from
nucleic acid 114-1373 including the stop codon.
[0042] FIG. 3. A) The amino acid sequence of 273P4B7 v.1 is shown
in FIG. 3A; it has 150 amino acids. B) The amino acid sequence of
273P4B7 v.2 is shown in FIG. 3B; it has 1127 amino acids. C) The
amino acid sequence of 273P4B7 v.4 is shown in FIG. 3C; it has 1250
amino acids. D) The amino acid sequence of 273P4B7 v.5 is shown in
FIG. 3D; it has 1250 amino acids. E) The amino acid sequence of
273P4B7 v.6 is shown in FIG. 3E; it has 1250 amino acids. F) The
amino acid sequence of 273P4B7 v.9 is shown in FIG. 3F; it has 1106
amino acids. G) The amino acid sequence of 273P4B7 v.10 is shown in
FIG. 3G; it has 419 amino acids. H) The amino acid sequence of
273P4B7 v.11 is shown in FIG. 3H; it has 419 amino acids. As used
herein, a reference to 273P4B7 includes all variants thereof,
including those shown in FIGS. 2, 3, 10, and 11, unless the context
clearly indicates otherwise.
[0043] FIG. 4. Alignment of 273P4B7 with known homologs. FIG. 4(A)
Alignment of 273P4B7 with human un-named protein
(gi.vertline.22760345). FIG. 4(B) Alignment of 273P4B7 with human
BJ-HCC-15 tumor antigen (gi.vertline.22002580). FIG. 4(C) Alignment
of 273P4B7 with Mouse Protein (gi.vertline.27706852).
[0044] FIG. 5. Hydrophilicity amino acid profile of 273P4B7 v.1
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.
[0045] FIG. 6. Hydropathicity amino acid profile of 273P4B7 v.1
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 through the ExPasy molecular biology server.
[0046] FIG. 7. Percent accessible residues amino acid profile of
273P4B7 v.1 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
through the ExPasy molecular biology server.
[0047] FIG. 8. Average flexibility amino acid profile of 273P4B7
v.1 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 through the ExPasy
molecular biology server.
[0048] FIG. 9. Beta-turn amino acid profile of 273P4B7 v.1
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 through the ExPasy molecular biology server.
[0049] FIG. 10. Structures of transcript variants of 273P4B07.
Variant 273P4B07 v.2 was identified as a transcript variant of
273P4B07 v.1. Variant 273P4B07 v.2 extended exon 1 by 22 bp as
compared to v.1 and added an exon in between exons 1 and 2 of
variant v.1. Variants v.9, v.10 and v.11 were part of the last exon
of v.1 or v.2. Poly A tails and SNP are not shown here. Numbers in
"( )" underneath the boxes correspond to those of 273P4B07 v.1.
Lengths of introns and exons are not proportional.
[0050] FIG. 11. Schematic alignment of protein variants of
273P4B07. Protein variants correspond to nucleotide variants.
Nucleotide variants 273P4B07 v.3, v.7, and v.8 coded for the same
protein as v.1. Variant v.2 coded a protein that was 123 amino
acids shorter than v.1. Nucleotide variant 273P4B07 v.2 was a
transcript variant of v.1, as shown in FIG. 10. Variants v.9 and
v.10 were shorter and had some different amino acid as compared
with v.1 in the corresponding positions shown in the figure.
Variant v.11 was the same as the C-terminal part of v.1 and
different from v.10 by one amino acid at position 158. SNP in v.1
could also appear in v.2. Single amino acid differences were
indicated above the boxes. Black boxes represent the same sequence
as 273P4B07 v.1. Numbers underneath the box correspond to 273P4B07
v.1.
[0051] FIG. 12. Schematic alignment of SNP variants of 273P4B07.
Variants 273P4B07 v.3 through v.8 were variants with single
nucleotide differences as compared to variant v.1 (ORF:29-1858).
Though these SNP variants were shown separately, they could also
occur in any combinations and in any transcript variants that
contained the base pairs, such as v.2 shown in FIG. 10. Numbers
correspond to those of 273P4B07 v.1. Black box shows the same
sequence as 273P4B07 v.1. SNPs are indicated above the box.
[0052] FIG. 13. Secondary structure and transmembrane domains
prediction for 273P4B7 protein variant 1. FIG. 13A: The secondary
structure of 273P4B7 protein variant 1 (FIG. 13A) (SEQ ID NO: 134)
was predicted using the HNN--Hierarchical Neural Network method
(NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25,
No 3 [291]:147-150, accessed from the ExPasy molecular biology
server located on the World Wide Web. 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 in a given secondary structure is also listed. FIG. 13B:
Schematic representation of the probability of existence of
transmembrane regions of 273P4B7 variant 1 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).
[0053] FIG. 13C: Schematic representation of the probability of the
existence of transmembrane regions of 273P4B7 variant 1, 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.
[0054] FIG. 14. 273P4B7 expression by RT-PCR. First strand cDNA was
prepared from normal tissues (bladder, brain, heart, kidney, liver,
lung, prostate, spleen, skeletal muscle, testis, pancreas, colon
and stomach), and from pools of patient cancer specimens (prostate
cancer pool, bladder cancer pool, kidney cancer pool, colon cancer
pool, lung cancer pool, ovary cancer pool, breast cancer pool,
cancer metastasis pool, pancreas cancer pool, prostate cancer
xenograft pool, prostate metastasis to lymph node, bone and
melanoma cancer pool, cervical cancer pool, lymphoma cancer pool,
stomach cancer pool, uterus cancer pool, and multi-xenograft pool).
Normalization was performed by PCR using primers to actin.
Semi-quantitative PCR, using primers to 273P4B7, was performed at
22, 26 and 30 cycles of amplification. In (FIG. 14A) picture of the
RT-PCR agarose gel is shown. In (FIG. 14B) PCR products were
quantitated using the AlphaImager software. Results show strong of
expression of 273P4B7 in prostate cancer pool, bladder cancer pool,
kidney cancer pool, colon cancer pool, lung cancer pool, ovary
cancer pool, breast cancer pool, cancer metastasis pool, pancreas
cancer pool, prostate cancer xenograft pool, prostate metastasis to
lymph node, bone and melanoma cancer pool, cervical cancer pool,
lymphoma cancer pool, stomach cancer pool, uterus cancer pool and
multi-xenograft pool (prostate cancer, kidney cancer and bladder
cancer xenograft pool). In normal tissues, 273P4B7 is predominantly
expressed in testis and not in any other normal tissue tested.
[0055] FIG. 15. 273P4B7 expression in normal tissues. Two multiple
tissue northern blots (Clontech) both with 2 ug of mRNA/lane were
probed with the 273P4B7 sequence. Size standards in kilobases (kb)
are indicated on the side. Results show expression of an
approximately 7 kb 273P4B7 transcript in normal testis but not in
the other normal tissues tested.
[0056] FIG. 16. Expression of 273P4B7 in pancreas, ovary, and
testis cancer patient specimens. RNA was extracted from normal
pancreas (NPa), normal ovary (NO), normal testis (NTe), pancreas
cancer patient specimen (P1), ovary cancer patient specimen (P2,
P3, P4), and testis cancer patient specimen (P5, P6, P7). Northern
blot with 10 ug of total RNA/lane was probed with 273P4B7 SSH
sequence. Size standards in kilobases (kb) are indicated on the
side. 273P4B7 transcript was detected in the patient specimens, but
not in the normal tissues.
[0057] FIG. 17. Expression of 273P4B7 in cervical cancer patient
specimens. FIG. 17(A): Total RNA was extracted from cervical cancer
patient specimens (T1-T7), and HeLa cell line. Northern blot with
10 ug of total RNA/lane was probed with 273P4B7 SSH sequence. Size
standards in kilobases (kb) are indicated on the side. 273P4B7
transcript was detected in all patient specimens tested as well as
in the Hela cell line. FIG. 17(B): First strand cDNA was prepared
from a panel of cervical cancer patient specimens, normal cervix
and HeLa cervical cell line. Normalization was performed by PCR
using primers to actin. Semi-quantitative PCR, using primers to
273P4B7, was performed at 26 and 30 cycles of amplification.
Samples were run on an agarose gel, and PCR products were
quantitated using the AlphaImager software. Expression was recorded
as absent, low, medium or high. Results show expression of 273P4B7
in most of the cervical cancer tissues tested.
[0058] FIG. 18. Expression of 273P4B7 in bladder cancer patient
specimens. First strand cDNA was prepared from a panel of bladder
cancer patient specimens, normal bladder (N) and bladder cancer
cell lines (UM-UC-3, TCCSUP, J82). Normalization was performed by
PCR using primers to actin. Semi-quantitative PCR, using primers to
273P4B7, was performed at 26 and 30 cycles of amplification.
Samples were run on an agarose gel (FIG. 18(A)), and PCR products
were quantitated using the AlphaImager software (FIG. 18(B)).
Expression was recorded as absent, low, medium or high. Results
show expression of 273P4B7 in most of the bladder cancer tissues
tested, but not in the normal bladder tissues.
[0059] FIG. 19. Expression of 273P4B7 in colon cancer patient
specimens. First strand cDNA was prepared from a panel of colon
cancer patient specimens, normal colon, and colon cancer cell lines
(LoVo, CaCo-2, SK-CO1, Colo205, and T284). Normalization was
performed by PCR using primers to actin. Semi-quantitative PCR,
using primers to 273P4B7, was performed at 26 and 30 cycles of
amplification. Samples were run on an agarose gel, and PCR products
were quantitated using the AlphaImager software. Expression was
recorded as absent, low, medium or high. Results show expression of
273P4B7 in the majority of the colon cancer tissues tested, but not
in the normal colon tissues. Expression was also detected in the
cell lines LoVo, CaCo-2, SK-CO1, Colo205, but not in the T284 cell
line.
[0060] FIG. 20. Expression of 273P4B7 in ovary cancer patient
specimens. First strand cDNA was prepared from a panel of ovarian
cancer patient specimens, normal ovary and ovarian cancer cell
lines (OV-1063, PA-1, SW626). Normalization was performed by PCR
using primers to actin. Semi-quantitative PCR, using primers to
273P4B7, was performed at 26 and 30 cycles of amplification.
Samples were run on an agarose gel, and PCR products were
quantitated using the AlphaImager software. Expression was recorded
as absent, low, medium or high. Results show expression of 273P4B7
in the majority of ovary cancer tissues tested as well as in the
cell lines, but not in normal ovary.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Outline of Sections:
[0062] I.) Definitions
[0063] II.) 273P4B7 Polynucleotides
[0064] II.A.) Uses of 273P4B7 Polynucleotides
[0065] II.A.1.) Monitoring of Genetic Abnormalities
[0066] II.A.2.) Antisense Embodiments
[0067] II.A.3.) Primers and Primer Pairs
[0068] II.A.4.) Isolation of 273P4B7-Encoding Nucleic Acid
Molecules
[0069] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0070] III.) 273P4B7-Related Proteins
[0071] III.A.) Motif-bearing Protein Embodiments
[0072] III.B.) Expression of 273P4B7-Related Proteins
[0073] III.C.) Modifications of 273P4B7-Related Proteins
[0074] III.D.) Uses of 273P4B7-Related Proteins
[0075] IV.) 273P4B7 Antibodies
[0076] V.) 273P4B7 Cellular Immune Responses
[0077] VI.) 273P4B7 Transgenic Animals
[0078] VII.) Methods for the Detection of 273P4B7
[0079] VIII.) Methods for Monitoring the Status of 273P4B7-related
Genes and Their Products
[0080] IX.) Identification of Molecules That Interact With
273P4B7
[0081] X.) Therapeutic Methods and Compositions
[0082] X.A.) Anti-Cancer Vaccines
[0083] X.B.) 273P4B7 as a Target for Antibody-Based Therapy
[0084] X.C.) 273P4B7 as a Target for Cellular Immune Responses
[0085] X.C.1. Minigene Vaccines
[0086] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0087] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0088] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0089] X.D.) Adoptive Immunotherapy
[0090] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0091] XI.) Diagnostic and Prognostic Embodiments of 273P4B7.
[0092] XII.) Inhibition of 273P4B7 Protein Function
[0093] XII.A.) Inhibition of 273P4B7 With Intracellular
Antibodies
[0094] XII.B.) Inhibition of 273P4B7 with Recombinant Proteins
[0095] XII.C.) Inhibition of 273P4B7 Transcription or
Translation
[0096] XII.D.) General Considerations for Therapeutic
Strategies
[0097] XIII.) Identification, Characterization and Use of
Modulators of 273P4B7
[0098] XIV.) KITS/Articles of Manufacture
[0099] I. Definitions:
[0100] 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.
[0101] The terms "advanced prostate cancer", "locally advanced
prostate cancer", "advanced disease" and "locally advanced disease"
mean prostate cancers that have extended through the prostate
capsule, and are meant to include stage C disease under the
American Urological Association (AUA) system, stage C.sub.1-C.sub.2
disease under the Whitmore-Jewett system, and stage T3-T4 and N+
disease under the TNM (tumor, node, metastasis) system. In general,
surgery is not recommended for patients with locally advanced
disease, and these patients have substantially less favorable
outcomes compared to patients having clinically localized
(organ-confined) prostate cancer. Locally advanced disease is
clinically identified by palpable evidence of induration beyond the
lateral border of the prostate, or asymmetry or induration above
the prostate base. Locally advanced prostate cancer is presently
diagnosed pathologically following radical prostatectomy if the
tumor invades or penetrates the prostatic capsule, extends into the
surgical margin, or invades the seminal vesicles.
[0102] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 273P4B7 (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 273P4B7. 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.
[0103] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 273P4B7-related protein). For example, an analog
of a 273P4B7 protein can be specifically bound by an antibody or T
cell that specifically binds to 273P4B7.
[0104] 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-273P4B7 antibodies 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.
[0105] 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-273P4B7 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-273P4B7 antibody compositions with polyepitopic
specificity.
[0106] The term "codon optimized sequences" refers to nucleotide
sequences that have been optimized for a particular host species by
replacing any codons having a usage frequency of less than about
20%. 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."
[0107] A "combinatorial library" is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide (e.g., mutein) library, is
formed by combining a set of chemical building blocks called amino
acids in every possible way for a given compound length (i.e., the
number of amino acids in a polypeptide compound). Numerous chemical
compounds are synthesized through such combinatorial mixing of
chemical building blocks (Gallop et al., J. Med. Chem. 37(9):
1233-1251 (1994)).
[0108] Preparation and screening of combinatorial libraries is well
known to those of skill in the art. Such combinatorial chemical
libraries include, but are not limited to, peptide libraries (see,
e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493
(1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT
Publication No WO 91/19735), encoded peptides (PCT Publication WO
93/20242), random bio-oligomers (PCT Publication WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et
al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic
syntheses of small compound libraries (Chen et al., J. Amer. Chem.
Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science
261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J.
Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med.
Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,
Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S.
Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,
Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al., Science
274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small
organic molecule libraries (see, e.g., benzodiazepines, Baum,
C&EN, Jan. 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
U.S. Pat. No. 5,288,514; and the like).
[0109] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced
Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A,
Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore,
Bedford, NIA). A number of well-known robotic systems have also
been developed for solution phase chemistries. These systems
include automated workstations such as the automated synthesis
apparatus developed by Takeda Chemical Industries, LTD. (Osaka,
Japan) and many robotic systems utilizing robotic arms (Zymate H,
Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo
Alto, Calif.), which mimic the manual synthetic operations
performed by a chemist. Any of the above devices are suitable for
use with the present invention. The nature and implementation of
modifications to these devices (if any) so that they can operate as
discussed herein will be apparent to persons skilled in the
relevant art. In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar,
Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md.; etc.).
[0110] The term "cytotoxic agent" refers to a substance that
inhibits or prevents the expression activity of cells, 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 auristatins, auromycins, maytansinoids, yttrium,
bismuth, ricin, ricin A-chain, combrestatin, duocarmycins,
dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065,
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,
retstrictocin, phenomycin, enomycin, curicin, crotin,
calicheamicin, Sapaonana 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 or 213, P.sup.32 and radioactive isotopes of
Lu including Lu.sup.177. Antibodies may also be conjugated to an
anti-cancer pro-drug activating enzyme capable of converting the
pro-drug to its active form.
[0111] The "gene product" is sometimes referred to herein as a
protein or mRNA. For example, a "gene product of the invention" is
sometimes referred to herein as a "cancer amino acid sequence",
"cancer protein", "protein of a cancer listed in Table I", a
"cancer mRNA", "mRNA of a cancer listed in Table I", etc. In one
embodiment, the cancer protein is encoded by a nucleic acid of FIG.
2. The cancer protein can be a fragment, or alternatively, be the
full-length protein to the fragment encoded by the nucleic acids of
FIG. 2. In one embodiment, a cancer amino acid sequence is used to
determine sequence identity or similarity. In another embodiment,
the sequences are naturally occurring allelic variants of a protein
encoded by a nucleic acid of FIG. 2. In another embodiment, the
sequences are sequence variants as further described herein. "High
throughput screening" assays for the presence, absence,
quantification, or other properties of particular nucleic acids or
protein products are well known to those of skill in the art.
Similarly, binding assays and reporter gene assays are similarly
well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high
throughput screening methods for proteins; U.S. Pat. No. 5,585,639
discloses high throughput screening methods for nucleic acid
binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and
5,541,061 disclose high throughput methods of screening for
ligand/antibody binding.
[0112] In addition, high throughput screening systems are
commercially available (see, e.g., Amersham Biosciences,
Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These
systems typically automate entire procedures, including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols for
various high throughput systems. Thus, e.g., Zymark Corp. provides
technical bulletins describing screening systems for detecting the
modulation of gene transcription, ligand binding, and the like.
[0113] 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.
[0114] "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).
[0115] 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.
[0116] 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 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 genes other than the 273P4B7 genes or that encode
polypeptides other than 273P4B7 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 273P4B7 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 273P4B7 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 273P4B7 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0117] 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.
[0118] The terms "metastatic prostate cancer" and "metastatic
disease" mean prostate cancers that have spread to regional lymph
nodes or to distant sites, and are meant to include stage D disease
under the AUA system and stage T.times.N.times.M+under the TNM
system. As is the case with locally advanced prostate cancer,
surgery is generally not indicated for patients with metastatic
disease, and hormonal (androgen ablation) therapy is a preferred
treatment modality. Patients with metastatic prostate cancer
eventually develop an androgen-refractory state within 12 to 18
months of treatment initiation. Approximately half of these
androgen-refractory patients die within 6 months after developing
that status. The most common site for prostate cancer metastasis is
bone. Prostate cancer bone metastases are often osteoblastic rather
than osteolytic (i.e., resulting in net bone formation). Bone
metastases are found most frequently in the spine, followed by the
femur, pelvis, rib cage, skull and humerus. Other common sites for
metastasis include lymph nodes, lung, liver and brain. Metastatic
prostate cancer is typically diagnosed by open or laparoscopic
pelvic lymphadenectomy, whole body radionuclide scans, skeletal
radiography, and/or bone lesion biopsy.
[0119] The term "modulator" or "test compound" or "drug candidate"
or grammatical equivalents as used herein describe any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., to be tested for the capacity
to directly or indirectly alter the cancer phenotype or the
expression of a cancer sequence, e.g., a nucleic acid or protein
sequences, or effects of cancer sequences (e.g., signaling, gene
expression, protein interaction, etc.) In one aspect, a modulator
will neutralize the effect of a cancer protein of the invention. By
"neutralize" is meant that an activity of a protein is inhibited or
blocked, along with the consequent effect on the cell. In another
aspect, a modulator will neutralize the effect of a gene, and its
corresponding protein, of the invention by normalizing levels of
said protein. In preferred embodiments, modulators alter expression
profiles, or expression profile nucleic acids or proteins provided
herein, or downstream effector pathways. In one embodiment, the
modulator suppresses a cancer phenotype, e.g. to a normal tissue
fingerprint. In another embodiment, a modulator induced a cancer
phenotype. Generally, a plurality of assay mixtures is run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection.
[0120] Modulators, drug candidates or test compounds encompass
numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular
weight of more than 100 and less than about 2,500 Daltons.
Preferred small molecules are less than 2000, or less than 1500 or
less than 1000 or less than 500 D. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Modulators also comprise biomolecules
such as peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof. Particularly preferred are peptides. One class of
modulators are peptides, for example of from about five to about 35
amino acids, with from about five to about 20 amino acids being
preferred, and from about 7 to about 15 being particularly
preferred. Preferably, the cancer modulatory protein is soluble,
includes a non-transmembrane region, and/or, has an N-terminal Cys
to aid in solubility. In one embodiment, the C-terminus of the
fragment is kept as a free acid and the N-terminus is a free amine
to aid in coupling, i.e., to cysteine. In one embodiment, a cancer
protein of the invention is conjugated to an immunogenic agent as
discussed herein. In one embodiment, the cancer protein is
conjugated to BSA. The peptides of the invention, e.g., of
preferred lengths, can be linked to each other or to other amino
acids to create a longer peptide/protein. The modulatory peptides
can be digests of naturally occurring proteins as is outlined
above, random peptides, or "biased" random peptides. In a preferred
embodiment, peptide/protein-based modulators are antibodies, and
fragments thereof, as defined herein.
[0121] Modulators of cancer can also be nucleic acids. Nucleic acid
modulating agents can be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of prokaryotic or eukaryotic genomes can be used in an
approach analogous to that outlined above for proteins.
[0122] 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.
[0123] A "motif", as in biological motif of a 273P4B7-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.
[0124] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, welting agents, preservative, and the like.
[0125] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0126] The term "polynucleotide" means a polymeric form of
nucleotides of at least 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 if often
used interchangeably with "oligonucleotide". A polynucleotide can
comprise a nucleotide sequence disclosed herein wherein thymidine
(T), as shown for example in FIG. 2, 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).
[0127] 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".
[0128] 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.
Alternatively, in another embodiment, the primary anchor residues
of a peptide binds 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.
[0129] "Radioisotopes" include, but are not limited to the
following (non-limiting exemplary uses are also set forth):
[0130] 1 Examples of Medical Isotopes: Isotope Description of use
Actinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227 Parent
of Radium-223 (Ra-223) which is an (AC-227) alpha emitter used to
treat metastases in the skeleton resulting from cancer (i.e.,
breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212 See Thorium-228 (Th-228) (Bi-212) Bismuth-213 See
Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancer detection (Cd-109)
Cobalt-60 Radiation source for radiotherapy of cancer, (Co-60) for
food irradiators, and for sterilization of medical supplies
Copper-64 A positron emitter used for cancer therapy (Cu-64) and
SPECT imaging Copper-67 Beta/gamma emitter used in cancer (Cu-67)
radioimmunotherapy and diagnostic studies (i.e., breast and colon
cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy
(Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly
(Er-169) for the small joints associated with fingers and toes
Europium-152 Radiation source for food irradiation and for (Eu-152)
sterilization of medical supplies Europium-154 Radiation source for
food irradiation and for (Eu-154) sterilization of medical supplies
Gadolinium-153 Osteoporosis detection and nuclear medical (Gd-153)
quality assurance devices Gold-198 Implant and intracavity therapy
of ovarian, (Au-198) prostate, and brain cancers Holmium-166
Multiple myeloma treatment in targeted (Ho-166) skeletal therapy,
cancer radioimmunotherapy, bone marrow ablation, and rheumatoid
arthritis treatment Iodine-125 Osteoporosis detection, diagnostic
imaging, (1-125) tracer drugs, brain cancer treatment,
radiolabeling, tumor imaging, mapping of receptors in the brain,
interstitial radiation therapy, brachytherapy for treatment of
prostate cancer, determination of glomerular filtration rate (GFR),
determination of plasma volume, detection of deep vein thrombosis
of the legs Iodine-131 Thyroid function evaluation, thyroid disease
(1-131) detection, treatment of thyroid cancer as well as other
non-malignant thyroid diseases (i.e., Graves disease, goiters, and
hyperthyroidism), treatment of leukemia, lymphoma, and other forms
of cancer (e.g., breast cancer) using radioimmunotherapy
Iridium-192 Brachytherapy, brain and spinal cord tumor (Ir-192)
treatment, treatment of blocked arteries (i.e., arteriosclerosis
and restenosis), and implants for breast and prostate tumors
Lutetium-177 Cancer radioimmunotherapy and treatment of (Lu-177)
blocked arteries (i.e., arteriosclerosis and restenosis)
Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is (Mo-99)
used for imaging the brain, liver, lungs, heart, and other organs.
Currently, Tc-99m is the most widely used radioisotope used for
diagnostic imaging of various cancers and diseases involving the
brain, heart, liver, lungs; also used in detection of deep vein
thrombosis of the legs Osmium-194 Cancer radioimmunotherapy
(Os-194) Palladium-103 Prostate cancer treatment (Pd-103)
Platinum-195m Studies on biodistribution and metabolism of
(Pt-195m) cisplatin, a chemotherapeutic drug Phosphorus-32
Polycythemia rubra vera (blood cell disease) (P-32) and leukemia
treatment, bone cancer diagnosis/treatment; colon, pancreatic, and
liver cancer treatment; radiolabeling nucleic acids for in vitro
research, diagnosis of superficial tumors, treatment of blocked
arteries (i.e., arteriosclerosis and restenosis), and intracavity
therapy Phosphorus-33 Leukemia treatment, bone disease (P-33)
diagnosis/treatment, radiolabeling, and treatment of blocked
arteries (i.e., arteriosclerosis and restenosis) Radium-223 See
Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,
rheumatoid arthritis (Re-186) treatment, and diagnosis and
treatment of lymphoma and bone, breast, colon, and liver cancers
using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment
using (Re-188) radioimmunotherapy, bone cancer pain relief,
treatment of rheumatoid arthritis, and treatment of prostate cancer
Rhodium-105 Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular
cancer treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy
and bone cancer (Sm-153) pain relief Scandium-47 Cancer
radioimmunotherapy and bone cancer (Sc-47) pain relief Selenium-75
Radiotracer used in brain studies, imaging of (Se-75) adrenal
cortex by gamma-scintigraphy, lateral locations of steroid
secreting tumors, pancreatic scanning, detection of hyperactive
parathyroid glands, measure rate of bile acid loss from the
endogenous pool Strontium-85 Bone cancer detection and brain scans
(Sr-85) Strontium-89 Bone cancer pain relief, multiple myeloma
(Sr-89) treatment, and osteoblastic therapy Technetium-99m See
Molybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212
(Bi-212) which is an (Th-228) alpha emitter used in cancer
radioimmunotherapy Thorium-229 Parent of Actinium-225 (Ac-225) and
(Th-229) grandparent of Bismuth-213 (Bi-213) which are alpha
emitters used in cancer radioimmunotherapy Thulium-170 Gamma source
for blood irradiators, energy (Tm-170) source for implanted medical
devices Tin-117m Cancer immunotherapy and bone cancer (Sn-117m)
pain relief Tungsten-188 Parent for Rhenium-188 (Re-188) which is
used (W-188) for cancer diagnostics/treatment, bone cancer pain
relief, rheumatoid arthritis treatment, and treatment of blocked
arteries (i.e., arteriosclerosis and restenosis) Xenon-127
Neuroimaging of brain disorders, high (Xe-127) resolution SPECT
studies, pulmonary function tests, and cerebral blood flow studies
Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90
Microseeds obtained from irradiating (Y-90) Yttrium-89 (Y-89) for
liver cancer treatment Yttrium-91 A gamma-emitting label for
Yttrium-90 (Y-90) (Y-91) which is used for cancer
radioimmunotherapy (i.e., lymphoma, breast, colon, kidney, lung,
ovarian, prostate, pancreatic, and inoperable liver cancers)
[0131] By "randomized" or grammatical equivalents as herein applied
to nucleic acids and proteins is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. These random peptides (or nucleic acids, discussed
herein) can incorporate any nucleotide or amino acid at any
position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the
sequence, thus forming a library of randomized candidate bioactive
proteinaceous agents.
[0132] In one embodiment, a library is "fully randomized," with no
sequence preferences or constants at any position. In another
embodiment, the library is a "biased random" library. That is, some
positions within the sequence either are held constant, or are
selected from a limited number of possibilities. For example, the
nucleotides or amino acid residues are randomized within a defined
class, e.g., of hydrophobic amino acids, hydrophilic residues,
sterically biased (either small or large) residues, towards the
creation of nucleic acid binding domains, the creation of
cysteines, for cross-linking, prolines for SH-3 domains, serines,
threonines, tyrosines or histidines for phosphorylation sites,
etc., or to purines, etc.
[0133] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0134] Non-limiting examples of small molecules include compounds
that bind or interact with 273P4B7, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit 273P4B7
protein function. Such non-limiting small molecules preferably have
a molecular weight of less than about 10 kDa, more preferably below
about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In
certain embodiments, small molecules physically associate with, or
bind, 273P4B7 protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions
[0135] "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).
[0136] "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.
[0137] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles. Overall
phenotypic frequencies of HLA-supertypes in different ethnic
populations are set forth in Table IV (F). The non-limiting
constituents of various supetypes are as follows:
A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901,
A*0207
A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101
B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601,
B*6701, B*7801, B*0702, B*5101, B*5602
B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
A1: A*0102, A*2604, A*3601, A*4301, A*8001
A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901,
B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
[0138] Calculated population coverage afforded by different
HLA-supertype combinations are set forth in Table IV (G).
[0139] As used herein "to treat" or "therapeutic" and grammatically
related terms, refer to any improvement of any consequence of
disease, such as prolonged survival, less morbidity, and/or a
lessening of side effects which are the byproducts of an
alternative therapeutic modality; full eradication of disease is
not required.
[0140] 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.
[0141] 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.
[0142] The term "variant" refers to a molecule that exhibits a
variable 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 273P4B7
protein shown in FIG. 2 or FIG. 3. An analog is an example of a
variant protein. Splice isoforms and single nucleotides
polymorphisms (SNPs) are further examples of variants.
[0143] The "273P4B7-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 273P4B7 proteins or fragments thereof, as well as fusion
proteins of a 273P4B7 protein and a heterologous polypeptide are
also included. Such 273P4B7 proteins are collectively referred to
as the 273P4B7-related proteins, the proteins of the invention, or
273P4B7. The term "273P4B7-related protein" refers to a polypeptide
fragment or a 273P4B7 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 30, 35, 40, 45, 50, 55, 60, 65,
70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,
575, or 576 or more amino acids.
[0144] II.) 273P4B7 Polynucleotides
[0145] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of a 273P4B7 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding a 273P4B7-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to a 273P4B7 gene
or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to a 273P4B7 gene, mRNA, or to a
273P4B7 encoding polynucleotide (collectively, "273P4B7
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0146] Embodiments of a 273P4B7 polynucleotide include: a 273P4B7
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 273P4B7 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 273P4B7 nucleotides comprise, without
limitation:
(I) a polynucleotide comprising, consisting essentially of, or
consisting of a sequence as shown in FIG. 2, wherein T can also be
U;
(II) a polynucleotide comprising, consisting essentially of, or
consisting of the sequence as shown in FIG. 2A, from nucleotide
residue number 95 through nucleotide residue number 3847, including
the stop codon, wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or
consisting of the sequence as shown in FIG. 2B, from nucleotide
residue number 604 through nucleotide residue number 3987,
including the stop codon, wherein T can also be U;
(IV) a polynucleotide that encodes a 273P4B7-related protein that
is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%
homologous to an entire amino acid sequence shown in FIG. 2A-F;
(V) a polynucleotide that encodes a 273P4B7-related protein that is
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical
to an entire amino acid sequence shown in FIG. 2A-F;
(VI) a polynucleotide that encodes at least one peptide set forth
in Tables VIII-XXI and XXII-XLIX;
[0147] (VII) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIGS. 3A and 3C-3E in any whole number
increment up to 1250 that includes at least 1, 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 amino acid position(s)
having a value greater than 0.5 in the Hydrophilicity profile of
FIG. 5;
[0148] (VIII) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIGS. 3A and 3C-3E in any whole number
increment up to 1250 that includes 1, 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 amino acid position(s) having a
value less than 0.5 in the Hydropathicity profile of FIG. 6;
[0149] (IX) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIGS. 3A and 3C-3E in any whole number
increment up to 1250 that includes 1, 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 amino acid position(s) having a
value greater than 0.5 in the Percent Accessible Residues profile
of FIG. 7;
[0150] (X) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIGS. 3A and 3C-3E in any whole number
increment up to 1250 that includes 1, 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 amino acid position(s) having a
value greater than 0.5 in the Average Flexibility profile of FIG.
8;
[0151] (XI) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIGS. 3A and 3C-3E in any whole number
increment up to 1250 that includes 1, 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 amino acid position(s) having a
value greater than 0.5 in the Beta-turn profile of FIG. 9;
[0152] (XII) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIG. 3B in any whole number increment up to
1127 that includes 1, 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, amino acid position(s) having a value greater than 0.5
in the Hydrophilicity profile of FIG. 5;
[0153] (XIII) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIG. 3B in any whole number increment up to
1127 that includes 1, 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, amino acid position(s) having a value less than 0.5 in
the Hydropathicity profile of FIG. 6;
[0154] (XIV) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIG. 3B in any whole number increment up to
1127 that includes 1, 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, amino acid position(s) having a value greater than 0.5
in the Percent Accessible Residues profile of FIG. 7;
[0155] (XV) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIG. 3B in any whole number increment up to
1127 that includes 1, 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, amino acid position(s) having a value greater than 0.5
in the Average Flexibility profile of FIG. 8;
[0156] (XVI) a polynucleotide that encodes a peptide region of at
least 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 amino
acids of a peptide of FIG. 3B in any whole number increment up to
1127 that includes 1, 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, amino acid position(s) having a value greater than 0.5
in the Beta-turn profile of FIG. 9;
(XVII) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XVI);
(XVIII) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XVII);
(XIX) a peptide that is encoded by any of (I) to (XVIII); and;
(XX) a composition comprising a polynucleotide of any of
(I)-(XVIII) or peptide of (XIX) together with a pharmaceutical
excipient and/or in a human unit dose form;
(XXI) a method of using a polynucleotide of any (I)-(XVIII) or
peptide of (XIX) or a composition of (XX) in a method to modulate a
cell expressing 273P4B7;
(XXII) a method of using a polynucleotide of any (I)-(XVIII) or
peptide of (XIX) or a composition of (XX) in a method to diagnose,
prophylax, prognose, or treat an individual who bears a cell
expressing 273P4B7;
[0157] (XXIII) a method of using a polynucleotide of any
(I)-(XVIII) or peptide of (XIX) or a composition of (XX) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 273P4B7, said cell from a cancer of a
tissue listed in Table I;
(XXIV) a method of using a polynucleotide of any (I)-(XXVIII) or
peptide of (XIX) or a composition of (XX) in a method to diagnose,
prophylax, prognose, or treat a cancer;
(XXV) a method of using a polynucleotide of any (I)-(XXVIII) or
peptide of (XIX) or a composition of (XX) in a method to diagnose,
prophylax, prognose, or treat a cancer of a tissue listed in Table
I; and;
(XXVI) a method of using a polynucleotide of any (I)-(XXVIII) or
peptide of (XIX) or a composition of (XX) in a method to identify
or characterize a modulator of a cell expressing 273P4B7.
[0158] As used herein, a range is understood to disclose
specifically all whole unit positions thereof.
[0159] Typical embodiments of the invention disclosed herein
include 273P4B7 polynucleotides that encode specific portions of
273P4B7 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0160] (a) 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, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 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, 850, 875, 900,
925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175,
1200, 1225, 1235, 1240, 1245 1250 or more contiguous amino acids of
273P4B7 variant 1; the maximal lengths relevant for other variants
are: variant 2, 1127 amino acids; variant 4, 1250 amino acids,
variant 5, 1250 amino acids, variant 6, 1250 amino acids, variant
9, 1106 amino acids; variant 10, 419 amino acids; and variant 11,
419 amino acids.
[0161] 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 273P4B7 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 273P4B7 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 273P4B7
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 273P4B7 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 273P4B7 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 273P4B7 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 273P4B7 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 273P4B7
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 273P4B7 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 273P4B7 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 273P4B7 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.
[0162] Polynucleotides encoding relatively long portions of a
273P4B7 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 273P4B7 protein "or variant" 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 273P4B7
sequence as shown in FIG. 2.
[0163] Additional illustrative embodiments of the invention
disclosed herein include 273P4B7 polynucleotide fragments encoding
one or more of the biological motifs contained within a 273P4B7
protein "or variant" sequence, including one or more of the
motif-bearing subsequences of a 273P4B7 protein "or variant" set
forth in Tables VIII-XXI and XXII-XLIX. In another embodiment,
typical polynucleotide fragments of the invention encode one or
more of the regions of 273P4B7 protein or variant that exhibit
homology to a known molecule. In another embodiment of the
invention, typical polynucleotide fragments can encode one or more
of the 273P4B7 protein or variant N-glycosylation sites, cAMP and
cGMP-dependent protein kinase phosphorylation sites, casein kinase
11 phosphorylation sites or N-myristoylation site and amidation
sites.
[0164] Note that to determine the starting position of any peptide
set forth in Tables VIII-XXI and Tables 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 listed in Table VII. Generally, a
unique Search Peptide is used to obtain HLA peptides for a
particular variant. The position of each Search Peptide relative to
its respective parent molecule is listed in Table VII. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X minus 1" to each position in Tables VIII-XXI and Tables XXII-IL
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.
[0165] II.A.) Uses of 273P4B7 Polynucleotides
[0166] II.A.1.) Monitoring of Genetic Abnormalities
[0167] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 273P4B7 gene maps to
the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 273P4B7." For example, because the 273P4B7
gene maps to this chromosome, polynucleotides that encode different
regions of the 273P4B7 proteins 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 273P4B7 proteins provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 273P4B7 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)).
[0168] Furthermore, as 273P4B7 was shown to be highly expressed in
prostate and other cancers, 273P4B7 polynucleotides are used in
methods assessing the status of 273P4B7 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 273P4B7 proteins 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 273P4B7 gene, such as 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.
[0169] II.A.2.) Antisense Embodiments
[0170] 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 273P4B7. 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 273P4B7 polynucleotides and polynucleotide
sequences disclosed herein.
[0171] 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., 273P4B7. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 273P4B7 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, e.g., 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 273P4B7 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).
[0172] The 273P4B7 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 a 273P4B7 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 273P4B7 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 273P4B7 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 273P4B7 mRNA. Optionally, 273P4B7 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
273P4B7. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 273P4B7 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet. 12:
510-515 (1996).
[0173] II.A.3.) Primers and Primer Pairs
[0174] Further specific embodiments of these 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. Probes 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 273P4B7 polynucleotide in a sample and as a means for
detecting a cell expressing a 273P4B7 protein.
[0175] Examples of such probes include polypeptides comprising all
or part of the human 273P4B7 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 273P4B7
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 273P4B7 mRNA.
[0176] The 273P4B7 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
273P4B7 gene(s), mRNA(s), or fragments thereof; as reagents for the
diagnosis and/or prognosis of prostate cancer and other cancers; as
coding sequences capable of directing the expression of 273P4B7
polypeptides; as tools for modulating or inhibiting the expression
of the 273P4B7 gene(s) and/or translation of the 273P4B7
transcript(s); and as therapeutic agents.
[0177] The present invention includes the use of any probe as
described herein to identify and isolate a 273P4B7 or 273P4B7
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.
[0178] II.A.4.) Isolation of 273P4B7-Encoding Nucleic Acid
Molecules
[0179] The 273P4B7 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 273P4B7 gene
product(s), as well as the isolation of polynucleotides encoding
273P4B7 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 273P4B7 gene product as
well as polynucleotides that encode analogs of 273P4B7-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding a 273P4B7 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 273P4B7 gene cDNAs can be identified by
probing with a labeled 273P4B7 cDNA or a fragment thereof. For
example, in one embodiment, a 273P4B7 cDNA (e.g., FIG. 2) or a
portion thereof can be synthesized and used as a probe to retrieve
overlapping and full-length cDNAs corresponding to a 273P4B7 gene.
A 273P4B7 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 273P4B7
DNA probes or primers.
[0180] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0181] The invention also provides recombinant DNA or RNA molecules
containing a 273P4B7 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).
[0182] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a 273P4B7
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 prostate
cancer cell lines such as DU145 and TsuPr1, other transfectable or
transducible prostate cancer cell lines, primary cells (PrEC), 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 273P4B7 or a fragment, analog or homolog thereof can be
used to generate 273P4B7 proteins or fragments thereof using any
number of host-vector systems routinely used and widely known in
the art.
[0183] A wide range of host-vector systems suitable for the
expression of 273P4B7 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 111:1785). Using these expression vectors, 273P4B7
can be expressed in several prostate cancer and non-prostate cell
lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1.
The host-vector systems of the invention are useful for the
production of a 273P4B7 protein or fragment thereof. Such
host-vector systems can be employed to study the functional
properties of 273P4B7 and 273P4B7 mutations or analogs.
[0184] Recombinant human 273P4B7 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 273P4B7-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 273P4B7 or fragment, analog or homolog thereof, a
273P4B7-related protein is expressed in the 293T cells, and the
recombinant 273P4B7 protein is isolated using standard purification
methods (e.g., affinity purification using anti-273P4B7
antibodies). In another embodiment, a 273P4B7 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 273P4B7 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 a 273P4B7 coding sequence can be used for the generation
of a secreted form of recombinant 273P4B7 protein.
[0185] As discussed herein, redundancy in the genetic code permits
variation in 273P4B7 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 such as at URL
dna.affrc.go.jp/.about.nakamura/codon.html.
[0186] 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)).
[0187] III.) 273P4B7-Related Proteins
[0188] Another aspect of the present invention provides
273P4B7-related proteins. Specific embodiments of 273P4B7 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 273P4B7 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 273P4B7 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 273P4B7 shown in FIG. 2 or FIG. 3.
[0189] Embodiments of a 273P4B7 polypeptide include: a 273P4B7
polypeptide having a sequence shown in FIG. 2, a peptide sequence
of a 273P4B7 as shown in FIG. 2 wherein T is U; at least 10
contiguous nucleotides of a polypeptide having the sequence as
shown in FIG. 2; or, at least 10 contiguous peptides of a
polypeptide having the sequence as shown in FIG. 2 where T is U.
For example, embodiments of 273P4B7 peptides comprise, without
limitation:
(I) a protein comprising, consisting essentially of, or consisting
of an amino acid sequence as shown in FIG. 2A-F or FIG. 3A-H;
(II) a 273P4B7-related protein that is at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100% homologous to an entire amino acid
sequence shown in FIG. 2A-F or 3A-H;
(III) a 273P4B7-related protein that is at least 90, 91, 92, 93,
94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid
sequence shown in FIG. 2A-F or 3A-H;
(IV) a protein that comprises at least one peptide set forth in
Tables VIII to XLIX, optionally with a proviso that it is not an
entire protein of FIG. 2;
(V) a protein that comprises at least one peptide set forth in
Tables VIII-XXI, collectively, which peptide is also set forth in
Tables XXII to XLIX, collectively, optionally with a proviso that
it is not an entire protein of FIG. 2;
(VI) a protein that comprises at least two peptides selected from
the peptides set forth in Tables VIII-XLIX, optionally with a
proviso that it is not an entire protein of FIG. 2;
(VII) a protein that comprises at least two peptides selected from
the peptides set forth in Tables VIII to XLIX collectively, with a
proviso that the protein is not a contiguous sequence from an amino
acid sequence of FIG. 2;
[0190] (VIII) a protein that comprises at least one peptide
selected from the peptides set forth in Tables VIII-XXI; and at
least one peptide selected from the peptides set forth in Tables
XXII to XLIX, with a proviso that the protein is not a contiguous
sequence from an amino acid sequence of FIG. 2;
[0191] (IX) a polypeptide comprising at least 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 amino acids of a protein of FIGS.
3A, 3C-3E in any whole number increment up to 1250 respectively
that includes at least 1, 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, amino acid position(s) having a value greater than
0.5 in the Hydrophilicity profile of FIG. 5;
[0192] (X) a polypeptide comprising at least 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 amino acids of a protein of FIGS. 3A,
3C-3E, in any whole number increment up to 1250 respectively that
includes at least at least 1, 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 amino acid position(s) having a value less
than 0.5 in the Hydropathicity profile of FIG. 6;
[0193] (XI) a polypeptide comprising at least 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 amino acids of a protein of FIGS.
3A, 3C-3E, in any whole number increment up to 1250 respectively
that includes at least at least 1, 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 amino acid position(s) having a value
greater than 0.5 in the Percent Accessible Residues profile of FIG.
7;
[0194] (XII) a polypeptide comprising at least 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 amino acids of a protein of FIGS.
3A, 3C-3E, in any whole number increment up to 1250 respectively
that includes at least at least 1, 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 amino acid position(s) having a value
greater than 0.5 in the Average Flexibility profile of FIG. 8;
[0195] (XIII) a polypeptide comprising at least 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, amino acids of a protein of FIGS. 3A,
3C-3E in any whole number increment up to 1250 respectively that
includes at least at least 1, 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 amino acid position(s) having a value
greater than 0.5 in the Beta-turn profile of FIG. 9;
[0196] (XIV) a polypeptide comprising at least 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 amino acids of a protein of FIG. 3B,
in any whole number increment up to 1127 respectively that includes
at least 1, 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 amino acid position(s) having a value greater than 0.5 in the
Hydrophilicity profile of FIG. 5;
[0197] (XV) a polypeptide comprising at least 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 amino acids of a protein of FIG. 3B,
in any whole number increment up to 1127 respectively that includes
at least at least 1, 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 amino acid position(s) having a value less than 0.5
in the Hydropathicity profile of FIG. 6;
[0198] (XVI) a polypeptide comprising at least 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 amino acids of a protein of FIG. 3B,
in any whole number increment up to 1127 respectively that includes
at least at least 1, 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 amino acid position(s) having a value greater than
0.5 in the Percent Accessible Residues profile of FIG. 7;
[0199] (XVII) a polypeptide comprising at least 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 amino acids of a protein of FIG. 3B,
in any whole number increment up to 1127 respectively that includes
at least at least 1, 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 amino acid position(s) having a value greater than
0.5 in the Average Flexibility profile of FIG. 8;
[0200] (XVIII) a polypeptide comprising at least 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, amino acids of a protein of FIG. 3B in
any whole number increment up to 1127 respectively that includes at
least at least 1, 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 amino acid position(s) having a value greater than 0.5
in the Beta-turn profile of FIG. 9;
(XIX) a peptide that occurs at least twice in Tables VIII-XXI and
XXII to XLIX, collectively;
(XX) a peptide that occurs at least three times in Tables VIII-XXI
and XXII to XLIX, collectively;
(XXI) a peptide that occurs at least four times in Tables VIII-XXI
and XXII to XLIX, collectively;
(XXII) a peptide that occurs at least five times in Tables VIII-XXI
and XXII to XLIX, collectively;
(XXIII) a peptide that occurs at least once in Tables VIII-XXI, and
at least once in tables XXII to XLIX;
(XXIV) a peptide that occurs at least once in Tables VIII-XXI, and
at least twice in tables XXII to XLIX;
(XXV) a peptide that occurs at least twice in Tables VIII-XXI, and
at least once in tables XXII to XLIX;
(XXVI) a peptide that occurs at least twice in Tables VIII-XXI, and
at least twice in tables XXII to XLIX;
(XXVII) a peptide which comprises one two, three, four, or five of
the following characteristics, or an oligonucleotide encoding such
peptide:
[0201] i) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or greater than 0.5, 0.6, 0.7,
0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity
profile of FIG. 5;
[0202] ii) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or less than 0.5, 0.4, 0.3, 0.2,
0.1, or having a value equal to 0.0, in the Hydropathicity profile
of FIG. 6;
[0203] iii) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or greater than 0.5, 0.6, 0.7,
0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible
Residues profile of FIG. 7;
[0204] iv) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or greater than 0.5, 0.6, 0.7,
0.8, 0.9, or having a value equal to 1.0, in the Average
Flexibility profile of FIG. 8; or,
[0205] v) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or greater than 0.5, 0.6, 0.7,
0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile
of FIG. 9;
(XXVIII) a composition comprising a peptide of (I)-(XXVII) or an
antibody or binding region thereof together with a pharmaceutical
excipient and/or in a human unit dose form.
(XXIX) a method of using a peptide of (I)-(XXVII), or an antibody
or binding region thereof or a composition of (XXVIII) in a method
to modulate a cell expressing 273P4B7;
(XXX) a method of using a peptide of (I)-(XXVII) or an antibody or
binding region thereof or a composition of (XXVIII) in a method to
diagnose, prophylax, prognose, or treat an individual who bears a
cell expressing 273P4B7;
[0206] (XXXI) a method of using a peptide of (I)-(XXVII) or an
antibody or binding region thereof or a composition (XXVIII) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 273P4B7, said cell from a cancer of a
tissue listed in Table I;
(XXXII) a method of using a peptide of (I)-(XXVII) or an antibody
or binding region thereof or a composition of (XXVIII) in a method
to diagnose, prophylax, prognose, or treat a cancer;
(XXXIII) a method of using a peptide of (I)-(XXVII) or an antibody
or binding region thereof or a composition of (XXVIII) in a method
to diagnose, prophylax, prognose, or treat a cancer of a tissue
listed in Table I; and;
(XXXIV) a method of using a peptide of (I)-(XXVII) or an antibody
or binding region thereof or a composition (XXVIII) in a method to
identify or characterize a modulator of a cell expressing
273P4B7.
[0207] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0208] Typical embodiments of the invention disclosed herein
include 273P4B7 polynucleotides that encode specific portions of
273P4B7 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0209] (a)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, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225,
1235, 1240, 1245, and 1250, or more contiguous amino acids of
273P4B7 variant 1; the maximal lengths relevant for other variants
are: variant 2, 1127 amino acids; variant 4, 1250 amino acids,
variant 5, 1250 amino acids; variant 6, 1250 amino acids; variant
9, 1106 amino acids; variant 10, 419 amino acids; and variant 11,
419 amino acids.
[0210] In general, naturally occurring allelic variants of human
273P4B7 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
273P4B7 protein contain conservative amino acid substitutions
within the 273P4B7 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 273P4B7. One class of 273P4B7 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 273P4B7 amino acid sequence, but further
contain a radical departure from the sequence, such as a
nonconservative 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.
[0211] 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 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" 2.sup.nd 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).
[0212] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 273P4B7 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 273P4B7 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
273P4B7 variant DNA.
[0213] 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., New
York); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine
substitution does not yield adequate amounts of variant, an
isosteric amino acid can be used.
[0214] As defined herein, 273P4B7 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 273P4B7 protein having an amino
acid sequence of FIG. 3. As used in this sentence, "cross reactive"
means that an antibody or T cell that specifically binds to a
273P4B7 variant also specifically binds to a 273P4B7 protein having
an amino acid sequence set forth in FIG. 3. A polypeptide ceases to
be a variant of a protein shown in FIG. 3, when it no longer
contains any epitope capable of being recognized by an antibody or
T cell that specifically binds to the starting 273P4B7 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.
[0215] Other classes of 273P4B7-related protein variants share 70%,
75%, 80%, 85% or 90% or more similarity with an amino acid sequence
of FIG. 3, or a fragment thereof. Another specific class of 273P4B7
protein variants or analogs comprises one or more of the 273P4B7
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of 273P4B7
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.
[0216] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a 273P4B7 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 a 273P4B7 protein shown in
FIG. 2 or FIG. 3.
[0217] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of a 273P4B7 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a 273P4B7 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a 273P4B7 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of a
273P4B7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of a 273P4B7 protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 273P4B7 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 60 to about
amino acid 70 of a 273P4B7 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 70 to about amino acid
80 of a 273P4B7 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 80 to about amino acid 90 of a
273P4B7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 90 to about amino acid 100 of a 273P4B7 protein
shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a
273P4B7 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 a 273P4B7 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.
[0218] 273P4B7-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
273P4B7-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a 273P4B7 protein
(or variants, homologs or analogs thereof).
[0219] Moreover the invention comprises 273P4B7 nucleic acid and
amino acid sequences. Further, the invention comprises variants of
273P4B7, and fragments thereof. In an embodiment of the invention a
protein fragment is: a subsequence of at least 158, or 262, or 420
contiguous amino acids of a protein of 273P4B7 v. 1; is an amino
acid subsequence of a protein of 273P4B7 v. 1 with a proviso that
273P4B7 v. 1 protein is such that it does not include an valine (V)
or methionine (M) at position 145; arginine (R) or glycine (G) at
position 172; isoleucine (I) or valine (V) at position 889; or,
lysine (K) or arginine (R) at position 989. An embodiment of an
amino acid sequence of the invention is a fragment of a protein of
273P4B7 v. 1 with a proviso that it is not a protein of 273P4B7 v.
9, v. 10 or v.11. In an embodiment, an amino acid fragment of the
invention is 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, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 170,
175, 180, 185, 190, 195, 200, 225, 250, 260, 261, 262, 263, 264,
265, 270, 275, 300, 325, 350, 375, 400, 418, 419, 420, 421, 422,
423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 422, 434, 435,
450, 475, 500, 525, 550, 575, 600, 650, 675, 700, 705, 710, 715,
716, 717, 718, 719, 720, 725, 750, 775, 800, 825, 850, 875, 900,
925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1127, 1150,
1175, 1200, 1025, or 1250 contiguous amino acids of a protein of
FIG. 2; in certain embodiments the fragment/subsequence comprises a
functional or structural motif, e.g., as set forth herein, or
comprises an immune system (antibody or T cell) epitope.
Embodiments of a nucleic acid sequence of the invention comprise a
sequence that encodes an amino acid sequence as set forth
herein.
[0220] III.A.) Motif-Bearing Protein Embodiments
[0221] Additional illustrative embodiments of the invention
disclosed herein include 273P4B7 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a 273P4B7 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., URL addresses:
pfam.wustl.edu/;
searchlauncher.bcm.tmc.edu/seqsearch/struc-pr-edict.html;
psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/;
ebi.ac.uk/interpro/scan-.html; expasy.ch/tools/scnpsit1.html;
Epimatrix.TM. and Epimer.TM., Brown University,
brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dcrt.nih.gov/.).
[0222] Motif bearing subsequences of all 273P4B7 variant proteins
are set forth and identified in Tables VIII-XXI and XXII-XLIX.
[0223] Table V sets forth several frequently occurring motifs based
on pfam searches (see URL address pfam.wustl.edu/). The columns of
Table V 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.
[0224] Polypeptides comprising one or more of the 273P4B7 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 273P4B7 motifs discussed above are associated with growth
dysregulation and because 273P4B7 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)).
[0225] 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
VIII-XXI and XXII-XLIX. CTL epitopes can be determined using
specific algorithms to identify peptides within a 273P4B7 protein
that are capable of optimally binding to specified HLA alleles
(e.g., Table IV; Epimatrix.TM. and Epimer.TM., Brown University,
URL brown.edu/Research/TB-HIV_Lab/epima-trix/epimatrix.html; and
BIMAS, URL bimas.dcrt.nih.gov/.) 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.
[0226] 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, on the basis of residues
defined in Table IV, one can substitute out a deleterious residue
in favor of any other residue, such as a preferred residue;
substitute a less-preferred residue with a preferred residue; or
substitute an originally-occurring preferred residue with another
preferred residue. Substitutions can occur at primary anchor
positions or at other positions in a peptide; see, e.g., Table
IV.
[0227] 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 97/33602 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.
[0228] Related embodiments of the invention include polypeptides
comprising combinations of the different motifs set forth in Table
VI, and/or, one or more of the predicted CTL epitopes of Tables
VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL
epitopes of Tables XLVI-XLIX, 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
within 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.
[0229] 273P4B7-related proteins are embodied in many forms,
preferably in isolated form. A purified 273P4B7 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 273P4B7 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 273P4B7-related proteins include
purified 273P4B7-related proteins and functional, soluble
273P4B7-related proteins. In one embodiment, a functional, soluble
273P4B7 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0230] The invention also provides 273P4B7 proteins comprising
biologically active fragments of a 273P4B7 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting 273P4B7 protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting 273P4B7 protein; to be bound by such
antibodies; to elicit the activation of HTL or CTL; and/or, to be
recognized by HTL or CTL that also specifically bind to the
starting protein.
[0231] 273P4B7-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 based on immunogenicity. Fragments that contain such
structures are particularly useful in generating subunit-specific
anU-273P4B7 antibodies or T cells or in identifying cellular
factors that bind to 273P4B7. For example, hydrophilicity profiles
can be generated, and immunogenic peptide fragments identified,
using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be
generated, and immunogenic peptide fragments identified, using the
method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.
157:105-132. Percent (%) Accessible Residues profiles can be
generated, and immunogenic peptide fragments identified, using the
method of Janin J., 1979, Nature 277:491-492. Average Flexibility
profiles can be generated, and immunogenic peptide fragments
identified, using the method of Bhaskaran R., Ponnuswamy P. K.,
1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can
be generated, and immunogenic peptide fragments identified, using
the method of Deleage, G., Roux B., 1987, Protein Engineering
1:289-294.
[0232] CTL epitopes can be determined using specific algorithms to
identify peptides within a 273P4B7 protein that are capable of
optimally binding to specified HLA alleles (e.g., by using the
SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/;
the listings in Table IV(A)-(E); Epimatrix.TM. and Epimer.TM.,
Brown University, URL
(brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and
BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide
epitopes from 273P4B7 that are presented in the context of human
MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35
were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX).
Specifically, the complete amino acid sequence of the 273P4B7
protein and relevant portions of other variants, i.e., for HLA
Class I predictions 9 flanking residues on either side of a point
mutation or exon junction, and for HLA Class II predictions 14
flanking residues on either side of a point mutation or exon
junction corresponding to that variant, were entered into the HLA
Peptide Motif Search algorithm found in the Bioinformatics and
Molecular Analysis Section (BIMAS) web site listed above; in
addition to the site SYFPEITHI, at URL
syfpeithi.bmi-heidelberg.com/.
[0233] 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 I 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 FILA 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 273P4B7 predicted binding peptides are
shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI
and XXII-XLVII, selected 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.
In Tables XLVI-XLIX, selected candidates, 15-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.
[0234] 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.
[0235] 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 site URL syfpeithi.bmi-heidelberg.com/, or BIMAS,
bimas.dcrt.nih.gov/) are to be "applied" to a 273P4B7 protein in
accordance with the invention. As used in this context "applied"
means that a 273P4B7 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 a 273P4B7 protein
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.
[0236] III.B.) Expression of 273P4B7-Related Proteins
[0237] In an embodiment described in the examples that follow,
73P4B7 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 273P4B7 with a C-terminal
6xHis 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 273P4B7 protein in transfected cells. The secreted
HIS-tagged 273P4B7 in the culture media can be purified, e.g.,
using a nickel column using standard techniques.
[0238] III.C.) Modifications of 273P4B7-Related Proteins
[0239] Modifications of 273P4B7-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 273P4B7 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a 273P4B7 protein. Another type of
covalent modification of a 273P4B7 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 273P4B7 comprises linking a 273P4B7 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. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0240] The 273P4B7-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 273P4B7
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 a 273P4B7 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 273P4B7. A chimeric
molecule can comprise a fusion of a 273P4B7-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 a 273P4B7 protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
273P4B7-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 273P4B7 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 IgG1
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0241] III.D.) Uses of 273P4B7-Related Proteins
[0242] The proteins of the invention have a number of different
specific uses. As 273P4B7 is highly expressed in prostate and other
cancers, 273P4B7-related proteins are used in methods that assess
the status of 273P4B7 gene products in normal versus cancerous
tissues, thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of a 273P4B7 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 273P4B7-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within a 273P4B7 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,
273P4B7-related proteins that contain the amino acid residues of
one or more of the biological motifs in a 273P4B7 protein are used
to screen for factors that interact with that region of
273P4B7.
[0243] 273P4B7 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of a 273P4B7 protein), for identifying agents or cellular
factors that bind to 273P4B7 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.
[0244] Proteins encoded by the 273P4B7 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 a 273P4B7 gene product. Antibodies raised against a 273P4B7
protein or fragment thereof are useful in diagnostic and prognostic
assays, and imaging methodologies in the management of human
cancers characterized by expression of 273P4B7 protein, such as
those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 273P4B7-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0245] Various immunological assays useful for the detection of
273P4B7 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
273P4B7-expressing cells (e.g., in radioscintigraphic imaging
methods). 273P4B7 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
[0246] IV.) 273P4B7 Antibodies
[0247] Another aspect of the invention provides antibodies that
bind to 273P4B7-related proteins. Preferred antibodies specifically
bind to a 273P4B7-related protein and do not bind (or bind weakly)
to peptides or proteins that are not 273P4B7-related proteins under
physiological conditions. In this context, examples of
physiological conditions include: 1) phosphate buffered saline; 2)
Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or
normal saline (0.9% NaCl); 4) animal serum such as human serum; or,
5) a combination of any of 1) through 4); these reactions
preferably taking place at pH 7.5, alternatively in a range of pH
7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also,
these reactions taking place at a temperature between 4.degree. C.
to 370 C. For example, antibodies that bind 273P4B7 can bind
273P4B7-related proteins such as the homologs or analogs
thereof.
[0248] 273P4B7 antibodies of the invention are particularly useful
in cancer (see, e.g., Table I) 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 273P4B7 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 273P4B7 is involved, such as
advanced or metastatic prostate cancers.
[0249] The invention also provides various immunological assays
useful for the detection and quantification of 273P4B7 and mutant
273P4B7-related proteins. Such assays can comprise one or more
273P4B7 antibodies capable of recognizing and binding a
273P4B7-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.
[0250] 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.
[0251] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing 273P4B7 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 273P4B7
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 273P4B7 expressing cancers such as
prostate cancer.
[0252] 273P4B7 antibodies are also used in methods for purifying a
273P4B7-related protein and for isolating 273P4B7 homologues and
related molecules. For example, a method of purifying a
273P4B7-related protein comprises incubating a 273P4B7 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 273P4B7-related protein under conditions that
permit the 273P4B7 antibody to bind to the 273P4B7-related protein;
washing the solid matrix to eliminate impurities; and eluting the
273P4B7-related protein from the coupled antibody. Other uses of
273P4B7 antibodies in accordance with the invention include
generating anti-idiotypic antibodies that mimic a 273P4B7
protein.
[0253] 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 273P4B7-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 273P4B7 can also be used, such as a
273P4B7 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 273P4B7-related
protein is synthesized and used as an immunogen.
[0254] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 273P4B7-related protein or
273P4B7 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0255] The amino acid sequence of a 273P4B7 protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
273P4B7 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 273P4B7 amino acid
sequence are used to identify hydrophilic regions in the 273P4B7
structure. Regions of a 273P4B7 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, Garnier-Robson, Kyte-Doolitle, Eisenberg,
Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles
can be generated using the method of Hopp, T. P. and Woods, K. R.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity
profiles can be generated using the method of Kyte, J. and
Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%)
Accessible Residues profiles can be generated using the method of
Janin J., 1979, Nature 277:491-492. Average Flexibility profiles
can be generated using the method of Bhaskaran R., Ponnuswamy P.
K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles
can be generated using the method of Deleage, G., Roux B., 1987,
Protein Engineering 1:289-294. Thus, each region identified by any
of these programs or methods is within the scope of the present
invention. Methods for the generation of 273P4B7 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
273P4B7 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.
[0256] 273P4B7 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
273P4B7-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.
[0257] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a 273P4B7 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
273P4B7 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.
[0258] 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 273P4B7 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 273P4B7
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. No. 6,162,963 issued
19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and,
U.S. Pat. No. 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.
[0259] Reactivity of 273P4B7 antibodies with a 273P4B7-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 273P4B7-related proteins,
273P4B7-expressing cells or extracts thereof. A 273P4B7 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 273P4B7 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).
[0260] V.) 273P4B7 Cellular Immune Responses
[0261] 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.
[0262] 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; Babbill, 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); 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):201-1Z Review).
[0263] 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.)
[0264] 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).
[0265] 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.
[0266] Various strategies can be utilized to evaluate cellular
immunogenicity, including: 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 .sup.51Cr-release assay
involving peptide sensitized target cells.
[0267] 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 .sup.51 Cr-release assay involving peptide
sensitized target cells and target cells expressing endogenously
generated antigen.
[0268] 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 .sup.51Cr release involving
peptide-sensitized targets, T cell proliferation, or lymphokine
release.
[0269] VI.) 273P4B7 Transgenic Animals
[0270] Nucleic acids that encode a 273P4B7-related protein can also
be used to generate either transgenic animals or "knock out"
animals that, in turn, are useful in the development and screening
of therapeutically useful reagents. In accordance with established
techniques, cDNA encoding 273P4B7 can be used to clone genomic DNA
that encodes 273P4B7. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 273P4B7. 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. No.
4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued
26 Sep. 1989. Typically, particular cells would be targeted for
273P4B7 transgene incorporation with tissue-specific enhancers.
[0271] Transgenic animals that include a copy of a transgene
encoding 273P4B7 can be used to examine the effect of increased
expression of DNA that encodes 273P4B7. 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.
[0272] Alternatively, non-human homologues of 273P4B7 can be used
to construct a 273P4B7 "knock out" animal that has a defective or
altered gene encoding 273P4B7 as a result of homologous
recombination between the endogenous gene encoding 273P4B7 and
altered genomic DNA encoding 273P4B7 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 273P4B7 can be
used to clone genomic DNA encoding 273P4B7 in accordance with
established techniques. A portion of the genomic DNA encoding
273P4B7 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 a 273P4B7
polypeptide.
[0273] VII.) Methods for the Detection of 273P4B7
[0274] Another aspect of the present invention relates to methods
for detecting 273P4B7 polynucleotides and 273P4B7-related proteins,
as well as methods for identifying a cell that expresses 273P4B7.
The expression profile of 273P4B7 makes it a diagnostic marker for
metastasized disease. Accordingly, the status of 273P4B7 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 273P4B7 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.
[0275] More particularly, the invention provides assays for the
detection of 273P4B7 polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 273P4B7 polynucleotides
include, for example, a 273P4B7 gene or fragment thereof, 273P4B7
mRNA, alternative splice variant 273P4B7 mRNAs, and recombinant DNA
or RNA molecules that contain a 273P4B7 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 273P4B7
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0276] In one embodiment, a method for detecting a 273P4B7 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 a 273P4B7 polynucleotides as sense and
antisense primers to amplify 273P4B7 cDNAs therein; and detecting
the presence of the amplified 273P4B7 cDNA. Optionally, the
sequence of the amplified 273P4B7 cDNA can be determined.
[0277] In another embodiment, a method of detecting a 273P4B7 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 273P4B7
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 273P4B7 gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
273P4B7 nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0278] The invention also provides assays for detecting the
presence of a 273P4B7 protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a 273P4B7-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 273P4B7-related
protein in a biological sample comprises first contacting the
sample with a 273P4B7 antibody, a 273P4B7-reactive fragment
thereof, or a recombinant protein containing an antigen-binding
region of a 273P4B7 antibody; and then detecting the binding of
273P4B7-related protein in the sample.
[0279] Methods for identifying a cell that expresses 273P4B7 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 273P4B7 gene comprises
detecting the presence of 273P4B7 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 273P4B7 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 273P4B7, 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 273P4B7 gene comprises detecting the presence of
273P4B7-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 273P4B7-related proteins
and cells that express 273P4B7-related proteins.
[0280] 273P4B7 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 273P4B7 gene
expression. For example, 273P4B7 expression is significantly
upregulated in prostate cancer, and is expressed in cancers of the
tissues listed in Table I. Identification of a molecule or
biological agent that inhibits 273P4B7 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 273P4B7 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0281] VIII.) Methods for Monitoring the Status of 273P4B7-Related
Genes and their Products
[0282] 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 273P4B7 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 273P4B7 in a biological
sample of interest can be compared, for example, to the status of
273P4B7 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 273P4B7 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., Grever et
al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No.
5,837,501) to compare 273P4B7 status in a sample.
[0283] 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 273P4B7
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 273P4B7 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 273P4B7
comprises a change in the location of 273P4B7 and/or 273P4B7
expressing cells and/or an increase in 273P4B7 mRNA and/or protein
expression.
[0284] 273P4B7 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 a 273P4B7 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 273P4B7 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
a 273P4B7 gene), Northern analysis and/or PCR analysis of 273P4B7
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 273P4B7 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
273P4B7 proteins and/or associations of 273P4B7 proteins with
polypeptide binding partners). Detectable 273P4B7 polynucleotides
include, for example, a 273P4B7 gene or fragment thereof, 273P4B7
mRNA, alternative splice variants, 273P4B7 mRNAs, and recombinant
DNA or RNA molecules containing a 273P4B7 polynucleotide.
[0285] The expression profile of 273P4B7 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 273P4B7 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 273P4B7 status and diagnosing
cancers that express 273P4B7, such as cancers of the tissues listed
in Table I. For example, because 273P4B7 mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of 273P4B7 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 273P4B7 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0286] The expression status of 273P4B7 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 273P4B7 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.
[0287] As described above, the status of 273P4B7 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 273P4B7 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 273P4B7
expressing cells (e.g. those that express 273P4B7 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 273P4B7-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 273P4B7 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 prostate) to a different area of
the body (such as a lymph node). In this context, evidence of
dysregulated cellular growth is important for example 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).
[0288] In one aspect, the invention provides methods for monitoring
273P4B7 gene products by determining the status of 273P4B7 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 273P4B7 gene products in a corresponding normal
sample. The presence of aberrant 273P4B7 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.
[0289] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 273P4B7 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
273P4B7 mRNA can, for example, be evaluated in tissues including
but not limited to those listed in Table I. The presence of
significant 273P4B7 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 273P4B7 mRNA or
express it at lower levels.
[0290] In a related embodiment, 273P4B7 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 273P4B7 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 273P4B7 expressed in a corresponding
normal sample. In one embodiment, the presence of 273P4B7 protein
is evaluated, for example, using immunohistochemical methods.
273P4B7 antibodies or binding partners capable of detecting 273P4B7
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0291] In a further embodiment, one can evaluate the status of
273P4B7 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
273P4B7 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 273P4B7 indicates a potential loss of function or
increase in tumor growth.
[0292] 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 273P4B7 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. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No.
5,952,170 issued 17 Jan. 1995).
[0293] Additionally, one can examine the methylation status of a
273P4B7 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 pi-class glutathione S-transferase
(a protein expressed in normal prostate but not expressed in
>90% of prostate carcinomas) appears to permanently silence
transcription of this gene and is the most frequently detected
genomic alteration in prostate carcinomas (De Marzo et al., Am. J.
Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is
present in at least 70% of cases of high-grade prostatic
intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.
Biomarkers Prev., 1998, 7:531-536). In another example, expression
of the LAGE-1 tumor specific gene (which is not expressed in normal
prostate but is expressed in 25-50% of prostate cancers) is induced
by deoxy-azacytidine in lymphoblastoid cells, suggesting that
tumoral expression is due to demethylation (Lethe et al., Int. J.
Cancer 76(6): 903-908 (1998)). 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 that 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.
[0294] Gene amplification is an additional method for assessing the
status of 273P4B7. 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.
[0295] 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 273P4B7 expression.
The presence of RT-PCR amplifiable 273P4B7 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. In the prostate
cancer field, these include RT-PCR assays for the detection of
cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res.
25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;
Heston et al., 1995, Clin. Chem. 41:1687-1688).
[0296] 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 273P4B7 mRNA or 273P4B7 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 273P4B7 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 273P4B7
in prostate or other tissue is examined, with the presence of
273P4B7 in the sample providing an indication of prostate cancer
susceptibility (or the emergence or existence of a prostate tumor).
Similarly, one can evaluate the integrity 273P4B7 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 273P4B7 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0297] 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
273P4B7 mRNA or 273P4B7 protein expressed by tumor cells, comparing
the level so determined to the level of 273P4B7 mRNA or 273P4B7
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 273P4B7 mRNA or 273P4B7 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 273P4B7 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 273P4B7 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.
[0298] 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 273P4B7 mRNA or 273P4B7 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 273P4B7 mRNA or 273P4B7 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 273P4B7 mRNA or 273P4B7 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 273P4B7 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
273P4B7 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.
[0299] 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 273P4B7 gene and 273P4B7 gene products (or
perturbations in 273P4B7 gene and 273P4B7 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.
PSA, PSCA and PSM expression for prostate cancer 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 273P4B7 gene
and 273P4B7 gene products (or perturbations in 273P4B7 gene and
273P4B7 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.
[0300] In one embodiment, methods for observing a coincidence
between the expression of 273P4B7 gene and 273P4B7 gene products
(or perturbations in 273P4B7 gene and 273P4B7 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 273P4B7 mRNA or protein in a tissue sample,
detecting the overexpression of PSA mRNA or protein in a issue
sample (or PSCA or PSM expression), and observing a coincidence of
273P4B7 mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of 273P4B7 and PSA mRNA in prostate tissue is examined, where the
coincidence of 273P4B7 and PSA mRNA overexpression in the sample
indicates the existence of prostate cancer, prostate cancer
susceptibility or the emergence or status of a prostate tumor.
[0301] Methods for detecting and quantifying the expression of
273P4B7 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 273P4B7 mRNA include in situ hybridization using
labeled 273P4B7 riboprobes, Northern blot and related techniques
using 273P4B7 polynucleotide probes, RT-PCR analysis using primers
specific for 273P4B7, 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 273P4B7 mRNA expression. Any number of primers
capable of amplifying 273P4B7 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
273P4B7 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0302] IX.) Identification of Molecules that Interact with
273P4B7
[0303] The 273P4B7 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 273P4B7, as well as
pathways activated by 273P4B7 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. No. 5,955,280 issued 21 Sep. 1999,
U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No.
5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 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 November 1999, 83-86).
[0304] Alternatively one can screen peptide libraries to identify
molecules that interact with 273P4B7 protein sequences. In such
methods, peptides that bind to 273P4B7 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 273P4B7 protein(s).
[0305] 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 273P4B7 protein sequences are disclosed for example
in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No.
5,733,731 issued 31 Mar. 1998.
[0306] Alternatively, cell lines that express 273P4B7 are used to
identify protein-protein interactions mediated by 273P4B7. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 273P4B7 protein can be immunoprecipitated from
273P4B7-expressing cell lines using anti-273P4B7 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 273P4B7 and a His-tag
(vectors mentioned above). The immunoprecipitated complex can be
examined for protein association by procedures such as Western
blotting, .sup.35S-methionine labeling of proteins, protein
microsequencing, silver staining and two-dimensional gel
electrophoresis.
[0307] Small molecules and ligands that interact with 273P4B7 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 273P4B7'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
273P4B7-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses 273P4B7 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass.,
1992). Moreover, ligands that regulate 273P4B7 function can be
identified based on their ability to bind 273P4B7 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 273P4B7 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
273P4B7.
[0308] An embodiment of this invention comprises a method of
screening for a molecule that interacts with a 273P4B7 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a 273P4B7 amino acid
sequence, allowing the population of molecules and the 273P4B7
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 273P4B7 amino acid sequence, and then separating molecules
that do not interact with the 273P4B7 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 273P4B7 amino acid sequence. The identified
molecule can be used to modulate a function performed by 273P4B7.
In a preferred embodiment, the 273P4B7 amino acid sequence is
contacted with a library of peptides.
[0309] X.) Therapeutic Methods and Compositions
[0310] The identification of 273P4B7 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in cancers such as those listed in Table I, opens a
number of therapeutic approaches to the treatment of such
cancers.
[0311] Of note, targeted antitumor therapies have been useful even
when the targeted protein is expressed on normal tissues, even
vital normal organ tissues. A vital organ is one that is necessary
to sustain life, such as the heart or colon. A non-vital organ is
one that can be removed whereupon the individual is still able to
survive. Examples of non-vital organs are ovary, breast, and
prostate.
[0312] For example, Herceptin.RTM. is an FDA approved
pharmaceutical that has as its active ingredient an antibody which
is immunoreactive with the protein variously known as HER2,
HER2/neu, and erb-b-2. It is marketed by Genentech and has been a
commercially successful antitumor agent. Herceptin sales reached
almost $400 million in 2002. Herceptin is a treatment for HER2
positive metastatic breast cancer. However, the expression of HER2
is not limited to such tumors. The same protein is expressed in a
number of normal tissues. In particular, it is known that HER2/neu
is present in normal kidney and heart, thus these tissues are
present in all human recipients of Herceptin. The presence of
HER2/neu in normal kidney is also confirmed by Latif, Z., et al.,
B.J.U. International (2002) 89:5-9. As shown in this article (which
evaluated whether renal cell carcinoma should be a preferred
indication for anti-HER2 antibodies such as Herceptin) both protein
and mRNA are produced in benign renal tissues. Notably, HER2/neu
protein was strongly overexpressed in benign renal tissue. Despite
the fact that HER2/neu is expressed in such vital tissues as heart
and kidney, Herceptin is a very useful, FDA approved, and
commercially successful drug. The effect of Herceptin on cardiac
tissue, i.e., "cardiotoxicity," has merely been a side effect to
treatment. When patients were treated with Herceptin alone,
significant cardiotoxicity occurred in a very low percentage of
patients.
[0313] Of particular note, although kidney tissue is indicated to
exhibit normal expression, possibly even higher expression than
cardiac tissue, kidney has no appreciable Herceptin side effect
whatsoever. Moreover, of the diverse array of normal tissues in
which HER2 is expressed, there is very little occurrence of any
side effect. Only cardiac tissue has manifested any appreciable
side effect at all. A tissue such as kidney, where HER2/neu
expression is especially notable, has not been the basis for any
side effect.
[0314] Furthermore, favorable therapeutic effects have been found
for antitumor therapies that target epidermal growth factor
receptor (EGFR). EGFR is also expressed in numerous normal tissues.
There have been very limited side effects in normal tissues
following use of anti-EGFR therapeutics.
[0315] Thus, expression of a target protein in normal tissue, even
vital normal tissue, does not defeat the utility of a targeting
agent for the protein as a therapeutic for certain tumors in which
the protein is also overexpressed.
[0316] Accordingly, therapeutic approaches that inhibit the
activity of a 273P4B7 protein are useful for patients suffering
from a cancer that expresses 273P4B7. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a 273P4B7
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a 273P4B7 gene or translation of 273P4B7 mRNA.
[0317] X.A.) Anti-Cancer Vaccines
[0318] The invention provides cancer vaccines comprising a
273P4B7-related protein or 273P4B7-related nucleic acid. In view of
the expression of 273P4B7, cancer vaccines prevent and/or treat
273P4B7-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 and has been employed in prostate
cancer using human PSMA and rodent PAP immunogens (Hodge et al.,
1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol.
159:3113-3117).
[0319] Such methods can be readily practiced by employing a
273P4B7-related protein, or a 273P4B7-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 273P4B7 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 a
273P4B7 protein shown in FIG. 3 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, a 273P4B7 immunogen contains
a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a
peptide of a size range from 273P4B7 indicated in FIG. 5, FIG. 6,
FIG. 7, FIG. 8, and FIG. 9.
[0320] The entire 273P4B7 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.
[0321] In patients with 273P4B7-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.
[0322] Cellular Vaccines:
[0323] CTL epitopes can be determined using specific algorithms to
identify peptides within 273P4B7 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University (URL
brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and,
BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL
syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a
273P4B7 immunogen contains one or more amino acid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables VIII-XXI and XXII-XLIX 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.
[0324] Antibody-Based Vaccines
[0325] 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. a 273P4B7 protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to 273P4B7 in a host,
by contacting the host with a sufficient amount of at least one
273P4B7 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval thereafter re-contacting the host
with the 273P4B7 B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a 273P4B7-related protein or a man-made
multiepitopic peptide comprising: administering 273P4B7 immunogen
(e.g. a 273P4B7 protein or a peptide fragment thereof, a 273P4B7
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 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 273P4B7 immunogen by: administering in vivo to
muscle or skin of the individual's body a DNA molecule that
comprises a DNA sequence that encodes a 273P4B7 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. In addition, an antiidiotypic antibody can be
administered that mimics 273P4B7, in order to generate a response
to the target antigen.
[0326] Nucleic Acid Vaccines:
[0327] 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 273P4B7. Constructs comprising DNA encoding a
273P4B7-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 273P4B7 protein/immunogen.
Alternatively, a vaccine comprises a 273P4B7-related protein.
Expression of the 273P4B7-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a 273P4B7 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 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).
[0328] 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
273P4B7-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0329] 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.
[0330] Thus, gene delivery systems are used to deliver a
273P4B7-related nucleic acid molecule. In one embodiment, the
full-length human 273P4B7 cDNA is employed. In another embodiment,
273P4B7 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0331] Ex Vivo Vaccines
[0332] 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
273P4B7 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
prostate cancer, autologous dendritic cells pulsed with peptides of
the prostate-specific membrane antigen (PSMA) are being used in a
Phase I clinical trial to stimulate prostate cancer patients'
immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et
al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used
to present 273P4B7 peptides to T cells in the context of MHC class
I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with 273P4B7 peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete 273P4B7 protein. Yet another
embodiment involves engineering the overexpression of a 273P4B7
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 273P4B7 can also be engineered to express immune
modulators, such as GM-CSF, and used as immunizing agents.
[0333] X.B.) 273P4B7 as a Target for Antibody-Based Therapy
[0334] 273P4B7 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 273P4B7 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 273P4B7-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 273P4B7 are useful
to treat 273P4B7-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0335] 273P4B7 antibodies can be introduced into a patient such
that the antibody binds to 273P4B7 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 273P4B7, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0336] Those skilled in the art understand that antibodies can be
used to specifically target and bind immunogenic molecules such as
an immunogenic region of a 273P4B7 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. 273P4B7), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0337] 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-273P4B7
antibody) that binds to a marker (e.g. 273P4B7) 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 273P4B7, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
273P4B7 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.
[0338] Cancer immunotherapy using anti-273P4B7 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 or radioisotope, such as
the conjugation of Y.sup.91 or I.sup.131 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.). The antibodies can
be conjugated to a therapeutic agent. To treat prostate cancer, for
example, 273P4B7 antibodies can be administered in conjunction with
radiation, chemotherapy or hormone ablation. Also, antibodies can
be conjugated to a toxin such as calicheamicin (e.g., Mylotarg.TM.,
Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG.sub.4
kappa antibody conjugated to antitumor antibiotic calicheamicin) or
a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP,
platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No.
5,416,064).
[0339] Although 273P4B7 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. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al.
(International J. of Onco. 9:217-224, 1996), and Hancock et al.
(Cancer Res. 51:4575-4580, 1991) describe the use of various
antibodies together with chemotherapeutic agents.
[0340] Although 273P4B7 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.
[0341] Cancer patients can be evaluated for the presence and level
of 273P4B7 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 273P4B7 imaging, or other
techniques that reliably indicate the presence and degree of
273P4B7 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.
[0342] Anti-273P4B7 monoclonal antibodies that treat prostate and
other cancers include those that initiate a potent immune response
against the tumor or those that are directly cytotoxic. In this
regard, anti-273P4B7 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-273P4B7 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 273P4B7.
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-273P4B7 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.
[0343] 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 273P4B7 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0344] Therapeutic methods of the invention contemplate the
administration of single anti-273P4B7 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-273P4B7 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-273P4B7 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0345] Anti-273P4B7 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-273P4B7 antibody preparation, via an acceptable route of
administration such as intravenous injection (IV), typically at a
dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body
weight. In general, doses in the range of 10-1000 mg mAb per week
are effective and well tolerated.
[0346] Based on clinical experience with the Herceptin.TM. 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-273P4B7 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 273P4B7 expression in the patient, the
extent of circulating shed 273P4B7 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.
[0347] Optionally, patients should be evaluated for the levels of
273P4B7 in a given sample (e.g. the levels of circulating 273P4B7
antigen and/or 273P4B7 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).
[0348] Anti-idiotypic anti-273P4B7 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 273P4B7-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-273P4B7 antibodies that mimic an epitope on a 273P4B7-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.
[0349] X.C.) 273P4B7 as a Target for Cellular Immune Responses
[0350] 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.
[0351] 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 L-lysine, poly L-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))
[0352] 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 273P4B7 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0353] 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).
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC.sub.50 of 500 nM or less, often 200 nM or less; and
for Class II an IC.sub.50 of 1000 nM or less.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] X.C.1. Minigene Vaccines
[0364] 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.
[0365] 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 273P4B7, the PADRE.RTM. universal helper T cell
epitope or multiple HTL epitopes from 273P4B7 (see e.g., Tables
VIII-XXI and XXII to XLIX), and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0366] 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.
[0367] 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 1 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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-P) may be
beneficial in certain diseases.
[0374] 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.
[0375] 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, Bio
Techniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309;
and Feigner, 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.
[0376] 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 (.sup.51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by .sup.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.
[0377] 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., 1M 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.
[0378] 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.
[0379] 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.
[0380] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0381] 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.
[0382] 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.
[0383] 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: 26), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 27), and Streptococcus 1 8 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 28).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0384] 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, most preferably,
to bind most HLA-DR (human HLA class II) molecules. For instance, a
pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAa
(SEQ ID NO: 29), where "X" is either cyclohexylalanine,
phenylalanine, or tyrosine, and a is either D-alanine or L-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.
[0385] 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.
[0386] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0387] 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 E- and a-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 E- and
a-amino groups of Lys, which is attached via linkage, e.g.,
Ser-Ser, to the amino terminus of the immunogenic peptide.
[0388] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) 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 P.sub.3CSS, for
example, and the lipopeptide administered to an individual to prime
specifically 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.
[0389] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0390] 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.
[0391] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 273P4B7. 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 273P4B7.
[0392] X.D. Adoptive Immunotherapy
[0393] Antigenic 273P4B7-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.
[0394] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0395] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 273P4B7. 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.
[0396] 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 273P4B7. 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.
[0397] For therapeutic use, administration should generally begin
at the first diagnosis of 273P4B7-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 273P4B7, a vaccine comprising
273P4B7-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0398] It is generally important to provide an amount of the
peptide epitope delivered by a mode of administration sufficient to
stimulate effectively a cytotoxic T cell response; compositions
which stimulate helper T cell responses can also be given in
accordance with this embodiment of the invention.
[0399] 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.
[0400] 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.
[0401] 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.
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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.
[0406] A human unit dose form of a composition is typically
included in a pharmaceutical composition that comprises a human
unit dose of an acceptable carrier, in one embodiment an aqueous
carrier, and is administered in a volume/quantity 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). For example a peptide dose for initial
immunization can be from about 1 to about 50,000 .mu.g, generally
100-5,000 .mu.g, for a 70 kg patient. For example, for nucleic
acids an initial immunization may be performed using an expression
vector 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.
[0407] For antibodies, a treatment generally involves repeated
administration of the anti-273P4B7 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. Moreover, 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-273P4B7
mAb preparation represents an acceptable dosing regimen. As
appreciated by those of skill in the art, various factors can
influence the ideal dose in a particular case. Such factors
include, for example, half life of a composition, the binding
affinity of an Ab, the immunogenicity of a substance, the degree of
273P4B7 expression in the patient, the extent of circulating shed
273P4B7 antigen, the desired steady-state 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.
Non-limiting preferred human unit doses are, for example, 500
.mu.g-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg,
400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800
mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the
dose is in a range of 2-5 mg/kg body weight, e.g., with follow on
weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
mg/kg body weight followed, e.g., in two, three or four weeks by
weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two,
three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350,
375, 400 mg m.sup.2 of body area weekly; 1-600 mg m.sup.2 of body
area weekly; 225-400 mg m.sup.2 of body area weekly; these does can
be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12
or more weeks.
[0408] In one embodiment, human unit dose forms of polynucleotides
comprise a suitable dosage range or effective amount that provides
any therapeutic effect. As appreciated by one of ordinary skill in
the art a therapeutic effect depends on a number of factors,
including the sequence of the polynucleotide, molecular weight of
the polynucleotide and route of administration. Dosages are
generally selected by the physician or other health care
professional in accordance with a variety of parameters known in
the art, such as severity of symptoms, history of the patient and
the like. Generally, for a polynucleotide of about 20 bases, a
dosage range may be selected from, for example, an independently
selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to
an independently selected upper limit, greater than the lower
limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500,
2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For
example, a dose may be about any of the following: 0.1 to 100
mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500
mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to
200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg,
500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral
routes of administration may require higher doses of polynucleotide
compared to more direct application to the nucleotide to diseased
tissue, as do polynucleotides of increasing length.
[0409] In one embodiment, human unit dose forms of T-cells comprise
a suitable dosage range or effective amount that provides any
therapeutic effect. As appreciated by one of ordinary skill in the
art, a therapeutic effect depends on a number of factors. Dosages
are generally selected by the physician or other health care
professional in accordance with a variety of parameters known in
the art, such as severity of symptoms, history of the patient and
the like. A dose may be about 10.sup.4 cells to about 10.sup.6
cells, about 10.sup.6 cells to about 10.sup.8 cells, about 10.sup.8
to about 10.sup.11 cells, or about 10.sup.8 to about
5.times.10.sup.10 cells. A dose may also about 10.sup.6
cells/m.sup.2 to about 10.sup.10 cells/m.sup.2, or about 10.sup.6
cells/m.sup.2 to about 10.sup.8 cells/m.sup.2.
[0410] 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.
[0411] 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.
[0412] 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%.
[0413] 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.
[0414] XI.) Diagnostic and Prognostic Embodiments of 273P4B7.
[0415] As disclosed herein, 273P4B7 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 the Example entitled
"Expression analysis of 273P4B7 in normal tissues, and patient
specimens").
[0416] 273P4B7 can be analogized to a prostate associated antigen
PSA, the archetypal marker that has been used by medical
practitioners for years to identify and monitor the presence of
prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2):
503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306
(1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):
1635-1640(1999)). A variety of other diagnostic markers are also
used in similar contexts including p53 and K-ras (see, e.g.,
Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto
et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this
disclosure of 273P4B7 polynucleotides and polypeptides (as well as
273P4B7 polynucleotide probes and anti-273P4B7 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.
[0417] Typical embodiments of diagnostic methods which utilize the
273P4B7 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
273P4B7 polynucleotides described herein can be utilized in the
same way to detect 273P4B7 overexpression or the metastasis of
prostate 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 273P4B7
polypeptides described herein can be utilized to generate
antibodies for use in detecting 273P4B7 overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0418] Specifically, because metastases involves the movement of
cancer cells from an organ of origin (such as the lung or prostate
gland 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 273P4B7 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
273P4B7-expressing cells (lymph node) is found to contain
273P4B7-expressing cells such as the 273P4B7 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0419] Alternatively 273P4B7 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 273P4B7 or
express 273P4B7 at a different level are found to express 273P4B7
or have an increased expression of 273P4B7 (see, e.g., the 273P4B7
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 273P4B7) such as PSA, PSCA
etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237
(1996)).
[0420] The use of immunohistochemistry to identify the presence of
a 273P4B7 polypeptide within a tissue section can indicate an
altered state of certain cells within that tissue. It is well
understood in the art that the ability of an antibody to localize
to a polypeptide that is expressed in cancer cells is a way of
diagnosing presence of disease, disease stage, progression and/or
tumor aggressiveness. Such an antibody can also detect an altered
distribution of the polypeptide within the cancer cells, as
compared to corresponding non-malignant tissue.
[0421] The 273P4B7 polypeptide and immunogenic compositions are
also useful in view of the phenomena of altered subcellular protein
localization in disease states. Alteration of cells from normal to
diseased state causes changes in cellular morphology and is often
associated with changes in subcellular protein
localization/distribution. For example, cell membrane proteins that
are expressed in a polarized manner in normal cells can be altered
in disease, resulting in distribution of the protein in a non-polar
manner over the whole cell surface.
[0422] The phenomenon of altered subcellular protein localization
in a disease state has been demonstrated with MUC1 and Her2 protein
expression by use of immunohistochemical means. Normal epithelial
cells have a typical apical distribution of MUC1, in addition to
some supranuclear localization of the glycoprotein, whereas
malignant lesions often demonstrate an apolar staining pattern
(Diaz et al., The Breast Journal, 7; 40-45 (2001); Zhang et al.,
Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al., The
Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)).
In addition, normal breast epithelium is either negative for Her2
protein or exhibits only a basolateral distribution whereas
malignant cells can express the protein over the whole cell surface
(De Potter, et al., International Journal of Cancer, 44; 969-974
(1989): McCormick, et al, 117; 935-943 (2002)). Alternatively,
distribution of the protein may be altered from a surface only
localization to include diffuse cytoplasmic expression in the
diseased state. Such an example can be seen with MUC1 (Diaz, et
al., The Breast Journal, 7: 40-45 (2001)).
[0423] Alteration in the localization/distribution of a protein in
the cell, as detected by immunohistochemical methods, can also
provide valuable information concerning the favorability of certain
treatment modalities. This last point is illustrated by a situation
where a protein may be intracellular in normal tissue, but cell
surface in malignant cells; the cell surface location makes the
cells favorably amenable to antibody-based diagnostic and treatment
regimens. When such an alteration of protein localization occurs
for 273P4B7, the 273P4B7 protein and immune responses related
thereto are very useful. Accordingly, the ability to determine
whether alteration of subcellular protein localization occurred for
24P4C12 make the 273P4B7 protein and immune responses related
thereto very useful. Use of the 273P4B7 compositions allows those
skilled in the art to make important diagnostic and therapeutic
decisions. Immunohistochemical reagents specific to 273P4B7 are
also useful to detect metastases of tumors expressing 273P4B7 when
the polypeptide appears in tissues where 273P4B7 is not normally
produced.
[0424] Thus, 273P4B7 polypeptides and antibodies resulting from
immune responses thereto are useful in a variety of important
contexts such as diagnostic, prognostic, preventative and/or
therapeutic purposes known to those skilled in the art.
[0425] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, 273P4B7 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 the Example entitled
"Expression analysis of 273P4B7 in normal tissues, and patient
specimens, where a 273P4B7 polynucleotide fragment is used as a
probe to show the expression of 273P4B7 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., a 273P4B7 polynucleotide shown in FIG. 2 or variant
thereof) under conditions of high stringency.
[0426] 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. 273P4B7
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
273P4B7 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. a 273P4B7
polypeptide shown in FIG. 3).
[0427] As shown herein, the 273P4B7 polynucleotides and
polypeptides (as well as the 273P4B7 polynucleotide probes and
anti-273P4B7 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 273P4B7 gene products, in order
to evaluate the presence or onset of a disease condition described
herein, such as prostate cancer, are used to identify patients for
preventive measures or further monitoring, as has been done so
successfully with PSA. Moreover, these materials satisfy a need in
the art for molecules having similar or complementary
characteristics to PSA in situations where, for example, a definite
diagnosis of metastasis of prostatic origin cannot be made on the
basis of a test for PSA alone (see, e.g., Alanen et al., Pathol.
Res. Pract. 192(3): 233-237 (1996)), and consequently, materials
such as 273P4B7 polynucleotides and polypeptides (as well as the
273P4B7 polynucleotide probes and anti-273P4B7 antibodies used to
identify the presence of these molecules) need to be employed to
confirm a metastases of prostatic origin.
[0428] Finally, in addition to their use in diagnostic assays, the
273P4B7 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 273P4B7 gene maps (see the Example entitled "Chromosomal
Mapping of 273P4B7" below). Moreover, in addition to their use in
diagnostic assays, the 273P4B7-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).
[0429] Additionally, 273P4B7-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 273P4B7. 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
a 273P4B7 antigen. Antibodies or other molecules that react with
273P4B7 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0430] XII.) Inhibition of 273P4B7 Protein Function
[0431] The invention includes various methods and compositions for
inhibiting the binding of 273P4B7 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
273P4B7 function.
[0432] XII.A.) Inhibition of 273P4B7 with Intracellular
Antibodies
[0433] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 273P4B7 are introduced
into 273P4B7 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-273P4B7 antibody is
expressed intracellularly, binds to 273P4B7 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).
[0434] 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 target
precisely 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.
[0435] In one embodiment, intrabodies are used to capture 273P4B7
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 273P4B7
intrabodies in order to achieve the desired targeting. Such 273P4B7
intrabodies are designed to bind specifically to a particular
273P4B7 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a 273P4B7 protein are used to prevent 273P4B7
from gaining access to the nucleus, thereby preventing it from
exerting any biological activity within the nucleus (e.g.,
preventing 273P4B7 from forming transcription complexes with other
factors).
[0436] 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 prostate, for example, the PSA
promoter and/or promoter/enhancer can be utilized (See, for
example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).
[0437] XII.B.) Inhibition of 273P4B7 with Recombinant Proteins
[0438] In another approach, recombinant molecules bind to 273P4B7
and thereby inhibit 273P4B7 function. For example, these
recombinant molecules prevent or inhibit 273P4B7 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 273P4B7 specific antibody
molecule. In a particular embodiment, the 273P4B7 binding domain of
a 273P4B7 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 273P4B7 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 patents suffering from a cancer associated with the
expression of 273P4B7, whereby the dimeric fusion protein
specifically binds to 273P4B7 and blocks 273P4B7 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0439] XII.C.) Inhibition of 273P4B7 Transcription or
Translation
[0440] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 273P4B7 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 273P4B7 mRNA into protein.
[0441] In one approach, a method of inhibiting the transcription of
the 273P4B7 gene comprises contacting the 273P4B7 gene with a
273P4B7 antisense polynucleotide. In another approach, a method of
inhibiting 273P4B7 mRNA translation comprises contacting a 273P4B7
mRNA with an antisense polynucleotide. In another approach, a
273P4B7 specific ribozyme is used to cleave a 273P4B7 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
273P4B7 gene, such as 273P4B7 promoter and/or enhancer elements.
Similarly, proteins capable of inhibiting a 273P4B7 gene
transcription factor are used to inhibit 273P4B7 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.
[0442] Other factors that inhibit the transcription of 273P4B7 by
interfering with 273P4B7 transcriptional activation are also useful
to treat cancers expressing 273P4B7. Similarly, factors that
interfere with 273P4B7 processing are useful to treat cancers that
express 273P4B7. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0443] XII.D.) General Considerations for Therapeutic
Strategies
[0444] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 273P4B7 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 273P4B7 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 273P4B7 antisense polynucleotides, ribozymes,
factors capable of interfering with 273P4B7 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0445] 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.
[0446] 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 273P4B7 to a binding partner, etc.
[0447] In vivo, the effect of a 273P4B7 therapeutic composition can
be evaluated in a suitable animal model. For example, xenogenic
prostate cancer models can be used, wherein human prostate cancer
explants or passaged xenograft tissues are introduced into immune
compromised animals, such as nude or SCID mice (Klein et al., 1997,
Nature Medicine 3: 402-408). For example, PCT Patent Application
WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft
models of human prostate cancer capable of recapitulating the
development of primary tumors, micrometastasis, and the formation
of osteoblastic metastases characteristic of late stage disease.
Efficacy can be predicted using assays that measure inhibition of
tumor formation, tumor regression or metastasis, and the like.
[0448] 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.
[0449] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carder 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 16.sup.th
Edition, A. Osal., Ed., 1980).
[0450] 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.
[0451] 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.
[0452] XIII.) Identification, Characterization and Use of
Modulators of 273P4B7
[0453] Methods to Identify and Use Modulators
[0454] 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.
[0455] 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.
[0456] Modulator-Related Identification and Screening Assays:
[0457] Gene Expression-Related Assays
[0458] 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); Zlokamik, et al., Science 279:84-8 (1998); Heid,
Genome Res 6:986-94, 1996).
[0459] 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 Zlokamik, supra.
[0460] 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 1 0-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.
[0461] 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.
[0462] Expression Monitoring to Identify Compounds that Modify Gene
Expression
[0463] 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.
[0464] 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.
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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.
[0469] 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.
[0470] 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.
[0471] 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.
[0472] 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.
[0473] Biological Activity-Related Assays
[0474] 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.
[0475] 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.
[0476] 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.
[0477] 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.
[0478] High Throughput Screening to Identify Modulators
[0479] 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.
[0480] 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.
[0481] Use of Soft Agar Growth and Colony Formation to Identify and
Characterize Modulators
[0482] 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.
[0483] 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.
[0484] Evaluation of Contact Inhibition and Growth Density
Limitation to Identify and Characterize Modulators
[0485] 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 (.sup.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.
[0486] In this assay, labeling index with .sup.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.
[0487] 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.
[0488] Evaluation of Growth Factor or Serum Dependence to Identify
and Characterize Modulators
[0489] 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.
[0490] Use of Tumor-Specific Marker Levels to Identify and
Characterize Modulators
[0491] 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).
[0492] 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.
[0493] Invasiveness into Matrigel to Identify and Characterize
Modulators
[0494] 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. 1251 and counting the
radioactivity on the distal side of the filter or bottom of the
dish. See, e.g., Freshney (1984), supra.
[0495] Evaluation of Tumor Growth In Vivo to Identify and
Characterize Modulators
[0496] 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.
[0497] 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).
[0498] 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 thymectomized 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 10.sup.6 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.
[0499] In Vitro Assays to Identify and Characterize Modulators
[0500] 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.
[0501] 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 G F,
supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol.
1998: 9:624).
[0502] 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.
[0503] 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.
[0504] Binding Assays to Identify and Characterize Modulators
[0505] In binding assays in accordance with the invention, a
purified or isolated gene 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.
[0506] 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.
[0507] 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.
[0508] 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, 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.
[0509] 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.
[0510] 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.
[0511] 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.
[0512] 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., 1.sup.125, for the proteins and a fluorophor for the
compound. Proximity reagents, e.g., quenching or energy transfer
reagents are also useful.
[0513] Competitive Binding to Identify and Characterize
Modulators
[0514] 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.
[0515] 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.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] 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.
[0520] 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.
[0521] Use of Polynucleotides to Down-Regulate or Inhibit a Protein
of the Invention.
[0522] 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.
[0523] Inhibitory and Antisense Nucleotides
[0524] 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.
[0525] 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.
[0526] 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.
[0527] 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. (Bio Techniques 6:958 (1988)).
[0528] Ribozymes
[0529] 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).
[0530] 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)).
[0531] Use of Modulators in Phenotypic Screening
[0532] 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
[0533] 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.
[0534] Use of Modulators to Affect Peptides of the Invention
[0535] 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.
[0536] Methods of Identifying Characterizing Cancer-Associated
Sequences
[0537] 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.
[0538] 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.
[0539] XIV.) Kits/Articles of Manufacture
[0540] 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.
[0541] 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; carder, package, container, vial and/or
tube labels listing contents and/or instructions for use, and
package inserts with instructions for use.
[0542] 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 vftm 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.
[0543] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0544] 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 282P1 G3 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.
[0545] 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 282P1 G3 and modulating the function of 282P1
G3.
[0546] 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
[0547] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which is intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the 273P4B7 Gene
[0548] To isolate genes that are over-expressed in lung cancer the
Suppression Subtractive Hybridization (SSH) procedure was used
using cDNA derived from lung cancer tissues. The 273P4B7 SSH cDNA
sequence was derived from lung tumor minus cDNAs derived from
normal lung. The 273P4B7 cDNA was identified as highly expressed in
cancer.
[0549] Materials and Methods
[0550] Human Tissues:
[0551] The patient cancer and normal tissues were purchased from
different sources such as the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues were purchased from Clontech, Palo Alto,
Calif.
[0552] RNA Isolation:
[0553] 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.
[0554] Oligonucleotides:
[0555] The following HPLC purified oligonucleotides were used.
TABLE-US-00001 2 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 30)
5'TTTTGATCAAGCTT3-' Adaptor 1: (SEQ ID NO: 31)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 32)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 33)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 34)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 35)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO: 36)
5'TCGAGCGGCCGCCCGGGCAGGA3' Nested primer (NP)2: (SEQ ID NO: 37)
5'AGCGTGGTCGCGGCCGAGGA3'
[0556] Suppression Subtractive Hybridization:
[0557] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in cancer. The SSH reaction utilized cDNA from lung
cancer and normal tissues.
[0558] The gene 273P4B7 sequence was derived from lung cancer minus
normal lung and a mix of 9 normal tissues cDNA subtraction. The SSH
DNA sequence (FIG. 1) was identified.
[0559] The cDNA derived from normal lung mixed with a pool of 9
normal tissues was used as the source of the "driver" cDNA, while
the cDNA from lung cancer 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 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.
[0560] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from normal lung with a mix of digested cDNAs derived
from the nine normal tissues: stomach, skeletal muscle, lung,
brain, liver, kidney, pancreas, small intestine, and heart.
[0561] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 511 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.
[0562] 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.
[0563] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0564] 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 .mu.l
of PCR primer 1 (10 pM), 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.
[0565] 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 .mu.l of bacterial culture using the conditions of PCR1 and NP1
and NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0566] 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.
[0567] RT-PCR Expression Analysis:
[0568] 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.
[0569] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 38) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 39) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 5011 containing 0.4 .mu.M primers, 0.2 .mu.M each dNTPs,
1.times.PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 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 M-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.
[0570] To determine expression levels of the 273P4B7 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 are listed
below:
[0571] 3 273P4B7.1 5'-GCTAGTGCTCAGAATACCAGACTATGG-3' (SEQ ID NO:
40) 272P4B7.2 5'-CGCTTGACATAAAAAGTGCAGATCC-3' (SEQ ID NO: 41)
[0572] A typical RT-PCR expression analysis is shown in FIGS. 14(A)
and 14(B). First strand cDNA was prepared from vital pool 1 (liver,
lung and kidney), vital pool 2 (pancreas, colon and stomach),
normal pancreas, ovary cancer pool, and pancreas cancer pool.
Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using primers to 273P4B7, was
performed at 26 and 30 cycles of amplification. Expression of
273P4B7 was detected in ovary cancer pool, pancreas cancer pool
vital pool 1, but not in vital pool 2 nor in normal pancreas.
Example 2
Full Length Cloning of 273P4B7
[0573] The 273P4B7 SSH cDNA sequence was derived from a subtraction
consisting of lung cancer minus a normal tissues. The SSH cDNA
sequence of 170 bp (FIG. 1) was designated 273P4B7.
[0574] 273P4B7 v.1 of 4194 bp was cloned from lung cancer,
revealing an ORF of 1250 amino acids (FIG. 2 and FIG. 3). Other
variants of 273P4B7 were also identified and these are listed in
FIG. 2 and FIG. 3.
[0575] 273P4B7 v.1, v.3, v.7, and v.8 code for identical proteins
of 1250 amino acids in length. 273P4B7 v.4, v.5 and v.6 differ from
273P4B7 v.1 by one amino acid as shown in FIG. 2. 273P4B7 v.2 is a
splice variant of 273P4B7 v.1 and code for a protein of 1127 amino
acids.
[0576] 273P4B7 v.1 shows 99% over only 1106 amino acids to the
unnamed protein AK074719. The nucleic acid sequence of 273P4B7 v.1
aligns with 99% identity to the nucleotide position 159-4194, to
cDNA FLJ31932 fis, clone NT2RP7006296, weakly similar to EXCISION
REPAIR PROTEIN ERCC-6.
[0577] 273P4B7 v.1 shows 72% identity to the mouse protein BC004701
shown to be a member of the family of DEAD-like helicase
superfamily. Members of this family include the DEAD and DEAH box
helicases. Helicases are involved in unwinding nucleic acids. The
DEAD box helicases are involved in various aspects of RNA
metabolism, including nuclear transcription, pre mRNA splicing,
ribosome biogenesis, nucleocytoplasmic transport, translation, RNA
decay and organellar gene expression.
Example 3
Chromosomal Mapping of 273P4B7
[0578] 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.).
[0579] 273P4B7 maps to chromosome Xq13.1 using 273P4B7 sequence and
the NCBI BLAST tool located on the World Wide Web.
Example 4
Expression Analysis of 273P4B7 in Normal Tissues and Patient
Specimens
[0580] Expression analysis by RT-PCR demonstrated that 273P4B7 is
strongly expressed in patient cancer specimens (FIG. 14). First
strand cDNA was prepared from normal tissues (bladder, brain,
heart, kidney, liver, lung, prostate, spleen, skeletal muscle,
testis, pancreas, colon and stomach), and from pools of patient
cancer specimens (pancreas cancer pool, bladder cancer pool, kidney
cancer pool, colon cancer pool, lung cancer pool, ovary cancer
pool, breast cancer pool, cancer metastasis pool, pancreas cancer
pool, prostate cancer xenograft pool, prostate metastasis to lymph
node, bone and melanoma cancer pool, cervical cancer pool, lymphoma
cancer pool, stomach cancer pool, uterus cancer pool, and
multi-xenograft pool). Normalization was performed by PCR using
primers to actin. Semi-quantitative PCR, using primers to 273P4B7,
was performed at 22, 26 and 30 cycles of amplification. In FIG.
14(A) picture of the RT-PCR agarose gel is shown. In FIG. 14(B) PCR
products were quantitated using the ALPHALMAGER software. Results
show strong of expression of 273P4B7 in prostate cancer pool,
bladder cancer pool, kidney cancer pool, colon cancer pool, lung
cancer pool, ovary cancer pool, breast cancer pool, cancer
metastasis pool, pancreas cancer pool, prostate cancer xenograft
pool, prostate metastasis to lymph node, bone and melanoma cancer
pool, cervical cancer pool, lymphoma cancer pool, stomach cancer
pool, uterus cancer pool and multi-xenograft pool (prostate cancer,
kidney cancer and bladder cancer xenograft pool). In normal
tissues, 273P4B7 is predominantly expressed in testis and not in
any other normal tissue tested.
[0581] Extensive expression of 273P4B7 in normal tissues is shown
in FIG. 15. Two multiple tissue northern blots (Clontech) both with
2 .mu.g of mRNA/lane were probed with the 273P4B7 sequence. Size
standards in kilobases (kb) are indicated on the side. Results show
expression of an approximately 7 kb 273P4B7 transcript in normal
testis but not in the other normal tissues tested.
[0582] Expression of 273P4B7 in pancreas, ovary and testis cancer
patient specimens is shown in FIG. 16. RNA was extracted from
normal pancreas (NPa), normal ovary (NO), normal testis (NTe),
pancreas cancer patient specimen (P1), ovary cancer patient
specimen (P2, P3, P4), and testis cancer patient specimen (P5, P6,
P7). Northern blot with 10 .mu.g of total RNA/lane was probed with
273P4B7 SSH sequence. Size standards in kilobases (kb) are
indicated on the side. 273P4B7 transcript was detected in the
patient specimens, but not in the normal tissues.
[0583] FIG. 17 shows 273P4B7 expression in cervical cancer patient
specimens. In FIG. 17(A), total RNA was extracted from cervical
cancer patient specimens (T1-T7), and HeLa cell line. Northern blot
with 10 .mu.g of total RNA/lane was probed with 273P4B7 SSH
sequence. Size standards in kilobases (kb) are indicated on the
side. 273P4B7 transcript was detected in all patient specimens
tested as well as in the HeLa cell line. In FIG. 17(B), first
strand cDNA was prepared from a panel of cervical cancer patient
specimens, normal cervix and HeLa cervical cell line. Normalization
was performed by PCR using primers to actin. Semi-quantitative PCR,
using primers to 273P4B7, was performed at 26 and 30 cycles of
amplification. Samples were run on an agarose gel, and PCR products
were quantitated using the ALPHALMAGER software. Expression was
recorded as absent, low, medium or strong. Results show expression
of 273P4B7 in most of the cervical cancer tissues tested.
[0584] Expression of 273P4B7 in bladder cancer patient specimens is
shown in FIG. 18. First strand cDNA was prepared from a panel of
bladder cancer patient specimens, normal bladder (N) and bladder
cancer cell lines (UM-UC-3, TCCSUP, J82). Normalization was
performed by PCR using primers to actin. Semi-quantitative PCR,
using primers to 273P4B7, was performed at 26 and 30 cycles of
amplification. Samples were run on an agarose gel FIG. 18(A), and
PCR products were quantitated using the ALPHALMAGER software FIG.
18(B). Expression was recorded as absent, low, medium or high.
Results show expression of 273P4B7 in most of the bladder cancer
tissues tested, but not in the normal bladder tissues.
[0585] Expression of 273P4B7 in colon cancer patient specimens is
shown in FIG. 19. First strand cDNA was prepared from a panel of
colon cancer patient specimens, normal colon, and colon cancer cell
lines (LoVo, CaCo-2, SK-CO1, Colo205, and T284). Normalization was
performed by PCR using primers to actin. Semi-quantitative PCR,
using primers to 273P4B7, was performed at 26 and 30 cycles of
amplification. Samples were run on an agarose gel, and PCR products
were quantitated using the ALPHALMAGER software. Expression was
recorded as absent, low, medium or high. Results show expression of
273P4B7 in the majority of the colon cancer tissues tested, but not
in the normal colon tissues. Expression was also detected in the
cell lines LoVo, CaCo-2, SK-CO1, Colo2O5, but not in the T284 cell
line.
[0586] FIG. 20 shows 273P4B7 expression in ovary cancer patient
specimens. First strand cDNA was prepared from a panel of ovarian
cancer patient specimens, normal ovary and ovarian cancer cell
lines (OV-1063, PA-1, SW626). Normalization was performed by PCR
using primers to actin. Semi-quantitative PCR, using primers to
273P4B7, was performed at 26 and 30 cycles of amplification.
Samples were run on an agarose gel, and PCR products were
quantitated using the ALPHALMAGER software. Expression was recorded
as absent, low, medium or high. Results show expression of 273P4B7
in the majority of ovary cancer tissues tested as well as in the
cell lines, but not in normal ovary.
[0587] The restricted expression of 273P4B7 in normal tissues and
the expression detected in cancer patient specimens suggest that
273P4B7 is a therapeutic target and a diagnostic marker for human
cancers.
Example 5
Transcript Variants of 273P4B7
[0588] As used herein, the term variant comprises Transcript
variants and Single Nucleotide Polymorphisms (SNPs). 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 the
same, 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 subcellular or
extracellular localizations, e.g., secreted versus
intracellular.
[0589] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified by full-length cloning experiments,
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. Even when a variant is
identified that is not yet a full-length clone, that portion of the
variant is very useful as a research tool, e.g., for antigen
generation and for further cloning of the full-length splice
variant, using techniques known to those skilled in the art.
[0590] Moreover, computer programs are available to those skilled
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 and Gen Scan. 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.
[0591] 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).
[0592] 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 273P4B7 has a particular expression profile related to cancer
(See, e.g., Table I). Alternative transcripts and splice variants
of 273P4B7 are also be involved in cancers in the same or different
tissues, thus serving as tumor-associated markers/antigens.
[0593] Using the full-length gene and EST sequences, four
additional transcript variants were identified, designated as
273P4B7 v.2, v.9 and v.10. The boundaries of exons in the original
transcript, 273P4B7 v.1 are shown in Table LI. The structures of
the transcript variants are shown in FIG. 10. Variant 273P4B7 v.2
added 22 bases to the 5' end of exon1 and an additional exon in the
first intron of variant v.1. Variants v.9 and v.10 were shorter and
matched part of the last exon of v.1, with a few different base
pairs. Tables LII(a)-(d) through LV(a)-(c) are set forth on a
variant-by-variant bases. Tables LII(a)-(d) show nucleotide
sequence of the transcript variants. Tables LIII(a)-(d) show the
alignment of the transcript variant with nucleic acid sequence of
273P4B7 v.1. Table LIV(a)-(d) lay out amino acid translation of the
transcript variant for the identified reading frame orientation.
Table LV(a)-(d) display alignments of the amino acid sequence
encoded by the splice variant with that of 273P4B7 v. 1.
Example 6
Single Nucleotide Polymorphisms of 273P4B7
[0594] 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(I):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The
predictive power of haplotypes in clinical response,"
Pharmacogenomics. 2000 February; 1(1): 15-26).
[0595] 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," Annul.
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).
[0596] Using the methods described above, six SNP were identified
in the transcript, 273P4B7 v. 1, as shown in Table LVI. The
transcripts or proteins with alternative allele were designated as
variant 273P4B7 v.3 through v.8, as shown in Table LVI and FIG. 12.
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 273P4B7 v.2, as listed in table LVI)
that contains the site of the SNP, as laid out in FIGS. 11 and
12.
Example 7
Production of Recombinant 273P4B7 in Prokaryotic Systems
[0597] To express recombinant 273P4B7 and 273P4B7 variants in
prokaryotic cells, the full or partial length 273P4B7 and 273P4B7
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 273P4B7 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 273P4B7, variants, or analogs
thereof.
[0598] A. In Vitro Transcription and Translation Constructs:
[0599] pCRII: To generate 273P4B7 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 273P4B7 cDNA. The pCRII vector has Sp6 and T7 promoters
flanking the insert to drive the transcription of 273P4B7 RNA for
use as probes in RNA in situ hybridization experiments. These
probes are used to analyze the cell and tissue expression of
273P4B7 at the RNA level. Transcribed 273P4B7 RNA representing the
cDNA amino acid coding region of the 273P4B7 gene is used in in
vitro translation systems such as the TNT COUPLED RETICULOLYSATE
SYSTEM (Promega, Corp., Madison, Wis.) to synthesize 273P4B7
protein.
[0600] B. Bacterial Constructs:
[0601] pGEX Constructs: To generate recombinant 273P4B7 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the 273P4B7 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 273P4B7 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
PRECISION recognition site in pGEX-6P-1, may be employed such that
it permits cleavage of the GST tag from 273P4B7-related protein.
The ampicillin resistance gene and pBR322 origin permits selection
and maintenance of the pGEX plasmids in E. coli.
[0602] pMAL Constructs: To generate, in bacteria, recombinant
273P4B7 proteins that are fused to maltose-binding protein (MBP),
all or parts of the 273P4B7 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 273P4B7 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 273P4B7. 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.
[0603] pET Constructs: To express 273P4B7 in bacterial cells, all
or parts of the 273P4B7 cDNA protein coding sequence are cloned
into the pET family of vectors (Novagen, Madison, Wis.). These
vectors allow tightly controlled expression of recombinant 273P4B7
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
273P4B7 protein are expressed as amino-terminal fusions to
NusA.
[0604] C. Yeast Constructs:
[0605] pESC Constructs: To express 273P4B7 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 273P4B7 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 or Myc epitope tags in the
same yeast cell. This system is useful to confirm protein-protein
interactions of 273P4B7. In addition, expression in yeast yields
similar post-translational modifications, such as glycosylations
and phosphorylations that are found when expressed in eukaryotic
cells.
[0606] pESP Constructs: To express 273P4B7 in the yeast species
Saccharomyces pombe, all or parts of the 273P4B7 cDNA protein
coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of expression of a 273P4B7
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 epitope tag allows detection of the
recombinant protein with anti-FLAG antibody.
Example 8
Production of Recombinant 273P4B7 in Higher Eukaryotic Systems
[0607] A. Mammalian Constructs:
[0608] To express recombinant 273P4B7 in eukaryotic cells, the full
or partial length 273P4B7 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 273P4B7 were expressed in these
constructs, amino acids 1 to 1250 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 273P4B7 v.1, v.4, v.5, and v.6;
amino acids 1 to 1127 of v.2 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 273P4B7 variants, or analogs
thereof.
[0609] 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-273P4B7 polyclonal serum,
described herein.
[0610] pcDNA4/HisMax Constructs: To express 273P4B7 in mammalian
cells, a 273P4B7 ORF, or portions thereof, of 273P4B7 are cloned
into pcDNA4/HisMax 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/HisMax 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.
[0611] pcDNA3.11MycHis Constructs: To express 273P4B7 in mammalian
cells, 273P4B7 ORF, or portions thereof, of 273P4B7 with a
consensus Kozak translation initiation site was cloned into
pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein
expression is 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.
[0612] pcDNA3.11CT-GFP-TOPO Construct: To express 273P4B7 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, a 273P4B7 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 273P4B7
protein.
[0613] PAPtag: A 273P4B7 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 273P4B7 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 273P4B7 protein. The resulting
recombinant 273P4B7 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 273P4B7
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.
[0614] pTag5: A 273P4B7 ORF, or portions thereof, is cloned into
pTag-5. This vector is similar to pAPtag but without the alkaline
phosphatase fusion. This construct generates 273P4B7 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
273P4B7 protein is optimized for secretion into the media of
transfected mammalian cells, and is used as immunogen or ligand to
identify proteins such as ligands or receptors that interact with
the 273P4B7 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.
[0615] PsecFc: A 273P4B7 ORF, or portions thereof, is 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 273P4B7 proteins, while
fusing the IgGK signal sequence to N-terminus. 273P4B7 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant 273P4B7 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 273P4B7 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. 1
[0616] pSR.alpha. Constructs: To generate mammalian cell lines that
express 273P4B7 constitutively, 273P4B7 ORF, or portions thereof,
of 273P4B7 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, 273P4B7, 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.
[0617] Additional pSR.alpha. constructs were made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
273P4B7 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: 42) 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 273P4B7 proteins.
[0618] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 273P4B7. High virus titer
leading to high level expression of 273P4B7 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. A 273P4B7 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, 273P4B7 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.
[0619] Regulated Expression Systems: To control expression of
273P4B7 in mammalian cells, coding sequences of 273P4B7, 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 273P4B7. These
vectors are thereafter used to control expression of 273P4B7 in
various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0620] B. Baculovirus Expression Systems
[0621] To generate recombinant 273P4B7 proteins in a baculovirus
expression system, 273P4B7 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-273P4B7 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.
[0622] Recombinant 273P4B7 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 273P4B7 protein can be detected using anti-273P4B7 or
anti-His-tag antibody. 273P4B7 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 273P4B7.
Example 9
Antigenicity Profiles and Secondary Structure
[0623] FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict
graphically five amino acid profiles of 273P4B7 variant 1, each
assessment available by accessing the ProtScale website located on
the World Wide Web at (www.expasy.ch/cgi-bin/protscale.pl) on the
ExPasy molecular biology server.
[0624] These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods
K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6,
Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.
157:105-132); FIG. 7, Percentage Accessible Residues (Janin J.,
1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran
R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.
32:242-255); FIG. 9, 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 273P4B7 variant proteins. Each of the above
amino acid profiles of 273P4B7 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.
[0625] Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and
Percentage Accessible Residues (FIG. 7) 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.
[0626] Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) 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.
[0627] Antigenic sequences of the 273P4B7 variant proteins
indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG.
7, FIG. 8, and/or FIG. 9 are used to prepare immunogens, either
peptides or nucleic acids that encode them, to generate therapeutic
and diagnostic anti-273P4B7 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
273P4B7 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. 5; 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. 6; 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. 7; 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. 8; 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. 9. Peptide immunogens of the
invention can also comprise nucleic acids that encode any of the
forgoing.
[0628] 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.
[0629] The secondary structure of 273P4B7 protein variant 1, namely
the predicted presence and location of alpha helices, extended
strands, and random coils, is 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., Geourjon C. and Delage G.),
accessed from the ExPasy molecular biology server located on the
World Wide Web. The analysis indicates that 273P4B7 variant 1 is
composed of 41.60% alpha helix, 11.12% extended strand, and 47.28%
random coil (FIG. 13A).
[0630] Analysis for the potential presence of transmembrane domains
in the 273P4B7 variant protein 1 was carried out using a variety of
transmembrane prediction algorithms accessed from the ExPasy
molecular biology server located on the World Wide Web. Shown
graphically in FIG. 13B and FIG. 13C are the results of analysis of
variant 1 using the TMpred program (FIG. 13B) and TMHMM program
(FIG. 13C). The TMpred program predicts the presence of 2
transmembrane domains, whereas the TMHMM program does not predict
transmembrane domains. Taken together with analysis using other
programs summarized in Table VI and Table L, the data suggest that
273P4B7 is most likely a soluble protein.
Example 10
Generation of 273P4B7 Polyclonal Antibodies
[0631] 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 273P4B7 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. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for
amino acid profiles that indicate such regions of 273P4B7 protein
variant 1).
[0632] For example, recombinant bacterial fusion proteins or
peptides containing hydrophilic, flexible, beta-turn regions of
273P4B7 protein variants are used as antigens to generate
polyclonal antibodies in New Zealand White rabbits or monoclonal
antibodies as described (see the Example entitled "Generation of
273P4B7 Monoclonal Antibodies (mAbs)"). For example, in 273P4B7
variant 1, such regions include, but are not limited to, amino
acids 1-16, amino acids 23-43, amino acids 170-194, amino acids
324-368, amino acids 430-461, amino acids 735-753, amino acids
774-792, amino acids 1002-1043, and amino acids 1105-1158. 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 1-16 of 273P4B7 variant 1 was conjugated to KLH and
used to immunize a rabbit. Alternatively the immunizing agent may
include all or portions of the 273P4B7 variant proteins, analogs or
fusion proteins thereof. For example, the 273P4B7 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 1000-1250 of
273P4B7 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.
[0633] Other recombinant bacterial fusion proteins that may be
employed include maltose binding protein, LacZ, thioredoxin, NusA,
or an immunoglobulin constant region (see the Example entitled
"Production of 273P4B7 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., Urnes, M.,
Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med.
174, 561-566).
[0634] 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 Example entitled "Production of
Recombinant 273P4B7 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, the complete cDNA of 273P4B7
variant 1 is cloned into the Tag5 mammalian secretion vector, and
expressed in 293T cells. The recombinant protein is purified by
metal chelate chromatography from tissue culture supernatants of
293T cells stably expressing the recombinant vector. The purified
Tag5 273P4B7 protein is then used as immunogen.
[0635] 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).
[0636] 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.
[0637] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with the GST-fusion of
273P4B7 variant 1 protein, the full-length 273P4B7 variant 1 cDNA
is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see
the Example entitled "Production of Recombinant 273P4B7 in
Eukaryotic Systems"). After transfection of the constructs into
293T cells, cell lysates are probed with the anti-273P4B7 serum and
with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz,
Calif.) to determine specific reactivity to denatured 273P4B7
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
273P4B7-expressing cells to determine specific recognition of
native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and flow cytometric techniques using cells that
endogenously express 273P4B7 are also carried out to test
reactivity and specificity.
[0638] Anti-serum from rabbits immunized with 273P4B7 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-273P4B7 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-273P4B7 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 11
Generation of 273P4B7 Monoclonal Antibodies (mAbs)
[0639] In one embodiment, therapeutic mAbs to 273P4B7 variants
comprise those that react with epitopes specific for each variant
protein or specific to sequences in common between the variants
that would disrupt or modulate the biological function of the
273P4B7 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 entire 273P4B7 protein variant sequence, regions predicted to
contain functional motifs, and regions of the 273P4B7 protein
variants predicted to be antigenic from computer analysis of the
amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or
FIG. 9, and the Example entitled "Antigenicity Profiles").
Immunogens include peptides, recombinant bacterial proteins, and
mammalian expressed Tag 5 proteins and human and murine IgG FC
fusion proteins. In addition, cells engineered to express high
levels of a respective 273P4B7 variant, such as 293T-273P4B7
variant 1 or 300.19-273P4B7 variant 1 murine Pre-B cells, are used
to immunize mice.
[0640] To generate mAbs to a 273P4B7 variant, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen or 10.sup.7 273P4B7-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 273P4B7 variant sequence
is used to immunize mice by direct injection of the plasmid DNA.
For example, the complete cDNA of 273P4B7 of variant 1 is cloned
into the Tag5 mammalian secretion vector and the recombinant vector
will then be used as immunogen. In another example the same amino
acids are cloned into an Fc-fusion secretion vector in which the
273P4B7 variant 2 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 is
then used as immunogen. The plasmid immunization protocols are used
in combination with purified proteins expressed from the same
vector and with cells expressing the respective 273P4B7
variant.
[0641] 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).
[0642] In one embodiment for generating 273P4B7 monoclonal
antibodies, a GST-fusion of variant 1 antigen encoding amino acids
1000-1250 is expressed and purified from bacteria. Balb C mice are
initially immunized intraperitoneally with 25 .mu.g of the
GST-273P4B7 variant 1 protein mixed in complete Freund's adjuvant.
Mice are subsequently immunized every two weeks with 25 .mu.g of
the antigen mixed in incomplete Freund's adjuvant for a total of
three immunizations. ELISA using the GST-fusion antigen and a
cleavage product from which the GST portion is removed determines
the titer of serum from immunized mice. Reactivity and specificity
of serum to full length 273P4B7 variant 1 protein is monitored by
Western blotting, immunoprecipitation and flow cytometry using 293T
cells transfected with an expression vector encoding the 273P4B7
variant 1 cDNA (see e.g., the Example entitled "Production of
Recombinant 273P4B7 in Eukaryotic Systems"). Other recombinant
273P4B7 variant 1-expressing cells or cells endogenously expressing
273P4B7 variant 1 are also used. Mice showing the strongest
reactivity are rested and given a final injection of antigen in PBS
and then sacrificed four days later. The spleens of the sacrificed
mice are harvested and fused to SPO/2 myeloma cells using standard
procedures (Harlow and Lane, 1988). Supernatants from HAT selected
growth wells are screened by ELISA, Western blot,
immunoprecipitation, fluorescent microscopy, and flow cytometry to
identify 273P4B7 specific antibody-producing clones. The binding
affinity of a 273P4B7 variant monoclonal antibody is determined
using standard technologies. Affinity measurements quantify the
strength of antibody to epitope binding and are used to help define
which 273P4B7 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.
Example 12
HLA Class I and Class II Binding Assays
[0643] 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.
[0644] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq[HLA], the measured IC50 values are reasonable
approximations of the true KD 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 IC50 of a positive control for inhibition
by the IC50 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 IC50 nM values
by dividing the IC50 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.
[0645] Binding assays as outlined above may be used to analyze HLA
supermotif and/or HLA motif-bearing peptides (see Table IV).
Example 13
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate
Epitopes
[0646] 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.
[0647] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0648] The searches performed to identify the motif-bearing peptide
sequences in the Example entitled "Antigenicity Profiles" and
Tables VIII-XXI and XXII-XLIX employ the protein sequence data from
the gene product of 273P4B7 set forth in FIGS. 2 and 3, the
specific search peptides used to generate the tables are listed in
Table VII.
[0649] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
273P4B7 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.
[0650] 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 AG) of peptide-HLA molecule interactions
can be approximated as a linear polynomial function of the type:
.DELTA.G''=a.sub.1ixa2ixa3i . . . xani
[0651] where aji 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 ji to the
free energy of binding of the peptide irrespective of the sequence
of the rest of the peptide.
[0652] 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 ji. 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.
Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0653] Protein sequences from 273P4B7 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).
[0654] 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.
Selection of HLA-A3 Supermotif-Bearing Epitopes
[0655] The 273P4B7 protein sequence(s) 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.
[0656] Selection of HLA-B7 Supermotif Bearing Epitopes
[0657] The 273P4B7 protein(s) scanned above 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.
Selection of A1 and A24 Motif-Bearing Epitopes
[0658] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 273P4B7 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0659] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs are identified using analogous
methodology.
Example 14
Confirmation of Immunogenicity
[0660] 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:
Target Cell Lines for Cellular Screening:
[0661] The 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.
Primary CTL Induction Cultures:
[0662] Generation of Dendritic Cells (DC): 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.
[0663] Induction of CTL with DC and Peptide: CD8+ T-cells are
isolated by positive selection with Dynal immunomagnetic beads
(DYNABEADS M-450) and the DETACHABEAD reagent. Typically about
200-250.times.10.sup.6 PBMC are processed to obtain
24.times.10.sup.6 CD8+ 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 (140 .mu.l
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
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 92-microglobulin for 4 hours at 20.degree.
C. The DC are then irradiated (4,200 rads), washed 1 time with
medium and counted again.
[0664] Setting up induction cultures: 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.
[0665] Restimulation of the induction cultures with peptide-pulsed
adherent cells: 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 -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 501 U/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 IFNy 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.
[0666] 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.
[0667] 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.
[0668] Maximum and spontaneous release are determined by incubating
the labeled targets with 1% Triton 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.
In Situ Measurement of Human IFN.gamma. Production as an Indicator
of Peptide-Specific and Endogenous Recognition
[0669] 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.
[0670] Recombinant human IFN-gamma is added to the standard wells
starting at 400 .mu.g or 1200 .mu.g/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 OD.sub.450.
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.
[0671] CTL Expansion.
[0672] 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 (autologues 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 501 U/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.
[0673] Cultures are expanded in the absence of anti-CD.sup.3+ as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8, 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.
Immunogenicity of A2 Supermotif-Bearing Peptides
[0674] 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.
[0675] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 273P4B7. 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.
[0676] Evaluation of A*03/A11 Immunogenicity
[0677] 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.
[0678] Evaluation of B7 Immunogenicity
[0679] 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.
[0680] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also confirmed using similar methodology
Example 15
Implementation of the Extended Supermotif to Improve the Binding
Capacity of Native Epitopes by Creating Analogs
[0681] 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.
[0682] Analoging at Primary Anchor Residues
[0683] 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.
[0684] 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.
[0685] Alternatively, a peptide is confirmed as binding one or all
supertype members and then analoged to modulate binding affinity to
any one (or more) of the supertype members to add population
coverage.
[0686] 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).
[0687] 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.
[0688] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0689] 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.
[0690] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate .ltoreq.500 nM binding capacity are then confirmed as
having A3-supertype cross-reactivity.
[0691] 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).
[0692] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0693] 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.
[0694] Analoging at Secondary Anchor Residues
[0695] 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.
[0696] 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. Analoged peptides are additionally
tested for the ability to stimulate a recall response using PBMC
from patients with 273P4B7-expressing tumors.
[0697] Other Analoging Strategies
[0698] Another form of peptide analoging, 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 disultide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substitution of a-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
1. Chen, John Wiley & Sons, England, 1999).
[0699] 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 16
Identification and Confirmation of 273P4B7-Derived Sequences with
HLA-DR Binding Motifs
[0700] 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.
[0701] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0702] To identify 273P4B7-derived, HLA class II HTL epitopes, a
273P4B7 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).
[0703] 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.
[0704] The 273P4B7-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. 273P4B7-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0705] Selection of DR3Motif Peptides
[0706] 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.
[0707] To efficiently identify peptides that bind DR3, target
273P4B7 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.
[0708] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0709] 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 17
Immunogenicity of 273P4B7-Derived HTL Epitopes
[0710] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0711] 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 273P4B7-expressing
tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0712] 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.
[0713] 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].
[0714] 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: 87, 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).
[0715] 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%, see, e.g., Table IV
(G). An analogous approach can be used to estimate population
coverage achieved with combinations of class II motif-bearing
epitopes.
[0716] 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.
[0717] 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 19
CTL Recognition of Endogenously Processed Antigens after
Priming
[0718] 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.
[0719] 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 273P4B7
expression vectors.
[0720] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
273P4B7 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 20
Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
[0721] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 273P4B7-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 273P4B7-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.
[0722] Immunization procedures: 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 CTUHTL 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.
[0723] Cell lines: 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)
[0724] In vitro CTL activation: One week after priming, spleen
cells (30.times.10.sup.6 cells/flask) are co-cultured at 37.degree.
C. with syngenic, 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.
[0725] Assay for cytotoxic activity.: Target cells (1.0 to
1.5.times.10.sup.6) are incubated at 37*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, 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)]10.sup.6=18 LU.
[0726] 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 21
Selection of CTL and HTL Epitopes for Inclusion in a
273P4B7-Specific Vaccine
[0727] 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.
[0728] 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.
[0729] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 273P4B7 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 273P4B7. For example, if it has been observed
that patients who spontaneously clear 273P4B7-expressing cells
generate an immune response to at least three (3) epitopes from
273P4B7 antigen, then at least three epitopes should be included
for HLA class I. A similar rationale is used to determine HLA class
II epitopes.
[0730] 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.
[0731] 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.
[0732] 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 273P4B7, 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.
[0733] 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 273P4B7.
Example 22
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0734] 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.
[0735] 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 273P4B7, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 273P4B7 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.
[0736] 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.
[0737] 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.
[0738] 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 H is antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0739] 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.
[0740] 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
(1x=10 mM KCL, 10 mM (NH4).sub.2SO.sub.4, 20 mM Tris-chloride, pH
8.75, 2 mM MgSO.sub.4, 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 23
The Plasmid Construct and the Degree to which it Induces
Immunogenicity
[0741] 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.
156:683692, 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).
[0742] 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.
[0743] 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/K.sup.b 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.
[0744] 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 .sup.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.
[0745] 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.
[0746] 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 .sup.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.
[0747] 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. Aced. 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).
[0748] 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/K.sup.b 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 10.sup.7 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.
[0749] 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 24
Peptide Compositions for Prophylactic Uses
[0750] Vaccine compositions of the present invention can be used to
prevent 273P4B7 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
273P4B7-associated tumor.
[0751] 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 273P4B7-associated disease.
[0752] 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 25
Polyepitopic Vaccine Compositions Derived from Native 273P4B7
Sequences
[0753] A native 273P4B7 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 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.
[0754] The vaccine composition will include, for example, multiple
CTL epitopes from 273P4B7 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.
[0755] 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(s) 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 273P4B7, 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.
[0756] 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 26
Polyepitopic Vaccine Compositions from Multiple Antigens
[0757] The 273P4B7 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
273P4B7 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 273P4B7 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 273P4B7
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 27
Use of Peptides to Evaluate an Immune Response
[0758] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 273P4B7. 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.
[0759] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 273P4B7 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising a 273P4B7
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 P2-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, P2-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.
[0760] 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 273P4B7 epitope, and thus the
status of exposure to 273P4B7, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
[0761] 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 273P4B7-associated disease or who have been
vaccinated with a 273P4B7 vaccine.
[0762] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
273P4B7 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.
[0763] 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.
[0764] 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 .mu.l 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).
[0765] 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).
[0766] 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 100 .mu.Ci of .sup.51Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0767] 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.
[0768] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 273P4B7 or a 273P4B7 vaccine.
[0769] 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 273P4B7
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 29
Induction of Specific CTL Response in Humans
[0770] 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:
[0771] A total of about 27 individuals are enrolled and divided
into 3 groups:
Group I: 3 subjects are injected with placebo and 6 subjects are
injected with 5 .mu.g of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are
injected with 50 .mu.g peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are
injected with 500 .mu.g of peptide composition.
[0772] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0773] 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.
[0774] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0775] 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.
[0776] The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 273P4B7
[0777] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 273P4B7. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 273P4B7, 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:
[0778] 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.
[0779] 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 273P4B7.
[0780] 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 273P4B7-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
[0781] 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.
[0782] 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-1-Hypaque
density gradient centrifugation, aliquoted in freezing media and
stored frozen. Samples are assayed for CTL and HTL activity.
[0783] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 273P4B7 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0784] 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
273P4B7 protein from which the epitopes in the vaccine are
derived.
[0785] 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 (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.
[0786] 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.
[0787] 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 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
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
is typically estimated to be between 2-10%, but can vary as
appreciated by one of skill in the art.
[0788] Ex Vivo Activation of CTL/HTL Responses
[0789] Alternatively, ex vivo CTL or HTL responses to 273P4B7
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 33
An Alternative Method of Identifying and Confirming Motif-Bearing
Peptides
[0790] 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. 273P4B7.
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.
[0791] 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
273P4B7 to isolate peptides corresponding to 273P4B7 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.
[0792] 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 34
Complementary Polynucleotides
[0793] Sequences complementary to the 273P4B7-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 273P4B7. 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 273P4B7. 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 a 273P4B7-encoding
transcript.
Example 35
Purification of Naturally-Occurring or Recombinant 273P4B7 Using
273P4B7-Specific Antibodies
[0794] Naturally occurring or recombinant 273P4B7 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 273P4B7. An immunoaffinity column is constructed by covalently
coupling anti-273P4B7 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.
[0795] Media containing 273P4B7 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 273P4B7 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/273P4B7 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 GCRP is collected.
Example 36
Identification of Molecules which Interact with 273P4B7
[0796] 273P4B7, or biologically active fragments thereof, are
labeled with 121 1 Bolton-Hunter reagent. (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
273P4B7, washed, and any wells with labeled 273P4B7 complex are
assayed. Data obtained using different concentrations of 273P4B7
are used to calculate values for the number, affinity, and
association of 273P4B7 with the candidate molecules.
Example 37
In Vivo Assay for 273P4B7 Tumor Growth Promotion
[0797] The effect of the 273P4B7 protein on tumor cell growth is
evaluated in vivo by evaluating tumor development and growth of
cells expressing or lacking 273P4B7. For example, SCID mice are
injected subcutaneously on each flank with 1.times.10.sup.6 of
either 3T3, cancer cell lines expressing 273P4B7 (Table I), or
cancer cell lines containing tkNeo empty vector. At least two
strategies may be used: (1) Constitutive 273P4B7 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, and (2)
Regulated expression under control of an inducible vector system,
such as ecdysone, tetracycline, etc., provided such promoters are
compatible with the host cell systems. Tumor volume is then
monitored by caliper measurement at the appearance of palpable
tumors and followed over time to determine if 273P4B7-expressing
cells grow at a faster rate and whether tumors produced by
273P4B7-expressing cells demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs).
[0798] Additionally, mice can be implanted with 1.times.10.sup.5 of
the same cells orthotopically to determine if 273P4B7 has an effect
on local growth, and whether 273P4B7 affects the ability of the
cells to metastasize, specifically to lymph nodes, and bone (Azuma
H et al., J. Urol. 2003, 169:2372; Fu X et al., Int J. Cancer.
1991, 49:938). The effect of 273P4B7 on bone tumor formation and
growth may be assessed by injecting tumor cells intratibially. The
assay is also useful to determine the 273P4B7 inhibitory effect of
candidate therapeutic compositions, such as for example, 273P4B7
intrabodies, 273P4B7 antisense molecules and ribozymes.
Example 38
273P4B7 Monoclonal Antibody-Mediated Inhibition of Tumors In
Vivo
[0799] The significant expression of 273P4B7 in cancer tissues,
together with its restricted expression in normal tissues, makes
273P4B7 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 on
the World Wide Web. In cases where the target is not on the cell
surface, such as for 273P4B7, and including 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, 273P4B7 is a
target for T cell-based immunotherapy.
[0800] Accordingly, the therapeutic efficacy of anti-273P4B7 mAbs
in human xenograft mouse models, including bladder, pancreas,
cervix, lung and the other cancers set forth in Table I, is modeled
in 273P4B7-expressing cancer xenografts or cancer cell lines, such
as those described in the Example entitled "In Vivo Assay for
273P4B7 Tumor Growth Promotion", that endogenously express 273P4B7
or that have been engineered to express 273P4B7.
[0801] Antibody efficacy on tumor growth and metastasis formation
is confirmed, e.g., in a mouse orthotopic 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-273P4B7 mAbs inhibit formation
of 273P4B7-expressing tumors. Anti-273P4B7 mAbs also retard the
growth of established orthotopic tumors and prolong survival of
tumor-bearing mice. These results indicate the utility of
anti-273P4B7 mAbs in the treatment of local and advanced stages of
cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or on
the World Wide Web.
[0802] Administration of anti-273P4B7 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 273P4B7 is an
attractive target for immunotherapy and demonstrate the therapeutic
potential of anti-273P4B7 mAbs for the treatment of local and
metastatic cancer.
[0803] This example demonstrates that unconjugated 273P4B7
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.
Tumor Inhibition Using Multiple Unconjugated 273P4B7 mAbs Materials
and Methods
[0804] 273P4B7 Monoclonal Antibodies
[0805] Monoclonal antibodies are raised against 273P4B7 as
described in the Example entitled "Generation of 273P4B7 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 273P4B7.
Epitope mapping data for the anti-273P4B7 mAbs, as determined by
ELISA and Western analysis, recognize epitopes on the 273P4B7
protein. Immunohistochemical analysis of cancer tissues and cells
with these antibodies is performed.
[0806] 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.
[0807] Cancer Cell Lines
[0808] Cancer cell lines expressing 273P4B7 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. Cancer cell lines endogenously expressing 273P4B7,
including prostate, bladder, kidney, and the other tissues set
forth in Table I are also used for in vivo and in vitro models.
Anti-273P4B7 staining is detected by using an FITC-conjugated goat
anti-mouse antibody (Southern Biotechnology Associates) followed by
analysis on a Coulter Epics-XL flow cytometer.
[0809] In Vivo Mouse Models
[0810] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10.sup.6 273P4B7-expressing 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-273P4B7 mAbs are determined by a capture ELISA kit (Bethyl
Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al.,
PNAS 10:1073-1078)
[0811] 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 cancer cells is administered directly (Peralta, E. A.,
et al., J. Urol., 1999. 162:1806-1811). In a second embodiment, an
incision is made through the abdominal wall, the tissue is exposed,
and tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor
are surgically glued onto the exterior wall, 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.
[0812] Anti-273P4B7 mAbs Inhibit Growth of 273P4B7-Expressing
Tumors
[0813] In one embodiment, the effect of anti-273P4B7 mAbs on tumor
formation is investigated in subcutaneous models of the cancers
listed in Table I, by inoculating the right flank of SCID mice with
the appropriate 273P4B7-expressing cell line, and comparing its
growth in the presence or absence of anti-273P4B7 mAb, as described
below.
[0814] In another embodiment, the effect of anti-273P4B7 mAbs on
tumor formation is tested by using the orthotopic model. As
compared with the s.c. tumor model, the orthotopic model, which
requires surgical attachment of tumor tissue directly, 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 feature 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.
[0815] Accordingly, 273P4B7-expressing tumor cells are onplanted
orthotopically, and 2 days later, the mice are segregated into two
groups and treated with either: a) 50-2000 gp, usually 200-500
.mu.g, of anti-273P4B7 Ab, or b) PBS, three times per week for two
to five weeks. Mice are monitored weekly for indications of tumor
growth.
[0816] As noted, a major advantage of the orthotopic 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-273P4B7 antibodies can be
performed on the tissue sections.
[0817] Mice bearing established orthotopic 273P4B7-expressing
tumors are administered 1 OOOpg injections of either anti-273P4B7
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 tumor and lung and lymph
node tissue are analyzed for the presence of tumor cells by
histology and IHC analysis.
[0818] These studies demonstrate a broad anti-tumor efficacy of
anti-273P4B7 antibodies on initiation and progression of cancers in
mouse models. Anti-273P4B7 antibodies inhibit tumor formation and
retard the growth of already established tumors and prolong the
survival of treated mice. Moreover, anti-273P4B7 mAbs demonstrate a
dramatic inhibitory effect on the spread of local tumor to distal
sites, even in the presence of a large tumor burden. Thus,
anti-273P4B7 mAbs are efficacious on major clinically relevant end
points including lessened tumor growth, lessened metastasis, and
prolongation of survival.
Example 39
Therapeutic and Diagnostic use of Anti-273P4B7 Antibodies in
Humans
[0819] Anti-273P4B7 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-273P4B7 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 273P4B7 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-273P4B7 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0820] As determined by flow cytometry, anti-273P4B7 mAb
specifically binds to carcinoma cells. Thus, anti-273P4B7
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 273P4B7. Shedding or release of an extracellular
domain of 273P4B7 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 273P4B7 by
anti-273P4B7 antibodies in serum and/or urine samples from suspect
patients.
[0821] Anti-273P4B7 antibodies that specifically bind 273P4B7 are
used in therapeutic applications for the treatment of cancers that
express 273P4B7. Anti-273P4B7 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-273P4B7 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 "273P4B7 Monoclonal Antibody-mediated Inhibition
of Bladder and Lung Tumors In Vivo"). Either conjugated and
unconjugated anti-273P4B7 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 40
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of Human Anti-273P4B7 Antibodies In Vivo
[0822] Antibodies are used in accordance with the present invention
which recognize an epitope on 273P4B7, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 273P4B7 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.
[0823] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-273P4B7 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-273P4B7
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-273P4B7 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).
[0824] II.) Monotherapy: In connection with the use of the
anti-273P4B7 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.
[0825] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (1131, Y.sup.90) to anti-273P4B7 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
273P4B7. In connection with the use of the anti-273P4B7 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)-273P4B7
antibody is used as an imaging agent in a Phase I human clinical
trial in patients having a carcinoma that expresses 273P4B7 (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.
[0826] Dose and Route of Administration
[0827] 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-273P4B7
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-273P4B7 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-273P4B7 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-273P4B7 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.
[0828] Three distinct delivery approaches are useful for delivery
of anti-273P4B7 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.
[0829] Clinical Development Plan (CDP)
[0830] Overview: The CDP follows and develops treatments of
anti-273P4B7 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-273P4B7 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
273P4B7 expression levels in their tumors as determined by
biopsy.
[0831] 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 273P4B7. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-273P4B7 antibodies are
found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-273P4B7
Antibody and Chemotherapeutic Agent
[0832] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-273P4B7 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-273P4B7 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-273P4B7 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-00002 Day 0 Day
7 Day 14 Day 21 Day 28 Day 35 mAb 25 75 125 175 225 275 Dose
mg/m.sup.2 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2
Chemotherapy + + + + + + (standard dose)
[0833] 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 273P4B7. 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.
[0834] The anti-273P4B7 antibodies are demonstrated to be safe and
efficacious, Phase II trials confirm the efficacy and refine
optimum dosing.
Example 42
Human Clinical Trial: Monotherapy with Human Anti-273P4B7
Antibody
[0835] Anti-273P4B7 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-273P4B7
antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-273P4B7
Antibody
[0836] 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-273P4B7 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 44
Homology Comparison of 273P4B7 to Known Sequences
[0837] The 273P4B7 protein of FIG. 3 has 1250 amino acids with
calculated molecular weight of 141.1 kDa, and pl of 5.19. 273P4B7
is predicted to be a nuclear protein (65% by PSORT) with a
possibility of being a cytoplasmic protein (50% PSORT). Although
some prediction programs indicate that 273P4B7 may have a
transmembrane domain, it is equally likely that the 273P4B7 protein
is a soluble intracellular protein.
[0838] By use of the PubMed website of the N.C.B.I. available on
the World Wide Web, it was found at the protein level that 273P4B7
shows best homology to an un-named protein (gi.vertline.22760345)
of unknown function, with 99% identity and 99% homology over the
entire length of the protein (FIG. 4A). The 273P4B7 protein
demonstrates similarity to a hypothetical human protein named
BJ-HCC-15 tumor antigen (gi.vertline.22002580) with 99% identity
and 100% homology over the last 419aa of the 273P4B7 protein (FIG.
4B). The mouse ortholog of 273P4B7 has been identified showing 72%
identity and 81% homology to 273P4B7 (FIG. 4C). Bioinformatic
analysis revealed the presence of a SNF2 motif at aa 99-417 and a
helicase motif at aa 490-574 of the 273P4B7 protein. These motifs
are also found in the mouse SNF2/RAD54 family protein
(gi.vertline.27414501) which carries 72% identity to 273P4B7.
[0839] The SNF2 domain is often found in proteins involved in
transcription regulation, DNA repair, DNA recombination, and
chromatin unwinding (Alexeev A, Mazin A, Kowalczykowski S C. Nat
Struct Biol. 2003, 10:182; Solinger J A, Kiianitsa K, Heyer W D.
Mol. Cell. 2002, 10:1175; Martens J A, Winston F.: Genes Dev. 2002,
16:2231). By remodeling DNA complexes, SNF2 makes nucleosomal DNA
accessible to regulatory factors, thereby regulating gene
expression (Fan H Y et al., Mol. Cell. 2003, 11:1311). Evidence in
Saccharomyces cerevisiae indicates that SNF2 regulates
transcription in these organisms. It has been shown that SNF
complexes with SWI and the SWI/SNF is recruited to the promoter of
specific genes inducing their transcriptional activation (Kingston,
R. E. and Narlikar, G. J. Genes & Dev. 1999, 13: 2339-2352). A
similar chromatin remodeling complex has been identified in
mammalian cells, known as Brm/Brg1. This complex was found to
regulate gene expression as well as cell cycle (Muchardt C and
Yaniv M, Oncogene 2001, 20:3067). Finally, a
"proliferation-associated SNF2-like genes which contains SNF2
motifs has been associated with AML (Lee D et al., Cancer Res.
2000, 60:3612).
[0840] Our findings that 273P4B7 is highly expressed in several
cancers while showing a restricted expression pattern in normal
tissues suggests that the 273P4B7 gene may play an important role
in various cancers, including the cancers set forth in Table I. It
is provided by the present invention that 273P4B7 controls tumor
growth and progression by regulating proliferation, cell cycle,
gene expression as well as cell survival. Accordingly, when 273P4B7
functions as a regulator of proliferation, cell cycle, gene
expression, and cell survival, 273P4B7 is used for therapeutic,
diagnostic, prognostic or preventative purposes.
Example 45
Identification and Confirmation of Signal Transduction Pathways
[0841] 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,
transcription factors have been shown to regulate mitogenic and
survival pathways (Neeley K, Biochim Biophys Acta. 2002, 1603:19).
Using immunoprecipitation and Western blotting techniques, proteins
are identified that associate with 273P4B7 and mediate signaling
events. Several pathways known to play a role in cancer biology can
be regulated by 273P4B7, including phospholipid pathways such as
P13K, 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 273P4B7 can become
phosphorylated by serine/threonine as well as tyrosine kinases.
Thus, the phosphorylation of 273P4B7 is provided by the present
invention to lead to activation of the above listed pathways.
[0842] Using, e.g., Western blotting techniques the ability of
273P4B7 to regulate these pathways is confirmed. Cells expressing
or lacking 273P4B7 are 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, P13K, PLC and other signaling molecules.
[0843] To confirm that 273P4B7 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.
[0844] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0845] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0846] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0847] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0848] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0849] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0850] Gene-mediated effects can be assayed in cells showing mRNA
expression. Luciferase reporter plasmids can be 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.
[0851] Signaling pathways activated by 273P4B7 are mapped and used
for the identification and validation of therapeutic targets. When
273P4B7 plays a role in the regulation of signaling pathways,
mitogenic and survival pathways, phospholipid pathways and adhesion
and migration pathways whether individually or communally, it is
used as a target for diagnostic, prognostic, preventative and
therapeutic purposes. Additionally, when 273P4B7 is involved in
cell signaling, it is used as target for diagnostic, prognostic,
preventative and therapeutic purposes.
Example 46
Involvement in Tumor Progression
[0852] The 273P4B7 gene can contribute to the growth of cancer
cells. The role of 273P4B7 in tumor growth is confirmed in a
variety of primary and transfected cell lines including pancreas,
cervix, bladder, lung, prostate, kidney, colon, ovary, breast,
bone, skin, lymph node, stomach, and uterus cell lines as well as
NIH 3T3 cells engineered to stably express 273P4B7. Parental cells
lacking 273P4B7 and cells expressing 273P4B7 are evaluated for cell
growth using a well-documented proliferation assay (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).
[0853] To confirm the role of 273P4B7 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH3T3 cells lacking 273P4B7 are compared to NHI3T3 cells
expressing 273P4B7, using a soft agar assay under stringent and
more permissive conditions (Song Z. et al. Cancer Res. 2000,
60:6730).
[0854] To confirm the role of 273P4B7 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, but not limited to prostate, colon,
bladder and kidney cell lines lacking 273P4B7 are compared to cells
expressing 273P4B7. 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. 273P4B7 can also play a
role in cell cycle and apoptosis. Parental cells and cells
expressing 273P4B7 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
273P4B7. 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 273P4B7 can play a
critical role in regulating tumor progression and tumor load.
[0855] When 273P4B7 plays a role in cell growth, transformation,
invasion and metastasis, and cell cycle and apoptosis, it is used
as a target for diagnostic, prognostic, preventative and
therapeutic purposes.
Example 47
Involvement in Angiogenesis
[0856] 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 273P4B7 on angiogenesis
is confirmed.
[0857] For example, endothelial cells engineered to express 273P4B7
are evaluated using tube formation and proliferation assays. The
effect of 273P4B7 is also confirmed in animal models in vivo. For
example, cells either expressing or lacking 273P4B7 are implanted
subcutaneously in immunocompromised mice. Endothelial cell
migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques.
[0858] When 273P4B7 affects angiogenesis, it is used as a target
for diagnostic, prognostic, preventative and therapeutic
purposes.
Example 48
Regulation of Transcription
[0859] The localization of 273P4B7 to the nucleus and its
similarity to SNF2 containing proteins known to regulate gene
expression and chromatin structure, support the present invention
use of 273P4B7 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
273P4B7. For this purpose, two types of experiments are
performed.
[0860] In the first set of experiments, RNA from parental and
273P4B7-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).
[0861] In the second set of experiments, specific transcriptional
pathway activation is evaluated using commercially available
(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.
[0862] Thus, when 273P4B7 plays a role in gene regulation, it is
used as a target for diagnostic, prognostic, preventative and
therapeutic purposes.
Example 49
Protein-Protein Association
[0863] SNF2 containing proteins have been shown to interact with
other proteins, thereby forming protein complexes that can regulate
protein localization, chromatin structure, gene transcription, and
cell transformation (Papoulas et al., Development, 1998, 125:3955;
Cao et al., Mol. Cell. Biol. 1997, 17:3323). Using
immunoprecipitation techniques as well as two yeast hybrid systems,
proteins are identified that associate with 273P4B7.
Immunoprecipitates from cells expressing 273P4B7 and cells lacking
273P4B7 are compared for specific protein-protein associations.
[0864] Studies are performed to determine the extent of the
association of 273P4B7 with receptors, such as the EGF and IGF
receptors, and with intracellular proteins, such as IGF-BP,
cytoskeletal proteins etc. Studies comparing 273P4B7 positive and
273P4B7 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 interactions.
[0865] 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 273P4B7-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 273P4B7, 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 273P4B7. When 273P4B7 associates with proteins
to regulate protein localization, chromatin structure, gene
transcription, and cell transformation or associates with small
molecules it is used as a target for diagnostic, prognostic,
preventative and therapeutic purposes.
[0866] Throughout this application, various website data content,
publications, patent applications and patents are referenced. The
disclosures of each of these references are hereby incorporated by
reference herein in their entireties.
[0867] 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.
Sequence CWU 1
1
134 1 170 DNA Homo Sapiens misc_feature 151, 161 n = A,T,C or G 1
gatcctgaag ttatgctctt gactttaagt ttgtataagc aacttaataa caattgagaa
60 tgtaacctgt ttactgtatt ttaaagtgaa actgaatatg agggaatttt
tgttcccata 120 attggattct ttgggaacat gaagcattta ngcttaaggc
nagaaagatc 170 2 4194 DNA Homo Sapiens 2 aaattcaagc tccaaactct
aagctccaag ctccaagctc caagctccaa gctccaaact 60 cccgccgggg
taactggaac ccaatccgag ggtcatggag gcatcccgaa ggtttccgga 120
agccgaggcc ttgagcccag agcaggctgc tcattaccta agatatgtga aagaggccaa
180 agaagcaact aagaatggag acctggaaga agcatttaaa cttttcaatt
tggcaaagga 240 catttttccc aatgaaaaag tgctgagcag aatccaaaaa
atacaggaag ccttggagga 300 gttggcagaa cagggagatg atgaatttac
agatgtgtgc aactctggct tgctacttta 360 tcgagaactg cacaaccaac
tctttgagca ccagaaggaa ggcatagctt tcctctatag 420 cctgtatagg
gatggaagaa aaggtggtat attggctgat gatatgggat tagggaagac 480
tgttcaaatc attgctttcc tttccggtat gtttgatgca tcacttgtga atcatgtgct
540 gctgatcatg ccaaccaatc ttattaacac atgggtaaaa gaattcatca
agtggactcc 600 aggaatgaga gtcaaaacct ttcatggtcc tagcaaggat
gaacggacca gaaacctcaa 660 tcggattcag caaaggaatg gtgttattat
cactacatac caaatgttaa tcaataactg 720 gcagcaactt tcaagcttta
ggggccaaga gtttgtgtgg gactatgtca tcctcgatga 780 agcacataaa
ataaaaacct catctactaa gtcagcaata tgtgctcgtg ctattcctgc 840
aagtaatcgc ctcctcctca caggaacccc aatccagaat aatttacaag aactatggtc
900 cctatttgat tttgcttgtc aagggtccct gctgggaaca ttaaaaactt
ttaagatgga 960 gtatgaaaat cctattacta gagcaagaga gaaggatgct
accccaggag aaaaagcctt 1020 gggatttaaa atatctgaaa acttaatggc
aatcataaaa ccctattttc tcaggaggac 1080 taaagaagac gtacagaaga
aaaagtcaag caacccagag gccagactta atgaaaagaa 1140 tccagatgtt
gatgccattt gtgaaatgcc ttccctttcc aggaaaaatg atttaattat 1200
ttggatacga cttgtgcctt tacaagaaga aatatacagg aaatttgtgt ctttagatca
1260 tatcaaggag ttgctaatgg agacgcgctc acctttggct gagctaggtg
tcttaaagaa 1320 gctgtgtgat catcctaggc tgctgtctgc acgggcttgt
tgtttgctaa atcttgggac 1380 attctctgct caagatggaa atgaggggga
agattcccca gatgtggacc atattgatca 1440 agtaactgat gacacattga
tggaagaatc tggaaaaatg atattcctaa tggacctact 1500 taagaggctg
cgagatgagg gacatcaaac tctggtgttt tctcaatcga ggcaaattct 1560
aaacatcatt gaacgcctct taaagaatag gcactttaag acattgcgaa tcgatgggac
1620 agttactcat cttttggaac gagaaaaaag aattaactta ttccagcaaa
ataaagatta 1680 ctctgttttt ctgcttacca ctcaagtagg tggtgtcggt
ttaacattaa ctgcagcaac 1740 tagagtggtc atttttgacc ctagctggaa
tcctgcaact gatgctcaag ctgtggatag 1800 agtttaccga attggacaaa
aagagaatgt tgtggtttat aggctaatca cttgtgggac 1860 tgtagaggaa
aaaatataca gaagacaggt tttcaaggac tcattaataa gacaaactac 1920
tggtgaaaaa aagaaccctt tccgatattt tagtaaacaa gaattaagag agctctttac
1980 aatcgaggat cttcagaact ctgtaaccca gctgcagctt cagtctttgc
atgctgctca 2040 gaggaaatct gatataaaac tagatgaaca tattgcctac
ctgcagtctt tggggatagc 2100 tggaatctca gaccatgatt tgatgtacac
atgtgatctg tctgttaaag aagagcttga 2160 tgtggtagaa gaatctcact
atattcaaca aagggttcag aaagctcaat tcctcgttga 2220 attcgagtct
caaaataaag agttcctgat ggaacaacaa agaactagaa atgagggggc 2280
ctggctaaga gaacctgtat ttccttcttc aacaaagaag aaatgcccta aattgaataa
2340 accacagcct cagccttcac ctcttctaag tactcatcat actcaggaag
aagatatcag 2400 ttccaaaatg gcaagtgtag tcattgatga tctgcccaaa
gagggtgaga aacaagatct 2460 ctccagtata aaggtgaatg ttaccacctt
gcaagatggt aaaggtacag gtagtgctga 2520 ctctatagct actttaccaa
aggggtttgg aagtgtagaa gaactttgta ctaactcttc 2580 attgggaatg
gaaaaaagct ttgcaactaa aaatgaagct gtacaaaaag agacattaca 2640
agaggggcct aagcaagagg cactgcaaga ggatcctctg gaaagtttta attatgtact
2700 tagcaaatca accaaagctg atattgggcc aaatttagat caactaaagg
atgatgagat 2760 tttacgtcat tgcaatcctt ggcccattat ttccataaca
aatgaaagtc aaaatgcaga 2820 atcaaatgta tccattattg aaatagctga
tgacctttca gcatcccata gtgcactgca 2880 ggatgctcaa gcaagtgagg
ccaagttgga agaggaacct tcagcatctt caccacagta 2940 tgcatgtgat
ttcaatcttt tcttggaaga ctcagcagac aacagacaaa atttttccag 3000
tcagtcttta gagcatgttg agaaagaaaa tagcttgtgt ggctctgcac ctaattccag
3060 agcagggttt gtgcatagca aaacatgtct cagttgggag ttttctgaga
aagacgatga 3120 accagaagaa gtagtagtta aagcaaaaat cagaagtaaa
gctagaagga ttgtttcaga 3180 tggcgaagat gaagatgatt cttttaaaga
tacctcaagc ataaatccat tcaacacatc 3240 tctctttcaa ttctcatctg
tgaaacaatt tgatgcttca actcccaaaa atgacatcag 3300 tccaccagga
aggttctttt catctcaaat acccagtagt gtaaataagt ctatgaactc 3360
tagaagatct ctggcttcta ggaggtctct tattaatatg gttttagacc acgtggagga
3420 catggaggaa agacttgacg acagcagtga agcaaagggt cctgaagatt
atccagaaga 3480 aggggtggag gaaagcagtg gcgaagcctc caagtataca
gaagaggatc cttccggaga 3540 aacactgtct tcagaaaaca agtccagctg
gttaatgacg tctaagccta gtgctctagc 3600 tcaagagacc tctcttggtg
cccctgagcc tttgtctggt gaacagttgg ttggttctcc 3660 ccaggataag
gcggcagagg ctacaaatga ctatgagact cttgtaaagc gtggaaaaga 3720
actaaaagag tgtggaaaaa tccaggaggc cctaaactgc ttagttaaag cgcttgacat
3780 aaaaagtgca gatcctgaag ttatgctctt gactttaagt ttgtataagc
aacttaataa 3840 caattgagaa tgtaacctgt ttattgtatt ttaaagtgaa
actgaatatg agggaatttt 3900 tgttcccata attggattct ttgggaacat
gaagcattca ggcttaaggc aagaaagatc 3960 tcaaaaagca acttctgccc
tgcaacgccc cccactccat agtctggtat tctgagcact 4020 agcttaatat
ttcttcactt gaatattctt atattttagg catattctat aaatttaact 4080
gtgttgtttc ttggaaagtt ttgtaaaatt attctggtca ttcttaattt tactctgaaa
4140 gtgatcatct ttgtatataa cagttcagat aagaaaatta aagttacttt tctc
4194 3 1250 PRT Homo Sapiens 3 Met Glu Ala Ser Arg Arg Phe Pro Glu
Ala Glu Ala Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala His Tyr Leu Arg
Tyr Val Lys Glu Ala Lys Glu Ala Thr 20 25 30 Lys Asn Gly Asp Leu
Glu Glu Ala Phe Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp Ile Phe
Pro Asn Glu Lys Val Leu Ser Arg Ile Gln Lys Ile Gln 50 55 60 Glu
Ala Leu Glu Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe Thr Asp 65 70
75 80 Val Cys Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu His Asn Gln
Leu 85 90 95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr Ser
Leu Tyr Arg 100 105 110 Asp Gly Arg Lys Gly Gly Ile Leu Ala Asp Asp
Met Gly Leu Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu Ser
Gly Met Phe Asp Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu Ile
Met Pro Thr Asn Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu Phe
Ile Lys Trp Thr Pro Gly Met Arg Val Lys Thr Phe 165 170 175 His Gly
Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 180 185 190
Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn 195
200 205 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp Asp
Tyr 210 215 220 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser Ser
Thr Lys Ser 225 230 235 240 Ala Ile Cys Ala Arg Ala Ile Pro Ala Ser
Asn Arg Leu Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn Leu
Gln Glu Leu Trp Ser Leu Phe Asp 260 265 270 Phe Ala Cys Gln Gly Ser
Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 275 280 285 Glu Tyr Glu Asn
Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro 290 295 300 Gly Glu
Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met Ala Ile 305 310 315
320 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val Gln Lys Lys
325 330 335 Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn Glu Lys Asn Pro
Asp Val 340 345 350 Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg Lys
Asn Asp Leu Ile 355 360 365 Ile Trp Ile Arg Leu Val Pro Leu Gln Glu
Glu Ile Tyr Arg Lys Phe 370 375 380 Val Ser Leu Asp His Ile Lys Glu
Leu Leu Met Glu Thr Arg Ser Pro 385 390 395 400 Leu Ala Glu Leu Gly
Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 405 410 415 Leu Ser Ala
Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala 420 425 430 Gln
Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp His Ile Asp 435 440
445 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser Gly Lys Met Ile Phe
450 455 460 Leu Met Asp Leu Leu Lys Arg Leu Arg Asp Glu Gly His Gln
Thr Leu 465 470 475 480 Val Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile
Ile Glu Arg Leu Leu 485 490 495 Lys Asn Arg His Phe Lys Thr Leu Arg
Ile Asp Gly Thr Val Thr His 500 505 510 Leu Leu Glu Arg Glu Lys Arg
Ile Asn Leu Phe Gln Gln Asn Lys Asp 515 520 525 Tyr Ser Val Phe Leu
Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 530 535 540 Leu Thr Ala
Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn Pro 545 550 555 560
Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly Gln Lys 565
570 575 Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly Thr Val Glu
Glu 580 585 590 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu Ile
Arg Gln Thr 595 600 605 Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe
Ser Lys Gln Glu Leu 610 615 620 Arg Glu Leu Phe Thr Ile Glu Asp Leu
Gln Asn Ser Val Thr Gln Leu 625 630 635 640 Gln Leu Gln Ser Leu His
Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 645 650 655 Asp Glu His Ile
Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 660 665 670 Asp His
Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu 675 680 685
Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln Lys Ala 690
695 700 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu Phe Leu Met
Glu 705 710 715 720 Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg
Glu Pro Val Phe 725 730 735 Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys
Leu Asn Lys Pro Gln Pro 740 745 750 Gln Pro Ser Pro Leu Leu Ser Thr
His His Thr Gln Glu Glu Asp Ile 755 760 765 Ser Ser Lys Met Ala Ser
Val Val Ile Asp Asp Leu Pro Lys Glu Gly 770 775 780 Glu Lys Gln Asp
Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln 785 790 795 800 Asp
Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro Lys 805 810
815 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser Leu Gly Met
820 825 830 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu
Thr Leu 835 840 845 Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp
Pro Leu Glu Ser 850 855 860 Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys
Ala Asp Ile Gly Pro Asn 865 870 875 880 Leu Asp Gln Leu Lys Asp Asp
Glu Ile Leu Arg His Cys Asn Pro Trp 885 890 895 Pro Ile Ile Ser Ile
Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 900 905 910 Ser Ile Ile
Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 915 920 925 Gln
Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser Ala 930 935
940 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp Ser
945 950 955 960 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu
His Val Glu 965 970 975 Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn
Ser Arg Ala Gly Phe 980 985 990 Val His Ser Lys Thr Cys Leu Ser Trp
Glu Phe Ser Glu Lys Asp Asp 995 1000 1005 Glu Pro Glu Glu Val Val
Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 1010 1015 1020 Arg Ile Val
Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 1025 1030 1035
1040 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser
Val 1045 1050 1055 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile
Ser Pro Pro Gly 1060 1065 1070 Arg Phe Phe Ser Ser Gln Ile Pro Ser
Ser Val Asn Lys Ser Met Asn 1075 1080 1085 Ser Arg Arg Ser Leu Ala
Ser Arg Arg Ser Leu Ile Asn Met Val Leu 1090 1095 1100 Asp His Val
Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 1105 1110 1115
1120 Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser
Gly 1125 1130 1135 Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly
Glu Thr Leu Ser 1140 1145 1150 Ser Glu Asn Lys Ser Ser Trp Leu Met
Thr Ser Lys Pro Ser Ala Leu 1155 1160 1165 Ala Gln Glu Thr Ser Leu
Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln 1170 1175 1180 Leu Val Gly
Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr 1185 1190 1195
1200 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys
Ile 1205 1210 1215 Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp
Ile Lys Ser Ala 1220 1225 1230 Asp Pro Glu Val Met Leu Leu Thr Leu
Ser Leu Tyr Lys Gln Leu Asn 1235 1240 1245 Asn Asn 1250 4 4334 DNA
Homo Sapiens 4 atgcgcgggg cgggagtgag cgaaattcaa gctccaaact
ctaagctcca agctccaagc 60 tccaagctcc aagctccaaa ctcccgccgg
ggtaactgga acccaatccg agggtcatgg 120 aggcatcccg aaggtttccg
gaagccgagg ccttgagccc agagcaggct gctcattacc 180 taagggtctt
gctgtgtcgc ccagactgga attcagtggc ctgatcatag ttcactgcag 240
cctcgaactc ctgggctcaa gcagtcctcc tgccccagcc tccctagtag ctgggactta
300 agatatgtga aagaggccaa agaagcaact aagaatggag acctggaaga
agcatttaaa 360 cttttcaatt tggcaaagga catttttccc aatgaaaaag
tgctgagcag aatccaaaaa 420 atacaggaag ccttggagga gttggcagaa
cagggagatg atgaatttac agatgtgtgc 480 aactctggct tgctacttta
tcgagaactg cacaaccaac tctttgagca ccagaaggaa 540 ggcatagctt
tcctctatag cctgtatagg gatggaagaa aaggtggtat attggctgat 600
gatatgggat tagggaagac tgttcaaatc attgctttcc tttccggtat gtttgatgca
660 tcacttgtga atcatgtgct gctgatcatg ccaaccaatc ttattaacac
atgggtaaaa 720 gaattcatca agtggactcc aggaatgaga gtcaaaacct
ttcatggtcc tagcaaggat 780 gaacggacca gaaacctcaa tcggattcag
caaaggaatg gtgttattat cactacatac 840 caaatgttaa tcaataactg
gcagcaactt tcaagcttta ggggccaaga gtttgtgtgg 900 gactatgtca
tcctcgatga agcacataaa ataaaaacct catctactaa gtcagcaata 960
tgtgctcgtg ctattcctgc aagtaatcgc ctcctcctca caggaacccc aatccagaat
1020 aatttacaag aactatggtc cctatttgat tttgcttgtc aagggtccct
gctgggaaca 1080 ttaaaaactt ttaagatgga gtatgaaaat cctattacta
gagcaagaga gaaggatgct 1140 accccaggag aaaaagcctt gggatttaaa
atatctgaaa acttaatggc aatcataaaa 1200 ccctattttc tcaggaggac
taaagaagac gtacagaaga aaaagtcaag caacccagag 1260 gccagactta
atgaaaagaa tccagatgtt gatgccattt gtgaaatgcc ttccctttcc 1320
aggaaaaatg atttaattat ttggatacga cttgtgcctt tacaagaaga aatatacagg
1380 aaatttgtgt ctttagatca tatcaaggag ttgctaatgg agacgcgctc
acctttggct 1440 gagctaggtg tcttaaagaa gctgtgtgat catcctaggc
tgctgtctgc acgggcttgt 1500 tgtttgctaa atcttgggac attctctgct
caagatggaa atgaggggga agattcccca 1560 gatgtggacc atattgatca
agtaactgat gacacattga tggaagaatc tggaaaaatg 1620 atattcctaa
tggacctact taagaggctg cgagatgagg gacatcaaac tctggtgttt 1680
tctcaatcga ggcaaattct aaacatcatt gaacgcctct taaagaatag gcactttaag
1740 acattgcgaa tcgatgggac agttactcat cttttggaac gagaaaaaag
aattaactta 1800 ttccagcaaa ataaagatta ctctgttttt ctgcttacca
ctcaagtagg tggtgtcggt 1860 ttaacattaa ctgcagcaac tagagtggtc
atttttgacc ctagctggaa tcctgcaact 1920 gatgctcaag ctgtggatag
agtttaccga attggacaaa aagagaatgt tgtggtttat 1980 aggctaatca
cttgtgggac tgtagaggaa aaaatataca gaagacaggt tttcaaggac 2040
tcattaataa gacaaactac tggtgaaaaa aagaaccctt tccgatattt tagtaaacaa
2100 gaattaagag agctctttac aatcgaggat cttcagaact ctgtaaccca
gctgcagctt 2160 cagtctttgc atgctgctca gaggaaatct gatataaaac
tagatgaaca tattgcctac 2220 ctgcagtctt tggggatagc tggaatctca
gaccatgatt tgatgtacac atgtgatctg 2280 tctgttaaag aagagcttga
tgtggtagaa gaatctcact atattcaaca aagggttcag 2340 aaagctcaat
tcctcgttga attcgagtct caaaataaag agttcctgat ggaacaacaa 2400
agaactagaa atgagggggc ctggctaaga gaacctgtat ttccttcttc aacaaagaag
2460 aaatgcccta aattgaataa accacagcct cagccttcac ctcttctaag
tactcatcat 2520 actcaggaag aagatatcag ttccaaaatg gcaagtgtag
tcattgatga tctgcccaaa 2580 gagggtgaga aacaagatct ctccagtata
aaggtgaatg ttaccacctt gcaagatggt 2640 aaaggtacag gtagtgctga
ctctatagct actttaccaa aggggtttgg aagtgtagaa 2700 gaactttgta
ctaactcttc attgggaatg gaaaaaagct ttgcaactaa aaatgaagct 2760
gtacaaaaag agacattaca agaggggcct aagcaagagg cactgcaaga ggatcctctg
2820 gaaagtttta attatgtact
tagcaaatca accaaagctg atattgggcc aaatttagat 2880 caactaaagg
atgatgagat tttacgtcat tgcaatcctt ggcccattat ttccataaca 2940
aatgaaagtc aaaatgcaga atcaaatgta tccattattg aaatagctga tgacctttca
3000 gcatcccata gtgcactgca ggatgctcaa gcaagtgagg ccaagttgga
agaggaacct 3060 tcagcatctt caccacagta tgcatgtgat ttcaatcttt
tcttggaaga ctcagcagac 3120 aacagacaaa atttttccag tcagtcttta
gagcatgttg agaaagaaaa tagcttgtgt 3180 ggctctgcac ctaattccag
agcagggttt gtgcatagca aaacatgtct cagttgggag 3240 ttttctgaga
aagacgatga accagaagaa gtagtagtta aagcaaaaat cagaagtaaa 3300
gctagaagga ttgtttcaga tggcgaagat gaagatgatt cttttaaaga tacctcaagc
3360 ataaatccat tcaacacatc tctctttcaa ttctcatctg tgaaacaatt
tgatgcttca 3420 actcccaaaa atgacatcag tccaccagga aggttctttt
catctcaaat acccagtagt 3480 gtaaataagt ctatgaactc tagaagatct
ctggcttcta ggaggtctct tattaatatg 3540 gttttagacc acgtggagga
catggaggaa agacttgacg acagcagtga agcaaagggt 3600 cctgaagatt
atccagaaga aggggtggag gaaagcagtg gcgaagcctc caagtataca 3660
gaagaggatc cttccggaga aacactgtct tcagaaaaca agtccagctg gttaatgacg
3720 tctaagccta gtgctctagc tcaagagacc tctcttggtg cccctgagcc
tttgtctggt 3780 gaacagttgg ttggttctcc ccaggataag gcggcagagg
ctacaaatga ctatgagact 3840 cttgtaaagc gtggaaaaga actaaaagag
tgtggaaaaa tccaggaggc cctaaactgc 3900 ttagttaaag cgcttgacat
aaaaagtgca gatcctgaag ttatgctctt gactttaagt 3960 ttgtataagc
aacttaataa caattgagaa tgtaacctgt ttattgtatt ttaaagtgaa 4020
actgaatatg agggaatttt tgttcccata attggattct ttgggaacat gaagcattca
4080 ggcttaaggc aagaaagatc tcaaaaagca acttctgccc tgcaacgccc
cccactccat 4140 agtctggtat tctgagcact agcttaatat ttcttcactt
gaatattctt atattttagg 4200 catattctat aaatttaact gtgttgtttc
ttggaaagtt ttgtaaaatt attctggtca 4260 ttcttaattt tactctgaaa
gtgatcatct ttgtatataa cagttcagat aagaaaatta 4320 aagttacttt tctc
4334 5 1127 PRT Homo Sapiens 5 Met Gly Leu Gly Lys Thr Val Gln Ile
Ile Ala Phe Leu Ser Gly Met 1 5 10 15 Phe Asp Ala Ser Leu Val Asn
His Val Leu Leu Ile Met Pro Thr Asn 20 25 30 Leu Ile Asn Thr Trp
Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met 35 40 45 Arg Val Lys
Thr Phe His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn 50 55 60 Leu
Asn Arg Ile Gln Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln 65 70
75 80 Met Leu Ile Asn Asn Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln
Glu 85 90 95 Phe Val Trp Asp Tyr Val Ile Leu Asp Glu Ala His Lys
Ile Lys Thr 100 105 110 Ser Ser Thr Lys Ser Ala Ile Cys Ala Arg Ala
Ile Pro Ala Ser Asn 115 120 125 Arg Leu Leu Leu Thr Gly Thr Pro Ile
Gln Asn Asn Leu Gln Glu Leu 130 135 140 Trp Ser Leu Phe Asp Phe Ala
Cys Gln Gly Ser Leu Leu Gly Thr Leu 145 150 155 160 Lys Thr Phe Lys
Met Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu 165 170 175 Lys Asp
Ala Thr Pro Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu 180 185 190
Asn Leu Met Ala Ile Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu 195
200 205 Asp Val Gln Lys Lys Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn
Glu 210 215 220 Lys Asn Pro Asp Val Asp Ala Ile Cys Glu Met Pro Ser
Leu Ser Arg 225 230 235 240 Lys Asn Asp Leu Ile Ile Trp Ile Arg Leu
Val Pro Leu Gln Glu Glu 245 250 255 Ile Tyr Arg Lys Phe Val Ser Leu
Asp His Ile Lys Glu Leu Leu Met 260 265 270 Glu Thr Arg Ser Pro Leu
Ala Glu Leu Gly Val Leu Lys Lys Leu Cys 275 280 285 Asp His Pro Arg
Leu Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu 290 295 300 Gly Thr
Phe Ser Ala Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp 305 310 315
320 Val Asp His Ile Asp Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser
325 330 335 Gly Lys Met Ile Phe Leu Met Asp Leu Leu Lys Arg Leu Arg
Asp Glu 340 345 350 Gly His Gln Thr Leu Val Phe Ser Gln Ser Arg Gln
Ile Leu Asn Ile 355 360 365 Ile Glu Arg Leu Leu Lys Asn Arg His Phe
Lys Thr Leu Arg Ile Asp 370 375 380 Gly Thr Val Thr His Leu Leu Glu
Arg Glu Lys Arg Ile Asn Leu Phe 385 390 395 400 Gln Gln Asn Lys Asp
Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly 405 410 415 Gly Val Gly
Leu Thr Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp 420 425 430 Pro
Ser Trp Asn Pro Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr 435 440
445 Arg Ile Gly Gln Lys Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys
450 455 460 Gly Thr Val Glu Glu Lys Ile Tyr Arg Arg Gln Val Phe Lys
Asp Ser 465 470 475 480 Leu Ile Arg Gln Thr Thr Gly Glu Lys Lys Asn
Pro Phe Arg Tyr Phe 485 490 495 Ser Lys Gln Glu Leu Arg Glu Leu Phe
Thr Ile Glu Asp Leu Gln Asn 500 505 510 Ser Val Thr Gln Leu Gln Leu
Gln Ser Leu His Ala Ala Gln Arg Lys 515 520 525 Ser Asp Ile Lys Leu
Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly 530 535 540 Ile Ala Gly
Ile Ser Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser 545 550 555 560
Val Lys Glu Glu Leu Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln 565
570 575 Arg Val Gln Lys Ala Gln Phe Leu Val Glu Phe Glu Ser Gln Asn
Lys 580 585 590 Glu Phe Leu Met Glu Gln Gln Arg Thr Arg Asn Glu Gly
Ala Trp Leu 595 600 605 Arg Glu Pro Val Phe Pro Ser Ser Thr Lys Lys
Lys Cys Pro Lys Leu 610 615 620 Asn Lys Pro Gln Pro Gln Pro Ser Pro
Leu Leu Ser Thr His His Thr 625 630 635 640 Gln Glu Glu Asp Ile Ser
Ser Lys Met Ala Ser Val Val Ile Asp Asp 645 650 655 Leu Pro Lys Glu
Gly Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn 660 665 670 Val Thr
Thr Leu Gln Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile 675 680 685
Ala Thr Leu Pro Lys Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn 690
695 700 Ser Ser Leu Gly Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val 705 710 715 720 Gln Lys Glu Thr Leu Gln Glu Gly Pro Lys Gln Glu
Ala Leu Gln Glu 725 730 735 Asp Pro Leu Glu Ser Phe Asn Tyr Val Leu
Ser Lys Ser Thr Lys Ala 740 745 750 Asp Ile Gly Pro Asn Leu Asp Gln
Leu Lys Asp Asp Glu Ile Leu Arg 755 760 765 His Cys Asn Pro Trp Pro
Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn 770 775 780 Ala Glu Ser Asn
Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala 785 790 795 800 Ser
His Ser Ala Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu 805 810
815 Glu Glu Pro Ser Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu
820 825 830 Phe Leu Glu Asp Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser
Gln Ser 835 840 845 Leu Glu His Val Glu Lys Glu Asn Ser Leu Cys Gly
Ser Ala Pro Asn 850 855 860 Ser Arg Ala Gly Phe Val His Ser Lys Thr
Cys Leu Ser Trp Glu Phe 865 870 875 880 Ser Glu Lys Asp Asp Glu Pro
Glu Glu Val Val Val Lys Ala Lys Ile 885 890 895 Arg Ser Lys Ala Arg
Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp 900 905 910 Ser Phe Lys
Asp Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe 915 920 925 Gln
Phe Ser Ser Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp 930 935
940 Ile Ser Pro Pro Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val
945 950 955 960 Asn Lys Ser Met Asn Ser Arg Arg Ser Leu Ala Ser Arg
Arg Ser Leu 965 970 975 Ile Asn Met Val Leu Asp His Val Glu Asp Met
Glu Glu Arg Leu Asp 980 985 990 Asp Ser Ser Glu Ala Lys Gly Pro Glu
Asp Tyr Pro Glu Glu Gly Val 995 1000 1005 Glu Glu Ser Ser Gly Glu
Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser 1010 1015 1020 Gly Glu Thr
Leu Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser 1025 1030 1035
1040 Lys Pro Ser Ala Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu
Pro 1045 1050 1055 Leu Ser Gly Glu Gln Leu Val Gly Ser Pro Gln Asp
Lys Ala Ala Glu 1060 1065 1070 Ala Thr Asn Asp Tyr Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys 1075 1080 1085 Glu Cys Gly Lys Ile Gln
Glu Ala Leu Asn Cys Leu Val Lys Ala Leu 1090 1095 1100 Asp Ile Lys
Ser Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu 1105 1110 1115
1120 Tyr Lys Gln Leu Asn Asn Asn 1125 6 3671 DNA Homo Sapiens 6
aaaatgaatc atgtgctgct gatcatgcca accaatctta ttaacacttg ggtaaaagaa
60 ttcatcaagt ggactccagg aatgggagtc aaaacctttc atggtcctag
caaggatgaa 120 cggaccagaa acctcaatcg gattcagcaa aggaatggtg
ttattatcac tacataccaa 180 atgttaatca ataactggca gcaactttca
agctttaggg gccaagagtt tgtgtgggac 240 tatgtcatcc tcgatgaagc
acataaaata aaaacctcat ctactaagtc agcaatatgt 300 gctcgtgcta
ttcctgcaag taatcgcctc ctcctcacag gaaccccaat ccagaataat 360
ttacaagaac tatggtccct atttgatttt gcttgtcaag ggtccctgct gggaacatta
420 aaaactttta agatggagta tgaaaatcct attactagag caagagagaa
ggatgctacc 480 ccaggagaaa aagccttggg atttaaaata tctgaaaact
taatggcaat cataaaaccc 540 tattttctca ggaggactaa agaagacgta
cagaagaaaa agtcaagcaa cccagaggcc 600 agacttaatg aaaagaatcc
agatgttgat gccatttgtg aaatgccttc cctttccagg 660 aaaaatgatt
taattatttg gatacgactt gtgcctttac aagaagaaat atacaggaaa 720
tttgtgtctt tagatcatat caaggagttg ctaatggaga cgcgctcacc tttggctgag
780 ctaggtgtct taaagaagct gtgtgatcat cctaggctgc tgtctgcacg
ggcttgttgt 840 ttgctaaatc ttgggacatt ctctgctcaa gatggaaatg
agggggaaga ttccccagat 900 gtggaccata ttgatcaagt aactgatgac
acattgatgg aagaatctgg aaaaatgata 960 ttcctaatgg acctacttaa
gaggctgcga gatgagggac atcaaactct ggtgttttct 1020 caatcgaggc
aaattctaaa catcattgaa cgcctcttaa agaataggca ctttaagaca 1080
ttgcgaatcg atgggacagt tactcatctt ttggaacgag aaaaaagaat taacttattc
1140 cagcaaaata aagattactc tgtttttctg cttaccactc aagtaggtgg
tgtcggttta 1200 acattaactg cagcaactag agtggtcatt tttgacccta
gctggaatcc tgcaactgat 1260 gctcaagctg tggatagagt ttaccgaatt
ggacaaaaag agaatgttgt ggtttatagg 1320 ctaatcactt gtgggactgt
agaggaaaaa atatacagaa gacaggtttt caaggactca 1380 ttaataagac
aaactactgg tgaaaaaaag aaccctttcc gatattttag taaacaagaa 1440
ttaagagagc tctttacaat cgaggatctt cagaactctg taacccagct gcagcttcag
1500 tctttgcatg ctgctcagag gaaatctgat ataaaactag atgaacatat
tgcctacctg 1560 cagtctttgg ggatagctgg aatctcagac catgatttga
tgtacacatg tgatctgtct 1620 gttaaagaag agcttgatgt ggtagaagaa
tctcactata ttcaacaaag ggttcagaaa 1680 gctcaattcc tcgttgaatt
cgagtctcaa aataaagagt tcctgatgga acaacaaaga 1740 actagaaatg
agggggcctg gctaagagaa cctgtatttc cttcttcaac aaagaagaaa 1800
tgccctaaat tgaataaacc acagcctcag ccttcacctc ttctaagtac tcatcatact
1860 caggaagaag atatcagttc caaaatggca agtgtagtca ttgatgatct
gcccaaagag 1920 ggtgagaaac aagatctctc cagtataaag gtgaatgtta
ccaccttgca agatggtaaa 1980 ggtacaggta gtgctgactc tatagctact
ttaccaaagg ggtttggaag tgtagaagaa 2040 ctttgtacta actcttcatt
gggaatggaa aaaagctttg caactaaaaa tgaagctgta 2100 caaaaagaga
cattacaaga ggggcctaag caagaggcac tgcaagagga tcctctggaa 2160
agttttaatt atgtacttag caaatcaacc aaagctgata ttgggccaaa tttagatcaa
2220 ctaaaggatg atgaggtttt acgtcattgc aatccttggc ccattatttc
cataacaaat 2280 gaaagtcaaa atgcagaatc aaatgtatcc attattgaaa
tagctgatga cctttcagca 2340 tcccatagtg cactgcagga tgctcaagca
agtgaggcca agttggaaga ggaaccttca 2400 gcatcttcac cacagtatgc
atgtgatttc aatcttttct tggaagactc agcagacaac 2460 agacaaaatt
tttccagtca gtctttagag catgttgaga aagaaaatag cttgtgtggc 2520
tctgcaccta attccagagc agggtttgtg catagcaaaa catgtctcag ttgggagttt
2580 tctgagaaag acgatgaacc agaagaagta gtagttaaag caaaaatcag
aagtaaagct 2640 agaaggattg tttcagatgg cgaagatgaa gatgattctt
ttaaagatac ctcaagcata 2700 aatccattca acacatctct ctttcaattc
tcatctgtga aacaatttga tgcttcaact 2760 cccaaaaatg acatcagtcc
accaggaagg ttcttttcat ctcaaatacc cagtagtgta 2820 aataagtcta
tgaactctag aagatctctg gcttctagga ggtctcttat taatatggtt 2880
ttagaccacg tggaggacat ggaggaaaga cttgacgaca gcagtgaagc aaagggtcct
2940 gaagattatc cagaagaagg ggtggaggaa agcagtggcg aagcctccaa
gtatacagaa 3000 gaggatcctt ccggagaaac actgtcttca gaaaacaagt
ccagctggtt aatgacgtct 3060 aagcctagtg ctctagctca agagacctct
cttggtgccc ctgagccttt gtctggtgaa 3120 cagttggttg gttcccccca
ggataaggcg gcagaggcta caaatgacta tgagactctt 3180 gtaaagcgtg
gaaaagaact aaaagagtgt ggaaaaatcc aggaggccct aaactgctta 3240
gttaaagcgc ttgacataaa aagtgcagat cctgaagtta tgctcttgac tttaagtttg
3300 tataagcaac ttaataacaa ttgagaatgt aacctgttta ttgtatttta
aagtgaaact 3360 gaatatgagg gaatttttgt tcccataatt ggattctttg
ggaacatgaa gcattcaggc 3420 ttaaggcaag aaagatctca aaaagcaact
tctgccctgc aacgcccccc actccatagt 3480 ctggtattct gagcactagc
ttaatatttc ttcacttgaa tattcttata ttttaggcat 3540 attctataaa
tttaactgtg ttgtttcttg gaaagttttg taaaattatt ctggtcattc 3600
ttaattttac tctgaaagtg atcatctttg tatataacag ttcagataag aaaattaaag
3660 ttacttttct c 3671 7 1106 PRT Homo Sapiens 7 Met Asn His Val
Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 1 5 10 15 Val Lys
Glu Phe Ile Lys Trp Thr Pro Gly Met Gly Val Lys Thr Phe 20 25 30
His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 35
40 45 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn
Asn 50 55 60 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val
Trp Asp Tyr 65 70 75 80 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr
Ser Ser Thr Lys Ser 85 90 95 Ala Ile Cys Ala Arg Ala Ile Pro Ala
Ser Asn Arg Leu Leu Leu Thr 100 105 110 Gly Thr Pro Ile Gln Asn Asn
Leu Gln Glu Leu Trp Ser Leu Phe Asp 115 120 125 Phe Ala Cys Gln Gly
Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 130 135 140 Glu Tyr Glu
Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro 145 150 155 160
Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met Ala Ile 165
170 175 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val Gln Lys
Lys 180 185 190 Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn Glu Lys Asn
Pro Asp Val 195 200 205 Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg
Lys Asn Asp Leu Ile 210 215 220 Ile Trp Ile Arg Leu Val Pro Leu Gln
Glu Glu Ile Tyr Arg Lys Phe 225 230 235 240 Val Ser Leu Asp His Ile
Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 245 250 255 Leu Ala Glu Leu
Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 260 265 270 Leu Ser
Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala 275 280 285
Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp His Ile Asp 290
295 300 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser Gly Lys Met Ile
Phe 305 310 315 320 Leu Met Asp Leu Leu Lys Arg Leu Arg Asp Glu Gly
His Gln Thr Leu 325 330 335 Val Phe Ser Gln Ser Arg Gln Ile Leu Asn
Ile Ile Glu Arg Leu Leu 340 345 350 Lys Asn Arg His Phe Lys Thr Leu
Arg Ile Asp Gly Thr Val Thr His 355 360 365 Leu Leu Glu Arg Glu Lys
Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 370 375 380 Tyr Ser Val Phe
Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 385 390 395 400 Leu
Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn Pro 405 410
415 Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly Gln Lys
420 425 430 Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly Thr Val
Glu Glu 435 440 445 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu
Ile Arg Gln
Thr 450 455 460 Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys
Gln Glu Leu 465 470 475 480 Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln
Asn Ser Val Thr Gln Leu 485 490 495 Gln Leu Gln Ser Leu His Ala Ala
Gln Arg Lys Ser Asp Ile Lys Leu 500 505 510 Asp Glu His Ile Ala Tyr
Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 515 520 525 Asp His Asp Leu
Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu 530 535 540 Asp Val
Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln Lys Ala 545 550 555
560 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu Phe Leu Met Glu
565 570 575 Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro
Val Phe 580 585 590 Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn
Lys Pro Gln Pro 595 600 605 Gln Pro Ser Pro Leu Leu Ser Thr His His
Thr Gln Glu Glu Asp Ile 610 615 620 Ser Ser Lys Met Ala Ser Val Val
Ile Asp Asp Leu Pro Lys Glu Gly 625 630 635 640 Glu Lys Gln Asp Leu
Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln 645 650 655 Asp Gly Lys
Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro Lys 660 665 670 Gly
Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser Leu Gly Met 675 680
685 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr Leu
690 695 700 Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu
Glu Ser 705 710 715 720 Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala
Asp Ile Gly Pro Asn 725 730 735 Leu Asp Gln Leu Lys Asp Asp Glu Val
Leu Arg His Cys Asn Pro Trp 740 745 750 Pro Ile Ile Ser Ile Thr Asn
Glu Ser Gln Asn Ala Glu Ser Asn Val 755 760 765 Ser Ile Ile Glu Ile
Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 770 775 780 Gln Asp Ala
Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser Ala 785 790 795 800
Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp Ser 805
810 815 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val
Glu 820 825 830 Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg
Ala Gly Phe 835 840 845 Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe
Ser Glu Lys Asp Asp 850 855 860 Glu Pro Glu Glu Val Val Val Lys Ala
Lys Ile Arg Ser Lys Ala Arg 865 870 875 880 Arg Ile Val Ser Asp Gly
Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 885 890 895 Ser Ser Ile Asn
Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val 900 905 910 Lys Gln
Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro Gly 915 920 925
Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met Asn 930
935 940 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val
Leu 945 950 955 960 Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp
Ser Ser Glu Ala 965 970 975 Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly
Val Glu Glu Ser Ser Gly 980 985 990 Glu Ala Ser Lys Tyr Thr Glu Glu
Asp Pro Ser Gly Glu Thr Leu Ser 995 1000 1005 Ser Glu Asn Lys Ser
Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1010 1015 1020 Ala Gln
Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln 1025 1030
1035 1040 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp Tyr 1045 1050 1055 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys
Glu Cys Gly Lys Ile 1060 1065 1070 Gln Glu Ala Leu Asn Cys Leu Val
Lys Ala Leu Asp Ile Lys Ser Ala 1075 1080 1085 Asp Pro Glu Val Met
Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1090 1095 1100 Asn Asn
1105 8 2312 DNA Homo Sapiens 8 tcattaataa gacaaactac tggtgaaaaa
aagaaccctt tccgatattt tagtaaacaa 60 gaattaagag agctctttac
aatcgaggat cttcagaact ctgtaaccca gctgcagctt 120 cagtctttgc
atgctgctca gaggaaatct gatataaaac tagatgaaca tattgcctac 180
ctgcagtctt tggggatagc tggaatctca gaccatgatt tgatgtacac atgtgatctg
240 tctgttaaag aagagcttga tgtggtagaa gaatctcact atattcaaca
aagggttcag 300 aaagctcaat tcctcgttga attcgagtct caaaataaag
agttcctgat ggaacaacaa 360 agaactagaa atgagggggc ctggctaaga
gaacctgtat ttccttcttc aacaaagaag 420 aaatgcccta aattgaataa
accacagcct cagccttcac ctcttctaag tactcatcat 480 actcaggaag
aagatatcag ttccaaaatg gcaagtgtag tcattgatga tctgcccaaa 540
gagggtgaga aacaagatct ctccagtata aaggtgaatg ttaccacctt gcaagatggg
600 taaggtacag gtagtgctga ctctataact actttaccaa aggggtttgg
aagtgtagaa 660 gaactttgta ctaactcttc attgggaatg gaaaaaagct
ttgcaactaa aaatgaagct 720 gtacaaaaag agacattaca agaggggcct
aagcaggagg cactgcaaga ggatcctctg 780 gaaagtttta attatgtact
tagcaaatca accaaagctg atattgggcc aaatttagat 840 caactaaagg
atgatgagat tttacgtcat tgcaatcctt ggcccattat ttccataaca 900
aatgaaagtc aaaatgcaga atcaaatgta tccattattg aaatagctga tgacctttca
960 gcatcccata gtgcactgca ggatgctcaa gcaagtgagg ccaagttgga
agaggaacct 1020 tcagcatctt caccacagta tgcatgtgat ttcaatcttt
tcttggaaga ctcagcagac 1080 aacagacaaa atttttccag tcagtcttta
gagcatgttg agaaagaaaa tagcttgtgt 1140 ggctctgcac ctaattccaa
agcagggttt gtgcatagca aaacatgtct cagttgggag 1200 ttttctgaga
aagacgatga accagaagaa gtagtagtta aagcaaaaat cagaagtaaa 1260
gctagaagga ttgtttcaga tggcgaagat gaagatgatt cttttaaaga tacctcaagc
1320 ataaatccat tcaacacatc tctctttcaa ttctcatctg tgaaacaatt
tgatgcttca 1380 actcccaaaa atgacatcag tccaccagga aggttctttt
catctcaaat acccagtagt 1440 gtaaataagt ctatgaactc tagaagatct
ctggcttcta ggaggtctct tattaatatg 1500 gttttagacc acgtggagga
catggaggaa agacttgacg acagcagtga agcaaagggt 1560 cctgaagatt
atccagaaga aggggtggag gaaagcagtg gcgaagcctc caagtataca 1620
gaagaggatc cttccggaga aacactgtct tcagaaaaca agtccagctg gttaatgacg
1680 tctaagccta gtgctctagc tcaagagacc tctcttggtg cccctgagcc
tttgtctggt 1740 gaacagttgg ttggttctcc ccaggataag gcggcagagg
ctacaaatga ctatgagact 1800 cttgtaaagc gtggaaaaga actaaaagag
tgtggaaaaa tccaggaggc cctaaactgc 1860 ttagttaaag cgcttgacat
aaaaagtgca gatcctgaag ttatgctctt gactttaagt 1920 ttgtataagc
aacttaataa caattgagaa tgtaacctgt ttattgtatt ttaaagtgaa 1980
actgaatatg agggaatttt tgttcccata attggattct ttgggaacat gaagcattca
2040 ggcttaaggc aagaaagatc tcaaaaagca acttctgccc tgcaacgccc
cccactccat 2100 agtctggtat tctgagcact agcttaatat ttcttcactt
gaatattctt atattttagg 2160 catattctat aaatttaact gtgttgtttc
ttggaaagtt ttgtaaaatt attctggtca 2220 ttcttaattt tactctgaaa
gtgatcatct ttgtatataa cagttcagat aagaaaatta 2280 aagttacttt
tctcaaaaaa aaaaaaaaaa aa 2312 9 419 PRT Homo Sapiens 9 Met Glu Lys
Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr 1 5 10 15 Leu
Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu 20 25
30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro
35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys
Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn
Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile Glu Ile Ala Asp Asp Leu
Ser Ala Ser His Ser Ala 85 90 95 Leu Gln Asp Ala Gln Ala Ser Glu
Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110 Ala Ser Ser Pro Gln Tyr
Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115 120 125 Ser Ala Asp Asn
Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val 130 135 140 Glu Lys
Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Lys Ala Gly 145 150 155
160 Phe Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp
165 170 175 Asp Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser
Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp
Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser
Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys Gln Phe Asp Ala Ser Thr
Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235 240 Gly Arg Phe Phe Ser
Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met 245 250 255 Asn Ser Arg
Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val 260 265 270 Leu
Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu 275 280
285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser
290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu
Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr
Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln Glu Thr Ser Leu Gly Ala
Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln Leu Val Gly Ser Pro Gln
Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360 365 Tyr Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys 370 375 380 Ile Gln Glu
Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser 385 390 395 400
Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu 405
410 415 Asn Asn Asn 10 1720 DNA Homo Sapiens 10 ggcacgaggc
caccttgcaa gatggtaaag gtacaggtag tgctgactct atagctactt 60
taccaaaggg gtttggaagt gtagaagaac tttgtactaa ctcttcattg ggaatggaaa
120 aaagctttgc aactaaaaat gaagctgtac aaaaagagac attacaagag
gggcctaagc 180 aagaggcact gcaagaggat cctctggaaa gttttaatta
tgtacttagc aaatcaacca 240 aagctgatat tgggccaaat ttagatcaac
taaaggatga tgagatttta cgtcattgca 300 atccttggcc cattatttcc
ataacaaatg aaagtcaaaa tgcagaatca aatgtatcca 360 ttattgaaat
agctgatgac ctttcagcat cccatagtgc actgcaggat gctcaagcaa 420
gtgaggccaa gttggaagag gaaccttcag catcttcacc acagtatgca tgtgatttca
480 atcttttctt ggaagactca gcagacaaca gacaaaattt ttccagtcag
tctttagagc 540 atgttgagaa agaaaatagc ttgtgtggct ctgcacctaa
ttccagagca gggtttgtgc 600 atagcaaaac atgtctcagt tgggagtttt
ctgagaaaga cgatgaacca gaagaagtag 660 tagttaaagc aaaaatcaga
agtaaagcta gaaggattgt ttcagatggc gaagatgaag 720 atgattcttt
taaagatacc tcaagcataa atccattcaa cacatctctc tttcaattct 780
catctgtgaa acaatttgat gcttcaactc ccaaaaatga catcagtcca ccaggaaggt
840 tcttttcatc tcaaataccc agtagtgtaa ataagtctat gaactctaga
agatctctgg 900 cttctaggag gtctcttatt aatatggttt tagaccacgt
ggaggacatg gaggaaagac 960 ttgacgacag cagtgaagca aagggtcctg
aagattatcc agaagaaggg gtggaggaaa 1020 gcagtggcga agcctccaag
tatacagaag aggatccttc cggagaaaca ctgtcttcag 1080 aaaacaagtc
cagctggtta atgacgtcta agcctagtgc tctagctcaa gagacctctc 1140
ttggtgcccc tgagcctttg tctggtgaac agttggttgg ttctccccag gataaggcgg
1200 cagaggctac aaatgactat gagactcttg taaagcgtgg aaaagaacta
aaagagtgtg 1260 gaaaaatcca ggaggcccta aactgcttag ttaaagcgct
tgacataaaa agtgcagatc 1320 ctgaagttat gctcttgact ttaagtttgt
ataagcaact taataacaat tgagaatgta 1380 acctgtttat tgtattttaa
agtgaaactg aatatgaggg aatttttgtt cccataattg 1440 gattctttgg
gaacatgaag cattcaggct taaggcaaga aagatctcaa aaagcaactt 1500
ctgccctgca acgcccccca ctccatagtc tggtattctg agcactagct taatatttct
1560 tcacttgaat attcttatat tttaggcata ttctataaat ttaactgtgt
tgtttcttgg 1620 aaagttttgt aaaattattc tggtcattct taattttact
ctgaaagtga tcatctttgt 1680 atataacagt tcagataaga aaattaaagt
tacttttctc 1720 11 419 PRT Homo Sapiens 11 Met Glu Lys Ser Phe Ala
Thr Lys Asn Glu Ala Val Gln Lys Glu Thr 1 5 10 15 Leu Gln Glu Gly
Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu 20 25 30 Ser Phe
Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro 35 40 45
Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro 50
55 60 Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser
Asn 65 70 75 80 Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser
His Ser Ala 85 90 95 Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu
Glu Glu Glu Pro Ser 100 105 110 Ala Ser Ser Pro Gln Tyr Ala Cys Asp
Phe Asn Leu Phe Leu Glu Asp 115 120 125 Ser Ala Asp Asn Arg Gln Asn
Phe Ser Ser Gln Ser Leu Glu His Val 130 135 140 Glu Lys Glu Asn Ser
Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly 145 150 155 160 Phe Val
His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp 165 170 175
Asp Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala 180
185 190 Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys
Asp 195 200 205 Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln
Phe Ser Ser 210 215 220 Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn
Asp Ile Ser Pro Pro 225 230 235 240 Gly Arg Phe Phe Ser Ser Gln Ile
Pro Ser Ser Val Asn Lys Ser Met 245 250 255 Asn Ser Arg Arg Ser Leu
Ala Ser Arg Arg Ser Leu Ile Asn Met Val 260 265 270 Leu Asp His Val
Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu 275 280 285 Ala Lys
Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser 290 295 300
Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu 305
310 315 320 Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro
Ser Ala 325 330 335 Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro
Leu Ser Gly Glu 340 345 350 Gln Leu Val Gly Ser Pro Gln Asp Lys Ala
Ala Glu Ala Thr Asn Asp 355 360 365 Tyr Glu Thr Leu Val Lys Arg Gly
Lys Glu Leu Lys Glu Cys Gly Lys 370 375 380 Ile Gln Glu Ala Leu Asn
Cys Leu Val Lys Ala Leu Asp Ile Lys Ser 385 390 395 400 Ala Asp Pro
Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu 405 410 415 Asn
Asn Asn 12 1250 PRT Homo Sapiens 12 Met Glu Ala Ser Arg Arg Phe Pro
Glu Ala Glu Ala Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala His Tyr Leu
Arg Tyr Val Lys Glu Ala Lys Glu Ala Thr 20 25 30 Lys Asn Gly Asp
Leu Glu Glu Ala Phe Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp Ile
Phe Pro Asn Glu Lys Val Leu Ser Arg Ile Gln Lys Ile Gln 50 55 60
Glu Ala Leu Glu Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe Thr Asp 65
70 75 80 Val Cys Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu His Asn
Gln Leu 85 90 95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr
Ser Leu Tyr Arg 100 105 110 Asp Gly Arg Lys Gly Gly Ile Leu Ala Asp
Asp Met Gly Leu Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu
Ser Gly Met Phe Asp Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu
Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu
Phe Ile Lys Trp Thr Pro Gly Met Arg Val Lys Thr Phe 165 170 175 His
Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 180 185
190 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn
195 200 205 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp
Asp Tyr 210 215 220 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser
Ser Thr Lys Ser 225 230 235 240 Ala Ile Cys Ala Arg Ala Ile Pro Ala
Ser Asn Arg Leu Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn
Leu Gln Glu Leu Trp Ser Leu Phe Asp 260 265 270 Phe Ala Cys Gln Gly
Ser Leu Leu Gly Thr Leu Lys Thr Phe
Lys Met 275 280 285 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys
Asp Ala Thr Pro 290 295 300 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser
Glu Asn Leu Met Ala Ile 305 310 315 320 Ile Lys Pro Tyr Phe Leu Arg
Arg Thr Lys Glu Asp Val Gln Lys Lys 325 330 335 Lys Ser Ser Asn Pro
Glu Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 340 345 350 Asp Ala Ile
Cys Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 355 360 365 Ile
Trp Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370 375
380 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro
385 390 395 400 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His
Pro Arg Leu 405 410 415 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu
Gly Thr Phe Ser Ala 420 425 430 Gln Asp Gly Asn Glu Gly Glu Asp Ser
Pro Asp Val Asp His Ile Asp 435 440 445 Gln Val Thr Asp Asp Thr Leu
Met Glu Glu Ser Gly Lys Met Ile Phe 450 455 460 Leu Met Asp Leu Leu
Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 465 470 475 480 Val Phe
Ser Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 485 490 495
Lys Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 500
505 510 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys
Asp 515 520 525 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val
Gly Leu Thr 530 535 540 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp
Pro Ser Trp Asn Pro 545 550 555 560 Ala Thr Asp Ala Gln Ala Val Asp
Arg Val Tyr Arg Ile Gly Gln Lys 565 570 575 Glu Asn Val Val Val Tyr
Arg Leu Ile Thr Cys Gly Thr Val Glu Glu 580 585 590 Lys Ile Tyr Arg
Arg Gln Val Phe Lys Asp Ser Leu Ile Arg Gln Thr 595 600 605 Thr Gly
Glu Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 610 615 620
Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 625
630 635 640 Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile
Lys Leu 645 650 655 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile
Ala Gly Ile Ser 660 665 670 Asp His Asp Leu Met Tyr Thr Cys Asp Leu
Ser Val Lys Glu Glu Leu 675 680 685 Asp Val Val Glu Glu Ser His Tyr
Ile Gln Gln Arg Val Gln Lys Ala 690 695 700 Gln Phe Leu Val Glu Phe
Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 705 710 715 720 Gln Gln Arg
Thr Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 725 730 735 Pro
Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 740 745
750 Gln Pro Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile
755 760 765 Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys
Glu Gly 770 775 780 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val
Thr Thr Leu Gln 785 790 795 800 Asp Gly Lys Gly Thr Gly Ser Ala Asp
Ser Ile Ala Thr Leu Pro Lys 805 810 815 Gly Phe Gly Ser Val Glu Glu
Leu Cys Thr Asn Ser Ser Leu Gly Met 820 825 830 Glu Lys Ser Phe Ala
Thr Lys Asn Glu Ala Val Gln Lys Glu Thr Leu 835 840 845 Gln Glu Gly
Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 850 855 860 Phe
Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 865 870
875 880 Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro
Trp 885 890 895 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu
Ser Asn Val 900 905 910 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala
Ser His Ser Ala Leu 915 920 925 Gln Asp Ala Gln Ala Ser Glu Ala Lys
Leu Glu Glu Glu Pro Ser Ala 930 935 940 Ser Ser Pro Gln Tyr Ala Cys
Asp Phe Asn Leu Phe Leu Glu Asp Ser 945 950 955 960 Ala Asp Asn Arg
Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val Glu 965 970 975 Lys Glu
Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 980 985 990
Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 995
1000 1005 Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys
Ala Arg 1010 1015 1020 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp
Ser Phe Lys Asp Thr 1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe Asn
Thr Ser Leu Phe Gln Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe Asp
Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070 Arg
Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met Asn 1075
1080 1085 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met
Val Leu 1090 1095 1100 Asp His Val Glu Asp Met Glu Glu Arg Leu Asp
Asp Ser Ser Glu Ala 1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr Pro
Glu Glu Gly Val Glu Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser Lys
Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150 Ser
Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1155
1160 1165 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly
Glu Gln 1170 1175 1180 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu
Ala Thr Asn Asp Tyr 1185 1190 1195 1200 Glu Thr Leu Val Lys Arg Gly
Lys Glu Leu Lys Glu Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala Leu
Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230 Asp
Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1235
1240 1245 Asn Asn 1250 13 1127 PRT Homo Sapiens 13 Met Gly Leu Gly
Lys Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met 1 5 10 15 Phe Asp
Ala Ser Leu Val Asn His Val Leu Leu Ile Met Pro Thr Asn 20 25 30
Leu Ile Asn Thr Trp Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met 35
40 45 Arg Val Lys Thr Phe His Gly Pro Ser Lys Asp Glu Arg Thr Arg
Asn 50 55 60 Leu Asn Arg Ile Gln Gln Arg Asn Gly Val Ile Ile Thr
Thr Tyr Gln 65 70 75 80 Met Leu Ile Asn Asn Trp Gln Gln Leu Ser Ser
Phe Arg Gly Gln Glu 85 90 95 Phe Val Trp Asp Tyr Val Ile Leu Asp
Glu Ala His Lys Ile Lys Thr 100 105 110 Ser Ser Thr Lys Ser Ala Ile
Cys Ala Arg Ala Ile Pro Ala Ser Asn 115 120 125 Arg Leu Leu Leu Thr
Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu 130 135 140 Trp Ser Leu
Phe Asp Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu 145 150 155 160
Lys Thr Phe Lys Met Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu 165
170 175 Lys Asp Ala Thr Pro Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser
Glu 180 185 190 Asn Leu Met Ala Ile Ile Lys Pro Tyr Phe Leu Arg Arg
Thr Lys Glu 195 200 205 Asp Val Gln Lys Lys Lys Ser Ser Asn Pro Glu
Ala Arg Leu Asn Glu 210 215 220 Lys Asn Pro Asp Val Asp Ala Ile Cys
Glu Met Pro Ser Leu Ser Arg 225 230 235 240 Lys Asn Asp Leu Ile Ile
Trp Ile Arg Leu Val Pro Leu Gln Glu Glu 245 250 255 Ile Tyr Arg Lys
Phe Val Ser Leu Asp His Ile Lys Glu Leu Leu Met 260 265 270 Glu Thr
Arg Ser Pro Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys 275 280 285
Asp His Pro Arg Leu Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu 290
295 300 Gly Thr Phe Ser Ala Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro
Asp 305 310 315 320 Val Asp His Ile Asp Gln Val Thr Asp Asp Thr Leu
Met Glu Glu Ser 325 330 335 Gly Lys Met Ile Phe Leu Met Asp Leu Leu
Lys Arg Leu Arg Asp Glu 340 345 350 Gly His Gln Thr Leu Val Phe Ser
Gln Ser Arg Gln Ile Leu Asn Ile 355 360 365 Ile Glu Arg Leu Leu Lys
Asn Arg His Phe Lys Thr Leu Arg Ile Asp 370 375 380 Gly Thr Val Thr
His Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe 385 390 395 400 Gln
Gln Asn Lys Asp Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly 405 410
415 Gly Val Gly Leu Thr Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp
420 425 430 Pro Ser Trp Asn Pro Ala Thr Asp Ala Gln Ala Val Asp Arg
Val Tyr 435 440 445 Arg Ile Gly Gln Lys Glu Asn Val Val Val Tyr Arg
Leu Ile Thr Cys 450 455 460 Gly Thr Val Glu Glu Lys Ile Tyr Arg Arg
Gln Val Phe Lys Asp Ser 465 470 475 480 Leu Ile Arg Gln Thr Thr Gly
Glu Lys Lys Asn Pro Phe Arg Tyr Phe 485 490 495 Ser Lys Gln Glu Leu
Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn 500 505 510 Ser Val Thr
Gln Leu Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys 515 520 525 Ser
Asp Ile Lys Leu Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly 530 535
540 Ile Ala Gly Ile Ser Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser
545 550 555 560 Val Lys Glu Glu Leu Asp Val Val Glu Glu Ser His Tyr
Ile Gln Gln 565 570 575 Arg Val Gln Lys Ala Gln Phe Leu Val Glu Phe
Glu Ser Gln Asn Lys 580 585 590 Glu Phe Leu Met Glu Gln Gln Arg Thr
Arg Asn Glu Gly Ala Trp Leu 595 600 605 Arg Glu Pro Val Phe Pro Ser
Ser Thr Lys Lys Lys Cys Pro Lys Leu 610 615 620 Asn Lys Pro Gln Pro
Gln Pro Ser Pro Leu Leu Ser Thr His His Thr 625 630 635 640 Gln Glu
Glu Asp Ile Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp 645 650 655
Leu Pro Lys Glu Gly Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn 660
665 670 Val Thr Thr Leu Gln Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser
Ile 675 680 685 Ala Thr Leu Pro Lys Gly Phe Gly Ser Val Glu Glu Leu
Cys Thr Asn 690 695 700 Ser Ser Leu Gly Met Glu Lys Ser Phe Ala Thr
Lys Asn Glu Ala Val 705 710 715 720 Gln Lys Glu Thr Leu Gln Glu Gly
Pro Lys Gln Glu Ala Leu Gln Glu 725 730 735 Asp Pro Leu Glu Ser Phe
Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala 740 745 750 Asp Ile Gly Pro
Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg 755 760 765 His Cys
Asn Pro Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn 770 775 780
Ala Glu Ser Asn Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala 785
790 795 800 Ser His Ser Ala Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys
Leu Glu 805 810 815 Glu Glu Pro Ser Ala Ser Ser Pro Gln Tyr Ala Cys
Asp Phe Asn Leu 820 825 830 Phe Leu Glu Asp Ser Ala Asp Asn Arg Gln
Asn Phe Ser Ser Gln Ser 835 840 845 Leu Glu His Val Glu Lys Glu Asn
Ser Leu Cys Gly Ser Ala Pro Asn 850 855 860 Ser Arg Ala Gly Phe Val
His Ser Lys Thr Cys Leu Ser Trp Glu Phe 865 870 875 880 Ser Glu Lys
Asp Asp Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile 885 890 895 Arg
Ser Lys Ala Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp 900 905
910 Ser Phe Lys Asp Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe
915 920 925 Gln Phe Ser Ser Val Lys Gln Phe Asp Ala Ser Thr Pro Lys
Asn Asp 930 935 940 Ile Ser Pro Pro Gly Arg Phe Phe Ser Ser Gln Ile
Pro Ser Ser Val 945 950 955 960 Asn Lys Ser Met Asn Ser Arg Arg Ser
Leu Ala Ser Arg Arg Ser Leu 965 970 975 Ile Asn Met Val Leu Asp His
Val Glu Asp Met Glu Glu Arg Leu Asp 980 985 990 Asp Ser Ser Glu Ala
Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val 995 1000 1005 Glu Glu
Ser Ser Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser 1010 1015
1020 Gly Glu Thr Leu Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr
Ser 1025 1030 1035 1040 Lys Pro Ser Ala Leu Ala Gln Glu Thr Ser Leu
Gly Ala Pro Glu Pro 1045 1050 1055 Leu Ser Gly Glu Gln Leu Val Gly
Ser Pro Gln Asp Lys Ala Ala Glu 1060 1065 1070 Ala Thr Asn Asp Tyr
Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys 1075 1080 1085 Glu Cys
Gly Lys Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu 1090 1095
1100 Asp Ile Lys Ser Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser
Leu 1105 1110 1115 1120 Tyr Lys Gln Leu Asn Asn Asn 1125 14 1250
PRT Homo Sapiens 14 Met Glu Ala Ser Arg Arg Phe Pro Glu Ala Glu Ala
Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala His Tyr Leu Arg Tyr Val Lys
Glu Ala Lys Glu Ala Thr 20 25 30 Lys Asn Gly Asp Leu Glu Glu Ala
Phe Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp Ile Phe Pro Asn Glu
Lys Val Leu Ser Arg Ile Gln Lys Ile Gln 50 55 60 Glu Ala Leu Glu
Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe Thr Asp 65 70 75 80 Val Cys
Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu His Asn Gln Leu 85 90 95
Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr Ser Leu Tyr Arg 100
105 110 Asp Gly Arg Lys Gly Gly Ile Leu Ala Asp Asp Met Gly Leu Gly
Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met Phe Asp
Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu Ile Met Pro Thr Asn
Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu Phe Ile Lys Trp Thr
Pro Gly Met Gly Val Lys Thr Phe 165 170 175 His Gly Pro Ser Lys Asp
Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 180 185 190 Gln Arg Asn Gly
Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn 195 200 205 Trp Gln
Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp Asp Tyr 210 215 220
Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser Ser Thr Lys Ser 225
230 235 240 Ala Ile Cys Ala Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu
Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu Trp
Ser Leu Phe Asp 260 265 270 Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr
Leu Lys Thr Phe Lys Met 275 280 285 Glu Tyr Glu Asn Pro Ile Thr Arg
Ala Arg Glu Lys Asp Ala Thr Pro 290 295 300 Gly Glu Lys Ala Leu Gly
Phe Lys Ile Ser Glu Asn Leu Met Ala Ile 305 310 315 320 Ile Lys Pro
Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val Gln Lys Lys 325 330 335 Lys
Ser Ser Asn
Pro Glu Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 340 345 350 Asp Ala
Ile Cys Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 355 360 365
Ile Trp Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370
375 380 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser
Pro 385 390 395 400 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp
His Pro Arg Leu 405 410 415 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn
Leu Gly Thr Phe Ser Ala 420 425 430 Gln Asp Gly Asn Glu Gly Glu Asp
Ser Pro Asp Val Asp His Ile Asp 435 440 445 Gln Val Thr Asp Asp Thr
Leu Met Glu Glu Ser Gly Lys Met Ile Phe 450 455 460 Leu Met Asp Leu
Leu Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 465 470 475 480 Val
Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 485 490
495 Lys Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His
500 505 510 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn
Lys Asp 515 520 525 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly
Val Gly Leu Thr 530 535 540 Leu Thr Ala Ala Thr Arg Val Val Ile Phe
Asp Pro Ser Trp Asn Pro 545 550 555 560 Ala Thr Asp Ala Gln Ala Val
Asp Arg Val Tyr Arg Ile Gly Gln Lys 565 570 575 Glu Asn Val Val Val
Tyr Arg Leu Ile Thr Cys Gly Thr Val Glu Glu 580 585 590 Lys Ile Tyr
Arg Arg Gln Val Phe Lys Asp Ser Leu Ile Arg Gln Thr 595 600 605 Thr
Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 610 615
620 Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu
625 630 635 640 Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp
Ile Lys Leu 645 650 655 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly
Ile Ala Gly Ile Ser 660 665 670 Asp His Asp Leu Met Tyr Thr Cys Asp
Leu Ser Val Lys Glu Glu Leu 675 680 685 Asp Val Val Glu Glu Ser His
Tyr Ile Gln Gln Arg Val Gln Lys Ala 690 695 700 Gln Phe Leu Val Glu
Phe Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 705 710 715 720 Gln Gln
Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 725 730 735
Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 740
745 750 Gln Pro Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp
Ile 755 760 765 Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro
Lys Glu Gly 770 775 780 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn
Val Thr Thr Leu Gln 785 790 795 800 Asp Gly Lys Gly Thr Gly Ser Ala
Asp Ser Ile Ala Thr Leu Pro Lys 805 810 815 Gly Phe Gly Ser Val Glu
Glu Leu Cys Thr Asn Ser Ser Leu Gly Met 820 825 830 Glu Lys Ser Phe
Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr Leu 835 840 845 Gln Glu
Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 850 855 860
Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 865
870 875 880 Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn
Pro Trp 885 890 895 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala
Glu Ser Asn Val 900 905 910 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser
Ala Ser His Ser Ala Leu 915 920 925 Gln Asp Ala Gln Ala Ser Glu Ala
Lys Leu Glu Glu Glu Pro Ser Ala 930 935 940 Ser Ser Pro Gln Tyr Ala
Cys Asp Phe Asn Leu Phe Leu Glu Asp Ser 945 950 955 960 Ala Asp Asn
Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val Glu 965 970 975 Lys
Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 980 985
990 Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp
995 1000 1005 Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser
Lys Ala Arg 1010 1015 1020 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp
Asp Ser Phe Lys Asp Thr 1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe
Asn Thr Ser Leu Phe Gln Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe
Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070
Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met Asn
1075 1080 1085 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn
Met Val Leu 1090 1095 1100 Asp His Val Glu Asp Met Glu Glu Arg Leu
Asp Asp Ser Ser Glu Ala 1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr
Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser
Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150
Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu
1155 1160 1165 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser
Gly Glu Gln 1170 1175 1180 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala
Glu Ala Thr Asn Asp Tyr 1185 1190 1195 1200 Glu Thr Leu Val Lys Arg
Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala
Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230
Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn
1235 1240 1245 Asn Asn 1250 15 1250 PRT Homo Sapiens 15 Met Glu Ala
Ser Arg Arg Phe Pro Glu Ala Glu Ala Leu Ser Pro Glu 1 5 10 15 Gln
Ala Ala His Tyr Leu Arg Tyr Val Lys Glu Ala Lys Glu Ala Thr 20 25
30 Lys Asn Gly Asp Leu Glu Glu Ala Phe Lys Leu Phe Asn Leu Ala Lys
35 40 45 Asp Ile Phe Pro Asn Glu Lys Val Leu Ser Arg Ile Gln Lys
Ile Gln 50 55 60 Glu Ala Leu Glu Glu Leu Ala Glu Gln Gly Asp Asp
Glu Phe Thr Asp 65 70 75 80 Val Cys Asn Ser Gly Leu Leu Leu Tyr Arg
Glu Leu His Asn Gln Leu 85 90 95 Phe Glu His Gln Lys Glu Gly Ile
Ala Phe Leu Tyr Ser Leu Tyr Arg 100 105 110 Asp Gly Arg Lys Gly Gly
Ile Leu Ala Asp Asp Met Gly Leu Gly Lys 115 120 125 Thr Val Gln Ile
Ile Ala Phe Leu Ser Gly Met Phe Asp Ala Ser Leu 130 135 140 Val Asn
His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 145 150 155
160 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Arg Val Lys Thr Phe
165 170 175 His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg
Ile Gln 180 185 190 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met
Leu Ile Asn Asn 195 200 205 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln
Glu Phe Val Trp Asp Tyr 210 215 220 Val Ile Leu Asp Glu Ala His Lys
Ile Lys Thr Ser Ser Thr Lys Ser 225 230 235 240 Ala Ile Cys Ala Arg
Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 245 250 255 Gly Thr Pro
Ile Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 260 265 270 Phe
Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 275 280
285 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro
290 295 300 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met
Ala Ile 305 310 315 320 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu
Asp Val Gln Lys Lys 325 330 335 Lys Ser Ser Asn Pro Glu Ala Arg Leu
Asn Glu Lys Asn Pro Asp Val 340 345 350 Asp Ala Ile Cys Glu Met Pro
Ser Leu Ser Arg Arg Asn Asp Leu Ile 355 360 365 Ile Trp Ile Arg Leu
Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370 375 380 Val Ser Leu
Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 385 390 395 400
Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 405
410 415 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser
Ala 420 425 430 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp
His Ile Asp 435 440 445 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser
Gly Lys Met Ile Phe 450 455 460 Leu Met Asp Leu Leu Lys Arg Leu Arg
Asp Glu Gly His Gln Thr Leu 465 470 475 480 Val Phe Ser Gln Ser Arg
Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 485 490 495 Lys Asn Arg His
Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 500 505 510 Leu Leu
Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 515 520 525
Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 530
535 540 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn
Pro 545 550 555 560 Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg
Ile Gly Gln Lys 565 570 575 Glu Asn Val Val Val Tyr Arg Leu Ile Thr
Cys Gly Thr Val Glu Glu 580 585 590 Lys Ile Tyr Arg Arg Gln Val Phe
Lys Asp Ser Leu Ile Arg Gln Thr 595 600 605 Thr Gly Glu Lys Lys Asn
Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 610 615 620 Arg Glu Leu Phe
Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 625 630 635 640 Gln
Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 645 650
655 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser
660 665 670 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys Glu
Glu Leu 675 680 685 Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln Arg
Val Gln Lys Ala 690 695 700 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn
Lys Glu Phe Leu Met Glu 705 710 715 720 Gln Gln Arg Thr Arg Asn Glu
Gly Ala Trp Leu Arg Glu Pro Val Phe 725 730 735 Pro Ser Ser Thr Lys
Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 740 745 750 Gln Pro Ser
Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 755 760 765 Ser
Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly 770 775
780 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln
785 790 795 800 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr
Leu Pro Lys 805 810 815 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn
Ser Ser Leu Gly Met 820 825 830 Glu Lys Ser Phe Ala Thr Lys Asn Glu
Ala Val Gln Lys Glu Thr Leu 835 840 845 Gln Glu Gly Pro Lys Gln Glu
Ala Leu Gln Glu Asp Pro Leu Glu Ser 850 855 860 Phe Asn Tyr Val Leu
Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 865 870 875 880 Leu Asp
Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp 885 890 895
Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 900
905 910 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala
Leu 915 920 925 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu
Pro Ser Ala 930 935 940 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu
Phe Leu Glu Asp Ser 945 950 955 960 Ala Asp Asn Arg Gln Asn Phe Ser
Ser Gln Ser Leu Glu His Val Glu 965 970 975 Lys Glu Asn Ser Leu Cys
Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 980 985 990 Val His Ser Lys
Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 995 1000 1005 Glu
Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 1010
1015 1020 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys
Asp Thr 1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu
Phe Gln Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe Asp Ala Ser Thr
Pro Lys Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070 Arg Phe Phe Ser
Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met Asn 1075 1080 1085 Ser
Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val Leu 1090
1095 1100 Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser
Glu Ala 1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly
Val Glu Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser Lys Tyr Thr Glu
Glu Asp Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150 Ser Glu Asn Lys
Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1155 1160 1165 Ala
Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln 1170
1175 1180 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp Tyr 1185 1190 1195 1200 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu
Lys Glu Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala Leu Asn Cys Leu
Val Lys Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230 Asp Pro Glu Val
Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1235 1240 1245 Asn
Asn 1250 16 1250 PRT Homo Sapiens 16 Met Glu Ala Ser Arg Arg Phe
Pro Glu Ala Glu Ala Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala His Tyr
Leu Arg Tyr Val Lys Glu Ala Lys Glu Ala Thr 20 25 30 Lys Asn Gly
Asp Leu Glu Glu Ala Phe Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp
Ile Phe Pro Asn Glu Lys Val Leu Ser Arg Ile Gln Lys Ile Gln 50 55
60 Glu Ala Leu Glu Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe Thr Asp
65 70 75 80 Val Cys Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu His Asn
Gln Leu 85 90 95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr
Ser Leu Tyr Arg 100 105 110 Asp Gly Arg Lys Gly Gly Ile Leu Ala Asp
Asp Met Gly Leu Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu
Ser Gly Met Phe Asp Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu
Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu
Phe Ile Lys Trp Thr Pro Gly Met Arg Val Lys Thr Phe 165 170 175 His
Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 180 185
190 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn
195 200 205 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp
Asp Tyr 210 215 220 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser
Ser Thr Lys Ser 225 230 235 240 Ala Ile Cys Ala Arg Ala Ile Pro Ala
Ser Asn Arg Leu Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn
Leu Gln Glu Leu Trp Ser Leu Phe Asp 260 265
270 Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met
275 280 285 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala
Thr Pro 290 295 300 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn
Leu Met Ala Ile 305 310 315 320 Ile Lys Pro Tyr Phe Leu Arg Arg Thr
Lys Glu Asp Val Gln Lys Lys 325 330 335 Lys Ser Ser Asn Pro Glu Ala
Arg Leu Asn Glu Lys Asn Pro Asp Val 340 345 350 Asp Ala Ile Cys Glu
Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 355 360 365 Ile Trp Ile
Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370 375 380 Val
Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 385 390
395 400 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg
Leu 405 410 415 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr
Phe Ser Ala 420 425 430 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp
Val Asp His Ile Asp 435 440 445 Gln Val Thr Asp Asp Thr Leu Met Glu
Glu Ser Gly Lys Met Ile Phe 450 455 460 Leu Met Asp Leu Leu Lys Arg
Leu Arg Asp Glu Gly His Gln Thr Leu 465 470 475 480 Val Phe Ser Gln
Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 485 490 495 Lys Asn
Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 500 505 510
Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 515
520 525 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu
Thr 530 535 540 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser
Trp Asn Pro 545 550 555 560 Ala Thr Asp Ala Gln Ala Val Asp Arg Val
Tyr Arg Ile Gly Gln Lys 565 570 575 Glu Asn Val Val Val Tyr Arg Leu
Ile Thr Cys Gly Thr Val Glu Glu 580 585 590 Lys Ile Tyr Arg Arg Gln
Val Phe Lys Asp Ser Leu Ile Arg Gln Thr 595 600 605 Thr Gly Glu Lys
Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 610 615 620 Arg Glu
Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 625 630 635
640 Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu
645 650 655 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly
Ile Ser 660 665 670 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser Val
Lys Glu Glu Leu 675 680 685 Asp Val Val Glu Glu Ser His Tyr Ile Gln
Gln Arg Val Gln Lys Ala 690 695 700 Gln Phe Leu Val Glu Phe Glu Ser
Gln Asn Lys Glu Phe Leu Met Glu 705 710 715 720 Gln Gln Arg Thr Arg
Asn Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 725 730 735 Pro Ser Ser
Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 740 745 750 Gln
Pro Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 755 760
765 Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly
770 775 780 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr
Leu Gln 785 790 795 800 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile
Ala Thr Leu Pro Lys 805 810 815 Gly Phe Gly Ser Val Glu Glu Leu Cys
Thr Asn Ser Ser Leu Gly Met 820 825 830 Glu Lys Ser Phe Ala Thr Lys
Asn Glu Ala Val Gln Lys Glu Thr Leu 835 840 845 Gln Glu Gly Pro Lys
Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 850 855 860 Phe Asn Tyr
Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 865 870 875 880
Leu Asp Gln Leu Lys Asp Asp Glu Val Leu Arg His Cys Asn Pro Trp 885
890 895 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn
Val 900 905 910 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His
Ser Ala Leu 915 920 925 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu
Glu Glu Pro Ser Ala 930 935 940 Ser Ser Pro Gln Tyr Ala Cys Asp Phe
Asn Leu Phe Leu Glu Asp Ser 945 950 955 960 Ala Asp Asn Arg Gln Asn
Phe Ser Ser Gln Ser Leu Glu His Val Glu 965 970 975 Lys Glu Asn Ser
Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 980 985 990 Val His
Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 995 1000
1005 Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala
Arg 1010 1015 1020 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser
Phe Lys Asp Thr 1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe Asn Thr
Ser Leu Phe Gln Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe Asp Ala
Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070 Arg Phe
Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met Asn 1075 1080
1085 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val
Leu 1090 1095 1100 Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp
Ser Ser Glu Ala 1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr Pro Glu
Glu Gly Val Glu Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser Lys Tyr
Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150 Ser Glu
Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1155 1160
1165 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu
Gln 1170 1175 1180 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala
Thr Asn Asp Tyr 1185 1190 1195 1200 Glu Thr Leu Val Lys Arg Gly Lys
Glu Leu Lys Glu Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala Leu Asn
Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230 Asp Pro
Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1235 1240
1245 Asn Asn 1250 17 1106 PRT Homo Sapiens 17 Met Asn His Val Leu
Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 1 5 10 15 Val Lys Glu
Phe Ile Lys Trp Thr Pro Gly Met Gly Val Lys Thr Phe 20 25 30 His
Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 35 40
45 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn
50 55 60 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp
Asp Tyr 65 70 75 80 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser
Ser Thr Lys Ser 85 90 95 Ala Ile Cys Ala Arg Ala Ile Pro Ala Ser
Asn Arg Leu Leu Leu Thr 100 105 110 Gly Thr Pro Ile Gln Asn Asn Leu
Gln Glu Leu Trp Ser Leu Phe Asp 115 120 125 Phe Ala Cys Gln Gly Ser
Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 130 135 140 Glu Tyr Glu Asn
Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro 145 150 155 160 Gly
Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met Ala Ile 165 170
175 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val Gln Lys Lys
180 185 190 Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn Glu Lys Asn Pro
Asp Val 195 200 205 Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg Lys
Asn Asp Leu Ile 210 215 220 Ile Trp Ile Arg Leu Val Pro Leu Gln Glu
Glu Ile Tyr Arg Lys Phe 225 230 235 240 Val Ser Leu Asp His Ile Lys
Glu Leu Leu Met Glu Thr Arg Ser Pro 245 250 255 Leu Ala Glu Leu Gly
Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 260 265 270 Leu Ser Ala
Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala 275 280 285 Gln
Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp His Ile Asp 290 295
300 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser Gly Lys Met Ile Phe
305 310 315 320 Leu Met Asp Leu Leu Lys Arg Leu Arg Asp Glu Gly His
Gln Thr Leu 325 330 335 Val Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile
Ile Glu Arg Leu Leu 340 345 350 Lys Asn Arg His Phe Lys Thr Leu Arg
Ile Asp Gly Thr Val Thr His 355 360 365 Leu Leu Glu Arg Glu Lys Arg
Ile Asn Leu Phe Gln Gln Asn Lys Asp 370 375 380 Tyr Ser Val Phe Leu
Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 385 390 395 400 Leu Thr
Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn Pro 405 410 415
Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly Gln Lys 420
425 430 Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly Thr Val Glu
Glu 435 440 445 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu Ile
Arg Gln Thr 450 455 460 Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe
Ser Lys Gln Glu Leu 465 470 475 480 Arg Glu Leu Phe Thr Ile Glu Asp
Leu Gln Asn Ser Val Thr Gln Leu 485 490 495 Gln Leu Gln Ser Leu His
Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 500 505 510 Asp Glu His Ile
Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 515 520 525 Asp His
Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu 530 535 540
Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln Lys Ala 545
550 555 560 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu Phe Leu
Met Glu 565 570 575 Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg
Glu Pro Val Phe 580 585 590 Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys
Leu Asn Lys Pro Gln Pro 595 600 605 Gln Pro Ser Pro Leu Leu Ser Thr
His His Thr Gln Glu Glu Asp Ile 610 615 620 Ser Ser Lys Met Ala Ser
Val Val Ile Asp Asp Leu Pro Lys Glu Gly 625 630 635 640 Glu Lys Gln
Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln 645 650 655 Asp
Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro Lys 660 665
670 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser Leu Gly Met
675 680 685 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu
Thr Leu 690 695 700 Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp
Pro Leu Glu Ser 705 710 715 720 Phe Asn Tyr Val Leu Ser Lys Ser Thr
Lys Ala Asp Ile Gly Pro Asn 725 730 735 Leu Asp Gln Leu Lys Asp Asp
Glu Val Leu Arg His Cys Asn Pro Trp 740 745 750 Pro Ile Ile Ser Ile
Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 755 760 765 Ser Ile Ile
Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 770 775 780 Gln
Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser Ala 785 790
795 800 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp
Ser 805 810 815 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu
His Val Glu 820 825 830 Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn
Ser Arg Ala Gly Phe 835 840 845 Val His Ser Lys Thr Cys Leu Ser Trp
Glu Phe Ser Glu Lys Asp Asp 850 855 860 Glu Pro Glu Glu Val Val Val
Lys Ala Lys Ile Arg Ser Lys Ala Arg 865 870 875 880 Arg Ile Val Ser
Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 885 890 895 Ser Ser
Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val 900 905 910
Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro Gly 915
920 925 Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met
Asn 930 935 940 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn
Met Val Leu 945 950 955 960 Asp His Val Glu Asp Met Glu Glu Arg Leu
Asp Asp Ser Ser Glu Ala 965 970 975 Lys Gly Pro Glu Asp Tyr Pro Glu
Glu Gly Val Glu Glu Ser Ser Gly 980 985 990 Glu Ala Ser Lys Tyr Thr
Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 995 1000 1005 Ser Glu Asn
Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1010 1015 1020
Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln
1025 1030 1035 1040 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala
Thr Asn Asp Tyr 1045 1050 1055 Glu Thr Leu Val Lys Arg Gly Lys Glu
Leu Lys Glu Cys Gly Lys Ile 1060 1065 1070 Gln Glu Ala Leu Asn Cys
Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1075 1080 1085 Asp Pro Glu
Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1090 1095 1100
Asn Asn 1105 18 419 PRT Homo Sapiens 18 Met Glu Lys Ser Phe Ala Thr
Lys Asn Glu Ala Val Gln Lys Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro
Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu 20 25 30 Ser Phe Asn
Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro 35 40 45 Asn
Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro 50 55
60 Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn
65 70 75 80 Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His
Ser Ala 85 90 95 Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu
Glu Glu Pro Ser 100 105 110 Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe
Asn Leu Phe Leu Glu Asp 115 120 125 Ser Ala Asp Asn Arg Gln Asn Phe
Ser Ser Gln Ser Leu Glu His Val 130 135 140 Glu Lys Glu Asn Ser Leu
Cys Gly Ser Ala Pro Asn Ser Lys Ala Gly 145 150 155 160 Phe Val His
Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp 165 170 175 Asp
Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala 180 185
190 Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp
195 200 205 Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe
Ser Ser 210 215 220 Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp
Ile Ser Pro Pro 225 230 235 240 Gly Arg Phe Phe Ser Ser Gln Ile Pro
Ser Ser Val Asn Lys Ser Met 245 250 255 Asn Ser Arg Arg Ser Leu Ala
Ser Arg Arg Ser Leu Ile Asn Met Val 260 265 270 Leu Asp His Val Glu
Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu 275 280 285 Ala Lys Gly
Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser 290 295 300 Gly
Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu 305 310
315 320 Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser
Ala 325 330 335 Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu
Ser Gly Glu 340 345 350
Gln Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp 355
360 365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly
Lys 370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp
Ile Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu Thr Leu
Ser Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 19 419 PRT Homo
Sapiens 19 Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys
Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu
Asp Pro Leu Glu 20 25 30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr
Lys Ala Asp Ile Gly Pro 35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp
Glu Ile Leu Arg His Cys Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile
Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile
Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala 85 90 95 Leu Gln
Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110
Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115
120 125 Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His
Val 130 135 140 Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser
Arg Ala Gly 145 150 155 160 Phe Val His Ser Lys Thr Cys Leu Ser Trp
Glu Phe Ser Glu Lys Asp 165 170 175 Asp Glu Pro Glu Glu Val Val Val
Lys Ala Lys Ile Arg Ser Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp
Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile
Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys
Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235
240 Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met
245 250 255 Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn
Met Val 260 265 270 Leu Asp His Val Glu Asp Met Glu Glu Arg Leu Asp
Asp Ser Ser Glu 275 280 285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu
Gly Val Glu Glu Ser Ser 290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu
Glu Asp Pro Ser Gly Glu Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys
Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln
Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln
Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360
365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys
370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile
Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser
Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 20 1106 PRT Homo
Sapiens 20 Val Asn His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn
Thr Trp 1 5 10 15 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Arg
Val Lys Thr Phe 20 25 30 His Gly Pro Ser Lys Asp Glu Arg Thr Arg
Asn Leu Asn Arg Ile Gln 35 40 45 Gln Arg Asn Gly Val Ile Ile Thr
Thr Tyr Gln Met Leu Ile Asn Asn 50 55 60 Trp Gln Gln Leu Ser Ser
Phe Arg Gly Gln Glu Phe Val Trp Asp Tyr 65 70 75 80 Val Ile Leu Asp
Glu Ala His Lys Ile Lys Thr Ser Ser Thr Lys Ser 85 90 95 Ala Ile
Cys Ala Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 100 105 110
Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 115
120 125 Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys
Met 130 135 140 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp
Ala Thr Pro 145 150 155 160 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser
Glu Asn Leu Met Ala Ile 165 170 175 Ile Lys Pro Tyr Phe Leu Arg Arg
Thr Lys Glu Asp Val Gln Lys Lys 180 185 190 Lys Ser Ser Asn Pro Glu
Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 195 200 205 Asp Ala Ile Cys
Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 210 215 220 Ile Trp
Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 225 230 235
240 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro
245 250 255 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro
Arg Leu 260 265 270 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly
Thr Phe Ser Ala 275 280 285 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro
Asp Val Asp His Ile Asp 290 295 300 Gln Val Thr Asp Asp Thr Leu Met
Glu Glu Ser Gly Lys Met Ile Phe 305 310 315 320 Leu Met Asp Leu Leu
Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 325 330 335 Val Phe Ser
Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 340 345 350 Lys
Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 355 360
365 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp
370 375 380 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly
Leu Thr 385 390 395 400 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp
Pro Ser Trp Asn Pro 405 410 415 Ala Thr Asp Ala Gln Ala Val Asp Arg
Val Tyr Arg Ile Gly Gln Lys 420 425 430 Glu Asn Val Val Val Tyr Arg
Leu Ile Thr Cys Gly Thr Val Glu Glu 435 440 445 Lys Ile Tyr Arg Arg
Gln Val Phe Lys Asp Ser Leu Ile Arg Gln Thr 450 455 460 Thr Gly Glu
Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 465 470 475 480
Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 485
490 495 Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys
Leu 500 505 510 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala
Gly Ile Ser 515 520 525 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser
Val Lys Glu Glu Leu 530 535 540 Asp Val Val Glu Glu Ser His Tyr Ile
Gln Gln Arg Val Gln Lys Ala 545 550 555 560 Gln Phe Leu Val Glu Phe
Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 565 570 575 Gln Gln Arg Thr
Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 580 585 590 Pro Ser
Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 595 600 605
Gln Pro Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 610
615 620 Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu
Gly 625 630 635 640 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val
Thr Thr Leu Gln 645 650 655 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser
Ile Ala Thr Leu Pro Lys 660 665 670 Gly Phe Gly Ser Val Glu Glu Leu
Cys Thr Asn Ser Ser Leu Gly Met 675 680 685 Glu Lys Ser Phe Ala Thr
Lys Asn Glu Ala Val Gln Lys Glu Thr Leu 690 695 700 Gln Glu Gly Pro
Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 705 710 715 720 Phe
Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 725 730
735 Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp
740 745 750 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser
Asn Val 755 760 765 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser
His Ser Ala Leu 770 775 780 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu
Glu Glu Glu Pro Ser Ala 785 790 795 800 Ser Ser Pro Gln Tyr Ala Cys
Asp Phe Asn Leu Phe Leu Glu Asp Ser 805 810 815 Ala Asp Asn Arg Gln
Asn Phe Ser Ser Gln Ser Leu Glu His Val Glu 820 825 830 Lys Glu Asn
Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 835 840 845 Val
His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 850 855
860 Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg
865 870 875 880 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe
Lys Asp Thr 885 890 895 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe
Gln Phe Ser Ser Val 900 905 910 Lys Gln Phe Asp Ala Ser Thr Pro Lys
Asn Asp Ile Ser Pro Pro Gly 915 920 925 Arg Phe Phe Ser Ser Gln Ile
Pro Ser Ser Val Asn Lys Ser Met Asn 930 935 940 Ser Arg Arg Ser Leu
Ala Ser Arg Arg Ser Leu Ile Asn Met Val Leu 945 950 955 960 Asp His
Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 965 970 975
Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 980
985 990 Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu
Ser 995 1000 1005 Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys
Pro Ser Ala Leu 1010 1015 1020 Ala Gln Glu Thr Ser Leu Gly Ala Pro
Glu Pro Leu Ser Gly Glu Gln 1025 1030 1035 1040 Leu Val Gly Ser Pro
Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr 1045 1050 1055 Glu Thr
Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1060 1065
1070 Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser
Ala 1075 1080 1085 Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr
Lys Gln Leu Asn 1090 1095 1100 Asn Asn 1105 21 1106 PRT Homo
Sapiens 21 Met Asn His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn
Thr Trp 1 5 10 15 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Gly
Val Lys Thr Phe 20 25 30 His Gly Pro Ser Lys Asp Glu Arg Thr Arg
Asn Leu Asn Arg Ile Gln 35 40 45 Gln Arg Asn Gly Val Ile Ile Thr
Thr Tyr Gln Met Leu Ile Asn Asn 50 55 60 Trp Gln Gln Leu Ser Ser
Phe Arg Gly Gln Glu Phe Val Trp Asp Tyr 65 70 75 80 Val Ile Leu Asp
Glu Ala His Lys Ile Lys Thr Ser Ser Thr Lys Ser 85 90 95 Ala Ile
Cys Ala Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 100 105 110
Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 115
120 125 Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys
Met 130 135 140 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp
Ala Thr Pro 145 150 155 160 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser
Glu Asn Leu Met Ala Ile 165 170 175 Ile Lys Pro Tyr Phe Leu Arg Arg
Thr Lys Glu Asp Val Gln Lys Lys 180 185 190 Lys Ser Ser Asn Pro Glu
Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 195 200 205 Asp Ala Ile Cys
Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 210 215 220 Ile Trp
Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 225 230 235
240 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro
245 250 255 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro
Arg Leu 260 265 270 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly
Thr Phe Ser Ala 275 280 285 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro
Asp Val Asp His Ile Asp 290 295 300 Gln Val Thr Asp Asp Thr Leu Met
Glu Glu Ser Gly Lys Met Ile Phe 305 310 315 320 Leu Met Asp Leu Leu
Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 325 330 335 Val Phe Ser
Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 340 345 350 Lys
Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 355 360
365 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp
370 375 380 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly
Leu Thr 385 390 395 400 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp
Pro Ser Trp Asn Pro 405 410 415 Ala Thr Asp Ala Gln Ala Val Asp Arg
Val Tyr Arg Ile Gly Gln Lys 420 425 430 Glu Asn Val Val Val Tyr Arg
Leu Ile Thr Cys Gly Thr Val Glu Glu 435 440 445 Lys Ile Tyr Arg Arg
Gln Val Phe Lys Asp Ser Leu Ile Arg Gln Thr 450 455 460 Thr Gly Glu
Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 465 470 475 480
Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 485
490 495 Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys
Leu 500 505 510 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala
Gly Ile Ser 515 520 525 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser
Val Lys Glu Glu Leu 530 535 540 Asp Val Val Glu Glu Ser His Tyr Ile
Gln Gln Arg Val Gln Lys Ala 545 550 555 560 Gln Phe Leu Val Glu Phe
Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 565 570 575 Gln Gln Arg Thr
Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 580 585 590 Pro Ser
Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 595 600 605
Gln Pro Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 610
615 620 Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu
Gly 625 630 635 640 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val
Thr Thr Leu Gln 645 650 655 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser
Ile Ala Thr Leu Pro Lys 660 665 670 Gly Phe Gly Ser Val Glu Glu Leu
Cys Thr Asn Ser Ser Leu Gly Met 675 680 685 Glu Lys Ser Phe Ala Thr
Lys Asn Glu Ala Val Gln Lys Glu Thr Leu 690 695 700 Gln Glu Gly Pro
Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 705 710 715 720 Phe
Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 725 730
735 Leu Asp Gln Leu Lys Asp Asp Glu Val Leu Arg His Cys Asn Pro Trp
740 745 750 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser
Asn Val 755 760 765 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser
His Ser Ala Leu 770 775 780 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu
Glu Glu Glu Pro Ser Ala 785 790 795 800 Ser Ser Pro Gln Tyr Ala Cys
Asp Phe Asn Leu Phe Leu Glu Asp Ser 805 810 815 Ala Asp Asn Arg Gln
Asn Phe Ser Ser Gln Ser Leu Glu His Val Glu 820 825 830 Lys Glu Asn
Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 835 840 845 Val
His Ser
Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 850 855 860 Glu
Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 865 870
875 880 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp
Thr 885 890 895 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe
Ser Ser Val 900 905 910 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp
Ile Ser Pro Pro Gly 915 920 925 Arg Phe Phe Ser Ser Gln Ile Pro Ser
Ser Val Asn Lys Ser Met Asn 930 935 940 Ser Arg Arg Ser Leu Ala Ser
Arg Arg Ser Leu Ile Asn Met Val Leu 945 950 955 960 Asp His Val Glu
Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 965 970 975 Lys Gly
Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 980 985 990
Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 995
1000 1005 Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser
Ala Leu 1010 1015 1020 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro
Leu Ser Gly Glu Gln 1025 1030 1035 1040 Leu Val Gly Ser Pro Gln Asp
Lys Ala Ala Glu Ala Thr Asn Asp Tyr 1045 1050 1055 Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1060 1065 1070 Gln
Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1075
1080 1085 Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln
Leu Asn 1090 1095 1100 Asn Asn 1105 22 419 PRT Homo Sapiens 22 Met
Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr 1 5 10
15 Leu Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu
20 25 30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile
Gly Pro 35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg
His Cys Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser
Gln Asn Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile Glu Ile Ala Asp
Asp Leu Ser Ala Ser His Ser Ala 85 90 95 Leu Gln Asp Ala Gln Ala
Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110 Ala Ser Ser Pro
Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115 120 125 Ser Ala
Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val 130 135 140
Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly 145
150 155 160 Phe Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu
Lys Asp 165 170 175 Asp Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile
Arg Ser Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp Gly Glu Asp Glu
Asp Asp Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile Asn Pro Phe Asn
Thr Ser Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys Gln Phe Asp Ala
Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235 240 Gly Arg Phe
Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met 245 250 255 Asn
Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val 260 265
270 Leu Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu
275 280 285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu
Ser Ser 290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser
Gly Glu Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys Ser Ser Trp Leu
Met Thr Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln Glu Thr Ser Leu
Gly Ala Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln Leu Val Gly Ser
Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360 365 Tyr Glu Thr
Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys 370 375 380 Ile
Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser 385 390
395 400 Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln
Leu 405 410 415 Asn Asn Asn 23 419 PRT Homo Sapiens 23 Met Glu Lys
Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr 1 5 10 15 Leu
Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu 20 25
30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro
35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg His Cys
Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn
Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile Glu Ile Ala Asp Asp Leu
Ser Ala Ser His Ser Ala 85 90 95 Leu Gln Asp Ala Gln Ala Ser Glu
Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110 Ala Ser Ser Pro Gln Tyr
Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115 120 125 Ser Ala Asp Asn
Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val 130 135 140 Glu Lys
Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Lys Ala Gly 145 150 155
160 Phe Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp
165 170 175 Asp Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser
Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp
Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser
Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys Gln Phe Asp Ala Ser Thr
Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235 240 Gly Arg Phe Phe Ser
Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met 245 250 255 Asn Ser Arg
Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val 260 265 270 Leu
Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu 275 280
285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser
290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu
Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr
Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln Glu Thr Ser Leu Gly Ala
Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln Leu Val Gly Ser Pro Gln
Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360 365 Tyr Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys 370 375 380 Ile Gln Glu
Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser 385 390 395 400
Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu 405
410 415 Asn Asn Asn 24 1248 PRT Homo Sapiens 24 Met Glu Ala Ser Arg
Arg Phe Pro Glu Ala Glu Ala Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala
His Tyr Leu Arg Tyr Val Lys Glu Ala Lys Glu Ala Thr 20 25 30 Lys
Asn Gly Asp Leu Glu Glu Ala Phe Lys Leu Phe Asn Leu Ala Lys 35 40
45 Asp Ile Phe Pro Asn Glu Lys Val Leu Ser Arg Ile Gln Lys Ile Gln
50 55 60 Glu Ala Leu Glu Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe
Thr Asp 65 70 75 80 Val Cys Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu
His Asn Gln Leu 85 90 95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe
Leu Tyr Ser Leu Tyr Arg 100 105 110 Asp Gly Arg Lys Gly Gly Ile Leu
Ala Asp Asp Met Gly Leu Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala
Phe Leu Ser Gly Met Phe Asp Ala Ser Leu 130 135 140 Val Asn His Val
Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 145 150 155 160 Val
Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Arg Val Lys Thr Phe 165 170
175 His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln
180 185 190 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile
Asn Asn 195 200 205 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe
Val Trp Asp Tyr 210 215 220 Val Ile Leu Asp Glu Ala His Lys Ile Lys
Thr Ser Ser Thr Lys Ser 225 230 235 240 Ala Ile Cys Ala Arg Ala Ile
Pro Ala Ser Asn Arg Leu Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln
Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 260 265 270 Phe Ala Cys
Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 275 280 285 Glu
Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro 290 295
300 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met Ala Ile
305 310 315 320 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val
Gln Lys Lys 325 330 335 Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn Glu
Lys Asn Pro Asp Val 340 345 350 Asp Ala Ile Cys Glu Met Pro Ser Leu
Ser Arg Lys Asn Asp Leu Ile 355 360 365 Ile Trp Ile Arg Leu Val Pro
Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370 375 380 Val Ser Leu Asp His
Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 385 390 395 400 Leu Ala
Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 405 410 415
Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala 420
425 430 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp His Ile
Asp 435 440 445 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser Gly Lys
Met Ile Phe 450 455 460 Leu Met Asp Leu Leu Lys Arg Leu Arg Asp Glu
Gly His Gln Thr Leu 465 470 475 480 Val Phe Ser Gln Ser Arg Gln Ile
Leu Asn Ile Ile Glu Arg Leu Leu 485 490 495 Lys Asn Arg His Phe Lys
Thr Leu Arg Ile Asp Gly Thr Val Thr His 500 505 510 Leu Leu Glu Arg
Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 515 520 525 Tyr Ser
Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 530 535 540
Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn Pro 545
550 555 560 Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly
Gln Lys 565 570 575 Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly
Thr Val Glu Glu 580 585 590 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp
Ser Leu Ile Arg Gln Thr 595 600 605 Thr Gly Glu Lys Lys Asn Pro Phe
Arg Tyr Phe Ser Lys Gln Glu Leu 610 615 620 Arg Glu Leu Phe Thr Ile
Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 625 630 635 640 Gln Leu Gln
Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 645 650 655 Asp
Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 660 665
670 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu
675 680 685 Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln
Lys Ala 690 695 700 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu
Phe Leu Met Glu 705 710 715 720 Gln Gln Arg Thr Arg Asn Glu Gly Ala
Trp Leu Arg Glu Pro Val Phe 725 730 735 Pro Ser Ser Thr Lys Lys Lys
Cys Pro Lys Leu Asn Lys Pro Gln Pro 740 745 750 Gln Pro Ser Pro Leu
Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 755 760 765 Ser Ser Lys
Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly 770 775 780 Glu
Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln 785 790
795 800 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro
Lys 805 810 815 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser
Leu Gly Met 820 825 830 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val
Gln Lys Glu Thr Leu 835 840 845 Gln Glu Gly Pro Lys Gln Glu Ala Leu
Gln Glu Asp Pro Leu Glu Ser 850 855 860 Phe Asn Tyr Val Leu Ser Lys
Ser Thr Lys Ala Asp Ile Gly Pro Asn 865 870 875 880 Leu Asp Gln Leu
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp 885 890 895 Pro Ile
Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 900 905 910
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 915
920 925 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
Ala 930 935 940 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp Ser 945 950 955 960 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln
Ser Leu Glu His Val Glu 965 970 975 Lys Glu Asn Ser Leu Cys Gly Ser
Ala Pro Asn Ser Arg Ala Gly Phe 980 985 990 Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 995 1000 1005 Glu Pro Glu
Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 1010 1015 1020
Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr
1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln
Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe Asp Ala Ser Thr Pro Lys
Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070 Arg Phe Phe Ser Ser Gln
Ile Pro Ser Ser Val Asn Lys Ser Met Asn 1075 1080 1085 Ser Arg Arg
Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val Leu 1090 1095 1100
Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala
1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu
Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser Lys Tyr Thr Glu Glu Asp
Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150 Ser Glu Asn Lys Ser Ser
Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1155 1160 1165 Ala Gln Glu
Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln 1170 1175 1180
Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr
1185 1190 1195 1200 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu
Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala Leu Asn Cys Leu Val Lys
Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230 Asp Pro Glu Val Met Leu
Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1235 1240 1245 25 1229 PRT
Homo Sapiens 25 Met Glu Ala Ser Gln Gly Leu Ala Glu Val Gly Thr Leu
Ser Pro Gln 1 5 10 15 Leu Ala Glu Ser Tyr Leu Arg Tyr Ser Gln Glu
Ala Lys Glu Ala Ala 20 25 30 Lys Asn Gly Asp Leu Glu Glu Ala Leu
Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp Ile Phe Pro Thr Lys Lys
Val Ile Ser Arg Ile Gln Lys Leu Gln 50 55 60 Glu Ala Leu Glu Gln
Leu Ala Glu Glu Glu Asp Asp Glu Phe Thr Asp 65 70 75 80 Val Cys Asn
Ser Gly Leu Leu Leu Tyr Arg Glu Leu Tyr Glu Lys Leu 85 90
95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr Ser Leu Tyr Lys
100 105 110 Asn Gly Arg Lys Gly Gly Ile Leu Ala Asp Asp Met Gly Leu
Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met Phe
Asp Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu Ile Met Pro Thr
Asn Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu Phe Ala Lys Trp
Thr Pro Gly Met Arg Val Lys Thr Phe 165 170 175 His Gly Ser Ser Lys
Asn Glu Arg Ile Arg Asn Leu Thr Arg Ile Gln 180 185 190 Gln Arg Asn
Gly Val Val Ile Thr Thr Tyr Gln Met Leu Leu Asn Asn 195 200 205 Trp
Gln Gln Leu Ala Ser Phe Asn Gly Gln Ala Phe Val Trp Asp Tyr 210 215
220 Val Ile Leu Asp Glu Ala His Lys Ile Lys Ser Ala Ser Thr Lys Ser
225 230 235 240 Ala Val Cys Ala Arg Ala Val Pro Ala Ser Asn Arg Leu
Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu
Trp Ser Leu Phe Asp 260 265 270 Phe Ala Cys Gln Gly Ser Leu Leu Gly
Thr Leu Lys Thr Phe Lys Met 275 280 285 Glu Tyr Glu Asn Pro Ile Ile
Arg Ala Arg Glu Lys Asp Ala Thr Pro 290 295 300 Gly Glu Lys Ala Leu
Gly Phe Lys Met Ser Glu Asn Leu Met Glu Ile 305 310 315 320 Ile Lys
Pro Tyr Phe Leu Arg Arg Thr Lys Glu Glu Val His Met Lys 325 330 335
Lys Ala Asp Lys Pro Glu Val Arg Pro Gly Glu Lys Asn Ser Gly Val 340
345 350 Glu Asp Ile Cys Glu Met Leu Ser Leu Thr Arg Lys Asn Asp Leu
Ile 355 360 365 Val Trp Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr
Arg Lys Phe 370 375 380 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met
Glu Thr Arg Ser Pro 385 390 395 400 Leu Ala Glu Leu Gly Val Leu Lys
Lys Leu Cys Asp His Pro Arg Leu 405 410 415 Leu Ser Ala Arg Ala Cys
His Leu Leu Asn Leu Gly Thr Val Thr Phe 420 425 430 Ser Ala Glu Asp
Glu Asn Glu Gln Glu Asp Ala Ser Asn Met Gly Ser 435 440 445 Ile Asp
His Leu Ser Asp Asn Ala Leu Met Gln Glu Ser Gly Lys Met 450 455 460
Ile Phe Leu Met Ala Leu Leu Glu Arg Leu Gln Asp Glu Gly His Gln 465
470 475 480 Thr Leu Val Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile Ile
Glu Arg 485 490 495 Leu Leu Lys Asn Lys His Phe Lys Thr Leu Arg Ile
Asp Gly Thr Val 500 505 510 Thr His Leu Trp Glu Arg Glu Lys Arg Ile
Gln Leu Phe Gln Gln Asn 515 520 525 Lys Glu Tyr Ser Val Phe Leu Leu
Thr Thr Gln Val Gly Gly Val Gly 530 535 540 Leu Thr Leu Thr Ala Ala
Ser Arg Val Val Ile Phe Asp Pro Ser Trp 545 550 555 560 Asn Pro Ala
Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly 565 570 575 Gln
Lys Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly Thr Val 580 585
590 Glu Glu Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu Ile Arg
595 600 605 Gln Thr Thr Gly Asp Lys Lys Asn Pro Phe Arg Tyr Phe Thr
Lys Gln 610 615 620 Glu Leu Lys Glu Leu Phe Thr Val Gly Asp Leu Leu
Asn Ser Ala Thr 625 630 635 640 Gln Met Gln Leu Gln Cys Leu His Ala
Ala Gln Arg Lys Ser Asp Glu 645 650 655 Lys Leu Asp Glu His Ile Ala
Tyr Leu His Ser Leu Gly Ile Ala Gly 660 665 670 Ile Ser Asp His Asp
Leu Met Phe Thr Cys Asp Leu Ser Val Lys Glu 675 680 685 Glu Leu Asp
Met Leu Glu Asp Ala Gln Tyr Ile Gln His Arg Val Gln 690 695 700 Lys
Ala Gln Phe Leu Val Glu Ser Glu Ser Gln Asn Thr Lys Glu Arg 705 710
715 720 Pro Ser Glu Glu Asn Trp Leu Lys Ala Gln Glu Phe Pro Ser Gln
Gln 725 730 735 Arg Lys Lys Gly Asn Lys Pro Gln Pro Gln Pro Ser Arg
Leu Leu Ala 740 745 750 Asn Pro Thr Gln Val Glu Ala Ile Ser Ser Gln
Met Ala Ser Ile Ser 755 760 765 Ile Tyr Asp Gln Ser Thr Glu Ser Glu
Pro Gln Glu Leu Ser Glu Ala 770 775 780 His Asp Val Thr Ser Leu Gln
Gly Asp Gln His His Phe Glu Ser Thr 785 790 795 800 Ser Asp Ala Gly
Thr Ile Ala Ser Leu Pro Gln Gly Ala Glu Ser Ile 805 810 815 Gly Gly
Val Trp Thr Asp Ser Leu Leu Ser Pro Ala Lys Gly Phe Ala 820 825 830
Ala Asp Asn Glu Ala Val Gln Lys Asn Glu Leu Gln Ala Ser Pro Gly 835
840 845 Gln Glu Ala Leu Ser Glu Asn Leu Gly Ser Phe His Tyr Leu Pro
Arg 850 855 860 Gln Ser Ser Lys Ala Asp Leu Glu Pro Asn Leu Asp Val
Gln Asp Ser 865 870 875 880 Val Val Leu Tyr His Gln Ser Pro Thr Ala
Asn Glu Asn Gln Asn Leu 885 890 895 Glu Ser Asn Glu Pro Met Ile Glu
Ile Ser Asp Asp Leu Ser Ala Ser 900 905 910 Pro Ser Ala Leu Gln Gly
Ala Gln Ala Met Glu Ala Gln Leu Glu Leu 915 920 925 Lys Lys Asp Pro
Leu Glu Ser Pro Pro Gln Tyr Glu Cys Asp Phe Asn 930 935 940 Leu Phe
Leu Glu Asp Ser Ala Asp Thr Arg Gln Asn Leu Ser Ser Lys 945 950 955
960 Phe Leu Glu His Val Glu Lys Glu Asn Ser Leu Gln Arg Pro Ala Gly
965 970 975 Asn Ser Gly Glu Glu Ser Ala His Asn Leu Ser Leu Asp Ser
Ser Asn 980 985 990 Lys Ile Asp Glu Glu Ser Glu Val Ile Thr Val Lys
Thr Lys Asn Lys 995 1000 1005 Ala Arg Arg Ile Leu Ser Asp Asp Glu
Asp Glu Glu Asp Ala Phe Lys 1010 1015 1020 Asp Thr Ser Thr Asn Ser
Phe Ser Val Ser Pro Leu Thr Phe Ser Ser 1025 1030 1035 1040 Val Lys
His Phe Asp Ala Ser Thr Pro Gln Asn Asp Ser Asn Pro Ser 1045 1050
1055 Arg Arg Phe Phe Ser Pro Lys Ile Pro Asp Asp Val Asn Thr Ser
Leu 1060 1065 1070 His Pro Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu
Ile Asn Val Val 1075 1080 1085 Leu Asp Asp Met Glu Asp Met Glu Glu
Arg Leu Asp Thr Ser Ser Glu 1090 1095 1100 Glu Glu Ser Glu Pro Glu
Leu Ser Glu Asp Ser Asn Glu Glu Ala Val 1105 1110 1115 1120 Ala Cys
Thr Glu Glu Gln Arg Ser Gly Ala Thr Leu Ala Ser Gly Asn 1125 1130
1135 Lys His Ser Ser Leu Thr Glu Ser Glu Pro Thr Ser Pro Ala Pro
Gln 1140 1145 1150 Ser Ser Pro Cys Ala Pro Glu Pro Ser Ser Ser Asp
Pro Leu Leu Asp 1155 1160 1165 Pro Pro Gln Asp Pro Ala Val Glu Ala
Ala Asn Asp Tyr Glu Ser Leu 1170 1175 1180 Val Ala Arg Gly Lys Glu
Leu Lys Glu Cys Gly Lys Thr Gln Glu Ala 1185 1190 1195 1200 Leu Asn
Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala Asp Pro Glu 1205 1210
1215 Val Met Arg Met Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1220 1225
26 14 PRT Clostridium tetani 26 Gln Tyr Ile Lys Ala Asn Ser Lys Phe
Ile Gly Ile Thr Glu 1 5 10 27 21 PRT Plasmodium falciparum 27 Asp
Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe 1 5 10
15 Asn Val Val Asn Ser 20 28 16 PRT Streptococcus aureus 28 Gly Ala
Val Asp Ser Ile Leu Gly Gly Val Ala Thr Tyr Gly Ala Ala 1 5 10 15
29 12 PRT Artificial Sequence VARIANT 3 Xaa is either
cyclohexylalanine, phenylalanine, or tyrosine VARIANT 1, 13 Xaa is
is either D-alanine or L-alanine Artificially Synthesized Peptide
29 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Xaa 1 5 10 30 14 DNA
Homo Sapiens 30 ttttgatcaa gctt 14 31 42 DNA Homo Sapiens 31
ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag 42 32 12 DNA Homo
Sapiens 32 gatcctgccc gg 12 33 40 DNA Homo Sapiens 33 gtaatacgac
tcactatagg gcagcgtggt cgcggccgag 40 34 10 DNA Homo Sapiens 34
gatcctcggc 10 35 22 DNA Homo Sapiens 35 ctaatacgac tcactatagg gc 22
36 22 DNA Homo Sapiens 36 tcgagcggcc gcccgggcag ga 22 37 20 DNA
Homo Sapiens 37 agcgtggtcg cggccgagga 20 38 25 DNA Homo Sapiens 38
atatcgccgc gctcgtcgtc gacaa 25 39 26 DNA Homo Sapiens 39 agccacacgc
agctcattgt agaagg 26 40 27 DNA Homo Sapiens 40 gctagtgctc
agaataccag actatgg 27 41 25 DNA Homo Sapiens 41 cgcttgacat
aaaaagtgca gatcc 25 42 24 DNA Homo Sapiens 42 gattacaagg atgacgacga
taag 24 43 4 PRT Homo Sapiens 43 Asn Val Thr Thr 1 44 4 PRT Homo
Sapiens 44 Asn Ser Ser Leu 1 45 4 PRT Homo Sapiens 45 Asn Glu Ser
Gln 1 46 4 PRT Homo Sapiens 46 Asn Val Ser Ile 1 47 4 PRT Homo
Sapiens 47 Asn Phe Ser Ser 1 48 4 PRT Homo Sapiens 48 Asn Thr Ser
Leu 1 49 4 PRT Homo Sapiens 49 Asn Lys Ser Met 1 50 4 PRT Homo
Sapiens 50 Asn Lys Ser Ser 1 51 15 PRT Homo Sapiens 51 Glu Ala Lys
Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu 1 5 10 15 52 15 PRT
Homo Sapiens 52 Ser Ser Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro
Ser Gly 1 5 10 15 53 19 PRT Homo Sapiens 53 Ala Ala Glu Ala Thr Asn
Asp Tyr Glu Thr Leu Val Lys Arg Gly Lys 1 5 10 15 Lys Lys Ser 54 4
PRT Homo Sapiens 54 Lys Lys Lys Ser 1 55 4 PRT Homo Sapiens 55 Lys
Lys Ser Ser 1 56 4 PRT Homo Sapiens 56 Ser Lys Asp Glu 1 57 4 PRT
Homo Sapiens 57 Ser Leu Phe Asp 1 58 4 PRT Homo Sapiens 58 Thr Pro
Gly Glu 1 59 4 PRT Homo Sapiens 59 Thr Lys Glu Asp 1 60 4 PRT Homo
Sapiens 60 Ser Asn Pro Glu 1 61 4 PRT Homo Sapiens 61 Ser Ala Gln
Asp 1 62 4 PRT Homo Sapiens 62 Thr Leu Met Glu 1 63 4 PRT Homo
Sapiens 63 Thr Val Glu Glu 1 64 4 PRT Homo Sapiens 64 Thr Thr Gly
Glu 1 65 4 PRT Homo Sapiens 65 Ser Lys Gln Glu 1 66 4 PRT Homo
Sapiens 66 Thr Ile Glu Asp 1 67 4 PRT Homo Sapiens 67 Ser Asp His
Asp 1 68 4 PRT Homo Sapiens 68 Ser Val Lys Glu 1 69 4 PRT Homo
Sapiens 69 Thr Arg Asn Glu 1 70 4 PRT Homo Sapiens 70 Thr Gln Glu
Glu 1 71 4 PRT Homo Sapiens 71 Thr Leu Gln Asp 1 72 4 PRT Homo
Sapiens 72 Ser Val Glu Glu 1 73 4 PRT Homo Sapiens 73 Thr Lys Asn
Glu 1 74 4 PRT Homo Sapiens 74 Thr Leu Gln Glu 1 75 4 PRT Homo
Sapiens 75 Thr Lys Ala Asp 1 76 4 PRT Homo Sapiens 76 Ser Ile Ile
Glu 1 77 4 PRT Homo Sapiens 77 Ser Glu Lys Asp 1 78 4 PRT Homo
Sapiens 78 Ser Asp Gly Glu 1 79 4 PRT Homo Sapiens 79 Ser Phe Lys
Asp 1 80 4 PRT Homo Sapiens 80 Ser Ser Gly Glu 1 81 4 PRT Homo
Sapiens 81 Thr Glu Glu Asp 1 82 4 PRT Homo Sapiens 82 Ser Pro Gln
Asp 1 83 8 PRT Homo Sapiens 83 Lys Leu Asp Glu His Ile Ala Tyr 1 5
84 6 PRT Homo Sapiens 84 Gly Gly Ile Leu Ala Asp 1 5 85 6 PRT Homo
Sapiens 85 Gly Leu Gly Lys Thr Val 1 5 86 6 PRT Homo Sapiens 86 Gly
Met Phe Asp Ala Ser 1 5 87 6 PRT Homo Sapiens 87 Gly Val Ile Ile
Thr Thr 1 5 88 6 PRT Homo Sapiens 88 Gly Ser Leu Leu Gly Thr 1 5 89
6 PRT Homo Sapiens 89 Gly Thr Leu Lys Thr Phe 1 5 90 6 PRT Homo
Sapiens 90 Gly Thr Phe Ser Ala Gln 1 5 91 6 PRT Homo Sapiens 91 Gly
Val Gly Leu Thr Leu 1 5 92 6 PRT Homo Sapiens 92 Gly Leu Thr Leu
Thr Ala 1 5 93 6 PRT Homo Sapiens 93 Gly Gln Lys Glu Asn Val 1 5 94
6 PRT Homo Sapiens 94 Gly Thr Gly Ser Ala Asp 1 5 95 6 PRT Homo
Sapiens 95 Gly Ser Ala Asp Ser Ile 1 5 96 6 PRT Homo Sapiens 96 Gly
Met Glu Lys Ser Phe 1 5 97 6 PRT Homo Sapiens 97 Gly Ser Ala Pro
Asn Ser 1 5 98 6 PRT Homo Sapiens 98 Gly Val Glu Glu Ser Ser 1 5 99
4 PRT Homo Sapiens 99 Asp Gly Arg Lys 1 100 1250 PRT Homo Sapiens
100 Met Glu Ala Ser Arg Arg Phe Pro Glu Ala Glu Ala Leu Ser Pro Glu
1 5 10 15 Gln Ala Ala His Tyr Leu Arg Tyr Val Lys Glu Ala Lys Glu
Ala Thr 20 25 30 Lys Asn Gly Asp Leu Glu Glu Ala Phe Lys Leu Phe
Asn Leu Ala Lys 35 40 45 Asp Ile Phe Pro Asn Glu Lys Val Leu Ser
Arg Ile Gln Lys Ile Gln 50 55 60 Glu Ala Leu Glu Glu Leu Ala Glu
Gln Gly Asp Asp Glu Phe Thr Asp 65 70 75 80 Val Cys Asn Ser Gly Leu
Leu Leu Tyr Arg Glu Leu His Asn Gln Leu 85 90 95 Phe Glu His Gln
Lys Glu Gly Ile Ala Phe Leu Tyr Ser Leu Tyr Arg 100 105 110 Asp Gly
Arg Lys Gly Gly Ile Leu Ala Asp Asp Met Gly Leu Gly Lys 115 120 125
Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met Phe Asp Ala Ser Leu 130
135 140 Val Asn His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr
Trp 145 150 155 160 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Arg
Val Lys Thr Phe 165 170 175 His Gly Pro Ser Lys Asp Glu Arg Thr Arg
Asn Leu Asn Arg Ile Gln 180 185 190 Gln Arg Asn Gly Val Ile Ile Thr
Thr Tyr Gln Met Leu Ile Asn Asn 195 200 205 Trp Gln Gln Leu Ser Ser
Phe Arg Gly Gln Glu Phe Val Trp Asp Tyr 210 215 220 Val Ile Leu Asp
Glu Ala His Lys Ile Lys Thr Ser Ser Thr Lys Ser 225 230 235 240 Ala
Ile Cys Ala Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 245 250
255 Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp
260 265 270 Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe
Lys Met 275 280 285 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys
Asp Ala Thr Pro 290 295 300 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser
Glu Asn Leu Met Ala Ile 305 310 315 320 Ile Lys Pro Tyr Phe Leu Arg
Arg Thr Lys Glu Asp Val Gln Lys Lys 325 330 335 Lys Ser Ser Asn Pro
Glu Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 340 345 350 Asp Ala Ile
Cys Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 355 360 365 Ile
Trp Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 370 375
380 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro
385 390 395 400 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His
Pro Arg Leu 405 410 415 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu
Gly Thr Phe Ser Ala 420 425 430 Gln Asp Gly Asn Glu Gly Glu Asp Ser
Pro Asp Val Asp His Ile Asp 435 440 445 Gln Val Thr Asp Asp Thr Leu
Met Glu Glu Ser Gly Lys Met Ile Phe 450 455 460 Leu Met Asp Leu Leu
Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 465 470 475 480 Val Phe
Ser Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 485 490 495
Lys Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 500
505 510 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys
Asp 515 520 525 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val
Gly Leu Thr 530 535 540 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp
Pro Ser Trp Asn Pro 545 550 555 560 Ala Thr Asp Ala Gln Ala Val Asp
Arg Val Tyr Arg Ile Gly Gln Lys 565 570 575 Glu Asn Val Val Val Tyr
Arg Leu Ile Thr Cys Gly Thr Val Glu Glu 580
585 590 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu Ile Arg Gln
Thr 595 600 605 Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys
Gln Glu Leu 610 615 620 Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn
Ser Val Thr Gln Leu 625 630 635 640 Gln Leu Gln Ser Leu His Ala Ala
Gln Arg Lys Ser Asp Ile Lys Leu 645 650 655 Asp Glu His Ile Ala Tyr
Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 660 665 670 Asp His Asp Leu
Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu 675 680 685 Asp Val
Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln Lys Ala 690 695 700
Gln Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 705
710 715 720 Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro
Val Phe 725 730 735 Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn
Lys Pro Gln Pro 740 745 750 Gln Pro Ser Pro Leu Leu Ser Thr His His
Thr Gln Glu Glu Asp Ile 755 760 765 Ser Ser Lys Met Ala Ser Val Val
Ile Asp Asp Leu Pro Lys Glu Gly 770 775 780 Glu Lys Gln Asp Leu Ser
Ser Ile Lys Val Asn Val Thr Thr Leu Gln 785 790 795 800 Asp Gly Lys
Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro Lys 805 810 815 Gly
Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser Leu Gly Met 820 825
830 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr Leu
835 840 845 Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu
Glu Ser 850 855 860 Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp
Ile Gly Pro Asn 865 870 875 880 Leu Asp Gln Leu Lys Asp Asp Glu Ile
Leu Arg His Cys Asn Pro Trp 885 890 895 Pro Ile Ile Ser Ile Thr Asn
Glu Ser Gln Asn Ala Glu Ser Asn Val 900 905 910 Ser Ile Ile Glu Ile
Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 915 920 925 Gln Asp Ala
Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser Ala 930 935 940 Ser
Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp Ser 945 950
955 960 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His Val
Glu 965 970 975 Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser Arg
Ala Gly Phe 980 985 990 Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe
Ser Glu Lys Asp Asp 995 1000 1005 Glu Pro Glu Glu Val Val Val Lys
Ala Lys Ile Arg Ser Lys Ala Arg 1010 1015 1020 Arg Ile Val Ser Asp
Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 1025 1030 1035 1040 Ser
Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val 1045
1050 1055 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro
Pro Gly 1060 1065 1070 Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val
Asn Lys Ser Met Asn 1075 1080 1085 Ser Arg Arg Ser Leu Ala Ser Arg
Arg Ser Leu Ile Asn Met Val Leu 1090 1095 1100 Asp His Val Glu Asp
Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 1105 1110 1115 1120 Lys
Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 1125
1130 1135 Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr
Leu Ser 1140 1145 1150 Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser
Lys Pro Ser Ala Leu 1155 1160 1165 Ala Gln Glu Thr Ser Leu Gly Ala
Pro Glu Pro Leu Ser Gly Glu Gln 1170 1175 1180 Leu Val Gly Ser Pro
Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr 1185 1190 1195 1200 Glu
Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1205
1210 1215 Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys
Ser Ala 1220 1225 1230 Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu
Tyr Lys Gln Leu Asn 1235 1240 1245 Asn Asn 1250 101 17 PRT Homo
Sapiens 101 Phe Ile Lys Trp Thr Pro Gly Met Gly Val Lys Thr Phe His
Gly Pro 1 5 10 15 Ser 102 19 PRT Homo Sapiens 102 Glu Phe Ile Lys
Trp Thr Pro Gly Met Gly Val Lys Thr Phe His Gly 1 5 10 15 Pro Ser
Lys 103 29 PRT Homo Sapiens 103 Asn Thr Trp Val Lys Glu Phe Ile Lys
Trp Thr Pro Gly Met Gly Val 1 5 10 15 Lys Thr Phe His Gly Pro Ser
Lys Asp Glu Arg Thr Arg 20 25 104 17 PRT Homo Sapiens 104 Cys Glu
Met Pro Ser Leu Ser Arg Arg Asn Asp Leu Ile Ile Trp Ile 1 5 10 15
Arg 105 19 PRT Homo Sapiens 105 Ile Cys Glu Met Pro Ser Leu Ser Arg
Arg Asn Asp Leu Ile Ile Trp 1 5 10 15 Ile Arg Leu 106 29 PRT Homo
Sapiens 106 Pro Asp Val Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg
Arg Asn 1 5 10 15 Asp Leu Ile Ile Trp Ile Arg Leu Val Pro Leu Gln
Glu 20 25 107 17 PRT Homo Sapiens 107 Leu Asp Gln Leu Lys Asp Asp
Glu Val Leu Arg His Cys Asn Pro Trp 1 5 10 15 Pro 108 19 PRT Homo
Sapiens 108 Asn Leu Asp Gln Leu Lys Asp Asp Glu Val Leu Arg His Cys
Asn Pro 1 5 10 15 Trp Pro Ile 109 29 PRT Homo Sapiens 109 Ala Asp
Ile Gly Pro Asn Leu Asp Gln Leu Lys Asp Asp Glu Val Leu 1 5 10 15
Arg His Cys Asn Pro Trp Pro Ile Ile Ser Ile Thr Asn 20 25 110 4334
DNA Homo Sapiens 110 atgcgcgggg cgggagtgag cgaaattcaa gctccaaact
ctaagctcca agctccaagc 60 tccaagctcc aagctccaaa ctcccgccgg
ggtaactgga acccaatccg agggtcatgg 120 aggcatcccg aaggtttccg
gaagccgagg ccttgagccc agagcaggct gctcattacc 180 taagggtctt
gctgtgtcgc ccagactgga attcagtggc ctgatcatag ttcactgcag 240
cctcgaactc ctgggctcaa gcagtcctcc tgccccagcc tccctagtag ctgggactta
300 agatatgtga aagaggccaa agaagcaact aagaatggag acctggaaga
agcatttaaa 360 cttttcaatt tggcaaagga catttttccc aatgaaaaag
tgctgagcag aatccaaaaa 420 atacaggaag ccttggagga gttggcagaa
cagggagatg atgaatttac agatgtgtgc 480 aactctggct tgctacttta
tcgagaactg cacaaccaac tctttgagca ccagaaggaa 540 ggcatagctt
tcctctatag cctgtatagg gatggaagaa aaggtggtat attggctgat 600
gatatgggat tagggaagac tgttcaaatc attgctttcc tttccggtat gtttgatgca
660 tcacttgtga atcatgtgct gctgatcatg ccaaccaatc ttattaacac
atgggtaaaa 720 gaattcatca agtggactcc aggaatgaga gtcaaaacct
ttcatggtcc tagcaaggat 780 gaacggacca gaaacctcaa tcggattcag
caaaggaatg gtgttattat cactacatac 840 caaatgttaa tcaataactg
gcagcaactt tcaagcttta ggggccaaga gtttgtgtgg 900 gactatgtca
tcctcgatga agcacataaa ataaaaacct catctactaa gtcagcaata 960
tgtgctcgtg ctattcctgc aagtaatcgc ctcctcctca caggaacccc aatccagaat
1020 aatttacaag aactatggtc cctatttgat tttgcttgtc aagggtccct
gctgggaaca 1080 ttaaaaactt ttaagatgga gtatgaaaat cctattacta
gagcaagaga gaaggatgct 1140 accccaggag aaaaagcctt gggatttaaa
atatctgaaa acttaatggc aatcataaaa 1200 ccctattttc tcaggaggac
taaagaagac gtacagaaga aaaagtcaag caacccagag 1260 gccagactta
atgaaaagaa tccagatgtt gatgccattt gtgaaatgcc ttccctttcc 1320
aggaaaaatg atttaattat ttggatacga cttgtgcctt tacaagaaga aatatacagg
1380 aaatttgtgt ctttagatca tatcaaggag ttgctaatgg agacgcgctc
acctttggct 1440 gagctaggtg tcttaaagaa gctgtgtgat catcctaggc
tgctgtctgc acgggcttgt 1500 tgtttgctaa atcttgggac attctctgct
caagatggaa atgaggggga agattcccca 1560 gatgtggacc atattgatca
agtaactgat gacacattga tggaagaatc tggaaaaatg 1620 atattcctaa
tggacctact taagaggctg cgagatgagg gacatcaaac tctggtgttt 1680
tctcaatcga ggcaaattct aaacatcatt gaacgcctct taaagaatag gcactttaag
1740 acattgcgaa tcgatgggac agttactcat cttttggaac gagaaaaaag
aattaactta 1800 ttccagcaaa ataaagatta ctctgttttt ctgcttacca
ctcaagtagg tggtgtcggt 1860 ttaacattaa ctgcagcaac tagagtggtc
atttttgacc ctagctggaa tcctgcaact 1920 gatgctcaag ctgtggatag
agtttaccga attggacaaa aagagaatgt tgtggtttat 1980 aggctaatca
cttgtgggac tgtagaggaa aaaatataca gaagacaggt tttcaaggac 2040
tcattaataa gacaaactac tggtgaaaaa aagaaccctt tccgatattt tagtaaacaa
2100 gaattaagag agctctttac aatcgaggat cttcagaact ctgtaaccca
gctgcagctt 2160 cagtctttgc atgctgctca gaggaaatct gatataaaac
tagatgaaca tattgcctac 2220 ctgcagtctt tggggatagc tggaatctca
gaccatgatt tgatgtacac atgtgatctg 2280 tctgttaaag aagagcttga
tgtggtagaa gaatctcact atattcaaca aagggttcag 2340 aaagctcaat
tcctcgttga attcgagtct caaaataaag agttcctgat ggaacaacaa 2400
agaactagaa atgagggggc ctggctaaga gaacctgtat ttccttcttc aacaaagaag
2460 aaatgcccta aattgaataa accacagcct cagccttcac ctcttctaag
tactcatcat 2520 actcaggaag aagatatcag ttccaaaatg gcaagtgtag
tcattgatga tctgcccaaa 2580 gagggtgaga aacaagatct ctccagtata
aaggtgaatg ttaccacctt gcaagatggt 2640 aaaggtacag gtagtgctga
ctctatagct actttaccaa aggggtttgg aagtgtagaa 2700 gaactttgta
ctaactcttc attgggaatg gaaaaaagct ttgcaactaa aaatgaagct 2760
gtacaaaaag agacattaca agaggggcct aagcaagagg cactgcaaga ggatcctctg
2820 gaaagtttta attatgtact tagcaaatca accaaagctg atattgggcc
aaatttagat 2880 caactaaagg atgatgagat tttacgtcat tgcaatcctt
ggcccattat ttccataaca 2940 aatgaaagtc aaaatgcaga atcaaatgta
tccattattg aaatagctga tgacctttca 3000 gcatcccata gtgcactgca
ggatgctcaa gcaagtgagg ccaagttgga agaggaacct 3060 tcagcatctt
caccacagta tgcatgtgat ttcaatcttt tcttggaaga ctcagcagac 3120
aacagacaaa atttttccag tcagtcttta gagcatgttg agaaagaaaa tagcttgtgt
3180 ggctctgcac ctaattccag agcagggttt gtgcatagca aaacatgtct
cagttgggag 3240 ttttctgaga aagacgatga accagaagaa gtagtagtta
aagcaaaaat cagaagtaaa 3300 gctagaagga ttgtttcaga tggcgaagat
gaagatgatt cttttaaaga tacctcaagc 3360 ataaatccat tcaacacatc
tctctttcaa ttctcatctg tgaaacaatt tgatgcttca 3420 actcccaaaa
atgacatcag tccaccagga aggttctttt catctcaaat acccagtagt 3480
gtaaataagt ctatgaactc tagaagatct ctggcttcta ggaggtctct tattaatatg
3540 gttttagacc acgtggagga catggaggaa agacttgacg acagcagtga
agcaaagggt 3600 cctgaagatt atccagaaga aggggtggag gaaagcagtg
gcgaagcctc caagtataca 3660 gaagaggatc cttccggaga aacactgtct
tcagaaaaca agtccagctg gttaatgacg 3720 tctaagccta gtgctctagc
tcaagagacc tctcttggtg cccctgagcc tttgtctggt 3780 gaacagttgg
ttggttctcc ccaggataag gcggcagagg ctacaaatga ctatgagact 3840
cttgtaaagc gtggaaaaga actaaaagag tgtggaaaaa tccaggaggc cctaaactgc
3900 ttagttaaag cgcttgacat aaaaagtgca gatcctgaag ttatgctctt
gactttaagt 3960 ttgtataagc aacttaataa caattgagaa tgtaacctgt
ttattgtatt ttaaagtgaa 4020 actgaatatg agggaatttt tgttcccata
attggattct ttgggaacat gaagcattca 4080 ggcttaaggc aagaaagatc
tcaaaaagca acttctgccc tgcaacgccc cccactccat 4140 agtctggtat
tctgagcact agcttaatat ttcttcactt gaatattctt atattttagg 4200
catattctat aaatttaact gtgttgtttc ttggaaagtt ttgtaaaatt attctggtca
4260 ttcttaattt tactctgaaa gtgatcatct ttgtatataa cagttcagat
aagaaaatta 4320 aagttacttt tctc 4334 111 4312 DNA Homo Sapiens 111
aaattcaagc tccaaactct aagctccaag ctccaagctc caagctccaa gctccaaact
60 cccgccgggg taactggaac ccaatccgag ggtcatggag gcatcccgaa
ggtttccgga 120 agccgaggcc ttgagcccag agcaggctgc tcattaccta
agggtcttgc tgtgtcgccc 180 agactggaat tcagtggcct gatcatagtt
cactgcagcc tcgaactcct gggctcaagc 240 agtcctcctg ccccagcctc
cctagtagct gggacttaag atatgtgaaa gaggccaaag 300 aagcaactaa
gaatggagac ctggaagaag catttaaact tttcaatttg gcaaaggaca 360
tttttcccaa tgaaaaagtg ctgagcagaa tccaaaaaat acaggaagcc ttggaggagt
420 tggcagaaca gggagatgat gaatttacag atgtgtgcaa ctctggcttg
ctactttatc 480 gagaactgca caaccaactc tttgagcacc agaaggaagg
catagctttc ctctatagcc 540 tgtataggga tggaagaaaa ggtggtatat
tggctgatga tatgggatta gggaagactg 600 ttcaaatcat tgctttcctt
tccggtatgt ttgatgcatc acttgtgaat catgtgctgc 660 tgatcatgcc
aaccaatctt attaacacat gggtaaaaga attcatcaag tggactccag 720
gaatgagagt caaaaccttt catggtccta gcaaggatga acggaccaga aacctcaatc
780 ggattcagca aaggaatggt gttattatca ctacatacca aatgttaatc
aataactggc 840 agcaactttc aagctttagg ggccaagagt ttgtgtggga
ctatgtcatc ctcgatgaag 900 cacataaaat aaaaacctca tctactaagt
cagcaatatg tgctcgtgct attcctgcaa 960 gtaatcgcct cctcctcaca
ggaaccccaa tccagaataa tttacaagaa ctatggtccc 1020 tatttgattt
tgcttgtcaa gggtccctgc tgggaacatt aaaaactttt aagatggagt 1080
atgaaaatcc tattactaga gcaagagaga aggatgctac cccaggagaa aaagccttgg
1140 gatttaaaat atctgaaaac ttaatggcaa tcataaaacc ctattttctc
aggaggacta 1200 aagaagacgt acagaagaaa aagtcaagca acccagaggc
cagacttaat gaaaagaatc 1260 cagatgttga tgccatttgt gaaatgcctt
ccctttccag gaaaaatgat ttaattattt 1320 ggatacgact tgtgccttta
caagaagaaa tatacaggaa atttgtgtct ttagatcata 1380 tcaaggagtt
gctaatggag acgcgctcac ctttggctga gctaggtgtc ttaaagaagc 1440
tgtgtgatca tcctaggctg ctgtctgcac gggcttgttg tttgctaaat cttgggacat
1500 tctctgctca agatggaaat gagggggaag attccccaga tgtggaccat
attgatcaag 1560 taactgatga cacattgatg gaagaatctg gaaaaatgat
attcctaatg gacctactta 1620 agaggctgcg agatgaggga catcaaactc
tggtgttttc tcaatcgagg caaattctaa 1680 acatcattga acgcctctta
aagaataggc actttaagac attgcgaatc gatgggacag 1740 ttactcatct
tttggaacga gaaaaaagaa ttaacttatt ccagcaaaat aaagattact 1800
ctgtttttct gcttaccact caagtaggtg gtgtcggttt aacattaact gcagcaacta
1860 gagtggtcat ttttgaccct agctggaatc ctgcaactga tgctcaagct
gtggatagag 1920 tttaccgaat tggacaaaaa gagaatgttg tggtttatag
gctaatcact tgtgggactg 1980 tagaggaaaa aatatacaga agacaggttt
tcaaggactc attaataaga caaactactg 2040 gtgaaaaaaa gaaccctttc
cgatatttta gtaaacaaga attaagagag ctctttacaa 2100 tcgaggatct
tcagaactct gtaacccagc tgcagcttca gtctttgcat gctgctcaga 2160
ggaaatctga tataaaacta gatgaacata ttgcctacct gcagtctttg gggatagctg
2220 gaatctcaga ccatgatttg atgtacacat gtgatctgtc tgttaaagaa
gagcttgatg 2280 tggtagaaga atctcactat attcaacaaa gggttcagaa
agctcaattc ctcgttgaat 2340 tcgagtctca aaataaagag ttcctgatgg
aacaacaaag aactagaaat gagggggcct 2400 ggctaagaga acctgtattt
ccttcttcaa caaagaagaa atgccctaaa ttgaataaac 2460 cacagcctca
gccttcacct cttctaagta ctcatcatac tcaggaagaa gatatcagtt 2520
ccaaaatggc aagtgtagtc attgatgatc tgcccaaaga gggtgagaaa caagatctct
2580 ccagtataaa ggtgaatgtt accaccttgc aagatggtaa aggtacaggt
agtgctgact 2640 ctatagctac tttaccaaag gggtttggaa gtgtagaaga
actttgtact aactcttcat 2700 tgggaatgga aaaaagcttt gcaactaaaa
atgaagctgt acaaaaagag acattacaag 2760 aggggcctaa gcaagaggca
ctgcaagagg atcctctgga aagttttaat tatgtactta 2820 gcaaatcaac
caaagctgat attgggccaa atttagatca actaaaggat gatgagattt 2880
tacgtcattg caatccttgg cccattattt ccataacaaa tgaaagtcaa aatgcagaat
2940 caaatgtatc cattattgaa atagctgatg acctttcagc atcccatagt
gcactgcagg 3000 atgctcaagc aagtgaggcc aagttggaag aggaaccttc
agcatcttca ccacagtatg 3060 catgtgattt caatcttttc ttggaagact
cagcagacaa cagacaaaat ttttccagtc 3120 agtctttaga gcatgttgag
aaagaaaata gcttgtgtgg ctctgcacct aattccagag 3180 cagggtttgt
gcatagcaaa acatgtctca gttgggagtt ttctgagaaa gacgatgaac 3240
cagaagaagt agtagttaaa gcaaaaatca gaagtaaagc tagaaggatt gtttcagatg
3300 gcgaagatga agatgattct tttaaagata cctcaagcat aaatccattc
aacacatctc 3360 tctttcaatt ctcatctgtg aaacaatttg atgcttcaac
tcccaaaaat gacatcagtc 3420 caccaggaag gttcttttca tctcaaatac
ccagtagtgt aaataagtct atgaactcta 3480 gaagatctct ggcttctagg
aggtctctta ttaatatggt tttagaccac gtggaggaca 3540 tggaggaaag
acttgacgac agcagtgaag caaagggtcc tgaagattat ccagaagaag 3600
gggtggagga aagcagtggc gaagcctcca agtatacaga agaggatcct tccggagaaa
3660 cactgtcttc agaaaacaag tccagctggt taatgacgtc taagcctagt
gctctagctc 3720 aagagacctc tcttggtgcc cctgagcctt tgtctggtga
acagttggtt ggttctcccc 3780 aggataaggc ggcagaggct acaaatgact
atgagactct tgtaaagcgt ggaaaagaac 3840 taaaagagtg tggaaaaatc
caggaggccc taaactgctt agttaaagcg cttgacataa 3900 aaagtgcaga
tcctgaagtt atgctcttga ctttaagttt gtataagcaa cttaataaca 3960
attgagaatg taacctgttt attgtatttt aaagtgaaac tgaatatgag ggaatttttg
4020 ttcccataat tggattcttt gggaacatga agcattcagg cttaaggcaa
gaaagatctc 4080 aaaaagcaac ttctgccctg caacgccccc cactccatag
tctggtattc tgagcactag 4140 cttaatattt cttcacttga atattcttat
attttaggca tattctataa atttaactgt 4200 gttgtttctt ggaaagtttt
gtaaaattat tctggtcatt cttaatttta ctctgaaagt 4260 gatcatcttt
gtatataaca gttcagataa gaaaattaaa gttacttttc tc 4312 112 4194 DNA
Homo Sapiens 112 aaattcaagc tccaaactct aagctccaag ctccaagctc
caagctccaa gctccaaact 60 cccgccgggg taactggaac ccaatccgag
ggtcatggag gcatcccgaa ggtttccgga 120 agccgaggcc ttgagcccag
agcaggctgc tcattaccta agatatgtga aagaggccaa 180 agaagcaact
aagaatggag acctggaaga agcatttaaa cttttcaatt tggcaaagga 240
catttttccc aatgaaaaag tgctgagcag aatccaaaaa atacaggaag ccttggagga
300 gttggcagaa cagggagatg atgaatttac agatgtgtgc aactctggct
tgctacttta 360 tcgagaactg cacaaccaac tctttgagca ccagaaggaa
ggcatagctt tcctctatag 420 cctgtatagg gatggaagaa aaggtggtat
attggctgat gatatgggat tagggaagac 480 tgttcaaatc attgctttcc
tttccggtat gtttgatgca tcacttgtga atcatgtgct 540 gctgatcatg
ccaaccaatc ttattaacac atgggtaaaa gaattcatca agtggactcc 600
aggaatgaga gtcaaaacct ttcatggtcc tagcaaggat gaacggacca gaaacctcaa
660 tcggattcag caaaggaatg gtgttattat cactacatac caaatgttaa
tcaataactg 720 gcagcaactt tcaagcttta ggggccaaga gtttgtgtgg
gactatgtca tcctcgatga 780 agcacataaa ataaaaacct
catctactaa gtcagcaata tgtgctcgtg ctattcctgc 840 aagtaatcgc
ctcctcctca caggaacccc aatccagaat aatttacaag aactatggtc 900
cctatttgat tttgcttgtc aagggtccct gctgggaaca ttaaaaactt ttaagatgga
960 gtatgaaaat cctattacta gagcaagaga gaaggatgct accccaggag
aaaaagcctt 1020 gggatttaaa atatctgaaa acttaatggc aatcataaaa
ccctattttc tcaggaggac 1080 taaagaagac gtacagaaga aaaagtcaag
caacccagag gccagactta atgaaaagaa 1140 tccagatgtt gatgccattt
gtgaaatgcc ttccctttcc aggaaaaatg atttaattat 1200 ttggatacga
cttgtgcctt tacaagaaga aatatacagg aaatttgtgt ctttagatca 1260
tatcaaggag ttgctaatgg agacgcgctc acctttggct gagctaggtg tcttaaagaa
1320 gctgtgtgat catcctaggc tgctgtctgc acgggcttgt tgtttgctaa
atcttgggac 1380 attctctgct caagatggaa atgaggggga agattcccca
gatgtggacc atattgatca 1440 agtaactgat gacacattga tggaagaatc
tggaaaaatg atattcctaa tggacctact 1500 taagaggctg cgagatgagg
gacatcaaac tctggtgttt tctcaatcga ggcaaattct 1560 aaacatcatt
gaacgcctct taaagaatag gcactttaag acattgcgaa tcgatgggac 1620
agttactcat cttttggaac gagaaaaaag aattaactta ttccagcaaa ataaagatta
1680 ctctgttttt ctgcttacca ctcaagtagg tggtgtcggt ttaacattaa
ctgcagcaac 1740 tagagtggtc atttttgacc ctagctggaa tcctgcaact
gatgctcaag ctgtggatag 1800 agtttaccga attggacaaa aagagaatgt
tgtggtttat aggctaatca cttgtgggac 1860 tgtagaggaa aaaatataca
gaagacaggt tttcaaggac tcattaataa gacaaactac 1920 tggtgaaaaa
aagaaccctt tccgatattt tagtaaacaa gaattaagag agctctttac 1980
aatcgaggat cttcagaact ctgtaaccca gctgcagctt cagtctttgc atgctgctca
2040 gaggaaatct gatataaaac tagatgaaca tattgcctac ctgcagtctt
tggggatagc 2100 tggaatctca gaccatgatt tgatgtacac atgtgatctg
tctgttaaag aagagcttga 2160 tgtggtagaa gaatctcact atattcaaca
aagggttcag aaagctcaat tcctcgttga 2220 attcgagtct caaaataaag
agttcctgat ggaacaacaa agaactagaa atgagggggc 2280 ctggctaaga
gaacctgtat ttccttcttc aacaaagaag aaatgcccta aattgaataa 2340
accacagcct cagccttcac ctcttctaag tactcatcat actcaggaag aagatatcag
2400 ttccaaaatg gcaagtgtag tcattgatga tctgcccaaa gagggtgaga
aacaagatct 2460 ctccagtata aaggtgaatg ttaccacctt gcaagatggt
aaaggtacag gtagtgctga 2520 ctctatagct actttaccaa aggggtttgg
aagtgtagaa gaactttgta ctaactcttc 2580 attgggaatg gaaaaaagct
ttgcaactaa aaatgaagct gtacaaaaag agacattaca 2640 agaggggcct
aagcaagagg cactgcaaga ggatcctctg gaaagtttta attatgtact 2700
tagcaaatca accaaagctg atattgggcc aaatttagat caactaaagg atgatgagat
2760 tttacgtcat tgcaatcctt ggcccattat ttccataaca aatgaaagtc
aaaatgcaga 2820 atcaaatgta tccattattg aaatagctga tgacctttca
gcatcccata gtgcactgca 2880 ggatgctcaa gcaagtgagg ccaagttgga
agaggaacct tcagcatctt caccacagta 2940 tgcatgtgat ttcaatcttt
tcttggaaga ctcagcagac aacagacaaa atttttccag 3000 tcagtcttta
gagcatgttg agaaagaaaa tagcttgtgt ggctctgcac ctaattccag 3060
agcagggttt gtgcatagca aaacatgtct cagttgggag ttttctgaga aagacgatga
3120 accagaagaa gtagtagtta aagcaaaaat cagaagtaaa gctagaagga
ttgtttcaga 3180 tggcgaagat gaagatgatt cttttaaaga tacctcaagc
ataaatccat tcaacacatc 3240 tctctttcaa ttctcatctg tgaaacaatt
tgatgcttca actcccaaaa atgacatcag 3300 tccaccagga aggttctttt
catctcaaat acccagtagt gtaaataagt ctatgaactc 3360 tagaagatct
ctggcttcta ggaggtctct tattaatatg gttttagacc acgtggagga 3420
catggaggaa agacttgacg acagcagtga agcaaagggt cctgaagatt atccagaaga
3480 aggggtggag gaaagcagtg gcgaagcctc caagtataca gaagaggatc
cttccggaga 3540 aacactgtct tcagaaaaca agtccagctg gttaatgacg
tctaagccta gtgctctagc 3600 tcaagagacc tctcttggtg cccctgagcc
tttgtctggt gaacagttgg ttggttctcc 3660 ccaggataag gcggcagagg
ctacaaatga ctatgagact cttgtaaagc gtggaaaaga 3720 actaaaagag
tgtggaaaaa tccaggaggc cctaaactgc ttagttaaag cgcttgacat 3780
aaaaagtgca gatcctgaag ttatgctctt gactttaagt ttgtataagc aacttaataa
3840 caattgagaa tgtaacctgt ttattgtatt ttaaagtgaa actgaatatg
agggaatttt 3900 tgttcccata attggattct ttgggaacat gaagcattca
ggcttaaggc aagaaagatc 3960 tcaaaaagca acttctgccc tgcaacgccc
cccactccat agtctggtat tctgagcact 4020 agcttaatat ttcttcactt
gaatattctt atattttagg catattctat aaatttaact 4080 gtgttgtttc
ttggaaagtt ttgtaaaatt attctggtca ttcttaattt tactctgaaa 4140
gtgatcatct ttgtatataa cagttcagat aagaaaatta aagttacttt tctc 4194
113 1127 PRT Homo Sapiens 113 Met Gly Leu Gly Lys Thr Val Gln Ile
Ile Ala Phe Leu Ser Gly Met 1 5 10 15 Phe Asp Ala Ser Leu Val Asn
His Val Leu Leu Ile Met Pro Thr Asn 20 25 30 Leu Ile Asn Thr Trp
Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met 35 40 45 Arg Val Lys
Thr Phe His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn 50 55 60 Leu
Asn Arg Ile Gln Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln 65 70
75 80 Met Leu Ile Asn Asn Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln
Glu 85 90 95 Phe Val Trp Asp Tyr Val Ile Leu Asp Glu Ala His Lys
Ile Lys Thr 100 105 110 Ser Ser Thr Lys Ser Ala Ile Cys Ala Arg Ala
Ile Pro Ala Ser Asn 115 120 125 Arg Leu Leu Leu Thr Gly Thr Pro Ile
Gln Asn Asn Leu Gln Glu Leu 130 135 140 Trp Ser Leu Phe Asp Phe Ala
Cys Gln Gly Ser Leu Leu Gly Thr Leu 145 150 155 160 Lys Thr Phe Lys
Met Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu 165 170 175 Lys Asp
Ala Thr Pro Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu 180 185 190
Asn Leu Met Ala Ile Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu 195
200 205 Asp Val Gln Lys Lys Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn
Glu 210 215 220 Lys Asn Pro Asp Val Asp Ala Ile Cys Glu Met Pro Ser
Leu Ser Arg 225 230 235 240 Lys Asn Asp Leu Ile Ile Trp Ile Arg Leu
Val Pro Leu Gln Glu Glu 245 250 255 Ile Tyr Arg Lys Phe Val Ser Leu
Asp His Ile Lys Glu Leu Leu Met 260 265 270 Glu Thr Arg Ser Pro Leu
Ala Glu Leu Gly Val Leu Lys Lys Leu Cys 275 280 285 Asp His Pro Arg
Leu Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu 290 295 300 Gly Thr
Phe Ser Ala Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp 305 310 315
320 Val Asp His Ile Asp Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser
325 330 335 Gly Lys Met Ile Phe Leu Met Asp Leu Leu Lys Arg Leu Arg
Asp Glu 340 345 350 Gly His Gln Thr Leu Val Phe Ser Gln Ser Arg Gln
Ile Leu Asn Ile 355 360 365 Ile Glu Arg Leu Leu Lys Asn Arg His Phe
Lys Thr Leu Arg Ile Asp 370 375 380 Gly Thr Val Thr His Leu Leu Glu
Arg Glu Lys Arg Ile Asn Leu Phe 385 390 395 400 Gln Gln Asn Lys Asp
Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly 405 410 415 Gly Val Gly
Leu Thr Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp 420 425 430 Pro
Ser Trp Asn Pro Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr 435 440
445 Arg Ile Gly Gln Lys Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys
450 455 460 Gly Thr Val Glu Glu Lys Ile Tyr Arg Arg Gln Val Phe Lys
Asp Ser 465 470 475 480 Leu Ile Arg Gln Thr Thr Gly Glu Lys Lys Asn
Pro Phe Arg Tyr Phe 485 490 495 Ser Lys Gln Glu Leu Arg Glu Leu Phe
Thr Ile Glu Asp Leu Gln Asn 500 505 510 Ser Val Thr Gln Leu Gln Leu
Gln Ser Leu His Ala Ala Gln Arg Lys 515 520 525 Ser Asp Ile Lys Leu
Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly 530 535 540 Ile Ala Gly
Ile Ser Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser 545 550 555 560
Val Lys Glu Glu Leu Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln 565
570 575 Arg Val Gln Lys Ala Gln Phe Leu Val Glu Phe Glu Ser Gln Asn
Lys 580 585 590 Glu Phe Leu Met Glu Gln Gln Arg Thr Arg Asn Glu Gly
Ala Trp Leu 595 600 605 Arg Glu Pro Val Phe Pro Ser Ser Thr Lys Lys
Lys Cys Pro Lys Leu 610 615 620 Asn Lys Pro Gln Pro Gln Pro Ser Pro
Leu Leu Ser Thr His His Thr 625 630 635 640 Gln Glu Glu Asp Ile Ser
Ser Lys Met Ala Ser Val Val Ile Asp Asp 645 650 655 Leu Pro Lys Glu
Gly Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn 660 665 670 Val Thr
Thr Leu Gln Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile 675 680 685
Ala Thr Leu Pro Lys Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn 690
695 700 Ser Ser Leu Gly Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val 705 710 715 720 Gln Lys Glu Thr Leu Gln Glu Gly Pro Lys Gln Glu
Ala Leu Gln Glu 725 730 735 Asp Pro Leu Glu Ser Phe Asn Tyr Val Leu
Ser Lys Ser Thr Lys Ala 740 745 750 Asp Ile Gly Pro Asn Leu Asp Gln
Leu Lys Asp Asp Glu Ile Leu Arg 755 760 765 His Cys Asn Pro Trp Pro
Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn 770 775 780 Ala Glu Ser Asn
Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala 785 790 795 800 Ser
His Ser Ala Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu 805 810
815 Glu Glu Pro Ser Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu
820 825 830 Phe Leu Glu Asp Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser
Gln Ser 835 840 845 Leu Glu His Val Glu Lys Glu Asn Ser Leu Cys Gly
Ser Ala Pro Asn 850 855 860 Ser Arg Ala Gly Phe Val His Ser Lys Thr
Cys Leu Ser Trp Glu Phe 865 870 875 880 Ser Glu Lys Asp Asp Glu Pro
Glu Glu Val Val Val Lys Ala Lys Ile 885 890 895 Arg Ser Lys Ala Arg
Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp 900 905 910 Ser Phe Lys
Asp Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe 915 920 925 Gln
Phe Ser Ser Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp 930 935
940 Ile Ser Pro Pro Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val
945 950 955 960 Asn Lys Ser Met Asn Ser Arg Arg Ser Leu Ala Ser Arg
Arg Ser Leu 965 970 975 Ile Asn Met Val Leu Asp His Val Glu Asp Met
Glu Glu Arg Leu Asp 980 985 990 Asp Ser Ser Glu Ala Lys Gly Pro Glu
Asp Tyr Pro Glu Glu Gly Val 995 1000 1005 Glu Glu Ser Ser Gly Glu
Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser 1010 1015 1020 Gly Glu Thr
Leu Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser 1025 1030 1035
1040 Lys Pro Ser Ala Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu
Pro 1045 1050 1055 Leu Ser Gly Glu Gln Leu Val Gly Ser Pro Gln Asp
Lys Ala Ala Glu 1060 1065 1070 Ala Thr Asn Asp Tyr Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys 1075 1080 1085 Glu Cys Gly Lys Ile Gln
Glu Ala Leu Asn Cys Leu Val Lys Ala Leu 1090 1095 1100 Asp Ile Lys
Ser Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu 1105 1110 1115
1120 Tyr Lys Gln Leu Asn Asn Asn 1125 114 1127 PRT Homo Sapiens 114
Met Gly Leu Gly Lys Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met 1 5
10 15 Phe Asp Ala Ser Leu Val Asn His Val Leu Leu Ile Met Pro Thr
Asn 20 25 30 Leu Ile Asn Thr Trp Val Lys Glu Phe Ile Lys Trp Thr
Pro Gly Met 35 40 45 Arg Val Lys Thr Phe His Gly Pro Ser Lys Asp
Glu Arg Thr Arg Asn 50 55 60 Leu Asn Arg Ile Gln Gln Arg Asn Gly
Val Ile Ile Thr Thr Tyr Gln 65 70 75 80 Met Leu Ile Asn Asn Trp Gln
Gln Leu Ser Ser Phe Arg Gly Gln Glu 85 90 95 Phe Val Trp Asp Tyr
Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr 100 105 110 Ser Ser Thr
Lys Ser Ala Ile Cys Ala Arg Ala Ile Pro Ala Ser Asn 115 120 125 Arg
Leu Leu Leu Thr Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu 130 135
140 Trp Ser Leu Phe Asp Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu
145 150 155 160 Lys Thr Phe Lys Met Glu Tyr Glu Asn Pro Ile Thr Arg
Ala Arg Glu 165 170 175 Lys Asp Ala Thr Pro Gly Glu Lys Ala Leu Gly
Phe Lys Ile Ser Glu 180 185 190 Asn Leu Met Ala Ile Ile Lys Pro Tyr
Phe Leu Arg Arg Thr Lys Glu 195 200 205 Asp Val Gln Lys Lys Lys Ser
Ser Asn Pro Glu Ala Arg Leu Asn Glu 210 215 220 Lys Asn Pro Asp Val
Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg 225 230 235 240 Lys Asn
Asp Leu Ile Ile Trp Ile Arg Leu Val Pro Leu Gln Glu Glu 245 250 255
Ile Tyr Arg Lys Phe Val Ser Leu Asp His Ile Lys Glu Leu Leu Met 260
265 270 Glu Thr Arg Ser Pro Leu Ala Glu Leu Gly Val Leu Lys Lys Leu
Cys 275 280 285 Asp His Pro Arg Leu Leu Ser Ala Arg Ala Cys Cys Leu
Leu Asn Leu 290 295 300 Gly Thr Phe Ser Ala Gln Asp Gly Asn Glu Gly
Glu Asp Ser Pro Asp 305 310 315 320 Val Asp His Ile Asp Gln Val Thr
Asp Asp Thr Leu Met Glu Glu Ser 325 330 335 Gly Lys Met Ile Phe Leu
Met Asp Leu Leu Lys Arg Leu Arg Asp Glu 340 345 350 Gly His Gln Thr
Leu Val Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile 355 360 365 Ile Glu
Arg Leu Leu Lys Asn Arg His Phe Lys Thr Leu Arg Ile Asp 370 375 380
Gly Thr Val Thr His Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe 385
390 395 400 Gln Gln Asn Lys Asp Tyr Ser Val Phe Leu Leu Thr Thr Gln
Val Gly 405 410 415 Gly Val Gly Leu Thr Leu Thr Ala Ala Thr Arg Val
Val Ile Phe Asp 420 425 430 Pro Ser Trp Asn Pro Ala Thr Asp Ala Gln
Ala Val Asp Arg Val Tyr 435 440 445 Arg Ile Gly Gln Lys Glu Asn Val
Val Val Tyr Arg Leu Ile Thr Cys 450 455 460 Gly Thr Val Glu Glu Lys
Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser 465 470 475 480 Leu Ile Arg
Gln Thr Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe 485 490 495 Ser
Lys Gln Glu Leu Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn 500 505
510 Ser Val Thr Gln Leu Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys
515 520 525 Ser Asp Ile Lys Leu Asp Glu His Ile Ala Tyr Leu Gln Ser
Leu Gly 530 535 540 Ile Ala Gly Ile Ser Asp His Asp Leu Met Tyr Thr
Cys Asp Leu Ser 545 550 555 560 Val Lys Glu Glu Leu Asp Val Val Glu
Glu Ser His Tyr Ile Gln Gln 565 570 575 Arg Val Gln Lys Ala Gln Phe
Leu Val Glu Phe Glu Ser Gln Asn Lys 580 585 590 Glu Phe Leu Met Glu
Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu 595 600 605 Arg Glu Pro
Val Phe Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu 610 615 620 Asn
Lys Pro Gln Pro Gln Pro Ser Pro Leu Leu Ser Thr His His Thr 625 630
635 640 Gln Glu Glu Asp Ile Ser Ser Lys Met Ala Ser Val Val Ile Asp
Asp 645 650 655 Leu Pro Lys Glu Gly Glu Lys Gln Asp Leu Ser Ser Ile
Lys Val Asn 660 665 670 Val Thr Thr Leu Gln Asp Gly Lys Gly Thr Gly
Ser Ala Asp Ser Ile 675 680 685 Ala Thr Leu Pro Lys Gly Phe Gly Ser
Val Glu Glu Leu Cys Thr Asn 690 695 700 Ser Ser Leu Gly Met Glu Lys
Ser Phe Ala Thr Lys Asn Glu Ala Val 705 710 715 720 Gln Lys Glu Thr
Leu Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu 725 730 735 Asp Pro
Leu Glu Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala 740 745 750
Asp Ile Gly Pro Asn Leu Asp Gln Leu Lys Asp Asp Glu Ile
Leu Arg 755 760 765 His Cys Asn Pro Trp Pro Ile Ile Ser Ile Thr Asn
Glu Ser Gln Asn 770 775 780 Ala Glu Ser Asn Val Ser Ile Ile Glu Ile
Ala Asp Asp Leu Ser Ala 785 790 795 800 Ser His Ser Ala Leu Gln Asp
Ala Gln Ala Ser Glu Ala Lys Leu Glu 805 810 815 Glu Glu Pro Ser Ala
Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu 820 825 830 Phe Leu Glu
Asp Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser 835 840 845 Leu
Glu His Val Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn 850 855
860 Ser Arg Ala Gly Phe Val His Ser Lys Thr Cys Leu Ser Trp Glu Phe
865 870 875 880 Ser Glu Lys Asp Asp Glu Pro Glu Glu Val Val Val Lys
Ala Lys Ile 885 890 895 Arg Ser Lys Ala Arg Arg Ile Val Ser Asp Gly
Glu Asp Glu Asp Asp 900 905 910 Ser Phe Lys Asp Thr Ser Ser Ile Asn
Pro Phe Asn Thr Ser Leu Phe 915 920 925 Gln Phe Ser Ser Val Lys Gln
Phe Asp Ala Ser Thr Pro Lys Asn Asp 930 935 940 Ile Ser Pro Pro Gly
Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val 945 950 955 960 Asn Lys
Ser Met Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu 965 970 975
Ile Asn Met Val Leu Asp His Val Glu Asp Met Glu Glu Arg Leu Asp 980
985 990 Asp Ser Ser Glu Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly
Val 995 1000 1005 Glu Glu Ser Ser Gly Glu Ala Ser Lys Tyr Thr Glu
Glu Asp Pro Ser 1010 1015 1020 Gly Glu Thr Leu Ser Ser Glu Asn Lys
Ser Ser Trp Leu Met Thr Ser 1025 1030 1035 1040 Lys Pro Ser Ala Leu
Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro 1045 1050 1055 Leu Ser
Gly Glu Gln Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu 1060 1065
1070 Ala Thr Asn Asp Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu
Lys 1075 1080 1085 Glu Cys Gly Lys Ile Gln Glu Ala Leu Asn Cys Leu
Val Lys Ala Leu 1090 1095 1100 Asp Ile Lys Ser Ala Asp Pro Glu Val
Met Leu Leu Thr Leu Ser Leu 1105 1110 1115 1120 Tyr Lys Gln Leu Asn
Asn Asn 1125 115 1127 PRT Homo Sapiens 115 Met Gly Leu Gly Lys Thr
Val Gln Ile Ile Ala Phe Leu Ser Gly Met 1 5 10 15 Phe Asp Ala Ser
Leu Val Asn His Val Leu Leu Ile Met Pro Thr Asn 20 25 30 Leu Ile
Asn Thr Trp Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met 35 40 45
Arg Val Lys Thr Phe His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn 50
55 60 Leu Asn Arg Ile Gln Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr
Gln 65 70 75 80 Met Leu Ile Asn Asn Trp Gln Gln Leu Ser Ser Phe Arg
Gly Gln Glu 85 90 95 Phe Val Trp Asp Tyr Val Ile Leu Asp Glu Ala
His Lys Ile Lys Thr 100 105 110 Ser Ser Thr Lys Ser Ala Ile Cys Ala
Arg Ala Ile Pro Ala Ser Asn 115 120 125 Arg Leu Leu Leu Thr Gly Thr
Pro Ile Gln Asn Asn Leu Gln Glu Leu 130 135 140 Trp Ser Leu Phe Asp
Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu 145 150 155 160 Lys Thr
Phe Lys Met Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu 165 170 175
Lys Asp Ala Thr Pro Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu 180
185 190 Asn Leu Met Ala Ile Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys
Glu 195 200 205 Asp Val Gln Lys Lys Lys Ser Ser Asn Pro Glu Ala Arg
Leu Asn Glu 210 215 220 Lys Asn Pro Asp Val Asp Ala Ile Cys Glu Met
Pro Ser Leu Ser Arg 225 230 235 240 Lys Asn Asp Leu Ile Ile Trp Ile
Arg Leu Val Pro Leu Gln Glu Glu 245 250 255 Ile Tyr Arg Lys Phe Val
Ser Leu Asp His Ile Lys Glu Leu Leu Met 260 265 270 Glu Thr Arg Ser
Pro Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys 275 280 285 Asp His
Pro Arg Leu Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu 290 295 300
Gly Thr Phe Ser Ala Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp 305
310 315 320 Val Asp His Ile Asp Gln Val Thr Asp Asp Thr Leu Met Glu
Glu Ser 325 330 335 Gly Lys Met Ile Phe Leu Met Asp Leu Leu Lys Arg
Leu Arg Asp Glu 340 345 350 Gly His Gln Thr Leu Val Phe Ser Gln Ser
Arg Gln Ile Leu Asn Ile 355 360 365 Ile Glu Arg Leu Leu Lys Asn Arg
His Phe Lys Thr Leu Arg Ile Asp 370 375 380 Gly Thr Val Thr His Leu
Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe 385 390 395 400 Gln Gln Asn
Lys Asp Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly 405 410 415 Gly
Val Gly Leu Thr Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp 420 425
430 Pro Ser Trp Asn Pro Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr
435 440 445 Arg Ile Gly Gln Lys Glu Asn Val Val Val Tyr Arg Leu Ile
Thr Cys 450 455 460 Gly Thr Val Glu Glu Lys Ile Tyr Arg Arg Gln Val
Phe Lys Asp Ser 465 470 475 480 Leu Ile Arg Gln Thr Thr Gly Glu Lys
Lys Asn Pro Phe Arg Tyr Phe 485 490 495 Ser Lys Gln Glu Leu Arg Glu
Leu Phe Thr Ile Glu Asp Leu Gln Asn 500 505 510 Ser Val Thr Gln Leu
Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys 515 520 525 Ser Asp Ile
Lys Leu Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly 530 535 540 Ile
Ala Gly Ile Ser Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser 545 550
555 560 Val Lys Glu Glu Leu Asp Val Val Glu Glu Ser His Tyr Ile Gln
Gln 565 570 575 Arg Val Gln Lys Ala Gln Phe Leu Val Glu Phe Glu Ser
Gln Asn Lys 580 585 590 Glu Phe Leu Met Glu Gln Gln Arg Thr Arg Asn
Glu Gly Ala Trp Leu 595 600 605 Arg Glu Pro Val Phe Pro Ser Ser Thr
Lys Lys Lys Cys Pro Lys Leu 610 615 620 Asn Lys Pro Gln Pro Gln Pro
Ser Pro Leu Leu Ser Thr His His Thr 625 630 635 640 Gln Glu Glu Asp
Ile Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp 645 650 655 Leu Pro
Lys Glu Gly Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn 660 665 670
Val Thr Thr Leu Gln Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile 675
680 685 Ala Thr Leu Pro Lys Gly Phe Gly Ser Val Glu Glu Leu Cys Thr
Asn 690 695 700 Ser Ser Leu Gly Met Glu Lys Ser Phe Ala Thr Lys Asn
Glu Ala Val 705 710 715 720 Gln Lys Glu Thr Leu Gln Glu Gly Pro Lys
Gln Glu Ala Leu Gln Glu 725 730 735 Asp Pro Leu Glu Ser Phe Asn Tyr
Val Leu Ser Lys Ser Thr Lys Ala 740 745 750 Asp Ile Gly Pro Asn Leu
Asp Gln Leu Lys Asp Asp Glu Ile Leu Arg 755 760 765 His Cys Asn Pro
Trp Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn 770 775 780 Ala Glu
Ser Asn Val Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala 785 790 795
800 Ser His Ser Ala Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu
805 810 815 Glu Glu Pro Ser Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe
Asn Leu 820 825 830 Phe Leu Glu Asp Ser Ala Asp Asn Arg Gln Asn Phe
Ser Ser Gln Ser 835 840 845 Leu Glu His Val Glu Lys Glu Asn Ser Leu
Cys Gly Ser Ala Pro Asn 850 855 860 Ser Arg Ala Gly Phe Val His Ser
Lys Thr Cys Leu Ser Trp Glu Phe 865 870 875 880 Ser Glu Lys Asp Asp
Glu Pro Glu Glu Val Val Val Lys Ala Lys Ile 885 890 895 Arg Ser Lys
Ala Arg Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp 900 905 910 Ser
Phe Lys Asp Thr Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe 915 920
925 Gln Phe Ser Ser Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp
930 935 940 Ile Ser Pro Pro Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser
Ser Val 945 950 955 960 Asn Lys Ser Met Asn Ser Arg Arg Ser Leu Ala
Ser Arg Arg Ser Leu 965 970 975 Ile Asn Met Val Leu Asp His Val Glu
Asp Met Glu Glu Arg Leu Asp 980 985 990 Asp Ser Ser Glu Ala Lys Gly
Pro Glu Asp Tyr Pro Glu Glu Gly Val 995 1000 1005 Glu Glu Ser Ser
Gly Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser 1010 1015 1020 Gly
Glu Thr Leu Ser Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser 1025
1030 1035 1040 Lys Pro Ser Ala Leu Ala Gln Glu Thr Ser Leu Gly Ala
Pro Glu Pro 1045 1050 1055 Leu Ser Gly Glu Gln Leu Val Gly Ser Pro
Gln Asp Lys Ala Ala Glu 1060 1065 1070 Ala Thr Asn Asp Tyr Glu Thr
Leu Val Lys Arg Gly Lys Glu Leu Lys 1075 1080 1085 Glu Cys Gly Lys
Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu 1090 1095 1100 Asp
Ile Lys Ser Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu 1105
1110 1115 1120 Tyr Lys Gln Leu Asn Asn Asn 1125 116 3671 DNA Homo
Sapiens 116 aaaatgaatc atgtgctgct gatcatgcca accaatctta ttaacacttg
ggtaaaagaa 60 ttcatcaagt ggactccagg aatgggagtc aaaacctttc
atggtcctag caaggatgaa 120 cggaccagaa acctcaatcg gattcagcaa
aggaatggtg ttattatcac tacataccaa 180 atgttaatca ataactggca
gcaactttca agctttaggg gccaagagtt tgtgtgggac 240 tatgtcatcc
tcgatgaagc acataaaata aaaacctcat ctactaagtc agcaatatgt 300
gctcgtgcta ttcctgcaag taatcgcctc ctcctcacag gaaccccaat ccagaataat
360 ttacaagaac tatggtccct atttgatttt gcttgtcaag ggtccctgct
gggaacatta 420 aaaactttta agatggagta tgaaaatcct attactagag
caagagagaa ggatgctacc 480 ccaggagaaa aagccttggg atttaaaata
tctgaaaact taatggcaat cataaaaccc 540 tattttctca ggaggactaa
agaagacgta cagaagaaaa agtcaagcaa cccagaggcc 600 agacttaatg
aaaagaatcc agatgttgat gccatttgtg aaatgccttc cctttccagg 660
aaaaatgatt taattatttg gatacgactt gtgcctttac aagaagaaat atacaggaaa
720 tttgtgtctt tagatcatat caaggagttg ctaatggaga cgcgctcacc
tttggctgag 780 ctaggtgtct taaagaagct gtgtgatcat cctaggctgc
tgtctgcacg ggcttgttgt 840 ttgctaaatc ttgggacatt ctctgctcaa
gatggaaatg agggggaaga ttccccagat 900 gtggaccata ttgatcaagt
aactgatgac acattgatgg aagaatctgg aaaaatgata 960 ttcctaatgg
acctacttaa gaggctgcga gatgagggac atcaaactct ggtgttttct 1020
caatcgaggc aaattctaaa catcattgaa cgcctcttaa agaataggca ctttaagaca
1080 ttgcgaatcg atgggacagt tactcatctt ttggaacgag aaaaaagaat
taacttattc 1140 cagcaaaata aagattactc tgtttttctg cttaccactc
aagtaggtgg tgtcggttta 1200 acattaactg cagcaactag agtggtcatt
tttgacccta gctggaatcc tgcaactgat 1260 gctcaagctg tggatagagt
ttaccgaatt ggacaaaaag agaatgttgt ggtttatagg 1320 ctaatcactt
gtgggactgt agaggaaaaa atatacagaa gacaggtttt caaggactca 1380
ttaataagac aaactactgg tgaaaaaaag aaccctttcc gatattttag taaacaagaa
1440 ttaagagagc tctttacaat cgaggatctt cagaactctg taacccagct
gcagcttcag 1500 tctttgcatg ctgctcagag gaaatctgat ataaaactag
atgaacatat tgcctacctg 1560 cagtctttgg ggatagctgg aatctcagac
catgatttga tgtacacatg tgatctgtct 1620 gttaaagaag agcttgatgt
ggtagaagaa tctcactata ttcaacaaag ggttcagaaa 1680 gctcaattcc
tcgttgaatt cgagtctcaa aataaagagt tcctgatgga acaacaaaga 1740
actagaaatg agggggcctg gctaagagaa cctgtatttc cttcttcaac aaagaagaaa
1800 tgccctaaat tgaataaacc acagcctcag ccttcacctc ttctaagtac
tcatcatact 1860 caggaagaag atatcagttc caaaatggca agtgtagtca
ttgatgatct gcccaaagag 1920 ggtgagaaac aagatctctc cagtataaag
gtgaatgtta ccaccttgca agatggtaaa 1980 ggtacaggta gtgctgactc
tatagctact ttaccaaagg ggtttggaag tgtagaagaa 2040 ctttgtacta
actcttcatt gggaatggaa aaaagctttg caactaaaaa tgaagctgta 2100
caaaaagaga cattacaaga ggggcctaag caagaggcac tgcaagagga tcctctggaa
2160 agttttaatt atgtacttag caaatcaacc aaagctgata ttgggccaaa
tttagatcaa 2220 ctaaaggatg atgaggtttt acgtcattgc aatccttggc
ccattatttc cataacaaat 2280 gaaagtcaaa atgcagaatc aaatgtatcc
attattgaaa tagctgatga cctttcagca 2340 tcccatagtg cactgcagga
tgctcaagca agtgaggcca agttggaaga ggaaccttca 2400 gcatcttcac
cacagtatgc atgtgatttc aatcttttct tggaagactc agcagacaac 2460
agacaaaatt tttccagtca gtctttagag catgttgaga aagaaaatag cttgtgtggc
2520 tctgcaccta attccagagc agggtttgtg catagcaaaa catgtctcag
ttgggagttt 2580 tctgagaaag acgatgaacc agaagaagta gtagttaaag
caaaaatcag aagtaaagct 2640 agaaggattg tttcagatgg cgaagatgaa
gatgattctt ttaaagatac ctcaagcata 2700 aatccattca acacatctct
ctttcaattc tcatctgtga aacaatttga tgcttcaact 2760 cccaaaaatg
acatcagtcc accaggaagg ttcttttcat ctcaaatacc cagtagtgta 2820
aataagtcta tgaactctag aagatctctg gcttctagga ggtctcttat taatatggtt
2880 ttagaccacg tggaggacat ggaggaaaga cttgacgaca gcagtgaagc
aaagggtcct 2940 gaagattatc cagaagaagg ggtggaggaa agcagtggcg
aagcctccaa gtatacagaa 3000 gaggatcctt ccggagaaac actgtcttca
gaaaacaagt ccagctggtt aatgacgtct 3060 aagcctagtg ctctagctca
agagacctct cttggtgccc ctgagccttt gtctggtgaa 3120 cagttggttg
gttcccccca ggataaggcg gcagaggcta caaatgacta tgagactctt 3180
gtaaagcgtg gaaaagaact aaaagagtgt ggaaaaatcc aggaggccct aaactgctta
3240 gttaaagcgc ttgacataaa aagtgcagat cctgaagtta tgctcttgac
tttaagtttg 3300 tataagcaac ttaataacaa ttgagaatgt aacctgttta
ttgtatttta aagtgaaact 3360 gaatatgagg gaatttttgt tcccataatt
ggattctttg ggaacatgaa gcattcaggc 3420 ttaaggcaag aaagatctca
aaaagcaact tctgccctgc aacgcccccc actccatagt 3480 ctggtattct
gagcactagc ttaatatttc ttcacttgaa tattcttata ttttaggcat 3540
attctataaa tttaactgtg ttgtttcttg gaaagttttg taaaattatt ctggtcattc
3600 ttaattttac tctgaaagtg atcatctttg tatataacag ttcagataag
aaaattaaag 3660 ttacttttct c 3671 117 3694 DNA Homo Sapiens 117
tttccggtat gtttgatgca tcacttgtga atcatgtgct gctgatcatg ccaaccaatc
60 ttattaacac atgggtaaaa gaattcatca agtggactcc aggaatgaga
gtcaaaacct 120 ttcatggtcc tagcaaggat gaacggacca gaaacctcaa
tcggattcag caaaggaatg 180 gtgttattat cactacatac caaatgttaa
tcaataactg gcagcaactt tcaagcttta 240 ggggccaaga gtttgtgtgg
gactatgtca tcctcgatga agcacataaa ataaaaacct 300 catctactaa
gtcagcaata tgtgctcgtg ctattcctgc aagtaatcgc ctcctcctca 360
caggaacccc aatccagaat aatttacaag aactatggtc cctatttgat tttgcttgtc
420 aagggtccct gctgggaaca ttaaaaactt ttaagatgga gtatgaaaat
cctattacta 480 gagcaagaga gaaggatgct accccaggag aaaaagcctt
gggatttaaa atatctgaaa 540 acttaatggc aatcataaaa ccctattttc
tcaggaggac taaagaagac gtacagaaga 600 aaaagtcaag caacccagag
gccagactta atgaaaagaa tccagatgtt gatgccattt 660 gtgaaatgcc
ttccctttcc aggaaaaatg atttaattat ttggatacga cttgtgcctt 720
tacaagaaga aatatacagg aaatttgtgt ctttagatca tatcaaggag ttgctaatgg
780 agacgcgctc acctttggct gagctaggtg tcttaaagaa gctgtgtgat
catcctaggc 840 tgctgtctgc acgggcttgt tgtttgctaa atcttgggac
attctctgct caagatggaa 900 atgaggggga agattcccca gatgtggacc
atattgatca agtaactgat gacacattga 960 tggaagaatc tggaaaaatg
atattcctaa tggacctact taagaggctg cgagatgagg 1020 gacatcaaac
tctggtgttt tctcaatcga ggcaaattct aaacatcatt gaacgcctct 1080
taaagaatag gcactttaag acattgcgaa tcgatgggac agttactcat cttttggaac
1140 gagaaaaaag aattaactta ttccagcaaa ataaagatta ctctgttttt
ctgcttacca 1200 ctcaagtagg tggtgtcggt ttaacattaa ctgcagcaac
tagagtggtc atttttgacc 1260 ctagctggaa tcctgcaact gatgctcaag
ctgtggatag agtttaccga attggacaaa 1320 aagagaatgt tgtggtttat
aggctaatca cttgtgggac tgtagaggaa aaaatataca 1380 gaagacaggt
tttcaaggac tcattaataa gacaaactac tggtgaaaaa aagaaccctt 1440
tccgatattt tagtaaacaa gaattaagag agctctttac aatcgaggat cttcagaact
1500 ctgtaaccca gctgcagctt cagtctttgc atgctgctca gaggaaatct
gatataaaac 1560 tagatgaaca tattgcctac ctgcagtctt tggggatagc
tggaatctca gaccatgatt 1620 tgatgtacac atgtgatctg tctgttaaag
aagagcttga tgtggtagaa gaatctcact 1680 atattcaaca aagggttcag
aaagctcaat tcctcgttga attcgagtct caaaataaag 1740 agttcctgat
ggaacaacaa agaactagaa atgagggggc ctggctaaga gaacctgtat 1800
ttccttcttc aacaaagaag aaatgcccta aattgaataa accacagcct cagccttcac
1860 ctcttctaag tactcatcat actcaggaag aagatatcag ttccaaaatg
gcaagtgtag 1920 tcattgatga tctgcccaaa gagggtgaga aacaagatct
ctccagtata aaggtgaatg 1980 ttaccacctt gcaagatggt aaaggtacag
gtagtgctga ctctatagct actttaccaa 2040 aggggtttgg aagtgtagaa
gaactttgta ctaactcttc attgggaatg gaaaaaagct 2100 ttgcaactaa
aaatgaagct gtacaaaaag agacattaca agaggggcct aagcaagagg 2160
cactgcaaga ggatcctctg gaaagtttta attatgtact tagcaaatca accaaagctg
2220 atattgggcc aaatttagat caactaaagg atgatgagat tttacgtcat
tgcaatcctt 2280 ggcccattat ttccataaca aatgaaagtc aaaatgcaga
atcaaatgta tccattattg 2340 aaatagctga tgacctttca gcatcccata
gtgcactgca ggatgctcaa gcaagtgagg 2400 ccaagttgga agaggaacct
tcagcatctt caccacagta tgcatgtgat ttcaatcttt 2460 tcttggaaga
ctcagcagac aacagacaaa atttttccag tcagtcttta gagcatgttg 2520
agaaagaaaa tagcttgtgt ggctctgcac ctaattccag agcagggttt gtgcatagca
2580 aaacatgtct cagttgggag ttttctgaga aagacgatga accagaagaa
gtagtagtta 2640 aagcaaaaat cagaagtaaa gctagaagga ttgtttcaga
tggcgaagat gaagatgatt 2700 cttttaaaga tacctcaagc ataaatccat
tcaacacatc tctctttcaa ttctcatctg 2760 tgaaacaatt tgatgcttca
actcccaaaa atgacatcag tccaccagga aggttctttt 2820 catctcaaat
acccagtagt gtaaataagt ctatgaactc tagaagatct ctggcttcta 2880
ggaggtctct tattaatatg gttttagacc acgtggagga catggaggaa agacttgacg
2940 acagcagtga agcaaagggt cctgaagatt atccagaaga aggggtggag
gaaagcagtg 3000 gcgaagcctc caagtataca gaagaggatc cttccggaga
aacactgtct tcagaaaaca 3060 agtccagctg gttaatgacg tctaagccta
gtgctctagc tcaagagacc tctcttggtg 3120 cccctgagcc tttgtctggt
gaacagttgg ttggttctcc ccaggataag gcggcagagg 3180 ctacaaatga
ctatgagact cttgtaaagc gtggaaaaga actaaaagag tgtggaaaaa 3240
tccaggaggc cctaaactgc ttagttaaag cgcttgacat aaaaagtgca gatcctgaag
3300 ttatgctctt gactttaagt ttgtataagc aacttaataa caattgagaa
tgtaacctgt 3360 ttattgtatt ttaaagtgaa actgaatatg agggaatttt
tgttcccata attggattct 3420 ttgggaacat gaagcattca ggcttaaggc
aagaaagatc tcaaaaagca acttctgccc 3480 tgcaacgccc cccactccat
agtctggtat tctgagcact agcttaatat ttcttcactt 3540 gaatattctt
atattttagg catattctat aaatttaact gtgttgtttc ttggaaagtt 3600
ttgtaaaatt attctggtca ttcttaattt tactctgaaa gtgatcatct ttgtatataa
3660 cagttcagat aagaaaatta aagttacttt tctc 3694 118 3671 DNA Homo
Sapiens 118 aaaatgaatc atgtgctgct gatcatgcca accaatctta ttaacacttg
ggtaaaagaa 60 ttcatcaagt ggactccagg aatgggagtc aaaacctttc
atggtcctag caaggatgaa 120 cggaccagaa acctcaatcg gattcagcaa
aggaatggtg ttattatcac tacataccaa 180 atgttaatca ataactggca
gcaactttca agctttaggg gccaagagtt tgtgtgggac 240 tatgtcatcc
tcgatgaagc acataaaata aaaacctcat ctactaagtc agcaatatgt 300
gctcgtgcta ttcctgcaag taatcgcctc ctcctcacag gaaccccaat ccagaataat
360 ttacaagaac tatggtccct atttgatttt gcttgtcaag ggtccctgct
gggaacatta 420 aaaactttta agatggagta tgaaaatcct attactagag
caagagagaa ggatgctacc 480 ccaggagaaa aagccttggg atttaaaata
tctgaaaact taatggcaat cataaaaccc 540 tattttctca ggaggactaa
agaagacgta cagaagaaaa agtcaagcaa cccagaggcc 600 agacttaatg
aaaagaatcc agatgttgat gccatttgtg aaatgccttc cctttccagg 660
aaaaatgatt taattatttg gatacgactt gtgcctttac aagaagaaat atacaggaaa
720 tttgtgtctt tagatcatat caaggagttg ctaatggaga cgcgctcacc
tttggctgag 780 ctaggtgtct taaagaagct gtgtgatcat cctaggctgc
tgtctgcacg ggcttgttgt 840 ttgctaaatc ttgggacatt ctctgctcaa
gatggaaatg agggggaaga ttccccagat 900 gtggaccata ttgatcaagt
aactgatgac acattgatgg aagaatctgg aaaaatgata 960 ttcctaatgg
acctacttaa gaggctgcga gatgagggac atcaaactct ggtgttttct 1020
caatcgaggc aaattctaaa catcattgaa cgcctcttaa agaataggca ctttaagaca
1080 ttgcgaatcg atgggacagt tactcatctt ttggaacgag aaaaaagaat
taacttattc 1140 cagcaaaata aagattactc tgtttttctg cttaccactc
aagtaggtgg tgtcggttta 1200 acattaactg cagcaactag agtggtcatt
tttgacccta gctggaatcc tgcaactgat 1260 gctcaagctg tggatagagt
ttaccgaatt ggacaaaaag agaatgttgt ggtttatagg 1320 ctaatcactt
gtgggactgt agaggaaaaa atatacagaa gacaggtttt caaggactca 1380
ttaataagac aaactactgg tgaaaaaaag aaccctttcc gatattttag taaacaagaa
1440 ttaagagagc tctttacaat cgaggatctt cagaactctg taacccagct
gcagcttcag 1500 tctttgcatg ctgctcagag gaaatctgat ataaaactag
atgaacatat tgcctacctg 1560 cagtctttgg ggatagctgg aatctcagac
catgatttga tgtacacatg tgatctgtct 1620 gttaaagaag agcttgatgt
ggtagaagaa tctcactata ttcaacaaag ggttcagaaa 1680 gctcaattcc
tcgttgaatt cgagtctcaa aataaagagt tcctgatgga acaacaaaga 1740
actagaaatg agggggcctg gctaagagaa cctgtatttc cttcttcaac aaagaagaaa
1800 tgccctaaat tgaataaacc acagcctcag ccttcacctc ttctaagtac
tcatcatact 1860 caggaagaag atatcagttc caaaatggca agtgtagtca
ttgatgatct gcccaaagag 1920 ggtgagaaac aagatctctc cagtataaag
gtgaatgtta ccaccttgca agatggtaaa 1980 ggtacaggta gtgctgactc
tatagctact ttaccaaagg ggtttggaag tgtagaagaa 2040 ctttgtacta
actcttcatt gggaatggaa aaaagctttg caactaaaaa tgaagctgta 2100
caaaaagaga cattacaaga ggggcctaag caagaggcac tgcaagagga tcctctggaa
2160 agttttaatt atgtacttag caaatcaacc aaagctgata ttgggccaaa
tttagatcaa 2220 ctaaaggatg atgaggtttt acgtcattgc aatccttggc
ccattatttc cataacaaat 2280 gaaagtcaaa atgcagaatc aaatgtatcc
attattgaaa tagctgatga cctttcagca 2340 tcccatagtg cactgcagga
tgctcaagca agtgaggcca agttggaaga ggaaccttca 2400 gcatcttcac
cacagtatgc atgtgatttc aatcttttct tggaagactc agcagacaac 2460
agacaaaatt tttccagtca gtctttagag catgttgaga aagaaaatag cttgtgtggc
2520 tctgcaccta attccagagc agggtttgtg catagcaaaa catgtctcag
ttgggagttt 2580 tctgagaaag acgatgaacc agaagaagta gtagttaaag
caaaaatcag aagtaaagct 2640 agaaggattg tttcagatgg cgaagatgaa
gatgattctt ttaaagatac ctcaagcata 2700 aatccattca acacatctct
ctttcaattc tcatctgtga aacaatttga tgcttcaact 2760 cccaaaaatg
acatcagtcc accaggaagg ttcttttcat ctcaaatacc cagtagtgta 2820
aataagtcta tgaactctag aagatctctg gcttctagga ggtctcttat taatatggtt
2880 ttagaccacg tggaggacat ggaggaaaga cttgacgaca gcagtgaagc
aaagggtcct 2940 gaagattatc cagaagaagg ggtggaggaa agcagtggcg
aagcctccaa gtatacagaa 3000 gaggatcctt ccggagaaac actgtcttca
gaaaacaagt ccagctggtt aatgacgtct 3060 aagcctagtg ctctagctca
agagacctct cttggtgccc ctgagccttt gtctggtgaa 3120 cagttggttg
gttcccccca ggataaggcg gcagaggcta caaatgacta tgagactctt 3180
gtaaagcgtg gaaaagaact aaaagagtgt ggaaaaatcc aggaggccct aaactgctta
3240 gttaaagcgc ttgacataaa aagtgcagat cctgaagtta tgctcttgac
tttaagtttg 3300 tataagcaac ttaataacaa ttgagaatgt aacctgttta
ttgtatttta aagtgaaact 3360 gaatatgagg gaatttttgt tcccataatt
ggattctttg ggaacatgaa gcattcaggc 3420 ttaaggcaag aaagatctca
aaaagcaact tctgccctgc aacgcccccc actccatagt 3480 ctggtattct
gagcactagc ttaatatttc ttcacttgaa tattcttata ttttaggcat 3540
attctataaa tttaactgtg ttgtttcttg gaaagttttg taaaattatt ctggtcattc
3600 ttaattttac tctgaaagtg atcatctttg tatataacag ttcagataag
aaaattaaag 3660 ttacttttct c 3671 119 1106 PRT Homo Sapiens 119 Met
Asn His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp 1 5 10
15 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Gly Val Lys Thr Phe
20 25 30 His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu Asn Arg
Ile Gln 35 40 45 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr Gln Met
Leu Ile Asn Asn 50 55 60 Trp Gln Gln Leu Ser Ser Phe Arg Gly Gln
Glu Phe Val Trp Asp Tyr 65 70 75 80 Val Ile Leu Asp Glu Ala His Lys
Ile Lys Thr Ser Ser Thr Lys Ser 85 90 95 Ala Ile Cys Ala Arg Ala
Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 100 105 110 Gly Thr Pro Ile
Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 115 120 125 Phe Ala
Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 130 135 140
Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr Pro 145
150 155 160 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn Leu Met
Ala Ile 165 170 175 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp
Val Gln Lys Lys 180 185 190 Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn
Glu Lys Asn Pro Asp Val 195 200 205 Asp Ala Ile Cys Glu Met Pro Ser
Leu Ser Arg Lys Asn Asp Leu Ile 210 215 220 Ile Trp Ile Arg Leu Val
Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 225 230 235 240 Val Ser Leu
Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 245 250 255 Leu
Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu 260 265
270 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala
275 280 285 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val Asp His
Ile Asp 290 295 300 Gln Val Thr Asp Asp Thr Leu Met Glu Glu Ser Gly
Lys Met Ile Phe 305 310 315 320 Leu Met Asp Leu Leu Lys Arg Leu Arg
Asp Glu Gly His Gln Thr Leu 325 330 335 Val Phe Ser Gln Ser Arg Gln
Ile Leu Asn Ile Ile Glu Arg Leu Leu 340 345 350 Lys Asn Arg His Phe
Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 355 360 365 Leu Leu Glu
Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 370 375 380 Tyr
Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr 385 390
395 400 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser Trp Asn
Pro 405 410 415 Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr Arg Ile
Gly Gln Lys 420 425 430 Glu Asn Val Val Val Tyr Arg Leu Ile Thr Cys
Gly Thr Val Glu Glu 435 440 445 Lys Ile Tyr Arg Arg Gln Val Phe Lys
Asp Ser Leu Ile Arg Gln Thr 450 455 460 Thr Gly Glu Lys Lys Asn Pro
Phe Arg Tyr Phe Ser Lys Gln Glu Leu 465 470 475 480 Arg Glu Leu Phe
Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 485 490 495 Gln Leu
Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 500 505 510
Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser 515
520 525 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu
Leu 530 535 540 Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln Arg Val
Gln Lys Ala 545 550 555 560 Gln Phe Leu Val Glu Phe Glu Ser Gln Asn
Lys Glu Phe Leu Met Glu 565 570 575 Gln Gln Arg Thr Arg Asn Glu Gly
Ala Trp Leu Arg Glu Pro Val Phe 580 585 590 Pro Ser Ser Thr Lys Lys
Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 595 600 605 Gln Pro Ser Pro
Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 610 615 620 Ser Ser
Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly 625 630 635
640 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln
645 650 655 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu
Pro Lys 660 665 670 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser
Ser Leu Gly Met 675 680 685 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr Leu 690 695 700 Gln Glu Gly Pro Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu Ser 705 710 715 720 Phe Asn Tyr Val Leu
Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 725 730 735 Leu Asp Gln
Leu Lys Asp Asp Glu Val Leu Arg His Cys Asn Pro Trp 740 745 750 Pro
Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 755 760
765 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu
770 775 780 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro
Ser Ala 785 790 795 800 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu
Phe Leu Glu Asp Ser 805 810 815 Ala Asp Asn Arg Gln Asn Phe Ser Ser
Gln Ser Leu Glu His Val Glu 820 825 830 Lys Glu Asn Ser Leu Cys Gly
Ser Ala Pro Asn Ser Arg Ala Gly Phe 835 840 845 Val His Ser Lys Thr
Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 850 855 860 Glu Pro Glu
Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 865 870 875 880
Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 885
890 895 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser
Val 900 905 910 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser
Pro Pro Gly 915 920 925 Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val
Asn Lys Ser Met Asn 930 935 940 Ser Arg Arg Ser Leu Ala Ser Arg Arg
Ser Leu Ile Asn Met Val Leu 945 950 955 960 Asp His Val Glu Asp Met
Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 965 970 975 Lys Gly Pro Glu
Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 980 985 990 Glu Ala
Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 995 1000
1005 Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala
Leu 1010 1015 1020 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu
Ser Gly Glu Gln 1025 1030 1035 1040 Leu Val Gly Ser Pro Gln Asp Lys
Ala Ala Glu Ala Thr Asn Asp Tyr 1045 1050 1055 Glu Thr Leu Val Lys
Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1060 1065 1070 Gln Glu
Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1075 1080
1085 Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln Leu
Asn 1090 1095 1100 Asn Asn 1105 120 1150 PRT Homo Sapiens 120 Lys
Glu Gly Ile Ala Phe Leu Tyr Ser Leu Tyr Arg Asp Gly Arg Lys 1 5 10
15 Gly Gly Ile Leu Ala Asp Asp Met Gly Leu Gly Lys Thr Val Gln Ile
20 25 30 Ile Ala Phe Leu Ser Gly Met Phe Asp Ala Ser Leu Val Asn
His Val 35 40 45 Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp
Val Lys Glu Phe 50 55 60 Ile Lys Trp Thr Pro Gly Met Arg Val Lys
Thr Phe His Gly Pro Ser 65 70 75 80 Lys Asp Glu Arg Thr Arg Asn Leu
Asn Arg Ile Gln Gln Arg Asn Gly 85 90 95 Val Ile Ile Thr Thr Tyr
Gln Met Leu Ile Asn Asn Trp Gln Gln Leu 100 105 110 Ser Ser Phe Arg
Gly Gln Glu Phe Val Trp Asp Tyr Val Ile Leu Asp 115 120 125 Glu Ala
His Lys Ile Lys Thr Ser Ser Thr Lys Ser Ala Ile Cys Ala 130 135 140
Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr Gly Thr Pro Ile 145
150 155 160 Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp Phe Ala
Cys Gln 165 170 175 Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met
Glu Tyr Glu Asn 180 185 190 Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala
Thr Pro Gly Glu Lys Ala 195 200 205 Leu Gly Phe Lys Ile Ser Glu Asn
Leu Met Ala Ile Ile Lys Pro Tyr 210 215 220 Phe Leu Arg Arg Thr Lys
Glu Asp Val Gln Lys Lys Lys Ser Ser Asn 225 230 235 240 Pro Glu Ala
Arg Leu Asn Glu Lys Asn Pro Asp Val Asp Ala Ile Cys 245 250 255 Glu
Met Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile Ile Trp Ile Arg 260 265
270 Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe Val Ser Leu Asp
275 280 285 His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro Leu Ala
Glu Leu 290 295 300 Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu
Leu Ser Ala Arg 305 310 315 320 Ala Cys Cys Leu Leu Asn Leu Gly Thr
Phe Ser Ala Gln Asp Gly Asn 325 330 335 Glu Gly Glu Asp Ser Pro Asp
Val Asp His Ile Asp Gln Val Thr Asp 340 345 350 Asp Thr Leu Met Glu
Glu Ser Gly Lys Met Ile Phe Leu Met Asp Leu 355 360 365 Leu Lys Arg
Leu Arg Asp Glu Gly His Gln Thr Leu Val Phe Ser Gln 370 375 380 Ser
Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu Lys Asn Arg His 385 390
395 400 Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His Leu Leu Glu
Arg 405 410 415 Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp Tyr
Ser Val Phe 420 425 430 Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu
Thr Leu Thr Ala Ala 435 440 445 Thr Arg Val Val Ile Phe Asp Pro Ser
Trp Asn Pro
Ala Thr Asp Ala 450 455 460 Gln Ala Val Asp Arg Val Tyr Arg Ile Gly
Gln Lys Glu Asn Val Val 465 470 475 480 Val Tyr Arg Leu Ile Thr Cys
Gly Thr Val Glu Glu Lys Ile Tyr Arg 485 490 495 Arg Gln Val Phe Lys
Asp Ser Leu Ile Arg Gln Thr Thr Gly Glu Lys 500 505 510 Lys Asn Pro
Phe Arg Tyr Phe Ser Lys Gln Glu Leu Arg Glu Leu Phe 515 520 525 Thr
Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu Gln Leu Gln Ser 530 535
540 Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu Asp Glu His Ile
545 550 555 560 Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile Ser Asp
His Asp Leu 565 570 575 Met Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu
Leu Asp Val Val Glu 580 585 590 Glu Ser His Tyr Ile Gln Gln Arg Val
Gln Lys Ala Gln Phe Leu Val 595 600 605 Glu Phe Glu Ser Gln Asn Lys
Glu Phe Leu Met Glu Gln Gln Arg Thr 610 615 620 Arg Asn Glu Gly Ala
Trp Leu Arg Glu Pro Val Phe Pro Ser Ser Thr 625 630 635 640 Lys Lys
Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro Gln Pro Ser Pro 645 650 655
Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile Ser Ser Lys Met 660
665 670 Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly Glu Lys Gln
Asp 675 680 685 Leu Ser Ser Ile Lys Val Asn Val Thr Thr Leu Gln Asp
Gly Lys Gly 690 695 700 Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro
Lys Gly Phe Gly Ser 705 710 715 720 Val Glu Glu Leu Cys Thr Asn Ser
Ser Leu Gly Met Glu Lys Ser Phe 725 730 735 Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr Leu Gln Glu Gly Pro 740 745 750 Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu Ser Phe Asn Tyr Val 755 760 765 Leu Ser
Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn Leu Asp Gln Leu 770 775 780
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp Pro Ile Ile Ser 785
790 795 800 Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val Ser Ile
Ile Glu 805 810 815 Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu
Gln Asp Ala Gln 820 825 830 Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro
Ser Ala Ser Ser Pro Gln 835 840 845 Tyr Ala Cys Asp Phe Asn Leu Phe
Leu Glu Asp Ser Ala Asp Asn Arg 850 855 860 Gln Asn Phe Ser Ser Gln
Ser Leu Glu His Val Glu Lys Glu Asn Ser 865 870 875 880 Leu Cys Gly
Ser Ala Pro Asn Ser Arg Ala Gly Phe Val His Ser Lys 885 890 895 Thr
Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp Glu Pro Glu Glu 900 905
910 Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg Arg Ile Val Ser
915 920 925 Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr Ser Ser
Ile Asn 930 935 940 Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val
Lys Gln Phe Asp 945 950 955 960 Ala Ser Thr Pro Lys Asn Asp Ile Ser
Pro Pro Gly Arg Phe Phe Ser 965 970 975 Ser Gln Ile Pro Ser Ser Val
Asn Lys Ser Met Asn Ser Arg Arg Ser 980 985 990 Leu Ala Ser Arg Arg
Ser Leu Ile Asn Met Val Leu Asp His Val Glu 995 1000 1005 Asp Met
Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala Lys Gly Pro Glu 1010 1015
1020 Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly Glu Ala Ser
Lys 1025 1030 1035 1040 Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu
Ser Ser Glu Asn Lys 1045 1050 1055 Ser Ser Trp Leu Met Thr Ser Lys
Pro Ser Ala Leu Ala Gln Glu Thr 1060 1065 1070 Ser Leu Gly Ala Pro
Glu Pro Leu Ser Gly Glu Gln Leu Val Gly Ser 1075 1080 1085 Pro Gln
Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr Glu Thr Leu Val 1090 1095
1100 Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile Gln Glu Ala
Leu 1105 1110 1115 1120 Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser
Ala Asp Pro Glu Val 1125 1130 1135 Met Leu Leu Thr Leu Ser Leu Tyr
Lys Gln Leu Asn Asn Asn 1140 1145 1150 121 1106 PRT Homo Sapiens
121 Met Asn His Val Leu Leu Ile Met Pro Thr Asn Leu Ile Asn Thr Trp
1 5 10 15 Val Lys Glu Phe Ile Lys Trp Thr Pro Gly Met Gly Val Lys
Thr Phe 20 25 30 His Gly Pro Ser Lys Asp Glu Arg Thr Arg Asn Leu
Asn Arg Ile Gln 35 40 45 Gln Arg Asn Gly Val Ile Ile Thr Thr Tyr
Gln Met Leu Ile Asn Asn 50 55 60 Trp Gln Gln Leu Ser Ser Phe Arg
Gly Gln Glu Phe Val Trp Asp Tyr 65 70 75 80 Val Ile Leu Asp Glu Ala
His Lys Ile Lys Thr Ser Ser Thr Lys Ser 85 90 95 Ala Ile Cys Ala
Arg Ala Ile Pro Ala Ser Asn Arg Leu Leu Leu Thr 100 105 110 Gly Thr
Pro Ile Gln Asn Asn Leu Gln Glu Leu Trp Ser Leu Phe Asp 115 120 125
Phe Ala Cys Gln Gly Ser Leu Leu Gly Thr Leu Lys Thr Phe Lys Met 130
135 140 Glu Tyr Glu Asn Pro Ile Thr Arg Ala Arg Glu Lys Asp Ala Thr
Pro 145 150 155 160 Gly Glu Lys Ala Leu Gly Phe Lys Ile Ser Glu Asn
Leu Met Ala Ile 165 170 175 Ile Lys Pro Tyr Phe Leu Arg Arg Thr Lys
Glu Asp Val Gln Lys Lys 180 185 190 Lys Ser Ser Asn Pro Glu Ala Arg
Leu Asn Glu Lys Asn Pro Asp Val 195 200 205 Asp Ala Ile Cys Glu Met
Pro Ser Leu Ser Arg Lys Asn Asp Leu Ile 210 215 220 Ile Trp Ile Arg
Leu Val Pro Leu Gln Glu Glu Ile Tyr Arg Lys Phe 225 230 235 240 Val
Ser Leu Asp His Ile Lys Glu Leu Leu Met Glu Thr Arg Ser Pro 245 250
255 Leu Ala Glu Leu Gly Val Leu Lys Lys Leu Cys Asp His Pro Arg Leu
260 265 270 Leu Ser Ala Arg Ala Cys Cys Leu Leu Asn Leu Gly Thr Phe
Ser Ala 275 280 285 Gln Asp Gly Asn Glu Gly Glu Asp Ser Pro Asp Val
Asp His Ile Asp 290 295 300 Gln Val Thr Asp Asp Thr Leu Met Glu Glu
Ser Gly Lys Met Ile Phe 305 310 315 320 Leu Met Asp Leu Leu Lys Arg
Leu Arg Asp Glu Gly His Gln Thr Leu 325 330 335 Val Phe Ser Gln Ser
Arg Gln Ile Leu Asn Ile Ile Glu Arg Leu Leu 340 345 350 Lys Asn Arg
His Phe Lys Thr Leu Arg Ile Asp Gly Thr Val Thr His 355 360 365 Leu
Leu Glu Arg Glu Lys Arg Ile Asn Leu Phe Gln Gln Asn Lys Asp 370 375
380 Tyr Ser Val Phe Leu Leu Thr Thr Gln Val Gly Gly Val Gly Leu Thr
385 390 395 400 Leu Thr Ala Ala Thr Arg Val Val Ile Phe Asp Pro Ser
Trp Asn Pro 405 410 415 Ala Thr Asp Ala Gln Ala Val Asp Arg Val Tyr
Arg Ile Gly Gln Lys 420 425 430 Glu Asn Val Val Val Tyr Arg Leu Ile
Thr Cys Gly Thr Val Glu Glu 435 440 445 Lys Ile Tyr Arg Arg Gln Val
Phe Lys Asp Ser Leu Ile Arg Gln Thr 450 455 460 Thr Gly Glu Lys Lys
Asn Pro Phe Arg Tyr Phe Ser Lys Gln Glu Leu 465 470 475 480 Arg Glu
Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser Val Thr Gln Leu 485 490 495
Gln Leu Gln Ser Leu His Ala Ala Gln Arg Lys Ser Asp Ile Lys Leu 500
505 510 Asp Glu His Ile Ala Tyr Leu Gln Ser Leu Gly Ile Ala Gly Ile
Ser 515 520 525 Asp His Asp Leu Met Tyr Thr Cys Asp Leu Ser Val Lys
Glu Glu Leu 530 535 540 Asp Val Val Glu Glu Ser His Tyr Ile Gln Gln
Arg Val Gln Lys Ala 545 550 555 560 Gln Phe Leu Val Glu Phe Glu Ser
Gln Asn Lys Glu Phe Leu Met Glu 565 570 575 Gln Gln Arg Thr Arg Asn
Glu Gly Ala Trp Leu Arg Glu Pro Val Phe 580 585 590 Pro Ser Ser Thr
Lys Lys Lys Cys Pro Lys Leu Asn Lys Pro Gln Pro 595 600 605 Gln Pro
Ser Pro Leu Leu Ser Thr His His Thr Gln Glu Glu Asp Ile 610 615 620
Ser Ser Lys Met Ala Ser Val Val Ile Asp Asp Leu Pro Lys Glu Gly 625
630 635 640 Glu Lys Gln Asp Leu Ser Ser Ile Lys Val Asn Val Thr Thr
Leu Gln 645 650 655 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala
Thr Leu Pro Lys 660 665 670 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr
Asn Ser Ser Leu Gly Met 675 680 685 Glu Lys Ser Phe Ala Thr Lys Asn
Glu Ala Val Gln Lys Glu Thr Leu 690 695 700 Gln Glu Gly Pro Lys Gln
Glu Ala Leu Gln Glu Asp Pro Leu Glu Ser 705 710 715 720 Phe Asn Tyr
Val Leu Ser Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 725 730 735 Leu
Asp Gln Leu Lys Asp Asp Glu Val Leu Arg His Cys Asn Pro Trp 740 745
750 Pro Ile Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val
755 760 765 Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser
Ala Leu 770 775 780 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu
Glu Pro Ser Ala 785 790 795 800 Ser Ser Pro Gln Tyr Ala Cys Asp Phe
Asn Leu Phe Leu Glu Asp Ser 805 810 815 Ala Asp Asn Arg Gln Asn Phe
Ser Ser Gln Ser Leu Glu His Val Glu 820 825 830 Lys Glu Asn Ser Leu
Cys Gly Ser Ala Pro Asn Ser Arg Ala Gly Phe 835 840 845 Val His Ser
Lys Thr Cys Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 850 855 860 Glu
Pro Glu Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 865 870
875 880 Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp
Thr 885 890 895 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe
Ser Ser Val 900 905 910 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp
Ile Ser Pro Pro Gly 915 920 925 Arg Phe Phe Ser Ser Gln Ile Pro Ser
Ser Val Asn Lys Ser Met Asn 930 935 940 Ser Arg Arg Ser Leu Ala Ser
Arg Arg Ser Leu Ile Asn Met Val Leu 945 950 955 960 Asp His Val Glu
Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala 965 970 975 Lys Gly
Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 980 985 990
Glu Ala Ser Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 995
1000 1005 Ser Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser
Ala Leu 1010 1015 1020 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro
Leu Ser Gly Glu Gln 1025 1030 1035 1040 Leu Val Gly Ser Pro Gln Asp
Lys Ala Ala Glu Ala Thr Asn Asp Tyr 1045 1050 1055 Glu Thr Leu Val
Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys Ile 1060 1065 1070 Gln
Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile Lys Ser Ala 1075
1080 1085 Asp Pro Glu Val Met Leu Leu Thr Leu Ser Leu Tyr Lys Gln
Leu Asn 1090 1095 1100 Asn Asn 1105 122 2294 DNA Homo Sapiens 122
tcattaataa gacaaactac tggtgaaaaa aagaaccctt tccgatattt tagtaaacaa
60 gaattaagag agctctttac aatcgaggat cttcagaact ctgtaaccca
gctgcagctt 120 cagtctttgc atgctgctca gaggaaatct gatataaaac
tagatgaaca tattgcctac 180 ctgcagtctt tggggatagc tggaatctca
gaccatgatt tgatgtacac atgtgatctg 240 tctgttaaag aagagcttga
tgtggtagaa gaatctcact atattcaaca aagggttcag 300 aaagctcaat
tcctcgttga attcgagtct caaaataaag agttcctgat ggaacaacaa 360
agaactagaa atgagggggc ctggctaaga gaacctgtat ttccttcttc aacaaagaag
420 aaatgcccta aattgaataa accacagcct cagccttcac ctcttctaag
tactcatcat 480 actcaggaag aagatatcag ttccaaaatg gcaagtgtag
tcattgatga tctgcccaaa 540 gagggtgaga aacaagatct ctccagtata
aaggtgaatg ttaccacctt gcaagatggg 600 taaggtacag gtagtgctga
ctctataact actttaccaa aggggtttgg aagtgtagaa 660 gaactttgta
ctaactcttc attgggaatg gaaaaaagct ttgcaactaa aaatgaagct 720
gtacaaaaag agacattaca agaggggcct aagcaggagg cactgcaaga ggatcctctg
780 gaaagtttta attatgtact tagcaaatca accaaagctg atattgggcc
aaatttagat 840 caactaaagg atgatgagat tttacgtcat tgcaatcctt
ggcccattat ttccataaca 900 aatgaaagtc aaaatgcaga atcaaatgta
tccattattg aaatagctga tgacctttca 960 gcatcccata gtgcactgca
ggatgctcaa gcaagtgagg ccaagttgga agaggaacct 1020 tcagcatctt
caccacagta tgcatgtgat ttcaatcttt tcttggaaga ctcagcagac 1080
aacagacaaa atttttccag tcagtcttta gagcatgttg agaaagaaaa tagcttgtgt
1140 ggctctgcac ctaattccaa agcagggttt gtgcatagca aaacatgtct
cagttgggag 1200 ttttctgaga aagacgatga accagaagaa gtagtagtta
aagcaaaaat cagaagtaaa 1260 gctagaagga ttgtttcaga tggcgaagat
gaagatgatt cttttaaaga tacctcaagc 1320 ataaatccat tcaacacatc
tctctttcaa ttctcatctg tgaaacaatt tgatgcttca 1380 actcccaaaa
atgacatcag tccaccagga aggttctttt catctcaaat acccagtagt 1440
gtaaataagt ctatgaactc tagaagatct ctggcttcta ggaggtctct tattaatatg
1500 gttttagacc acgtggagga catggaggaa agacttgacg acagcagtga
agcaaagggt 1560 cctgaagatt atccagaaga aggggtggag gaaagcagtg
gcgaagcctc caagtataca 1620 gaagaggatc cttccggaga aacactgtct
tcagaaaaca agtccagctg gttaatgacg 1680 tctaagccta gtgctctagc
tcaagagacc tctcttggtg cccctgagcc tttgtctggt 1740 gaacagttgg
ttggttctcc ccaggataag gcggcagagg ctacaaatga ctatgagact 1800
cttgtaaagc gtggaaaaga actaaaagag tgtggaaaaa tccaggaggc cctaaactgc
1860 ttagttaaag cgcttgacat aaaaagtgca gatcctgaag ttatgctctt
gactttaagt 1920 ttgtataagc aacttaataa caattgagaa tgtaacctgt
ttattgtatt ttaaagtgaa 1980 actgaatatg agggaatttt tgttcccata
attggattct ttgggaacat gaagcattca 2040 ggcttaaggc aagaaagatc
tcaaaaagca acttctgccc tgcaacgccc cccactccat 2100 agtctggtat
tctgagcact agcttaatat ttcttcactt gaatattctt atattttagg 2160
catattctat aaatttaact gtgttgtttc ttggaaagtt ttgtaaaatt attctggtca
2220 ttcttaattt tactctgaaa gtgatcatct ttgtatataa cagttcagat
aagaaaatta 2280 aagttacttt tctc 2294 123 2294 DNA Homo Sapiens 123
tcattaataa gacaaactac tggtgaaaaa aagaaccctt tccgatattt tagtaaacaa
60 gaattaagag agctctttac aatcgaggat cttcagaact ctgtaaccca
gctgcagctt 120 cagtctttgc atgctgctca gaggaaatct gatataaaac
tagatgaaca tattgcctac 180 ctgcagtctt tggggatagc tggaatctca
gaccatgatt tgatgtacac atgtgatctg 240 tctgttaaag aagagcttga
tgtggtagaa gaatctcact atattcaaca aagggttcag 300 aaagctcaat
tcctcgttga attcgagtct caaaataaag agttcctgat ggaacaacaa 360
agaactagaa atgagggggc ctggctaaga gaacctgtat ttccttcttc aacaaagaag
420 aaatgcccta aattgaataa accacagcct cagccttcac ctcttctaag
tactcatcat 480 actcaggaag aagatatcag ttccaaaatg gcaagtgtag
tcattgatga tctgcccaaa 540 gagggtgaga aacaagatct ctccagtata
aaggtgaatg ttaccacctt gcaagatggt 600 aaaggtacag gtagtgctga
ctctatagct actttaccaa aggggtttgg aagtgtagaa 660 gaactttgta
ctaactcttc attgggaatg gaaaaaagct ttgcaactaa aaatgaagct 720
gtacaaaaag agacattaca agaggggcct aagcaagagg cactgcaaga ggatcctctg
780 gaaagtttta attatgtact tagcaaatca accaaagctg atattgggcc
aaatttagat 840 caactaaagg atgatgagat tttacgtcat tgcaatcctt
ggcccattat ttccataaca 900 aatgaaagtc aaaatgcaga atcaaatgta
tccattattg aaatagctga tgacctttca 960 gcatcccata gtgcactgca
ggatgctcaa gcaagtgagg ccaagttgga agaggaacct 1020 tcagcatctt
caccacagta tgcatgtgat ttcaatcttt tcttggaaga ctcagcagac 1080
aacagacaaa atttttccag tcagtcttta gagcatgttg agaaagaaaa tagcttgtgt
1140 ggctctgcac ctaattccag agcagggttt gtgcatagca aaacatgtct
cagttgggag 1200 ttttctgaga aagacgatga accagaagaa gtagtagtta
aagcaaaaat cagaagtaaa 1260 gctagaagga ttgtttcaga tggcgaagat
gaagatgatt cttttaaaga tacctcaagc 1320 ataaatccat tcaacacatc
tctctttcaa ttctcatctg tgaaacaatt tgatgcttca 1380 actcccaaaa
atgacatcag tccaccagga aggttctttt catctcaaat acccagtagt 1440
gtaaataagt ctatgaactc tagaagatct ctggcttcta ggaggtctct tattaatatg
1500 gttttagacc acgtggagga catggaggaa agacttgacg
acagcagtga agcaaagggt 1560 cctgaagatt atccagaaga aggggtggag
gaaagcagtg gcgaagcctc caagtataca 1620 gaagaggatc cttccggaga
aacactgtct tcagaaaaca agtccagctg gttaatgacg 1680 tctaagccta
gtgctctagc tcaagagacc tctcttggtg cccctgagcc tttgtctggt 1740
gaacagttgg ttggttctcc ccaggataag gcggcagagg ctacaaatga ctatgagact
1800 cttgtaaagc gtggaaaaga actaaaagag tgtggaaaaa tccaggaggc
cctaaactgc 1860 ttagttaaag cgcttgacat aaaaagtgca gatcctgaag
ttatgctctt gactttaagt 1920 ttgtataagc aacttaataa caattgagaa
tgtaacctgt ttattgtatt ttaaagtgaa 1980 actgaatatg agggaatttt
tgttcccata attggattct ttgggaacat gaagcattca 2040 ggcttaaggc
aagaaagatc tcaaaaagca acttctgccc tgcaacgccc cccactccat 2100
agtctggtat tctgagcact agcttaatat ttcttcactt gaatattctt atattttagg
2160 catattctat aaatttaact gtgttgtttc ttggaaagtt ttgtaaaatt
attctggtca 2220 ttcttaattt tactctgaaa gtgatcatct ttgtatataa
cagttcagat aagaaaatta 2280 aagttacttt tctc 2294 124 2294 DNA Homo
Sapiens 124 tcattaataa gacaaactac tggtgaaaaa aagaaccctt tccgatattt
tagtaaacaa 60 gaattaagag agctctttac aatcgaggat cttcagaact
ctgtaaccca gctgcagctt 120 cagtctttgc atgctgctca gaggaaatct
gatataaaac tagatgaaca tattgcctac 180 ctgcagtctt tggggatagc
tggaatctca gaccatgatt tgatgtacac atgtgatctg 240 tctgttaaag
aagagcttga tgtggtagaa gaatctcact atattcaaca aagggttcag 300
aaagctcaat tcctcgttga attcgagtct caaaataaag agttcctgat ggaacaacaa
360 agaactagaa atgagggggc ctggctaaga gaacctgtat ttccttcttc
aacaaagaag 420 aaatgcccta aattgaataa accacagcct cagccttcac
ctcttctaag tactcatcat 480 actcaggaag aagatatcag ttccaaaatg
gcaagtgtag tcattgatga tctgcccaaa 540 gagggtgaga aacaagatct
ctccagtata aaggtgaatg ttaccacctt gcaagatggg 600 taaggtacag
gtagtgctga ctctataact actttaccaa aggggtttgg aagtgtagaa 660
gaactttgta ctaactcttc attgggaatg gaaaaaagct ttgcaactaa aaatgaagct
720 gtacaaaaag agacattaca agaggggcct aagcaggagg cactgcaaga
ggatcctctg 780 gaaagtttta attatgtact tagcaaatca accaaagctg
atattgggcc aaatttagat 840 caactaaagg atgatgagat tttacgtcat
tgcaatcctt ggcccattat ttccataaca 900 aatgaaagtc aaaatgcaga
atcaaatgta tccattattg aaatagctga tgacctttca 960 gcatcccata
gtgcactgca ggatgctcaa gcaagtgagg ccaagttgga agaggaacct 1020
tcagcatctt caccacagta tgcatgtgat ttcaatcttt tcttggaaga ctcagcagac
1080 aacagacaaa atttttccag tcagtcttta gagcatgttg agaaagaaaa
tagcttgtgt 1140 ggctctgcac ctaattccaa agcagggttt gtgcatagca
aaacatgtct cagttgggag 1200 ttttctgaga aagacgatga accagaagaa
gtagtagtta aagcaaaaat cagaagtaaa 1260 gctagaagga ttgtttcaga
tggcgaagat gaagatgatt cttttaaaga tacctcaagc 1320 ataaatccat
tcaacacatc tctctttcaa ttctcatctg tgaaacaatt tgatgcttca 1380
actcccaaaa atgacatcag tccaccagga aggttctttt catctcaaat acccagtagt
1440 gtaaataagt ctatgaactc tagaagatct ctggcttcta ggaggtctct
tattaatatg 1500 gttttagacc acgtggagga catggaggaa agacttgacg
acagcagtga agcaaagggt 1560 cctgaagatt atccagaaga aggggtggag
gaaagcagtg gcgaagcctc caagtataca 1620 gaagaggatc cttccggaga
aacactgtct tcagaaaaca agtccagctg gttaatgacg 1680 tctaagccta
gtgctctagc tcaagagacc tctcttggtg cccctgagcc tttgtctggt 1740
gaacagttgg ttggttctcc ccaggataag gcggcagagg ctacaaatga ctatgagact
1800 cttgtaaagc gtggaaaaga actaaaagag tgtggaaaaa tccaggaggc
cctaaactgc 1860 ttagttaaag cgcttgacat aaaaagtgca gatcctgaag
ttatgctctt gactttaagt 1920 ttgtataagc aacttaataa caattgagaa
tgtaacctgt ttattgtatt ttaaagtgaa 1980 actgaatatg agggaatttt
tgttcccata attggattct ttgggaacat gaagcattca 2040 ggcttaaggc
aagaaagatc tcaaaaagca acttctgccc tgcaacgccc cccactccat 2100
agtctggtat tctgagcact agcttaatat ttcttcactt gaatattctt atattttagg
2160 catattctat aaatttaact gtgttgtttc ttggaaagtt ttgtaaaatt
attctggtca 2220 ttcttaattt tactctgaaa gtgatcatct ttgtatataa
cagttcagat aagaaaatta 2280 aagttacttt tctc 2294 125 419 PRT Homo
Sapiens 125 Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys
Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu
Asp Pro Leu Glu 20 25 30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr
Lys Ala Asp Ile Gly Pro 35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp
Glu Ile Leu Arg His Cys Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile
Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile
Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala 85 90 95 Leu Gln
Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110
Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115
120 125 Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His
Val 130 135 140 Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser
Lys Ala Gly 145 150 155 160 Phe Val His Ser Lys Thr Cys Leu Ser Trp
Glu Phe Ser Glu Lys Asp 165 170 175 Asp Glu Pro Glu Glu Val Val Val
Lys Ala Lys Ile Arg Ser Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp
Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile
Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys
Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235
240 Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met
245 250 255 Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn
Met Val 260 265 270 Leu Asp His Val Glu Asp Met Glu Glu Arg Leu Asp
Asp Ser Ser Glu 275 280 285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu
Gly Val Glu Glu Ser Ser 290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu
Glu Asp Pro Ser Gly Glu Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys
Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln
Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln
Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360
365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys
370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile
Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser
Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 126 450 PRT Homo
Sapiens 126 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu
Pro Lys 1 5 10 15 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser
Ser Leu Gly Met 20 25 30 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr Leu 35 40 45 Gln Glu Gly Pro Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu Ser 50 55 60 Phe Asn Tyr Val Leu Ser
Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 65 70 75 80 Leu Asp Gln Leu
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp 85 90 95 Pro Ile
Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 100 105 110
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 115
120 125 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
Ala 130 135 140 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp Ser 145 150 155 160 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln
Ser Leu Glu His Val Glu 165 170 175 Lys Glu Asn Ser Leu Cys Gly Ser
Ala Pro Asn Ser Arg Ala Gly Phe 180 185 190 Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 195 200 205 Glu Pro Glu Glu
Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 210 215 220 Arg Ile
Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 225 230 235
240 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val
245 250 255 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro
Pro Gly 260 265 270 Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn
Lys Ser Met Asn 275 280 285 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser
Leu Ile Asn Met Val Leu 290 295 300 Asp His Val Glu Asp Met Glu Glu
Arg Leu Asp Asp Ser Ser Glu Ala 305 310 315 320 Lys Gly Pro Glu Asp
Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 325 330 335 Glu Ala Ser
Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 340 345 350 Ser
Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 355 360
365 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln
370 375 380 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp Tyr 385 390 395 400 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys
Glu Cys Gly Lys Ile 405 410 415 Gln Glu Ala Leu Asn Cys Leu Val Lys
Ala Leu Asp Ile Lys Ser Ala 420 425 430 Asp Pro Glu Val Met Leu Leu
Thr Leu Ser Leu Tyr Lys Gln Leu Asn 435 440 445 Asn Asn 450 127 419
PRT Homo Sapiens 127 Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu 20 25 30 Ser Phe Asn Tyr Val Leu Ser
Lys Ser Thr Lys Ala Asp Ile Gly Pro 35 40 45 Asn Leu Asp Gln Leu
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro 50 55 60 Trp Pro Ile
Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn 65 70 75 80 Val
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala 85 90
95 Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
100 105 110 Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp 115 120 125 Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser
Leu Glu His Val 130 135 140 Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala
Pro Asn Ser Lys Ala Gly 145 150 155 160 Phe Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp 165 170 175 Asp Glu Pro Glu Glu
Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala 180 185 190 Arg Arg Ile
Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp 195 200 205 Thr
Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser 210 215
220 Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro
225 230 235 240 Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn
Lys Ser Met 245 250 255 Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser
Leu Ile Asn Met Val 260 265 270 Leu Asp His Val Glu Asp Met Glu Glu
Arg Leu Asp Asp Ser Ser Glu 275 280 285 Ala Lys Gly Pro Glu Asp Tyr
Pro Glu Glu Gly Val Glu Glu Ser Ser 290 295 300 Gly Glu Ala Ser Lys
Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu 305 310 315 320 Ser Ser
Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala 325 330 335
Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu 340
345 350 Gln Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp 355 360 365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu
Cys Gly Lys 370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala
Leu Asp Ile Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu
Thr Leu Ser Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 128 1720
DNA Homo Sapiens 128 ggcacgaggc caccttgcaa gatggtaaag gtacaggtag
tgctgactct atagctactt 60 taccaaaggg gtttggaagt gtagaagaac
tttgtactaa ctcttcattg ggaatggaaa 120 aaagctttgc aactaaaaat
gaagctgtac aaaaagagac attacaagag gggcctaagc 180 aagaggcact
gcaagaggat cctctggaaa gttttaatta tgtacttagc aaatcaacca 240
aagctgatat tgggccaaat ttagatcaac taaaggatga tgagatttta cgtcattgca
300 atccttggcc cattatttcc ataacaaatg aaagtcaaaa tgcagaatca
aatgtatcca 360 ttattgaaat agctgatgac ctttcagcat cccatagtgc
actgcaggat gctcaagcaa 420 gtgaggccaa gttggaagag gaaccttcag
catcttcacc acagtatgca tgtgatttca 480 atcttttctt ggaagactca
gcagacaaca gacaaaattt ttccagtcag tctttagagc 540 atgttgagaa
agaaaatagc ttgtgtggct ctgcacctaa ttccagagca gggtttgtgc 600
atagcaaaac atgtctcagt tgggagtttt ctgagaaaga cgatgaacca gaagaagtag
660 tagttaaagc aaaaatcaga agtaaagcta gaaggattgt ttcagatggc
gaagatgaag 720 atgattcttt taaagatacc tcaagcataa atccattcaa
cacatctctc tttcaattct 780 catctgtgaa acaatttgat gcttcaactc
ccaaaaatga catcagtcca ccaggaaggt 840 tcttttcatc tcaaataccc
agtagtgtaa ataagtctat gaactctaga agatctctgg 900 cttctaggag
gtctcttatt aatatggttt tagaccacgt ggaggacatg gaggaaagac 960
ttgacgacag cagtgaagca aagggtcctg aagattatcc agaagaaggg gtggaggaaa
1020 gcagtggcga agcctccaag tatacagaag aggatccttc cggagaaaca
ctgtcttcag 1080 aaaacaagtc cagctggtta atgacgtcta agcctagtgc
tctagctcaa gagacctctc 1140 ttggtgcccc tgagcctttg tctggtgaac
agttggttgg ttctccccag gataaggcgg 1200 cagaggctac aaatgactat
gagactcttg taaagcgtgg aaaagaacta aaagagtgtg 1260 gaaaaatcca
ggaggcccta aactgcttag ttaaagcgct tgacataaaa agtgcagatc 1320
ctgaagttat gctcttgact ttaagtttgt ataagcaact taataacaat tgagaatgta
1380 acctgtttat tgtattttaa agtgaaactg aatatgaggg aatttttgtt
cccataattg 1440 gattctttgg gaacatgaag cattcaggct taaggcaaga
aagatctcaa aaagcaactt 1500 ctgccctgca acgcccccca ctccatagtc
tggtattctg agcactagct taatatttct 1560 tcacttgaat attcttatat
tttaggcata ttctataaat ttaactgtgt tgtttcttgg 1620 aaagttttgt
aaaattattc tggtcattct taattttact ctgaaagtga tcatctttgt 1680
atataacagt tcagataaga aaattaaagt tacttttctc 1720 129 1744 DNA Homo
Sapiens 129 aacaagatct ctccagtata aaggtgaatg ttaccacctt gcaagatggt
aaaggtacag 60 gtagtgctga ctctatagct actttaccaa aggggtttgg
aagtgtagaa gaactttgta 120 ctaactcttc attgggaatg gaaaaaagct
ttgcaactaa aaatgaagct gtacaaaaag 180 agacattaca agaggggcct
aagcaagagg cactgcaaga ggatcctctg gaaagtttta 240 attatgtact
tagcaaatca accaaagctg atattgggcc aaatttagat caactaaagg 300
atgatgagat tttacgtcat tgcaatcctt ggcccattat ttccataaca aatgaaagtc
360 aaaatgcaga atcaaatgta tccattattg aaatagctga tgacctttca
gcatcccata 420 gtgcactgca ggatgctcaa gcaagtgagg ccaagttgga
agaggaacct tcagcatctt 480 caccacagta tgcatgtgat ttcaatcttt
tcttggaaga ctcagcagac aacagacaaa 540 atttttccag tcagtcttta
gagcatgttg agaaagaaaa tagcttgtgt ggctctgcac 600 ctaattccag
agcagggttt gtgcatagca aaacatgtct cagttgggag ttttctgaga 660
aagacgatga accagaagaa gtagtagtta aagcaaaaat cagaagtaaa gctagaagga
720 ttgtttcaga tggcgaagat gaagatgatt cttttaaaga tacctcaagc
ataaatccat 780 tcaacacatc tctctttcaa ttctcatctg tgaaacaatt
tgatgcttca actcccaaaa 840 atgacatcag tccaccagga aggttctttt
catctcaaat acccagtagt gtaaataagt 900 ctatgaactc tagaagatct
ctggcttcta ggaggtctct tattaatatg gttttagacc 960 acgtggagga
catggaggaa agacttgacg acagcagtga agcaaagggt cctgaagatt 1020
atccagaaga aggggtggag gaaagcagtg gcgaagcctc caagtataca gaagaggatc
1080 cttccggaga aacactgtct tcagaaaaca agtccagctg gttaatgacg
tctaagccta 1140 gtgctctagc tcaagagacc tctcttggtg cccctgagcc
tttgtctggt gaacagttgg 1200 ttggttctcc ccaggataag gcggcagagg
ctacaaatga ctatgagact cttgtaaagc 1260 gtggaaaaga actaaaagag
tgtggaaaaa tccaggaggc cctaaactgc ttagttaaag 1320 cgcttgacat
aaaaagtgca gatcctgaag ttatgctctt gactttaagt ttgtataagc 1380
aacttaataa caattgagaa tgtaacctgt ttattgtatt ttaaagtgaa actgaatatg
1440 agggaatttt tgttcccata attggattct ttgggaacat gaagcattca
ggcttaaggc 1500 aagaaagatc tcaaaaagca acttctgccc tgcaacgccc
cccactccat agtctggtat 1560 tctgagcact agcttaatat ttcttcactt
gaatattctt atattttagg catattctat 1620 aaatttaact gtgttgtttc
ttggaaagtt ttgtaaaatt attctggtca ttcttaattt 1680 tactctgaaa
gtgatcatct ttgtatataa cagttcagat aagaaaatta aagttacttt 1740 tctc
1744 130 1711 DNA Homo Sapiens 130 ccaccttgca agatggtaaa ggtacaggta
gtgctgactc tatagctact ttaccaaagg 60 ggtttggaag tgtagaagaa
ctttgtacta actcttcatt gggaatggaa aaaagctttg 120 caactaaaaa
tgaagctgta caaaaagaga cattacaaga ggggcctaag caagaggcac 180
tgcaagagga tcctctggaa agttttaatt atgtacttag caaatcaacc aaagctgata
240 ttgggccaaa tttagatcaa ctaaaggatg atgagatttt acgtcattgc
aatccttggc 300 ccattatttc cataacaaat gaaagtcaaa atgcagaatc
aaatgtatcc attattgaaa 360 tagctgatga cctttcagca tcccatagtg
cactgcagga tgctcaagca agtgaggcca 420 agttggaaga ggaaccttca
gcatcttcac cacagtatgc atgtgatttc aatcttttct 480 tggaagactc
agcagacaac agacaaaatt tttccagtca gtctttagag catgttgaga 540
aagaaaatag cttgtgtggc tctgcaccta attccagagc agggtttgtg catagcaaaa
600 catgtctcag ttgggagttt tctgagaaag acgatgaacc agaagaagta
gtagttaaag 660 caaaaatcag aagtaaagct agaaggattg tttcagatgg
cgaagatgaa gatgattctt 720 ttaaagatac ctcaagcata aatccattca
acacatctct ctttcaattc tcatctgtga 780 aacaatttga tgcttcaact
cccaaaaatg acatcagtcc accaggaagg ttcttttcat 840 ctcaaatacc
cagtagtgta aataagtcta tgaactctag aagatctctg gcttctagga 900
ggtctcttat taatatggtt ttagaccacg tggaggacat ggaggaaaga cttgacgaca
960 gcagtgaagc aaagggtcct gaagattatc cagaagaagg ggtggaggaa
agcagtggcg 1020 aagcctccaa gtatacagaa gaggatcctt ccggagaaac
actgtcttca gaaaacaagt 1080 ccagctggtt aatgacgtct aagcctagtg
ctctagctca agagacctct cttggtgccc 1140 ctgagccttt gtctggtgaa
cagttggttg gttctcccca ggataaggcg gcagaggcta 1200 caaatgacta
tgagactctt gtaaagcgtg gaaaagaact aaaagagtgt ggaaaaatcc 1260
aggaggccct aaactgctta gttaaagcgc ttgacataaa aagtgcagat cctgaagtta
1320 tgctcttgac tttaagtttg tataagcaac ttaataacaa ttgagaatgt
aacctgttta 1380 ttgtatttta aagtgaaact gaatatgagg gaatttttgt
tcccataatt ggattctttg 1440 ggaacatgaa gcattcaggc ttaaggcaag
aaagatctca aaaagcaact tctgccctgc 1500 aacgcccccc actccatagt
ctggtattct gagcactagc ttaatatttc ttcacttgaa 1560 tattcttata
ttttaggcat attctataaa tttaactgtg ttgtttcttg gaaagttttg 1620
taaaattatt ctggtcattc ttaattttac tctgaaagtg atcatctttg tatataacag
1680 ttcagataag aaaattaaag ttacttttct c 1711 131 419 PRT Homo
Sapiens 131 Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys
Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu
Asp Pro Leu Glu 20 25 30 Ser Phe Asn Tyr Val Leu Ser Lys Ser Thr
Lys Ala Asp Ile Gly Pro 35 40 45 Asn Leu Asp Gln Leu Lys Asp Asp
Glu Ile Leu Arg His Cys Asn Pro 50 55 60 Trp Pro Ile Ile Ser Ile
Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn 65 70 75 80 Val Ser Ile Ile
Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala 85 90 95 Leu Gln
Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser 100 105 110
Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu Glu Asp 115
120 125 Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser Leu Glu His
Val 130 135 140 Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala Pro Asn Ser
Arg Ala Gly 145 150 155 160 Phe Val His Ser Lys Thr Cys Leu Ser Trp
Glu Phe Ser Glu Lys Asp 165 170 175 Asp Glu Pro Glu Glu Val Val Val
Lys Ala Lys Ile Arg Ser Lys Ala 180 185 190 Arg Arg Ile Val Ser Asp
Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp 195 200 205 Thr Ser Ser Ile
Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser 210 215 220 Val Lys
Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro 225 230 235
240 Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn Lys Ser Met
245 250 255 Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn
Met Val 260 265 270 Leu Asp His Val Glu Asp Met Glu Glu Arg Leu Asp
Asp Ser Ser Glu 275 280 285 Ala Lys Gly Pro Glu Asp Tyr Pro Glu Glu
Gly Val Glu Glu Ser Ser 290 295 300 Gly Glu Ala Ser Lys Tyr Thr Glu
Glu Asp Pro Ser Gly Glu Thr Leu 305 310 315 320 Ser Ser Glu Asn Lys
Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala 325 330 335 Leu Ala Gln
Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu 340 345 350 Gln
Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp 355 360
365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu Cys Gly Lys
370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala Leu Asp Ile
Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu Thr Leu Ser
Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 132 450 PRT Homo
Sapiens 132 Asp Gly Lys Gly Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu
Pro Lys 1 5 10 15 Gly Phe Gly Ser Val Glu Glu Leu Cys Thr Asn Ser
Ser Leu Gly Met 20 25 30 Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr Leu 35 40 45 Gln Glu Gly Pro Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu Ser 50 55 60 Phe Asn Tyr Val Leu Ser
Lys Ser Thr Lys Ala Asp Ile Gly Pro Asn 65 70 75 80 Leu Asp Gln Leu
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro Trp 85 90 95 Pro Ile
Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn Val 100 105 110
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 115
120 125 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
Ala 130 135 140 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp Ser 145 150 155 160 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln
Ser Leu Glu His Val Glu 165 170 175 Lys Glu Asn Ser Leu Cys Gly Ser
Ala Pro Asn Ser Arg Ala Gly Phe 180 185 190 Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 195 200 205 Glu Pro Glu Glu
Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 210 215 220 Arg Ile
Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr 225 230 235
240 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser Val
245 250 255 Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro
Pro Gly 260 265 270 Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn
Lys Ser Met Asn 275 280 285 Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser
Leu Ile Asn Met Val Leu 290 295 300 Asp His Val Glu Asp Met Glu Glu
Arg Leu Asp Asp Ser Ser Glu Ala 305 310 315 320 Lys Gly Pro Glu Asp
Tyr Pro Glu Glu Gly Val Glu Glu Ser Ser Gly 325 330 335 Glu Ala Ser
Lys Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu Ser 340 345 350 Ser
Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 355 360
365 Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln
370 375 380 Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp Tyr 385 390 395 400 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys
Glu Cys Gly Lys Ile 405 410 415 Gln Glu Ala Leu Asn Cys Leu Val Lys
Ala Leu Asp Ile Lys Ser Ala 420 425 430 Asp Pro Glu Val Met Leu Leu
Thr Leu Ser Leu Tyr Lys Gln Leu Asn 435 440 445 Asn Asn 450 133 419
PRT Homo Sapiens 133 Met Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala
Val Gln Lys Glu Thr 1 5 10 15 Leu Gln Glu Gly Pro Lys Gln Glu Ala
Leu Gln Glu Asp Pro Leu Glu 20 25 30 Ser Phe Asn Tyr Val Leu Ser
Lys Ser Thr Lys Ala Asp Ile Gly Pro 35 40 45 Asn Leu Asp Gln Leu
Lys Asp Asp Glu Ile Leu Arg His Cys Asn Pro 50 55 60 Trp Pro Ile
Ile Ser Ile Thr Asn Glu Ser Gln Asn Ala Glu Ser Asn 65 70 75 80 Val
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala 85 90
95 Leu Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
100 105 110 Ala Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp 115 120 125 Ser Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln Ser
Leu Glu His Val 130 135 140 Glu Lys Glu Asn Ser Leu Cys Gly Ser Ala
Pro Asn Ser Arg Ala Gly 145 150 155 160 Phe Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp 165 170 175 Asp Glu Pro Glu Glu
Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala 180 185 190 Arg Arg Ile
Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp 195 200 205 Thr
Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln Phe Ser Ser 210 215
220 Val Lys Gln Phe Asp Ala Ser Thr Pro Lys Asn Asp Ile Ser Pro Pro
225 230 235 240 Gly Arg Phe Phe Ser Ser Gln Ile Pro Ser Ser Val Asn
Lys Ser Met 245 250 255 Asn Ser Arg Arg Ser Leu Ala Ser Arg Arg Ser
Leu Ile Asn Met Val 260 265 270 Leu Asp His Val Glu Asp Met Glu Glu
Arg Leu Asp Asp Ser Ser Glu 275 280 285 Ala Lys Gly Pro Glu Asp Tyr
Pro Glu Glu Gly Val Glu Glu Ser Ser 290 295 300 Gly Glu Ala Ser Lys
Tyr Thr Glu Glu Asp Pro Ser Gly Glu Thr Leu 305 310 315 320 Ser Ser
Glu Asn Lys Ser Ser Trp Leu Met Thr Ser Lys Pro Ser Ala 325 330 335
Leu Ala Gln Glu Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu 340
345 350 Gln Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn
Asp 355 360 365 Tyr Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu
Cys Gly Lys 370 375 380 Ile Gln Glu Ala Leu Asn Cys Leu Val Lys Ala
Leu Asp Ile Lys Ser 385 390 395 400 Ala Asp Pro Glu Val Met Leu Leu
Thr Leu Ser Leu Tyr Lys Gln Leu 405 410 415 Asn Asn Asn 134 1250
PRT Homo Sapiens 134 Met Glu Ala Ser Arg Arg Phe Pro Glu Ala Glu
Ala Leu Ser Pro Glu 1 5 10 15 Gln Ala Ala His Tyr Leu Arg Tyr Val
Lys Glu Ala Lys Glu Ala Thr 20 25 30 Lys Asn Gly Asp Leu Glu Glu
Ala Phe Lys Leu Phe Asn Leu Ala Lys 35 40 45 Asp Ile Phe Pro Asn
Glu Lys Val Leu Ser Arg Ile Gln Lys Ile Gln 50 55 60 Glu Ala Leu
Glu Glu Leu Ala Glu Gln Gly Asp Asp Glu Phe Thr Asp 65 70 75 80 Val
Cys Asn Ser Gly Leu Leu Leu Tyr Arg Glu Leu His Asn Gln Leu 85 90
95 Phe Glu His Gln Lys Glu Gly Ile Ala Phe Leu Tyr Ser Leu Tyr Arg
100 105 110 Asp Gly Arg Lys Gly Gly Ile Leu Ala Asp Asp Met Gly Leu
Gly Lys 115 120 125 Thr Val Gln Ile Ile Ala Phe Leu Ser Gly Met Phe
Asp Ala Ser Leu 130 135 140 Val Asn His Val Leu Leu Ile Met Pro Thr
Asn Leu Ile Asn Thr Trp 145 150 155 160 Val Lys Glu Phe Ile Lys Trp
Thr Pro Gly Met Arg Val Lys Thr Phe 165 170 175 His Gly Pro Ser Lys
Asp Glu Arg Thr Arg Asn Leu Asn Arg Ile Gln 180 185 190 Gln Arg Asn
Gly Val Ile Ile Thr Thr Tyr Gln Met Leu Ile Asn Asn 195 200 205 Trp
Gln Gln Leu Ser Ser Phe Arg Gly Gln Glu Phe Val Trp Asp Tyr 210 215
220 Val Ile Leu Asp Glu Ala His Lys Ile Lys Thr Ser Ser Thr Lys Ser
225 230 235 240 Ala Ile Cys Ala Arg Ala Ile Pro Ala Ser Asn Arg Leu
Leu Leu Thr 245 250 255 Gly Thr Pro Ile Gln Asn Asn Leu Gln Glu Leu
Trp Ser Leu Phe Asp 260 265 270 Phe Ala Cys Gln Gly Ser Leu Leu Gly
Thr Leu Lys Thr Phe Lys Met 275 280 285 Glu Tyr Glu Asn Pro Ile Thr
Arg Ala Arg Glu Lys Asp Ala Thr Pro 290 295 300 Gly Glu Lys Ala Leu
Gly Phe Lys Ile Ser Glu Asn Leu Met Ala Ile 305 310 315 320 Ile Lys
Pro Tyr Phe Leu Arg Arg Thr Lys Glu Asp Val Gln Lys Lys 325 330 335
Lys Ser Ser Asn Pro Glu Ala Arg Leu Asn Glu Lys Asn Pro Asp Val 340
345 350 Asp Ala Ile Cys Glu Met Pro Ser Leu Ser Arg Lys Asn Asp Leu
Ile 355 360 365 Ile Trp Ile Arg Leu Val Pro Leu Gln Glu Glu Ile Tyr
Arg Lys Phe 370 375 380 Val Ser Leu Asp His Ile Lys Glu Leu Leu Met
Glu Thr Arg Ser Pro 385 390 395 400 Leu Ala Glu Leu Gly Val Leu Lys
Lys Leu Cys Asp His Pro Arg Leu 405 410 415 Leu Ser Ala Arg Ala Cys
Cys Leu Leu Asn Leu Gly Thr Phe Ser Ala 420 425 430 Gln Asp Gly Asn
Glu Gly Glu Asp Ser Pro Asp Val Asp His Ile Asp 435 440 445 Gln Val
Thr Asp Asp Thr Leu Met Glu Glu Ser Gly Lys Met Ile Phe 450 455 460
Leu Met Asp Leu Leu Lys Arg Leu Arg Asp Glu Gly His Gln Thr Leu 465
470 475 480 Val Phe Ser Gln Ser Arg Gln Ile Leu Asn Ile Ile Glu Arg
Leu Leu 485 490 495 Lys Asn Arg His Phe Lys Thr Leu Arg Ile Asp Gly
Thr Val Thr His 500 505 510 Leu Leu Glu Arg Glu Lys Arg Ile Asn Leu
Phe Gln Gln Asn Lys Asp 515 520 525 Tyr Ser Val Phe Leu Leu Thr Thr
Gln Val Gly Gly Val Gly Leu Thr 530 535 540 Leu Thr Ala Ala Thr Arg
Val Val Ile Phe Asp Pro Ser Trp Asn Pro 545 550 555 560 Ala Thr Asp
Ala Gln Ala Val Asp Arg Val Tyr Arg Ile Gly Gln Lys 565 570 575 Glu
Asn Val Val Val Tyr Arg Leu Ile Thr Cys Gly Thr Val Glu Glu 580 585
590 Lys Ile Tyr Arg Arg Gln Val Phe Lys Asp Ser Leu Ile Arg Gln Thr
595 600 605 Thr Gly Glu Lys Lys Asn Pro Phe Arg Tyr Phe Ser Lys Gln
Glu Leu 610 615 620 Arg Glu Leu Phe Thr Ile Glu Asp Leu Gln Asn Ser
Val Thr Gln Leu 625 630 635 640 Gln Leu Gln Ser Leu His Ala Ala Gln
Arg Lys Ser Asp Ile Lys Leu 645 650 655 Asp Glu His Ile Ala Tyr Leu
Gln Ser Leu Gly Ile Ala Gly Ile Ser 660 665 670 Asp His Asp Leu Met
Tyr Thr Cys Asp Leu Ser Val Lys Glu Glu Leu 675 680 685 Asp Val Val
Glu Glu Ser His Tyr Ile Gln Gln Arg Val Gln Lys Ala 690 695 700 Gln
Phe Leu Val Glu Phe Glu Ser Gln Asn Lys Glu Phe Leu Met Glu 705 710
715 720 Gln Gln Arg Thr Arg Asn Glu Gly Ala Trp Leu Arg Glu Pro Val
Phe 725 730 735 Pro Ser Ser Thr Lys Lys Lys Cys Pro Lys Leu Asn Lys
Pro Gln Pro 740 745 750 Gln Pro Ser Pro Leu Leu Ser Thr His His Thr
Gln Glu Glu Asp Ile 755 760 765 Ser Ser Lys Met Ala Ser Val Val Ile
Asp Asp Leu Pro Lys Glu Gly 770 775 780 Glu Lys Gln Asp Leu Ser Ser
Ile Lys Val Asn Val Thr Thr Leu Gln 785 790 795 800 Asp Gly Lys Gly
Thr Gly Ser Ala Asp Ser Ile Ala Thr Leu Pro Lys 805 810 815 Gly Phe
Gly Ser Val Glu Glu Leu Cys Thr Asn Ser Ser Leu Gly Met 820 825 830
Glu Lys Ser Phe Ala Thr Lys Asn Glu Ala Val Gln Lys Glu Thr Leu 835
840 845 Gln Glu Gly Pro Lys Gln Glu Ala Leu Gln Glu Asp Pro Leu Glu
Ser 850 855 860 Phe Asn Tyr Val Leu Ser Lys Ser Thr Lys Ala Asp Ile
Gly Pro Asn 865 870 875 880 Leu Asp Gln Leu Lys Asp Asp Glu Ile Leu
Arg His Cys Asn Pro Trp 885 890 895 Pro Ile Ile Ser Ile Thr Asn Glu
Ser Gln Asn Ala Glu Ser Asn Val 900 905 910
Ser Ile Ile Glu Ile Ala Asp Asp Leu Ser Ala Ser His Ser Ala Leu 915
920 925 Gln Asp Ala Gln Ala Ser Glu Ala Lys Leu Glu Glu Glu Pro Ser
Ala 930 935 940 Ser Ser Pro Gln Tyr Ala Cys Asp Phe Asn Leu Phe Leu
Glu Asp Ser 945 950 955 960 Ala Asp Asn Arg Gln Asn Phe Ser Ser Gln
Ser Leu Glu His Val Glu 965 970 975 Lys Glu Asn Ser Leu Cys Gly Ser
Ala Pro Asn Ser Arg Ala Gly Phe 980 985 990 Val His Ser Lys Thr Cys
Leu Ser Trp Glu Phe Ser Glu Lys Asp Asp 995 1000 1005 Glu Pro Glu
Glu Val Val Val Lys Ala Lys Ile Arg Ser Lys Ala Arg 1010 1015 1020
Arg Ile Val Ser Asp Gly Glu Asp Glu Asp Asp Ser Phe Lys Asp Thr
1025 1030 1035 1040 Ser Ser Ile Asn Pro Phe Asn Thr Ser Leu Phe Gln
Phe Ser Ser Val 1045 1050 1055 Lys Gln Phe Asp Ala Ser Thr Pro Lys
Asn Asp Ile Ser Pro Pro Gly 1060 1065 1070 Arg Phe Phe Ser Ser Gln
Ile Pro Ser Ser Val Asn Lys Ser Met Asn 1075 1080 1085 Ser Arg Arg
Ser Leu Ala Ser Arg Arg Ser Leu Ile Asn Met Val Leu 1090 1095 1100
Asp His Val Glu Asp Met Glu Glu Arg Leu Asp Asp Ser Ser Glu Ala
1105 1110 1115 1120 Lys Gly Pro Glu Asp Tyr Pro Glu Glu Gly Val Glu
Glu Ser Ser Gly 1125 1130 1135 Glu Ala Ser Lys Tyr Thr Glu Glu Asp
Pro Ser Gly Glu Thr Leu Ser 1140 1145 1150 Ser Glu Asn Lys Ser Ser
Trp Leu Met Thr Ser Lys Pro Ser Ala Leu 1155 1160 1165 Ala Gln Glu
Thr Ser Leu Gly Ala Pro Glu Pro Leu Ser Gly Glu Gln 1170 1175 1180
Leu Val Gly Ser Pro Gln Asp Lys Ala Ala Glu Ala Thr Asn Asp Tyr
1185 1190 1195 1200 Glu Thr Leu Val Lys Arg Gly Lys Glu Leu Lys Glu
Cys Gly Lys Ile 1205 1210 1215 Gln Glu Ala Leu Asn Cys Leu Val Lys
Ala Leu Asp Ile Lys Ser Ala 1220 1225 1230 Asp Pro Glu Val Met Leu
Leu Thr Leu Ser Leu Tyr Lys Gln Leu Asn 1235 1240 1245 Asn Asn
1250
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