U.S. patent application number 13/749461 was filed with the patent office on 2013-07-11 for nucleic acids and corresponding proteins entitled 202p5a5 useful in treatment and detection of cancer.
This patent application is currently assigned to Agensys, Inc.. The applicant listed for this patent is Agensys, Inc.. Invention is credited to Pia M. Challita-Eid, Mary Faris, Wangmao Ge, Aya Jakobovits, Arthur B. RAITANO.
Application Number | 20130177569 13/749461 |
Document ID | / |
Family ID | 31891426 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177569 |
Kind Code |
A1 |
RAITANO; Arthur B. ; et
al. |
July 11, 2013 |
NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 202P5A5 USEFUL IN
TREATMENT AND DETECTION OF CANCER
Abstract
A novel gene 202P5A5 and its encoded protein, and variants
thereof, are described wherein 202P5A5 exhibits tissue specific
expression in normal adult tissue, and is aberrantly expressed in
the cancers listed in Table I. Consequently, 202P5A5 provides a
diagnostic, prognostic, prophylactic and/or therapeutic target for
cancer. The 202P5A5 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 202P5A5 can be used in active or passive
immunization.
Inventors: |
RAITANO; Arthur B.; (Los
Alamitos, CA) ; Faris; Mary; (Los Angeles, CA)
; Challita-Eid; Pia M.; (Encino, CA) ; Jakobovits;
Aya; (Beverly Hills, CA) ; Ge; Wangmao;
(Tampa, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agensys, Inc.; |
Santa Monica |
CA |
US |
|
|
Assignee: |
Agensys, Inc.
Santa Monica
CA
|
Family ID: |
31891426 |
Appl. No.: |
13/749461 |
Filed: |
January 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13021202 |
Feb 4, 2011 |
8426571 |
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13749461 |
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11548626 |
Oct 11, 2006 |
8057996 |
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13021202 |
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10463782 |
Jun 16, 2003 |
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11548626 |
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60404306 |
Aug 16, 2002 |
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60423290 |
Nov 1, 2002 |
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Current U.S.
Class: |
424/139.1 ;
424/185.1; 435/188; 530/350; 530/387.3; 530/387.9; 530/391.3;
530/391.7 |
Current CPC
Class: |
A61K 47/6851 20170801;
Y02A 50/466 20180101; C07K 2319/00 20130101; A61P 35/00 20180101;
A01K 2217/075 20130101; C12N 5/0693 20130101; A01K 2217/05
20130101; A61P 37/04 20180101; C07K 14/435 20130101; Y02A 50/30
20180101; G01N 33/57484 20130101; C07K 14/47 20130101; A61K 38/00
20130101; A61K 2039/505 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/139.1 ;
530/350; 424/185.1; 530/387.9; 530/387.3; 530/391.3; 530/391.7;
435/188 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Claims
1. An isolated polypeptide having an amino acid sequence consisting
of SEQ ID NO: 12 or comprising SEQ ID NO: 14, 15, 16, or 17.
2. The polypeptide of claim 1, wherein the amino acid sequence
consists of SEQ ID NO: 12.
3. The polypeptide of claim 1, wherein the amino acid sequence
comprises SEQ ID NO: 14.
4. The polypeptide of claim 1, wherein the amino acid sequence
comprises SEQ ID NO: 15.
5. The polypeptide of claim 1, wherein the amino acid sequence
comprises SEQ ID NO: 16.
6. The polypeptide of claim 1, wherein the amino acid sequence
comprises SEQ ID NO: 17.
7. A composition, comprising the polypeptide of claim 1 and a
pharmaceutically acceptable carrier.
8. The polypeptide of claim 1, which is labeled with a detectable
marker, a therapeutic agent, or a cytotoxic agent.
9. An antibody or antigen-binding fragment thereof that
immunospecifically binds to an epitope on a protein having an amino
acid sequence consisting of SEQ ID NO: 12, 14, 15, 16, or 17.
10. A composition, comprising the antibody or fragment thereof of
claim 9 and a pharmaceutically acceptable carrier.
11. The antibody or fragment thereof of claim 9, wherein the
antibody or fragment is an Fab, F(ab').sub.2, Fv or Sfv
fragment.
12. The antibody or fragment thereof of claim 9, which is
monoclonal.
13. The antibody or fragment thereof of claim 12, which is
recombinant.
14. The antibody or fragment thereof of claim 12, which is a human
antibody or fragment, or a humanized antibody or fragment.
15. The antibody or fragment thereof of claim 9, which is labeled
with a detectable marker, a therapeutic agent, or a cytotoxic
agent.
16. The antibody or fragment thereof of claim 15, which is labeled
with the detectable marker, which is selected from the group
consisting of a radioisotope, a metal chelator, an enzyme, a
fluorescent compound, a bioluminescent compound, and a
chemiluminescent compound.
17. The antibody or fragment thereof of claim 15, which is labeled
with the cytotoxic agent, which is selected from the group
consisting of a radioactive isotope, a chemotherapeutic agent, and
a toxin.
18. The antibody or fragment thereof of claim 17, wherein the
cytotoxic agent is the radioactive isotope, which is selected from
the group consisting of .sup.211At, .sup.131I, .sup.125I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P, and
radioactive isotopes of Lu.
19. The antibody or fragment thereof of claim 17, wherein the
cytotoxic agent is the chemotherapeutic agent, which is selected
from the group consisting of taxol, actinomycin, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicine,
gelonin, and calicheamicin.
20. The antibody or fragment thereof of claim 17, wherein the
cytotoxic agent is the toxin, which is selected from the group
consisting of diphtheria toxin, enomycin, phenomycin, Pseudomonas
exotoxin (PE) A, PE40, abrin, abrin A chain, mitogellin, modeccin A
chain, and alpha-sarcin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/021,202 filed 4 Feb. 2011, now allowed, which is a
divisional of U.S. application Ser. No. 11/548,626 filed 11 Oct.
2006, now U.S. Pat. No. 8,057,996, which is a continuation of U.S.
application Ser. No. 10/463,782 filed 16 Jun. 2003, now abandoned,
which claims priority from U.S. Provisional Application No.
60/404,306, filed 16 Aug. 2002, and from U.S. Provisional
Application No. 60/423,290, filed 1 Nov. 2002. The contents of the
applications listed in this paragraph are fully incorporated by
reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
511582008603SeqList.txt, date recorded: Jan. 4, 2013, size: 229,788
bytes).
FIELD OF THE INVENTION
[0004] The invention described herein relates to genes and their
encoded proteins, termed 202P5A5 and variants thereof, expressed in
certain cancers, and to diagnostic and therapeutic methods and
compositions useful in the management of cancers that express
202P5A5.
BACKGROUND OF THE INVENTION
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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/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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] Taking into account the medical circumstances and the
patient's preferences, treatment of breast cancer may involve
lumpectomy (local removal of the tumor) and removal of the lymph
nodes under the arm; mastectomy (surgical removal of the breast)
and removal of the lymph nodes under the arm; radiation therapy;
chemotherapy; or hormone therapy. Often, two or more methods are
used in combination. Numerous studies have shown that, for early
stage disease, long-term survival rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical
mastectomy. Significant advances in reconstruction techniques
provide several options for breast reconstruction after mastectomy.
Recently, such reconstruction has been done at the same time as the
mastectomy.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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
[0028] The present invention relates to a gene, designated 202P5A5,
that has now been found to be over-expressed in the cancer(s)
listed in Table I. Northern blot expression analysis of 202P5A5
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 202P5A5 are provided. The
tissue-related profile of 202P5A5 in normal adult tissues, combined
with the over-expression observed in the tissues listed in Table I,
shows that 202P5A5 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.
[0029] The invention provides polynucleotides corresponding or
complementary to all or part of the 202P5A5 genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding 202P5A5-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 202P5A5-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 202P5A5
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the 202P5A5 genes, mRNAs, or to
202P5A5-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding 202P5A5. Recombinant DNA
molecules containing 202P5A5 polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of 202P5A5 gene products are also provided. The
invention further provides antibodies that bind to 202P5A5 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.
[0030] The invention further provides methods for detecting the
presence and status of 202P5A5 polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express 202P5A5. A typical embodiment of this invention
provides methods for monitoring 202P5A5 gene products in a tissue
or hematology sample having or suspected of having some form of
growth dysregulation such as cancer.
[0031] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 202P5A5 such as cancers of tissues listed in Table I,
including therapies aimed at inhibiting the transcription,
translation, processing or function of 202P5A5 as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
202P5A5 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 202P5A5.
Preferably, the carrier is a uniquely human carrier. In another
aspect of the invention, the agent is a moiety that is
immunoreactive with 202P5A5 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.
[0032] 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 202P5A5 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 202P5A5 as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
202P5A5. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of 202P5A5 (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for 202P5A5 production) or a ribozyme effective to lyse
202P5A5 mRNA.
[0033] 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.
[0034] 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.
[0035] 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:
[0036] 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;
[0037] 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;
[0038] 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;
[0039] 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 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.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1. The 202P5A5 SSH sequence of 186 nucleotides.
[0041] FIG. 2. A) The cDNA and amino acid sequence of 202P5A5
variant 1 (also called "202P5A5 v.1" or "202P5A5 variant 1") is
shown in FIG. 2A. The start methionine is underlined. The open
reading frame extends from nucleic acid 29-1858 including the stop
codon.
[0042] B) The cDNA and amino acid sequence of 202P5A5 variant 2
(also called "202P5A5 v.2") is shown in FIG. 2B. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 13-1890 including the stop codon.
[0043] C) The cDNA and amino acid sequence of 202P5A5 variant 3
(also called "202P5A5 v.3") is shown in FIG. 2C. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 121-1950 including the stop codon.
[0044] D) The cDNA and amino acid sequence of 202P5A5 variant 14
(also called "202P5A5 v.14") is shown in FIG. 2D. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 29-1858 including the stop codon.
[0045] E) The cDNA and amino acid sequence of 202P5A5 variant 22
(also called "202P5A5 v.22") is shown in FIG. 2E. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 29-1858 including the stop codon.
[0046] F) 202P5A5 v.4 through v.26, SNP variants of 202P5A5 v.1.
The 202P5A5 v.4 through v.26 are variants with single nucleotide
difference from 202P5A5 v.1. 202P5A5 v.4, v.5, v.6 and v.8 differ
from 202P5A5 v.1 by one amino acid. 202P5A5 v.7, and v.9 through
v.26 code for the same protein as v.1. 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 through
2C.
[0047] FIG. 3. A) The amino acid sequence of 202P5A5 v.1 is shown
in FIG. 3A; it has 609 amino acids.
[0048] B) The amino acid sequence of 202P5A5 v.2 is shown in FIG.
3B; it has 625 amino acids.
[0049] C) The amino acid sequence of 202P5A5 v.4 is shown in FIG.
3C; it has 609 amino acids.
[0050] D) The amino acid sequence of 202P5A5 v.5 is shown in FIG.
3D; it has 609 amino acids.
[0051] E) The amino acid sequence of 202P5A5 v.6 is shown in FIG.
3E; it has 609 amino acids.
[0052] F) The amino acid sequence of 202P5A5 v.8 is shown in FIG.
3F; it has 609 amino acids.
[0053] As used herein, a reference to 202P5A5 includes all variants
thereof, including those shown in FIGS. 2, 3, 10, and 11, unless
the context clearly indicates otherwise.
[0054] FIGS. 4A-C. Alignment of 202P5A5 with known homologs. A)
Alignment of 202P5A5 with human hypothetical protein FLJ13782 (gi
13376382). B) Alignment of 202P5A5 with mouse BOM (gi 20502771). C)
Alignment of 202P5A5 with mouse grainyhead-like protein (gi
21312674).
[0055] FIG. 5. Hydrophilicity amino acid profile of 202P5A5 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 at (expasy.ch/cgi-bin/protscale.pl) through
the ExPasy molecular biology server.
[0056] FIG. 6. Hydropathicity amino acid profile of 202P5A5 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 at (.expasy.ch/cgi-bin/protscale.pl) through the
ExPasy molecular biology server.
[0057] FIG. 7. Percent accessible residues amino acid profile of
202P5A5 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 at
(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0058] FIG. 8. Average flexibility amino acid profile of 202P5A5
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 at
(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0059] FIG. 9. Beta-turn amino acid profile of 202P5A5 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 at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0060] FIG. 10. Structures of transcript variants of 202P5A5.
Variants 202P5A5 v.2 and v.3 are transcript variants of 202P5A05
v.1. Variant 202P5A05 v.2 added an exon to the 5' end of variant
v.1. Variant v.3 further extended exon 1 of v.2 into intron 1. Poly
A tails are not shown in this figure. Numbers in "( )" underneath
the boxes correspond to those of 202P5A05 v.1. Lengths of introns
and exons are not proportional.
[0061] FIG. 11. Schematic alignment of protein variants of 202P5A5.
Protein variants correspond to nucleotide variants. Nucleotide
variants 202P5A5 v.3, v.7, and v.9 through v.26 coded the same
protein as v.1. Variant v.2 coded a protein that was 16 amino acids
longer and contained the whole protein of v.1. Nucleotide variants
202P5A5 v.2 and v.3 were transcript variants of v.1, as shown in
FIG. 10. SNP in v.1 also existed in v.2 and v.3. Single amino acid
differences were indicated above the boxes. Black boxes represent
the same sequence as 202P5A5 v.1. Numbers underneath the box
correspond to 202P5A5 v.1.
[0062] FIG. 12. Schematic alignment of SNP variants of 202P5A5.
Variants 202P5A5 v.4 through v.26 are variants with single
nucleotide differences as compared to variant v.1 (ORF:29-1858).
Variant v.14 inserted two base pairs at 2269-2270 while variant
v.22 deleted one base pair at 3427. Though these SNP variants were
shown separately, they could also occur in any combinations and in
any transcript variants, such as v.3 shown in FIG. 10, that
contained the base pairs. Numbers correspond to those of 202P5A5
v.1. The black box shows the same sequence as 202P5A5 v.1. SNPs are
indicated above the box.
[0063] FIGS. 13A-C. Secondary structure and transmembrane domains
prediction for 202P5A05 protein variant 1. A: The secondary
structure of 202P5A5 protein variant 1 (SEQ ID NO:108) was
predicted using the HNN--Hierarchical Neural Network method (NPS@:
Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3
[291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G.,
located on the World Wide Web at
pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed
from the ExPasy molecular biology server located on the World Wide
Web at (.expasy.ch/tools/). This method predicts the presence and
location of alpha helices, extended strands, and random coils from
the primary protein sequence. The percent of the protein in a given
secondary structure is also listed. B: Schematic representation of
the probability of existence of transmembrane regions of 202P5A5
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). C: Schematic representation of the probability of
the existence of transmembrane regions of 202P5A05 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 at (.expasy.ch/tools/). Both algorithms do not predict the
presence of transmembrane regions in 202P5A5 variant 1.
[0064] FIGS. 14A-B. Expression of 202P5A5 by RT-PCR. A: First
strand cDNA was prepared from vital pool 1 (liver, lung and
kidney), vital pool 2 (pancreas, colon and stomach), prostate
cancer metastasis to lymph node, prostate cancer pool, bladder
cancer pool, colon cancer pool, lung cancer pool, breast cancer
pool, and cancer metastasis pool. Normalization was performed by
PCR using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 202P5A5, was performed at 26 and 30 cycles of
amplification. Expression was detected in prostate cancer
metastasis to lymph node, prostate cancer pool, bladder cancer
pool, colon cancer pool, lung cancer pool, breast cancer pool, and
cancer metastasis pool. Low expression was also detected in vital
pool 1 but not in vital pool 2. B: Semi-quantitative PCR, using
primers to 202P5A5, was performed on a panel of 13 normal tissues
and 13 cancer pools. Samples were run on an agarose gel, and PCR
products were quantitated using the AlphaImager software. Results
show strong expression of 202P5A5 in prostate cancer, bladder
cancer, colon cancer, lung cancer, ovary cancer, breast cancer,
metastasis cancer, xenograft pool, prostate metastasis to lymph
node (PMLN), bone cancer/melanoma pool, cervical cancer, lymphoma
and stomach cancer compared to all normal tissues tested.
[0065] FIG. 15. Expression of 202P5A5 variants by RT-PCR. Primers
were designed to differentiate between 202P5A5 v.2 and 202P5A5 v.3.
202P5A5 leads to a PCR product of 173 bp, whereas 202P5A5 v.3 leads
to a PCR product of 233 bp in size. First strand cDNA was prepared
from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas,
colon and stomach), LAPC prostate xenograft pool (LAPC-4AD.
LAPC-4AI, LAPC-9AD and LAPC-9AI), prostate cancer pool, bladder
cancer pool, lung cancer pool, ovary cancer pool, breast cancer
pool, cancer metastasis pool, cervical cancer pool, stomach cancer
pool, uterus cancer pool, and master xenograft pool (LAPC xenograft
pool, bladder cancer xenograft, kidney cancer xenograft).
Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using the variant specific primers
was performed at 26 and 30 cycles of amplification. Stronger
expression of the 173 bp product was detected in all cancer pools
tested and weakly in vital pools. The larger 233 bp product was
mostly detected in the cancer pools and not in the vital tissues,
and at a frequency of 20-30% compared to the smaller product.
[0066] FIG. 16. Expression of 202P5A5 in normal tissues. Two
multiple tissue northern blots (Clontech) both with 2 ug of
mRNA/lane were probed with the 202P5A5 sequence. Size standards in
kilobases (kb) are indicated on the side. Results show expression
of an approximately 7 kb 202P5A5 transcript in normal prostate and
normal placenta but not in any other normal tissue tested.
[0067] FIG. 17. Expression of 202P5A5 in Prostate Cancer Patient
Specimens. RNA was extracted from prostate cancer xenografts
(LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI), prostate cancer cell
lines (LNCaP and PC3), normal prostate (N), and prostate cancer
patient tumors (T). Northern blots with 10 ug of total RNA were
probed with the 202P5A5 SSH fragment. Size standards in kilobases
are on the side. Results show expression of 202P5A5 in all prostate
cancer specimens tested as well as in the normal prostate, prostate
cancer xenografts and LNCaP, but not in the PC3 cell line.
[0068] FIG. 18. Expression of 202P5A5 in Bladder Cancer Patient
Specimens. RNA was extracted from bladder cancer cell lines (CL),
normal bladder (N), bladder cancer patient tumors (T) as well as
their adjacent normal tissues (Nat). Northern blots with 10 ug of
total RNA were probed with the 202P5A5 sequence. Size standards in
kilobases are on the side. Results show expression of 202P5A5 in
all bladder cancer patient tumor specimens tested but not in normal
bladder. Expression was also detected in SCABER but not in the
other cancer cell lines tested.
[0069] FIG. 19. Expression of 202P5A5 in Breast Cancer Patient
Specimens. RNA was extracted from breast cancer cell lines (CL),
normal breast (N), breast cancer patient tumors (T), and breast
cancer metastasis specimens (M). Northern blots with 10 ug of total
RNA were probed with the 202P5A5 sequence. Size standards in
kilobases are on the side. Results show expression of 202P5A5 in
the breast cancer patient tumors and metastasis specimens.
Expression was also detected in MCF-7 and CAMA-1 but not in the
DU4475 cell line. Lower level expression was also detected in
normal breast.
[0070] FIG. 20. Expression of 202P5A5 in Colon and Cervical Cancer
Patient Specimens. First strand cDNA was prepared from a panel of
patient cancer specimens. Normalization was performed by PCR using
primers to actin. Semi-quantitative PCR, using primers to 202P5A5,
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 strong. Results show expression of 202P5A5 in the
majority of patient cancer specimens tested.
[0071] FIG. 21. Expression of 202P5A5.pcDNA3.1/MycHis following
transfection into 293T cells. 293T cells were transfected with
either 202P5A5.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control.
Forty hours later, cell lysates were collected. Samples were run on
an SDS-PAGE acrylamide gel, blotted and stained with anti-his
antibody. The blot was developed using the ECL chemiluminescence
kit and visualized by autoradiography. Results show expression of
202P5A5 from the 202P5A5.pcDNA3.1/MycHis construct in the lysates
of transfected cells but not in the control pcDNA3.1/MycHis
transfected cells.
DETAILED DESCRIPTION OF THE INVENTION
[0072] Outline of Sections
[0073] I.) Definitions
[0074] II.) 202P5A5 Polynucleotides
[0075] II.A.) Uses of 202P5A5 Polynucleotides
[0076] II.A.1.) Monitoring of Genetic Abnormalities
[0077] II.A.2.) Antisense Embodiments
[0078] II.A.3.) Primers and Primer Pairs
[0079] II.A.4.) Isolation of 202P5A5-Encoding Nucleic Acid
Molecules
[0080] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0081] III.) 202P5A5-related Proteins
[0082] III.A.) Motif-bearing Protein Embodiments
[0083] III.B.) Expression of 202P5A5-related Proteins
[0084] III.C.) Modifications of 202P5A5-related Proteins
[0085] III.D.) Uses of 202P5A5-related Proteins
[0086] IV.) 202P5A5 Antibodies
[0087] V.) 202P5A5 Cellular Immune Responses
[0088] VI.) 202P5A5 Transgenic Animals
[0089] VII.) Methods for the Detection of 202P5A5
[0090] VIII.) Methods for Monitoring the Status of 202P5A5-related
Genes and Their Products
[0091] IX.) Identification of Molecules That Interact With
202P5A5
[0092] X.) Therapeutic Methods and Compositions
[0093] X.A.) Anti-Cancer Vaccines
[0094] X.B.) 202P5A5 as a Target for Antibody-Based Therapy
[0095] X.C.) 202P5A5 as a Target for Cellular Immune Responses
[0096] X.C.1. Minigene Vaccines
[0097] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0098] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0099] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0100] X.D.) Adoptive Immunotherapy
[0101] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0102] XI.) Diagnostic and Prognostic Embodiments of 202P5A5.
[0103] XII.) Inhibition of 202P5A5 Protein Function
[0104] XII.A.) Inhibition of 202P5A5 With Intracellular
Antibodies
[0105] XII.B.) Inhibition of 202P5A5 with Recombinant Proteins
[0106] XII.C.) Inhibition of 202P5A5 Transcription or
Translation
[0107] XII.D.) General Considerations for Therapeutic
Strategies
[0108] XIII.) Identification, Characterization and Use of
Modulators of 202P5A5
[0109] XIV.) KITS/Articles of Manufacture
[0110] I.) Definitions:
[0111] 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.
[0112] 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 C1-C2 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.
[0113] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 202P5A5 (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 202P5A5. 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.
[0114] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 202P5A5-related protein). For example, an analog
of a 202P5A5 protein can be specifically bound by an antibody or T
cell that specifically binds to 202P5A5.
[0115] 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-202P5A5 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.
[0116] 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-202P5A5 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-202P5A5 antibody compositions with polyepitopic
specificity.
[0117] 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."
[0118] 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)).
[0119] 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, January 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).
[0120] 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.).
[0121] 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, Sapaonaria officinalis inhibitor, and glucocorticoid
and other chemotherapeutic agents, as well as radioisotopes such as
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212 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.
[0122] 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.
[0123] "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.
[0124] 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.
[0125] 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.
[0126] "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).
[0127] 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.
[0128] 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 202P5A5 genes or that encode
polypeptides other than 202P5A5 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 202P5A5 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 202P5A5 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 202P5A5 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] A "motif", as in biological motif of a 202P5A5-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.
[0136] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
[0137] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0138] 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).
[0139] 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".
[0140] 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.
[0141] "Radioisotopes" include, but are not limited to the
following (non-limiting exemplary uses are also set forth):
Examples of Medical Isotopes:
TABLE-US-00001 [0142] Isotope Description of use Actinium-225 See
Thorium-229 (Th-229) (AC-225) Actinium-227 Parent of Radium-223
(Ra-223) which is an alpha emitter used to treat (AC-227)
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, for food irradiators, and for
(Co-60) sterilization of medical supplies Copper-64 A positron
emitter used for cancer therapy and SPECT imaging (Cu-64) Copper-67
Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic
(Cu-67) studies (i.e., breast and colon cancers, and lymphoma)
Dysprosium-166 Cancer radioimmunotherapy (Dy-166) Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints
associated (Er-169) with fingers and toes Europium-152 Radiation
source for food irradiation and for sterilization of medical
supplies (Eu-152) Europium-154 Radiation source for food
irradiation and for sterilization of medical supplies (Eu-154)
Gadolinium-153 Osteoporosis detection and nuclear medical quality
assurance devices (Gd-153) Gold-198 Implant and intracavity therapy
of ovarian, prostate, and brain cancers (Au-198) Holmium-166
Multiple myeloma treatment in targeted skeletal therapy, cancer
(Ho-166) radioimmunotherapy, bone marrow ablation, and rheumatoid
arthritis treatment Iodine-125 Osteoporosis detection, diagnostic
imaging, tracer drugs, brain cancer (I-125) 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
detection, treatment of thyroid (I-131) 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 treatment,
treatment of blocked (Ir-192) arteries (i.e., arteriosclerosis and
restenosis), and implants for breast and prostate tumors
Lutetium-177 Cancer radioimmunotherapy and treatment of blocked
arteries (i.e., (Lu-177) arteriosclerosis and restenosis)
Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used for
imaging the brain, (Mo-99) 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
cisplatin, a chemotherapeutic (Pt-195m) drug Phosphorus-32
Polycythemia rubra vera (blood cell disease) and leukemia
treatment, bone (P-32) 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
diagnosis/treatment, radiolabeling, and (P-33) 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 treatment, and diagnosis and (Re-186)
treatment of lymphoma and bone, breast, colon, and liver cancers
using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment
using radioimmunotherapy, bone cancer pain (Re-188) 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 pain relief (Sm-153) Scandium-47 Cancer
radioimmunotherapy and bone cancer pain relief (Sc-47) Selenium-75
Radiotracer used in brain studies, imaging of adrenal cortex by
gamma- (Se-75) 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 treatment,
and osteoblastic (Sr-89) therapy Technetium-99m See Molybdenum-99
(Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212 (Bi-212) which
is an alpha emitter used in cancer (Th-228) radioimmunotherapy
Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of
Bismuth-213 (Bi-213) (Th-229) which are alpha emitters used in
cancer radioimmunotherapy Thulium-170 Gamma source for blood
irradiators, energy source for implanted medical (Tm-170) devices
Tin-117m Cancer immunotherapy and bone cancer pain relief (Sn-117m)
Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for
cancer (W-188) 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 resolution SPECT studies, pulmonary (Xe-127)
function tests, and cerebral blood flow studies Ytterbium-175
Cancer radioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained
from irradiating Yttrium-89 (Y-89) for liver cancer (Y-90)
treatment Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90)
which is used for cancer (Y-91) radioimmunotherapy (i.e., lymphoma,
breast, colon, kidney, lung, ovarian, prostate, pancreatic, and
inoperable liver cancers)
[0143] 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.
[0144] 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.
[0145] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0146] Non-limiting examples of small molecules include compounds
that bind or interact with 202P5A5, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit 202P5A5
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, 202P5A5 protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions.
[0147] "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).
[0148] "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.
[0149] 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: [0150] A2:
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901,
A*0207 [0151] A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101,
A*3101 [0152] 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 [0153] B44:
B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006) [0154] A1:
A*0102, A*2604, A*3601, A*4301, A*8001 [0155] A24: A*24, A*30,
A*2403, A*2404, A*3002, A*3003 [0156] 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 [0157] B58: B*1516, B*1517, B*5701,
B*5702, B58 [0158] B62: B*4601, B52, B*1501 (B62), B*1502 (B75),
B*1513 (B77)
[0159] Calculated population coverage afforded by different
HLA-supertype combinations are set forth in Table IV (G).
[0160] 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.
[0161] 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.
[0162] 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.
[0163] The term "variant" refers to a molecule that exhibits a
variation from a described type or norm, such as a protein that has
one or more different amino acid residues in the corresponding
position(s) of a specifically described protein (e.g. the 202P5A5
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.
[0164] The "202P5A5-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 202P5A5 proteins or fragments thereof, as well as fusion
proteins of a 202P5A5 protein and a heterologous polypeptide are
also included. Such 202P5A5 proteins are collectively referred to
as the 202P5A5-related proteins, the proteins of the invention, or
202P5A5. The term "202P5A5-related protein" refers to a polypeptide
fragment or a 202P5A5 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.
II.) 202P5A5 Polynucleotides
[0165] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of a 202P5A5 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding a 202P5A5-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to a 202P5A5 gene
or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to a 202P5A5 gene, mRNA, or to a
202P5A5 encoding polynucleotide (collectively, "202P5A5
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0166] Embodiments of a 202P5A5 polynucleotide include: a 202P5A5
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 202P5A5 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 202P5A5 nucleotides comprise, without
limitation:
[0167] (I) a polynucleotide comprising, consisting essentially of,
or consisting of a sequence as shown in FIG. 2, wherein T can also
be U;
[0168] (II) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2A, from nucleotide
residue number 29 through nucleotide residue number 1858, including
the stop codon, wherein T can also be U;
[0169] (III) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2B, from
nucleotide residue number 13 through nucleotide residue number
1890, including the stop codon, wherein T can also be U;
[0170] (IV) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2C, from nucleotide
residue number 121 through nucleotide residue number 1950,
including the a stop codon, wherein T can also be U;
[0171] (V) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2D, from nucleotide
residue number 29 through nucleotide residue number 1858, including
the stop codon, wherein T can also be U;
[0172] (VI) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2E, from nucleotide
residue number 29 through nucleotide residue number 1858, including
the stop codon, wherein T can also be U;
[0173] (VII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIGS. 2F and 2A, from
nucleotide residue number 29 through nucleotide residue number
1858, including the stop codon, wherein T can also be U;
[0174] (VIII) a polynucleotide that encodes a 202P5A5-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;
[0175] (IX) a polynucleotide that encodes a 202P5A5-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;
[0176] (X) a polynucleotide that encodes at least one peptide set
forth in Tables VIII-XXI and XXII-XLIX;
[0177] (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-3F in any whole number
increment up to 609 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;
[0178] (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 FIGS. 3A and 3C-3F in any whole number
increment up to 609 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;
[0179] (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 FIGS. 3A and 3C-3F in any whole number
increment up to 609 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;
[0180] (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 FIGS. 3A and 3C-3F in any whole number
increment up to 609 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;
[0181] (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 FIGS. 3A and 3C-3F in any whole number
increment up to 609 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;
[0182] (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
625 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 Hydrophilicity profile of FIG. 5;
[0183] (XVII) 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
625 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;
[0184] (XVIII) 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
625 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;
[0185] (XIX) 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
625 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;
[0186] (XX) 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
625 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;
[0187] (XXI) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XX);
[0188] (XXII) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XXI);
[0189] (XXIII) a peptide that is encoded by any of (I) to (XXII);
and;
[0190] (XXIV) a composition comprising a polynucleotide of any of
(I)-(XXII) or peptide of (XXIII) together with a pharmaceutical
excipient and/or in a human unit dose form;
[0191] (XXV) a method of using a polynucleotide of any (I)-(XXII)
or peptide of (XXIII) or a composition of (XXIV) in a method to
modulate a cell expressing 202P5A5;
[0192] (XXVI) a method of using a polynucleotide of any (I)-(XXII)
or peptide of (XXIII) or a composition of (XXIV) in a method to
diagnose, prophylax, prognose, or treat an individual who bears a
cell expressing 202P5A5;
[0193] (XXVII) a method of using a polynucleotide of any (I)-(XXII)
or peptide of (XXIII) or a composition of (XXIV) in a method to
diagnose, prophylax, prognose, or treat an individual who bears a
cell expressing 202P5A5, said cell from a cancer of a tissue listed
in Table I;
[0194] (XXVIII) a method of using a polynucleotide of any
(I)-(XXII) or peptide of (XXIII) or a composition of (XXIV) in a
method to diagnose, prophylax, prognose, or treat a cancer;
[0195] (XXIX) a method of using a polynucleotide of any (I)-(XXII)
or peptide of (XXIII) or a composition of (XXIV) in a method to
diagnose, prophylax, prognose, or treat a cancer of a tissue listed
in Table I; and;
[0196] (XXX) a method of using a polynucleotide of any (I)-(XXII)
or peptide of (XXIII) or a composition of (XXIV) in a method to
identify or characterize a modulator of a cell expressing
202P5A5.
[0197] As used herein, a range is understood to disclose
specifically all whole unit positions thereof.
[0198] Typical embodiments of the invention disclosed herein
include 202P5A5 polynucleotides that encode specific portions of
202P5A5 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0199] (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, 605, 609 or more contiguous amino acids of 202P5A5 variant 1;
the maximal lengths relevant for other variants are: variant 2, 625
amino acids; variant 4, 609 amino acids, variant 5, 609 amino
acids, variant 6, 609 amino acids, and variant 8, 609 amoni
acids.
[0200] 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 202P5A5 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 202P5A5 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 202P5A5
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 202P5A5 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 202P5A5 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 202P5A5 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 202P5A5 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 202P5A5
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 202P5A5 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 202P5A5 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 202P5A5 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.
[0201] Polynucleotides encoding relatively long portions of a
202P5A5 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 202P5A5 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 202P5A5
sequence as shown in FIG. 2.
[0202] Additional illustrative embodiments of the invention
disclosed herein include 202P5A5 polynucleotide fragments encoding
one or more of the biological motifs contained within a 202P5A5
protein "or variant" sequence, including one or more of the
motif-bearing subsequences of a 202P5A5 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 202P5A5 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 202P5A5 protein or variant N-glycosylation sites, cAMP and
cGMP-dependent protein kinase phosphorylation sites, casein kinase
II phosphorylation sites or N-myristoylation site and amidation
sites.
[0203] 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.
[0204] II.A.) Uses of 202P5A5 Polynucleotides
[0205] II.A.1.) Monitoring of Genetic Abnormalities
[0206] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 202P5A5 gene maps to
the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 202P5A5." For example, because the 202P5A5
gene maps to this chromosome, polynucleotides that encode different
regions of the 202P5A5 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 202P5A5 proteins provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 202P5A5 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)).
[0207] Furthermore, as 202P5A5 was shown to be highly expressed in
prostate and other cancers, 202P5A5 polynucleotides are used in
methods assessing the status of 202P5A5 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 202P5A5 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 202P5A5 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.
[0208] II.A.2.) Antisense Embodiments
[0209] 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 202P5A5. 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 202P5A5 polynucleotides and polynucleotide
sequences disclosed herein.
[0210] 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., 202P5A5. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 202P5A5 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 202P5A5 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).
[0211] The 202P5A5 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 202P5A5 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 202P5A5 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 202P5A5 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 202P5A5 mRNA. Optionally, 202P5A5 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
202P5A5. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 202P5A5 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet.
12:510-515 (1996).
[0212] II.A.3.) Primers and Primer Pairs
[0213] 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 202P5A5 polynucleotide in a sample and as a means for
detecting a cell expressing a 202P5A5 protein.
[0214] Examples of such probes include polypeptides comprising all
or part of the human 202P5A5 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 202P5A5
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 202P5A5 mRNA.
[0215] The 202P5A5 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
202P5A5 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 202P5A5
polypeptides; as tools for modulating or inhibiting the expression
of the 202P5A5 gene(s) and/or translation of the 202P5A5
transcript(s); and as therapeutic agents.
[0216] The present invention includes the use of any probe as
described herein to identify and isolate a 202P5A5 or 202P5A5
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.
[0217] II.A.4.) Isolation of 202P5A5-Encoding Nucleic Acid
Molecules
[0218] The 202P5A5 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 202P5A5 gene
product(s), as well as the isolation of polynucleotides encoding
202P5A5 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 202P5A5 gene product as
well as polynucleotides that encode analogs of 202P5A5-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding a 202P5A5 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 202P5A5 gene cDNAs can be identified by
probing with a labeled 202P5A5 cDNA or a fragment thereof. For
example, in one embodiment, a 202P5A5 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 202P5A5 gene.
A 202P5A5 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 202P5A5
DNA probes or primers.
[0219] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0220] The invention also provides recombinant DNA or RNA molecules
containing a 202P5A5 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).
[0221] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a 202P5A5
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 202P5A5 or a fragment, analog or homolog thereof can be
used to generate 202P5A5 proteins or fragments thereof using any
number of host-vector systems routinely used and widely known in
the art.
[0222] A wide range of host-vector systems suitable for the
expression of 202P5A5 proteins or fragments thereof are available,
see for example, Sambrook et al., 1989, supra; Current Protocols in
Molecular Biology, 1995, supra). Preferred vectors for mammalian
expression include but are not limited to pcDNA 3.1 myc-His-tag
(Invitrogen) and the retroviral vector pSR.alpha.tkneo (Muller et
al., 1991, MCB 11:1785). Using these expression vectors, 202P5A5
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 202P5A5 protein or fragment thereof. Such
host-vector systems can be employed to study the functional
properties of 202P5A5 and 202P5A5 mutations or analogs.
[0223] Recombinant human 202P5A5 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 202P5A5-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 202P5A5 or fragment, analog or homolog thereof, a
202P5A5-related protein is expressed in the 293T cells, and the
recombinant 202P5A5 protein is isolated using standard purification
methods (e.g., affinity purification using anti-202P5A5
antibodies). In another embodiment, a 202P5A5 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 202P5A5 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 202P5A5 coding sequence can be used for the generation
of a secreted form of recombinant 202P5A5 protein.
[0224] As discussed herein, redundancy in the genetic code permits
variation in 202P5A5 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.
[0225] 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)).
[0226] III.) 202P5A5-Related Proteins
[0227] Another aspect of the present invention provides
202P5A5-related proteins. Specific embodiments of 202P5A5 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 202P5A5 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 202P5A5 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 202P5A5 shown in FIG. 2 or FIG. 3.
[0228] Embodiments of a 202P5A5 polypeptide include: a 202P5A5
polypeptide having a sequence shown in FIG. 2, a peptide sequence
of a 202P5A5 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 202P5A5 peptides comprise, without
limitation:
[0229] (I) a protein comprising, consisting essentially of, or
consisting of an amino acid sequence as shown in FIG. 2A-F or FIG.
3A-F;
[0230] (II) a 202P5A5-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-F;
[0231] (III) a 202P5A5-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-F;
[0232] (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;
[0233] (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;
[0234] (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;
[0235] (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;
[0236] (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;
[0237] (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 FIG. 3A,
3C-3F in any whole number increment up to 609 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;
[0238] (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 FIG. 3A,
3C-3F, in any whole number increment up to 609 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;
[0239] (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 FIG. 3A,
3C-3F, in any whole number increment up to 609 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;
[0240] (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 FIG. 3A,
3C-3F, in any whole number increment up to 609 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;
[0241] (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 FIG. 3A,
3C-3F in any whole number increment up to 609 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;
[0242] (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 625 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;
[0243] (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 625 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;
[0244] (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 625 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;
[0245] (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 625 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;
[0246] (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 625 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;
[0247] (XIX) a peptide that occurs at least twice in Tables
VIII-XXI and XXII to XLIX, collectively;
[0248] (XX) a peptide that occurs at least three times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0249] (XXI) a peptide that occurs at least four times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0250] (XXII) a peptide that occurs at least five times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0251] (XXIII) a peptide that occurs at least once in Tables
VIII-XXI, and at least once in tables XXII to XLIX;
[0252] (XXIV) a peptide that occurs at least once in Tables
VIII-XXI, and at least twice in tables XXII to XLIX;
[0253] (XXV) a peptide that occurs at least twice in Tables
VIII-XXI, and at least once in tables XXII to XLIX;
[0254] (XXVI) a peptide that occurs at least twice in Tables
VIII-XXI, and at least twice in tables XXII to XLIX;
[0255] (XXVII) a peptide which comprises one two, three, four, or
five of the following characteristics, or an oligonucleotide
encoding such peptide:
[0256] 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;
[0257] 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;
[0258] 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;
[0259] 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,
[0260] 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;
[0261] (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;
[0262] (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 202P5A5;
[0263] (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 202P5A5;
[0264] (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 202P5A5, said cell from a cancer of a
tissue listed in Table I;
[0265] (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;
[0266] (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;
[0267] (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
202P5A5.
[0268] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0269] Typical embodiments of the invention disclosed herein
include 202P5A5 polynucleotides that encode specific portions of
202P5A5 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0270] (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, 605, and 609 or more contiguous amino acids of 202P5A5 variant
1; the maximal lengths relevant for other variants are: variant 2,
625 amino acids; variant 4, 609 amino acids, variant 5, 609 amino
acids, variant 6, 609 amino acids, and variant 8, 609 amino
acids.
[0271] In general, naturally occurring allelic variants of human
202P5A5 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
202P5A5 protein contain conservative amino acid substitutions
within the 202P5A5 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 202P5A5. One class of 202P5A5 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 202P5A5 amino acid sequence, but further
contain a radical departure from the sequence, such as a
non-conservative substitution, truncation, insertion or frame
shift. In comparisons of protein sequences, the terms, similarity,
identity, and homology each have a distinct meaning as appreciated
in the field of genetics. Moreover, orthology and paralogy can be
important concepts describing the relationship of members of a
given protein family in one organism to the members of the same
family in other organisms.
[0272] 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).
[0273] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 202P5A5 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 202P5A5 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
202P5A5 variant DNA.
[0274] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence that
is involved in a specific biological activity such as a
protein-protein interaction. Among the preferred scanning amino
acids are relatively small, neutral amino acids. Such amino acids
include alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid among this group because
it eliminates the side-chain beyond the beta-carbon and is less
likely to alter the main-chain conformation of the variant. Alanine
is also typically preferred because it is the most common amino
acid. Further, it is frequently found in both buried and exposed
positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does
not yield adequate amounts of variant, an isosteric amino acid can
be used.
[0275] As defined herein, 202P5A5 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 202P5A5 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
202P5A5 variant also specifically binds to a 202P5A5 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 202P5A5 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.
[0276] Other classes of 202P5A5-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 202P5A5
protein variants or analogs comprises one or more of the 202P5A5
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of 202P5A5
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.
[0277] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a 202P5A5 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 202P5A5 protein shown in
FIG. 2 or FIG. 3.
[0278] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of a 202P5A5 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a 202P5A5 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a 202P5A5 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of a
202P5A5 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of a 202P5A5 protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 202P5A5 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 60 to about
amino acid 70 of a 202P5A5 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 70 to about amino acid
80 of a 202P5A5 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 80 to about amino acid 90 of a
202P5A5 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 90 to about amino acid 100 of a 202P5A5 protein
shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a
202P5A5 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 202P5A5 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.
[0279] 202P5A5-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
202P5A5-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a 202P5A5 protein
(or variants, homologs or analogs thereof).
[0280] III.A.) Motif-Bearing Protein Embodiments
[0281] Additional illustrative embodiments of the invention
disclosed herein include 202P5A5 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a 202P5A5 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/seq-search/struc-predict.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/.).
[0282] Motif bearing subsequences of all 202P5A5 variant proteins
are set forth and identified in Tables VIII-XXI and XXII-XLIX.
[0283] 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.
[0284] Polypeptides comprising one or more of the 202P5A5 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 202P5A5 motifs discussed above are associated with growth
dysregulation and because 202P5A5 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)).
[0285] 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 202P5A5 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/epimatrix/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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 202P5A5-related proteins are embodied in many forms,
preferably in isolated form. A purified 202P5A5 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 202P5A5 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 202P5A5-related proteins include
purified 202P5A5-related proteins and functional, soluble
202P5A5-related proteins. In one embodiment, a functional, soluble
202P5A5 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0290] The invention also provides 202P5A5 proteins comprising
biologically active fragments of a 202P5A5 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting 202P5A5 protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting 202P5A5 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.
[0291] 202P5A5-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
anti-202P5A5 antibodies or T cells or in identifying cellular
factors that bind to 202P5A5. 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.
[0292] CTL epitopes can be determined using specific algorithms to
identify peptides within a 202P5A5 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 202P5A5 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 202P5A5
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/.
[0293] 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 HLA Class I molecules. Many HLA class I
binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class
I HLA-A2, the epitopes preferably contain a leucine (L) or
methionine (M) at position 2 and a valine (V) or leucine (L) at the
C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7
(1992)). Selected results of 202P5A5 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.
[0294] 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.
[0295] 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 202P5A5 protein in
accordance with the invention. As used in this context "applied"
means that a 202P5A5 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 202P5A5 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.
[0296] III.B.) Expression of 202P5A5-Related Proteins
[0297] In an embodiment described in the examples that follow,
202P5A5 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 202P5A5 with a C-terminal
6.times.His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5,
GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides
an IgGK secretion signal that can be used to facilitate the
production of a secreted 202P5A5 protein in transfected cells. The
secreted HIS-tagged 202P5A5 in the culture media can be purified,
e.g., using a nickel column using standard techniques.
[0298] III.C.) Modifications of 202P5A5-Related Proteins
[0299] Modifications of 202P5A5-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 202P5A5 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a 202P5A5 protein. Another type of
covalent modification of a 202P5A5 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 202P5A5 comprises linking a 202P5A5 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.
[0300] The 202P5A5-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 202P5A5
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 202P5A5 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 202P5A5. A chimeric
molecule can comprise a fusion of a 202P5A5-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 202P5A5 protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
202P5A5-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 202P5A5 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0301] III.D.) Uses of 202P5A5-Related Proteins
[0302] The proteins of the invention have a number of different
specific uses. As 202P5A5 is highly expressed in prostate and other
cancers, 202P5A5-related proteins are used in methods that assess
the status of 202P5A5 gene products in normal versus cancerous
tissues, thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of a 202P5A5 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 202P5A5-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within a 202P5A5 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,
202P5A5-related proteins that contain the amino acid residues of
one or more of the biological motifs in a 202P5A5 protein are used
to screen for factors that interact with that region of
202P5A5.
[0303] 202P5A5 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of a 202P5A5 protein), for identifying agents or cellular
factors that bind to 202P5A5 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.
[0304] Proteins encoded by the 202P5A5 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 202P5A5 gene product. Antibodies raised against a 202P5A5
protein or fragment thereof are useful in diagnostic and prognostic
assays, and imaging methodologies in the management of human
cancers characterized by expression of 202P5A5 protein, such as
those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 202P5A5-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0305] Various immunological assays useful for the detection of
202P5A5 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
202P5A5-expressing cells (e.g., in radioscintigraphic imaging
methods). 202P5A5 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
[0306] IV.) 202P5A5 Antibodies
[0307] Another aspect of the invention provides antibodies that
bind to 202P5A5-related proteins. Preferred antibodies specifically
bind to a 202P5A5-related protein and do not bind (or bind weakly)
to peptides or proteins that are not 202P5A5-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 37.degree. C. For example, antibodies that bind 202P5A5 can bind
202P5A5-related proteins such as the homologs or analogs
thereof.
[0308] 202P5A5 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 202P5A5 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 202P5A5 is involved, such as
advanced or metastatic prostate cancers.
[0309] The invention also provides various immunological assays
useful for the detection and quantification of 202P5A5 and mutant
202P5A5-related proteins. Such assays can comprise one or more
202P5A5 antibodies capable of recognizing and binding a
202P5A5-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.
[0310] 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.
[0311] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing 202P5A5 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 202P5A5
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 202P5A5 expressing cancers such as
prostate cancer.
[0312] 202P5A5 antibodies are also used in methods for purifying a
202P5A5-related protein and for isolating 202P5A5 homologues and
related molecules. For example, a method of purifying a
202P5A5-related protein comprises incubating a 202P5A5 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 202P5A5-related protein under conditions that
permit the 202P5A5 antibody to bind to the 202P5A5-related protein;
washing the solid matrix to eliminate impurities; and eluting the
202P5A5-related protein from the coupled antibody. Other uses of
202P5A5 antibodies in accordance with the invention include
generating anti-idiotypic antibodies that mimic a 202P5A5
protein.
[0313] 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 202P5A5-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 202P5A5 can also be used, such as a
202P5A5 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 202P5A5-related
protein is synthesized and used as an immunogen.
[0314] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 202P5A5-related protein or
202P5A5 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0315] The amino acid sequence of a 202P5A5 protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
202P5A5 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 202P5A5 amino acid
sequence are used to identify hydrophilic regions in the 202P5A5
structure. Regions of a 202P5A5 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-Doolittle, 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 202P5A5 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
202P5A5 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.
[0316] 202P5A5 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
202P5A5-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.
[0317] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a 202P5A5 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
202P5A5 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.
[0318] 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 202P5A5 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 202P5A5
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued
19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and, 6,114598 issued 5
Sep. 2000). This method avoids the in vitro manipulation required
with phage display technology and efficiently produces high
affinity authentic human antibodies.
[0319] Reactivity of 202P5A5 antibodies with a 202P5A5-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 202P5A5-related proteins,
202P5A5-expressing cells or extracts thereof. A 202P5A5 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 202P5A5 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).
[0320] V.) 202P5A5 Cellular Immune Responses
[0321] 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.
[0322] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the
study of single amino acid substituted antigen analogs and the
sequencing of endogenously bound, naturally processed peptides,
critical residues that correspond to motifs required for specific
binding to HLA antigen molecules have been identified and are set
forth in Table IV (see also, e.g., Southwood, et al., J. Immunol.
160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al., SYFPEITHI, access via World Wide Web at URL
(134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and
Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H.,
Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr.
Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr.
Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et
al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol.
157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996;
Sette, A. and Sidney, J. Immunogenetics 1999 November;
50(3-4):201-12, Review).
[0323] 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.)
[0324] 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).
[0325] 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.
[0326] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0327] 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.
[0328] 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.51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0329] 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.
[0330] VI.) 202P5A5 Transgenic Animals
[0331] Nucleic acids that encode a 202P5A5-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 202P5A5 can be used to clone genomic DNA
that encodes 202P5A5. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 202P5A5. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. Nos.
4,736,866 issued 12 Apr. 1988, and 4,870,009 issued 26 Sep. 1989.
Typically, particular cells would be targeted for 202P5A5 transgene
incorporation with tissue-specific enhancers.
[0332] Transgenic animals that include a copy of a transgene
encoding 202P5A5 can be used to examine the effect of increased
expression of DNA that encodes 202P5A5. 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.
[0333] Alternatively, non-human homologues of 202P5A5 can be used
to construct a 202P5A5 "knock out" animal that has a defective or
altered gene encoding 202P5A5 as a result of homologous
recombination between the endogenous gene encoding 202P5A5 and
altered genomic DNA encoding 202P5A5 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 202P5A5 can be
used to clone genomic DNA encoding 202P5A5 in accordance with
established techniques. A portion of the genomic DNA encoding
202P5A5 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 202P5A5
polypeptide.
[0334] VII.) Methods for the Detection of 202P5A5
[0335] Another aspect of the present invention relates to methods
for detecting 202P5A5 polynucleotides and 202P5A5-related proteins,
as well as methods for identifying a cell that expresses 202P5A5.
The expression profile of 202P5A5 makes it a diagnostic marker for
metastasized disease. Accordingly, the status of 202P5A5 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 202P5A5 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.
[0336] More particularly, the invention provides assays for the
detection of 202P5A5 polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 202P5A5 polynucleotides
include, for example, a 202P5A5 gene or fragment thereof, 202P5A5
mRNA, alternative splice variant 202P5A5 mRNAs, and recombinant DNA
or RNA molecules that contain a 202P5A5 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 202P5A5
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0337] In one embodiment, a method for detecting a 202P5A5 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 202P5A5 polynucleotides as sense and
antisense primers to amplify 202P5A5 cDNAs therein; and detecting
the presence of the amplified 202P5A5 cDNA. Optionally, the
sequence of the amplified 202P5A5 cDNA can be determined.
[0338] In another embodiment, a method of detecting a 202P5A5 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 202P5A5
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 202P5A5 gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
202P5A5 nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0339] The invention also provides assays for detecting the
presence of a 202P5A5 protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a 202P5A5-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 202P5A5-related
protein in a biological sample comprises first contacting the
sample with a 202P5A5 antibody, a 202P5A5-reactive fragment
thereof, or a recombinant protein containing an antigen-binding
region of a 202P5A5 antibody; and then detecting the binding of
202P5A5-related protein in the sample.
[0340] Methods for identifying a cell that expresses 202P5A5 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 202P5A5 gene comprises
detecting the presence of 202P5A5 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 202P5A5 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 202P5A5, 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 202P5A5 gene comprises detecting the presence of
202P5A5-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 202P5A5-related proteins
and cells that express 202P5A5-related proteins.
[0341] 202P5A5 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 202P5A5 gene
expression. For example, 202P5A5 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 202P5A5 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 202P5A5 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0342] VIII.) Methods for Monitoring the Status of 202P5A5-Related
Genes and Their
Products
[0343] 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 202P5A5 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 202P5A5 in a biological
sample of interest can be compared, for example, to the status of
202P5A5 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 202P5A5 in the
biological sample (as compared to the normal sample) provides
evidence of dysregulated cellular growth. In addition to using a
biological sample that is not affected by a pathology as a normal
sample, one can also use a predetermined normative value such as a
predetermined normal level of mRNA expression (see, e.g., Greyer et
al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No.
5,837,501) to compare 202P5A5 status in a sample.
[0344] 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 202P5A5
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 202P5A5 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 202P5A5
comprises a change in the location of 202P5A5 and/or 202P5A5
expressing cells and/or an increase in 202P5A5 mRNA and/or protein
expression.
[0345] 202P5A5 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 202P5A5 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 202P5A5 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 202P5A5 gene), Northern analysis and/or PCR analysis of 202P5A5
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 202P5A5 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
202P5A5 proteins and/or associations of 202P5A5 proteins with
polypeptide binding partners). Detectable 202P5A5 polynucleotides
include, for example, a 202P5A5 gene or fragment thereof, 202P5A5
mRNA, alternative splice variants, 202P5A5 mRNAs, and recombinant
DNA or RNA molecules containing a 202P5A5 polynucleotide.
[0346] The expression profile of 202P5A5 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 202P5A5 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 202P5A5 status and diagnosing
cancers that express 202P5A5, such as cancers of the tissues listed
in Table I. For example, because 202P5A5 mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of 202P5A5 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 202P5A5 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0347] The expression status of 202P5A5 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 202P5A5 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.
[0348] As described above, the status of 202P5A5 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 202P5A5 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 202P5A5
expressing cells (e.g. those that express 202P5A5 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 202P5A5-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 202P5A5 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).
[0349] In one aspect, the invention provides methods for monitoring
202P5A5 gene products by determining the status of 202P5A5 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 202P5A5 gene products in a corresponding normal
sample. The presence of aberrant 202P5A5 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.
[0350] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 202P5A5 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
202P5A5 mRNA can, for example, be evaluated in tissues including
but not limited to those listed in Table I. The presence of
significant 202P5A5 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 202P5A5 mRNA or
express it at lower levels.
[0351] In a related embodiment, 202P5A5 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 202P5A5 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 202P5A5 expressed in a corresponding
normal sample. In one embodiment, the presence of 202P5A5 protein
is evaluated, for example, using immunohistochemical methods.
202P5A5 antibodies or binding partners capable of detecting 202P5A5
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0352] In a further embodiment, one can evaluate the status of
202P5A5 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
202P5A5 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 202P5A5 indicates a potential loss of function or
increase in tumor growth.
[0353] 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 202P5A5 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and 5,952,170
issued 17 Jan. 1995).
[0354] Additionally, one can examine the methylation status of a
202P5A5 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-I 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.
[0355] Gene amplification is an additional method for assessing the
status of 202P5A5. 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.
[0356] 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 202P5A5 expression.
The presence of RT-PCR amplifiable 202P5A5 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).
[0357] 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 202P5A5 mRNA or 202P5A5 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 202P5A5 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 202P5A5
in prostate or other tissue is examined, with the presence of
202P5A5 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 202P5A5 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 202P5A5 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0358] 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
202P5A5 mRNA or 202P5A5 protein expressed by tumor cells, comparing
the level so determined to the level of 202P5A5 mRNA or 202P5A5
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 202P5A5 mRNA or 202P5A5 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 202P5A5 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 202P5A5 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.
[0359] 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 202P5A5 mRNA or 202P5A5 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 202P5A5 mRNA or 202P5A5 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 202P5A5 mRNA or 202P5A5 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 202P5A5 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
202P5A5 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.
[0360] 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 202P5A5 gene and 202P5A5 gene products (or
perturbations in 202P5A5 gene and 202P5A5 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 202P5A5 gene
and 202P5A5 gene products (or perturbations in 202P5A5 gene and
202P5A5 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.
[0361] In one embodiment, methods for observing a coincidence
between the expression of 202P5A5 gene and 202P5A5 gene products
(or perturbations in 202P5A5 gene and 202P5A5 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 202P5A5 mRNA or protein in a tissue sample,
detecting the overexpression of PSA mRNA or protein in a tissue
sample (or PSCA or PSM expression), and observing a coincidence of
202P5A5 mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of 202P5A5 and PSA mRNA in prostate tissue is examined, where the
coincidence of 202P5A5 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.
[0362] Methods for detecting and quantifying the expression of
202P5A5 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 202P5A5 mRNA include in situ hybridization using
labeled 202P5A5 riboprobes, Northern blot and related techniques
using 202P5A5 polynucleotide probes, RT-PCR analysis using primers
specific for 202P5A5, 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 202P5A5 mRNA expression. Any number of primers
capable of amplifying 202P5A5 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
202P5A5 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0363] IX.) Identification of Molecules that Interact with
202P5A5
[0364] The 202P5A5 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 202P5A5, as well as
pathways activated by 202P5A5 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999,
5,925,523 issued 20 Jul. 1999, 5,846,722 issued 8 Dec. 1998 and
6,004,746 issued 21 Dec. 1999. Algorithms are also available in the
art for genome-based predictions of protein function (see, e.g.,
Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
[0365] Alternatively one can screen peptide libraries to identify
molecules that interact with 202P5A5 protein sequences. In such
methods, peptides that bind to 202P5A5 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 202P5A5 protein(s).
[0366] 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 202P5A5 protein sequences are disclosed for example
in U.S. Pat. Nos. 5,723,286 issued 3 Mar. 1998 and 5,733,731 issued
31 Mar. 1998.
[0367] Alternatively, cell lines that express 202P5A5 are used to
identify protein-protein interactions mediated by 202P5A5. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 202P5A5 protein can be immunoprecipitated from
202P5A5-expressing cell lines using anti-202P5A5 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 202P5A5 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.
[0368] Small molecules and ligands that interact with 202P5A5 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 202P5A5'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
202P5A5-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses 202P5A5 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2.sup.nd Ed., Sinauer Assoc., Sunderland,
Mass., 1992). Moreover, ligands that regulate 202P5A5 function can
be identified based on their ability to bind 202P5A5 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 202P5A5 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
202P5A5.
[0369] An embodiment of this invention comprises a method of
screening for a molecule that interacts with a 202P5A5 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a 202P5A5 amino acid
sequence, allowing the population of molecules and the 202P5A5
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 202P5A5 amino acid sequence, and then separating molecules
that do not interact with the 202P5A5 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 202P5A5 amino acid sequence. The identified
molecule can be used to modulate a function performed by 202P5A5.
In a preferred embodiment, the 202P5A5 amino acid sequence is
contacted with a library of peptides.
[0370] X.) Therapeutic Methods and Compositions
[0371] The identification of 202P5A5 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.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] Accordingly, therapeutic approaches that inhibit the
activity of a 202P5A5 protein are useful for patients suffering
from a cancer that expresses 202P5A5. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a 202P5A5
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a 202P5A5 gene or translation of 202P5A5 mRNA.
[0379] X.A.) Anti-Cancer Vaccines
[0380] The invention provides cancer vaccines comprising a
202P5A5-related protein or 202P5A5-related nucleic acid. In view of
the expression of 202P5A5, cancer vaccines prevent and/or treat
202P5A5-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).
[0381] Such methods can be readily practiced by employing a
202P5A5-related protein, or a 202P5A5-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 202P5A5 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 Feb. 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
202P5A5 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 202P5A5 immunogen contains
a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a
peptide of a size range from 202P5A5 indicated in FIG. 5, FIG. 6,
FIG. 7, FIG. 8, and FIG. 9.
[0382] The entire 202P5A5 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.
[0383] In patients with 202P5A5-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.
[0384] Cellular Vaccines:
[0385] CTL epitopes can be determined using specific algorithms to
identify peptides within 202P5A5 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
202P5A5 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.
[0386] Antibody-Based Vaccines
[0387] 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 202P5A5 protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to 202P5A5 in a host,
by contacting the host with a sufficient amount of at least one
202P5A5 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval thereafter re-contacting the host
with the 202P5A5 B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a 202P5A5-related protein or a man-made
multiepitopic peptide comprising: administering 202P5A5 immunogen
(e.g. a 202P5A5 protein or a peptide fragment thereof, a 202P5A5
fusion protein or analog etc.) in a vaccine preparation to a human
or another mammal. Typically, such vaccine preparations further
contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or
a universal helper epitope such as a PADRE.TM. peptide (Epimmune
Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol.
2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994
1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2):
79-92). An alternative method comprises generating an immune
response in an individual against a 202P5A5 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 202P5A5
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 202P5A5, in
order to generate a response to the target antigen.
[0388] Nucleic Acid Vaccines:
[0389] 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 202P5A5. Constructs comprising DNA encoding a
202P5A5-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 202P5A5 protein/immunogen.
Alternatively, a vaccine comprises a 202P5A5-related protein.
Expression of the 202P5A5-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a 202P5A5 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).
[0390] 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
202P5A5-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0391] 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.
[0392] Thus, gene delivery systems are used to deliver a
202P5A5-related nucleic acid molecule. In one embodiment, the
full-length human 202P5A5 cDNA is employed. In another embodiment,
202P5A5 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0393] Ex Vivo Vaccines
[0394] 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
202P5A5 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 202P5A5 peptides to T cells in the context of MHC class
I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with 202P5A5 peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete 202P5A5 protein. Yet another
embodiment involves engineering the overexpression of a 202P5A5
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 202P5A5 can also be engineered to express immune
modulators, such as GM-CSF, and used as immunizing agents.
[0395] X.B.) 202P5A5 as a Target for Antibody-Based Therapy
[0396] 202P5A5 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 202P5A5 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 202P5A5-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 202P5A5 are useful
to treat 202P5A5-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0397] 202P5A5 antibodies can be introduced into a patient such
that the antibody binds to 202P5A5 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 202P5A5, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0398] 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 202P5A5 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. 202P5A5), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0399] 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-202P5A5
antibody) that binds to a marker (e.g. 202P5A5) 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 202P5A5, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
202P5A5 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.
[0400] Cancer immunotherapy using anti-202P5A5 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, 202P5A5 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).
[0401] Although 202P5A5 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.
[0402] Although 202P5A5 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.
[0403] Cancer patients can be evaluated for the presence and level
of 202P5A5 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 202P5A5 imaging, or other
techniques that reliably indicate the presence and degree of
202P5A5 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.
[0404] Anti-202P5A5 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-202P5A5 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-202P5A5 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 202P5A5.
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-202P5A5 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.
[0405] 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 202P5A5 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0406] Therapeutic methods of the invention contemplate the
administration of single anti-202P5A5 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-202P5A5 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-202P5A5 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0407] Anti-202P5A5 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-202P5A5 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.
[0408] 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-202P5A5 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 202P5A5 expression in the patient, the
extent of circulating shed 202P5A5 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.
[0409] Optionally, patients should be evaluated for the levels of
202P5A5 in a given sample (e.g. the levels of circulating 202P5A5
antigen and/or 202P5A5 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).
[0410] Anti-idiotypic anti-202P5A5 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 202P5A5-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-202P5A5 antibodies that mimic an epitope on a 202P5A5-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.
[0411] X.C.) 202P5A5 as a Target for Cellular Immune Responses
[0412] 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.
[0413] 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 (P.sub.3CSS).
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))
[0414] 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 202P5A5 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0415] 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).
[0416] 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.
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] 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.
[0422] 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.
[0423] 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.
[0424] 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.
[0425] X.C.1. Minigene Vaccines
[0426] 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.
[0427] 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 202P5A5, the PADRE.RTM. universal helper T cell
epitope or multiple HTL epitopes from 202P5A5 (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.
[0428] 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.
[0429] For example, to create a DNA sequence encoding the selected
epitopes (minigene) for expression in human cells, the amino acid
sequences of the epitopes may be reverse translated. A human codon
usage table can be used to guide the codon choice for each amino
acid. These epitope-encoding DNA sequences may be directly
adjoined, so that when translated, a continuous polypeptide
sequence is created. To optimize expression and/or immunogenicity,
additional elements can be incorporated into the minigene design.
Examples of amino acid sequences that can be reverse translated and
included in the minigene sequence include: HLA class I epitopes,
HLA class II epitopes, antibody epitopes, a ubiquitination signal
sequence, and/or an endoplasmic reticulum targeting signal. In
addition, HLA presentation of CTL and HTL epitopes may be improved
by including synthetic (e.g. poly-alanine) or naturally-occurring
flanking sequences adjacent to the CTL or HTL epitopes; these
larger peptides comprising the epitope(s) are within the scope of
the invention.
[0430] 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.
[0431] 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.
[0432] 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.
[0433] 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.
[0434] 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.
[0435] In some embodiments, a bi-cistronic expression vector which
allows production of both the minigene-encoded epitopes and a
second protein (included to enhance or decrease immunogenicity) can
be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include
cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules
(e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR
binding proteins (PADRE.TM., Epimmune, San Diego, Calif.). Helper
(HTL) epitopes can be joined to intracellular targeting signals and
expressed separately from expressed CTL epitopes; this allows
direction of the HTL epitopes to a cell compartment different than
that of the CTL epitopes. If required, this could facilitate more
efficient entry of HTL epitopes into the HLA class II pathway,
thereby improving HTL induction. In contrast to HTL or CTL
induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0436] 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.
[0437] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0438] 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.
[0439] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human HLA proteins are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g., IM for DNA in PBS, intraperitoneal (i.p.) for
lipid-complexed DNA). Twenty-one days after immunization,
splenocytes are harvested and restimulated for one week in the
presence of peptides encoding each epitope being tested.
Thereafter, for CTL effector cells, assays are conducted for
cytolysis of peptide-loaded, .sup.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.
[0440] 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.
[0441] 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.
[0442] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0443] 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.
[0444] 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.
[0445] In certain embodiments, the T helper peptide is one that is
recognized by T helper cells present in a majority of a genetically
diverse population. This can be accomplished by selecting peptides
that bind to many, most, or all of the HLA class II molecules.
Examples of such amino acid bind many HLA Class II molecules
include sequences from antigens such as tetanus toxoid at positions
830-843 (QYIKANSKFIGITE; SEQ ID NO:24), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:25), and Streptococcus 18 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO:26).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0446] 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:27), 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.
[0447] 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.
[0448] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0449] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes B lymphocytes or T lymphocytes. Lipids have been
identified as agents capable of priming CTL in vivo. For example,
palmitic acid residues can be attached to the .epsilon.- and
.alpha.-amino groups of a lysine residue and then linked, e.g., via
one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser,
or the like, to an immunogenic peptide. The lipidated peptide can
then be administered either directly in a micelle or particle,
incorporated into a liposome, or emulsified in an adjuvant, e.g.,
incomplete Freund's adjuvant. In a preferred embodiment, a
particularly effective immunogenic composition comprises palmitic
acid attached to .epsilon.- and .alpha.-amino groups of Lys, which
is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the immunogenic peptide.
[0450] 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 P.sub.3CSS-conjugated epitopes, two such compositions can be
combined to more effectively elicit both humoral and cell-mediated
responses.
[0451] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0452] 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.
[0453] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 202P5A5. 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 202P5A5.
[0454] X.D. Adoptive Immunotherapy
[0455] Antigenic 202P5A5-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.
[0456] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0457] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 202P5A5. 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.
[0458] 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 202P5A5. 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.
[0459] For therapeutic use, administration should generally begin
at the first diagnosis of 202P5A5-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 202P5A5, a vaccine comprising
202P5A5-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] 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.
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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, 17.sup.th 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.
[0469] For antibodies, a treatment generally involves repeated
administration of the anti-202P5A5 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-202P5A5
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
202P5A5 expression in the patient, the extent of circulating shed
202P5A5 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.
[0470] 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.
[0471] 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.
[0472] 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.
[0473] 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.
[0474] 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%.
[0475] 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.
[0476] XI.) Diagnostic and Prognostic Embodiments of 202P5A5.
[0477] As disclosed herein, 202P5A5 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 202P5A5 in normal tissues, and patient
specimens").
[0478] 202P5A5 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 Prey 2000; 24(1):1-12). Therefore, this disclosure of
202P5A5 polynucleotides and polypeptides (as well as 202P5A5
polynucleotide probes and anti-202P5A5 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.
[0479] Typical embodiments of diagnostic methods which utilize the
202P5A5 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
202P5A5 polynucleotides described herein can be utilized in the
same way to detect 202P5A5 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 202P5A5
polypeptides described herein can be utilized to generate
antibodies for use in detecting 202P5A5 overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0480] 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 202P5A5 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
202P5A5-expressing cells (lymph node) is found to contain
202P5A5-expressing cells such as the 202P5A5 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0481] Alternatively 202P5A5 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 202P5A5 or
express 202P5A5 at a different level are found to express 202P5A5
or have an increased expression of 202P5A5 (see, e.g., the 202P5A5
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 202P5A5) such as PSA, PSCA
etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237
(1996)).
[0482] The use of immunohistochemistry to identify the presence of
a 202P5A5 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.
[0483] The 202P5A5 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.
[0484] 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)).
[0485] 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 202P5A5, the 202P5A5 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 202P5A5 protein and immune responses related
thereto very useful. Use of the 202P5A5 compositions allows those
skilled in the art to make important diagnostic and therapeutic
decisions.
[0486] Immunohistochemical reagents specific to 202P5A5 are also
useful to detect metastases of tumors expressing 202P5A5 when the
polypeptide appears in tissues where 202P5A5 is not normally
produced.
[0487] Thus, 202P5A5 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.
[0488] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, 202P5A5 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 202P5A5 in normal tissues, and patient
specimens," where a 202P5A5 polynucleotide fragment is used as a
probe to show the expression of 202P5A5 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 202P5A5 polynucleotide shown in
FIG. 2 or variant thereof) under conditions of high stringency.
[0489] 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. 202P5A5
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
202P5A5 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 202P5A5
polypeptide shown in FIG. 3).
[0490] As shown herein, the 202P5A5 polynucleotides and
polypeptides (as well as the 202P5A5 polynucleotide probes and
anti-202P5A5 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 202P5A5 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 202P5A5 polynucleotides and polypeptides (as well as the
202P5A5 polynucleotide probes and anti-202P5A5 antibodies used to
identify the presence of these molecules) need to be employed to
confirm a metastases of prostatic origin.
[0491] Finally, in addition to their use in diagnostic assays, the
202P5A5 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 202P5A5 gene maps (see the Example entitled "Chromosomal
Mapping of 202P5A5" below). Moreover, in addition to their use in
diagnostic assays, the 202P5A5-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).
[0492] Additionally, 202P5A5-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 202P5A5. 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 202P5A5 antigen. Antibodies or other molecules that react with
202P5A5 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0493] XII.) Inhibition of 202P5A5 Protein Function
[0494] The invention includes various methods and compositions for
inhibiting the binding of 202P5A5 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
202P5A5 function.
[0495] XII.A.) Inhibition of 202P5A5 with Intracellular
Antibodies
[0496] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 202P5A5 are introduced
into 202P5A5 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-202P5A5 antibody is
expressed intracellularly, binds to 202P5A5 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).
[0497] 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.
[0498] In one embodiment, intrabodies are used to capture 202P5A5
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 202P5A5
intrabodies in order to achieve the desired targeting. Such 202P5A5
intrabodies are designed to bind specifically to a particular
202P5A5 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a 202P5A5 protein are used to prevent 202P5A5
from gaining access to the nucleus, thereby preventing it from
exerting any biological activity within the nucleus (e.g.,
preventing 202P5A5 from forming transcription complexes with other
factors).
[0499] 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).
[0500] XII.B.) Inhibition of 202P5A5 with Recombinant Proteins
[0501] In another approach, recombinant molecules bind to 202P5A5
and thereby inhibit 202P5A5 function. For example, these
recombinant molecules prevent or inhibit 202P5A5 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 202P5A5 specific antibody
molecule. In a particular embodiment, the 202P5A5 binding domain of
a 202P5A5 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 202P5A5 ligand
binding domains linked to the Fc portion of a human IgG, such as
human IgG1. Such IgG portion can contain, for example, the C.sub.H2
and C.sub.H3 domains and the hinge region, but not the C.sub.H1
domain. Such dimeric fusion proteins are administered in soluble
form to patients suffering from a cancer associated with the
expression of 202P5A5, whereby the dimeric fusion protein
specifically binds to 202P5A5 and blocks 202P5A5 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0502] XII.C.) Inhibition of 202P5A5 Transcription or
Translation
[0503] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 202P5A5 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 202P5A5 mRNA into protein.
[0504] In one approach, a method of inhibiting the transcription of
the 202P5A5 gene comprises contacting the 202P5A5 gene with a
202P5A5 antisense polynucleotide. In another approach, a method of
inhibiting 202P5A5 mRNA translation comprises contacting a 202P5A5
mRNA with an antisense polynucleotide. In another approach, a
202P5A5 specific ribozyme is used to cleave a 202P5A5 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
202P5A5 gene, such as 202P5A5 promoter and/or enhancer elements.
Similarly, proteins capable of inhibiting a 202P5A5 gene
transcription factor are used to inhibit 202P5A5 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.
[0505] Other factors that inhibit the transcription of 202P5A5 by
interfering with 202P5A5 transcriptional activation are also useful
to treat cancers expressing 202P5A5. Similarly, factors that
interfere with 202P5A5 processing are useful to treat cancers that
express 202P5A5. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0506] XII.D.) General Considerations for Therapeutic
Strategies
[0507] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 202P5A5 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 202P5A5 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 202P5A5 antisense polynucleotides, ribozymes,
factors capable of interfering with 202P5A5 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0508] 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.
[0509] 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 202P5A5 to a binding partner, etc.
[0510] In vivo, the effect of a 202P5A5 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.
[0511] 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.
[0512] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th
Edition, A. Osal., Ed., 1980).
[0513] 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.
[0514] 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.
[0515] XIII.) Identification, Characterization and Use of
Modulators of 202P5A5
Methods to Identify and Use Modulators
[0516] 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.
[0517] 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.
Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
[0518] Proteins, nucleic acids, and antibodies of the invention are
used in screening assays. The cancer-associated proteins,
antibodies, nucleic acids, modified proteins and cells containing
these sequences are used in screening assays, such as evaluating
the effect of drug candidates on a "gene expression profile,"
expression profile of polypeptides or alteration of biological
function. In one embodiment, the expression profiles are used,
preferably in conjunction with high throughput screening techniques
to allow monitoring for expression profile genes after treatment
with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69
(2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome
Res 6:986-94, 1996).
[0519] The cancer proteins, antibodies, nucleic acids, modified
proteins and cells containing the native or modified cancer
proteins or genes are used in screening assays. That is, the
present invention comprises methods for screening for compositions
which modulate the cancer phenotype or a physiological function of
a cancer protein of the invention. This is done on a gene itself or
by evaluating the effect of drug candidates on a "gene expression
profile" or biological function. In one embodiment, expression
profiles are used, preferably in conjunction with high throughput
screening techniques to allow monitoring after treatment with a
candidate agent, see Zlokarnik, supra.
[0520] A variety of assays are executed directed to the genes and
proteins of the invention. Assays are run on an individual nucleic
acid or protein level. That is, having identified a particular gene
as up regulated in cancer, test compounds are screened for the
ability to modulate gene expression or for binding to the cancer
protein of the invention. "Modulation" in this context includes an
increase or a decrease in gene expression. The preferred amount of
modulation will depend on the original change of the gene
expression in normal versus tissue undergoing cancer, with changes
of at least 10%, preferably 50%, more preferably 100-300%, and in
some embodiments 300-1000% or greater. Thus, if a gene exhibits a
4-fold increase in cancer tissue compared to normal tissue, a
decrease of about four-fold is often desired; similarly, a 10-fold
decrease in cancer tissue compared to normal tissue a target value
of a 10-fold increase in expression by the test compound is often
desired. Modulators that exacerbate the type of gene expression
seen in cancer are also useful, e.g., as an upregulated target in
further analyses.
[0521] 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.
Expression Monitoring to Identify Compounds that Modify Gene
Expression
[0522] 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.
[0523] 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.
[0524] 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.
[0525] 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.
[0526] 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.
[0527] 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.
[0528] 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.
[0529] 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.
[0530] 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.
[0531] 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.
Biological Activity-Related Assays
[0532] 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.
[0533] 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.
[0534] 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.
[0535] 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.
High Throughput Screening to Identify Modulators
[0536] 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.
[0537] 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.
Use of Soft Agar Growth and Colony Formation to Identify and
Characterize Modulators
[0538] 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.
[0539] 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.
Evaluation of Contact Inhibition and Growth Density Limitation to
Identify and Characterize Modulators
[0540] 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.
[0541] 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 (.sup.3H)-thymidine is determined by incorporated
cpm.
[0542] 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.
Evaluation of Growth Factor or Serum Dependence to Identify and
Characterize Modulators
[0543] 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.
Use of Tumor-specific Marker Levels to Identify and Characterize
Modulators
[0544] 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).
[0545] 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.
Invasiveness into Matrigel to Identify and Characterize
Modulators
[0546] The degree of invasiveness into Matrigel or an extracellular
matrix constituent can be used as an assay to identify and
characterize compounds that modulate cancer associated sequences.
Tumor cells exhibit a positive correlation between malignancy and
invasiveness of cells into Matrigel or some other extracellular
matrix constituent. In this assay, tumorigenic cells are typically
used as host cells. Expression of a tumor suppressor gene in these
host cells would decrease invasiveness of the host cells.
Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994),
supra, can be used. Briefly, the level of invasion of host cells is
measured by using filters coated with Matrigel or some other
extracellular matrix constituent. Penetration into the gel, or
through to the distal side of the filter, is rated as invasiveness,
and rated histologically by number of cells and distance moved, or
by prelabeling the cells with .sup.125I and counting the
radioactivity on the distal side of the filter or bottom of the
dish. See, e.g., Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize
Modulators
[0547] 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.
[0548] 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).
[0549] Alternatively, various immune-suppressed or immune-deficient
host animals can be used. For example, a genetically athymic "nude"
mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921
(1974)), a SCID mouse, a thymectornized mouse, or an irradiated
mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978);
Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host.
Transplantable tumor cells (typically about 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.
In Vitro Assays to Identify and Characterize Modulators
[0550] 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.
[0551] Alternatively, a reporter gene system can be devised using a
cancer protein promoter operably linked to a reporter gene such as
luciferase, green fluorescent protein, CAT, or P-gal. The reporter
construct is typically transfected into a cell. After treatment
with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured according to
standard techniques known to those of skill in the art (Davis GF,
supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol.
1998: 9:624).
[0552] 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.
[0553] 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.
Binding Assays to Identify and Characterize Modulators
[0554] In binding assays in accordance with the invention, a
purified or isolated gene product of the invention is generally
used. For example, antibodies are generated to a protein of the
invention, and immunoassays are run to determine the amount and/or
location of protein. Alternatively, cells comprising the cancer
proteins are used in the assays.
[0555] 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.
[0556] 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.
[0557] Examples of suitable insoluble supports include microtiter
plates, arrays, membranes and beads. These are typically made of
glass, plastic (e.g., polystyrene), polysaccharide, nylon,
nitrocellulose, or Teflon.TM., etc. Microtiter plates and arrays
are especially convenient because a large number of assays can be
carried out simultaneously, using small amounts of reagents and
samples. The particular manner of binding of the composition to the
support is not crucial so long as it is compatible with the
reagents and overall methods of the invention, maintains the
activity of the composition and is nondiffusable. Preferred methods
of binding include the use of antibodies which do not sterically
block either the ligand binding site or activation sequence when
attaching the protein to the support, direct binding to "sticky" or
ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface, etc. Following binding of the protein or
ligand/binding agent to the support, excess unbound material is
removed by washing. The sample receiving areas may then be blocked
through incubation with bovine serum albumin (BSA), casein or other
innocuous protein or other moiety.
[0558] 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.
[0559] 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.
[0560] 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.
[0561] 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., I.sup.125, for the proteins and a fluorophor for the
compound. Proximity reagents, e.g., quenching or energy transfer
reagents are also useful.
Competitive Binding to Identify and Characterize Modulators
[0562] 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.
[0563] 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.
[0564] 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.
[0565] 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.
[0566] 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.
[0567] 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.
[0568] 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.
Use of Polynucleotides to Down-regulate or Inhibit a Protein of the
Invention.
[0569] 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.
Inhibitory and Antisense Nucleotides
[0570] 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.
[0571] 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.
[0572] 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.
[0573] Antisense molecules as used herein include antisense or
sense oligonucleotides. Sense oligonucleotides can, e.g., be
employed to block transcription by binding to the anti-sense
strand. The antisense and sense oligonucleotide comprise a single
stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target mRNA (sense) or DNA (antisense) sequences for
cancer molecules. Antisense or sense oligonucleotides, according to
the present invention, comprise a fragment generally at least about
12 nucleotides, preferably from about 12 to 30 nucleotides. The
ability to derive an antisense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in,
e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol
et al. (BioTechniques 6:958 (1988)).
Ribozymes
[0574] 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).
[0575] 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)).
Use of Modulators in Phenotypic Screening
[0576] 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
[0577] 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.
Use of Modulators to Affect Peptides of the Invention
[0578] 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.
Methods of Identifying Characterizing Cancer-associated
Sequences
[0579] 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.
[0580] In a preferred embodiment, the cancer genes are used as
probes to determine the number of copies of the cancer gene in the
genome. The cancer genes are used as probes to determine the
chromosomal localization of the cancer genes. Information such as
chromosomal localization finds use in providing a diagnosis or
prognosis in particular when chromosomal abnormalities such as
translocations, and the like are identified in the cancer gene
locus.
XIV.) Kits/Articles of Manufacture
[0581] 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.
[0582] The kit of the invention will typically comprise the
container described above and one or more other containers
associated therewith that comprise materials desirable from a
commercial and user standpoint, including buffers, diluents,
filters, needles, syringes; carrier, package, container, vial
and/or tube labels listing contents and/or instructions for use,
and package inserts with instructions for use.
[0583] A label can be present on or with the container to indicate
that the composition is used for a specific therapy or
non-therapeutic application, such as a prognostic, prophylactic,
diagnostic or laboratory application, and can also indicate
directions for either in vivo or in vitro use, such as those
described herein. Directions and or other information can also be
included on an insert(s) or label(s) which is included with or on
the kit. The label can be on or associated with the container. A
label a can be on a container when letters, numbers or other
characters forming the label are molded or etched into the
container itself; a label can be associated with a container when
it is present within a receptacle or carrier that also holds the
container, e.g., as a package insert. The label can indicate that
the composition is used for diagnosing, treating, prophylaxing or
prognosing a condition, such as a neoplasia of a tissue set forth
in Table I.
[0584] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0585] 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 282P1G3 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.
[0586] 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 282P1G3 and modulating the function of
282P1G3.
[0587] 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
[0588] 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 202P5A5 Gene
[0589] To isolate genes that are over-expressed in prostate cancer
the Suppression Subtractive Hybridization (SSH) procedure was
performed using cDNA derived from prostate cancer tissues. The
202P5A5 SSH cDNA sequence was derived from prostate tumor minus
cDNAs derived from normal prostate. The 202P5A5 cDNA was identified
as highly expressed in prostate cancer as well as in other cancers
listed in Table I.
Materials and Methods
[0590] Human Tissues:
[0591] The patient cancer and normal tissues were purchased from
different sources such as the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues was purchased from Clontech, Palo Alto,
Calif.
[0592] RNA Isolation:
[0593] Tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue to 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.
[0594] Oligonucleotides:
[0595] The following HPLC purified oligonucleotides were used.
TABLE-US-00002 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 28)
5'TTTTGATCAAGCTT.sub.303' Adaptor 1: (SEQ ID NO: 29)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 30)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 31)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 32)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 33)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO: 34)
5'TCGAGCGGCCGCCCGGGCAGGA3' Nested primer (NP)2: (SEQ ID NO: 35)
5'AGCGTGGTCGCGGCCGAGGA3'
[0596] Suppression Subtractive Hybridization:
[0597] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in prostate cancer. The SSH reaction utilized cDNA from
prostate cancer and normal tissues.
[0598] The gene 202P5A5 sequence was derived from prostate cancer
minus normal prostate cDNA subtraction. The SSH DNA sequence (FIG.
1) was identified.
[0599] The cDNA derived from normal prostate mixed with a pool of 9
normal tissues was used as the source of the "driver" cDNA, while
the cDNA from prostate 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).sup.+ RNA
isolated from the relevant xenograft tissue, as described above,
using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 .mu.g 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.
[0600] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from normal prostate with a mix of digested cDNAs
derived from the nine normal tissues: stomach, skeletal muscle,
lung, brain, liver, kidney, pancreas, small intestine, and
heart.
[0601] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from prostate cancer (400 ng) in 5 .mu.l of water.
The diluted cDNA (2 .mu.l, 160 ng) was then ligated to 2 .mu.l of
Adaptor 1 and Adaptor 2 (10 .mu.M), in separate ligation reactions,
in a total volume of 10 .mu.l at 16.degree. C. overnight, using 400
u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 .mu.l
of 0.2 M EDTA and heating at 72.degree. C. for 5 min.
[0602] 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.
[0603] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0604] 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 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5
.mu.l 10.times. reaction buffer (CLONTECH) and 0.5 .mu.l 50.times.
Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25
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.
[0605] 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.
[0606] 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.
[0607] RT-PCR Expression Analysis:
[0608] 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.
[0609] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO:36) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO:37) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1.times.PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM
MgCl.sub.2, 50 mM KCl, pH8.3) and 1.times. Klentaq DNA polymerase
(Clontech). Five .mu.l of the PCR reaction can be removed at 18,
20, and 22 cycles and used for agarose gel electrophoresis. PCR was
performed using an MJ Research thermal cycler under the following
conditions: Initial denaturation can be at 94.degree. C. for 15
sec, followed by a 18, 20, and 22 cycles of 94.degree. C. for 15,
65.degree. C. for 2 min, 72.degree. C. for 5 sec. A final extension
at 72.degree. C. was carried out for 2 min. After agarose gel
electrophoresis, the band intensities of the 283 b.p. .beta.-actin
bands from multiple tissues were compared by visual inspection.
Dilution factors for the first strand cDNAs were calculated to
result in equal .beta.-actin band intensities in all tissues after
22 cycles of PCR. Three rounds of normalization can be required to
achieve equal band intensities in all tissues after 22 cycles of
PCR.
[0610] To determine expression levels of the 202P5A5 gene, 5 .mu.l
of normalized first strand cDNA were analyzed by PCR using 26, and
30 cycles of amplification. Semi-quantitative expression analysis
can be achieved by comparing the PCR products at cycle numbers that
give light band intensities. The primers used for RT-PCR were
designed using the 202P5A5 SSH sequence and are listed below:
TABLE-US-00003 202P5A5.1 (SEQ ID NO: 38)
5'-CATTTCACATGTCCATGATCTTCC-3' 202P5A5.2 (SEQ ID NO: 39)
5'-CTTTGATGTGTCCGCTGTGTATGT-3'
[0611] A typical RT-PCR expression analysis is shown in FIG. 14A.
First strand cDNA was prepared from vital pool 1 (liver, lung and
kidney), vital pool 2 (pancreas, colon and stomach), prostate
cancer metastasis to lymph node, prostate cancer pool, bladder
cancer pool, colon cancer pool, lung cancer pool, breast cancer
pool, and cancer metastasis pool. Normalization was performed by
PCR using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 202P5A5, was performed at 26 and 30 cycles of
amplification. Expression was detected in prostate cancer
metastasis to lymph node, prostate cancer pool, bladder cancer
pool, colon cancer pool, lung cancer pool, breast cancer pool, and
cancer metastasis pool. Low expression was also detected in vital
pool 1 but not in vital pool 2.
Example 2
Full Length Cloning of 202P5A5
[0612] The 202P5A5 SSH cDNA sequence was derived from a
substraction consisting of prostate cancer minus normal prostate.
The SSH cDNA sequence of 186 bp (FIG. 1) was designated
202P5A5.
[0613] 202P5A5 v.3 of 4973 bp was cloned from a pool of bladder
cancer cDNA library, revealing an ORF of 609 amino acids (FIG. 2
and FIG. 3). Other variants of 202P5A5 were also identified and
these are listed in FIG. 2 and FIG. 3.
[0614] 202P5A5 v.1, v.4, v.5, v.6, and v.8 proteins are 609 amino
acids in length and differ from each other by one amino acid as
shown in FIG. 11. 202P5A5 v.7 and v.9 through v.26, are SNP
variants and code for the same protein as 202P5A5 v.1. 202P5A5 v.2
is a splice variant adding extra 16 amino acids to the amino
terminus of v.1 and thereby codes for a 625 amino acids
protein.
[0615] 202P5A5 v.1 shows 99% identity over 4760 nucleotides, and
99% identity over 609 amino acids, to cDNA FLJ13782, a gene similar
to gene coding for Grainy Head protein. 202P5A5 v.2 shows 99%
identity over 4792 nucleotides, and 99% identity over 625 amino
acids, to cDNA FLJ13782.
Example 3
Chromosomal Mapping of 202P5A5
[0616] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are known in
the art including fluorescent in situ hybridization (FISH),
human/hamster radiation hybrid (RH) panels (Walter et al., 1994;
Nature Genetics 7:22; Research Genetics, Huntsville Al),
human-rodent somatic cell hybrid panels available from the Cornell
Institute (Camden, N.J.), and genomic viewers utilizing BLAST
homologies to sequenced and mapped genomic clones (NCBI, Bethesda,
Md.).
[0617] Accordingly, 202P5A5 maps to chromosome 8q22.3 using 202P5A5
sequence and the NCBI BLAST tool located on the World Wide Web at
(.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4
Expression Analysis of 202P5A5 in Normal Tissues and Patient
Specimens
[0618] Expression analysis by RT-PCR demonstrated that 202P5A5 is
strongly expressed in patient cancer specimens (FIG. 14). In FIG.
14A, first strand cDNA was prepared from vital pool 1 (liver, lung
and kidney), vital pool 2 (pancreas, colon and stomach), prostate
cancer metastasis to lymph node, prostate cancer pool, bladder
cancer pool, colon cancer pool, lung cancer pool, breast cancer
pool, and cancer metastasis pool. Normalization was performed by
PCR using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 202P5A5, was performed at 26 and 30 cycles of
amplification. Expression was detected in prostate cancer
metastasis to lymph node, prostate cancer pool, bladder cancer
pool, colon cancer pool, lung cancer pool, breast cancer pool, and
cancer metastasis pool. Low expression was detected in vital pool 1
but not in vital pool 2.
[0619] In FIG. 14B, semi-quantitative PCR, using primers to
202P5A5, was performed on a panel of 13 normal tissues and 13
cancer pools. Samples were run on an agarose gel, and PCR products
were quantitated using the AlphaImager software. Results show
strong expression of 202P5A5 in prostate cancer, bladder cancer,
colon cancer, lung cancer, ovary cancer, breast cancer, metastasis
cancer, xenograft pool, prostate metastasis to lymph node (PMLN),
bone cancer/melanoma pool, cervical cancer, lymphoma and stomach
cancer compared to all normal tissues tested.
[0620] In order to assay relative expression of 202P5A5 v.2
compared to the other variants, primers were designed spanning the
60 bp insertion at position 32-92 of 202P5A5 v.3 (FIG. 15). 202P5A5
v.2 leads to a PCR product of 173 base pairs in size, whereas other
202P5A5 variants lead to a PCR product of 233 base pairs in size.
First strand cDNA was prepared from vital pool 1 (liver, lung and
kidney), vital pool 2 (pancreas, colon and stomach), LAPC prostate
xenograft pool (LAPC-4AD. LAPC-4AI, LAPC-9AD and LAPC-9AI),
prostate cancer pool, bladder cancer pool, lung cancer pool, ovary
cancer pool, breast cancer pool, cancer metastasis pool, cervical
cancer pool, stomach cancer pool, uterus cancer pool, and master
xenograft pool (LAPC xenograft pool, bladder cancer xenograft,
kidney cancer xenograft). Normalization was performed by PCR using
primers to actin and GAPDH. Semi-quantitative PCR, using the
variant specific primers was performed at 26 and 30 cycles of
amplification. Stronger expression of the 173 bp product was
detected in all cancer pools tested and weakly in vital pools. The
larger 233 bp product was mostly detected in the cancer pools and
not in the vital tissues, and at a frequency of 20-50% compared to
the smaller product
[0621] Extensive expression of 202P5A5 in normal tissues is shown
in FIG. 16. Two multiple tissue northern blots (Clontech) both with
2 .mu.g of mRNA/lane were probed with the 202P5A5 sequence. Size
standards in kilobases (kb) are indicated on the side. Results show
expression of an approximately 7 kb 202P5A5 transcript in normal
prostate and normal placenta but not in any other normal tissue
tested.
[0622] Expression of 202P5A5 in prostate cancer patient specimens
is shown in FIG. 17. RNA was extracted from prostate cancer
xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI), prostate
cancer cell lines (LNCaP and PC3), normal prostate (N), and
prostate cancer patient tumors (T). Northern blots with 10 .mu.g of
total RNA were probed with the 202P5A5 SSH fragment. Size standards
in kilobases are on the side. Results show expression of 202P5A5 in
all prostate cancer specimens tested as well as in the normal
prostate, prostate cancer xenografts and LNCaP, but not in the PC3
cell line.
[0623] Expression of 202P5A5 was also detected in bladder cancer
patient specimens (FIG. 18). RNA was extracted from bladder cancer
cell lines (CL), normal bladder (N), bladder cancer patient tumors
(T) as well as their adjacent normal tissues (Nat). Northern blots
with 10 .mu.g of total RNA were probed with the 202P5A5 sequence.
Size standards in kilobases are on the side. Results show
expression of 202P5A5 in all bladder cancer patient tumor specimens
tested but not in normal bladder. Expression was also detected in
SCABER but not in the other cancer cell lines tested.
[0624] FIG. 19 shows expression of 202P5A5 in breast cancer patient
specimens. RNA was extracted from breast cancer cell lines (CL),
normal breast (N), breast cancer patient tumors (T), and breast
cancer metastasis specimens (M). Northern blots with 10 .mu.g of
total RNA were probed with the 202P5A5 sequence. Size standards in
kilobases are on the side. Results show expression of 202P5A5 in
the breast cancer patient tumors and metastasis specimens.
Expression was also detected in MCF-7 and CAMA-1 but not in the
DU4475 cell line. Weaker expression was detected in normal
breast.
[0625] FIG. 20 shows expression of 202P5A5 in colon and cervical
cancer patient specimens. First strand cDNA was prepared from a
panel of patient cancer specimens. Normalization was performed by
PCR using primers to actin. Semi-quantitative PCR, using primers to
202P5A5, 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 strong. Results show expression of
202P5A5 in the majority of the colon and cervical cancer patient
specimens tested.
[0626] The restricted expression of 202P5A5 in normal tissues and
the expression detected in cancer patient specimens suggest that
202P5A5 is a potential therapeutic target and a diagnostic
prognostic, and/or preventative marker for human cancers.
Example 5
Transcript Variants of 202P5A5
[0627] Transcript variants are variants of mature mRNA from the
same gene which arise by alternative transcription or alternative
splicing. Alternative transcripts are transcripts from the same
gene but start transcription at different points. Splice variants
are mRNA variants spliced differently from the same transcript. In
eukaryotes, when a multi-exon gene is transcribed from genomic DNA,
the initial RNA is spliced to produce functional mRNA, which has
only exons and is used for translation into an amino acid sequence.
Accordingly, a given gene can have zero to many alternative
transcripts and each transcript can have zero to many splice
variants. Each transcript variant has a unique exon makeup, and can
have different coding and/or non-coding (5' or 3' end) portions,
from the original transcript. Transcript variants can code for
similar or different proteins with the same or a similar function
or can encode proteins with different functions, and can be
expressed in the same tissue at the same time, or in different
tissues at the same time, or in the same tissue at different times,
or in different tissues at different times. Proteins encoded by
transcript variants can have similar or different cellular or
extracellular localizations, e.g., secreted versus
intracellular.
[0628] 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 a full-length clone, that portion of the
variant is very useful for antigen generation and for further
cloning of the full-length splice variant, using techniques known
to those skilled in the art.
[0629] 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 (URL
compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL
genes.mit.edu/GENSCAN.html). For a general discussion of splice
variant identification protocols see., e.g., Southan, C., A genomic
perspective on human proteases, FEBS Lett. 2001 Jun. 8;
498(2-3):214-8; de Souza, S. J., et al., Identification of human
chromosome 22 transcribed sequences with ORF expressed sequence
tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.
[0630] 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).
[0631] 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 202P5A5 has a particular expression profile related to cancer
(See, Table I). Alternative transcripts and splice variants of
202P5A5 may also be involved in cancers in the same or different
tissues, thus serving as tumor-associated markers/antigens.
[0632] Using the full-length gene and EST sequences, two additional
transcript variants were identified, designated as 202P5A05 v.2 and
v.3. The boundaries of exons in the original transcript, 202P5A05
v.1 are shown in Table LI. The structures of the transcript
variants are shown in FIG. 10. Variant 202P5A05 v.2 added an exon
to the 5' end of variant v.1. Variants v.3 further extended exon 1
of v.2 into intron 1.
[0633] Tables LII(a)-(b) through LV(a)-(b) are set forth on a
variant-by-variant bases. LII(a)-(b) shows nucleotide sequence of
the transcript variant. Table LIII(a)-(b) shows the alignment of
the transcript variant with nucleic acid sequence of 202P5A05 v.1.
Table LIV(a)-(b) lays out amino acid translation of the transcript
variant for the identified reading frame orientation. Table
LV(a)-(b) displays alignments of the amino acid sequence encoded by
the splice variant with that of 202P5A05 v.1.
Example 6
Single Nucleotide Polymorphisms of 202P5A5
[0634] 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. SNPs that occur on a cDNA
are called cSNP. This cSNPs may change amino acids of the protein
encoded by the gene and thus change the functions of the protein.
Some SNP cause inherited diseases; others contribute to
quantitative variations in phenotype and reactions to environmental
factors including diet and drugs among individuals. Therefore, SNP
and/or combinations of alleles (called haplotypes) have many
applications, including diagnosis of inherited diseases,
determination of drug reactions and dosage, identification of genes
responsible for diseases, and analysis of the genetic relationship
between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, "SNP
analysis to dissect human traits," Curr. Opin. Neurobiol. 2001
October; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic
susceptibility to adverse drug reactions," Trends Pharmacol. Sci.
2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A.
Roses, "The use of single nucleotide polymorphisms in the isolation
of common disease genes," Pharmacogenomics. 2000 February;
1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The
predictive power of haplotypes in clinical response,"
Pharmacogenomics. 2000 February; 1(1):15-26).
[0635] SNPs 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, SNPs 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 SNPs 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). SNPs can be verified and genotype or haplotype of
an individual can be determined by a variety of methods including
direct sequencing and high throughput microarrays (P. Y. Kwok,
"Methods for genotyping single nucleotide polymorphisms," Annu.
Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.
Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A.
Duesterhoeft, "High-throughput SNP genotyping with the Masscode
system," Mol. Diagn. 2000 December; 5(4):329-340).
[0636] Using the methods described above, twenty-four SNPs were
identified in the transcript, 202P5A5 v.1, as shown in Table LVI.
The transcripts or proteins with alternative alleles were
designated as variant 202P5A5 v.4 through v.26, as shown in Table
LVI and FIG. 12. Table LVI also lists the amino acid changes of
protein sequence in the corresponding transcript variants v.2 and
v.3. 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 202P5A5 v.2 and v.3, as listed in
table LVI) that contains the site of the SNP, as set forth in FIGS.
11 and 12.
Example 7
Production of Recombinant 202P5A5 in Prokaryotic Systems
[0637] To express recombinant 202P5A5 and 202P5A5 variants in
prokaryotic cells, the full or partial length 202P5A5 and 202P5A5
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 202P5A5 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 202P5A5, variants, or analogs
thereof.
[0638] A. In Vitro Transcription and Translation Constructs:
[0639] pCR11:
[0640] To generate 202P5A05 sense and anti-sense RNA probes for RNA
in situ investigations, pCR11 constructs (Invitrogen, Carlsbad
Calif.) are generated encoding either all or fragments of the
202P5A05 cDNA. The pCR11 vector has Sp6 and T7 promoters flanking
the insert to drive the transcription of 202P5A05 RNA for use as
probes in RNA in situ hybridization experiments. These probes are
used to analyze the cell and tissue expression of 202P5A05 at the
RNA level. Transcribed 202P5A05 RNA representing the cDNA amino
acid coding region of the 202P5A05 gene is used in in vitro
translation systems such as the TnT.TM. Coupled Reticulolysate
System (Promega, Corp., Madison, Wis.) to synthesize 202P5A05
protein.
[0641] B. Bacterial Constructs:
[0642] pGEX Constructs:
[0643] To generate recombinant 202P5A5 proteins in bacteria that
are fused to the Glutathione S-transferase (GST) protein, all or
parts of the 202P5A5 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 202P5A5 protein sequences with GST fused at the
amino-terminus and a six histidine epitope (6.times.His) at the
carboxyl-terminus. The GST and 6.times.His tags permit purification
of the recombinant fusion protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-GST and anti-His antibodies. The 6.times.His tag
is generated by adding 6 histidine codons to the cloning primer at
the 3' end, e.g., of the open reading frame (ORF). A proteolytic
cleavage site, such as the PreScission.TM. recognition site in
pGEX-6P-1, may be employed such that it permits cleavage of the GST
tag from 202P5A05-related protein. The ampicillin resistance gene
and pBR322 origin permits selection and maintenance of the pGEX
plasmids in E. coli.
[0644] pMAL Constructs:
[0645] To generate, in bacteria, recombinant 202P5A5 proteins that
are fused to maltose-binding protein (MBP), all or parts of the
202P5A5 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 202P5A5 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 202P5A5. 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.
[0646] pET Constructs:
[0647] To express 202P5A05 in bacterial cells, all or parts of the
202P5A05 cDNA protein coding sequence are cloned into the pET
family of vectors (Novagen, Madison, Wis.). These vectors allow
tightly controlled expression of recombinant 202P5A05 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
202P5A05 protein are expressed as amino-terminal fusions to
NusA.
[0648] C. Yeast Constructs:
[0649] pESC Constructs:
[0650] To express 202P5A5 in the yeast species Saccharomyces
cerevisiae for generation of recombinant protein and functional
studies, all or parts of the 202P5A05 cDNA protein coding sequence
are cloned into the pESC family of vectors each of which contain 1
of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La
Jolla, Calif.). These vectors allow controlled expression from the
same plasmid of up to 2 different genes or cloned sequences
containing either Flag.TM. or Myc epitope tags in the same yeast
cell. This system is useful to confirm protein-protein interactions
of 202P5A5. In addition, expression in yeast yields similar
post-translational modifications, such as glycosylations and
phosphorylations that are found when expressed in eukaryotic
cells.
[0651] pESP Constructs:
[0652] To express 202P5A5 in the yeast species Saccharomyces pombe,
all or parts of the 202P5A5 cDNA protein coding sequence are cloned
into the pESP family of vectors. These vectors allow controlled
high level of expression of a 202P5A5 protein sequence that is
fused at either the amino terminus or at the carboxyl terminus to
GST which aids purification of the recombinant protein. A Flag.TM.
epitope tag allows detection of the recombinant protein with
anti-Flag.TM. antibody.
Example 8
Production of Recombinant 202P5A5 in Higher Eukaryotic Systems
[0653] A. Mammalian Constructs:
[0654] To express recombinant 202P5A5 in eukaryotic cells, the full
or partial length 202P5A5 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 202P5A5 were expressed in these
constructs, amino acids 1 to 609, 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 202P5A5 v.1, v.4, v.5, v.6 and
v.8; amino acids 1 to 625, 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 202P5A5 v.2 variants, or analogs
thereof.
[0655] 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-202P5A5 polyclonal serum,
described herein.
[0656] pcDNA4/HisMax Constructs:
[0657] To express 202P5A5 in mammalian cells, a 202P5A5 ORF, or
portions thereof, of 202P5A5 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.
[0658] pcDNA3.1/MycHis Constructs:
[0659] To express 202P5A5 in mammalian cells, a 202P5A5 ORF, or
portions thereof, of 202P5A5 with a consensus Kozak translation
initiation site is 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.
[0660] The complete ORF of 202P5A5 v.1 was cloned into the
pcDNA3.1/MycHis construct to generate 202P5A5.pcDNA3.1/MycHis. FIG.
21 shows expression of 202P5A5.pcDNA3.1/MycHis. 293T cells were
transfected with either 202P5A5.pcDNA3.1/MycHis or pcDNA3.1/MycHis
vector control. Forty hours later, cell lysates were collected.
Samples were run on an SDS-PAGE acrylamide gel, blotted and stained
with anti-his antibody. The blot was developed using the ECL
chemiluminescence kit and visualized by autoradiography. Results
show expression of 202P5A5 from the 202P5A5.pcDNA3.1/MycHis
construct in the lysates of transfected cells.
[0661] pcDNA3.1/CT-GFP-TOPO Construct:
[0662] To express 202P5A5 in mammalian cells and to allow detection
of the recombinant proteins using fluorescence, a 202P5A5 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
202P5A5 protein.
[0663] PAPtag:
[0664] A 202P5A5 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 202P5A5
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 202P5A5 protein. The resulting
recombinant 202P5A5 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 202P5A5
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.
[0665] pTag5:
[0666] A 202P5A5 ORF, or portions thereof, is cloned into pTag-5.
This vector is similar to pAPtag but without the alkaline
phosphatase fusion. This construct generates 202P5A5 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
202P5A5 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 202P5A5 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.
[0667] PsecFc:
[0668] A 202P5A5 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 202P5A5 proteins, while
fusing the IgGK signal sequence to N-terminus. 202P5A5 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant 202P5A5 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 202P5A5 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.
[0669] Retroviral Constructs:
[0670] To generate mammalian cell lines that express 202P5A5
constitutively, 202P5A5 ORF, or portions thereof, of 202P5A5 were
cloned into pQCXIN (Clontech) constructs. Amphotropic and ecotropic
retroviruses were generated by transfection of pQCXIN constructs
into the 293T-10A1 packaging line or co-transfection of pQCXIN 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, 202P5A5, into the host cell-lines. Protein expression is
driven from the CMV promoter. 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.
[0671] Additional pQCXIN constructs are made that fuse an epitope
tag such as the FLAG.TM. tag to the carboxyl-terminus of 202P5A5
sequences to allow detection using anti-Flag antibodies. For
example, the FLAG sequence 5' gat tac aag gat gac gac gat aag 3'
(SEQ ID NO:40) is added to cloning primer at the 3' end of the ORF.
Additional retroviral constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length 202P5A5 proteins and under various
selection methods.
[0672] Additional Viral Vectors:
[0673] Additional constructs are made for viral-mediated delivery
and expression of 202P5A5. High virus titer leading to high level
expression of 202P5A5 is achieved in viral delivery systems such as
adenoviral vectors and herpes amplicon vectors. A 202P5A5 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, 202P5A5
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.
[0674] Regulated Expression Systems:
[0675] To control expression of 202P5A5 in mammalian cells, coding
sequences of 202P5A5, 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 202P5A5. These vectors are thereafter used to control
expression of 202P5A5 in various cell lines such as PC3, NIH 3T3,
293 or rat-1 cells.
[0676] B. Baculovirus Expression Systems
[0677] To generate recombinant 202P5A5 proteins in a baculovirus
expression system, 202P5A5 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-202P5A5 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.
[0678] Recombinant 202P5A5 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 202P5A5 protein can be detected using anti-202P5A5 or
anti-His-tag antibody. 202P5A5 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 202P5A5.
Example 9
Antigenicity Profiles and Secondary Structure
[0679] FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict
graphically five amino acid profiles of 202P5A5 variant 1, each
assessment available by accessing the ProtScale website located on
the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the
ExPasy molecular biology server.
[0680] 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 202P5A5 variant proteins. Each of the above
amino acid profiles of 202P5A5 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.
[0681] 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.
[0682] 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.
[0683] Antigenic sequences of the 202P5A5 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-202P5A5 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
202P5A5 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.
[0684] 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.
[0685] The secondary structure of 202P5A5 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 Deleage G.,
located on the World Wide Web at
pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed
from the ExPasy molecular biology server located on the World Wide
Web at (.expasy.ch/tools/). The analysis indicates that 202P5A5
variant 1 is composed of 31.69% alpha helix, 19.87% extended
strand, and 48.44% random coil (FIG. 13A).
[0686] Analysis for the potential presence of transmembrane domains
in the 202P5A5 variant proteins was carried out using a variety of
transmembrane prediction algorithms accessed from the ExPasy
molecular biology server located on the World Wide Web at
(.expasy.ch/tools/). Shown graphically in FIGS. 13B and 13C are the
results of analysis of 202P5A5 variant 1 using the TMpred program
(FIG. 13B) and TMHMM program (FIG. 13C). Neither of the programs
predicted the presence of transmembrane domains, suggesting that
202P5A5 is a soluble protein. The results of structural analysis
programs are summarized in Table VI.
Example 10
Generation of 202P5A5 Polyclonal Antibodies
[0687] 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 202P5A5 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 Structures").
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 202P5A5 protein
variant 1).
[0688] For example, recombinant bacterial fusion proteins or
peptides containing hydrophilic, flexible, beta-turn regions of
202P5A5 protein variants are used as antigens to generate
polyclonal antibodies in New Zealand White rabbits or monoclonal
antibodies as described in the Example entitled "Generation of
202P5A5 Monoclonal Antibodies (mAbs)". For example, in 202P5A5
variant 1, such regions include, but are not limited to, amino
acids 1-22, amino acids 55-84, amino acids 181-225, amino acids
399-450, and amino acids 496-536. 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 1-22 of 202P5A5 variant
1 was conjugated to KLH and used to immunize a rabbit.
Alternatively the immunizing agent may include all or portions of
the 202P5A5 variant proteins, analogs or fusion proteins thereof.
For example, the 202P5A5 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, the complete cDNA of 202P5A5 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.
[0689] Other recombinant bacterial fusion proteins that may be
employed include maltose binding protein, LacZ, thioredoxin, NusA,
or an immunoglobulin constant region (see the section entitled
"Production of 202P5A5 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).
[0690] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the section entitled "Production of
Recombinant 202P5A5 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, the complete cDNA of 202P5A5
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 202P5A5 protein is then used as immunogen.
[0691] 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).
[0692] 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.
[0693] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with the GSTfusion of
202P5A5 variant 1 protein, the full-length 202P5A5 variant 1 cDNA
is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see
the Example entitled "Production of Recombinant 202P5A05 in
Eukaryotic Systems"). After transfection of the constructs into
293T cells, cell lysates are probed with the anti-202P5A5 serum and
with anti-His antibody (FIG. 21); Santa Cruz Biotechnologies, Santa
Cruz, Calif.) to determine specific reactivity to denatured 202P5A5
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
202P5A5-expressing cells to determine specific recognition of
native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and flow cytometric techniques using cells that
endogenously express 202P5A5 are also carried out to test
reactivity and specificity.
[0694] Anti-serum from rabbits immunized with 202P5A5 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-202P5A5 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-202P5A5 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 202P5A5 Monoclonal Antibodies (mAbs)
[0695] In one embodiment, therapeutic mAbs to 202P5A5 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
202P5A5 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 202P5A5 protein variant sequence, regions of the 202P5A5
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 and
Secondary Structures"). 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 202P5A5 variant,
such as 293T-202P5A05 variant 1 or 300.19-202P5A5 variant 1 murine
Pre-B cells, are used to immunize mice.
[0696] To generate mAbs to a 202P5A5 variant, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen or 10.sup.7 202P5A5-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 202P5A5 variant sequence
is used to immunize mice by direct injection of the plasmid DNA.
For example, the complete cDNA of 202P5A5 of variant 1 (amino acids
1-609) 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 202P5A5 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 202P5A5 variant.
[0697] 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).
[0698] In one embodiment for generating 202P5A5 monoclonal
antibodies, a GST-fusion of variant 1 antigen encoding amino acids
1-609, is expressed and then purified from stably transfected 293T
cells. Balb C mice are initially immunized intraperitoneally with
25 .mu.g of the Tag5-202P5A05 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 202P5A5 variant
1 protein is monitored by Western blotting, immunoprecipitation and
flow cytometry using 293T cells transfected with an expression
vector encoding the 202P5A5 variant 1 cDNA (see e.g., the Example
entitled "Production of Recombinant 202P5A05 in Eukaryotic Systems"
and FIG. 21). Other recombinant 202P5A5 variant 1-expressing cells
or cells endogenously expressing 202P5A5 variant 1 are also used.
Mice showing the strongest reactivity are rested and given a final
injection of Tag5 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 202P5A5 specific antibody-producing
clones.
[0699] To generate monoclonal antibodies that are specific for
202P5A5 variant 2 protein, immunogens are designed to encode the
sequence unique to that variant. For example, a peptide encoding
amino acids 1-16 of 202P5A5 variant 2 is synthesized, conjugated to
KLH and used as immunogen. Hybridoma supernatants are then screened
on the peptide antigen and then further screened on cells
expressing the 202P5A5 variant 2 and cross-screened on cells
expressing 202P5A5 variant 1 to derive variant 2-specific
monoclonal antibodies.
[0700] The binding affinity of a 202P5A5 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 202P5A5 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
[0701] 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.
[0702] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq.[HLA], the measured IC.sub.50 values are
reasonable approximations of the true K.sub.D values. Peptide
inhibitors are typically tested at concentrations ranging from 120
.mu.g/ml to 1.2 ng/ml, and are tested in two to four completely
independent experiments. To allow comparison of the data obtained
in different experiments, a relative binding Figure is calculated
for each peptide by dividing the IC.sub.50 of a positive control
for inhibition by the IC.sub.50 for each tested peptide (typically
unlabeled versions of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values
are compiled. These values can subsequently be converted back into
IC.sub.50 nM values by dividing the IC.sub.50 nM of the positive
controls for inhibition by the relative binding of the peptide of
interest. This method of data compilation is accurate and
consistent for comparing peptides that have been tested on
different days, or with different lots of purified MHC.
[0703] 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
[0704] 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.
[0705] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0706] 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 202P5A5 set forth in FIGS. 2 and 3, the
specific search peptides used to generate the tables are listed in
Table VII.
[0707] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
202P5A5 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.
[0708] 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.1i.times.a.sub.2i.times.a.sub.3i.times.a.sub.ni
[0709] where a.sub.ji is a coefficient which represents the effect
of the presence of a given amino acid (j) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial
assumption of this method is that the effects at each position are
essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at position i in the
peptide, it is assumed to contribute a constant amount j, to the
free energy of binding of the peptide irrespective of the sequence
of the rest of the peptide.
[0710] The method of derivation of specific algorithm coefficients
has been described in Gulukota et al., J. Mol. Biol. 267:1258-126,
1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and
Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for
all i positions, anchor and non-anchor alike, the geometric mean of
the average relative binding (ARB) of all peptides carrying j is
calculated relative to the remainder of the group, and used as the
estimate of j.sub.i. For Class II peptides, if multiple alignments
are possible, only the highest scoring alignment is utilized,
following an iterative procedure. To calculate an algorithm score
of a given peptide in a test set, the ARB values corresponding to
the sequence of the peptide are multiplied. If this product exceeds
a chosen threshold, the peptide is predicted to bind. Appropriate
thresholds are chosen as a function of the degree of stringency of
prediction desired.
[0711] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0712] Protein sequences from 202P5A5 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).
[0713] 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.
[0714] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0715] The 202P5A5 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.
[0716] Selection of HLA-B7 Supermotif Bearing Epitopes
[0717] The 202P5A5 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.
[0718] Selection of A1 and A24 Motif-Bearing Epitopes
[0719] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 202P5A5 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0720] 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
[0721] 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:
[0722] Target Cell Lines for Cellular Screening:
[0723] The 0.221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of HLA-A2.1-restricted CTL. This cell
line is grown in RPMI-1640 medium supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (v/v) heat
inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the antigen of interest,
can be used as target cells to confirm the ability of
peptide-specific CTLs to recognize endogenous antigen.
[0724] Primary CTL Induction Cultures:
[0725] Generation of Dendritic Cells (DC):
[0726] 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.
[0727] Induction of CTL with DC and Peptide:
[0728] CD8+ T-cells are isolated by positive selection with Dynal
immunomagnetic beads (Dynabeads.RTM. M-450) and the
Detacha-Bead.RTM. reagent. Typically about 200-250.times.10.sup.6
PBMC are processed to obtain 24.times.10.sup.6 CD8.sup.+ T-cells
(enough for a 48-well plate culture). Briefly, the PBMCs are thawed
in RPMI with 30 .mu.g/ml DNAse, washed once with PBS containing 1%
human AB serum and resuspended in PBS/1% AB serum at a
concentration of 20.times.10.sup.6 cells/ml. The magnetic beads are
washed 3 times with PBS/AB serum, added to the cells (1400
beads/20.times.10.sup.6 cells) and incubated for 1 hour at
4.degree. C. with continuous mixing. The beads and cells are washed
4.times. with PBS/AB serum to remove the nonadherent cells and
resuspended at 100.times.10.sup.6 cells/ml (based on the original
cell number) in PBS/AB serum containing 100 .mu.l/ml
Detacha-Bead.RTM. reagent and 30 .mu.g/ml DNAse. The mixture is
incubated for 1 hour at room temperature with continuous mixing.
The beads are washed again with PBS/AB/DNAse to collect the CD8+
T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7
minutes, washed once with PBS with 1% BSA, counted and pulsed with
40 .mu.g/ml of peptide at a cell concentration of
1-2.times.10.sup.6/ml in the presence of 3 .mu.g/ml
.beta..sub.2-microglobulin for 4 hours at 20.degree. C. The DC are
then irradiated (4,200 rads), washed 1 time with medium and counted
again.
[0729] Setting Up Induction Cultures:
[0730] 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.
[0731] Restimulation of the Induction Cultures with Peptide-Pulsed
Adherent Cells:
[0732] Seven and fourteen days after the primary induction, the
cells are restimulated with peptide-pulsed adherent cells. The
PBMCs are thawed and washed twice with RPMI and DNAse. The cells
are resuspended at 5.times.10.sup.6 cells/ml and irradiated at
.about.4200 rads. The PBMCs are plated at 2.times.10.sup.6 in 0.5
ml complete medium per well and incubated for 2 hours at 37.degree.
C. The plates are washed twice with RPMI by tapping the plate
gently to remove the nonadherent cells and the adherent cells
pulsed with 10 .mu.g/ml of peptide in the presence of 3 .mu.g/ml
.beta..sub.2 microglobulin in 0.25 ml RPMI/5% AB per well for 2
hours at 37.degree. C. Peptide solution from each well is aspirated
and the wells are washed once with RPMI. Most of the media is
aspirated from the induction cultures (CD8+ cells) and brought to
0.5 ml with fresh media. The cells are then transferred to the
wells containing the peptide-pulsed adherent cells. Twenty four
hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant human IL2 is added the
next day and again 2-3 days later at 50 IU/ml (Tsai et al.,
Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days
later, the cultures are assayed for CTL activity in a .sup.51Cr
release assay. In some experiments the cultures are assayed for
peptide-specific recognition in the in situ IFN.gamma. ELISA at the
time of the second restimulation followed by assay of endogenous
recognition 7 days later. After expansion, activity is measured in
both assays for a side-by-side comparison.
[0733] Measurement of CTL Lytic Activity by .sup.51Cr Release.
[0734] 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.
[0735] 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 (1000) 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.
[0736] 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.
[0737] In Situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-Specific and Endogenous Recognition
[0738] 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.
[0739] 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 OD450. A
culture is considered positive if it measured at least 50 pg of
IFN-gamma/well above background and is twice the background level
of expression.
[0740] CTL Expansion.
[0741] Those cultures that demonstrate specific lytic activity
against peptide-pulsed targets and/or tumor targets are expanded
over a two week period with anti-CD3. Briefly, 5.times.10.sup.4
CD8+ cells are added to a T25 flask containing the following:
1.times.10.sup.6 irradiated (4,200 rad) PBMC (autologous or
allogeneic) per ml, 2.times.10.sup.5 irradiated (8,000 rad)
EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml
in RPMI-1640 containing 10% (v/v) human AB serum, non-essential
amino acids, sodium pyruvate, 25 .mu.M 2-mercaptoethanol,
L-glutamine and penicillin/streptomycin. Recombinant human IL2 is
added 24 hours later at a final concentration of 200 IU/ml and
every three days thereafter with fresh media at 50 IU/ml. The cells
are split if the cell concentration exceeds 1.times.10.sup.6/ml and
the cultures are assayed between days 13 and 15 at E:T ratios of
30, 10, 3 and 1:1 in the .sup.51Cr release assay or at
1.times.10.sup.6/ml in the in situ IFN.gamma. assay using the same
targets as before the expansion.
[0742] Cultures are expanded in the absence of anti-CD3.sup.+ as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8.sup.+ cells are added to a T25 flask
containing the following: 1.times.10.sup.6 autologous PBMC per ml
which have been peptide-pulsed with 10 .mu.g/ml peptide for two
hours at 37.degree. C. and irradiated (4,200 rad); 2.times.10.sup.5
irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640
containing 10% (v/v) human AB serum, non-essential AA, sodium
pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0743] Immunogenicity of A2 Supermotif-Bearing Peptides
[0744] 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.
[0745] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 202P5A5. 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.
[0746] Evaluation of A*03/A11 Immunogenicity
[0747] 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.
[0748] Evaluation of B7 Immunogenicity
[0749] 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.
[0750] 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
[0751] 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.
[0752] Analoging at Primary Anchor Residues
[0753] 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.
[0754] 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.
[0755] 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.
[0756] 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).
[0757] 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.
[0758] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0759] 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.
[0760] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate 500 nM binding capacity are then confirmed as having
A3-supertype cross-reactivity.
[0761] 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).
[0762] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0763] 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.
[0764] Analoging at Secondary Anchor Residues
[0765] 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.
[0766] 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 202P5A5-expressing tumors.
[0767] Other Analoging Strategies
[0768] 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 disulfide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substitution of .alpha.-amino butyric acid for cysteine not only
alleviates this problem, but has been shown to improve binding and
crossbinding capabilities in some instances (see, e.g., the review
by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and
I. Chen, John Wiley & Sons, England, 1999).
[0769] 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 202P5A5-Derived Sequences with
HLA-DR Binding Motifs
[0770] 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.
[0771] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0772] To identify 202P5A5-derived, HLA class II HTL epitopes, a
202P5A5 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).
[0773] 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.
[0774] The 202P5A5-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. 202P5A5-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0775] Selection of DR3 Motif Peptides
[0776] 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.
[0777] To efficiently identify peptides that bind DR3, target
202P5A5 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.
[0778] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0779] 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 202P5A5-Derived HTL Epitopes
[0780] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0781] 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 202P5A5-expressing
tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0782] 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.
[0783] 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].
[0784] Where frequency data is not available at the level of DNA
typing, correspondence to the serologically defined antigen
frequencies is assumed. To obtain total potential supertype
population coverage no linkage disequilibrium is assumed, and only
alleles confirmed to belong to each of the supertypes are included
(minimal estimates). Estimates of total potential coverage achieved
by inter-loci combinations are made by adding to the A coverage the
proportion of the non-A covered population that could be expected
to be covered by the B alleles considered (e.g., total=A+B*(1-A)).
Confirmed members of the A3-like supertype are A3, A11, A31,
A*3301, and A*6801. Although the A3-like supertype may also include
A34, A66, and A*7401, these alleles were not included in overall
frequency calculations Likewise, confirmed members of the A2-like
supertype family are A*0201, A*0202, A*0203, A*0204, A*0205,
A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like
supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301,
B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also
B*1401, B*3504-06, B*4201, and B*5602).
[0785] 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.
[0786] 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.
[0787] 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
[0788] 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.
[0789] 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 202P5A5
expression vectors.
[0790] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
202P5A5 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
[0791] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 202P5A5-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 202P5A5-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.
[0792] Immunization Procedures:
[0793] Immunization of transgenic mice is performed as described
(Alexander et al., J. Immunol. 159:4753-4761, 1997). For example,
A2/K.sup.b mice, which are transgenic for the human HLA A2.1 allele
and are used to confirm the immunogenicity of HLA-A*0201 motif- or
HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously
(base of the tail) with a 0.1 ml of peptide in Incomplete Freund's
Adjuvant, or if the peptide composition is a lipidated CTL/HTL
conjugate, in DMSO/saline, or if the peptide composition is a
polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days
after priming, splenocytes obtained from these animals are
restimulated with syngenic irradiated LPS-activated lymphoblasts
coated with peptide.
[0794] Cell Lines:
[0795] 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)
[0796] In Vitro CTL Activation:
[0797] One week after priming, spleen cells (30.times.10.sup.6
cells/flask) are co-cultured at 37.degree. C. with syngeneic,
irradiated (3000 rads), peptide coated lymphoblasts
(10.times.10.sup.6 cells/flask) in 10 ml of culture medium/T25
flask. After six days, effector cells are harvested and assayed for
cytotoxic activity.
[0798] Assay for Cytotoxic Activity:
[0799] Target cells (1.0 to 1.5.times.10.sup.6) are incubated at
37.degree. C. in the presence of 200 .mu.l of .sup.51Cr. After 60
minutes, cells are washed three times and resuspended in R10
medium. Peptide is added where required at a concentration of 1
.mu.g/ml. For the assay, 10.sup.4 51Cr-labeled target cells are
added to different concentrations of effector cells (final volume
of 200 .mu.l) in U-bottom 96-well plates. After a six hour
incubation period at 37.degree. C., a 0.1 ml aliquot of supernatant
is removed from each well and radioactivity is determined in a
Micromedic automatic gamma counter. The percent specific lysis is
determined by the formula: percent specific release=100.times.
(experimental release-spontaneous release)/(maximum
release-spontaneous release). To facilitate comparison between
separate CTL assays run under the same conditions, % .sup.51Cr
release data is expressed as lytic units/10.sup.6 cells. One lytic
unit is arbitrarily defined as the number of effector cells
required to achieve 30% lysis of 10,000 target cells in a six hour
.sup.51Cr release assay. To obtain specific lytic units/10.sup.6,
the lytic units/10.sup.6 obtained in the absence of peptide is
subtracted from the lytic units/10.sup.6 obtained in the presence
of peptide. For example, if 30% .sup.51Cr release is obtained at
the effector (E):target (T) ratio of 50:1 (i.e., 5.times.10.sup.5
effector cells for 10,000 targets) in the absence of peptide and
5:1 (i.e., 5.times.10.sup.4 effector cells for 10,000 targets) in
the presence of peptide, the specific lytic units would be:
[(1/50,000)-(1/500,000)].times.10.sup.6=18 LU.
[0800] 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
202P5A5-Specific Vaccine
[0801] 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.
[0802] 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.
[0803] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 202P5A5 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 202P5A5. For example, if it has been observed
that patients who spontaneously clear 202P5A5-expressing cells
generate an immune response to at least three (3) epitopes from
202P5A5 antigen, then at least three epitopes should be included
for HLA class I. A similar rationale is used to determine HLA class
II epitopes.
[0804] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 nM or less for an HLA class I molecule, or for
class II, an IC.sub.50 of 1000 nM or less; or HLA Class I peptides
with high binding scores from the BIMAS web site, at URL
bimas.dcrt.nih.gov/.
[0805] 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.
[0806] 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 202P5A5, 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.
[0807] 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 202P5A5.
Example 22
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0808] 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.
[0809] 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 202P5A5, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 202P5A5 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.
[0810] 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.
[0811] 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.
[0812] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa Ig-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disclosed herein. The sequence
encodes an open reading frame fused to the Myc and His antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0813] 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.
[0814] For example, a minigene is prepared as follows. For a first
PCR reaction, 5 .mu.g of each of two oligonucleotides are annealed
and extended: In an example using eight oligonucleotides, i.e.,
four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are
combined in 100 .mu.l reactions containing Pfu polymerase buffer
(1.times.=10 mM KCL, 10 mM (NH4).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
[0815] The degree to which a plasmid construct, for example a
plasmid constructed in accordance with the previous Example, is
able to induce immunogenicity is confirmed in vitro by determining
epitope presentation by APC following transduction or transfection
of the APC with an epitope-expressing nucleic acid construct. Such
a study determines "antigenicity" and allows the use of human APC.
The assay determines the ability of the epitope to be presented by
the APC in a context that is recognized by a T cell by quantifying
the density of epitope-HLA class I complexes on the cell surface.
Quantitation can be performed by directly measuring the amount of
peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol.
156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the
number of peptide-HLA class I complexes can be estimated by
measuring the amount of lysis or lymphokine release induced by
diseased or transfected target cells, and then determining the
concentration of peptide necessary to obtain equivalent levels of
lysis or lymphokine release (see, e.g., Kageyama et al., J.
Immunol. 154:567-576, 1995).
[0816] 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.
[0817] 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.
[0818] 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.
[0819] 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.
[0820] 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-A.sup.b-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.
[0821] DNA minigenes, constructed as described in the previous
Example, can also be confirmed as a vaccine in combination with a
boosting agent using a prime boost protocol. The boosting agent can
consist of recombinant protein (e.g., Barnett et al., Aids Res. and
Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant
vaccinia, for example, expressing a minigene or DNA encoding the
complete protein of interest (see, e.g., Hanke et al., Vaccine
16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci. USA
95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181,
1999; and Robinson et al., Nature Med. 5:526-34, 1999).
[0822] 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.
[0823] 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
[0824] Vaccine compositions of the present invention can be used to
prevent 202P5A5 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
202P5A5-associated tumor.
[0825] 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 202P5A5-associated disease.
[0826] 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 202P5A5
Sequences
[0827] A native 202P5A5 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.
[0828] The vaccine composition will include, for example, multiple
CTL epitopes from 202P5A5 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.
[0829] 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 202P5A5, 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.
[0830] 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
[0831] The 202P5A5 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
202P5A5 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 202P5A5 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 202P5A5
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
[0832] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 202P5A5. 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.
[0833] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 202P5A5 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising a 202P5A5
peptide containing an A*0201 motif. Tetrameric complexes are
synthesized as described (Musey et al., N. Engl. J. Med. 337:1267,
1997). Briefly, purified HLA heavy chain (A*0201 in this example)
and .beta.2-microglobulin are synthesized by means of a prokaryotic
expression system. The heavy chain is modified by deletion of the
transmembrane-cytosolic tail and COOH-terminal addition of a
sequence containing a BirA enzymatic biotinylation site. The heavy
chain, .beta.2-microglobulin, and peptide are refolded by dilution.
The 45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0834] 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 202P5A5 epitope, and thus the
status of exposure to 202P5A5, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
[0835] 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 202P5A5-associated disease or who have been
vaccinated with a 202P5A5 vaccine.
[0836] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
202P5A5 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.
[0837] 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.
[0838] 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).
[0839] 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).
[0840] 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.
[0841] 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.
[0842] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 202P5A5 or a 202P5A5 vaccine.
[0843] 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 202P5A5
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
[0844] 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:
[0845] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0846] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0847] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0848] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0849] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0850] 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.
[0851] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0852] 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.
[0853] The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 202P5A5
[0854] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 202P5A5. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 202P5A5, 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:
[0855] 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.
[0856] 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 202P5A5.
[0857] 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 202P5A5-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
[0858] 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.
[0859] For example, the initial immunization may be performed using
an expression vector, such as that constructed in the Example
entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids" in
the form of naked nucleic acid administered IM (or SC or ID) in the
amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to
1000 .mu.g) can also be administered using a gene gun. Following an
incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-10.sup.7 to 5.times.10.sup.9 pfu. An
alternative recombinant virus, such as an MVA, canarypox,
adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient
blood samples are obtained before immunization as well as at
intervals following administration of the initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells
are isolated from fresh heparinized blood by Ficoll-Hypaque density
gradient centrifugation, aliquoted in freezing media and stored
frozen. Samples are assayed for CTL and HTL activity.
[0860] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 202P5A5 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0861] 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
202P5A5 protein from which the epitopes in the vaccine are
derived.
[0862] For example, a cocktail of epitope-comprising peptides is
administered ex vivo to PBMC, or isolated DC therefrom. A
pharmaceutical to facilitate harvesting of DC can be used, such as
Progenipoietin.TM. (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After
pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
[0863] 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.
[0864] In some embodiments, peptide-loaded PBMC are injected into
patients without purification of the DC. For example, PBMC
generated after treatment with an agent such as Progenipoietin.TM.
are injected into patients without purification of the DC. The
total number of PBMC that are administered often ranges from
10.sup.8 to 10.sup.10. Generally, the cell doses injected into
patients is based on the percentage of DC in the blood of each
patient, as determined, for example, by immunofluorescence analysis
with specific anti-DC antibodies. Thus, for example, if
Progenipoietin.TM. mobilizes 2% DC in the peripheral blood of a
given patient, and that patient is to receive 5.times.10.sup.6 DC,
then the patient will be injected with a total of
2.5.times.10.sup.8 peptide-loaded PBMC. The percent DC mobilized by
an agent such as Progenipoietin.TM. is typically estimated to be
between 2-10%, but can vary as appreciated by one of skill in the
art.
[0865] Ex Vivo Activation of CTL/HTL Responses
[0866] Alternatively, ex vivo CTL or HTL responses to 202P5A5
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
[0867] 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. 202P5A5.
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.
[0868] 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
202P5A5 to isolate peptides corresponding to 202P5A5 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.
[0869] 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
[0870] Sequences complementary to the 202P5A5-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 202P5A5. 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 202P5A5. 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 202P5A5-encoding
transcript.
Example 35
Purification of Naturally-occurring or Recombinant 202P5A5 Using
202P5A5-Specific Antibodies
[0871] Naturally occurring or recombinant 202P5A5 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 202P5A5. An immunoaffinity column is constructed by covalently
coupling anti-202P5A5 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.
[0872] Media containing 202P5A5 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 202P5A5 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/202P5A5 binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and GCR.P is collected.
Example 36
Identification of Molecules which Interact with 202P5A5
[0873] 202P5A5, or biologically active fragments thereof, are
labeled with 121 l 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
202P5A5, washed, and any wells with labeled 202P5A5 complex are
assayed. Data obtained using different concentrations of 202P5A5
are used to calculate values for the number, affinity, and
association of 202P5A5 with the candidate molecules.
Example 37
In Vivo Assay for 202P5A5 Tumor Growth Promotion
[0874] The effect of the 202P5A5 protein on tumor cell growth is
evaluated in vivo by evaluating tumor development and growth of
cells expressing or lacking 202P5A5. For example, SCID mice are
injected subcutaneously on each flank with 1.times.10.sup.6 of
either 3T3, prostate (e.g. PC3 cells), bladder (e.g. UM-UC3 cells)
or breast (e.g. DU4475 cells) cancer cell lines containing tkNeo
empty vector or 202P5A5. At least two strategies may be used: (1)
Constitutive 202P5A5 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
202P5A5-expressing cells grow at a faster rate and whether tumors
produced by 202P5A5-expressing cells demonstrate characteristics of
altered aggressiveness (e.g. enhanced metastasis, vascularization,
reduced responsiveness to chemotherapeutic drugs).
[0875] Additionally, mice can be implanted with 1.times.10.sup.5 of
the same cells orthotopically to determine if 202P5A5 has an effect
on local growth in the pancreas, and whether 202P5A5 affects the
ability of the cells to metastasize, specifically to lymph nodes,
and bone (Miki T et al, Oncol Res. 2001; 12:209; Fu X et al, Int J.
Cancer. 1991, 49:938). The effect of 202P5A5 on bone tumor
formation and growth may be assessed by injecting tumor cells
intratibially.
[0876] The assay is also useful to determine the 202P5A5 inhibitory
effect of candidate therapeutic compositions, such as for example,
202P5A5 intrabodies, 202P5A5 antisense molecules and ribozymes.
Example 38
202P5A5 Monoclonal Antibody-mediated Inhibition of Tumors In
Vivo
[0877] The significant expression of 202P5A5 in cancer tissues,
together with its restrictive expression in normal tissues makes
202P5A5 a good target for antibody therapy. Similarly, 202P5A5 is a
target for T cell-based immunotherapy. Thus, the therapeutic
efficacy of anti-202P5A5 mAbs in human cancer xenograft mouse
models, including prostate, bladder and breast (e.g. DU4475 cells)
and other 202P5A5 cancers listed in table I, is evaluated by using
recombinant cell lines such as PC3-202P5A5, UM-UC3-202P5A5,
DU4475-202P5A5, and 3T3-202P5A5 (see, e.g., Kaighn, M. E., et al.,
Invest Urol. 1979. 17(1): 16-23), as well as human xenograft models
(Saffran et al PNAS 1999, 10:1073-1078).
[0878] Antibody efficacy on tumor growth and metastasis formation
is studied, e.g., in a mouse orthotopic ovary, pancreas, or blood
cancer xenograft models. The antibodies can be unconjugated, as
discussed in this Example, or can be conjugated to a therapeutic
modality, as appreciated in the art. Anti-202P5A5 mAbs inhibit
formation of tumors in mouse xenografts. Anti-202P5A5 mAbs also
retard the growth of established orthotopic tumors and prolonged
survival of tumor-bearing mice. These results indicate the utility
of anti-202P5A5 mAbs in the treatment of local and advanced stages
several solid tumors. (See, e.g., Saffran, D., et al., PNAS
10:1073-1078 or world wide web URL
pnas.org/cgi/doi/10.1073/pnas.051624698).
[0879] Administration of the anti-202P5A5 mAbs led to retardation
of established orthotopic tumor growth and inhibition of metastasis
to distant sites, resulting in a significant prolongation in the
survival of tumor-bearing mice. These studies indicate that 202P5A5
as an attractive target for immunotherapy and demonstrate the
therapeutic potential of anti-202P5A5 mAbs for the treatment of
local and metastatic cancer. This example indicates that
unconjugated 202P5A5 monoclonal antibodies are effective to inhibit
the growth of human pancreatic, ovarian, and lymphomas tumor
xenografts grown in SCID mice; accordingly a combination of such
efficacious monoclonal antibodies is also effective.
Tumor Inhibition Using Multiple Unconjugated 202P5A5 mAbs
[0880] Materials and Methods
[0881] 202P5A5 Monoclonal Antibodies:
[0882] Monoclonal antibodies are raised against 202P5A5 as
described in the Example entitled "Generation of 202P5A5 Monoclonal
Antibodies (mAbs)." The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation for their capacity to
bind 202P5A5. Epitope mapping data for the anti-202P5A5 mAbs, as
determined by ELISA and Western analysis, recognize epitopes on the
202P5A5 protein. Immunohistochemical analysis of cancer tissues and
cells with these antibodies is performed.
[0883] 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 PC3, UM-UC3, CaKi and A427 tumor xenografts.
[0884] Cell Lines and Xenografts
[0885] The LAPC-9 xenograft, which expresses a wild-type androgen
receptor and produces prostate-specific antigen (PSA), is passaged
in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID)
mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al.,
1999, Cancer Res. 59:5030-5036). The AGS-K3 and AGS-K6 kidney
xenografts are also passaged by subcutaneous implants in 6- to
8-week old SCID mice. Single-cell suspensions of tumor cells are
prepared as described in Craft, et al.
[0886] The cancer cell lines PC3, UM-UC3 and DU4475 cell lines, as
well as the fibroblast line NIH 3T3 (American Type Culture
Collection). The prostate carcinoma cell line PC3 is maintained in
RPMI supplemented with L-glutamine and 10% FBS, and the bladder and
breast carcinoma lines, UM-UC3 and DU4475 respectively, are
maintained in DMEM supplemented with L-glutamine and 10% FBS.
PC3-202P5A5, UM-UC3-202P5A5, DU4475-202P5A5 and 3T3-202P5A5 cell
populations are generated by retroviral gene transfer as described
in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25):
14523.
[0887] Xenograft Mouse Models.
[0888] Subcutaneous (s.c.) tumors are generated by injection of
2.times.10.sup.6 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, 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 caliper measurements, and the tumor volume is
calculated as length.times.width.times.height. Mice with
Subcutaneous tumors greater than 1.5 cm in diameter are
sacrificed.
[0889] Orthotopic injections are performed under anesthesia by
using ketamine/xylazine. For prostate orthotopic studies, an
incision is made through the abdominal muscles to expose the
bladder and seminal vesicles, which then are delivered through the
incision to expose the dorsal prostate. LAPC-9 cells (5.times.105)
mixed with Matrigel are injected into each dorsal lobe in a 10
.mu.l volume. To monitor tumor growth, mice are bled on a weekly
basis for determination of PSA levels. For the breast orthopotic
model, an incision is made through the abdominal muscles to expose
the mammary tissues and a single cell suspension of breast cancer
cells is injected into the mammary pad. For the bladder orthotopic
model, AGS-B 1 bladder cancer tissue is adhered onto the bladder
wall. Following tumor implantation, the mice are segregated into
groups for the appropriate treatments, with anti-202P5A5 or control
mAbs being injected i.p. To monitor tumor growth, mice are palpated
and blood is collected on a weekly basis to measure hCG levels.
[0890] Anti-202P5A5 mAbs Inhibit Growth of 202P5A5-Expressing
Xenograft-Cancer Tumors
[0891] The effect of anti-202P5A5 mAbs on tumor formation is tested
by using cell line (e.g. PC3, UM-UC3, DU4475 and 3T3) and
patient-derived tumor orthotopic models. As compared with the s.c.
tumor model, the orthotopic model, which requires injection of
tumor cells directly in the mouse organ that results in a local
tumor growth, development of metastasis in distal sites,
deterioration of mouse health, and subsequent death (Saffran, D.,
et al., PNAS supra). The features make the orthotopic model more
representative of human disease progression and allowed the
therapeutic effect of mAbs on clinically relevant end points to be
followed more easily.
[0892] A major advantage of the orthotopic cancer models is the
ability to study the development of metastases. Formation of
metastasis in mice bearing established orthotopic tumors is studies
by IHC analysis on lung sections using an antibody against a
tumor-specific cell-surface protein such as anti-CK20 for prostate
cancer (Lin S et al, Cancer Detect Prev. 2001; 25:202).
[0893] Another advantage of xenograft cancer models is the ability
to study neovascularization and angiogenesis. Tumor growth is
partly dependent on new blood vessel development. Although the
capillary system and developing blood network is of host origin,
the initiation and architecture of the neovasculature is regulated
by the xenograft tumor (Davidoff A M et al, Clin Cancer Res. 2001;
7:2870; Solesvik O et al, Eur J Cancer Clin Oncol. 1984, 20:1295).
The effect of antibody and small molecule on neovascularization is
studied in accordance with procedures known in the art, such as by
IHC analysis of tumor tissues and their surrounding
microenvironment.
[0894] Mice bearing established orthotopic tumors are administered
1000 .mu.g injections of either anti-202P5A5 mAb or PBS over a
4-week period. Mice in both groups are allowed to establish a high
tumor burden, to ensure a high frequency of metastasis formation in
mouse lungs. Mice then are killed and their bladders, livers, bone,
and lungs are analyzed for the presence of tumor cells by IHC
analysis. These studies demonstrate a broad anti-tumor efficacy of
anti-202P5A5 antibodies on initiation and progression of prostate
cancer in xenograft mouse models. Anti-202P5A5 antibodies inhibit
tumor formation of tumors as well as retarding the growth of
already established tumors and prolong the survival of treated
mice. Moreover, anti-202P5A5 mAbs demonstrate a dramatic inhibitory
effect on the spread of local prostate tumor to distal sites, even
in the presence of a large tumor burden.
[0895] Thus, anti-202P5A5 mAbs are efficacious on major clinically
relevant end points (tumor growth), prolongation of survival, and
health.
Example 39
Therapeutic and Diagnostic Use of Anti-202P5A5 Antibodies in
Humans
[0896] Anti-202P5A5 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-202P5A5 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 202P5A5 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-202P5A5 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0897] As determined by flow cytometry, anti-202P5A5 mAb
specifically binds to carcinoma cells. Thus, anti-202P5A5
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 202P5A5. Shedding or release of an extracellular
domain of 202P5A5 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 202P5A5 by
anti-202P5A5 antibodies in serum and/or urine samples from suspect
patients.
[0898] Anti-202P5A5 antibodies that specifically bind 202P5A5 are
used in therapeutic applications for the treatment of cancers that
express 202P5A5. Anti-202P5A5 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-202P5A5 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 "202P5A5 Monoclonal Antibody-mediated Inhibition
of Bladder and Lung Tumors In Vivo"). Either conjugated and
unconjugated anti-202P5A5 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-202P5A5 Antibodies In vivo
[0899] Antibodies are used in accordance with the present invention
which recognize an epitope on 202P5A5, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 202P5A5 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.
[0900] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-202P5A5 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-202P5A5
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-202P5A5 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).
[0901] II.) Monotherapy: In connection with the use of the
anti-202P5A5 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.
[0902] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-202P5A5 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 202P5A5. In connection with the use of the anti-202P5A5
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 (202P5A5
(.sup.111In)-202P5A5 antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses 202P5A5 (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.
[0903] Dose and Route of Administration
[0904] 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-202P5A5
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-202P5A5 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-202P5A5 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-202P5A5 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.
[0905] Three distinct delivery approaches are useful for delivery
of anti-202P5A5 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.
[0906] Clinical Development Plan (CDP)
[0907] Overview: The CDP follows and develops treatments of
anti-202P5A5 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-202P5A5 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
202P5A5 expression levels in their tumors as determined by
biopsy.
[0908] 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 202P5A5. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-202P5A5 antibodies are
found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-202P5A5
Antibody and Chemotherapeutic Agent
[0909] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-202P5A5 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-202P5A5 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-202P5A5 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-00004 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 MAb Dose 25
75 125 175 225 275 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)
[0910] 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 202P5A5. 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.
[0911] The anti-202P5A5 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-202P5A5
Antibody
[0912] Anti-202P5A5 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-202P5A5
antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-202P5A5
Antibody
[0913] 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-202P5A5 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 202P5A5 to Known Sequences
[0914] The 202P5A5 gene encodes a 609 amino acid protein. The human
202P5A5 protein exhibits a high degree of homology to a human
protein of unknown function, namely hypothetical protein FLJ13782
(gi 13376382), exhibiting 99% identity to 202P5A5 at the protein
level (FIG. 4A). The mouse homolog of 202P5A5 has been identified
as murine BOM (gi 20502771), and shows 94% identity and 97%
homology to 202P5A5 (FIG. 4B). Mouse BOM and human 202P5A5 show
significant homology to a slightly smaller protein named grainyhead
protein or NTF1 (gi 21312674; Shirra M K, Hansen U. J. Biol. Chem.
1998, 273:19260) (FIG. 4C).
[0915] Grainyhead proteins were first identified in Drosophila
melanogaster, where they were found to function as transcription
factors regulating embryo development (Uv A E, Thompson C R, Bray S
J. Mol Cell Biol. 1994, 24:4020; Uv A E, Harrison E J, Bray S J.
Mol Cell Biol. 1997, 17:6727). Similarly, mammalian grainyhead-like
proteins have been identified in mammalian cells and found to
function as transcription factors in these cells. For example, CP2
(LBP-1c) and LBP-1a regulate transcription of diverse genes
involved in hematopoietic differentiation, T-cell activation,
metabolism and cell growth (Ramamurthy L et al, J Biol. Chem. 2001,
276:7836; Volker J L., et. al., Genes Dev. 1997, 11: 1435).
Grainyhead proteins have recently been shown to participate in the
Notch pathways as they participate in the regulation of
Notch-mediated gene expression (Fusse B, Hoch M. Curr Biol. 2002,
12:171).
[0916] The 202P5A5 protein has several variants (FIG. 11). These
include five SNPs, namely 202P5A5 v.1, v.4, v.5, v.6 and v.8, in
addition to splice variants, namely 202P5A5 v.2 and v.3. The
202P5A5 v.2 protein encompasses 16 additional aa at the N-terminus
of the protein relative to 202P5A5v.1. 202P5A5 v.3 further extended
exon 1 of v.2 into intron 1 (FIG. 10). Bioinformatic analysis using
topology prediction programs indicate that 202P5A5 is a soluble
protein with no transmembrane domains (Table L).
[0917] Motif analysis revealed the presence of several protein
functional motifs in the 202P5A5 protein (Table L). A fibronectin
type III repeat has been identified in addition to a CP2
transcription factor motif.
[0918] Fibronectin type III repeats are 100 amino acid domains with
binding sites for various molecules, including DNA, heparin,
basement membrane, and cell surface proteins (Kimizuka et al, J
Biol. Chem. 1991, 266:3045; Yokosaki et al, J Biol. Chem. 1998,
273:11423). Proteins containing fibronectin III motifs participate
in cell surface binding, binding to specific substrates including
heparin, collagen, DNA, actin, and fibrin, are involved in binding
to fibronectin receptors. Fibronectins have been reported to
function in wound healing; cell adhesion, cell differentiation,
cell migration, and tumor metastasis (Bloom et al, Mol Biol Cell.
1999, 10:1521; Brodt P. Cancer Met Rev 1991, 10:23).
[0919] CP2-related proteins are DNA-binding transcription factors.
They regulate transcription by homo-oligomerizing and
hetero-oligomerizing with transcription factors, thereby forming a
stable DNA-protein complex (Shirra, J. Biol. Chem. 1998,
273:19260). In addition, transcriptional activation of LBP-1, a
member of the CP2 family, is regulated by phosphorylation (Volker
J, et al. Genes Dev 1997, 11:1435). As indicated above, CP2
proteins regulate transcription of diverse genes, including those
regulating hematopoietic differentiation, immune response, and cell
growth (Ramamurthy L et al, J Biol. Chem. 2001, 276:7836; Volker J
L., Rameh L E. et al, Genes Dev. 1997, 11: 1435). Recent studies
have implicated CP2 in Alzheimer's disease (Taylor et al, J Med
Genet. 2001, 38:232).
[0920] The motifs found in 202P5A5 indicate that 202P5A5
participates in tumor growth, and progression by transcriptionally
regulating the expression of tumor-related genes, thereby
regulating tumor establishment, tumor growth, adhesion, migration,
metastasis, differentiation, immune response, and cell growth.
[0921] Accordingly, when 202P5A5 functions as a transcription
factor regulating embryo development, a regulator of tumor
establishment, tumor growth, tumor invasion, cell survival, cell
signaling, differentiation, immune response, and cell growth,
202P5A5 is used for therapeutic, diagnostic, prognostic, and/or
preventative purposes. In addition, when a molecule, such as a
splice variant or SNP of 202P5A5 is expressed in cancerous tissues,
such as those listed in Table I, they are used for therapeutic,
diagnostic, prognostic and/or preventative purposes.
Example 45
Regulation of Transcription
[0922] The nuclear localization of 202P5A5 coupled to the presence
of CP2 domains within its sequence indicate that 202P5A5 modulates
the transcriptional regulation of eukaryotic genes. Regulation of
gene expression is confirmed, e.g., by studying gene expression in
cells expressing or lacking 202P5A5. For this purpose, two types of
experiments are performed.
[0923] In the first set of experiments, RNA from parental and
202P5A5-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, androgen or growth factors 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.).
[0924] 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.
[0925] Thus, 202P5A5 plays a role in gene regulation, and it is
used as a target for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 46
Identification and Confirmation of Potential Signal Transduction
Pathways
[0926] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J. Neurochem. 2001; 76:217-223). Using
immunoprecipitation and Western blotting techniques, proteins are
identified that associate with 202P5A5 and mediate signaling
events. Several pathways known to play a role in cancer biology can
be regulated by 202P5A5, including phospholipid pathways such as
PI3K, AKT, etc, adhesion and migration pathways, including FAK,
Rho, Rac-1, catenin, 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.). In order to determine whether expression of 202P5A5 is
sufficient to regulate specific signaling pathways not otherwise
active in resting cancer cells, the effect of 202P5A5 on the
activation of the signaling cascade is investigated in the cancer
cell lines PA-1, Panc1 and Daudi. Cancer cells stably expressing
202P5A5 or neo are stimulated with growth factor, FBS or other
activating molecules. Whole cell lysates are analyzed by western
blotting.
[0927] To confirm that 202P5A5 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.
[0928] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0929] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0930] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0931] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0932] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0933] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0934] 7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion
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.
[0935] Signaling pathways activated by 202P5A5 are mapped and used
for the identification and validation of therapeutic targets. When
202P5A5 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 47
Involvement in Tumor Progression
[0936] Based on the role of CP2 domains and fibronectin motifs in
cell growth and protein interactions, the 202P5A5 gene can
contribute to the growth, invasion, and transformation of cancer
cells. The role of 202P5A5 in tumor growth is confirmed in a
variety of primary and transfected cell lines including prostate
cell lines, as well as NIH 3T3 cells engineered to stably express
202P5A5. Parental cells lacking 202P5A5 and cells expressing
202P5A5 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).
[0937] To confirm the role of 202P5A5 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH-3T3 cells lacking 202P5A5 are compared to NIH-3T3
cells expressing 202P5A5, using a soft agar assay under stringent
and more permissive conditions (Song Z. et al. Cancer Res. 2000;
60:6730).
[0938] To confirm the role of 202P5A5 in invasion and metastasis of
cancer cells, a well-established assay is used, e.g., a Transwell
Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010).
Control cells, including prostate, breast, and kidney cell lines
lacking 202P5A5 are compared to cells expressing 202P5A5. 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.
[0939] 202P5A5 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 202P5A5 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 202P5A5, including normal and tumor prostate
cells. Engineered and parental cells are treated with various
chemotherapeutic agents, such as etoposide, taxol, 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 202P5A5 can play a critical role in
regulating tumor progression and tumor load.
[0940] When 202P5A5 plays a role in cell growth, transformation,
invasion or apoptosis, it is used as a target for diagnostic,
prognostic, preventative and/or therapeutic purposes.
Example 48
Involvement in Angiogenesis
[0941] 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). Based on the
effect of fibronectins on tumor cell adhesion and their interaction
with endothelial cells, 202P5A5 plays a role in angiogenesis
(Mareel and Leroy: Physiol Rev, 83:337; DeFouw L et al, Microvasc
Res 2001, 62:263). 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 role of 202P5A5 in angiogenesis,
enhancement or inhibition, is confirmed.
[0942] For example, endothelial cells engineered to express 202P5A5
are evaluated using tube formation and proliferation assays. The
effect of 202P5A5 is also confirmed in animal models in vivo. For
example, cells either expressing or lacking 202P5A5 are implanted
subcutaneously in immunocompromised mice. Endothelial cell
migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. Thus, 202P5A5 affects
angiogenesis, and it is used as a target for diagnostic,
prognostic, preventative and/or therapeutic purposes.
Example 49
Involvement in Protein-Protein Interactions
[0943] CP2 domains and fibronectin motifs have been shown to
mediate interaction with other proteins. Using immunoprecipitation
techniques as well as two yeast hybrid systems, proteins are
identified that associate with 202P5A5. Immunoprecipitates from
cells expressing 202P5A5 and cells lacking 202P5A5 are compared for
specific protein-protein associations.
[0944] Studies are performed to confirm the extent of association
of 202P5A5 with effector molecules, such as nuclear proteins,
transcription factors, kinases, phosphates etc. Studies comparing
202P5A5 positive and 202P5A5 negative cells as well as studies
comparing unstimulated/resting cells and cells treated with
epithelial cell activators, such as cytokines, growth factors,
androgen and anti-integrin Ab reveal unique interactions.
[0945] 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 202P5A5-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
colorimetric reporter activity. Specific association with effector
molecules and transcription factors directs one of skill to the
mode of action of 202P5A5, 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 202P5A5.
[0946] Thus, it is found that 202P5A5 associates with proteins and
small molecules. Accordingly, 202P5A5 and these proteins and small
molecules are used for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 50
Involvement of 202P5A5 in Cell-Cell Communication
[0947] Cell-cell communication is essential in maintaining organ
integrity and homeostasis, both of which become deregulated during
tumor formation and progression. Based on the presence of a
fibronectin motif in 202P5A5, a motif known to be involved in cell
interaction and cell-cell adhesion, as well as the role of CP2 in
gene expression, 202P5A5 can regulate cell communication.
Intercellular communications can be measured using two types of
assays (J. Biol. Chem. 2000, 275:25207). In the first assay, cells
loaded with a fluorescent dye are incubated in the presence of
unlabeled recipient cells and the cell populations are examined
under fluorescent microscopy. This qualitative assay measures the
exchange of dye between adjacent cells. In the second assay system,
donor and recipient cell populations are treated as above and
quantitative measurements of the recipient cell population are
performed by FACS analysis. Using these two assay systems, cells
expressing 202P5A5 are compared to controls that do not express
202P5A5, and it is found that 202P5A5 enhances cell communications.
Small molecules and/or antibodies that modulate cell-cell
communication mediated by 202P5A5 are used as therapeutics for
cancers that express 202P5A5.
[0948] Thus, 202P5A5 functions in cell-cell communication and small
molecule transport, it is used as a target or marker for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
[0949] Throughout this application, various website data content,
publications, patent applications and patents are referenced.
(Websites are referenced by their Uniform Resource Locator, or URL,
addresses on the World Wide Web.) The disclosures of each of these
references are hereby incorporated by reference herein in their
entireties.
[0950] 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.
Tables:
TABLE-US-00005 [0951] TABLE I Tissues that Express 202P5A5: a.
Malignant Tissues Prostate Bladder Colon Lung Ovary Breast Stomach
Cervix Lymphoma Bone Skin
TABLE-US-00006 TABLE II Amino Acid Abbreviations SINGLE LETTER
THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser
serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline
H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M
Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val
valine A Ala alanine D Asp asp artic acid E Glu glutamic acid G Gly
glycine
TABLE-US-00007 TABLE III Amino Acid Substitution Matrix Adapted
from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix
(block substitution matrix.) The higher the value, the more likely
a substitution is found in related, natural proteins. (See world
wide web URL ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L
M N P Q R S T V W Y . 4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0
0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C
6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D 5 -3 -2 0 -3 1
-3 -2 0 -1 2 0 0 -1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2
-1 1 3 F 6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G 8 -3 -1 -3 -2
1 -2 0 0 -1 -2 -3 -2 2 H 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I 5 -2
-1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L 5 -2 -2
0 -1 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N 7 -1 -2 -1 -1 -2 -4 -3
P 5 1 0 -1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 5 0 -2 -2 T
4 -3 -1 V 11 2 W 7 Y
Table IV:
HLA Class I/II Motifs/Supermotifs
TABLE-US-00008 [0952] TABLE IV (A) HLA Class I Supermotifs/Motifs
POSITION POSITION POSITION C Terminus 2 (Primary 3 (Primary
(Primary Anchor) Anchor) Anchor) SUPERMOTIF A1 TILVMS FWY A2
LIVMATQ IVMATL A3 VSMATLI RK A24 YFWIVLMT FIYWLM B7 P VILFMWYA B27
RHK FYLWMIVA B44 ED FWYLIMVA B58 ATS FWYLIVMA B62 QLIVMP FWYMIVLA
MOTIFS A1 TSM Y A1 DEAS Y A2.1 LMVQIAT VLIMAT A3 LMVISATFCGD KYRHFA
A11 VTMLISAGNCDF KRYH A24 YFWM FLIW A*3101 MVTALIS RK A*3301
MVALFIST RK A*6801 AVTMSLI RK B*0702 P LMFWYAIV B*3501 P LMFWYIVA
B51 P LIVFWYAM B*5301 P IMFWYALV B*5401 P ATIVLMFWY Bolded residues
are preferred, italicized residues are less preferred: A peptide is
considered motif-bearing if it has primary anchors at each primary
anchor position for a motif or supermotif as specified in the above
table.
TABLE-US-00009 TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y,
V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y
TABLE-US-00010 TABLE IV (C) HLA Class II Motifs MOTIFS 1.degree.
anchor 1 2 3 4 5 1.degree. anchor 6 7 8 9 DR4 preferred FMYLIVW M T
I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ
VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY
M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1.degree.
anchor 1 2 3 1.degree. anchor 4 5 1.degree. anchor 6 Motif a
preferred LIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR
Supermotif MFLIVWY VMSTACPLI Italicized residues indicate less
preferred or "tolerated" residues
TABLE-US-00011 TABLE IV (D) HLA Class I Supermotifs SUPER- C-
MOTIFS POSITION: 1 2 3 4 5 6 7 8 terminus A1 1.degree. Anchor
1.degree. Anchor TILVMS FWY A2 1.degree. Anchor 1.degree. Anchor
LIVMATQ LIVMAT A3 Preferred 1.degree. Anchor YFW (4/5) YFW (3/5)
YFW (4/5) P (4/5) 1.degree. Anchor VSMATLI RK deleterious DE (3/5);
DE (4/5) P (5/5) A24 1.degree. Anchor 1.degree. Anchor YFWIVLMT
FIYWLM B7 Preferred FWY (5/5) 1.degree. Anchor FWY (4/5) FWY (3/5)
1.degree. Anchor LIVM (3/5) P VILFMWYA deleterious DE (3/5); DE
(3/5) G (4/5) QN (4/5) DE (4/5) P(5/5); G(4/5); A(3/5); QN(3/5) B27
1.degree. Anchor 1.degree. Anchor RHK FYLWMIVA B44 1.degree. Anchor
1.degree. Anchor ED FWYLIMVA B58 1.degree. Anchor 1.degree. Anchor
ATS FWYLIVMA B62 1.degree. Anchor 1.degree. Anchor QLIVMP FWYMIVLA
Italicized residues indicate less preferred or "tolerated"
residues
TABLE-US-00012 TABLE IV (E) HLA Class I Motifs 9 or C- C- position
1 2 3 4 5 6 7 8 terminus terminus A1 preferred GFYW 1.degree.
Anchor DEA YFW P DEQN YFW 1.degree. Anchor 9-mer STM Y deleterious
DE RHKLI- A G A VMP A1 preferred GRHK ASTCL- 1.degree. Anchor GSTC
ASTC LIVM DE 1.degree. Anchor 9-mer IVM DEAS Y deleterious A
RHKDEP- DE PQN RHK PG GP YFW A1 preferred YFW 1.degree. Anchor
DEAQN A YFWQN PASTC GDE P 1.degree. Anchor 10-mer STM Y deleterious
GP RHKGLIVM DE RHK QNA RHKYFW RHK A A1 preferred YFW STCLIVM
1.degree. Anchor A YFW PG G YFW 1.degree. Anchor 10-mer DEAS Y
deleterious RHK RHKDEP- P G PRHK QN YFW A2.1 preferred YFW
1.degree. Anchor YFW STC YFW A P 1.degree. Anchor 9-mer LMIVQAT
VLIMAT deleterious DEP DERKH RKH DERKH A2.1 preferred AYFW
1.degree. Anchor LVIM G G FYWL 1.degree. Anchor 10-mer LMIVQAT VIM
VLIMAT deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK
1.degree. Anchor YFW PRHK- A YFW P 1.degree. Anchor LMVISA- YFW
KYRHFA deleterious DEP TFCGD DE A11 preferred A 1.degree. Anchor
YFW YFW A YFW YFW P 1.degree. Anchor VTLMIS- KRYH AGNCDF
deleterious DEP A G A24 preferred YFWRHK 1.degree. Anchor STC YFW
YFW 1.degree. Anchor 9-mer YFWM FLIW deleterious DEG DE G QNP DERHK
G AQN A24 preferred 1.degree. Anchor P YFWP P 1.degree. Anchor
10-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 preferred
RHK 1.degree.Anchor YFW P YFW YFW AP 1.degree. Anchor MVTALIS RK
deleterious DEP DE ADE DE DE DE A3301 preferred 1.degree. Anchor
YFW AYFW 1.degree. Anchor MVALFIST RK deleterious GP DE A6801
preferred YFWSTC 1.degree. Anchor YFWLI- YFW P 1.degree. Anchor
AVTMSLI VM RK deleterious GP DEG RHK A B0702 preferred RHKFWY
1.degree. Anchor RHK RHK RHK RHK PA 1.degree. Anchor P LMFWYAIV
deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYL-
1.degree. Anchor FWY FWY 1.degree. Anchor IVM P LMFWYIVA
deleterious AGP G G B51 preferred LIVMF- 1.degree. Anchor FWY STC
FWY G FWY 1.degree. Anchor WY P LIVFWYAM deleterious AGPDER- DE G
DEQN GDE HKSTC B5301 preferred LIVMF- 1.degree. Anchor FWY STC FWY
LIVMF- FWY 1.degree. Anchor WY P WY IMFWYALV deleterious AGPQN G
RHKQN DE B5401 preferred FWY 1.degree. Anchor FWYL- LIVM ALIVM
FWYAP 1.degree. Anchor P IVM ATIVLMFWY deleterious GPQNDE GDESTC
RHKDE DE QNDGE DE
TABLE-US-00013 TABLE IV (F) Summary of HLA-supertypes Overall
phenotypic frequencies of HLA-supertypes in different ethnic
populations Specificity Phenotypic frequency Supertype Position 2
C-Terminus Caucasian N.A. Black Japanese Chinese Hispanic Average
B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3 AILMVST RK 37.5 42.1
45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2
A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D)
FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1
21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4
B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY
(LIV) 10.0 25.1 1.6 9.0 5.9 10.3
TABLE-US-00014 TABLE IV (G) Calculated population coverage afforded
by different HLA-supertype combinations Phenotypic frequency
HLA-supertypes Caucasian N.A Blacks Japanese Chinese Hispanic
Average A2, A3 and B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7,
A24, 99.5 98.1 100.0 99.5 99.4 99.3 B44 and A1 A2, A3, 99.9 99.6
100.0 99.8 99.9 99.8 B7, A24, B44, A1, B27, B62, and B58 Motifs
indicate the residues defining supertype specificites. The motifs
incorporate residues determined on the basis of published data to
be recognized by multiple alleles within the supertype. Residues
within brackets are additional residues also predicted to be
tolerated by multiple alleles within the supertype.
TABLE-US-00015 TABLE V Frequently Occurring Motifs avrg. % Name
identity Description Potential Function zf-C2H2 34% Zinc finger,
C2H2 Nucleic acid-binding protein functions as type transcription
factor, nuclear location probable cytochrome_b_N 68% Cytochrome
b(N- membrane bound oxidase, generate terminal)/b6/petB superoxide
Ig 19% Immunoglobulin domains are one hundred amino acids long
domain and include a conserved intradomain disulfide bond. WD40 18%
WD domain, G-beta tandem repeats of about 40 residues, each repeat
containing a Trp-Asp motif. Function in signal transduction and
protein interaction PDZ 23% PDZ domain may function in targeting
signaling molecules to sub-membranous sites LRR 28% Leucine Rich
Repeat short sequence motifs involved in protein- protein
interactions Pkinase 23% Protein kinase domain conserved catalytic
core common to both serine/threonine and tyrosine protein kinases
containing an ATP binding site and a catalytic site PH 16% PH
domain pleckstrin homology involved in intracellular signaling or
as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40
amino-acid long found in the extracellular domain of membrane-bound
proteins or in secreted proteins Rvt 49% Reverse transcriptase
(RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic
protein, associates integral membrane proteins to the cytoskeleton
Oxidored_q1 32% NADH- membrane associated. Involved in proton
Ubiquinone/plastoqui- translocation across the membrane none
(complex I), various chains Efhand 24% EF hand calcium-binding
domain, consists of a 12 residue loop flanked on both sides by a 12
residue alpha-helical domain Rvp 79% Retroviral aspartyl Aspartyl
or acid proteases, centered on a protease catalytic aspartyl
residue Collagen 42% Collagen triple helix extracellular structural
proteins involved in repeat (20 copies) formation of connective
tissue. The sequence consists of the G-X-Y and the polypeptide
chains forms a triple helix. Fn3 20% Fibronectin type III Located
in the extracellular ligand-binding domain region of receptors and
is about 200 amino acid residues long with two pairs of cysteines
involved in disulfide bonds 7tm_1 19% 7 transmembrane seven
hydrophobic transmembrane regions, receptor (rhodopsin with the
N-terminus located extracellularly family) while the C-terminus is
cytoplasmic. Signal through G proteins
TABLE-US-00016 TABLE VI Post-translational modifications of 202P5A5
N-glycosylation site 90-93 NLSG (SEQ ID NO: 41) 107-110 NLSL (SEQ
ID NO: 42) 384-387 NRSN (SEQ ID NO: 43) 431-434 NSSS (SEQ ID NO:
44) Tyrosine sulfation site 215-229 ASVGAEEYMYDQTSS (SEQ ID NO: 45)
217-231 VGAEEYMYDQTSSGT (SEQ ID NO: 46) 314-328 RVLDIADYKESFNTI
(SEQ ID NO: 47) 578-592 DDNIIEHYSNEDTFI (SEQ ID NO: 48) cAMP- and
cGMP-dependent protein kinase phosphorylation site 527-530 RKET
(SEQ ID NO: 49) Protein kinase C phosphorylation site 9-11 TRR
118-120 SKR 203-205 SFK 209-211 TEK 241-243 SLR 310-312 TAK 364-366
SQK 386-388 SNK 519-521 TKR 543-545 TVK 552-554 SEK 569-571 SKK
Casein kinase II phosphorylation site 14-17 TSED (SEQ ID NO: 50)
15-18 SEDE (SEQ ID NO: 51) 22-25 SYLE (SEQ ID NO: 52) 72-75 SQED
(SEQ ID NO: 53) 92-95 SGGE (SEQ ID NO: 54) 118-121 SKRE (SEQ ID NO:
55) 126-129 SFPE (SEQ ID NO: 56) 174-177 TQYD (SEQ ID NO: 57)
194-197 STPD (SEQ ID NO: 58) 199-202 TYSE (SEQ ID NO: 59) 203-206
SFKD (SEQ ID NO: 60) 263-266 TLSE (SEQ ID NO: 61) 432-435 SSSD (SEQ
ID NO: 62) 454-457 TMPD (SEQ ID NO: 63) 484-487 TDDE (SEQ ID NO:
64) 586-589 SNED (SEQ ID NO: 65) 597-600 SMVE (SEQ ID NO: 66)
605-608 TLME (SEQ ID NO: 67) Tyrosine kinase phosphorylation site
193-200 RSTPDSTY (SEQ ID NO: 68) 292-300 KNRDEQLKY (SEQ ID NO: 69)
314-321 RVLDIADY (SEQ ID NO: 70) 445-451 KKSDITY (SEQ ID NO: 71)
N-myristoylation site 83-88 GTSEAQ (SEQ ID NO: 72) 257-262 GQFYAI
(SEQ ID NO: 73) 546-551 GLMEAI (SEQ ID NO: 74) 572-577 GILVNM (SEQ
ID NO: 75) Bipartite nuclear targeting sequence 407-423
RKIRDEERKQNRKKGKG (SEQ ID NO: 76) Cell attachment sequence 160-162
RGD
TABLE-US-00017 TABLE VII Search Peptides 202P5A05 v.1 9-mers,
10-mers and 15-mers (SEQ ID NO: 77) MPSDPPFNTR RAYTSEDEAW
KSYLENPLTA ATKAMMSING DEDSAAALGL LYDYYKVPRD 60 KRLLSVSKAS
DSQEDQEKRN CLGTSEAQSN LSGGENRVQV LKTVPVNLSL NQDHLENSKR 120
EQYSISFPES SAIIPVSGIT VVKAEDFTPV FMAPPVHYPR GDGEEQRVVI FEQTQYDVPS
180 LATHSAYLKD DQRSTPDSTY SESFKDAATE KFRSASVGAE EYMYDQTSSG
TFQYTLEATK 240 SLRQKQGEGP MTYLNKGQFY AITLSETGDN KCFRHPISKV
RSVVMVVFSE DKNRDEQLKY 300 WKYWHSRQHT AKQRVLDIAD YKESFNTIGN
IEEIAYNAVS FTWDVNEEAK IFITVNCLST 360 DFSSQKGVKG LPLMIQIDTY
SYNNRSNKPI HRAYCQIKVF CDKGAERKIR DEERKQNRKK 420 GKGQASQTQC
NSSSDGKLAA IPLQKKSDIT YFKTMPDLHS QPVLFIPDVH FANLQRTGQV 480
YYNTDDEREG GSVLVKRMFR PMEEEFGPVP SKQMKEEGTK RVLLYVRKET DDVFDALMLK
540 SPTVKGLMEA ISEKYGLPVE KIAKLYKKSK KGILVNMDDN IIEHYSNEDT
FILNMESMVE 600 GFKVTLMEI 609 202P5A5v.2 ORF: 13-1890 9-mers, aa1-24
MSQESDNNKR LVALVPMPSD PPFN (SEQ ID NO: 78) 10-mers, aa 1-25
MSQESDNNKR LVALVPMPSD PPFNT (SEQ ID NO: 79) 25-mers, aa 1-30
MSQESDNNKR LVALVPMPSD PPFNTRRAYT (SEQ ID NO: 80) 202P5A5v.4 ORF:
121-1950 9-mers, aa 29-45 TAATKAMMIINGDEDSA (SEQ ID NO: 81)
10-mers, aa 28-46 LTAATKAMMIINGDEDSAA (SEQ ID NO: 82) 14-mers, aa
22-51 YLENPLTAATKAMMIINGDEDSAAALGLL (SEQ ID NO: 83) 202P5A5v.5
9-mers, aa 406-422 ERKIRDEEQKQNRKKGK (SEQ ID NO: 84) 10-mers, aa
405-423 AERKIRDEEQKQNRKKGKG (SEQ ID NO: 85) 15-mers, aa400-428
FCDKGAERKIRDEEQKQNRKKGKGQASQT (SEQ ID NO: 86) 202P5A5v.6 9-mers, aa
412-428 EERKQNRKNGKGQASQT (SEQ ID NO: 87) 10-mers, aa 411-429
DEERKQNRKNGKGQASQTQ (SEQ ID NO: 88) 15-mers, aa 406-434
ERKIRDEERKQNRKNGKGQASQTQCNSSS (SEQ ID NO: 89) 202P5A5V5/6 9-mers,
aa412-422 EEQKQNRKNGK (SEQ ID NO: 90) 10-mers, aa411-423
DEEQKQNRKNGKG (SEQ ID NO: 91) 15-mers, aa406-428
ERKIRDEEQKQNRKNGKGQASQT (SEQ ID NO: 92) 202P5A5v.8 9-mers, aa
537-553 LMLKSPTVMGLMEAISE (SEQ ID NO: 93) 10-mers, aa 536-554
ALMLKSPTVMGLMEAISEK (SEQ ID NO: 94) 15-mers, aa 531-559
DDVFDALMLKSPTVMGLMEAISEKYGLPV (SEQ ID NO: 95)
Tables VIII-XXI:
TABLE-US-00018 [0953] TABLE VIII V1-HLA-A1-9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 547 LMEAISEKY 22.500 143 KAEDFTPVF 18.000 235
TLEATKSLR 9.000 2 PSDPPFNTR 7.500 226 QTSSGTFQY 6.250 594 NMESMVEGF
4.500 446 KSDITYFKT 3.750 44 SAAALGLLY 2.500 119 KREQYSISF 2.250
152 MAPPVHYPR 2.000 598 MVEGFKVTL 1.800 294 RDEQLKYWK 1.800 218
GAEEYMYDQ 1.800 330 NIEEIAYNA 1.800 433 SSDGKLAAI 1.500 111
NQDHLENSK 1.500 200 YSESFKDAA 1.350 264 LSETGDNKC 1.350 14
TSEDEAWKS 1.350 84 TSEAQSNLS 1.350 529 ETDDVFDAL 1.250 102
KTVPVNLSL 1.250 483 NTDDEREGG 1.250 576 NMDDNIIEH 1.250 15
SEDEAWKSY 1.250 577 MDDNIIEHY 1.250 359 STDFSSQKG 1.250 345
VNEEAKIFI 1.125 586 SNEDTFILN 1.125 46 AALGLLYDY 1.000 532
DVFDALMLK 1.000 400 FCDKGAERK 1.000 216 SVGAEEYMY 1.000 376
QIDTYSYNN 1.000 556 GLPVEKIAK 1.000 437 KLAAIPLQK 1.000 374
MIQIDTYSY 1.000 13 YTSEDEAWK 1.000 163 GEEQRVVIF 0.900 114
HLENSKREQ 0.900 23 YLENPLTAA 0.900 551 ISEKYGLPV 0.675 328
IGNIEEIAY 0.625 267 TGDNKCFRH 0.625 468 DVHFANLQR 0.500 168
VVIFEQTQY 0.500 342 TWDVNEEAK 0.500 214 SASVGAEEY 0.500 318
IADYKESFN 0.500 492 SVLVKRMFR 0.500 47 ALGLLYDYY 0.500 51 LYDYYKVPR
0.500 507 GPVPSKQMK 0.500 558 PVEKIAKLY 0.450 162 DGEEQRVVI 0.450
410 RDEERKQNR 0.450 181 LATHSAYLK 0.400 245 KQGEGPMTY 0.375 380
YSYNNRSNK 0.300 358 LSTDFSSQK 0.300 197 DSTYSESFK 0.300 453
KTMPDLHSQ 0.250 39 NGDEDSAAA 0.250 266 ETGDNKCFR 0.250 248
EGPMTYLNK 0.250 204 FKDAATEKF 0.250 466 IPDVHFANL 0.250 251
MTYLNKGQF 0.250 139 ITVVKAEDF 0.250 346 NEEAKIFIT 0.225 246
QGEGPMTYL 0.225 93 GGENRVQVL 0.225 208 ATEKFRSAS 0.225 263
TLSETGDNK 0.200 314 RVLDIADYK 0.200 546 GLMEAISEK 0.200 438
LAAIPLQKK 0.200 457 DLHSQPVLF 0.200 463 VLFIPDVHF 0.200 333
EIAYNAVSF 0.200 429 QCNSSSDGK 0.200 71 DSQEDQEKR 0.150 125
ISFPESSAI 0.150 491 GSVLVKRMF 0.150 193 RSTPDSTYS 0.150 288
FSEDKNRDE 0.135 72 SQEDQEKRN 0.135 589 DTFILNMES 0.125 533
VFDALMLKS 0.125 175 QYDVPSLAT 0.125 386 SNKPIHRAY 0.125 230
GTFQYTLEA 0.125 587 NEDTFILNM 0.125 369 KGLPLMIQI 0.125 455
MPDLHSQPV 0.125 195 TPDSTYSES 0.125 160 RGDGEEQRV 0.125 127
FPESSAIIP 0.113 315 VLDIADYKE 0.100 354 TVNCLSTDF 0.100 V2-HLA-A1-9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 5; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 2 SQESDNNKR 1.350 4 ESDNNKRLV 0.750 15
VPMPSDPPF 0.500 1 MSQESDNNK 0.300 11 LVALVPMPS 0.050 13 ALVPMPSDP
0.010 5 SDNNKRLVA 0.003 12 VALVPMPSD 0.002 10 RLVALVPMP 0.002 9
KRLVALVPM 0.001 16 PMPSDPPFN 0.001 14 LVPMPSDPP 0.001 3 QESDNNKRL
0.001 6 DNNKRLVAL 0.001 7 NNKRLVALV 0.000 8 NKRLVALVP 0.000
V4-HLA-A1-9 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Start Subsequence Score 1 TAATKAMMI 0.050 3 ATKAMMIIN
0.013 8 MIINGDEDS 0.010 9 IINGDEDSA 0.010 2 AATKAMMII 0.005 4
TKAMMIING 0.003 5 KAMMIINGD 0.001 6 AMMIINGDE 0.001 7 MMIINGDED
0.001 V5-HLA-A1-9 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 6 DEEQKQNRK 0.900 5
RDEEQKQNR 0.450 4 IRDEEQKQN 0.025 9 QKQNRKKGK 0.010 7 EEQKQNRKK
0.001 2 RKIRDEEQK 0.001 3 KIRDEEQKQ 0.001 8 EQKQNRKKG 0.000 1
ERKIRDEEQ 0.000 V5&6-HLA-A1-9 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 3
QKQNRKNGK 0.010 2 EQKQNRKNG 0.000 1 EEQKQNRKN 0.000 V6-HLA-A1-9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 3 RKQNRKNGK 0.010 8 KNGKGQASQ 0.001 7
RKNGKGQAS 0.001 9 NGKGQASQT 0.000 4 KQNRKNGKG 0.000 2 ERKQNRKNG
0.000 6 NRKNGKGQA 0.000 5 QNRKNGKGQ 0.000 1 EERKQNRKN 0.000
V8-HLA-A1-9 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Start Subsequence Score 3 LKSPTVMGL 0.025 4 KSPTVMGLM
0.015 6 PTVMGLMEA 0.013 7 TVMGLMEAI 0.010 8 VMGLMEAIS 0.010 2
MLKSPTVMG 0.002 9 MGLMEAISE 0.001 5 SPTVMGLME 0.001 1 LMLKSPTVM
0.001
TABLE-US-00019 TABLE IX V1-HLA-A1- 10 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 14
TSEDEAWKSY 67.500 114 HLENSKREQY 45.000 69 ASDSQEDQEK 15.000 264
LSETGDNKCF 13.500 576 NMDDNIIEHY 12.500 93 GGENRVQVLK 9.000 594
NMESMVEGFK 9.000 2 PSDPPFNTRR 7.500 84 TSEAQSNLSG 6.750 376
QIDTYSYNNR 5.000 235 TLEATKSLRQ 4.500 487 EREGGSVLVK 4.500 162
DGEEQRVVIF 4.500 43 DSAAALGLLY 3.750 200 YSESFKDAAT 2.700 327
TIGNIEEIAY 2.500 529 ETDDVFDALM 2.500 555 YGLPVEKIAK 2.500 318
IADYKESFNT 2.500 294 RDEQLKYWKY 2.250 503 EEEFGPVPSK 1.800 143
KAEDFTPVFM 1.800 23 YLENPLTAAT 1.800 215 ASVGAEEYMY 1.500 288
FSEDKNRDEQ 1.350 251 MTYLNKGQFY 1.250 359 STDFSSQKGV 1.250 195
TPDSTYSESF 1.250 483 NTDDEREGGS 1.250 345 VNEEAKIFIT 1.125 127
FPESSAIIPV 1.125 586 SNEDTFILNM 1.125 151 FMAPPVHYPR 1.000 293
NRDEQLKYWK 1.000 45 AAALGLLYDY 1.000 598 MVEGFKVTLM 0.900 410
RDEERKQNRK 0.900 218 GAEEYMYDQT 0.900 330 NIEEIAYNAV 0.900 558
PVEKIAKLYK 0.900 581 IIEHYSNEDT 0.900 213 RSASVGAEEY 0.750 446
KSDITYFKTM 0.750 491 GSVLVKRMFR 0.750 111 NQDHLENSKR 0.750 132
AIIPVSGITV 0.500 46 AALGLLYDYY 0.500 546 GLMEAISEKY 0.500 167
RVVIFEQTQY 0.500 315 VLDIADYKES 0.500 373 LMIQIDTYSY 0.500 262
ITLSETGDNK 0.500 247 GEGPMTYLNK 0.500 208 ATEKFRSASV 0.450 547
LMEAISEKYG 0.450 246 QGEGPMTYLN 0.450 180 SLATHSAYLK 0.400 47
ALGLLYDYYK 0.400 225 DQTSSGTFQY 0.375 125 ISFPESSAII 0.300 223
MYDQTSSGTF 0.250 39 NGDEDSAAAL 0.250 353 ITVNCLSTDF 0.250 234
YTLEATKSLR 0.250 204 FKDAATEKFR 0.250 102 KTVPVNLSLN 0.250 409
IRDEERKQNR 0.250 455 MPDLHSQPVL 0.250 346 NEEAKIFITV 0.225 527
RKETDDVFDA 0.225 321 YKESFNTIGN 0.225 536 ALMLKSPTVK 0.200 152
MAPPVHYPRG 0.200 24 LENPLTAATK 0.200 50 LLYDYYKVPR 0.200 283
VVMVVFSEDK 0.200 357 CLSTDFSSQK 0.200 394 YCQIKVFCDK 0.200 398
KVFCDKGAER 0.200 437 KLAAIPLQKK 0.200 88 QSNLSGGENR 0.150 184
HSAYLKDDQR 0.150 433 SSDGKLAAIP 0.150 72 SQEDQEKRNC 0.135 41
DEDSAAALGL 0.125 466 IPDVHFANLQ 0.125 244 QKQGEGPMTY 0.125 371
LPLMIQIDTY 0.125 557 LPVEKIAKLY 0.125 160 RGDGEEQRVV 0.125 28
LTAATKAMMS 0.125 144 AEDFTPVFMA 0.125 472 ANLQRTGQVY 0.125 530
TDDVFDALML 0.125 4 DPPFNTRRAY 0.125 516 EEGTKRVLLY 0.125 178
VPSLATHSAY 0.125 266 ETGDNKCFRH 0.125 134 IPVSGITVVK 0.100 149
PVFMAPPVHY 0.100 V2-HLA-A1- 10 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 4
ESDNNKRLVA 3.75E 14 LVPMPSDPPF 0.200 1 MSQESDNNKR 0.150 2
SQESDNNKRL 0.135 15 VPMPSDPPFN 0.050 10 RLVALVPMPS 0.050 13
ALVPMPSDPP 0.010 16 PMPSDPPFNT 0.003 11 LVALVPMPSD 0.002 5
SDNNKRLVAL 0.001 12 VALVPMPSDP 0.001
3 QESDNNKRLV 0.001 6 DNNKRLVALV 0.001 8 NKRLVALVPM 0.000 9
KRLVALVPMP 0.000 7 NNKRLVALVP 0.000 V4-HLA-A1- l0 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 1 LTAATKAMMI 0.125 2 TAATKAMMII 0.050 4
ATKAMMIING 0.013 9 MIINGDEDSA 0.010 10 IINGDEDSAA 0.010 8
MMIINGDEDS 0.005 3 AATKAMMIIN 0.005 6 KAMMIINGDE 0.001 7 AMMIINGDED
0.001 5 TKAMMIINGD 0.000 V5-HLA-A1- 10 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 6
RDEEQKQNRK 0.900 7 DEEQKQNRKK 0.900 5 IRDEEQKQNR 0.250 9 EQKQNRKKGK
0.003 2 ERKIRDEEQK 0.001 4 KIRDEEQKQN 0.001 10 QKQNRKKGKG 0.001 3
RKIRDEEQKQ 0.000 8 EEQKQNRKKG 0.000 1 AERKIRDEEQ 0.000
V5&6-HLA-A1- 10 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start ;position is specified, the length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Start Subsequence Score 1 DEEQKQNRKN
0.045 3 EQKQNRKNGK 0.003 4 QKQNRKNGKG 0.001 2 EEQKQNRKNG 0.000
V6-HLA-A1- 10 mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 2 ERKQNRKNGK 0.001 7
RKNGKGQASQ 0.001 3 RKQNRKNGKG 0.001 8 KNGKGQASQT 0.000 4 KQNRKNGKGQ
0.000 6 NRKNGKGQAS 0.000 5 QNRKNGKGQA 0.000 1 EERKQNRKNG 0.000
V8-HLA-A1- 10 mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 10 MGLMEAISEK 0.050 8
TVMGLMEAIS 0.020 6 SPTVMGLMEA 0.013 1 ALMLKSPTVM 0.010 5 KSPTVMGLME
0.007 3 MLKSPTVMGL 0.005 4 LKSPTVMGLM 0.005 7 PTVMGLMEAI 0.003 9
VMGLMEAISE 0.003 2 LMLKSPTVMG 0.001
TABLE-US-00020 TABLE X V1-HLA-A0201- 9 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of: peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
253 YLNKGQFYA 729.004 49 GLLYDYYKV 386.014 536 ALMLKSPTV 257.342
591 FILNMESMV 162.769 180 SLATHSAYL 117.493 350 KIFITVNCL 7.488 90
NLSGGENRV 69.552 522 VLLYVRKET 46.873 337 NAVSFTWDV 27.531 234
YTLEATKSL 20.704 528 KETDDVFDA 18.492 597 SMVEGFKVT 17.190 133
IIPVSGITV 16.258 98 VQVLKTVPV 11.988 107 NLSLNQDHL 10.468 573
ILVNMDDNI 8.691 334 IAYNAVSFT 7.122 307 RQHTAKQRV 7.052 174
TQYDVPSLA 6.609 585 YSNEDTFIL 6.254 341 FTWDVNEEA 5.293 352
FITVNCLST 4.713 461 QPVLFIPDV 3.968 23 YLENPLTAA 3.364 222
YMYDQTSSG 3.248 557 LPVEKIAKL 2.236 369 KGLPLMIQI 2.028 1 MPSDPPFNT
1.967 92 SGGENRVQV 1.861 140 TVVKAEDFT 1.757 279 KVRSVVMVV 1.527 56
KVPRDKRLL 1.308 142 VKAEDFTPV 1.279 100 VLKTVPVNL 1.271 83
GTSEAQSNL 1.216 465 FIPDVHFAN 1.121 525 YVRKETDDV 1.043 102
KTVPVNLSL 1.038 187 YLKDDQRST 0.984 564 KLYKKSKKG 0.835 598
MVEGFKVTL 0.773 596 ESMVEGFKV 0.731 134 IPVSGITVV 0.728 124
SISFPESSA 0.683 173 QTQYDVPSL 0.682 574 LVNMDDNII 0.636 151
FMAPPVHYP 0.626 446 KSDITYFKT 0.592 21 KSYLENPLT 0.545 228
SSGTFQYTL 0.530 546 GLMEAISEK 0.520 227 TSSGTFQYT 0.508 373
LMIQIDTYS 0.503 256 KGQFYAITL 0.488 463 VLFIPDVHF 0.469 148
TPVFMAPPV 0.454 375 IQIDTYSYN 0.434 441 IPLQKKSDI 0.428 125
ISFPESSAI 0.428 518 GTKRVLLYV 0.428 599 VEGFKVTLM 0.378 360
TDFSSQKGV 0.357 567 KKSKKGILV 0.338 493 VLVKRMFRP 0.338 587
NEDTFILNM 0.338 37 SINGDEDSA 0.335 131 SAIIPVSGI 0.333 30 AATKAMMSI
0.333 363 SSQKGVKGL 0.321 382 YNNRSNKPI 0.313 455 MPDLHSQPV 0.309
371 LPLMIQIDT 0.306 47 ALGLLYDYY 0.301 169 VIFEQTQYD 0.291 62
RLLSVSKAS 0.276 366 KGVKGLPLM 0.261 230 GTFQYTLEA 0.255 24
LENPLTAAT 0.246 555 YGLPVEKIA 0.226 38 INGDEDSAA 0.226 278
SKVRSVVMV 0.222 331 IEEIAYNAV 0.221 472 ANLQRTGQV 0.218 144
AEDFTPVFM 0.213 50 LLYDYYKVP 0.204 576 NMDDNIIEH 0.203 347
EEAKIFITV 0.193 319 ADYKESFNT 0.192 160 RGDGEEQRV 0.182 466
IPDVHFANL 0.180 330 NIEEIAYNA 0.179 345 VNEEAKIFI 0.167 326
NTIGNIEEI 0.163 458 LHSQPVLFI 0.156 476 RTGQVYYNT 0.155 506
FGPVPSKQM 0.149 201 SESFKDAAT 0.145 327 TIGNIEEIA 0.137 55
YKVPRDKRL 0.136 20 WKSYLENPL 0.136 V2-HLA-A0201- 9 mers-202P5A5
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 3 QESDNNKRL 1.703 7 NNKRLVALV 0.037 10 RLVALVPMP
0.034 6 DNNKRLVAL 0.024 9 KRLVALVPM 0.021 13 ALVPMPSDP 0.015 11
LVALVPMPS 0.011 16 PMPSDPPFN 0.007 15 VPMPSDPPF 0.003 12 VALVPMPSD
0.003 5 SDNNKRLVA 0.003
4 ESDNNKRLV 0.003 14 LVPMPSDPP 0.001 1 MSQESDNNK 0.001 2 SQESDNNKR
0.000 8 NKRLVALVP 0.000 V4-HLA-A0201- 9 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 9
IINGDEDSA 0.569 1 TAATKAMMI 0.145 2 AATKAMMII 0.137 7 MMIINGDED
0.009 8 MIINGDEDS 0.009 5 KAMMIINGD 0.006 6 AMMIINGDE 0.005 4
TKAMMIING 0.000 3 ATKAMMIIN 0.000 V5-HLA-A0201- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 3 KIRDEEQKQ 0.002 4 IRDEEQKQN 0.000 9 QKQNRKKGK
0.000 8 EQKQNRKKG 0.000 2 RKIRDEEQK 0.000 5 RDEEQKQNR 0.000 7
EEQKQNRKK 0.000 6 DEEQKQNRK 0.000 1 ERKIRDEEQ 0.000 V5&6-HLA-
A0201-9 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Start Subsequence Score 1 EEQKQNRKN 0.000 3 QKQNRKNGK
0.000 2 EQKQNRKNG 0.000 V6-HLA-A0201- 9 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 4
KQNRKNGKG 0.003 9 NGKGQASQT 0.002 8 KNGKGQASQ 0.000 7 RKNGKGQAS
0.000 3 RKQNRKNGK 0.000 5 QNRKNGKGQ 0.000 6 NRKNGKGQA 0.000 1
EERKQNRKN 0.000 2 ERKQNRKNG 0.000 V8-HLA-A0201- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 1 LMLKSPTVM 9.253 7 TVMGLMEAI 3.807 3 LKSPTVMGL
0.116 8 VMGLMEAIS 0.038 4 KSPTVMGLM 0.034 2 MLKSPTVMG 0.004 6
PTVMGLMEA 0.003 9 MGLMEAISE 0.001 5 SPTVMGLME 0.000
TABLE-US-00021 TABLE XI V1-HLA-A0201- 10 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score
463 VLFIPDVHFA 395.296 245 KQGEGPMTYL 393.172 222 YMYDQTSSGT
324.814 169 VIFEQTQYDV 246.631 597 SMVEGFKVTL 240.374 454
TMPDLHSQPV 205.951 564 KLYKKSKKGI 116.847 465 FIPDVHFANL 105.256
253 YLNKGQFYAI 91.183 V1-HLA-A0201- 10 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score
460 SQPVLFIPDV 61.633 336 YNAVSFTWDV 59.522 556 GLPVEKIAKL 49.134
550 AISEKYGLPV 39.210 263 TLSETGDNKC 20.369 133 IIPVSGITVV 15.331
271 KCFRHPISKV 13.523 595 MESMVEGFKV 13.335 174 TQYDVPSLAT 9.913
457 DLHSQPVLFI 9.898 132 AIIPVSGITV 9.563 370 GLPLMIQIDT 7.452 573
ILVNMDDNII 7.272 513 QMKEEGTKRV 7.208 99 QVLKTVPVNL 6.916 97
RVQVLKTVPV 6.086 528 KETDDVFDAL 5.549 147 FTPVFMAPPV 4.444 48
LGLLYDYYKV 4.284 330 NIEEIAYNAV 3.764 15 SEDEAWKSYL 3.747 449
ITYFKTMPDL 3.712 493 VLVKRMFRPM 3.209 538 MLKSPTVKGL 2.923 572
GILVNMDDNI 2.601 521 RVLLYVRKET 2.413 23 YLENPLTAAT 2.194 500
RPMEEEFGPV 1.701 89 SNLSGGENRV 1.680 344 DVNEEAKIFI 1.544 362
FSSQKGVKGL 1.475 440 AIPLQKKSDI 1.435 124 SISFPESSAI 1.435 445
KKSDITYFKT 1.292 392 RAYCQIKVFC 1.214 226 QTSSGTFQYT 1.082 333
EIAYNAVSFT 0.972 91 LSGGENRVQV 0.772 548 MEAISEKYGL 0.706 141
VVKAEDFTPV 0.688 37 SINGDEDSAA 0.683 432 SSSDGKLAAI 0.642 471
FANLQRTGQV 0.578 546 GLMEAISEKY 0.554 57 VPRDKRLLSV 0.553 92
SGGENRVQVL 0.539 227 TSSGTFQYTL 0.530 144 AEDFTPVFMA 0.515 139
ITVVKAEDFT 0.474 230 GTFQYTLEAT 0.432 517 EGTKRVLLYV 0.431 106
VNLSLNQDHL 0.430 198 STYSESFKDA 0.420 473 NLQRTGQVYY 0.410 318
IADYKESFNT 0.408 359 STDFSSQKGV 0.386 172 EQTQYDVPSL 0.374 21
KSYLENPLTA 0.363 388 KPIHRAYCQI 0.358 325 FNTIGNIEEI 0.353 143
KAEDFTPVFM 0.349 537 LMLKSPTVKG 0.339 29 TAATKAMMSI 0.333 94
GENRVQVLKT 0.333 50 LLYDYYKVPR 0.332 127 FPESSAIIPV 0.307 592
ILNMESMVEG 0.291 535 DALMLKSPTV 0.268 366 KGVKGLPLMI 0.238 82
LGTSEAQSNL 0.237 39 NGDEDSAAAL 0.229 38 INGDEDSAAA 0.226 322
KESFNTIGNI 0.212 214 SASVGAEEYM 0.186 307 RQHTAKQRVL 0.178 125
ISFPESSAII 0.176 73 QEDQEKRNCL 0.166 346 NEEAKIFITV 0.164 130
SSAIIPVSGI 0.157 301 WKYWHSRQHT 0.152 123 YSISFPESSA 0.149 13
YTSEDEAWKS 0.146 374 MIQIDTYSYN 0.144 329 GNIEEIAYNA 0.133 350
KIFITVNCLS 0.133 63 LLSVSKASDS 0.127 26 NPLTAATKAM 0.120 437
KLAAIPLQKK 0.120 345 VNEEAKIFIT 0.119 468 DVHFANLQRT 0.112 232
FQYTLEATKS 0.111 V2-HLA-A0201- 10 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 3
QESDNNKRLV 1.109 16 PMPSDPPFNT 0.687 2 SQESDNNKRL 0.139 6
DNNKRLVALV 0.078
10 RLVALVPMPS 0.075 5 SDNNKRLVAL 0.068 15 VPMPSDPPFN 0.017 14
LVPMPSDPPF 0.011 13 ALVPMPSDPP 0.007 11 LVALVPMPSD 0.005 12
VALVPMPSDP 0.001 8 NKRLVALVPM 0.001 1 MSQESDNNKR 0.001 4 ESDNNKRLVA
0.000 9 KRLVALVPMP 0.000 7 NNKRLVALVP 0.000 V4-HLA-A0201- 10
mers-202P5A5 the end position for Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Start Subsequence Score 10 IINGDEDSAA
1.161 9 MIINGDEDSA 0.569 1 LTAATKAMMI 0.246 2 TAATKAMMII 0.137 8
MMIINGDEDS 0.045 7 AMMIINGDED 0.020 3 AATKAMMIIN 0.001 6 KAMMIINGDE
0.000 4 ATKAMMIING 0.000 5 TKAMMIINGD 0.000 V5-HLA-A0201- 10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
each peptide is the start position plus nine. Start Subsequence
Score 4 KIRDEEQKQN 0.011 8 EEQKQNRKKG 0.000 3 RKIRDEEQKQ 0.000 10
QKQNRKKGKG 0.000 1 AERKIRDEEQ 0.000 5 IRDEEQKQNR 0.000 6 RDEEQKQNRK
0.000 9 EQKQNRKKGK 0.000 7 DEEQKQNRKK 0.000 2 ERKIRDEEQK 0.000
V5&6-HLA- A0201-10 mers-202P5A5 Each peptide is a portion of
SEQ ID NO: 3; each start ;position is specified, the length of
peptide is 10 amino acids, and the end position for each peptide is
the start position plus nine. Start Subsequence Score 2 EEQKQNRKNG
0.000 4 QKQNRKNGKG 0.000 3 EQKQNRKNGK 0.000 1 DEEQKQNRKN 0.000
V6-HLA-A0201- 10 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 8 KNGKGQASQT 0.170 4
KQNRKNGKGQ 0.005 5 QNRKNGKGQA 0.000 7 RKNGKGQASQ 0.000 3 RKQNRKNGKG
0.000 1 EERKQNRKNG 0.000 6 NRKNGKGQAS 0.000 2 ERKQNRKNGK 0.000
V8-HLA-A0201- 10 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 1 ALMLKSPTVM 7.536 3
MLKSPTVMGL 2.923 2 LMLKSPTVMG 0.339 6 SPTVMGLMEA 0.075 9 VMGLMEAISE
0.018 8 TVMGLMEAIS 0.010 7 PTVMGLMEAI 0.004 4 LKSPTVMGLM 0.004 10
MGLMEAISEK 0.001 5 KSPTVMGLME 0.000
TABLE-US-00022 TABLE XII V1-HLA-A3- 9 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
546 GLMEAISEK 202.500 437 KLAAIPLQK 180.000 556 GLPVEKIAK 120.000
284 VMVVFSEDK 90.000 263 TLSETGDNK 30.000 537 LMLKSPTVK 30.000 463
VLFIPDVHF 15.000 532 DVFDALMLK 9.000 594 NMESMVEGF 6.000 547
LMEAISEKY 6.000 513 QMKEEGTKR 6.000 271 KCFRHPISK 6.000 314
RVLDIADYK 4.500 395 CQIKVFCDK 4.050 235 TLEATKSLR 4.000 473
NLQRTGQVY 4.000 47 ALGLLYDYY 4.000 232 FQYTLEATK 3.000 350
KIFITVNCL 2.700 49 GLLYDYYKV 2.700 297 QLKYWKYWH 1.800 13 YTSEDEAWK
1.500 507 GPVPSKQMK 1.350 216 SVGAEEYMY 1.200 492 SVLVKRMFR 1.200
374 MIQIDTYSY 1.200 245 KQGEGPMTY 1.080 286 VVFSEDKNR 1.000 230
GTFQYTLEA 0.900 372 PLMIQIDTY 0.900 100 VLKTVPVNL 0.900 573
ILVNMDDNI 0.900 512 KQMKEEGTK 0.810 102 KTVPVNLSL 0.608 504
EEFGPVPSK 0.607 180 SLATHSAYL 0.600 457 DLHSQPVLF 0.600 226
QTSSGTFQY 0.600 107 NLSLNQDHL 0.600 168 VVIFEQTQY 0.600 111
NQDHLENSK 0.600 576 NMDDNIIEH 0.600 181 LATHSAYLK 0.600 253
YLNKGQFYA 0.600 279 KVRSVVMVV 0.540 367 GVKGLPLMI 0.540 488
REGGSVLVK 0.540 380 YSYNNRSNK 0.500 438 LAAIPLQKK 0.450 135
PVSGITVVK 0.450 23 YLENPLTAA 0.450 46 AALGLLYDY 0.405 597 SMVEGFKVT
0.338 358 LSTDFSSQK 0.300 562 IAKLYKKSK 0.300 536 ALMLKSPTV 0.300
90 NLSGGENRV 0.300 48 LGLLYDYYK 0.270 94 GENRVQVLK 0.270 445
KKSDITYFK 0.270 152 MAPPVHYPR 0.270 598 MVEGFKVTL 0.270 468
DVHFANLQR 0.240 443 LQKKSDITY 0.240 497 RMFRPMEEE 0.225 354
TVNCLSTDF 0.200 400 FCDKGAERK 0.200 185 SAYLKDDQR 0.200 429
QCNSSSDGK 0.200 299 KYWKYWHSR 0.180 143 KAEDFTPVF 0.180 317
DIADYKESF 0.180 564 KLYKKSKKG 0.150 50 LLYDYYKVP 0.150 251
MTYLNKGQF 0.150 139 ITVVKAEDF 0.150 543 TVKGLMEAI 0.135 151
FMAPPVHYP 0.135 83 GTSEAQSNL 0.135 518 GTKRVLLYV 0.135 330
NIEEIAYNA 0.135 493 VLVKRMFRP 0.135 559 VEKIAKLYK 0.120 474
LQRTGQVYY 0.120 522 VLLYVRKET 0.113 326 NTIGNIEEI 0.101 523
LLYVRKETD 0.100 222 YMYDQTSSG 0.100 520 KRVLLYVRK 0.090 373
LMIQIDTYS 0.090 370 GLPLMIQID 0.090 173 QTQYDVPSL 0.090 44
SAAALGLLY 0.080 341 FTWDVNEEA 0.075 392 RAYCQIKVF 0.075 294
RDEQLKYWK 0.060 390 IHRAYCQIK 0.060 333 EIAYNAVSF 0.060 574
LVNMDDNII 0.060 315 VLDIADYKE 0.060 V2-HLA-A3- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 1 MSQESDNNK 0.150 2 SQESDNNKR 0.120 10 RLVALVPMP
0.090 13 ALVPMPSDP 0.045 15 VPMPSDPPF 0.045 11 LVALVPMPS 0.012 14
LVPMPSDPP 0.003 16 PMPSDPPFN 0.002 9 KRLVALVPM 0.001 7 NNKRLVALV
0.001 3 QESDNNKRL 0.001
12 VALVPMPSD 0.001 6 DNNKRLVAL 0.001 5 SDNNKRLVA 0.000 8 NKRLVALVP
0.000 4 ESDNNKRLV 0.000 V4-HLA-A3- 9 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position fo each
peptide is the start position plus eight. Start Subsequence Score 9
IINGDEDSA 0.030 7 MMIINGDED 0.030 2 AATKAMMII 0.018 6 AMMIINGDE
0.0131 1 TAATKAMMI 0.012 8 MIINGDEDS 0.006 3 ATKAMMIIN 0.003 5
KAMMIINGD 0.002 4 TKAMMIING 0.000 V5-HLA-A3- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 2 RKIRDEEQK 0.030 6 DEEQKQNRK 0.018 9 QKQNRKKGK
0.010 5 RDEEQKQNR 0.006 3 KIRDEEQKQ 0.006 7 EEQKQNRKK 0.002 8
EQKQNRKKG 0.000 4 IRDEEQKQN 0.000 1 ERKIRDEEQ 0.000
V5&6-HLA-A3- 9 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start ;position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 3 QKQNRKNGK 0.020 2
EQKQNRKNG 0.000 1 EEQKQNRKN 0.000 V6-HLA-A3- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 3 RKQNRKNGK 0.020 4 KQNRKNGKG 0.001 9 NGKGQASQT
0.000 8 KNGKGQASQ 0.000 7 RKNGKGQAS 0.000 6 NRKNGKGQA 0.000 5
QNRKNGKGQ 0.000 2 ERKQNRKNG 0.000 1 EERKQNRKN 0.000 V8-HLA-A3- 9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 1 LMLKSPTVM 0.300 7 TVMGLMEAI 0.203 8
VMGLMEAIS 0.040 2 MLKSPTVMG 0.030 3 LKSPTVMGL 0.005 6 PTVMGLMEA
0.005 4 KSPTVMGLM 0.002 5 SPTVMGLME 0.000 9 MGLMEAISE 0.000
TABLE-US-00023 TABLE XIII V1-HLA-A3- 10 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 47
ALGLLYDYYK 180.000 437 KLAAIPLQKK 135.000 50 LLYDYYKVPR 60.000 180
SLATHSAYLK 60.000 357 CLSTDFSSQK 60.000 546 GLMEAISEKY 40.500 536
ALMLKSPTVK 30.000 151 FMAPPVHYPR 27.000 373 LMIQIDTYSY 18.000 497
RMFRPMEEEF 10.000 561 KIAKLYKKSK 9.000 283 VVMVVFSEDK 9.000 594
NMESMVEGFK 9.000 576 NMDDNIIEHY 9.000 398 KVFCDKGAER 6.000 253
YLNKGQFYAI 5.400 341 FTWDVNEEAK 5.000 564 KLYKKSKKGI 4.500 597
SMVEGFKVTL 4.050 473 NLQRTGQVYY 4.000 114 HLENSKREQY 4.000 463
VLFIPDVHFA 3.000 518 GTKRVLLYVR 2.700 556 GLPVEKIAKL 2.700 479
QVYYNTDDER 2.000 457 DLHSQPVLFI 1.620 262 ITLSETGDNK 1.500 279
KVRSVVMVVF 1.350 251 MTYLNKGQFY 1.000 573 ILVNMDDNII 0.900 394
YCQIKVFCDK 0.900 538 MLKSPTVKGL 0.900 370 GLPLMIQIDT 0.900 327
TIGNIEEIAY 0.800 442 PLQKKSDITY 0.800 428 TQCNSSSDGK 0.600 167
RVVIFEQTQY 0.600 389 PIHRAYCQIK 0.600 138 GITVVKAEDF 0.600 312
KQRVLDIADY 0.540 512 KQMKEEGTKR 0.540 247 GEGPMTYLNK 0.540 222
YMYDQTSSGT 0.500 234 YTLEATKSLR 0.450 134 IPVSGITVVK 0.450 169
VIFEQTQYDV 0.450 376 QIDTYSYNNR 0.400 558 PVEKIAKLYK 0.400 149
PVFMAPPVHY 0.300 285 MVVFSEDKNR 0.300 263 TLSETGDNKC 0.300 572
GILVNMDDNI 0.270 559 VEKIAKLYKK 0.270 436 GKLAAIPLQK 0.270 449
ITYFKTMPDL 0.225 562 IAKLYKKSKK 0.200 525 YVRKETDDVF 0.200 454
TMPDLHSQPV 0.200 245 KQGEGPMTYL 0.182 1 MPSDPPFNTR 0.180 491
GSVLVKRMFR 0.180 465 FIPDVHFANL 0.180 45 AAALGLLYDY 0.180 309
HTAKQRVLDI 0.180 550 AISEKYGLPV 0.180 100 VLKTVPVNLS 0.180 202
ESFKDAATEK 0.150 353 ITVNCLSTDF 0.150 513 QMKEEGTKRV 0.150 49
GLLYDYYKVP 0.135 215 ASVGAEEYMY 0.135 545 KGLMEAISEK 0.135 99
QVLKTVPVNL 0.135 297 QLKYWKYWHS 0.120 111 NQDHLENSKR 0.120 230
GTFQYTLEAT 0.113 225 DQTSSGTFQY 0.108 23 YLENPLTAAT 0.100 69
ASDSQEDQEK 0.100 523 LLYVRKETDD 0.100 293 NRDEQLKYWK 0.090 444
QKKSDITYFK 0.090 93 GGENRVQVLK 0.090 555 YGLPVEKIAK 0.090 46
AALGLLYDYY 0.090 598 MVEGFKVTLM 0.090 443 LQKKSDITYF 0.090 493
VLVKRMFRPM 0.090 350 KIFITVNCLS 0.090 132 AIIPVSGITV 0.090 371
LPLMIQIDTY 0.090 24 LENPLTAATK 0.090 503 EEEFGPVPSK 0.081 198
STYSESFKDA 0.075 557 LPVEKIAKLY 0.068 271 KCFRHPISKV 0.068 124
SISFPESSAI 0.060 552 SEKYGLPVEK 0.060 109 SLNQDHLENS 0.060 195
TPDSTYSESF 0.060 V2-HLA-A3- 10 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide' is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 14
LVPMPSDPPF 0.200 10 RLVALVPMPS 0.180 13 ALVPMPSDPP 0.068 16
PMPSDPPFNT 0.045 1 MSQESDNNKR 0.030 2 SQESDNNKRL 0.009 11
LVALVPMPSD 0.005 5 SDNNKRLVAL 0.001 4 ESDNNKRLVA 0.001 15
VPMPSDPPFN 0.000 6 DNNKRLVALV 0.000
8 NKRLVALVPM 0.000 12 VALVPMPSDP 0.000 9 KRLVALVPMP 0.000 7
NNKRLVALVP 0.000 3 QESDNNKRLV 0.000 V4-HLA-A3- is 10 amino acids,
and 10 mers-202P5A5 the end position for Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 10 amino acids, the end position for each peptide is the
start position plus nine. Start Subsequence Score 1 LTAATKAMMI
0.060 8 MMIINGDEDS 0.060 7 AMMIINGDED 0.030 9 MIINGDEDSA 0.030 10
IINGDEDSAA 0.030 2 TAATKAMMII 0.018 3 AATKAMMIIN 0.001 6 KAMMIINGDE
0.000 5 TKAMMIINGD 0.000 V5-HLA-A3- 10 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and each peptide is the start
position plus nine. Start Subsequence Score 9 EQKQNRKKGK 0.090 6
RDEEQKQNRK 0.030 5 IRDEEQKQNR 0.006 2 ERKIRDEEQK 0.006 4 KIRDEEQKQN
0.003 7 DEEQKQNRKK 0.002 1 AERKIRDEEQ 0.000 3 RKIRDEEQKQ 0.000 8
EEQKQNRKKG 0.000 10 QKQNRKKGKG 0.000 V5&6-HLA-
A3-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Start Subsequence Score 3 EQKQNRKNGK 0.180 2 EEQKQNRKNG
0.000 4 QKQNRKNGKG 0.000 1 DEEQKQNRKN 0.000 V6-HLA-A3- 10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Start Subsequence Score 2 ERKQNRKNGK 0.006 4 KQNRKNGKGQ 0.001 8
KNGKGQASQT 0.001 5 QNRKNGKGQA 0.000 6 NRKNGKGQAS 0.000 7 RKNGKGQASQ
0.000 1 EERKQNRKNG 0.000 3 RKQNRKNGKG 0.000 V8-HLA-A3- 10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Start Subsequence Score 3 MLKSPTVMGL 3.600 1 ALMLKSPTVM 0.300 10
MGLMEAISEK 0.045 2 LMLKSPTVMG 0.045 9 VMGLMEAISE 0.040 8 TVMGLMEAIS
0.009 7 PTVMGLMEAI 0.007 6 SPTVMGLMEA 0.006 5 KSPTVMGLME 0.001 4
LKSPTVMGLM 0.000
TABLE-US-00024 TABLE XIV V1-HLA-A1101-9mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
314 RVLDIADYK 9.000 512 KQMKEEGTK 3.600 556 GLPVEKIAK 2.400 271
KCFRHPISK 2.400 546 GLMEAISEK 2.400 532 DVFDALMLK 2.400 437
KLAAIPLQK 2.400 492 SVLVKRMFR 1.200 232 FQYTLEATK 1.200 13
YTSEDEAWK 1.000 395 CQIKVFCDK 0.900 507 GPVPSKQMK 0.900 111
NQDHLENSK 0.600 284 VMVVFSEDK 0.600 537 LMLKSPTVK 0.600 299
KYWKYWHSR 0.480 263 TLSETGDNK 0.400 286 VVFSEDKNR 0.400 181
LATHSAYLK 0.400 488 REGGSVLVK 0.360 468 DVHFANLQR 0.240 429
QCNSSSDGK 0.200 400 FCDKGAERK 0.200 135 PVSGITVVK 0.200 203
SFKDAATEK 0.200 438 LAAIPLQKK 0.200 94 GENRVQVLK 0.180 480
VYYNTDDER 0.160 445 KKSDITYFK 0.120 294 RDEQLKYWK 0.120 279
KVRSVVMVV 0.120 559 VEKIAKLYK 0.120 367 GVKGLPLMI 0.120 230
GTFQYTLEA 0.120 562 IAKLYKKSK 0.100 407 RKIRDEERK 0.090 520
KRVLLYVRK 0.090 102 KTVPVNLSL 0.090 185 SAYLKDDQR 0.080 54
YYKVPRDKR 0.080 152 MAPPVHYPR 0.080 51 LYDYYKVPR 0.080 513
QMKEEGTKR 0.080 235 TLEATKSLR 0.080 48 LGLLYDYYK 0.060 266
ETGDNKCFR 0.060 361 DFSSQKGVK 0.060 595 MESMVEGFK 0.060 518
GTKRVLLYV 0.060 399 VFCDKGAER 0.040 216 SVGAEEYMY 0.040 380
YSYNNRSNK 0.040 504 EEFGPVPSK 0.036 245 KQGEGPMTY 0.036 49
GLLYDYYKV 0.036 563 AKLYKKSKK 0.030 414 RKQNRKKGK 0.030 168
VVIFEQTQY 0.030 83 GTSEAQSNL 0.030 226 QTSSGTFQY 0.030 53 DYYKVPRDK
0.024 350 KIFITVNCL 0.024 341 FTWDVNEEA 0.020 390 IHRAYCQIK 0.020
304 WHSRQHTAK 0.020 543 TVKGLMEAI 0.020 354 TVNCLSTDF 0.020 342
TWDVNEEAK 0.020 70 SDSQEDQEK 0.020 251 MTYLNKGQF 0.020 574
LVNMDDNII 0.020 358 LSTDFSSQK 0.020 598 MVEGFKVTL 0.020 525
YVRKETDDV 0.020 243 RQKQGEGPM 0.018 411 DEERKQNRK 0.018 307
RQHTAKQRV 0.018 560 EKIAKLYKK 0.018 139 ITVVKAEDF 0.015 326
NTIGNIEEI 0.015 410 RDEERKQNR 0.012 89 SNLSGGENR 0.012 253
YLNKGQFYA 0.012 11 RAYTSEDEA 0.012 335 AYNAVSFTW 0.012 398
KVFCDKGAE 0.012 601 GFKVTLMEI 0.012 553 EKYGLPVEK 0.012 554
KYGLPVEKI 0.012 248 EGPMTYLNK 0.012 25 ENPLTAATK 0.012 174
TQYDVPSLA 0.012 22 SYLENPLTA 0.012 443 LQKKSDITY 0.012 28 LTAATKAMM
0.010 173 QTQYDVPSL 0.010 98 VQVLKTVPV 0.009 282 SVVMVVFSE 0.009
167 RVVIFEQTQ 0.009 536 ALMLKSPTV 0.008 V2-HLA-A1101-9mers-202P5A5
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 2 SQESDNNKR 0.120 1 MSQESDNNK 0.020 15 VPMPSDPPF
0.004 11 LVALVPMPS 0.004 14 LVPMPSDPP 0.002 10 RLVALVPMP 0.002 9
KRLVALVPM 0.001 13 ALVPMPSDP 0.001 7 NNKRLVALV 0.000 5 SDNNKRLVA
0.000 12 VALVPMPSD 0.000
3 QESDNNKRL 0.000 6 DNNKRLVAL 0.000 16 PMPSDPPFN 0.000 8 NKRLVALVP
0.000 4 ESDNNKRLV 0.000 V4-HLA-A1101-9mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 9
IINGDEDSA 0.004 1 TAATKAMMI 0.004 2 AATKAMMII 0.004 3 ATKAMMIIN
0.002 5 KAMMIINGD 0.001 6 AMMIINGDE 0.001 8 MIINGDEDS 0.001 7
MMIINGDED 0.001 4 TKAMMIING 0.000 V5-HLA-A1101-9mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 2 RKIRDEEQK 0.090 6 DEEQKQNRK 0.018 5 RDEEQKQNR
0.012 9 QKQNRKKGK 0.010 7 EEQKQNRKK 0.002 3 KIRDEEQKQ 0.001 8
EQKQNRKKG 0.000 4 IRDEEQKQN 0.000 V5-HLA-A1101-9mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 1 ERKIRDEEQ 0.000
V5&6-HLA-A1101-9mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 9 amino acids, and the end position for each peptide is the
start position plus eight. Start Subsequence Score 3 QKQNRKNGK
0.020 2 EQKQNRKNG 0.000 1 EEQKQNRKN 0.000
V6-HLA-A1101-9mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 3 RKQNRKNGK 0.060 4
KQNRKNGKG 0.002 6 NRKNGKGQA 0.000 8 KNGKGQASQ 0.000 7 RKNGKGQAS
0.000 9 NGKGQASQT 0.000 5 QNRKNGKGQ 0.000 2 ERKQNRKNG 0.000 1
EERKQNRKN 0.000 V8-HLA-A1101-9mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 7
TVMGLMEAI 0.040 1 LMLKSPTVM 0.006 6 PTVMGLMEA 0.003 4 KSPTVMGLM
0.001 8 VMGLMEAIS 0.000 5 SPTVMGLME 0.000 3 LKSPTVMGL 0.000 2
MLKSPTVMG 0.000 9 MGLMEAISE 0.000
TABLE-US-00025 TABLE XV V1-HLA-A1101-10mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score
283 VVMVVFSEDK 4.000 398 KVFCDKGAER 2.400 341 FTWDVNEEAK 2.000 262
ITLSETGDNK 1.500 518 GTKRVLLYVR 1.200 437 KLAAIPLQKK 1.200 536
ALMLKSPTVK 0.800 180 SLATHSAYLK 0.800 479 QVYYNTDDER 0.800 47
ALGLLYDYYK 0.800 512 KQMKEEGTKR 0.720 428 TQCNSSSDGK 0.600 561
KIAKLYKKSK 0.600 379 TYSYNNRSNK 0.400 12 AYTSEDEAWK 0.400 357
CLSTDFSSQK 0.400 594 NMESMVEGFK 0.400 558 PVEKIAKLYK 0.400 247
GEGPMTYLNK 0.360 234 YTLEATKSLR 0.300 134 IPVSGITVVK 0.300 285
MVVFSEDKNR 0.300 231 TFQYTLEATK 0.200 399 VFCDKGAERK 0.200 562
IAKLYKKSKK 0.200 394 YCQIKVFCDK 0.200 436 GKLAAIPLQK 0.180 151
FMAPPVHYPR 0.160 50 LLYDYYKVPR 0.160 559 VEKIAKLYKK 0.120 111
NQDHLENSKR 0.120 545 KGLMEAISEK 0.090 167 RVVIFEQTQY 0.090 376
QIDTYSYNNR 0.080 59 RDKRLLSVSK 0.060 555 YGLPVEKIAK 0.060 410
RDEERKQNRK 0.060 93 GGENRVQVLK 0.060 279 KVRSVVMVVF 0.060 24
LENPLTAATK 0.060 97 RVQVLKTVPV 0.060 552 SEKYGLPVEK 0.060 53
DYYKVPRDKR 0.048 270 NKCFRHPISK 0.040 389 PIHRAYCQIK 0.040 444
QKKSDITYFK 0.040 110 LNQDHLENSK 0.040 360 TDFSSQKGVK 0.040 293
NRDEQLKYWK 0.040 158 YPRGDGEEQR 0.040 1 MPSDPPFNTR 0.040 491
GSVLVKRMFR 0.036 488 REGGSVLVKR 0.036 313 QRVLDIADYK 0.030 99
QVLKTVPVNL 0.030 302 KYWHSRQHTA 0.024 497 RMFRPMEEEF 0.024 546
GLMEAISEKY 0.024 303 YWHSRQHTAK 0.020 449 ITYFKTMPDL 0.020 519
TKRVLLYVRK 0.020 272 CFRHPISKVR 0.020 251 MTYLNKGQFY 0.020 69
ASDSQEDQEK 0.020 506 FGPVPSKQMK 0.020 598 MVEGFKVTLM 0.020 511
SKQMKEEGTK 0.020 141 VVKAEDFTPV 0.020 309 HTAKQRVLDI 0.020 525
YVRKETDDVF 0.020 314 RVLDIADYKE 0.018 252 TYLNKGQFYA 0.018 572
GILVNMDDNI 0.018 312 KQRVLDIADY 0.018 531 DDVFDALMLK 0.018 245
KQGEGPMTYL 0.018 503 EEEFGPVPSK 0.018 169 VIFEQTQYDV 0.016 383
NNRSNKPIHR 0.016 326 NTIGNIEEIA 0.015 353 ITVNCLSTDF 0.015 556
GLPVEKIAKL 0.012 265 SETGDNKCFR 0.012 202 ESFKDAATEK 0.012 500
RPMEEEFGPV 0.012 487 EREGGSVLVK 0.012 344 DVNEEAKIFI 0.012 138
GITVVKAEDF 0.012 271 KCFRHPISKV 0.012 11 RAYTSEDEAW 0.012 564
KLYKKSKKGI 0.012 364 SQKGVKGLPL 0.012 405 AERKIRDEER 0.012 367
GVKGLPLMIQ 0.012 132 AIIPVSGITV 0.012 373 LMIQIDTYSY 0.012 56
KVPRDKRLLS 0.012 334 IAYNAVSFTW 0.012 584 HYSNEDTFIL 0.012 208
ATEKFRSASV 0.010 V2-HLA-A1101-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 14
LVPMPSDPPF 0.020 1 MSQESDNNKR 0.004 10 RLVALVPMPS 0.004 2
SQESDNNKRL 0.003 11 LVALVPMPSD 0.002 13 ALVPMPSDPP 0.001 15
VPMPSDPPFN 0.000 12 VALVPMPSDP 0.000 5 SDNNKRLVAL 0.000 8
NKRLVALVPM 0.000 16 PMPSDPPFNT 0.000
6 DNNKRLVALV 0.000 4 ESDNNKRLVA 0.000 9 KRLVALVPMP 0.000 7
NNKRLVALVP 0.000 3 QESDNNKRLV 0.000 V4-HLA-A1101-10mers-202P5A5
Each peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 1 LTAATKAMMI 0.020 9 MIINGDEDSA 0.006 10
IINGDEDSAA 0.004 2 TAATKAMMII 0.004 4 ATKAMMIING 0.002 6 KAMMIINGDE
0.001 7 AMMIINGDED 0.001 8 MMIINGDEDS 0.001 3 AATKAMMIIN 0.000 5
TKAMMIINGD 0.000 V5-HLA-A1101-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 9
EQKQNRKKGK 0.090 6 RDEEQKQNRK 0.060 2 ERKIRDEEQK 0.006 5 IRDEEQKQNR
0.006 7 DEEQKQNRKK 0.002 4 KIRDEEQKQN 0.001 3 RKIRDEEQKQ 0.001 1
AERKIRDEEQ 0.000 10 QKQNRKKGKG 0.000 8 EEQKQNRKKG 0.000
V5&6-HLA-A1101-10mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Start Subsequence Score 3 EQKQNRKNGK
0.180 4 QKQNRKNGKG 0.000 2 EEQKQNRKNG 0.000 1 DEEQKQNRKN 0.000
V6-HLA-A1101-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 3 RKQNRKNGK 0.060 4
KQNRKNGKG 0.002 6 NRKNGKGQA 0.000 8 KNGKGQASQ 0.000 7 RKNGKGQAS
0.000 9 NGKGQASQT 0.000 5 QNRKNGKGQ 0.000 2 ERKQNRKNG 0.000 1
EERKQNRKN 0.000 V8-HLA-A1101-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 10
MGLMEAISEK 0.030 1 ALMLKSPTVM 0.008 3 MLKSPTVMGL 0.008 8 TVMGLMEAIS
0.004 6 SPTVMGLMEA 0.004 7 PTVMGLMEAI 0.002 9 VMGLMEAISE 0.001 2
LMLKSPTVMG 0.001 4 LKSPTVMGLM 0.000 5 KSPTVMGLME 0.000
TABLE-US-00026 TABLE XVI V1-HLA-A24-9mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
450 TYFKTMPDL 200.000 554 KYGLPVEKI 110.000 320 DYKESFNTI 86.400
565 LYKKSKKGI 50.000 584 HYSNEDTFI 50.000 102 KTVPVNLSL 20.160 498
MFRPMEEEF 13.200 302 KYWHSRQHT 12.000 56 KVPRDKRLL 12.000 256
KGQFYAITL 12.000 335 AYNAVSFTW 10.500 350 KIFITVNCL 9.600 22
SYLENPLTA 9.000 186 AYLKDDQRS 9.000 126 SFPESSAII 9.000 252
TYLNKGQFY 9.000 529 ETDDVFDAL 8.064 557 LPVEKIAKL 7.920 221
EYMYDQTSS 7.500 234 YTLEATKSL 7.200 199 TYSESFKDA 7.200 93
GGENRVQVL 7.200 585 YSNEDTFIL 7.200 143 KAEDFTPVF 7.200 393
AYCQIKVFC 7.000 122 QYSISFPES 6.600 363 SSQKGVKGL 6.000 598
MVEGFKVTL 6.000 549 EAISEKYGL 6.000 246 QGEGPMTYL 6.000 173
QTQYDVPSL 6.000 100 VLKTVPVNL 5.600 259 FYAITLSET 5.500 233
QYTLEATKS 5.500 601 GFKVTLMEI 5.500 12 AYTSEDEAW 5.000 175
QYDVPSLAT 5.000 223 MYDQTSSGT 5.000 379 TYSYNNRSN 5.000 83
GTSEAQSNL 4.800 466 IPDVHFANL 4.800 430 CNSSSDGKL 4.400 369
KGLPLMIQI 4.320 491 GSVLVKRMF 4.200 180 SLATHSAYL 4.000 435
DGKLAAIPL 4.000 228 SSGTFQYTL 4.000 392 RAYCQIKVF 4.000 107
NLSLNQDHL 4.000 43 DSAAALGLL 4.000 590 TFILNMESM 3.750 344
DVNEEAKIF 3.600 354 TVNCLSTDF 3.600 139 ITVVKAEDF 3.000 594
NMESMVEGF 3.000 317 DIADYKESF 2.400 540 KSPTVKGLM 2.100 463
VLFIPDVHF 2.000 333 EIAYNAVSF 2.000 457 DLHSQPVLF 2.000 251
MTYLNKGQF 2.000 345 VNEEAKIFI 1.800 574 LVNMDDNII 1.800 162
DGEEQRVVI 1.800 326 NTIGNIEEI 1.650 382 YNNRSNKPI 1.500 573
ILVNMDDNI 1.500 131 SAIIPVSGI 1.500 366 KGVKGLPLM 1.500 441
IPLQKKSDI 1.500 515 KEEGTKRVL 1.200 543 TVKGLMEAI 1.200 125
ISFPESSAI 1.200 367 GVKGLPLMI 1.200 254 LNKGQFYAI 1.200 351
IFITVNCLS 1.050 30 AATKAMMSI 1.000 310 TAKQRVLDI 1.000 323
ESFNTIGNI 1.000 211 KFRSASVGA 1.000 117 NSKREQYSI 1.000 243
RQKQGEGPM 1.000 299 KYWKYWHSR 1.000 269 DNKCFRHPI 1.000 433
SSDGKLAAI 1.000 381 SYNNRSNKP 0.990 464 LFIPDVHFA 0.900 74
EDQEKRNCL 0.864 157 HYPRGDGEE 0.825 150 VFMAPPVHY 0.750 170
IFEQTQYDV 0.750 524 LYVRKETDD 0.750 215 ASVGAEEYM 0.750 481
YYNTDDERE 0.750 231 TFQYTLEAT 0.750 506 FGPVPSKQM 0.750 40
GDEDSAAAL 0.720 55 YKVPRDKRL 0.720 53 DYYKVPRDK 0.700 533 VFDALMLKS
0.660 V2-HLA-A24-9mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 5; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 6 DNNKRLVAL 6.000 15
VPMPSDPPF 3.600 3 QESDNNKRL 0.480 9 KRLVALVPM 0.150 11 LVALVPMPS
0.140 7 NNKRLVALV 0.120 4 ESDNNKRLV 0.100 10 RLVALVPMP 0.036 1
MSQESDNNK 0.022 2 SQESDNNKR 0.020 13 ALVPMPSDP 0.018 5 SDNNKRLVA
0.015
12 VALVPMPSD 0.015 16 PMPSDPPFN 0.015 14 LVPMPSDPP 0.015 8
NKRLVALVP 0.001 V4-HLA-A24-9mers-202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Start Subsequence Score 1 TAATKAMMI
1.000 2 AATKAMMII 1.000 8 MIINGDEDS 0.150 9 IINGDEDSA 0.150 3
ATKAMMIIN 0.100 5 KAMMIINGD 0.050 6 AMMIINGDE 0.021 7 MMIINGDED
0.017 4 TKAMMIING 0.001 V5-HLA-A24-9mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 3
KIRDEEQKQ 0.032 4 IRDEEQKQN 0.012 8 EQKQNRKKG 0.011 5 RDEEQKQNR
0.004 2 RKIRDEEQK 0.003 6 DEEQKQNRK 0.002 7 EEQKQNRKK 0.002 9
QKQNRKKGK 0.002 1 ERKIRDEEQ 0.001 V5&6-HLA-A24-9mers-202P5A5
Each peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 1 EEQKQNRKN 0.017 2 EQKQNRKNG 0.010 3 QKQNRKNGK
0.002 V6-HLA-A24-9mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 9 NGKGQASQT 0.100 7
RKNGKGQAS 0.036 4 KQNRKNGKG 0.033 8 KNGKGQASQ 0.020 1 EERKQNRKN
0.011 6 NRKNGKGQA 0.010 5 QNRKNGKGQ 0.010 3 RKQNRKNGK 0.004 2
ERKQNRKNG 0.001 V8-HLA-A24-9mers-202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Start Subsequence Score 4 KSPTVMGLM
2.100 7 TVMGLMEAI 1.800 1 LMLKSPTVM 0.750 3 LKSPTVMGL 0.480 8
VMGLMEAIS 0.100 6 PTVMGLMEA 0.017 9 MGLMEAISE 0.015 2 MLKSPTVMG
0.010 5 SPTVMGLME 0.010
TABLE-US-00027 TABLE XVII V1-HLA-A24-10mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score
565 LYKKSKKGIL 200.000 584 HYSNEDTFIL 200.000 54 YYKVPRDKRL 200.000
233 QYTLEATKSL 200.000 223 MYDQTSSGTF 100.000 381 SYNNRSNKPI 75.000
554 KYGLPVEKIA 14.000 203 SFKDAATEKF 13.200 302 KYWHSRQHTA 10.000
245 KQGEGPMTYL 9.600 22 SYLENPLTAA 9.000 465 FIPDVHFANL 8.640 99
QVLKTVPVNL 8.400 307 RQHTAKQRVL 8.000 252 TYLNKGQFYA 7.500 524
LYVRKETDDV 7.500 186 AYLKDDQRST 7.500 597 SMVEGFKVTL 7.200 556
GLPVEKIAKL 6.600 429 QCNSSSDGKL 6.600 106 VNLSLNQDHL 6.000 199
TYSESFKDAA 6.000 19 AWKSYLENPL 5.760 92 SGGENRVQVL 5.760 279
KVRSVVMVVF 5.600 122 QYSISFPESS 5.000 39 NGDEDSAAAL 4.800 497
RMFRPMEEEF 4.400 449 ITYFKTMPDL 4.000 227 TSSGTFQYTL 4.000 538
MLKSPTVKGL 4.000 455 MPDLHSQPVL 4.000 364 SQKGVKGLPL 4.000 172
EQTQYDVPSL 4.000 82 LGTSEAQSNL 4.000 362 FSSQKGVKGL 4.000 353
ITVNCLSTDF 3.600 162 DGEEQRVVIF 3.600 593 LNMESMVEGF 3.600 366
KGVKGLPLMI 3.600 388 KPIHRAYCQI 3.000 264 LSETGDNKCF 3.000 505
EFGPVPSKQM 3.000 490 GGSVLVKRMF 2.800 138 GITVVKAEDF 2.000 195
TPDSTYSESF 2.000 564 KLYKKSKKGI 2.000 443 LQKKSDITYF 2.000 525
YVRKETDDVF 2.000 253 YLNKGQFYAI 1.800 344 DVNEEAKIFI 1.800 573
ILVNMDDNII 1.800 143 KAEDFTPVFM 1.800 528 KETDDVFDAL 1.613 440
AIPLQKKSDI 1.500 572 GILVNMDDNI 1.500 568 KSKKGILVNM 1.400 464
LFIPDVHFAN 1.260 446 KSDITYFKTM 1.200 125 ISFPESSAII 1.200 515
KEEGTKRVLL 1.200 432 SSSDGKLAAI 1.200 325 FNTIGNIEEI 1.100 342
TWDVNEEAKI 1.100 400 FCDKGAERKI 1.100 600 EGFKVTLMEI 1.100 116
ENSKREQYSI 1.000 299 KYWKYWHSRQ 1.000 457 DLHSQPVLFI 1.000 309
HTAKQRVLDI 1.000 124 SISFPESSAI 1.000 29 TAATKAMMSI 1.000 130
SSAIIPVSGI 1.000 493 VLVKRMFRPM 0.900 586 SNEDTFILNM 0.900 335
AYNAVSFTWD 0.900 157 HYPRGDGEEQ 0.825 481 YYNTDDEREG 0.825 340
SFTWDVNEEA 0.770 26 NPLTAATKAM 0.750 221 EYMYDQTSSG 0.750 598
MVEGFKVTLM 0.750 351 IFITVNCLST 0.750 590 TFILNMESMV 0.750 55
YKVPRDKRLL 0.720 514 MKEEGTKRVL 0.720 349 AKIFITVNCL 0.720 529
ETDDVFDALM 0.600 485 DDEREGGSVL 0.600 259 FYAITLSETG 0.600 320
DYKESFNTIG 0.600 489 EGGSVLVKRM 0.600 179 PSLATHSAYL 0.600 101
LKTVPVNLSL 0.560 53 DYYKVPRDKR 0.550 258 QFYAITLSET 0.550 451
YFKTMPDLHS 0.500 175 QYDVPSLATH 0.500 379 TYSYNNRSNK 0.500 589
DTFILNMESM 0.500 V2-HLA-A24-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 2
SQESDNNKRL 6.000 14 LVPMPSDPPF 3.000 5 SDNNKRLVAL 0.600 10
RLVALVPMPS 0.420 6 DNNKRLVALV 0.180 15 VPMPSDPPFN 0.180 4
ESDNNKRLVA 0.100 8 NKRLVALVPM 0.050 1 MSQESDNNKR 0.024 13
ALVPMPSDPP 0.018 16 PMPSDPPFNT 0.015 12 VALVPMPSDP 0.015
7 NNKRLVALVP 0.014 3 QESDNNKRLV 0.012 11 LVALVPMPSD 0.010 9
KRLVALVPMP 0.004 V4-HLA-A24-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 1
LTAATKAMMI 1.000 2 TAATKAMMII 1.000 8 MMIINGDEDS 0.150 9 MIINGDEDSA
0.150 10 IINGDEDSAA 0.150 3 AATKAMMIIN 0.100 6 KAMMIINGDE 0.042 7
AMMIINGDED 0.017 4 ATKAMMIING 0.010 5 TKAMMIINGD 0.002
V5-HLA-A24-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 4 KIRDEEQKQN 0.240 9
EQKQNRKKGK 0.010 6 RDEEQKQNRK 0.004 3 RKIRDEEQKQ 0.004 8 EEQKQNRKKG
0.002 10 QKQNRKKGKG 0.002 7 DEEQKQNRKK 0.002 5 IRDEEQKQNR 0.001 1
AERKIRDEEQ 0.001 2 ERKIRDEEQK 0.001 V5&6-HLA-
A24-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Start Subsequence Score 1 DEEQKQNRKN 0.017 3 EQKQNRKNGK
0.012 4 QKQNRKNGKG 0.002 2 EEQKQNRKNG 0.002
V6-HLA-A24-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 8 KNGKGQASQT 0.200 5
QNRKNGKGQA 0.100 4 KQNRKNGKGQ 0.030 6 NRKNGKGQAS 0.012 3 RKQNRKNGKG
0.003 7 RKNGKGQASQ 0.003 2 ERKQNRKNGK 0.001 1 EERKQNRKNG 0.001
V8-HLA-A24-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Start Subsequence Score 3 MLKSPTVMGL 4.000 1
ALMLKSPTVM 0.750 7 PTVMGLMEAI 0.180 8 TVMGLMEAIS 0.150 6 SPTVMGLMEA
0.110 4 LKSPTVMGLM 0.084 5 KSPTVMGLME 0.030 10 MGLMEAISEK 0.017 2
LMLKSPTVMG 0.015 9 VMGLMEAISE 0.010
TABLE-US-00028 TABLE XVIII V1-HLA-B7- 9 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
557 LPVEKIAKL 0.000 466 IPDVHFANL 24.000 56 KVPRDKRLL 20.000 549
EAISEKYGL 12.000 525 YVRKETDDV 10.000 279 KVRSVVMVV 10.000 441
IPLQKKSDI 8.000 598 MVEGFKVTL 6.000 57 VPRDKRLLS 6.000 494
LVKRMFRPM 5.000 461 QPVLFIPDV 4.000 256 KGQFYAITL 4.000 43
DSAAALGLL 4.000 100 VLKTVPVNL 4.000 275 HPISKVRSV 4.000 148
TPVFMAPPV 4.000 107 NLSLNQDHL 4.000 435 DGKLAAIPL 4.000 363
SSQKGVKGL 4.000 486 DEREGGSVL 4.000 83 GTSEAQSNL 4.000 585
YSNEDTFIL 4.000 234 YTLEATKSL 4.000 180 SLATHSAYL 4.000 173
QTQYDVPSL 4.000 228 SSGTFQYTL 4.000 134 IPVSGITVV 4.000 102
KTVPVNLSL 4.000 350 KIFITVNCL 4.000 430 CNSSSDGKL 4.000 30
AATKAMMSI 3.600 215 ASVGAEEYM 3.000 1 MPSDPPFNT 3.000 26 NPLTAATKA
2.000 371 LPLMIQIDT 2.000 158 YPRGDGEEQ 2.000 543 TVKGLMEAI 2.000 4
DPPFNTRRA 2.000 178 VPSLATHSA 2.000 574 LVNMDDNII 2.000 367
GVKGLPLMI 2.000 246 QGEGPMTYL 1.800 131 SAIIPVSGI 1.800 536
ALMLKSPTV 1.800 506 FGPVPSKQM 1.500 529 ETDDVFDAL 1.200 310
TAKQRVLDI 1.200 93 GGENRVQVL 1.200 455 MPDLHSQPV 1.200 490
GGSVLVKRM 1.000 277 ISKVRSVVM 1.000 366 KGVKGLPLM 1.000 95
ENRVQVLKT 1.000 540 KSPTVKGLM 1.000 191 DQRSTPDST 1.000 28
LTAATKAMM 1.000 243 RQKQGEGPM 1.000 207 AATEKFRSA 0.900 472
ANLQRTGQV 0.600 74 EDQEKRNCL 0.600 269 DNKCFRHPI 0.600 153
APPVHYPRG 0.600 337 NAVSFTWDV 0.600 516 EEGTKRVLL 0.600 596
ESMVEGFKV 0.600 500 RPMEEEFGP 0.600 249 GPMTYLNKG 0.600 140
TVVKAEDFT 0.500 125 ISFPESSAI 0.400 531 DDVFDALML 0.400 539
LKSPTVKGL 0.400 573 ILVNMDDNI 0.400 290 EDKNRDEQL 0.400 566
YKKSKKGIL 0.400 254 LNKGQFYAI 0.400 382 YNNRSNKPI 0.400 326
NTIGNIEEI 0.400 369 KGLPLMIQI 0.400 55 YKVPRDKRL 0.400 308
QHTAKQRVL 0.400 20 WKSYLENPL 0.400 365 QKGVKGLPL 0.400 42 EDSAAALGL
0.400 450 TYFKTMPDL 0.400 323 ESFNTIGNI 0.400 117 NSKREQYSI 0.400
334 IAYNAVSFT 0.300 338 AVSFTWDVN 0.300 11 RAYTSEDEA 0.300 92
SGGENRVQV 0.300 272 CFRHPISKV 0.300 535 DALMLKSPT 0.300 132
AIIPVSGIT 0.300 541 SPTVKGLME 0.200 591 FILNMESMV 0.200 518
GTKRVLLYV 0.200 90 NLSGGENRV 0.200 104 VPVNLSLNQ 0.200 307
RQHTAKQRV 0.200 509 VPSKQMKEE 0.200 V2-HLA-B7- 9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 6 DNNKRLVAL 4.000 15 VPMPSDPPF 1.200 3 QESDNNKRL
0.400 7 NNKRLVALV 0.200 11 LVALVPMPS 0.100 9 KRLVALVPM 0.100 14
LVPMPSDPP 0.075 4 ESDNNKRLV 0.060 12 VALVPMPSD 0.045 13 ALVPMPSDP
0.030 5 SDNNKRLVA 0.015 10 RLVALVPMP 0.010 8 NKRLVALVP 0.010 1
MSQESDNNK 0.010 2 SQESDNNKR 0.003 16 PMPSDPPFN 0.002 V4-HLA-B7- 9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 2 AATKAMMII 3.600 1 TAATKAMMI 1.200 9
IINGDEDSA 0.100 6 AMMIINGDE 0.090 5 KAMMIINGD 0.090 3 ATKAMMIIN
0.060 8 MIINGDEDS 0.020 7 MMIINGDED 0.010 4 TKAMMIING 0.001
V5-HLA-B7- 9 mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 3 KIRDEEQKQ 0.100 8
EQKQNRKKG 0.015 7 EEQKQNRKK 0.001 9 QKQNRKKGK 0.001 1 ERKIRDEEQ
0.001 2 RKIRDEEQK 0.001 4 IRDEEQKQN 0.001 6 DEEQKQNRK 0.000 5
RDEEQKQNR 0.000 V5 & 6-HLA- B7-9 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 2
EQKQNRKNG 0.015 1 EEQKQNRKN 0.002 3 QKQNRKNGK 0.001 V6-HLA-B7- 9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 9 NGKGQASQT 0.100 5 QNRKNGKGQ 0.100 1
EERKQNRKN 0.020 6 NRKNGKGQA 0.010 8 KNGKGQASQ 0.010 4 KQNRKNGKG
0.010 7 RKNGKGQAS 0.002 2 ERKQNRKNG 0.002 3 RKQNRKNGK 0.001
V8-HLA-B7- 9 mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score
7 TVMGLMEAI 6.000 1 LMLKSPTVM 1.000 4 KSPTVMGLM 1.000 3 LKSPTVMGL
0.400 5 SPTVMGLME 0.200 8 VMGLMEAIS 0.020 2 MLKSPTVMG 0.015 6
PTVMGLMEA 0.010 9 MGLMEAISE 0.010
TABLE-US-00029 TABLE XIX V1-HLA-B7- 10 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 57
VPRDKRLLSV 40.000 455 MPDLHSQPVL 24.000 99 QVLKTVPVNL 20.000 26
NPLTAATKAM 20.000 500 RPMEEEFGPV 12.000 388 KPIHRAYCQI 8.000 245
KQGEGPMTYL 6.000 275 HPISKVRSVV 6.000 364 SQKGVKGLPL 4.000 92
SGGENRVQVL 4.000 429 QCNSSSDGKL 4.000 449 ITYFKTMPDL 4.000 556
GLPVEKIAKL 4.000 172 EQTQYDVPSL 4.000 538 MLKSPTVKGL 4.000 106
VNLSLNQDHL 4.000 227 TSSGTFQYTL 4.000 362 FSSQKGVKGL 4.000 465
FIPDVHFANL 4.000 597 SMVEGFKVTL 4.000 82 LGTSEAQSNL 4.000 307
RQHTAKQRVL 4.000 214 SASVGAEEYM 3.000 95 ENRVQVLKTV 2.000 158
YPRGDGEEQR 2.000 441 IPLQKKSDIT 2.000 541 SPTVKGLMEA 2.000 344
DVNEEAKIFI 2.000 598 MVEGFKVTLM 1.500 29 TAATKAMMSI 1.200 39
NGDEDSAAAL 1.200 440 AIPLQKKSDI 1.200 19 AWKSYLENPL 1.200 127
FPESSAIIPV 1.200 349 AKIFITVNCL 1.200 416 QNRKKGKGQA 1.000 279
KVRSVVMVVF 1.000 525 YVRKETDDVF 1.000 493 VLVKRMFRPM 1.000 165
EQRVVIFEQT 1.000 97 RVQVLKTVPV 1.000 568 KSKKGILVNM 1.000 589
DTFILNMESM 1.000 489 EGGSVLVKRM 1.000 141 VVKAEDFTPV 1.000 143
KAEDFTPVFM 0.900 521 RVLLYVRKET 0.750 153 APPVHYPRGD 0.600 249
GPMTYLNKGQ 0.600 457 DLHSQPVLFI 0.600 535 DALMLKSPTV 0.600 130
SSAIIPVSGI 0.600 4 DPPFNTRRAY 0.600 132 AIIPVSGITV 0.600 471
FANLQRTGQV 0.600 550 AISEKYGLPV 0.600 177 DVPSLATHSA 0.500 468
DVHFANLQRT 0.500 600 EGFKVTLMEI 0.400 564 KLYKKSKKGI 0.400 309
HTAKQRVLDI 0.400 54 YYKVPRDKRL 0.400 584 HYSNEDTFIL 0.400 255
NKGQFYAITL 0.400 366 KGVKGLPLMI 0.400 434 SDGKLAAIPL 0.400 565
LYKKSKKGIL 0.400 371 LPLMIQIDTY 0.400 528 KETDDVFDAL 0.400 325
FNTIGNIEEI 0.400 233 QYTLEATKSL 0.400 557 LPVEKIAKLY 0.400 178
VPSLATHSAY 0.400 42 EDSAAALGLL 0.400 179 PSLATHSAYL 0.400 432
SSSDGKLAAI 0.400 572 GILVNMDDNI 0.400 573 ILVNMDDNII 0.400 124
SISFPESSAI 0.400 55 YKVPRDKRLL 0.400 253 YLNKGQFYAI 0.400 548
MEAISEKYGL 0.400 116 ENSKREQYSI 0.400 125 ISFPESSAII 0.400 101
LKTVPVNLSL 0.400 271 KCFRHPISKV 0.300 408 KIRDEERKQN 0.300 5
PPFNTRRAYT 0.300 586 SNEDTFILNM 0.300 191 DQRSTPDSTY 0.300 392
RAYCQIKVFC 0.300 596 ESMVEGFKVT 0.300 1 MPSDPPFNTR 0.300 310
TAKQRVLDIA 0.300 131 SAIIPVSGIT 0.300 91 LSGGENRVQV 0.300 206
DAATEKFRSA 0.300 348 EAKIFITVNC 0.300 446 KSDITYFKTM 0.300 529
ETDDVFDALM 0.300 V2-HLA-B7- 10 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 2
SQESDNNKRL 1.200 15 VPMPSDPPFN 1.200 8 NKRLVALVPM 1.000 5
SDNNKRLVAL 0.400 6 DNNKRLVALV 0.200 14 LVPMPSDPPF 0.100 11
LVALVPMPSD 0.075 13 ALVPMPSDPP 0.045 4 ESDNNKRLVA 0.045 12
VALVPMPSDP 0.030 10 RLVALVPMPS 0.020 3 QESDNNKRLV 0.020 16
PMPSDPPFNT 0.015 7 NNKRLVALVP 0.010 1 MSQESDNNKR 0.010 9 KRLVALVPMP
0.001 V4-HLA-B7- 10 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Start Subsequence Score 2 TAATKAMMII
1.200 1 LTAATKAMMI 0.400 3 AATKAMMIIN 0.180 9 MIINGDEDSA 0.100 10
IINGDEDSAA 0.100 6 KAMMIINGDE 0.090 7 AMMIINGDED 0.090 4 ATKAMMIING
0.030 8 MMIINGDEDS 0.020 5 TKAMMIINGD 0.001 V5-HLA-B7- 10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Start Subsequence Score 4 KIRDEEQKQN 0.200 1 AERKIRDEEQ 0.030 9
EQKQNRKKGK 0.010 8 EEQKQNRKKG 0.002 2 ERKIRDEEQK 0.001 10
QKQNRKKGKG 0.001 3 RKIRDEEQKQ 0.001 7 DEEQKQNRKK 0.000 6 RDEEQKQNRK
0.000 5 IRDEEQKQNR 0.000 V5 & 6-HLA-B7- 10 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 3 EQKQNRKNGK 0.010 2 EEQKQNRKNG 0.002 4
QKQNRKNGKG 0.001 1 DEEQKQNRKN 0.001 V6-HLA-B7- 10 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 5 QNRKNGKGQA 1.000 8 KNGKGQASQT 0.100 1
EERKQNRKNG 0.015 4 KQNRKNGKGQ 0.010 6 NRKNGKGQAS 0.002 7 RKNGKGQASQ
0.001 2 ERKQNRKNGK 0.001 3 RKQNRKNGKG 0.001 V8-HLA-B7- 10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus
nine. Start Subsequence Score 1 ALMLKSPTVM 9.000 3 MLKSPTVMGL 4.000
6 SPTVMGLMEA 2.000 8 TVMGLMEAIS 0.300 4 LKSPTVMGLM 0.100 7
PTVMGLMEAI 0.040 2 LMLKSPTVMG 0.015 10 MGLMEAISEK 0.010 9
VMGLMEAISE 0.010 5 KSPTVMGLME 0.010
TABLE-US-00030 TABLE XX V1-HLA-B3501- 9 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score
557 LPVEKIAKL 40.000 292 KNRDEQLKY 36.000 277 ISKVRSVVM 30.000 540
KSPTVKGLM 20.000 585 YSNEDTFIL 15.000 243 RQKQGEGPM 12.000 57
VPRDKRLLS 12.000 215 ASVGAEEYM 10.000 117 NSKREQYSI 9.000 245
KQGEGPMTY 8.000 441 IPLQKKSDI 8.000 214 SASVGAEEY 6.000 46
AALGLLYDY 6.000 443 LQKKSDITY 6.000 466 IPDVHFANL 6.000 386
SNKPIHRAY 6.000 392 RAYCQIKVF 6.000 474 LQRTGQVYY 6.000 494
LVKRMFRPM 6.000 44 SAAALGLLY 6.000 43 DSAAALGLL 5.000 491 GSVLVKRMF
5.000 228 SSGTFQYTL 5.000 363 SSQKGVKGL 5.000 549 EAISEKYGL 4.500
148 TPVFMAPPV 4.000 134 IPVSGITVV 4.000 5 PPFNTRRAY 4.000 1
MPSDPPFNT 4.000 366 KGVKGLPLM 4.000 275 HPISKVRSV 4.000 461
QPVLFIPDV 4.000 143 KAEDFTPVF 3.600 310 TAKQRVLDI 3.600 216
SVGAEEYMY 3.000 56 KVPRDKRLL 3.000 374 MIQIDTYSY 3.000 344
DVNEEAKIF 3.000 100 VLKTVPVNL 3.000 125 ISFPESSAI 3.000 328
IGNIEEIAY 3.000 435 DGKLAAIPL 3.000 568 KSKKGILVN 3.000 168
VVIFEQTQY 3.000 226 QTSSGTFQY 2.000 490 GGSVLVKRM 2.000 350
KIFITVNCL 2.000 371 LPLMIQIDT 2.000 4 DPPFNTRRA 2.000 26 NPLTAATKA
2.000 234 YTLEATKSL 2.000 506 FGPVPSKQM 2.000 323 ESFNTIGNI 2.000
178 VPSLATHSA 2.000 28 LTAATKAMM 2.000 83 GTSEAQSNL 2.000 256
KGQFYAITL 2.000 517 EGTKRVLLY 2.000 47 ALGLLYDYY 2.000 317
DIADYKESF 2.000 473 NLQRTGQVY 2.000 102 KTVPVNLSL 2.000 596
ESMVEGFKV 1.500 193 RSTPDSTYS 1.500 173 QTQYDVPSL 1.500 21
KSYLENPLT 1.500 254 LNKGQFYAI 1.200 30 AATKAMMSI 1.200 269
DNKCFRHPI 1.200 500 RPMEEEFGP 1.200 367 GVKGLPLMI 1.200 455
MPDLHSQPV 1.200 279 KVRSVVMVV 1.200 543 TVKGLMEAI 1.200 131
SAIIPVSGI 1.200 333 EIAYNAVSF 1.000 385 RSNKPIHRA 1.000 107
NLSLNQDHL 1.000 354 TVNCLSTDF 1.000 430 CNSSSDGKL 1.000 251
MTYLNKGQF 1.000 281 RSVVMVVFS 1.000 179 PSLATHSAY 1.000 457
DLHSQPVLF 1.000 139 ITVVKAEDF 1.000 180 SLATHSAYL 1.000 432
SSSDGKLAA 1.000 463 VLFIPDVHF 1.000 187 YLKDDQRST 0.900 348
EAKIFITVN 0.900 525 YVRKETDDV 0.900 158 YPRGDGEEQ 0.900 369
KGLPLMIQI 0.800 431 NSSSDGKLA 0.750 486 DEREGGSVL 0.600 547
LMEAISEKY 0.600 207 AATEKFRSA 0.600 529 ETDDVFDAL 0.600 195
TPDSTYSES 0.600 526 VRKETDDVF 0.600 V2-HLA-B3501- 9 mers-202P5A5
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 15 VPMPSDPPF 20.000 6 DNNKRLVAL 1.000 7 NNKRLVALV
0.600 9 KRLVALVPM 0.400 4 ESDNNKRLV 0.300 3 QESDNNKRL 0.200 11
LVALVPMPS 0.100 1 MSQESDNNK 0.100 12 VALVPMPSD 0.030 10 RLVALVPMP
0.020 16 PMPSDPPFN 0.015 5 SDNNKRLVA 0.010 14 LVPMPSDPP 0.010 13
ALVPMPSDP 0.010 2 SQESDNNKR 0.004 8 NKRLVALVP 0.003 V4-HLA-B3501- 9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 1 TAATKAMMI 1.200 2 AATKAMMII 1.200 3
ATKAMMIIN 0.300 9 IINGDEDSA 0.150 8 MIINGDEDS 0.100 5 KAMMIINGD
0.060 6 AMMIINGDE 0.010 7 MMIINGDED 0.010 4 TKAMMIING 0.001
V5-HLA-B3501- 9 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 3 KIRDEEQKQ 0.180 8
EQKQNRKKG 0.030 4 IRDEEQKQN 0.009 1 ERKIRDEEQ 0.003 2 RKIRDEEQK
0.003 5 RDEEQKQNR 0.001 9 QKQNRKKGK 0.001 7 EEQKQNRKK 0.001 6
DEEQKQNRK 0.000 V5 & 6-HLA- B3501-9 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 2
EQKQNRKNG 0.030 1 EEQKQNRKN 0.010 3 QKQNRKNGK 0.001 V6-HLA-B3501- 9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Start Subsequence Score 9 NGKGQASQT 0.300 5 QNRKNGKGQ 0.030 6
NRKNGKGQA 0.030 1 EERKQNRKN 0.030 7 RKNGKGQAS 0.020 8 KNGKGQASQ
0.020 4 KQNRKNGKG 0.020 2 ERKQNRKNG 0.003 3 RKQNRKNGK 0.002
V8-HLA-B3501- 9 mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Start Subsequence Score 4 KSPTVMGLM 20.000
1 LMLKSPTVM 2.000 7 TVMGLMEAI 0.400 5 SPTVMGLME 0.200 3 LKSPTVMGL
0.100 8 VMGLMEAIS 0.100 2 MLKSPTVMG 0.030 9 MGLMEAISE 0.015 6
PTVMGLMEA 0.010
TABLE-US-00031 TABLE XXI V1-HLA-B3501-1 10 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 557 LPVEKIAKLY 80.000 568 KSKKGILVNM 60.000 178
VPSLATHSAY 40.000 4 DPPFNTRRAY 40.000 371 LPLMIQIDTY 40.000 26
NPLTAATKAM 40.000 57 VPRDKRLLSV 24.000 500 RPMEEEFGPV 24.000 213
RSASVGAEEY 20.000 385 RSNKPIHRAY 20.000 312 KQRVLDIADY 18.000 388
KPIHRAYCQI 16.000 215 ASVGAEEYMY 15.000 43 DSAAALGLLY 10.000 143
KAEDFTPVFM 7.200 45 AAALGLLYDY 6.000 292 KNRDEQLKYW 6.000 455
MPDLHSQPVL 6.000 46 AALGLLYDYY 6.000 214 SASVGAEEYM 6.000 279
KVRSVVMVVF 6.000 14 TSEDEAWKSY 6.000 195 TPDSTYSESF 6.000 167
RVVIFEQTQY 6.000 446 KSDITYFKTM 6.000 191 DQRSTPDSTY 6.000 227
TSSGTFQYTL 5.000 362 FSSQKGVKGL 5.000 11 RAYTSEDEAW 4.500 443
LQKKSDITYF 4.500 432 SSSDGKLAAI 4.000 275 HPISKVRSVV 4.000 245
KQGEGPMTYL 4.000 546 GLMEAISEKY 4.000 538 MLKSPTVKGL 3.000 373
LMIQIDTYSY 3.000 364 SQKGVKGLPL 3.000 277 ISKVRSVVMV 3.000 525
YVRKETDDVF 3.000 327 TIGNIEEIAY 3.000 264 LSETGDNKCF 2.250 497
RMFRPMEEEF 2.000 307 RQHTAKQRVL 2.000 593 LNMESMVEGF 2.000 541
SPTVKGLMEA 2.000 473 NLQRTGQVYY 2.000 465 FIPDVHFANL 2.000 441
IPLQKKSDIT 2.000 589 DTFILNMESM 2.000 472 ANLQRTGQVY 2.000 597
SMVEGFKVTL 2.000 251 MTYLNKGQFY 2.000 92 SGGENRVQVL 2.000 489
EGGSVLVKRM 2.000 225 DQTSSGTFQY 2.000 130 SSAIIPVSGI 2.000 125
ISFPESSAII 2.000 493 VLVKRMFRPM 2.000 408 KIRDEERKQN 1.800 172
EQTQYDVPSL 1.500 117 NSKREQYSIS 1.500 334 IAYNAVSFTW 1.500 529
ETDDVFDALM 1.200 29 TAATKAMMSI 1.200 513 QMKEEGTKRV 1.200 586
SNEDTFILNM 1.200 576 NMDDNIIEHY 1.200 127 FPESSAIIPV 1.200 82
LGTSEAQSNL 1.000 106 VNLSLNQDHL 1.000 490 GGSVLVKRMF 1.000 429
QCNSSSDGKL 1.000 353 ITVNCLSTDF 1.000 21 KSYLENPLTA 1.000 91
LSGGENRVQV 1.000 449 ITYFKTMPDL 1.000 556 GLPVEKIAKL 1.000 585
YSNEDTFILN 1.000 138 GITVVKAEDF 1.000 71 DSQEDQEKRN 1.000 99
QVLKTVPVNL 1.000 141 VVKAEDFTPV 0.900 243 RQKQGEGPMT 0.900 310
TAKQRVLDIA 0.900 348 EAKIFITVNC 0.900 366 KGVKGLPLMI 0.800 564
KLYKKSKKGI 0.800 344 DVNEEAKIFI 0.800 108 LSLNQDHLEN 0.750 36
MSINGDEDSA 0.750 598 MVEGFKVTLM 0.600 116 ENSKREQYSI 0.600 124
SISFPESSAI 0.600 392 RAYCQIKVFC 0.600 114 HLENSKREQY 0.600 95
ENRVQVLKTV 0.600 203 SFKDAATEKF 0.600 207 AATEKFRSAS 0.600 158
YPRGDGEEQR 0.600 118 SKREQYSISF 0.600 V2-HLA- B3501-10 mers-202P5A5
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 15 VPMPSDPPFN 3.000 14 LVPMPSDPPF 1.000 8
NKRLVALVPM 0.600 2 SQESDNNKRL 0.300 6 DNNKRLVALV 0.200 10
RLVALVPMPS 0.200 4 ESDNNKRLVA 0.150 1 MSQESDNNKR 0.150 5 SDNNKRLVAL
0.100 3 QESDNNKRLV 0.040 12 VALVPMPSDP 0.030 7 NNKRLVALVP 0.030 13
ALVPMPSDPP 0.010 11 LVALVPMPSD 0.010 16 PMPSDPPFNT 0.010 9
KRLVALVPMP 0.002 V4-HLA- B3501-10 mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Start Subsequence Score 2
TAATKAMMII 1.200 1 LTAATKAMMI 0.400 3 AATKAMMIIN 0.300 9 MIINGDEDSA
0.150 10 IINGDEDSAA 0.150 8 MMIINGDEDS 0.100 6 KAMMIINGDE 0.060 4
ATKAMMIING 0.030 7 AMMIINGDED 0.010 5 TKAMMIINGD 0.001 V5-HLA-
B3501-10 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Start Subsequence Score 4 KIRDEEQKQN 1.800 9 EQKQNRKKGK
0.030 2 ERKIRDEEQK 0.005 1 AERKIRDEEQ 0.003 3 RKIRDEEQKQ 0.003 6
RDEEQKQNRK 0.001 8 EEQKQNRKKG 0.001 10 QKQNRKKGKG 0.001 5
IRDEEQKQNR 0.001 7 DEEQKQNRKK 0.000 V5 & 6-HLA- B3501-10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Start Subsequence Score 3 EQKQNRKNGK 0.030 1 DEEQKQNRKN 0.003 4
QKQNRKNGKG 0.001 2 EEQKQNRKNG 0.001 V6-HLA- B3501-10 mers-202P5A5
Each peptide is a portionof SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 5 QNRKNGKGQA 0.300 8 KNGKGQASQT 0.200 6
NRKNGKGQAS 0.030 4 KQNRKNGKGQ 0.020 1 EERKQNRKNG 0.003 2 ERKQNRKNGK
0.003 7 RKNGKGQASQ 0.002 3 RKQNRKNGKG 0.002 V8-HLA- B3501-10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus
nine. Start Subsequence Score 3 MLKSPTVMGL 3.000 1 ALMLKSPTVM 2.000
6 SPTVMGLMEA 2.000 4 LKSPTVMGLM 0.200 8 TVMGLMEAIS 0.100 5
KSPTVMGLME 0.100 7 PTVMGLMEAI 0.040 9 VMGLMEAISE 0.015 10
MGLMEAISEK 0.010 2 LMLKSPTVMG 0.010
Tables XXII-XLIX:
TABLE-US-00032 [0954] TABLE XXII V1-HLA- A1-9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 15 SEDEAWKSY 27 547 LMEAISEKY 27 558 PVEKIAKLY 27
44 SAAALGLLY 26 577 MDDNIIEHY 25 517 EGTKRVLLY 24 226 QTSSGTFQY 23
292 KNRDEQLKY 23 2 PSDPPFNTR 22 551 ISEKYGLPV 22 386 SNKPIHRAY 21
529 ETDDVFDAL 21 586 SNEDTFILN 21 359 STDFSSQKG 20 102 KTVPVNLSL 19
179 PSLATHSAY 19 214 SASVGAEEY 19 328 IGNIEEIAY 19 443 LQKKSDITY 19
533 VFDALMLKS 19 84 TSEAQSNLS 18 208 ATEKFRSAS 18 216 SVGAEEYMY 18
264 LSETGDNKC 18 372 PLMIQIDTY 18 473 NLQRTGQVY 18 46 AALGLLYDY 17
47 ALGLLYDYY 17 69 ASDSQEDQE 17 150 VFMAPPVHY 17 175 QYDVPSLAT 17
245 KQGEGPMTY 17 295 DEQLKYWKY 17 433 SSDGKLAAI 17 474 LQRTGQVYY 17
483 NTDDEREGG 17 5 PPFNTRRAY 16 14 TSEDEAWKS 16 58 PRDKRLLSV 16 115
LENSKREQY 16 168 VVIFEQTQY 16 192 QRSTPDSTY 16 200 YSESFKDAA 16 252
TYLNKGQFY 16 288 FSEDKNRDE 16 446 KSDITYFKT 16 587 NEDTFILNM 16 119
KREQYSISF 15 127 FPESSAIIP 15 182 ATHSAYLKD 15 313 QRVLDIADY 15 345
VNEEAKIFI 15 374 MIQIDTYSY 15 487 EREGGSVLV 15 23 YLENPLTAA 14 72
SQEDQEKRN 14 162 DGEEQRVVI 14 315 VLDIADYKE 14 432 SSSDGKLAA 14 518
GTKRVLLYV 14 576 NMDDNIIEH 14 75 DQEKRNCLG 13 188 LKDDQRSTP 13 204
FKDAATEKF 13 230 GTFQYTLEA 13 235 TLEATKSLR 13 289 SEDKNRDEQ 13 321
YKESFNTIG 13 400 FCDKGAERK 13 466 IPDVHFANL 13 501 PMEEEFGPV 13 568
KSKKGILVN 13 V2-HLA- A1-9 mers-202P5A5 Each peptide is a portion of
SEQ ID NO: 5; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 4 ESDNNKRLV 16 2
SQESDNNKR 15 5 SDNNKRLVA 10 8 NKRLVALVP 9 V4-HLA- A1-9 mers-202P5A5
Each peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 3 ATKAMMIIN 11 V5-HLA- A1-9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 1 YLENPLTAA 14 19 DEDSAAALG 12 9 ATKAMMIIN 11 17
NGDEDSAAA 10 18 GDEDSAAAL 10 6 LTAATKAMM 7 20 EDSAAALGL 6 21
DSAAALGLL 6 V5 & 6- HLA-A1-9 mers- 202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 1
EEQKQNRKN 5 V6-HLA- A1-9 mers-202P5A5 Each peptide is a portion of
SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 EERKQNRKN 5 4
KQNRKNGKG 3 9 NGKGQASQT 3 6 NRKNGKGQA 2 7 RKNGKGQAS 2 8 KNGKGQASQ 2
V8-HLA- A1-9 mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 5 SPTVMGLME 10 4 KSPTVMGLM
8 6 PTVMGLMEA 8 3 LKSPTVMGL 5 9 MGLMEAISE 4 V1-HLA- A0201-9 mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 536 ALMLKSPTV 27 180 SLATHSAYL 26 49 GLLYDYYKV
25 90 NLSGGENRV 25 350 KIFITVNCL 25 234 YTLEATKSL 23 557 LPVEKIAKL
23 100 VLKTVPVNL 22 131 SAIIPVSGI 22 133 IIPVSGITV 22 326 NTIGNIEEI
22 23 YLENPLTAA 21 591 FILNMESMV 21 102 KTVPVNLSL 20
107 NLSLNQDHL 20 173 QTQYDVPSL 20 433 SSDGKLAAI 20 546 GLMEAISEK 20
573 ILVNMDDNI 20 598 MVEGFKVTL 20 37 SINGDEDSA 19 151 FMAPPVHYP 19
253 YLNKGQFYA 19 275 HPISKVRSV 19 279 KVRSVVMVV 19 518 GTKRVLLYV 19
132 AIIPVSGIT 18 134 IPVSGITVV 18 187 YLKDDQRST 18 278 SKVRSVVMV 18
363 SSQKGVKGL 18 525 YVRKETDDV 18 539 LKSPTVKGL 18 83 GTSEAQSNL 17
272 CFRHPISKV 17 337 NAVSFTWDV 17 522 VLLYVRKET 17 585 YSNEDTFIL 17
597 SMVEGFKVT 17 46 AALGLLYDY 16 63 LLSVSKASD 16 92 SGGENRVQV 16 98
VQVLKTVPV 16 109 SLNQDHLEN 16 124 SISFPESSA 16 222 YMYDQTSSG 16 228
SSGTFQYTL 16 230 GTFQYTLEA 16 310 TAKQRVLDI 16 367 GVKGLPLMI 16 458
LHSQPVLFI 16 461 QPVLFIPDV 16 472 ANLQRTGQV 16 501 PMEEEFGPV 16 538
MLKSPTVKG 16 554 KYGLPVEKI 16 574 LVNMDDNII 16 24 LENPLTAAT 15 30
AATKAMMSI 15 43 DSAAALGLL 15 45 AAALGLLYD 15 56 KVPRDKRLL 15 96
NRVQVLKTV 15 142 VKAEDFTPV 15 241 SLRQKQGEG 15 276 PISKVRSVV 15 334
IAYNAVSFT 15 352 FITVNCLST 15 370 GLPLMIQID 15 438 LAAIPLQKK 15 453
KTMPDLHSQ 15 463 VLFIPDVHF 15 464 LFIPDVHFA 15 523 LLYVRKETD 15 543
TVKGLMEAI 15 549 EAISEKYGL 15 576 NMDDNIIEH 15 28 LTAATKAMM 14 40
GDEDSAAAL 14 50 LLYDYYKVP 14 58 PRDKRLLSV 14 93 GGENRVQVL 14 125
ISFPESSAI 14 126 SFPESSAII 14 169 VIFEQTQYD 14 207 AATEKFRSA 14 315
VLDIADYKE 14 330 NIEEIAYNA 14 369 KGLPLMIQI 14 372 PLMIQIDTY 14 437
KLAAIPLQK 14 466 IPDVHFANL 14 514 MKEEGTKRV 14 564 KLYKKSKKG 14 601
GFKVTLMEI 14 22 SYLENPLTA 13 47 ALGLLYDYY 13 55 YKVPRDKRL 13 61
KRLLSVSKA 13 136 VSGITVVKA 13 161 GDGEEQRVV 13 254 LNKGQFYAI 13 256
KGQFYAITL 13 259 FYAITLSET 13 317 DIADYKESF 13 331 IEEIAYNAV 13 341
FTWDVNEEA 13 374 MIQIDTYSY 13 389 PIHRAYCQI 13 391 HRAYCQIKV 13 441
IPLQKKSDI 13 486 DEREGGSVL 13 521 RVLLYVRKE 13 529 ETDDVFDAL 13 542
PTVKGLMEA 13 550 AISEKYGLP 13 551 ISEKYGLPV 13 561 KIAKLYKKS 13 569
SKKGILVNM 13 581 IIEHYSNED 13 592 ILNMESMVE 13
TABLE-US-00033 TABLE XXIII V2-HLA- A0201-9mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 6 DNNKRLVAL 17 7 NNKRLVALV 16 10 RLVALVPMP 16 13
ALVPMPSDP 14 9 KRLVALVPM 13 12 VALVPMPSD 11 3 QESDNNKRL 10 5
SDNNKRLVA 9 11 LVALVPMPS 8 V4-HLA- A0201-9mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 9 IINGDEDSA 19 1 TAATKAMMI 15 2 AATKAMMII 13 5
KAMMIINGD 12 7 MMIINGDED 12 8 MIINGDEDS 12 6 AMMIINGDE 11 V5-HLA-
A0201-9mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 3 KIRDEEQKQ 12 V5&6-
HLA-A0201-9mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 3 QKQNRKNGK 2 V6-HLA- A0201-9mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 8 KNGKGQASQ 7 4 KQNRKNGKG 5 6 NRKNGKGQA 5 7
RKNGKGQAS 5 9 NGKGQASQT 5 V8-HLA- A0201-9mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 3
LKSPTVMGL 19 7 TVMGLMEAI 17 1 LMLKSPTVM 16 2 MLKSPTVMG 14 6
PTVMGLMEA 13 8 VMGLMEAIS 11
TABLE-US-00034 TABLE XXIV V1-HLA- A0203-9mers- 202P5A5 Pos
123456789 score NoResultsFound. V2-HLA- A0203-9mers- 202P5A5 Pos
123456789 score NoResultsFound. V3-HLA- A0203-9mers- 202P5A5 Pos
123456789 score NoResultsFound. V4-HLA- A0203-9mers- 202P5A5 Pos
123456789 score NoResultsFound. V5-HLA- A0203-9mers- 202P5A5 Pos
123456789 score NoResultsFound. V5&6- HLA-A0203-9mers- 202P5A5
Pos 123456789 score NoResultsFound. V6-HLA- A0203-9mers- 202P5A5
Pos 123456789 score NoResultsFound. V8-HLA- A0203-9mers- 202P5A5
Pos 123456789 score NoResultsFound.
TABLE-US-00035 TABLE XXV V1-HLA- A3-9mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 437
KLAAIPLQK 33 135 PVSGITVVK 30 314 RVLDIADYK 30 473 NLQRTGQVY 26 263
TLSETGDNK 24 546 GLMEAISEK 24 168 VVIFEQTQY 23 488 REGGSVLVK 23 279
KVRSVVMVV 22 333 EIAYNAVSF 22 532 DVFDALMLK 22 25 ENPLTAATK 21 232
FQYTLEATK 21 372 PLMIQIDTY 21 462 PVLFIPDVH 21 553 EKYGLPVEK 21 556
GLPVEKIAK 21 47 ALGLLYDYY 20 60 DKRLLSVSK 20 216 SVGAEEYMY 20 407
RKIRDEERK 20 468 DVHFANLQR 20 492 SVLVKRMFR 20 523 LLYVRKETD 20 598
MVEGFKVTL 20 50 LLYDYYKVP 19 97 RVQVLKTVP 19 132 AIIPVSGIT 19 149
PVFMAPPVH 19 167 RVVIFEQTQ 19 235 TLEATKSLR 19 271 KCFRHPISK 19 380
YSYNNRSNK 19 457 DLHSQPVLF 19 463 VLFIPDVHF 19 536 ALMLKSPTV 19 558
PVEKIAKLY 19 563 AKLYKKSKK 19 62 RLLSVSKAS 18 94 GENRVQVLK 18 99
QVLKTVPVN 18 245 KQGEGPMTY 18 297 QLKYWKYWH 18 344 DVNEEAKIF 18 367
GVKGLPLMI 18 512 KQMKEEGTK 18 520 KRVLLYVRK 18 537 LMLKSPTVK 18 564
KLYKKSKKG 18 100 VLKTVPVNL 17 133 IIPVSGITV 17 141 VVKAEDFTP 17 203
SFKDAATEK 17 276 PISKVRSVV 17 338 AVSFTWDVN 17 358 LSTDFSSQK 17 374
MIQIDTYSY 17 390 IHRAYCQIK 17 414 RKQNRKKGK 17 521 RVLLYVRKE 17 592
ILNMESMVE 17 44 SAAALGLLY 16 53 DYYKVPRDK 16 56 KVPRDKRLL 16 81
CLGTSEAQS 16 241 SLRQKQGEG 16 292 KNRDEQLKY 16 317 DIADYKESF 16 392
RAYCQIKVF 16 479 QVYYNTDDE 16 486 DEREGGSVL 16 560 EKIAKLYKK 16 562
IAKLYKKSK 16 V2-HLA- A3-9mers-202P5A5 Each peptide is a portion of
SEQ ID NO: 5; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 13 ALVPMPSDP 20
10 RLVALVPMP 17 8 NKRLVALVP 15 11 LVALVPMPS 15 1 MSQESDNNK 11 5
SDNNKRLVA 11 14 LVPMPSDPP 10 9 KRLVALVPM 9 V4-HLA- A3-9mers-202P5A5
Each peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 8 MIINGDEDS 14 9 IINGDEDSA 14 1 TAATKAMMI 7 7
MMIINGDED 7 3 ATKAMMIIN 6 V5-HLA- A3-9mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 3
ENPLTAATK 21 14 MIINGDEDS 14 15 IINGDEDSA 14 1 YLENPLTAA 13 5
PLTAATKAM 13 V5&6-HLA- A3-9mers-202P5A5
Each peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight Pos
123456789 score 3 QKQNRKNGK 15 V6-HLA- A3-9mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 3 RKQNRKNGK 17 9 NGKGQASQT 12 7 RKNGKGQAS 11 8
KNGKGQASQ 10 4 KQNRKNGKG 8 5 QNRKNGKGQ 8 6 NRKNGKGQA 8 V8-HLA- Each
peptide is a A3-9mers-202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 2 MLKSPTVMG 16 7 TVMGLMEAI
13 1 LMLKSPTVM 8 5 SPTVMGLME 8 9 MGLMEAISE 7
TABLE-US-00036 TABLE XXVI V1-HLA- A26-9mers-202P5A5 Each peptide is
a portion of SEQ ID NO:3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 344
DVNEEAKIF 28 529 ETDDVFDAL 28 532 DVFDALMLK 27 517 EGTKRVLLY 26 168
VVIFEQTQY 25 333 EIAYNAVSF 24 102 KTVPVNLSL 23 549 EAISEKYGL 23 216
SVGAEEYMY 22 317 DIADYKESF 22 531 DDVFDALML 22 558 PVEKIAKLY 22 43
DSAAALGLL 21 74 EDQEKRNCL 21 139 ITVVKAEDF 21 173 QTQYDVPSL 21 583
EHYSNEDTF 21 589 DTFILNMES 21 177 DVPSLATHS 20 226 QTSSGTFQY 20 266
ETGDNKCFR 20 282 SVVMVVFSE 20 295 DEQLKYWKY 20 350 KIFITVNCL 20 354
TVNCLSTDF 20 468 DVHFANLQR 20 486 DEREGGSVL 20 557 LPVEKIAKL 20 560
EKIAKLYKK 20 16 EDEAWKSYL 19 42 EDSAAALGL 19 83 GTSEAQSNL 19 145
EDFTPVFMA 19 234 YTLEATKSL 19 251 MTYLNKGQF 19 290 EDKNRDEQL 19 323
ESFNTIGNI 19 504 EEFGPVPSK 19 516 EEGTKRVLL 19 598 MVEGFKVTL 19 56
KVPRDKRLL 18 313 QRVLDIADY 18 353 ITVNCLSTD 18 588 EDTFILNME 18 86
EAQSNLSGG 17 103 TVPVNLSLN 17 326 NTIGNIEEI 17 378 DTYSYNNRS 17 435
DGKLAAIPL 17 457 DLHSQPVLF 17 77 EKRNCLGTS 16 95 ENRVQVLKT 16 129
ESSAIIPVS 16 237 EATKSLRQK 16 398 KVFCDKGAE 16 489 EGGSVLVKR 16 542
PTVKGLMEA 16 577 MDDNIIEHY 16 600 EGFKVTLME 16 5 PPFNTRRAY 15 46
AALGLLYDY 15 105 PVNLSLNQD 15 140 TVVKAEDFT 15 164 EEQRVVIFE 15 167
RVVIFEQTQ 15 202 ESFKDAATE 15 210 EKFRSASVG 15 279 KVRSVVMVV 15 285
MVVFSEDKN 15 286 VVFSEDKNR 15 372 PLMIQIDTY 15 374 MIQIDTYSY 15 386
SNKPIHRAY 15 521 RVLLYVRKE 15 15 SEDEAWKSY 14 44 SAAALGLLY 14 99
QVLKTVPVN 14 149 PVFMAPPVH 14 165 EQRVVIFEQ 14 332 EEIAYNAVS 14 347
EEAKIFITV 14 348 EAKIFITVN 14 448 DITYFKTMP 14 453 KTMPDLHSQ 14 55
YKVPRDKRL 13 115 LENSKREQY 13 121 EQYSISFPE 13 230 GTFQYTLEA 13 245
KQGEGPMTY 13 252 TYLNKGQFY 13 292 KNRDEQLKY 13 314 RVLDIADYK 13 363
SSQKGVKGL 13 367 GVKGLPLMI 13 444 QKKSDITYF 13 450 TYFKTMPDL 13 491
GSVLVKRMF 13 505 EFGPVPSKQ 13 508 PVPSKQMKE 13 518 GTKRVLLYV 13 539
LKSPTVKGL 13 543 TVKGLMEAI 13 547 LMEAISEKY 13 579 DNIIEHYSN 13 594
NMESMVEGF 13 V2-HLA- A26-9mers-202P545 Each peptide is a portion of
SEQ ID NO: 5; each start position is specified, the length of
peptide is 9 amino acids, peptide is the and the end position for
each start position plus eight. Pos 123456789 score 6 DNNKRLVAL 21
4 ESDNNKRLV 11 3 QESDNNKRL 10
10 RLVALVPMP 10 11 LVALVPMPS 10 14 LVPMPSDPP 10 V4-HLA-
A26-9mers-202P545 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 3 ATKAMMIIN 9 8 MIINGDEDS 8 4
TKAMMIING 6 5 KAMMIINGD 6 7 MMIINGDED 6 9 IINGDEDSA 5 V5-HLA-
A26-9mers-202P545 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight Pos 123456789 score 21 DSAAALGLL 21 20 EDSAAALGL 19 3
ENPLTAATK 11 18 GDEDSAAAL 10 V5&6- HLA-A26-9mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight Pos
123456789 score 1 EEQKQNRKN 11 2 EQKQNRKNG 11 V6-HLA-
A26-9mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 1 EERKQNRKN 11 2 ERKQNRKNG 11
V8-HLA- A26-9mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 6 PTVMGLMEA 16 3 LKSPTVMGL
13 7 TVMGLMEAI 13
TABLE-US-00037 TABLE XXVII V1-HLA- B0702-9mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 466 IPDVHFANL 24 1 MPSDPPFNT 21 557 LPVEKIAKL 21
134 IPVSGITVV 19 178 VPSLATHSA 19 148 TPVFMAPPV 18 455 MPDLHSQPV 18
4 DPPFNTRRA 17 26 NPLTAATKA 17 275 HPISKVRSV 17 441 IPLQKKSDI 17
461 QPVLFIPDV 17 42 EDSAAALGL 16 246 QGEGPMTYL 16 371 LPLMIQIDT 16
57 VPRDKRLLS 15 158 YPRGDGEEQ 15 365 QKGVKGLPL 15 458 LHSQPVLFI 15
516 EEGTKRVLL 15 102 KTVPVNLSL 14 486 DEREGGSVL 14 515 KEEGTKRVL 14
529 ETDDVFDAL 14 539 LKSPTVKGL 14 598 MVEGFKVTL 14 20 WKSYLENPL 13
153 APPVHYPRG 13 175 QYDVPSLAT 13 509 VPSKQMKEE 13 541 SPTVKGLME 13
5 PPFNTRRAY 12 16 EDEAWKSYL 12 40 GDEDSAAAL 12 43 DSAAALGLL 12 74
EDQEKRNCL 12 83 GTSEAQSNL 12 93 GGENRVQVL 12 95 ENRVQVLKT 12 100
VLKTVPVNL 12 104 VPVNLSLNQ 12 107 NLSLNQDHL 12 144 AEDFTPVFM 12 173
QTQYDVPSL 12 180 SLATHSAYL 12 308 QHTAKQRVL 12 350 KIFITVNCL 12 363
SSQKGVKGL 12 430 CNSSSDGKL 12 450 TYFKTMPDL 12 456 PDLHSQPVL 12 500
RPMEEEFGP 12 531 DDVFDALML 12 551 ISEKYGLPV 12 554 KYGLPVEKI 12 23
YLENPLTAA 11 55 YKVPRDKRL 11 56 KVPRDKRLL 11 124 SISFPESSA 11 145
EDFTPVFMA 11 195 TPDSTYSES 11 211 KFRSASVGA 11 227 TSSGTFQYT 11 228
SSGTFQYTL 11 234 YTLEATKSL 11 249 GPMTYLNKG 11 256 KGQFYAITL 11 272
CFRHPISKV 11 276 PISKVRSVV 11 279 KVRSVVMVV 11 280 VRSVVMVVF 11 290
EDKNRDEQL 11 385 RSNKPIHRA 11 388 KPIHRAYCQ 11 432 SSSDGKLAA 11 433
SSDGKLAAI 11 435 DGKLAAIPL 11 463 VLFIPDVHF 11 487 EREGGSVLV 11 507
GPVPSKQMK 11 549 EAISEKYGL 11 567 KKSKKGILV 11 585 YSNEDTFIL 11
V2-HLA- B0702-9mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 5; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 15 VPMPSDPPF 20 6
DNNKRLVAL 14 3 QESDNNKRL 12 5 SDNNKRLVA 10 9 KRLVALVPM 10 V4-HLA-
B0702-9mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 2 AATKAMMII 9 9 IINGDEDSA 9 1
TAATKAMMI 7 V5-HLA- B0702-9mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight.
Pos 123456789 score 4 NPLTAATKA 17 20 EDSAAALGL 16 18 GDEDSAAAL 12
21 DSAAALGLL 12 1 YLENPLTAA 11 2 LENPLTAAT 10 5 PLTAATKAM 9 6
LTAATKAMM 9 8 AATKAMMII 9 15 IINGDEDSA 9 16 INGDEDSAA 9 17
NGDEDSAAA 9 V5&6-HLA- B0702-9mers- 202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 1
EEQKQNRKN 2 2 EQKQNRKNG 2 V6-HLA- B0702-9mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 9
NGKGQASQT 8 6 NRKNGKGQA 6 7 RKNGKGQAS 5 1 EERKQNRKN 4 5 QNRKNGKGQ 4
8 KNGKGQASQ 4 V8-HLA- B0702-9mers-202P5A Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 3 LKSPTVMGL 14 5
SPTVMGLME 13 7 TVMGLMEAI 10 1 LMLKSPTVM 9 4 KSPTVMGLM 7 2 MLKSPTVMG
6 6 PTVMGLMEA 6 V1-HLA- B08-9mers-202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, position for each peptide is the plus
eight. Pos 123456789 score 290 EDKNRDEQL 30 566 YKKSKKGIL 30 310
TAKQRVLDI 29 557 LPVEKIAKL 29 100 VLKTVPVNL 28 241 SLRQKQGEG 25 74
EDQEKRNCL 24 57 VPRDKRLLS 23 441 IPLQKKSDI 23 516 EEGTKRVLL 23 277
ISKVRSVVM 21 365 QKGVKGLPL 21 406 ERKIRDEER 21 418 RKKGKGQAS 21 435
DGKLAAIPL 21 93 GGENRVQVL 20 308 QHTAKQRVL 20 55 YKVPRDKRL 19 433
SSDGKLAAI 19 526 VRKETDDVF 19 541 SPTVKGLME 19 601 GFKVTLMEI 19 139
ITVVKAEDF 18 180 SLATHSAYL 18 209 TEKFRSASV 18 275 HPISKVRSV 18 320
DYKESFNTI 18 466 IPDVHFANL 18 486 DEREGGSVL 18 107 NLSLNQDHL 17 163
GEEQRVVIF 17 185 SAYLKDDQR 17 254 LNKGQFYAI 17 297 QLKYWKYWH 17 348
EAKIFITVN 17 367 GVKGLPLMI 17 416 QNRKKGKGQ 17 444 QKKSDITYF 17 536
ALMLKSPTV 17 538 MLKSPTVKG 17 543 TVKGLMEAI 17 562 IAKLYKKSK 17 564
KLYKKSKKG 17 565 LYKKSKKGI 17 117 NSKREQYSI 16 187 YLKDDQRST 16 207
AATEKFRSA 16 269 DNKCFRHPI 16 318 IADYKESFN 16 401 CDKGAERKI 16 404
GAERKIRDE 16 442 PLQKKSDIT 16 549 EAISEKYGL 16 550 AISEKYGLP 16 350
KIFITVNCL 15 396 QIKVFCDKG 15 493 VLVKRMFRP 15 523 LLYVRKETD 15 29
TAATKAMMS 14 333 EIAYNAVSF 14 363 SSQKGVKGL 14 388 KPIHRAYCQ 14 463
VLFIPDVHF 14
TABLE-US-00038 TABLE XXVIII V2- HLA-B08-9mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 6
DNNKRLVAL 20 15 VPMPSDPPF 13 3 QESDNNKRL 12 5 SDNNKRLVA 12 7
NNKRLVALV 11 V4- HLA-B08-9mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 TAATKAMMI 20 2
AATKAMMII 10 3 ATKAMMIIN 10 V5- HLA-B08-9mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 7
TAATKAMMI 20 18 GDEDSAAAL 12 20 EDSAAALGL 12 8 AATKAMMII 10 9
ATKAMMIIN 10 21 DSAAALGLL 10 V5&6- HLA-B08-9mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 2 EQKQNRKNG 13 3 QKQNRKNGK 8 V6- HLA-B08-9mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 2 ERKQNRKNG 13 4 KQNRKNGKG 11 7 RKNGKGQAS 11 6
NRKNGKGQA 10 9 NGKGQASQT 10 1 EERKQNRKN 9 3 RKQNRKNGK 8 5 QNRKNGKGQ
7 V8- HLA-B08-9mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 2 MLKSPTVMG 17 3 LKSPTVMGL
11 5 SPTVMGLME 9
TABLE-US-00039 TABLE XXIX V1-HLA- B1510-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 308 QHTAKQRVL 24 583 EHYSNEDTF 19 515 KEEGTKRVL 17
598 MVEGFKVTL 16 55 YKVPRDKRL 15 93 GGENRVQVL 15 274 RHPISKVRS 15
40 GDEDSAAAL 14 113 DHLENSKRE 14 246 QGEGPMTYL 14 450 TYFKTMPDL 14
458 LHSQPVLFI 14 486 DEREGGSVL 14 516 EEGTKRVLL 14 539 LKSPTVKGL 14
16 EDEAWKSYL 13 56 KVPRDKRLL 13 74 EDQEKRNCL 13 83 GTSEAQSNL 13 100
VLKTVPVNL 13 277 ISKVRSVVM 13 280 VRSVVMVVF 13 363 SSQKGVKGL 13 456
PDLHSQPVL 13 466 IPDVHFANL 13 469 VHFANLQRT 13 529 ETDDVFDAL 13 42
EDSAAALGL 12 102 KTVPVNLSL 12 107 NLSLNQDHL 12 156 VHYPRGDGE 12 163
GEEQRVVIF 12 173 QTQYDVPSL 12 183 THSAYLKDD 12 234 YTLEATKSL 12 304
WHSRQHTAK 12 390 IHRAYCQIK 12 490 GGSVLVKRM 12 549 EAISEKYGL 12 557
LPVEKIAKL 12 566 YKKSKKGIL 12 585 YSNEDTFIL 12 20 WKSYLENPL 11 43
DSAAALGLL 11 143 KAEDFTPVF 11 180 SLATHSAYL 11 228 SSGTFQYTL 11 256
KGQFYAITL 11 290 EDKNRDEQL 11 333 EIAYNAVSF 11 350 KIFITVNCL 11 365
QKGVKGLPL 11 430 CNSSSDGKL 11 V2-HLA- B1510-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, length of peptide is 9 amino acids, and the end position
for each peptide is the start position plus eight. Pos 123456789
score 3 QESDNNKRL 15 6 DNNKRLVAL 14 9 KRLVALVPM 9 15 VPMPSDPPF 9
V4-HLA- B1510-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 5; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 4 TKAMMIING 3 9 IINGDEDSA
3 1 TAATKAMMI 2 2 AATKAMMII 2 7 MMIINGDED 2 3 ATKAMMIIN 1 5
KAMMIINGD 1 8 MIINGDEDS 1 V5-HLA- B1510-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start the position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 6 DEEQKQNRK 4 7 EEQKQNRKK 4 8 EQKQNRKKG 4 4
IRDEEQKQN 3 5 RDEEQKQNR 3 1 ERKIRDEEQ 2 3 KIRDEEQKQ 1 9 QKQNRKKGK 1
V5 & 6- HLA-B1510-9 mers- 202P5A5 Each peptide is a portion of
SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 EEQKQNRKK 4 2
EQKQNRKNG 4 V6-HLA- B1510-9 mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 EERKQNRKN 4 2
ERKQNRKNG 4 5 QNRKNGKGQ 2 6 NRKNGKGQA 2 7 RKNGKGQAS 2 8 KNGKGQASQ 2
3 RKQNRKNGK 1 9 NGKGQASQT 1 V8-HLA- B1510-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 3 LKSPTVMGL 14 1 LMLKSPTVM 10 4 KSPTVMGLM 7
TABLE-US-00040 TABLE XXX V1-HLA- B2705-9 mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 520
KRVLLYVRK 28 119 KREQYSISF 27 273 FRHPISKVR 24 313 QRVLDIADY 24 159
PRGDGEEQR 23 280 VRSVVMVVF 23 526 VRKETDDVF 23 192 QRSTPDSTY 22 306
SRQHTAKQR 22 384 NRSNKPIHR 22 406 ERKIRDEER 22 61 KRLLSVSKA 20 392
RAYCQIKVF 20 488 REGGSVLVK 20 83 GTSEAQSNL 19 294 RDEQLKYWK 19 504
EEFGPVPSK 19 546 GLMEAISEK 19 557 LPVEKIAKL 19 102 KTVPVNLSL 18 314
RVLDIADYK 18 407 RKIRDEERK 18 410 RDEERKQNR 18 537 LMLKSPTVK 18 563
AKLYKKSKK 18 94 GENRVQVLK 17 163 GEEQRVVIF 17 271 KCFRHPISK 17 286
VVFSEDKNR 17 350 KIFITVNCL 17 366 KGVKGLPLM 17 400 FCDKGAERK 17 456
PDLHSQPVL 17 491 GSVLVKRMF 17 507 GPVPSKQMK 17 553 EKYGLPVEK 17 560
EKIAKLYKK 17 3 SDPPFNTRR 16 40 GDEDSAAAL 16 46 AALGLLYDY 16 78
KRNCLGTSE 16 89 SNLSGGENR 16 96 NRVQVLKTV 16 112 QDHLENSKR 16 168
VVIFEQTQY 16 185 SAYLKDDQR 16 243 RQKQGEGPM 16 292 KNRDEQLKY 16 411
DEERKQNRK 16 417 NRKKGKGQA 16 450 TYFKTMPDL 16 463 VLFIPDVHF 16 487
EREGGSVLV 16 512 KQMKEEGTK 16 513 QMKEEGTKR 16 515 KEEGTKRVL 16 556
GLPVEKIAK 16 10 RRAYTSEDE 15 48 LGLLYDYYK 15 53 DYYKVPRDK 15 55
YKVPRDKRL 15 58 PRDKRLLSV 15 60 DKRLLSVSK 15 70 SDSQEDQEK 15 71
DSQEDQEKR 15 93 GGENRVQVL 15 135 PVSGITVVK 15 232 FQYTLEATK 15 237
EATKSLRQK 15 245 KQGEGPMTY 15 246 QGEGPMTYL 15 251 MTYLNKGQF 15 256
KGQFYAITL 15 299 KYWKYWHSR 15 363 SSQKGVKGL 15 377 IDTYSYNNR 15 380
YSYNNRSNK 15 391 HRAYCQIKV 15 395 CQIKVFCDK 15 412 EERKQNRKK 15 414
RKQNRKKGK 15 437 KLAAIPLQK 15 444 QKKSDITYF 15 486 DEREGGSVL 15 489
EGGSVLVKR 15 490 GGSVLVKRM 15 532 DVFDALMLK 15 549 EAISEKYGL 15 569
SKKGILVNM 15 598 MVEGFKVTL 15 2 PSDPPFNTR 14 25 ENPLTAATK 14 100
VLKTVPVNL 14 125 ISFPESSAI 14 139 ITVVKAEDF 14 143 KAEDFTPVF 14 176
YDVPSLATH 14 203 SFKDAATEK 14 228 SSGTFQYTL 14 234 YTLEATKSL 14 308
QHTAKQRVL 14 323 ESFNTIGNI 14 326 NTIGNIEEI 14 354 TVNCLSTDF 14 369
KGLPLMIQI 14 409 IRDEERKQN 14 413 ERKQNRKKG 14 430 CNSSSDGKL 14 438
LAAIPLQKK 14 441 IPLQKKSDI 14 443 LQKKSDITY 14 445 KKSDITYFK 14 466
IPDVHFANL 14 475 QRTGQVYYN 14 480 VYYNTDDER 14 492 SVLVKRMFR 14 496
KRMFRPMEE 14 519 TKRVLLYVR 14 562 IAKLYKKSK 14 587 NEDTFILNM 14 54
YYKVPRDKR 13 56 KVPRDKRLL 13 74 EDQEKRNCL 13 107 NLSLNQDHL 13 111
NQDHLENSK 13 149 PVFMAPPVH 13 152 MAPPVHYPR 13 166 QRVVIFEQT 13 173
QTQYDVPSL 13 181 LATHSAYLK 13 224 YDQTSSGTF 13 248 EGPMTYLNK 13 263
TLSETGDNK 13 265 SETGDNKCF 13 295 DEQLKYWKY 13 333 EIAYNAVSF 13 344
DVNEEAKIF 13 358 LSTDFSSQK 13 367 GVKGLPLMI 13 399 VFCDKGAER 13 435
DGKLAAIPL 13 468 DVHFANLQR 13 474 LQRTGQVYY 13 531 DDVFDALML 13 554
KYGLPVEKI 13 576 NMDDNIIEH 13 583 EHYSNEDTF 13 V2-HLA- B2705-9
mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 5; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight
Pos 123456789 score 9 KRLVALVPM 24 1 MSQESDNNK 16 2 SQESDNNKR 14 6
DNNKRLVAL 14 15 VPMPSDPPF 14 3 QESDNNKRL 13 V4-HLA- B2705-9 mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 2 AATKAMMII 10 1 TAATKAMMI 9 5 KAMMIINGD 7 4
TKAMMIING 6 8 MIINGDEDS 5 9 IINGDEDSA 4 V5-HLA- B2705-9 mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 5 RDEEQKQNR 18 6 DEEQKQNRK 18 2 RKIRDEEQK 17 4
IRDEEQKQN 16 7 EEQKQNRKK 15 1 ERKIRDEEQ 12 9 QKQNRKKGK 11 V5 &
6-HLA- B2705-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids,
and the end position for each peptide is the start position plus
eight. Pos 123456789 score 3 QKQNRKNGK 12 1 EEQKQNRKN 5 V6-HLA-
B2705-9 mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 3 RKQNRKNGK 16 2 ERKQNRKNG 14 6
NRKNGKGQA 14 8 KNGKGQASQ 10 7 RKNGKGQAS 7 V8-HLA- B2705-9 mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 1 LMLKSPTVM 16 3 LKSPTVMGL 13 4 KSPTVMGLM 11 7
TVMGLMEAI 9
TABLE-US-00041 TABLE XXXI V1-HLA- B2709-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 119 KREQYSISF 20 58 PRDKRLLSV 19 96 NRVQVLKTV 19
280 VRSVVMVVF 19 487 EREGGSVLV 19 391 HRAYCQIKV 18 526 VRKETDDVF 18
49 GLLYDYYKV 15 61 KRLLSVSKA 15 102 KTVPVNLSL 15 350 KIFITVNCL 15
520 KRVLLYVRK 15 10 RRAYTSEDE 14 40 GDEDSAAAL 14 83 GTSEAQSNL 14 93
GGENRVQVL 14 369 KGLPLMIQI 14 392 RAYCQIKVF 14 531 DDVFDALML 14 55
YKVPRDKRL 13 56 KVPRDKRLL 13 160 RGDGEEQRV 13 163 GEEQRVVIF 13 166
QRVVIFEQT 13 173 QTQYDVPSL 13 256 KGQFYAITL 13 307 RQHTAKQRV 13 450
TYFKTMPDL 13 456 PDLHSQPVL 13 475 QRTGQVYYN 13 490 GGSVLVKRM 13 549
EAISEKYGL 13 42 EDSAAALGL 12 78 KRNCLGTSE 12 100 VLKTVPVNL 12 125
ISFPESSAI 12 234 YTLEATKSL 12 243 RQKQGEGPM 12 273 FRHPISKVR 12 279
KVRSVVMVV 12 306 SRQHTAKQR 12 313 QRVLDIADY 12 366 KGVKGLPLM 12 367
GVKGLPLMI 12 409 IRDEERKQN 12 466 IPDVHFANL 12 472 ANLQRTGQV 12 491
GSVLVKRMF 12 496 KRMFRPMEE 12 515 KEEGTKRVL 12 518 GTKRVLLYV 12 539
LKSPTVKGL 12 134 IPVSGITVV 11 139 ITVVKAEDF 11 143 KAEDFTPVF 11 148
TPVFMAPPV 11 159 PRGDGEEQR 11 161 GDGEEQRVV 11 212 FRSASVGAE 11 228
SSGTFQYTL 11 251 MTYLNKGQF 11 275 HPISKVRSV 11 278 SKVRSVVMV 11 290
EDKNRDEQL 11 308 QHTAKQRVL 11 323 ESFNTIGNI 11 360 TDFSSQKGV 11 363
SSQKGVKGL 11 365 QKGVKGLPL 11 417 NRKKGKGQA 11 430 CNSSSDGKL 11 435
DGKLAAIPL 11 441 IPLQKKSDI 11 463 VLFIPDVHF 11 516 EEGTKRVLL 11 554
KYGLPVEKI 11 557 LPVEKIAKL 11 566 YKKSKKGIL 11 569 SKKGILVNM 11 573
ILVNMDDNI 11 583 EHYSNEDTF 11 585 YSNEDTFIL 11 598 MVEGFKVTL 11 601
GFKVTLMEI 11 9 TRRAYTSED 10 16 EDEAWKSYL 10 20 WKSYLENPL 10 30
AATKAMMSI 10 43 DSAAALGLL 10 74 EDQEKRNCL 10 92 SGGENRVQV 10 98
VQVLKTVPV 10 107 NLSLNQDHL 10 131 SAIIPVSGI 10 144 AEDFTPVFM 10 170
IFEQTQYDV 10 180 SLATHSAYL 10 192 QRSTPDSTY 10 215 ASVGAEEYM 10 242
LRQKQGEGP 10 246 QGEGPMTYL 10 265 SETGDNKCF 10 277 ISKVRSVVM 10 293
NRDEQLKYW 10 310 TAKQRVLDI 10 333 EIAYNAVSF 10 337 NAVSFTWDV 10 384
NRSNKPIHR 10 406 ERKIRDEER 10 413 ERKQNRKKG 10 444 QKKSDITYF 10 457
DLHSQPVLF 10 458 LHSQPVLFI 10 461 QPVLFIPDV 10 486 DEREGGSVL 10 499
FRPMEEEFG 10 529 ETDDVFDAL 10 536 ALMLKSPTV 10 540 KSPTVKGLM 10 551
ISEKYGLPV 10 567 KKSKKGILV 10 587 NEDTFILNM 10 591 FILNMESMV 10 594
NMESMVEGF 10 27 PLTAATKAM 9 90 NLSGGENRV 9 117 NSKREQYSI 9 128
PESSAIIPV 9 133 IIPVSGITV 9 142 VKAEDFTPV 9 162 DGEEQRVVI 9 254
LNKGQFYAI 9 326 NTIGNIEEI 9 331 IEEIAYNAV 9 343 WDVNEEAKI 9 344
DVNEEAKIF 9 347 EEAKIFITV 9 389 PIHRAYCQI 9 447 SDITYFKTM 9 498
MFRPMEEEF 9 506 FGPVPSKQM 9 514 MKEEGTKRV 9 574 LVNMDDNII 9 590
TFILNMESM 9 599 VEGFKVTLM 9 V2-HLA- B2709-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 9 KRLVALVPM 23 3 QESDNNKRL 11 6 DNNKRLVAL 11
V4-HLA- B2709-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 2 AATKAMMII 10 1 TAATKAMMI
9 V5-HLA- B2709-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 4 IRDEEQKQN 12 1 ERKIRDEEQ
10 2 RKIRDEEQK 7 5 RDEEQKQNR 5 V5 & 6- HLA-B2709-9 mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 2 EQKQNRKNG 1 V6-HLA- B2709-9 mers- 202P5A5
Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 2
ERKQNRKNG 11 6 NRKNGKGQA 11 3 RKQNRKNGK 4 7 RKNGKGQAS 4 V8-HLA-
B2709-9 mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. Pos 123456789 score 3 LKSPTVMGL 12 1 LMLKSPTVM 10 4
KSPTVMGLM 10 7 TVMGLMEAI 8
TABLE-US-00042 TABLE XXXII V1-HLA- B4402-9 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 516 EEGTKRVLL 26 15 SEDEAWKSY 24 265 SETGDNKCF 24
515 KEEGTKRVL 24 163 GEEQRVVIF 23 115 LENSKREQY 22 295 DEQLKYWKY 22
486 DEREGGSVL 21 386 SNKPIHRAY 18 5 PPFNTRRAY 17 326 NTIGNIEEI 17
529 ETDDVFDAL 17 539 LKSPTVKGL 17 587 NEDTFILNM 17 24 LENPLTAAT 16
46 AALGLLYDY 16 55 YKVPRDKRL 16 144 AEDFTPVFM 16 323 ESFNTIGNI 16
332 EEIAYNAVS 16 347 EEAKIFITV 16 392 RAYCQIKVF 16 504 EEFGPVPSK 16
517 EGTKRVLLY 16 577 MDDNIIEHY 16 12 AYTSEDEAW 15 42 EDSAAALGL 15
56 KVPRDKRLL 15 102 KTVPVNLSL 15 125 ISFPESSAI 15 128 PESSAIIPV 15
164 EEQRVVIFE 15 247 GEGPMTYLN 15 290 EDKNRDEQL 15 293 NRDEQLKYW 15
296 EQLKYWKYW 15 322 KESFNTIGN 15 333 EIAYNAVSF 15 350 KIFITVNCL 15
369 KGLPLMIQI 15 372 PLMIQIDTY 15 405 AERKIRDEE 15 549 EAISEKYGL 15
558 PVEKIAKLY 15 40 GDEDSAAAL 14 47 ALGLLYDYY 14 74 EDQEKRNCL 14 93
GGENRVQVL 14 107 NLSLNQDHL 14 143 KAEDFTPVF 14 168 VVIFEQTQY 14 201
SESFKDAAT 14 219 AEEYMYDQT 14 234 YTLEATKSL 14 256 KGQFYAITL 14 289
SEDKNRDEQ 14 280 VRSVVMVVF 13 328 IGNIEEIAY 13 331 IEEIAYNAV 13 344
DVNEEAKIF 13 435 DGKLAAIPL 13 444 QKKSDITYF 13 457 DLHSQPVLF 13 463
VLFIPDVHF 13 473 NLQRTGQVY 13 498 MFRPMEEEF 13 528 KETDDVFDA 13 552
SEKYGLPVE 13 598 MVEGFKVTL 13 16 EDEAWKSYL 12 20 WKSYLENPL 12 43
DSAAALGLL 12 73 QEDQEKRNC 12 85 SEAQSNLSG 12 119 KREQYSISF 12 171
FEQTQYDVP 12 180 SLATHSAYL 12 196 PDSTYSESF 12 224 YDQTSSGTF 12 236
LEATKSLRQ 12 252 TYLNKGQFY 12 254 LNKGQFYAI 12 308 QHTAKQRVL 12 411
DEERKQNRK 12 292 KNRDEQLKY 14 313 QRVLDIADY 14 335 AYNAVSFTW 14 346
NEEAKIFIT 14 363 SSQKGVKGL 14 412 EERKQNRKK 14 433 SSDGKLAAI 14 488
REGGSVLVK 14 503 EEEFGPVPS 14 557 LPVEKIAKL 14 583 EHYSNEDTF 14 41
DEDSAAALG 13 44 SAAALGLLY 13 76 QEKRNCLGT 13 94 GENRVQVLK 13 100
VLKTVPVNL 13 120 REQYSISFP 13 131 SAIIPVSGI 13 150 VFMAPPVHY 13 179
PSLATHSAY 13 192 QRSTPDSTY 13 204 FKDAATEKF 13 214 SASVGAEEY 13 220
EEYMYDQTS 13 226 QTSSGTFQY 13 228 SSGTFQYTL 13 245 KQGEGPMTY 13 251
MTYLNKGQF 13 430 CNSSSDGKL 12 439 AAIPLQKKS 12 443 LQKKSDITY 12 450
TYFKTMPDL 12 456 PDLHSQPVL 12 466 IPDVHFANL 12 474 LQRTGQVYY 12 491
GSVLVKRMF 12 531 DDVFDALML 12 547 LMEAISEKY 12 548 MEAISEKYG 12 554
KYGLPVEKI 12 594 NMESMVEGF 12 599 VEGFKVTLM 12 V2-HLA- B4402-9
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 5; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 3 QESDNNKRL 25 6 DNNKRLVAL 15 15 VPMPSDPPF 14
V4-HLA- B4402-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 2 AATKAMMII 11 1 TAATKAMMI
10 5 KAMMIINGD 7 6 AMMIINGDE 5 V5-HLA- B4402-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 7 EEQKQNRKK 14 6 DEEQKQNRK 11 8 EQKQNRKKG 7 V5
& 6HLA-B4402- 9 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 9 amino acids, and the end position for each peptide is the
start position plus eight. Pos 123456789 score 1 EEQKQNRKN 14 2
EQKQNRKNG 6 V6HLA- B4402-9 mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 EERKQNRKN 14 2
ERKQNRKNG 6 V8HLA- B4402-9 mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 3 LKSPTVMGL 15 7 TVMGLMEAI 12
TABLE-US-00043 TABLE XXXIIII V1-HLA-B5101-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 162 DGEEQRVVI 26 134 IPVSGITVV 25 441 IPLQKKSDI 25
310 TAKQRVLDI 24 557 LPVEKIAKL 24 131 SAIIPVSGI 22 275 HPISKVRSV 22
30 AATKAMMSI 21 148 TPVFMAPPV 21 320 DYKESFNTI 21 337 NAVSFTWDV 19
369 KGLPLMIQI 19 392 RAYCQIKVF 19 455 MPDLHSQPV 19 461 QPVLFIPDV 19
466 IPDVHFANL 19 26 NPLTAATKA 18 334 IAYNAVSFT 18 435 DGKLAAIPL 18
4 DPPFNTRRA 17 92 SGGENRVQV 17 93 GGENRVQVL 17 256 KGQFYAITL 17 11
RAYTSEDEA 16 160 RGDGEEQRV 16 206 DAATEKFRS 16 249 GPMTYLNKG 16 535
DALMLKSPT 16 549 EAISEKYGL 16 96 NRVQVLKTV 15 133 IIPVSGITV 15 185
SAYLKDDQR 15 207 AATEKFRSA 15 260 YAITLSETG 15 279 KVRSVVMVV 15 348
EAKIFITVN 15 371 LPLMIQIDT 15 382 YNNRSNKPI 15 438 LAAIPLQKK 15 458
LHSQPVLFI 15 486 DEREGGSVL 15 565 LYKKSKKGI 15 18 EAWKSYLEN 14 29
TAATKAMMS 14 46 AALGLLYDY 14 57 VPRDKRLLS 14 126 SFPESSAII 14 127
FPESSAIIP 14 143 KAEDFTPVF 14 152 MAPPVHYPR 14 161 GDGEEQRVV 14 181
LATHSAYLK 14 237 EATKSLRQK 14 246 QGEGPMTYL 14 254 LNKGQFYAI 14 269
DNKCFRHPI 14 276 PISKVRSVV 14 345 VNEEAKIFI 14 401 CDKGAERKI 14 554
KYGLPVEKI 14 562 IAKLYKKSK 14 1 MPSDPPFNT 13 5 PPFNTRRAY 13 33
KAMMSINGD 13 53 DYYKVPRDK 13 125 ISFPESSAI 13 142 VKAEDFTPV 13 154
PPVHYPRGD 13 158 YPRGDGEEQ 13 218 GAEEYMYDQ 13 234 YTLEATKSL 13 318
IADYKESFN 13 323 ESFNTIGNI 13 326 NTIGNIEEI 13 347 EEAKIFITV 13 367
GVKGLPLMI 13 404 GAERKIRDE 13 433 SSDGKLAAI 13 439 AAIPLQKKS 13 509
VPSKQMKEE 13 514 MKEEGTKRV 13 551 ISEKYGLPV 13 555 YGLPVEKIA 13 573
ILVNMDDNI 13 574 LVNMDDNII 13 43 DSAAALGLL 12 44 SAAALGLLY 12 45
AAALGLLYD 12 49 GLLYDYYKV 12 50 LLYDYYKVP 12 68 KASDSQEDQ 12 90
NLSGGENRV 12 100 VLKTVPVNL 12 104 VPVNLSLNQ 12 137 SGITVVKAE 12 153
APPVHYPRG 12 195 TPDSTYSES 12 278 SKVRSVVMV 12 308 QHTAKQRVL 12 343
WDVNEEAKI 12 360 TDFSSQKGV 12 378 DTYSYNNRS 12 456 PDLHSQPVL 12 471
FANLQRTGQ 12 485 DDEREGGSV 12 489 EGGSVLVKR 12 500 RPMEEEFGP 12 515
KEEGTKRVL 12 518 GTKRVLLYV 12 531 DDVFDALML 12 539 LKSPTVKGL 12 541
SPTVKGLME 12 543 TVKGLMEAI 12 591 FILNMESMV 12 601 GFKVTLMEI 12 V2-
HLA-B5101-9 mers- 202P5A5 Each peptide is a portion of SEQ ID NO:
5; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. Pos 123456789 score 6 DNNKRLVAL 13 12
VALVPMPSD 13 15 VPMPSDPPF 12 7 NNKRLVALV 11 3 QESDNNKRL 10 4
ESDNNKRLV 9 8 NKRLVALVP 8 V4- HLA-B5101-9 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 1 TAATKAMMI 24 2 AATKAMMII 21 5 KAMMIINGD 12 V5-
HLA-B5101-9 mers- 202P5A5 NO: 3; each start Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. Pos 123456789 score 6
DEEQKQNRK 7 4 IRDEEQKQN 5 7 EEQKQNRKK 5 3 KIRDEEQKQ 3 8 EQKQNRKKG 3
V5 & 6- HLA-B5101-9 mers- 202P5A5 Each peptide is a portion of
SEQ ID NO: 3; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. Pos 123456789 score 1 EEQKQNRKN 5
V6- HLA-B5101-9 mers- 202P5A5 Each peptide is a portion of SEQ ID
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
Pos 123456789 score 9 NGKGQASQT 9 1 EERKQNRKN 5 4 KQNRKNGKG 4 V8-
HLA-B5101-9 mers- 202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 5 SPTVMGLME 12 7 TVMGLMEAI 12 1 LMLKSPTVM 11 3
LKSPTVMGL 11 9 MGLMEAISE 11
TABLE-US-00044 TABLE XXXIV V1-HLA- A1-10mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 14
TSEDEAWKSY 29 43 DSAAALGLLY 28 114 HLENSKREQY 27 294 RDEQLKYWKY 27
576 NMDDNIIEHY 25 516 EEGTKRVLLY 24 291 DKNRDEQLKY 23 215
ASVGAEEYMY 22 251 MTYLNKGQFY 22 84 TSEAQSNLSG 21 442 PLQKKSDITY 20
2 PSDPPFNTRR 19 213 RSASVGAEEY 19 327 TIGNIEEIAY 19 385 RSNKPIHRAY
19 487 EREGGSVLVK 19 359 STDFSSQKGV 18 472 ANLQRTGQVY 18 529
ETDDVFDALM 18 546 GLMEAISEKY 18 586 SNEDTFILNM 18 46 AALGLLYDYY 17
208 ATEKFRSASV 17 235 TLEATKSLRQ 17 346 NEEAKIFITV 17 433
SSDGKLAAIP 17 446 KSDITYFKTM 17 530 TDDVFDALML 17 557 LPVEKIAKLY 17
4 DPPFNTRRAY 16 41 DEDSAAALGL 16 45 AAALGLLYDY 16 75 DQEKRNCLGT 16
178 VPSLATHSAY 16 200 YSESFKDAAT 16 244 QKQGEGPMTY 16 288
FSEDKNRDEQ 16 321 YKESFNTIGN 16 473 NLQRTGQVYY 16 483 NTDDEREGGS 16
558 PVEKIAKLYK 16 69 ASDSQEDQEK 15 127 FPESSAIIPV 15 149 PVFMAPPVHY
15 163 GEEQRVVIFE 15 167 RVVIFEQTQY 15 191 DQRSTPDSTY 15 225
DQTSSGTFQY 15 264 LSETGDNKCF 15 312 KQRVLDIADY 15 371 LPLMIQIDTY 15
373 LMIQIDTYSY 15 459 HSQPVLFIPD 15 551 ISEKYGLPVE 15 585
YSNEDTFILN 15 515 KEEGTKRVLL 14 598 MVEGFKVTLM 14 V2-HLA-
A1-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 5; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 4 ESDNNKRLVA 22 2 SQESDNNKRL 13
V4-HLA- A1-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Pos 1234567890 score 4 ATKAMMIING 11 1
LTAATKAMMI 7 3 AATKAMMIIN 5 V5-HLA- A1-10mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 7
DEEQKQNRKK 13 5 IRDEEQKQNR 10 6 RDEEQKQNRK 10 V5&6-
HLA-A1-10mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 1 DEEQKQNRKN 13 V6-HLA-
A1-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 1 DEERKQNRKN 13 V8-HLA-
A1-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 5 KSPTVMGLME 12
4 LKSPTVMGLM 6 7 PTVMGLMEAI 6 9 VMGLMEAISE 5
TABLE-US-00045 TABLE XXXV V1-HLA- A0201-10mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 556 GLPVEKIAKL 29 132 AIIPVSGITV 26 597 SMVEGFKVTL
26 133 IIPVSGITVV 24 169 VIFEQTQYDV 23 253 YLNKGQFYAI 23 538
MLKSPTVKGL 23 550 AISEKYGLPV 23 457 DLHSQPVLFI 22 465 FIPDVHFANL 22
330 NIEEIAYNAV 21 463 VLFIPDVHFA 21 573 ILVNMDDNII 21 564
KLYKKSKKGI 20 57 VPRDKRLLSV 19 208 ATEKFRSASV 19 309 HTAKQRVLDI 19
437 KLAAIPLQKK 19 454 TMPDLHSQPV 19 572 GILVNMDDNI 19 23 YLENPLTAAT
18 37 SINGDEDSAA 18 92 SGGENRVQVL 18 99 QVLKTVPVNL 18 124
SISFPESSAI 18 271 KCFRHPISKV 18 349 AKIFITVNCL 18 432 SSSDGKLAAI 18
440 AIPLQKKSDI 18 513 QMKEEGTKRV 18 535 DALMLKSPTV 18 48 LGLLYDYYKV
17 109 SLNQDHLENS 17 130 SSAIIPVSGI 17 222 YMYDQTSSGT 17 277
ISKVRSVVMV 17 449 ITYFKTMPDL 17 460 SQPVLFIPDV 17 592 ILNMESMVEG 17
29 TAATKAMMSI 16 50 LLYDYYKVPR 16 62 RLLSVSKASD 16 91 LSGGENRVQV 16
97 RVQVLKTVPV 16 147 FTPVFMAPPV 16 274 RHPISKVRSV 16 278 SKVRSVVMVV
16 471 FANLQRTGQV 16 537 LMLKSPTVKG 16 546 GLMEAISEKY 16 22
SYLENPLTAA 15 39 NGDEDSAAAL 15 89 SNLSGGENRV 15 100 VLKTVPVNLS 15
125 ISFPESSAII 15 135 PVSGITVVKA 15 180 SLATHSAYLK 15 227
TSSGTFQYTL 15 230 GTFQYTLEAT 15 263 TLSETGDNKC 15 359 STDFSSQKGV 15
362 FSSQKGVKGL 15 370 GLPLMIQIDT 15 373 LMIQIDTYSY 15 493
VLVKRMFRPM 15 580 NIIEHYSNED 15 15 SEDEAWKSYL 14 55 YKVPRDKRLL 14
101 LKTVPVNLSL 14 127 FPESSAIIPV 14 141 VVKAEDFTPV 14 151
FMAPPVHYPR 14 187 YLKDDQRSTP 14 319 ADYKESFNTI 14 325 FNTIGNIEEI 14
333 EIAYNAVSFT 14 336 YNAVSFTWDV 14 344 DVNEEAKIFI 14 352
FITVNCLSTD 14 364 SQKGVKGLPL 14 390 IHRAYCQIKV 14 484 TDDEREGGSV 14
515 KEEGTKRVLL 14 522 VLLYVRKETD 14 548 MEAISEKYGL 14 553
EKYGLPVEKI 14 V2-HLA- A0201-10mers- 202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 5
SDNNKRLVAL 20 6 DNNKRLVALV 16 13 ALVPMPSDPP 14 3 QESDNNKRLV 11 11
LVALVPMPSD 11 2 SQESDNNKRL 10 8 NKRLVALVPM 10 10 RLVALVPMPS 10 16
PMPSDPPFNT 9 V4-HLA- A0201-10mers- 202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 10
IINGDEDSAA 18 1 LTAATKAMMI 16 9 MIINGDEDSA 16 2 TAATKAMMII 14
7 AMMIINGDED 13 8 MMIINGDEDS 10 4 ATKAMMIING 9 5 TKAMMIINGD 8
V5-HLA- A0201-10mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. Pos 1234567890 score 4 KIRDEEQKQN 9 1
AERKIRDEEQ 4 3 RKIRDEEQKQ 4 5 IRDEEQKQNR 4 V5&6-HLA-
A0201-10mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 4 QKQNRKNGKG 2 V6-HLA-
A0201-10mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 8 RKNGKGQASQ 7 9 KNGKGQASQT 5 6
QNRKNGKGQA 4 10 NGKGQASQTQ 3 V8-HLA- A0201-10mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 3 MLKSPTVMGL 24 1 ALMLKSPTVM 17 2 LMLKSPTVMG 14 6
SPTVMGLMEA 11 9 VMGLMEAISE 11
TABLE-US-00046 TABLE XXXVI V1-HLA- A0203-10mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 38 INGDEDSAAA 27 22 SYLENPLTAA 19 37 SINGDEDSAA 19
199 TYSESFKDAA 19 431 NSSSDGKLAA 19 23 YLENPLTAAT 17 39 NGDEDSAAAL
17 200 YSESFKDAAT 17 432 SSSDGKLAAI 17 V2-HLA- A0203-10mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 5; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Pos 1234567890 score 4 ESDNNKRLVA 10 5 SDNNKRLVAL 9 6 DNNKRLVALV 8
V4-HLA- A0203-10mers- 202P5A5 Each peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. Pos 1234567890 score 10 IINGDEDSAA 19 9
MIINGDEDSA 10 V5-HLA- A0203-10mers-202P5A5 Pos 1234567890 score
NoResultsFound. V5&6-HLA- A0203-10mers-202P5A5 Pos 1234567890
score NoResultsFound. V6-HLA- A0203-10mers- Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 6
QNRKNGKGQA 10 7 NRKNGKGQAS 9 8 RKNGKGASQ 8 V8-HLA- A0203-10mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Pos 1234567890 score 6 SPTVMGLMEA 10 7 PTVMGLMEAI 9 8 TVMGLMEAIS
8
TABLE-US-00047 TABLE XXXVII V1-HLA- A3-10mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 536
ALMLKSPTVK 30 279 KVRSVVMVVF 28 357 CLSTDFSSQK 26 437 KLAAIPLQKK 26
167 RVVIFEQTQY 25 180 SLATHSAYLK 25 47 ALGLLYDYYK 24 59 RDKRLLSVSK
24 132 AIIPVSGITV 24 561 KIAKLYKKSK 24 24 LENPLTAATK 23 50
LLYDYYKVPR 23 398 KVFCDKGAER 23 473 NLQRTGQVYY 23 558 PVEKIAKLYK 23
62 RLLSVSKASD 21 114 HLENSKREQY 21 149 PVFMAPPVHY 21 389 PIHRAYCQIK
21 436 GKLAAIPLQK 21 525 YVRKETDDVF 21 56 KVPRDKRLLS 20 134
IPVSGITVVK 20 187 YLKDDQRSTP 20 283 VVMVVFSEDK 20 442 PLQKKSDITY 20
462 PVLFIPDVHF 20 479 QVYYNTDDER 20 99 QVLKTVPVNL 19 408 KIRDEERKQN
19 487 EREGGSVLVK 19 97 RVQVLKTVPV 18 332 EEIAYNAVSF 18 472
ANLQRTGQVY 18 521 RVLLYVRKET 18 545 KGLMEAISEK 18 550 AISEKYGLPV 18
12 AYTSEDEAWK 17 65 SVSKASDSQE 17 231 TFQYTLEATK 17 241 SLRQKQGEGP
17 262 ITLSETGDNK 17 314 RVLDIADYKE 17 338 AVSFTWDVNE 17 519
TKRVLLYVRK 17 546 GLMEAISEKY 17 552 SEKYGLPVEK 17 562 IAKLYKKSKK 17
564 KLYKKSKKGI 17 21 KSYLENPLTA 16 27 PLTAATKAMM 16 49 GLLYDYYKVP
16 63 LLSVSKASDS 16 90 NLSGGENRVQ 16 135 PVSGITVVKA 16 191
DQRSTPDSTY 16 244 QKQGEGPMTY 16 247 GEGPMTYLNK 16 276 PISKVRSVVM 16
313 QRVLDIADYK 16 327 TIGNIEEIAY 16 494 LVKRMFRPME 16 511
SKQMKEEGTK 16 522 VLLYVRKETD 16 543 TVKGLMEAIS 16 555 YGLPVEKIAK 16
591 FILNMESMVE 16 592 ILNMESMVEG 16 23 YLENPLTAAT 15 37 SINGDEDSAA
15 43 DSAAALGLLY 15 52 YDYYKVPRDK 15 81 CLGTSEAQSN 15 93 GGENRVQVLK
15 105 PVNLSLNQDH 15 133 IIPVSGITVV 15 138 GITVVKAEDF 15 235
TLEATKSLRQ 15 312 KQRVLDIADY 15 352 FITVNCLSTD 15 360 TDFSSQKGVK 15
410 RDEERKQNRK 15 440 AIPLQKKSDI 15 523 LLYVRKETDD 15 140
TVVKAEDFTP 14 141 VVKAEDFTPV 14 202 ESFKDAATEK 14 213 RSASVGAEEY 14
216 SVGAEEYMYD 14 236 LEATKSLRQK 14 251 MTYLNKGQFY 14 282
SVVMVVFSED 14 285 MVVFSEDKNR 14 367 GVKGLPLMIQ 14 376 QIDTYSYNNR 14
379 TYSYNNRSNK 14 399 VFCDKGAERK 14 488 REGGSVLVKR 14 532
DVFDALMLKS 14 V2-HLA- A3-10mers-202P5A5 Each peptide is a portion
of SEQ ID NO: 5; each start position is specified, the length of
peptide is 10 amino acids, and the end position for each peptide is
the start position plus nine. Pos 1234567890 score 10 RLVALVPMPS 18
13 ALVPMPSDPP 16 7 NNKRLVALVP 14 11 LVALVPMPSD 14 14 LVPMPSDPPF 14
4 ESDNNKRLVA 9 8 NKRLVALVPM 8 V4-HLA- A3-10mers-202P5A5 Each
peptide is a portion of SEQ ID
NO: 3; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. Pos 1234567890 score 10 IINGDEDSAA 16 9
MIINGDEDSA 14 1 LTAATKAMMI 8 7 AMMIINGDED 7 V5-HLA-
A3-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 4 KIRDEEQKQN 18 2 ERKIRDEEQK 15 6
RDEEQKQNRK 15 9 EQKQNRKKGK 11 3 RKIRDEEQKQ 10 7 DEEQKQNRKK 10
V5&6- HLA-A3-10mers- 202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Pos 1234567890 score 3 EQKQNRKNGK 11
V6-HLA- A3-10mers-202P5A5 Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. Pos 1234567890 score 9 KNGKGQASQT 12 3
ERKQNRKNGK 11 8 RKNGKGQASQ 11 5 KQNRKNGKGQ 9 6 QNRKNGKGQA 9 10
NGKGQASQTQ 7 4 RIQNRKNGKG 6 7 NRKNGKGQAS 6 V8-HLA-
A3-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each
start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 1 ALMLKSPTVM 20 8 TVMGLMEAIS 15 10
MGLMEAISEK 15 3 MLKSPTVMGL 12 5 KSPTVMGLME 10
TABLE-US-00048 TABLE XXXVIII V1- HLA-A26-10mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 167 RVVIFEQTQY 25 516 EEGTKRVLLY 25 532 DVFDALMLKS
25 149 PVFMAPPVHY 24 172 EQTQYDVPSL 24 42 EDSAAALGLL 23 332
EEIAYNAVSF 23 162 DGEEQRVVIF 22 468 DVHFANLQRT 22 191 DQRSTPDSTY 21
225 DQTSSGTFQY 21 344 DVNEEAKIFI 21 589 DTFILNMESM 21 43 DSAAALGLLY
20 177 DVPSLATHSA 20 291 DKNRDEQLKY 20 353 ITVNCLSTDF 20 4
DPPFNTRRAY 19 251 MTYLNKGQFY 19 266 ETGDNKCFRH 19 279 KVRSVVMVVF 19
333 EIAYNAVSFT 19 529 ETDDVFDALM 19 41 DEDSAAALGL 18 99 QVLKTVPVNL
18 102 KTVPVNLSLN 18 378 DTYSYNNRSN 18 462 PVLFIPDVHF 18 485
DDEREGGSVL 18 525 YVRKETDDVF 18 600 EGFKVTLMEI 18 230 GTFQYTLEAT 17
327 TIGNIEEIAY 17 140 TVVKAEDFTP 16 282 SVVMVVFSED 16 349
AKIFITVNCL 16 449 ITYFKTMPDL 16 504 EEFGPVPSKQ 16 517 EGTKRVLLYV 16
531 DDVFDALMLK 16 549 EAISEKYGLP 16 557 LPVEKIAKLY 16 597
SMVEGFKVTL 16 92 SGGENRVQVL 15 95 ENRVQVLKTV 15 135 PVSGITVVKA 15
145 EDFTPVFMAP 15 215 ASVGAEEYMY 15 248 EGPMTYLNKG 15 286
VVFSEDKNRD 15 323 ESFNTIGNIE 15 371 LPLMIQIDTY 15 373 LMIQIDTYSY 15
398 KVFCDKGAER 15 487 EREGGSVLVK 15 489 EGGSVLVKRM 15 503
EEEFGPVPSK 15 553 EKYGLPVEKI 15 560 EKIAKLYKKS 15 576 NMDDNIIEHY 15
18 EAWKSYLENP 14 45 AAALGLLYDY 14 60 DKRLLSVSKA 14 164 EEQRVVIFEQ
14 168 VVIFEQTQYD 14 202 ESFKDAATEK 14 210 EKFRSASVGA 14 285
MVVFSEDKNR 14 312 KQRVLDIADY 14 317 DIADYKESFN 14 347 EEAKIFITVN 14
367 GVKGLPLMIQ 14 457 DLHSQPVLFI 14 593 LNMESMVEGF 14 598
MVEGFKVTLM 14 14 TSEDEAWKSY 13 55 YKVPRDKRLL 13 65 SVSKASDSQE 13
118 SKREQYSISF 13 138 GITVVKAEDF 13 182 ATHSAYLKDD 13 309
HTAKQRVLDI 13 316 LDIADYKESF 13 448 DITYFKTMPD 13 465 FIPDVHFANL 13
538 MLKSPTVKGL 13 542 PTVKGLMEAI 13 546 GLMEAISEKY 13 556
GLPVEKIAKL 13 579 DNIIEHYSNE 13 15 SEDEAWKSYL 12 74 EDQEKRNCLG 12
77 EKRNCLGTSE 12 86 EAQSNLSGGE 12 103 TVPVNLSLNQ 12 132 AIIPVSGITV
12 139 ITVVKAEDFT 12 178 VPSLATHSAY 12 221 EYMYDQTSSG 12 237
EATKSLRQKQ 12 314 RVLDIADYKE 12 326 NTIGNIEEIA 12 343 WDVNEEAKIF 12
362 FSSQKGVKGL 12 391 HRAYCQIKVF 12 435 DGKLAAIPLQ 12 442
PLQKKSDITY 12 443 LQKKSDITYF 12 497 RMFRPMEEEF 12 505 EFGPVPSKQM 12
518 GTKRVLLYVR 12 V2- HLA-A26-10mers- 202P5A5 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide
is 10 amino acids, and the end position for each peptide is the
start position plus nine. Pos 1234567890 score 14 LVPMPSDPPF 18 5
SDNNKRLVAL 14 6 DNNKRLVALV 12 4 ESDNNKRLVA 11 11 LVALVPMPSD 11 2
SQESDNNKRL 10 V4- HLA-A26-10mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 10 amino acids, and the end position for each peptide is
the start position plus nine. Pos 1234567890 score 4 ATKAMMIING 13
1 LTAATKAMMI 9 9 MIINGDEDSA 9 10 IINGDEDSAA 6 V5- HLA-A26-10mers-
202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Pos 1234567890 score 2 ERKIRDEEQK 11 8 EEQKQNRKKG 11 9 EQKQNRKKGK
10 7 DEEQKQNRKK 9 4 KIRDEEQKQN 6 3 RKIRDEEQKQ 5 V5&6-
HLA-A26-10mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 2 EEQKQNRKNG 10 3 EQKQNRKNGK 10 1
DEEQKQNRKN 9 V6- HLA-A26-10mers- 202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 10 amino acids, and the end position for each peptide is
the start position plus nine. Pos 1234567890 score 2 EERKQNRKNG 10
3 ERKQNRKNGK 10 1 DEERKQNRKN 9 V8HLA- A26-10mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 3 MLKSPTVMGL 13 7 PTVMGLMEAI 13 8 TVMGLMEAIS 11 6
SPTVMGLMEA 6
TABLE-US-00049 TABLE XXXIX V1-HLA- B0702-10mers- 202P5A5 Each
peptide is a portion of SEQ ID NO:3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 455 MPDLHSQPVL 23 57 VPRDKRLLSV 20 500 RPMEEEFGPV
20 5 PPFNTRRAYT 18 26 NPLTAATKAM 18 275 HPISKVRSVV 18 127
FPESSAIIPV 17 195 TPDSTYSESF 17 388 KPIHRAYCQI 17 441 IPLQKKSDIT 17
515 KEEGTKRVLL 16 541 SPTVKGLMEA 16 134 IPVSGITVVK 15 364
SQKGVKGLPL 15 42 EDSAAALGLL 14 158 YPRGDGEEQR 14 227 TSSGTFQYTL 14
1 MPSDPPFNTR 13 19 AWKSYLENPL 13 39 NGDEDSAAAL 13 41 DEDSAAALGL 13
92 SGGENRVQVL 13 99 QVLKTVPVNL 13 135 PVSGITVVKA 13 172 EQTQYDVPSL
13 178 VPSLATHSAY 13 245 KQGEGPMTYL 13 349 AKIFITVNCL 13 362
FSSQKGVKGL 13 434 SDGKLAAIPL 13 509 VPSKQMKEEG 13 528 KETDDVFDAL 13
530 TDDVFDALML 13 550 AISEKYGLPV 13 584 HYSNEDTFIL 13 4 DPPFNTRRAY
12 73 QEDQEKRNCL 12 91 LSGGENRVQV 12 101 LKTVPVNLSL 12 148
TPVFMAPPVH 12 153 APPVHYPRGD 12 179 PSLATHSAYL 12 276 PISKVRSVVM 12
279 KVRSVVMVVF 12 289 SEDKNRDEQL 12 307 RQHTAKQRVL 12 449
ITYFKTMPDL 12 465 FIPDVHFANL 12 466 IPDVHFANLQ 12 597 SMVEGFKVTL 12
15 SEDEAWKSYL 11 55 YKVPRDKRLL 11 97 RVQVLKTVPV 11 154 PPVHYPRGDG
11 160 RGDGEEQRVV 11 161 GDGEEQRVVI 11 233 QYTLEATKSL 11 249
GPMTYLNKGQ 11 255 NKGQFYAITL 11 309 HTAKQRVLDI 11 431 NSSSDGKLAA 11
461 QPVLFIPDVH 11 485 DDEREGGSVL 11 507 GPVPSKQMKE 11 514
MKEEGTKRVL 11 538 MLKSPTVKGL 11 548 MEAISEKYGL 11 556 GLPVEKIAKL 11
565 LYKKSKKGIL 11 V2-HLA- B0702-10mers- 202P545 Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide and the end position for each peptide is the
start position plus nine. Pos 1234567890 score 15 VPMPSDPPFN 14 5
SDNNKRLVAL 13 4 ESDNNKRLVA 12 8 NKRLVALVPM 11 2 SQESDNNKRL 10 6
DNNKRLVALV 9 3 QESDNNKRLV 8 14 LVPMPSDPPF 8 16 PMPSDPPFNT 6 V4-HLA-
B0702-10mers 202P545 Each peptide is a portion of SEQ ID length of
peptide NO: 3; each start is 10 amino acids, position is specified,
the and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 10 IINGDEDSAA 10 1 LTAATKAMMI 8 2
TAATKAMMII 7 9 MIINGDEDSA 6 V5-HLA- B0702-10mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. score
1234567890 score 1 AERKIRDEEQ 5 4 KIRDEEQKQN 3 8 EEQKQNRKKG 3 5
IRDEEQKQNR 2 V5&6-HLA- B0702-10mers-202P5A5 Each peptide is a
portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine.
Pos 1234567890 score 2 EEQKQNRKNG 3 3 EQKQNRKNGK 1 V6-HLA-
B0702-10mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 6 QNRKNGKGQA 9 9 KNGKGQASQT 9 2
EERKQNRKNG 5 8 RKNGKGQASQ 4 V8-HLA- B0702-10mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 6 SPTVMGLMEA 16 1 ALMLKSPTVM 11 3 MLKSPTVMGL 11 4
LKSPTVMGLM 9 7 PTVMGLMEAI 7
TABLE-US-00050 TABLE XL V1-HLA-B08- 10 mers-202P5A5 Pos 1234567890
score No Results Found. V2-HLA-B08- 10 mers-202P5A5 Pos 1234567890
score No Results Found. V4-HLA-B08- 10 mers-202P5A5 Pos 1234567890
score No Results Found. V5-HLA-B08- 10 mers-202P5A5 Pos 1234567890
score No Results Found. V5 & 6-HLA- B08-10 mers-202P5A5 Pos
1234567890 score No Results Found. V6-HLA-B08- 10 mers-202P5A5 Pos
1234567890 score No Results Found. V8-HLA-B08- 10 mers-202P5A5 Pos
1234567890 score No Results Found.
TABLE-US-00051 TABLE XLI V1-HLA- B1510-10 mers- 202P5A5 Pos
1234567890 score No Results Found. V2-HLA- B1510-10 mers- 202P5A5
Pos 1234567890 score No Results Found. V4-HLA- B1510-10 mers-
202P5A5 Pos 1234567890 score No Results Found. V5-HLA- B1510-10
mers- 202P5A5 Pos 1234567890 score No Results Found. V5 &
6-HLA- B1510-10 mers- 202P5A5 Pos 1234567890 score No Results
Found. V6-HLA- B1510-10 mers- 202P5A5 Pos 1234567890 score No
Results Found. V8-HLA- B1510-10 mers- 202P5A5 Pos 1234567890 score
No Results Found.
TABLE-US-00052 TABLE XLII V1-HLA- B2705-10 mers- 202P5A5 Pos
1234567890 score No Results Found. V2-HLA- B2705-10 mers- 202P5A5
Pos 1234567890 score No Results Found. V4-HLA- B2705-10 mers-
202P5A5 Pos 1234567890 score No Results Found. V5-HLA- B2705-10
mers- 202P5A5 Pos 1234567890 score No Results Found. V5 &
6-HLA- B2705-10 mers- 202P5A5 Pos 1234567890 score No Results
Found. V6-HLA- B2705-10 mers- 202P5A5 Pos 1234567890 score No
Results Found. V8-HLA- B2705-10 mers- 202P5A5 Pos 1234567890 score
No Results Found.
TABLE-US-00053 TABLE XLIII V1-HLA- B2709-10 mers- 202P5A5 Pos
1234567890 score No Results Found. V2-HLA- B2709-10 mers- 202P5A5
Pos 1234567890 score No Results Found.
TABLE-US-00054 TABLE XLI V4-HLA- B1510-10 mers- 202P5A5 Pos
1234567890 score No Results Found. V5-HLA- B1510-10 mers- 202P5A5
Pos 1234567890 score No Results Found. V5 & 6-HLA- B1510-10
mers- 202P5A5 Pos 1234567890 score No Results Found. V6-HLA-
B1510-10 mers- 202P5A5 Pos 1234567890 score No Results Found.
V8-HLA- B1510-10 mers- 202P5A5 Pos 1234567890 score No Results
Found.
TABLE-US-00055 TABLE XLIV V1-HLA- B4402-10 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 332 EEIAYNAVSF 27 516 EEGTKRVLLY 26 515 KEEGTKRVLL
25 528 KETDDVFDAL 25 41 DEDSAAALGL 23 73 QEDQEKRNCL 23 289
SEDKNRDEQL 23 295 DEQLKYWKYW 23 322 KESFNTIGNI 23 15 SEDEAWKSYL 22
582 IEHYSNEDTF 21 548 MEAISEKYGL 20 349 AKIFITVNCL 18 504
EEFGPVPSKQ 17 164 EEQRVVIFEQ 16 347 EEAKIFITVN 16 391 HRAYCQIKVF 16
412 EERKQNRKKG 16 576 NMDDNIIEHY 16 4 DPPFNTRRAY 15 24 LENPLTAATK
15 39 NGDEDSAAAL 15 42 EDSAAALGLL 15 45 AAALGLLYDY 15 46 AALGLLYDYY
15 55 YKVPRDKRLL 15 128 PESSAIIPVS 15 144 AEDFTPVFMA 15 292
KNRDEQLKYW 15 371 LPLMIQIDTY 15 472 ANLQRTGQVY 15 556 GLPVEKIAKL 15
587 NEDTFILNME 15 19 AWKSYLENPL 14 94 GENRVQVLKT 14 149 PVFMAPPVHY
14 178 VPSLATHSAY 14 201 SESFKDAATE 14 203 SFKDAATEKF 14 215
ASVGAEEYMY 14 219 AEEYMYDQTS 14 233 QYTLEATKSL 14 236 LEATKSLRQK 14
247 GEGPMTYLNK 14 255 NKGQFYAITL 14 312 KQRVLDIADY 14 319
ADYKESFNTI 14 327 TIGNIEEIAY 14 343 WDVNEEAKIF 14 346 NEEAKIFITV 14
362 FSSQKGVKGL 14 373 LMIQIDTYSY 14 432 SSSDGKLAAI 14 456
PDLHSQPVLF 14 488 REGGSVLVKR 14 557 LPVEKIAKLY 14 11 RAYTSEDEAW 13
14 TSEDEAWKSY 13 76 QEKRNCLGTS 13 92 SGGENRVQVL 13 99 QVLKTVPVNL 13
114 HLENSKREQY 13 125 ISFPESSAII 13 172 EQTQYDVPSL 13 227
TSSGTFQYTL 13 244 QKQGEGPMTY 13 264 LSETGDNKCF 13 279 KVRSVVMVVF 13
291 DKNRDEQLKY 13 307 RQHTAKQRVL 13 316 LDIADYKESF 13 364
SQKGVKGLPL 13 385 RSNKPIHRAY 13 388 KPIHRAYCQI 13 405 AERKIRDEER 13
411 DEERKQNRKK 13 434 SDGKLAAIPL 13 440 AIPLQKKSDI 13 443
LQKKSDITYF 13 455 MPDLHSQPVL 13 465 FIPDVHFANL 13 473 NLQRTGQVYY 13
497 RMFRPMEEEF 13 503 EEEFGPVPSK 13 530 TDDVFDALML 13 538
MLKSPTVKGL 13 552 SEKYGLPVEK 13 553 EKYGLPVEKI 13 584 HYSNEDTFIL 13
597 SMVEGFKVTL 13 600 EGFKVTLMEI 13 V2-HLA- B4402-10 mers- 202P5A5
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 5 SDNNKRLVAL 16 3 QESDNNKRLV 14 2 SQESDNNKRL 13 14
LVPMPSDPPF 11 4 ESDNNKRLVA 7 V4-HLA- B4402-10 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 1 LTAATKAMMI 9 2 TAATKAMMII 9 4 ATKAMMIING 7 3
AATKAMMIIN 4 5 TKAMMIINGD 4 7 AMMIINGDED 4 9 MIINGDEDSA 4 V5-HLA-
B4402-10 mers- 202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. Pos 1234567890 score 8 EEQKQNRKKG 16 1 AERKIRDEEQ 13 7
DEEQKQNRKK 12 V5 & 6-HLA- B4402-10 mers- 202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. Pos 1234567890 score 2
EEQKQNRKNG 15 1 DEEQKQNRKN 12 V6-HLA- B4402-10 mers- 202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 2 EERKQNRKNG 15 1 DEERKQNRKN 13 V8-HLA- B4402-10
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
Pos 1234567890 score 3 MLKSPTVMGL 11 7 PTVMGLMEAI 10 1 ALMLKSPTVM 7
4 LKSPTVMGLM 5
TABLE-US-00056 TABLE XLV V1-HLA- B5101-10 mers- 202P5A5 Pos
1234567890 score No Results Found. V2-HLA- B5101-10 mers- 202P5A5
Pos 1234567890 score No Results Found. V4-HLA- B5101-10 mers-
202P5A5 Pos 1234567890 score No Results Found. V5HLA- B5101-10
mers- 202P5A5 Pos 1234567890 score No Results Found. V5 & 6HLA-
B5101-10 mers- 202P5A5 Pos 1234567890 score No Results Found.
V6HLA- B5101-10 mers- 202P5A5 Pos 1234567890 score No Results
Found. V8HLA- B5101-10 mers- 202P5A5 Pos 1234567890 score No
Results Found.
TABLE-US-00057 TABLE XLVI V1-HLA-DRB1- 0101-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 136 VSGITVVKAEDFTPV 31 352 FITVNCLSTDFSSQK 31
503 EEEFGPVPSKQMKEE 31 95 ENRVQVLKTVPVNLS 30 130 SSAIIPVSGITVVKA 30
175 QYDVPSLATHSAYLK 30 274 RHPISKVRSVVMVVF 30 452 FKTMPDLHSQPVLFI
30 239 TKSLRQKQGEGPMTY 28 4 DPPFNTRRAYTSEDE 27 120 REQYSISFPESSAII
27 230 GTFQYTLEATKSLRQ 27 231 TFQYTLEATKSLRQK 27 531
DDVFDALMLKSPTVK 27 124 SISFPESSAIIPVSG 26 201 SESFKDAATEKFRSA 26
370 GLPLMIQIDTYSYNN 26 432 SSSDGKLAAIPLQKK 26 522 VLLYVRKETDDVFDA
26 144 AEDFTPVFMAPPVHY 25 248 EGPMTYLNKGQFYAI 25 359
STDFSSQKGVKGLPL 25 449 ITYFKTMPDLHSQPV 25 511 SKQMKEEGTKRVLLY 25
541 SPTVKGLMEAISEKY 25 593 LNMESMVEGFKVTLM 25 61 KRLLSVSKASDSQED 24
76 QEKRNCLGTSEAQSN 24 97 RVQVLKTVPVNLSLN 24 122 QYSISFPESSAIIPV 24
221 EYMYDQTSSGTFQYT 24 277 ISKVRSVVMVVFSED 24 499 FRPMEEEFGPVPSKQ
24 548 MEAISEKYGLPVEKI 24 582 IEHYSNEDTFILNME 24 98 VQVLKTVPVNLSLNQ
23 133 IIPVSGITVVKAEDF 23 362 FSSQKGVKGLPLMIQ 23 438
LAAIPLQKKSDITYF 23 460 SQPVLFIPDVHFANL 23 533 VFDALMLKSPTVKGL 23
571 KGILVNMDDNIIEHY 23 32 TKAMMSINGDEDSAA 22 116 ENSKREQYSISFPES 22
167 RVVIFEQTQYDVPSL 22 220 EEYMYDQTSSGTFQY 22 227 TSSGTFQYTLEATKS
22 328 IGNIEEIAYNAVSFT 22 455 MPDLHSQPVLFIPDV 22 463
VLFIPDVHFANLQRT 22 469 VHFANLQRTGQVYYN 22 468 DVHFANLQRTGQVYY 21
562 IAKLYKKSKKGILVN 21 563 AKLYKKSKKGILVNM 21 589 DTFILNMESMVEGFK
21 33 KAMMSINGDEDSAAA 20 52 YDYYKVPRDKRLLSV 20 58 PRDKRLLSVSKASDS
20 138 GITVVKAEDFTPVFM 20 281 RSVVMVVFSEDKNRD 20 313
QRVLDIADYKESFNT 20 340 SFTWDVNEEAKIFIT 20 371 LPLMIQIDTYSYNNR 20
379 TYSYNNRSNKPIHRA 20 394 YCQIKVFCDKGAERK 20 491 GSVLVKRMFRPMEEE
20 523 LLYVRKETDDVFDAL 20 536 ALMLKSPTVKGLMEA 20 552
SEKYGLPVEKIAKLY 20 17 DEAWKSYLENPLTAA 19 20 WKSYLENPLTAATKA 19 23
YLENPLTAATKAMMS 19 51 LYDYYKVPRDKRLLS 19 128 PESSAIIPVSGITVV 19 250
PMTYLNKGQFYAITL 19 251 MTYLNKGQFYAITLS 19 257 GQFYAITLSETGDNK 19
270 NKCFRHPISKVRSVV 19 338 AVSFTWDVNEEAKIF 19 341 FTWDVNEEAKIFITV
19 348 EAKIFITVNCLSTDF 19 397 IKVFCDKGAERKIRD 19 415
KQNRKKGKGQASQTQ 19 461 QPVLFIPDVHFANLQ 19 496 KRMFRPMEEEFGPVP 19
559 VEKIAKLYKKSKKGI 19 587 NEDTFILNMESMVEG 19 588 EDTFILNMESMVEGF
19 11 RAYTSEDEAWKSYLE 18 35 MMSINGDEDSAAALG 18 39 NGDEDSAAALGLLYD
18 131 SAIIPVSGITVVKAE 18 139 ITVVKAEDFTPVFMA 18 148
TPVFMAPPVHYPRGD 18 153 APPVHYPRGDGEEQR 18 155 PVHYPRGDGEEQRVV 18
168 VVIFEQTQYDVPSLA 18 174 TQYDVPSLATHSAYL 18 197 DSTYSESFKDAATEK
18 298 LKYWKYWHSRQHTAK 18 300 YWKYWHSRQHTAKQR 18 322
KESFNTIGNIEEIAY 18 333 EIAYNAVSFTWDVNE 18 448 DITYFKTMPDLHSQP 18 22
SYLENPLTAATKAMM 17 25 ENPLTAATKAMMSIN 17 45 AAALGLLYDYYKVPR 17 47
ALGLLYDYYKVPRDK 17 79 RNCLGTSEAQSNLSG 17 93 GGENRVQVLKTVPVN 17 107
NLSLNQDHLENSKRE 17 185 SAYLKDDQRSTPDST 17 240 KSLRQKQGEGPMTYL 17
259 FYAITLSETGDNKCF 17 295 DEQLKYWKYWHSRQH 17 342 TWDVNEEAKIFITVN
17 364 SQKGVKGLPLMIQID 17 365 QKGVKGLPLMIQIDT 17 428
TQCNSSSDGKLAAIP 17 482 YNTDDEREGGSVLVK 17 483 NTDDEREGGSVLVKR 17
484 TDDEREGGSVLVKRM 17 488 REGGSVLVKRMFRPM 17 530 TDDVFDALMLKSPTV
17 535 DALMLKSPTVKGLME 17 551 ISEKYGLPVEKIAKL 17 554
KYGLPVEKIAKLYKK 17 564 KLYKKSKKGILVNMD 17 595 MESMVEGFKVTLMEI 17 26
NPLTAATKAMMSING 16 37 SINGDEDSAAALGLL 16 54 YYKVPRDKRLLSVSK 16 59
RDKRLLSVSKASDSQ 16 85 SEAQSNLSGGENRVQ 16 94 GENRVQVLKTVPVNL 16 104
VPVNLSLNQDHLENS 16 145 EDFTPVFMAPPVHYP 16 178 VPSLATHSAYLKDDQ 16
206 DAATEKFRSASVGAE 16 208 ATEKFRSASVGAEEY 16 219 AEEYMYDQTSSGTFQ
16 253 YLNKGQFYAITLSET 16 256 KGQFYAITLSETGDN 16 280
VRSVVMVVFSEDKNR 16 307 RQHTAKQRVLDIADY 16 327 TIGNIEEIAYNAVSF 16
330 NIEEIAYNAVSFTWD 16 349 AKIFITVNCLSTDFS 16 351 IFITVNCLSTDFSSQ
16 391 HRAYCQIKVFCDKGA 16 396 QIKVFCDKGAERKIR 16 431
NSSSDGKLAAIPLQK 16 458 LHSQPVLFIPDVHFA 16 490 GGSVLVKRMFRPMEE 16
495 VKRMFRPMEEEFGPV 16 528 KETDDVFDALMLKSP 16 534 FDALMLKSPTVKGLM
16 542 PTVKGLMEAISEKYG 16 556 GLPVEKIAKLYKKSK 16 570
KKGILVNMDDNIIEH 16 586 SNEDTFILNMESMVE 16 1 MPSDPPFNTRRAYTS 15 19
AWKSYLENPLTAATK 15 24 LENPLTAATKAMMSI 15 57 VPRDKRLLSVSKASD 15 147
FTPVFMAPPVHYPRG 15 166 QRVVIFEQTQYDVPS 15 190 DDQRSTPDSTYSESF 15
211 KFRSASVGAEEYMYD 15 312 KQRVLDIADYKESFN 15 325 FNTIGNIEEIAYNAV
15 347 EEAKIFITVNCLSTD 15 368 VKGLPLMIQIDTYSY 15 378
DTYSYNNRSNKPIHR 15 435 DGKLAAIPLQKKSDI 15 446 KSDITYFKTMPDLHS 15
487 EREGGSVLVKRMFRP 15 492 SVLVKRMFRPMEEEF 15 500 RPMEEEFGPVPSKQM
15 544 VKGLMEAISEKYGLP 15 545 KGLMEAISEKYGLPV 15 V2-HLA-DRB1-
0101-15 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 5;
each start position is specified, the length of peptide is 14 amino
acids, and the end position for each peptide is the start position
plus fourteen. Pos 123456789012345 score 8 NKRLVALVPMPSDPP 31 11
LVALVPMPSDPPFNT 29 5 SDNNKRLVALVPMPS 25 6 DNNKRLVALVPMPSD 17 12
VALVPMPSDPPFNTR 17 9 KRLVALVPMPSDPPF 15 V4-HLA-DRB1- 0101-15
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 14 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 10 TKAMMIINGDEDSAA 25 11
KAMMIINGDEDSAAA 20 1 YLENPLTAATKAMMI 19 13 MMIINGDEDSAAALG 18 3
ENPLTAATKAMMIIN 17 4 NPLTAATKAMMIING 16 12 AMMIINGDEDSAAAL 16 15
IINGDEDSAAALGLL 16 2 LENPLTAATKAMMII 15 14 MIINGDEDSAAALGL 14
V5-HLA-DRB1- 0101-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 15 QKQNRKKGKGQASQT 13 7 ERKIRDEEQKQNRKK 12 14 EQKQNRKKGKGQASQ
12 4 KGAERKIRDEEQKQN 10 11 RDEEQKQNRKKGKGQ 10 9 KIRDEEQKQNRKKGK 6
V5 & 6-HLA-DRB1- 0101-15 mers-202P5A5 Each peptide is a portion
of SEQ ID NO: 3; each start position is specified, the length of
peptide is 14 amino acids, and the end position for each peptide is
the start position plus fourteen. Pos 123456789012345 score 9
QKQNRKNGKGQASQT 13 1 ERKIRDEEQKQNRKN 12 8 EQKQNRKNGKGQASQ 12 5
RDEEQKQNRKNGKGQ 9 3 KIRDEEQKQNRKNGK 6 V6-HLA-DRB1- 0101-15
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 14 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 10 KQNRKNGKGQASQTQ 17 9
RKQNRKNGKGQASQT 13 13 RKNGKGQASQTQCNS 13 1 ERKIRDEERKQNRKN 12 8
ERKQNRKNGKGQASQ 12 5 RDEERKQNRKNGKGQ 11 12 NRKNGKGQASQTQCN 10
V8-HLA-DRB1- 0101-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 1
DDVFDALMLKSPTVM 27 4 FDALMLKSPTVMGLM 24 11 SPTVMGLMEAISEKY 24 12
PTVMGLMEAISEKYG 24 3 VFDALMLKSPTVMGL 23 6 ALMLKSPTVMGLMEA 19 5
DALMLKSPTVMGLME 16 9 LKSPTVMGLMEAISE 16 14 VMGLMEAISEKYGLP 15 15
MGLMEAISEKYGLPV 15 8 MLKSPTVMGLMEAIS 14
TABLE-US-00058 TABLE XLVII V1-HLA-DRB1- 0301-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 54 YYKVPRDKRLLSVSK 29 185 SAYLKDDQRSTPDST 27
289 SEDKNRDEQLKYWKY 27 572 GILVNMDDNIIEHYS 27 595 MESMVEGFKVTLMEI
27 165 EQRVVIFEQTQYDVP 26 285 MVVFSEDKNRDEQLK 26 325
FNTIGNIEEIAYNAV 26 471 FANLQRTGQVYYNTD 26 112 QDHLENSKREQYSIS 25
248 EGPMTYLNKGQFYAI 25 314 RVLDIADYKESFNTI 25 338 AVSFTWDVNEEAKIF
25 495 VKRMFRPMEEEFGPV 24 35 MMSINGDEDSAAALG 22 37 SINGDEDSAAALGLL
22 47 ALGLLYDYYKVPRDK 22 97 RVQVLKTVPVNLSLN 22 396 QIKVFCDKGAERKIR
22 460 SQPVLFIPDVHFANL 22 60 DKRLLSVSKASDSQE 21 107 NLSLNQDHLENSKRE
21 147 FTPVFMAPPVHYPRG 21 277 ISKVRSVVMVVFSED 21 462
PVLFIPDVHFANLQR 21 554 KYGLPVEKIAKLYKK 21 136 VSGITVVKAEDFTPV 20
355 VNCLSTDFSSQKGVK 20 372 PLMIQIDTYSYNNRS 20 429 QCNSSSDGKLAAIPL
20 463 VLFIPDVHFANLQRT 20 526 VRKETDDVFDALMLK 20 536
ALMLKSPTVKGLMEA 20 544 VKGLMEAISEKYGLP 20 103 TVPVNLSLNQDHLEN 19
140 TVVKAEDFTPVFMAP 19 282 SVVMVVFSEDKNRDE 19 371 LPLMIQIDTYSYNNR
19 405 AERKIRDEERKQNRK 19 440 AIPLQKKSDITYFKT 19 519
TKRVLLYVRKETDDV 19 523 LLYVRKETDDVFDAL 19 17 DEAWKSYLENPLTAA 18 25
ENPLTAATKAMMSIN 18 65 SVSKASDSQEDQEKR 18 156 VHYPRGDGEEQRVVI 18 184
HSAYLKDDQRSTPDS 18 201 SESFKDAATEKFRSA 18 284 VMVVFSEDKNRDEQL 18
406 ERKIRDEERKQNRKK 18 438 LAAIPLQKKSDITYF 18 480 VYYNTDDEREGGSVL
18 520 KRVLLYVRKETDDVF 18 541 SPTVKGLMEAISEKY 18 574
LVNMDDNIIEHYSNE 18 48 LGLLYDYYKVPRDKR 17 53 DYYKVPRDKRLLSVS 17 69
ASDSQEDQEKRNCLG 17 80 NCLGTSEAQSNLSGG 17 88 QSNLSGGENRVQVLK 17 104
VPVNLSLNQDHLENS 17 167 RVVIFEQTQYDVPSL 17 263 TLSETGDNKCFRHPI 17
315 VLDIADYKESFNTIG 17 342 TWDVNEEAKIFITVN 17 348 EAKIFITVNCLSTDF
17 368 VKGLPLMIQIDTYSY 17 492 SVLVKRMFRPMEEEF 17 496
KRMFRPMEEEFGPVP 17 513 QMKEEGTKRVLLYVR 17 521 RVLLYVRKETDDVFD 17
545 KGLMEAISEKYGLPV 17 556 GLPVEKIAKLYKKSK 17 562 IAKLYKKSKKGILVN
17 579 DNIIEHYSNEDTFIL 17 592 ILNMESMVEGFKVTL 17 71 DSQEDQEKRNCLGTS
16 99 QVLKTVPVNLSLNQD 16 213 RSASVGAEEYMYDQT 16 229 SGTFQYTLEATKSLR
16 453 KTMPDLHSQPVLFIP 16 479 QVYYNTDDEREGGSV 16 559
VEKIAKLYKKSKKGI 16 563 AKLYKKSKKGILVNM 16 588 EDTFILNMESMVEGF 16 2
PSDPPFNTRRAYTSE 15 13 YTSEDEAWKSYLENP 15 305 HSRQHTAKQRVLDIA 15 322
KESFNTIGNIEEIAY 15 512 KQMKEEGTKRVLLYV 15 535 DALMLKSPTVKGLME 15
546 GLMEAISEKYGLPVE 15 98 VQVLKTVPVNLSLNQ 14 225 DQTSSGTFQYTLEAT 14
261 AITLSETGDNKCFRH 14 262 ITLSETGDNKCFRHP 14 468 DVHFANLQRTGQVYY
14 488 REGGSVLVKRMFRPM 14 580 NIIEHYSNEDTFILN 14
TABLE-US-00059 TABLE XLVI V2-HLA-DRB1- 0301-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 14 LVPMPSDPPFNTRRA 23 12 VALVPMPSDPPFNTR 20
11 LVALVPMPSDPPFNT 13 9 KRLVALVPMPSDPPF 12 8 NKRLVALVPMPSDPP 11
V4-HLA-DRB1- 0301-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 13
MMIINGDEDSAAALG 22 15 IINGDEDSAAALGLL 22 3 ENPLTAATKAMMIIN 18 12
AMMIINGDEDSAAAL 17 11 KAMMIINGDEDSAAA 12 10 TKAMMIINGDEDSAA 10
V5-HLA-DRB1- 0301-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 6
AERKIRDEEQKQNRK 20 7 ERKIRDEEQKQNRKK 18 8 RKIRDEEQKQNRKKG 9 9
KIRDEEQKQNRKKGK 9 V5 & 6-HLA-DRB1- 0301-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 1 ERKIRDEEQKQNRKN 18 2 RKIRDEEQKQNRKNG 9 3
KIRDEEQKQNRKNGK 9 6 DEEQKQNRKNGKGQA 8 7 EEQKQNRKNGKGQAS 8 9
QKQNRKNGKGQASQT 8 V6-HLA-DRB1- 0301-15 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 1 ERKIRDEERKQNRKN 18 2 RKIRDEERKQNRKNG 9 3 KIRDEERKQNRKNGK 9
5 RDEERKQNRKNGKGQ 8 6 DEERKQNRKNGKGQA 8 7 EERKQNRKNGKGQAS 8 9
RKQNRKNGKGQASQT 8 11 QNRKNGKGQASQTQC 8 V8-HLA-DRB1- 0301-15
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 14 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 6 ALMLKSPTVMGLMEA 20 14
VMGLMEAISEKYGLP 20 V8-HLA-DRB1- 0301-15 mers-202P5A5 Each peptide
is a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 11 SPTVMGLMEAISEKY 17 15 MGLMEAISEKYGLPV 17 5 DALMLKSPTVMGLME
14 4 FDALMLKSPTVMGLM 12 12 PTVMGLMEAISEKYG 12
TABLE-US-00060 TABLE XLVIII V1-HLA-DR1- 0401-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 184 HSAYLKDDQRSTPDS 28 231 TFQYTLEATKSLRQK 28
338 AVSFTWDVNEEAKIF 28 462 PVLFIPDVHFANLQR 28 468 DVHFANLQRTGQVYY
28 503 EEEFGPVPSKQMKEE 28 582 IEHYSNEDTFILNME 28 54 YYKVPRDKRLLSVSK
26 95 ENRVQVLKTVPVNLS 26 175 QYDVPSLATHSAYLK 26 274 RHPISKVRSVVMVVF
26 281 RSVVMVVFSEDKNRD 26 352 FITVNCLSTDFSSQK 26 355
VNCLSTDFSSQKGVK 26 406 ERKIRDEERKQNRKK 26 452 FKTMPDLHSQPVLFI 26
511 SKQMKEEGTKRVLLY 26 10 RRAYTSEDEAWKSYL 22 51 LYDYYKVPRDKRLLS 22
201 SESFKDAATEKFRSA 22 219 AEEYMYDQTSSGTFQ 22 256 KGQFYAITLSETGDN
22 285 MVVFSEDKNRDEQLK 22 297 QLKYWKYWHSRQHTA 22 300
YWKYWHSRQHTAKQR 22 322 KESFNTIGNIEEIAY 22 349 AKIFITVNCLSTDFS 22
377 IDTYSYNNRSNKPIH 22 21 KSYLENPLTAATKAM 20 25 ENPLTAATKAMMSIN 20
35 MMSINGDEDSAAALG 20 45 AAALGLLYDYYKVPR 20 88 QSNLSGGENRVQVLK 20
98 VQVLKTVPVNLSLNQ 20 101 LKTVPVNLSLNQDHL 20 107 NLSLNQDHLENSKRE 20
122 QYSISFPESSAIIPV 20 130 SSAIIPVSGITVVKA 20 133 IIPVSGITVVKAEDF
20 139 ITVVKAEDFTPVFMA 20 165 EQRVVIFEQTQYDVP 20 166
QRVVIFEQTQYDVPS 20 167 RVVIFEQTQYDVPSL 20 185 SAYLKDDQRSTPDST 20
220 EEYMYDQTSSGTFQY 20 233 QYTLEATKSLRQKQG 20 259 FYAITLSETGDNKCF
20 282 SVVMVVFSEDKNRDE 20 313 QRVLDIADYKESFNT 20 328
IGNIEEIAYNAVSFT 20 342 TWDVNEEAKIFITVN 20 348 EAKIFITVNCLSTDF 20
365 QKGVKGLPLMIQIDT 20 368 VKGLPLMIQIDTYSY 20 371 LPLMIQIDTYSYNNR
20 372 PLMIQIDTYSYNNRS 20 387 NKPIHRAYCQIKVFC 20 394
YCQIKVFCDKGAERK 20 435 DGKLAAIPLQKKSDI 20 446 KSDITYFKTMPDLHS 20
463 VLFIPDVHFANLQRT 20 466 IPDVHFANLQRTGQV 20 519 TKRVLLYVRKETDDV
20 530 TDDVFDALMLKSPTV 20 541 SPTVKGLMEAISEKY 20 544
VKGLMEAISEKYGLP 20 554 KYGLPVEKIAKLYKK 20 556 GLPVEKIAKLYKKSK 20
559 VEKIAKLYKKSKKGI 20 572 GILVNMDDNIIEHYS 20 579 DNIIEHYSNEDTFIL
20 1 MPSDPPFNTRRAYTS 18 18 EAWKSYLENPLTAAT 18 24 LENPLTAATKAMMSI 18
36 MSINGDEDSAAALGL 18 57 VPRDKRLLSVSKASD 18 65 SVSKASDSQEDQEKR 18
71 DSQEDQEKRNCLGTS 18 76 QEKRNCLGTSEAQSN 18 80 NCLGTSEAQSNLSGG 18
81 CLGTSEAQSNLSGGE 18 89 SNLSGGENRVQVLKT 18 104 VPVNLSLNQDHLENS 18
123 YSISFPESSAIIPVS 18 158 YPRGDGEEQRVVIFE 18 159 PRGDGEEQRVVIFEQ
18 172 EQTQYDVPSLATHSA 18 206 DAATEKFRSASVGAE 18 267
TGDNKCFRHPISKVR 18 305 HSRQHTAKQRVLDIA 18 319 ADYKESFNTIGNIEE 18
329 GNIEEIAYNAVSFTW 18 442 PLQKKSDITYFKTMP 18 533 VFDALMLKSPTVKGL
18 576 NMDDNIIEHYSNEDT 18 586 SNEDTFILNMESMVE 18 250
PMTYLNKGQFYAITL 17 301 WKYWHSRQHTAKQRV 17 379 TYSYNNRSNKPIHRA 17
522 VLLYVRKETDDVFDA 17 17 DEAWKSYLENPLTAA 16 20 WKSYLENPLTAATKA 16
52 YDYYKVPRDKRLLSV 16 120 REQYSISFPESSAII 16 144 AEDFTPVFMAPPVHY 16
155 PVHYPRGDGEEQRVV 16 197 DSTYSESFKDAATEK 16 209 TEKFRSASVGAEEYM
16 221 EYMYDQTSSGTFQYT 16 229 SGTFQYTLEATKSLR 16 257
GQFYAITLSETGDNK 16 270 NKCFRHPISKVRSVV 16 298 LKYWKYWHSRQHTAK 16
318 IADYKESFNTIGNIE 16 333 EIAYNAVSFTWDVNE 16 340 SFTWDVNEEAKIFIT
16 391 HRAYCQIKVFCDKGA 16 449 ITYFKTMPDLHSQPV 16 479
QVYYNTDDEREGGSV 16 531 DDVFDALMLKSPTVK 16 552 SEKYGLPVEKIAKLY 16
563 AKLYKKSKKGILVNM 16 588 EDTFILNMESMVEGF 16 32 TKAMMSINGDEDSAA 14
33 KAMMSINGDEDSAAA 14 47 ALGLLYDYYKVPRDK 14 60 DKRLLSVSKASDSQE 14
61 KRLLSVSKASDSQED 14 63 LLSVSKASDSQEDQE 14 79 RNCLGTSEAQSNLSG 14
97 RVQVLKTVPVNLSLN 14 103 TVPVNLSLNQDHLEN 14 112 QDHLENSKREQYSIS 14
131 SAIIPVSGITVVKAE 14 136 VSGITVVKAEDFTPV 14 138 GITVVKAEDFTPVFM
14 147 FTPVFMAPPVHYPRG 14 149 PVFMAPPVHYPRGDG 14 178
VPSLATHSAYLKDDQ 14 214 SASVGAEEYMYDQTS 14 248 EGPMTYLNKGQFYAI 14
251 MTYLNKGQFYAITLS 14 277 ISKVRSVVMVVFSED 14 280 VRSVVMVVFSEDKNR
14 284 VMVVFSEDKNRDEQL 14 295 DEQLKYWKYWHSRQH 14 312
KQRVLDIADYKESFN 14 315 VLDIADYKESFNTIG 14 325 FNTIGNIEEIAYNAV 14
331 IEEIAYNAVSFTWDV 14 370 GLPLMIQIDTYSYNN 14 396 QIKVFCDKGAERKIR
14 438 LAAIPLQKKSDITYF 14 455 MPDLHSQPVLFIPDV 14 460
SQPVLFIPDVHFANL 14 461 QPVLFIPDVHFANLQ 14 471 FANLQRTGQVYYNTD 14
492 SVLVKRMFRPMEEEF 14 499 FRPMEEEFGPVPSKQ 14 520 KRVLLYVRKETDDVF
14 523 LLYVRKETDDVFDAL 14 535 DALMLKSPTVKGLME 14 536
ALMLKSPTVKGLMEA 14 545 KGLMEAISEKYGLPV 14 570 KKGILVNMDDNIIEH 14
571 KGILVNMDDNIIEHY 14 578 DDNIIEHYSNEDTFI 14 589 DTFILNMESMVEGFK
14 590 TFILNMESMVEGFKV 14 592 ILNMESMVEGFKVTL 14 595
MESMVEGFKVTLMEI 14 V2-HLA-DR1- 0401-15 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 8 NKRLVALVPMPSDPP 20 14 LVPMPSDPPFNTRRA 20 9 KRLVALVPMPSDPPF
14 11 LVALVPMPSDPPFNT 14 2 SQESDNNKRLVALVP 12 6 DNNKRLVALVPMPSD 12
V4-HLA-DR1- 0401-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 3
ENPLTAATKAMMIIN 20 13 MMIINGDEDSAAALG 20 2 LENPLTAATKAMMII 18 14
MIINGDEDSAAALGL 18 10 TKAMMIINGDEDSAA 14 11 KAMMIINGDEDSAAA 14 12
AMMIINGDEDSAAAL 14 6 LTAATKAMMIINGDE 12 9 ATKAMMIINGDEDSA 12 15
IINGDEDSAAALGLL 12 V5-HLA-DR1- 0401-15 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 7 ERKIRDEEQKQNRKK 26 4 KGAERKIRDEEQKQN 12 6 AERKIRDEEQKQNRK
12 9 KIRDEEQKQNRKKGK 12 V5 & 6-HLA-
DR1-0401-15 mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3;
each start position is specified, the length of peptide is 14 amino
acids, and the end position for each peptide is the start position
plus fourteen. Pos 123456789012345 score 1 ERKIRDEEQKQNRKN 26 3
KIRDEEQKQNRKNGK 12 V6-HLA-DR1- 0401-15 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 1 ERKIRDEERKQNRKN 26 13 RKNGKGQASQTQCNS 12 14 KNGKGQASQTQCNSS
12 15 NGKGQASQTQCNSSS 12 V8-HLA-DR1- 0401-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 11 SPTVMGLMEAISEKY 20 14 VMGLMEAISEKYGLP 20 3
VFDALMLKSPTVMGL 18 1 DDVFDALMLKSPTVM 16 5 DALMLKSPTVMGLME 14 6
ALMLKSPTVMGLMEA 14 15 MGLMEAISEKYGLPV 14 13 TVMGLMEAISEKYGL 12 4
FDALMLKSPTVMGLM 9
TABLE-US-00061 TABLE XLIX V1-HLA-DRB1- 1101-15 mers-202P5A5 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 14 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 51 LYDYYKVPRDKRLLS 25 144 AEDFTPVFMAPPVHY 24
197 DSTYSESFKDAATEK 24 468 DVHFANLQRTGQVYY 24 248 EGPMTYLNKGQFYAI
22 271 KCFRHPISKVRSVVM 22 449 ITYFKTMPDLHSQPV 22 496
KRMFRPMEEEFGPVP 22 531 DDVFDALMLKSPTVK 22 54 YYKVPRDKRLLSVSK 21 94
GENRVQVLKTVPVNL 21 60 DKRLLSVSKASDSQE 20 185 SAYLKDDQRSTPDST 20 297
QLKYWKYWHSRQHTA 20 338 AVSFTWDVNEEAKIF 20 462 PVLFIPDVHFANLQR 20
492 SVLVKRMFRPMEEEF 20 520 KRVLLYVRKETDDVF 20 556 GLPVEKIAKLYKKSK
20 559 VEKIAKLYKKSKKGI 20 98 VQVLKTVPVNLSLNQ 19 133 IIPVSGITVVKAEDF
19 541 SPTVKGLMEAISEKY 19 552 SEKYGLPVEKIAKLY 19 563
AKLYKKSKKGILVNM 19 175 QYDVPSLATHSAYLK 18 301 WKYWHSRQHTAKQRV 18
352 FITVNCLSTDFSSQK 18 377 IDTYSYNNRSNKPIH 18 488 REGGSVLVKRMFRPM
18 545 KGLMEAISEKYGLPV 18 124 SISFPESSAIIPVSG 17 168
VVIFEQTQYDVPSLA 17 284 VMVVFSEDKNRDEQL 17 318 IADYKESFNTIGNIE 17
359 STDFSSQKGVKGLPL 17 361 DFSSQKGVKGLPLMI 17 479 QVYYNTDDEREGGSV
17 4 DPPFNTRRAYTSEDE 16 20 WKSYLENPLTAATKA 16 25 ENPLTAATKAMMSIN 16
120 REQYSISFPESSAII 16 135 PVSGITVVKAEDFTP 16 148 TPVFMAPPVHYPRGD
16 149 PVFMAPPVHYPRGDG 16 203 SFKDAATEKFRSASV 16 205
KDAATEKFRSASVGA 16 219 AEEYMYDQTSSGTFQ 16 250 PMTYLNKGQFYAITL 16
256 KGQFYAITLSETGDN 16 257 GQFYAITLSETGDNK 16 300 YWKYWHSRQHTAKQR
16 322 KESFNTIGNIEEIAY 16 333 EIAYNAVSFTWDVNE 16 348
EAKIFITVNCLSTDF 16 349 AKIFITVNCLSTDFS 16 391 HRAYCQIKVFCDKGA 16
480 VYYNTDDEREGGSVL 16 503 EEEFGPVPSKQMKEE 16 519 TKRVLLYVRKETDDV
16 136 VSGITVVKAEDFTPV 15 267 TGDNKCFRHPISKVR 15 277
ISKVRSVVMVVFSED 15 410 RDEERKQNRKKGKGQ 15 412 EERKQNRKKGKGQAS 15
490 GGSVLVKRMFRPMEE 15 48 LGLLYDYYKVPRDKR 14 112 QDHLENSKREQYSIS 14
150 VFMAPPVHYPRGDGE 14 152 MAPPVHYPRGDGEEQ 14 280 VRSVVMVVFSEDKNR
14 342 TWDVNEEAKIFITVN 14 384 NRSNKPIHRAYCQIK 14 406
ERKIRDEERKQNRKK 14 414 RKQNRKKGKGQASQT 14 429 QCNSSSDGKLAAIPL 14
437 KLAAIPLQKKSDITY 14 438 LAAIPLQKKSDITYF 14 445 KKSDITYFKTMPDLH
14 451 YFKTMPDLHSQPVLF 14 489 EGGSVLVKRMFRPME 14 507
GPVPSKQMKEEGTKR 14 530 TDDVFDALMLKSPTV 14 532 DVFDALMLKSPTVKG 14
553 EKYGLPVEKIAKLYK 14 562 IAKLYKKSKKGILVN 14 576 NMDDNIIEHYSNEDT
14 586 SNEDTFILNMESMVE 14 592 ILNMESMVEGFKVTL 14 595
MESMVEGFKVTLMEI 14 47 ALGLLYDYYKVPRDK 13 52 YDYYKVPRDKRLLSV 13 58
PRDKRLLSVSKASDS 13 61 KRLLSVSKASDSQED 13 95 ENRVQVLKTVPVNLS 13 230
GTFQYTLEATKSLRQ 13 274 RHPISKVRSVVMVVF 13 278 SKVRSVVMVVFSEDK 13
281 RSVVMVVFSEDKNRD 13 312 KQRVLDIADYKESFN 13 365 QKGVKGLPLMIQIDT
13 393 AYCQIKVFCDKGAER 13 394 YCQIKVFCDKGAERK 13 435
DGKLAAIPLQKKSDI 13 461 QPVLFIPDVHFANLQ 13 499 FRPMEEEFGPVPSKQ 13
535 DALMLKSPTVKGLME 13 571 KGILVNMDDNIIEHY 13 589 DTFILNMESMVEGFK
13 10 RRAYTSEDEAWKSYL 12 29 TAATKAMMSINGDED 12 32 TKAMMSINGDEDSAA
12 33 KAMMSINGDEDSAAA 12 35 MMSINGDEDSAAALG 12 45 AAALGLLYDYYKVPR
12 49 GLLYDYYKVPRDKRL 12 76 QEKRNCLGTSEAQSN 12 107 NLSLNQDHLENSKRE
12
119 KREQYSISFPESSAI 12 127 FPESSAIIPVSGITV 12 130 SSAIIPVSGITVVKA
12 138 GITVVKAEDFTPVFM 12 217 VGAEEYMYDQTSSGT 12 231
TFQYTLEATKSLRQK 12 236 LEATKSLRQKQGEGP 12 239 TKSLRQKQGEGPMTY 12
295 DEQLKYWKYWHSRQH 12 313 QRVLDIADYKESFNT 12 325 FNTIGNIEEIAYNAV
12 328 IGNIEEIAYNAVSFT 12 340 SFTWDVNEEAKIFIT 12 368
VKGLPLMIQIDTYSY 12 370 GLPLMIQIDTYSYNN 12 371 LPLMIQIDTYSYNNR 12
397 IKVFCDKGAERKIRD 12 448 DITYFKTMPDLHSQP 12 452 FKTMPDLHSQPVLFI
12 460 SQPVLFIPDVHFANL 12 463 VLFIPDVHFANLQRT 12 478
GQVYYNTDDEREGGS 12 575 VNMDDNIIEHYSNED 12 590 TFILNMESMVEGFKV 12
V2-HLA-DRB1- 1101-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 5; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 8
NKRLVALVPMPSDPP 20 11 LVALVPMPSDPPFNT 18 2 SQESDNNKRLVALVP 15 5
SDNNKRLVALVPMPS 15 9 KRLVALVPMPSDPPF 12 1 MSQESDNNKRLVALV 10
V4-HLA-DRB1- 1101-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO: 3; each start position is specified, the length of peptide
is 14 amino acids, and the end position for each peptide is the
start position plus fourteen. Pos 123456789012345 score 3
ENPLTAATKAMMIIN 16 7 TAATKAMMIINGDED 12 10 TKAMMIINGDEDSAA 12 11
KAMMIINGDEDSAAA 12 12 AMMIINGDEDSAAAL 12 13 MMIINGDEDSAAALG 12 1
YLENPLTAATKAMMI 7 V5-HLA-DRB1- 1101-15 mers-202P5A5 Each peptide is
a portion of SEQ ID NO: 3; each start position is specified, the
length of peptide is 14 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 11 RDEEQKQNRKKGKGQ 15 13 EEQKQNRKKGKGQAS 15 15
QKQNRKKGKGQASQT 14 1 FCDKGAERKIRDEEQ 10 3 DKGAERKIRDEEQKQ 8 8
RKIRDEEQKQNRKKG 8 12 DEEQKQNRKKGKGQA 8 V5&6-HLA-DRB1- 1101-15
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 14 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 5 RDEEQKQNRKNGKGQ 14 9
QKQNRKNGKGQASQT 14 2 RKIRDEEQKQNRKNG 8 6 DEEQKQNRKNGKGQA 8 7
EEQKQNRKNGKGQAS 7 1 ERKIRDEEQKQNRKN 6 V6-HLA-DRB1- 1101-15
mers-202P5A5 Each peptide is a portion of SEQ ID NO: 3; each start
position is specified, the length of peptide is 14 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 1 ERKIRDEERKQNRKN 14 5
RDEERKQNRKNGKGQ 14 9 RKQNRKNGKGQASQT 14 2 RKIRDEERKQNRKNG 8 6
DEERKQNRKNGKGQA 8 7 EERKQNRKNGKGQAS 7 10 KQNRKNGKGQASQTQ 6
V8-HLA-DRB1- 1101-15 mers-202P5A5 Each peptide is a portion of SEQ
ID NO:3; each start position is specified, the length of peptide is
14 amino acids, and the end position for each peptide is the start
position plus fourteen. Pos 123456789012345 score 11
SPTVMGLMEAISEKY 20 14 VMGLMEAISEKYGLP 20 3 VFDALMLKSPTVMGL 18 1
DDVFDALMLKSPTVM 16 5 DALMLKSPTVMGLME 14 6 ALMLKSPTVMGLMEA 14 15
MGLMEAISEKYGLPV 14 13 TVMGLMEAISEKYGL 12 4 FDALMLKSPTVMGLM 9
TABLE-US-00062 TABLE L Protein Characteristics of 202P5A5
Bioinformatic Located on the World Wide Program Web at: Outcome ORF
ORF finder 1829 bp Protein length 609aa Transmembrane TM Pred
ch.embnet.org/ no TM region HMMTop enzim.hu/hmmtop/ no TM Sosui
genome.ad.jp/SOSui/ soluble protein TMHMM cbs.dtu.dk/services/TMHMM
no TM, extracellular Signal Peptide Signal P
cbs.dtu.dk/services/SignalP/ no signal peptide pI pI/MW tool
expasy.ch/tools/ pI 6.05 Molecular weight pI/MW tool
expasy.ch/tools/ 63.9 kD Localization PSORT psort.nibb.ac.jp/ 76%
nuclear, 30% microbody PSORT II psort.nibb.ac.jp/ 61% nuclear, 22%
mitochondrial Motifs Pfam sanger.ac.uk/Pfam/ CP2 transcription
factor Prints biochem.ucl.ac.uk/ Fibronetin type III repeat Blocks
blocks.fhcrc.org/ M protein repeat
TABLE-US-00063 TABLE LI Exon boundaries of transcript 202P5A05 v.1
Exon Number Start End Length 1 1 196 196 2 197 264 68 3 265 658 394
4 659 714 56 5 715 871 157 6 872 983 112 7 984 1078 95 8 1079 1237
159 9 1238 1325 88 10 1326 1465 140 11 1466 1497 32 12 1498 1592 95
13 1593 1678 86 14 1679 1743 65 15 1744 4746 3002
TABLE-US-00064 TABLE LII(a) Nucleotide sequence of transcript
variant 202P5A05 v.2 (SEQ ID NO: 96) attggatcaa acatgtcaca
agagtcggac aataataaaa gactagtggc cttagtgccc 60 atgcccagtg
accctccatt caatacccga agagcctaca ccagtgagga tgaagcctgg 120
aagtcatact tggagaatcc cctgacagca gccaccaagg ccatgatgag cattaatggt
180 gatgaggaca gtgctgctgc cctcggcctg ctctatgact actacaaggt
tcctcgagac 240 aagaggctgc tgtctgtaag caaagcaagt gacagccaag
aagaccagga gaaaagaaac 300 tgccttggca ccagtgaagc ccagagtaat
ttgagtggag gagaaaaccg agtgcaagtc 360 ctaaagactg ttccagtgaa
cctttcccta aatcaagatc acctggagaa ttccaagcgg 420 gaacagtaca
gcatcagctt ccccgagagc tctgccatca tcccggtgtc gggaatcacg 480
gtggtgaaag ctgaagattt cacaccagtt ttcatggccc cacctgtgca ctatccccgg
540 ggagatgggg aagagcaacg agtggttatc tttgaacaga ctcagtatga
cgtgccctcg 600 ctggccaccc acagcgccta tctcaaagac gaccagcgca
gcactccgga cagcacatac 660 agcgagagct tcaaggacgc agccacagag
aaatttcgga gtgcttcagt tggggctgag 720 gagtacatgt atgatcagac
atcaagtggc acatttcagt acaccctgga agccaccaaa 780 tctctccgtc
agaagcaggg ggagggcccc atgacctacc tcaacaaagg acagttctat 840
gccataacac tcagcgagac cggagacaac aaatgcttcc gacaccccat cagcaaagtc
900 aggagtgtgg tgatggtggt cttcagtgaa gacaaaaaca gagatgaaca
gctcaaatac 960 tggaaatact ggcactctcg gcagcatacg gcgaagcaga
gggtccttga cattgccgat 1020 tacaaggaga gctttaatac gattggaaac
attgaagaga ttgcatataa tgctgtttcc 1080 tttacctggg acgtgaatga
agaggcgaag attttcatca ccgtgaattg cttgagcaca 1140 gatttctcct
cccaaaaagg ggtgaaagga cttcctttga tgattcagat tgacacatac 1200
agttataaca atcgtagcaa taaacccatt catagagctt attgccagat caaggtcttc
1260 tgtgacaaag gagcagaaag aaaaatccga gatgaagagc ggaagcagaa
caggaagaaa 1320 gggaaaggcc aggcctccca aactcaatgc aacagctcct
ctgatgggaa gttggctgcc 1380 atacctttac agaagaagag tgacatcacc
tacttcaaaa ccatgcctga tctccactca 1440 cagccagttc tcttcatacc
tgatgttcac tttgcaaacc tgcagaggac cggacaggtg 1500 tattacaaca
cggatgatga acgagaaggt ggcagtgtcc ttgttaaacg gatgttccgg 1560
cccatggaag aggagtttgg tccagtgcct tcaaagcaga tgaaagaaga agggacaaag
1620 cgagtgctct tgtacgtgag gaaggagact gacgatgtgt tcgatgcatt
gatgttgaag 1680 tctcccacag tgaagggcct gatggaagcg atatctgaga
aatatgggct gcccgtggag 1740 aagatagcaa agctttacaa gaaaagcaaa
aaaggcatct tggtgaacat ggatgacaac 1800 atcatcgagc actactcgaa
cgaggacacc ttcatcctca acatggagag catggtggag 1860 ggcttcaagg
tcacgctcat ggaaatctag ccctgggttt ggcatccgct ttggctggag 1920
ctctcagtgc gttcctccct gagagagaca gaagccccag ccccagaacc tggagaccca
1980 tctcccccat ctcacaactg ctgttacaag accgtgctgg ggagtggggc
aagggacagg 2040 ccccactgtc ggtgtgcttg gcccatccac tggcacctac
cacggagctg aagcctgagc 2100 ccctcaggaa ggtgccttag gcctgttgga
ttcctattta ttgcccacct tttcctggag 2160 cccaggtcca ggcccgccag
gactctgcag gtcactgcta gctccagatg agaccgtcca 2220 gcgttccccc
ttcaagagaa acactcatcc cgaacagcct aaaaaattcc catcccttct 2280
ctctcacccc tccatatcta tctcccgagt ggctggacaa aatgagctac gtctgggtgc
2340 agtagttata ggtggggcaa gaggtggatg cccactttct ggtcagacac
ctttaggttg 2400 ctctggggaa ggctgtcttg ctaaatacct ccagggttcc
cagcaagtgg ccaccaggcc 2460 ttgtacagga agacattcag tcaccgtgta
attagtaaca cagaaagtct gcctgtctgc 2520 attgtacata gtgtttataa
tattgtaata atatatttta cctgtggtat gtgggcatgt 2580 ttactgccac
tggcctagag gagacacaga cctggagacc gttttaatgg gggtttttgc 2640
ctctgtgcct gttcaagaga cttgcagggc taggtagagg gcctttggga tgttaaggtg
2700 actgcagctg atgccaagat ggactctgca atgggcatac ctgggggctc
gttccctgtc 2760 cccagaggaa gccccctctc cttctccatg ggcatgactc
tccttcgagg ccaccacgtt 2820 tatctcacaa tgatgtgttt tgcttgactt
tccctttgcg ctgtctcgtg ggaaaggtca 2880 ttctgtctga gaccccagct
ccttctccag ctttggctgc gggcatggcc tgagctttct 2940 ggagagcctc
tgcagggggt ttgccatcag ggccctgtgg ctgggtctgc tgcagagctc 3000
cttggctatc aggagaatcc tggacactgt actgtgcctc ccagtttaca aacacgccct
3060 tcatctcaag tggcccttta aaaggcctgc tgccatgtga gagctgtgaa
cagctcagct 3120 ctgagtcggc aggctggggc ttcctcctgg gccaccagat
ggaaaggggg tattgtttgc 3180 ctcactcctg gatgctgcgt tttaaggaag
tgagtgagaa agaatgtgcc aagatacctg 3240 gctcctgtga aaccagcctc
aggagggaaa ctgggagaga gaagctgtgg tctcctgcta 3300 catgccctgg
gagctggaag agaaaaacac tcccctaaac aatcgcaaaa tgatgaacca 3360
tcatgggcca ctgttctctt tgaggggaca ggtttagggg tttgcgttcg cccttgtggg
3420 ctgaagcact agctttttgg tagctagaca catcctgcac ccaaaggttc
tctacaaagg 3480 cccagatttg tttgtaaagc actttgactc ttacctggag
gcccgctctc taagggcttc 3540 ctgcgctccc acctcatctg tccctgagat
gcagagcagg atggagggtc tgcttctagc 3600 tcagctgttt ctccttgagg
ttgcggagga attgaattga atgggacaga gggcaggtgc 3660 tgtggccaag
aagatctccg agcagcagtg acggggcacc ttgctgtgtg tcctctgggc 3720
atgttaaccc ttctgtgggg ccaaaggttt gcatcgtgga tccagctgtg ctccagtctg
3780 tcccctcctc ctccactctg actgccacgc cccggaccag cagcttgggg
accctccagg 3840 gtactaatgg ggctctgttc tgagatggac aaattcagtg
ttggaaatac atgttgtact 3900 atgcacttcc catgctccta gggttaggaa
tagtttcaaa catgattggc agacataaca 3960 acggcaaata ctcggactgg
ggcataggac tccagagtag gaaaaagaca aaagatttgg 4020 cagcctgaca
caggcaacct acccctctct ctccagcctc tttatgaaac tgtttgtttg 4080
ccagtcctgc cctaaggcag aagatgaatt gaagatgctg tgcatgtttc ctaagtcctt
4140 gagcaatcat ggtggtgaca attgccacaa gggatatgag gccagtgcca
ccagagggtg 4200 gtgccaagtg ccacatccct tccgatccat tcccctctgc
atcctcggag caccccagtt 4260 tgcctttgat gtgtccgctg tgtatgttag
ctgaactttg atgagcaaaa tttcctgagc 4320 gaaacactcc aaagagatag
gaaaacttgc cgcctcttct tttttgtccc ttaatcaaac 4380 tcaaataagc
ttaaaaaaaa tccatggaag atcatggaca tgtgaaatga gcattttttt 4440
cttttttttt tttaacaaag tctgaactga acagaacaag actttttcct catacatctc
4500 caaattgttt aaacttactt tatgagtgtt tgtttagaag ttcggaccaa
cagaaaaatg 4560 cagtcagatg tcatcttgga attggtttct aaaagagtaa
ggcatgtccc tgcccagaaa 4620 cttaggaagc atgaaataaa tcaaatgttt
attttccttc ttatttaaaa tcatgcaaat 4680 gcaacagaaa tagagggttt
gtgccaaatg ctatgaacgg ccctttctta aagacaagca 4740 agggagattg
atatatgtac aatttgctct catgttttaa aaaaaaaagg taaatgtaac 4800
ttaatagttt tgtaaatggg agagggggaa tctataaact ataaatacag ttattttatt
4860 ttttgtacat ttttaaggag aaaaaaataa atattcataa cataagagga aaa
4913
TABLE-US-00065 TABLE LIII(a) Nucleotide sequence alignment of
202P5A05 v.2 (SEQ ID NO: 97) and 202P5A05 v.1 (SEQ ID NO: 98) v.1 1
TAATAAAAGACTAGTGGCCTTAGTGCCCATGCCCAGTGACCCTCCATTCA 50
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 33
taataaaagactagtggccttagtgcccatgcccagtgaccctccattca 82 v.1 51
ATACCCGAAGAGCCTACACCAGTGAGGATGAAGCCTGGAAGTCATACTTG 100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 83
atacccgaagagcctacaccagtgaggatgaagcctggaagtcatacttg 132 v.1 101
GAGAATCCCCTGACAGCAGCCACCAAGGCCATGATGAGCATTAATGGTGA 150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 133
gagaatcccctgacagcagccaccaaggccatgatgagcattaatggtga 182 v.1 151
TGAGGACAGTGCTGCTGCCCTCGGCCTGCTCTATGACTACTACAAGGTTC 200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 183
tgaggacagtgctgctgccctcggcctgctctatgactactacaaggttc 232 v.1 201
CTCGAGACAAGAGGCTGCTGTCTGTAAGCAAAGCAAGTGACAGCCAAGAA 250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 233
ctcgagacaagaggctgctgtctgtaagcaaagcaagtgacagccaagaa 282 v.1 251
GACCAGGAGAAAAGAAACTGCCTTGGCACCAGTGAAGCCCAGAGTAATTT 300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 283
gaccaggagaaaagaaactgccttggcaccagtgaagcccagagtaattt 332 v.1 301
GAGTGGAGGAGAAAACCGAGTGCAAGTCCTAAAGACTGTTCCAGTGAACC 350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 333
gagtggaggagaaaaccgagtgcaagtcctaaagactgttccagtgaacc 382 v.1 351
TTTCCCTAAATCAAGATCACCTGGAGAATTCCAAGCGGGAACAGTACAGC 400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 383
tttccctaaatcaagatcacctggagaattccaagcgggaacagtacagc 432 v.1 401
ATCAGCTTCCCCGAGAGCTCTGCCATCATCCCGGTGTCGGGAATCACGGT 450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 433
atcagcttccccgagagctctgccatcatcccggtgtcgggaatcacggt 482 v.1 451
GGTGAAAGCTGAAGATTTCACACCAGTTTTCATGGCCCCACCTGTGCACT 500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 483
ggtgaaagctgaagatttcacaccagttttcatggccccacctgtgcact 532 v.1 501
ATCCCCGGGGAGATGGGGAAGAGCAACGAGTGGTTATCTTTGAACAGACT 550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 533
atccccggggagatggggaagagcaacgagtggttatctttgaacagact 582 v.1 551
CAGTATGACGTGCCCTCGCTGGCCACCCACAGCGCCTATCTCAAAGACGA 600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 583
cagtatgacgtgccctcgctggccacccacagcgcctatctcaaagacga 632 v.1 601
CCAGCGCAGCACTCCGGACAGCACATACAGCGAGAGCTTCAAGGACGCAG 650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 633
ccagcgcagcactccggacagcacatacagcgagagcttcaaggacgcag 682 v.1 651
CCACAGAGAAATTTCGGAGTGCTTCAGTTGGGGCTGAGGAGTACATGTAT 700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 683
ccacagagaaatttcggagtgcttcagttggggctgaggagtacatgtat 732 v.1 701
GATCAGACATCAAGTGGCACATTTCAGTACACCCTGGAAGCCACCAAATC 750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 733
gatcagacatcaagtggcacatttcagtacaccctggaagccaccaaatc 782 v.1 751
TCTCCGTCAGAAGCAGGGGGAGGGCCCCATGACCTACCTCAACAAAGGAC 800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 783
tctccgtcagaagcagggggagggccccatgacctacctcaacaaaggac 832 v.1 801
AGTTCTATGCCATAACACTCAGCGAGACCGGAGACAACAAATGCTTCCGA 850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 833
agttctatgccataacactcagcgagaccggagacaacaaatgcttccga 882 v.1 851
CACCCCATCAGCAAAGTCAGGAGTGTGGTGATGGTGGTCTTCAGTGAAGA 900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 883
caccccatcagcaaagtcaggagtgtggtgatggtggtcttcagtgaaga 932 v.1 901
CAAAAACAGAGATGAACAGCTCAAATACTGGAAATACTGGCACTCTCGGC 950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 933
caaaaacagagatgaacagctcaaatactggaaatactggcactctcggc 982 v.1 951
AGCATACGGCGAAGCAGAGGGTCCTTGACATTGCCGATTACAAGGAGAGC 1000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 983
agcatacggcgaagcagagggtccttgacattgccgattacaaggagagc 1032 v.1 1001
TTTAATACGATTGGAAACATTGAAGAGATTGCATATAATGCTGTTTCCTT 1050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1033
tttaatacgattggaaacattgaagagattgcatataatgctgtttcctt 1082 v.1 1051
TACCTGGGACGTGAATGAAGAGGCGAAGATTTTCATCACCGTGAATTGCT 1100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1083
tacctgggacgtgaatgaagaggcgaagattttcatcaccgtgaattgct 1132 v.1 1101
TGAGCACAGATTTCTCCTCCCAAAAAGGGGTGAAAGGACTTCCTTTGATG 1150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1133
tgagcacagatttctcctcccaaaaaggggtgaaaggacttcctttgatg 1182 v.1 1151
ATTCAGATTGACACATACAGTTATAACAATCGTAGCAATAAACCCATTCA 1200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1183
attcagattgacacatacagttataacaatcgtagcaataaacccattca 1232 v.1 1201
TAGAGCTTATTGCCAGATCAAGGTCTTCTGTGACAAAGGAGCAGAAAGAA 1250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1233
tagagcttattgccagatcaaggtcttctgtgacaaaggagcagaaagaa 1282 v.1 1251
AAATCCGAGATGAAGAGCGGAAGCAGAACAGGAAGAAAGGGAAAGGCCAG 1300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1283
aaatccgagatgaagagcggaagcagaacaggaagaaagggaaaggccag 1332 v.1 1301
GCCTCCCAAACTCAATGCAACAGCTCCTCTGATGGGAAGTTGGCTGCCAT 1350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1333
gcctcccaaactcaatgcaacagctcctctgatgggaagttggctgccat 1382 v.1 1351
ACCTTTACAGAAGAAGAGTGACATCACCTACTTCAAAACCATGCCTGATC 1400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1383
acctttacagaagaagagtgacatcacctacttcaaaaccatgcctgatc 1432 v.1 1401
TCCACTCACAGCCAGTTCTCTTCATACCTGATGTTCACTTTGCAAACCTG 1450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1433
tccactcacagccagttctcttcatacctgatgttcactttgcaaacctg 1482 v.1 1451
CAGAGGACCGGACAGGTGTATTACAACACGGATGATGAACGAGAAGGTGG 1500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1483
cagaggaccggacaggtgtattacaacacggatgatgaacgagaaggtgg 1532 v.1 1501
CAGTGTCCTTGTTAAACGGATGTTCCGGCCCATGGAAGAGGAGTTTGGTC 1550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1533
cagtgtccttgttaaacggatgttccggcccatggaagaggagtttggtc 1582 v.1 1551
CAGTGCCTTCAAAGCAGATGAAAGAAGAAGGGACAAAGCGAGTGCTCTTG 1600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1583
cagtgccttcaaagcagatgaaagaagaagggacaaagcgagtgctcttg 1632 v.1 1601
TACGTGAGGAAGGAGACTGACGATGTGTTCGATGCATTGATGTTGAAGTC 1650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1633
tacgtgaggaaggagactgacgatgtgttcgatgcattgatgttgaagtc 1682 v.1 1651
TCCCACAGTGAAGGGCCTGATGGAAGCGATATCTGAGAAATATGGGCTGC 1700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1683
tcccacagtgaagggcctgatggaagcgatatctgagaaatatgggctgc 1732 v.1 1701
CCGTGGAGAAGATAGCAAAGCTTTACAAGAAAAGCAAAAAAGGCATCTTG 1750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1733
ccgtggagaagatagcaaagctttacaagaaaagcaaaaaaggcatcttg 1782 v.1 1751
GTGAACATGGATGACAACATCATCGAGCACTACTCGAACGAGGACACCTT 1800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1783
gtgaacatggatgacaacatcatcgagcactactcgaacgaggacacctt 1832 v.1 1801
CATCCTCAACATGGAGAGCATGGTGGAGGGCTTCAAGGTCACGCTCATGG 1850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1833
catcctcaacatggagagcatggtggagggcttcaaggtcacgctcatgg 1882 v.1 1851
AAATCTAGCCCTGGGTTTGGCATCCGCTTTGGCTGGAGCTCTCAGTGCGT 1900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1883
aaatctagccctgggtttggcatccgctttggctggagctctcagtgcgt 1932 v.1 1901
TCCTCCCTGAGAGAGACAGAAGCCCCAGCCCCAGAACCTGGAGACCCATC 1950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1933
tcctccctgagagagacagaagccccagccccagaacctggagacccatc 1982 v.1 1951
TCCCCCATCTCACAACTGCTGTTACAAGACCGTGCTGGGGAGTGGGGCAA 2000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 1983
tcccccatctcacaactgctgttacaagaccgtgctggggagtggggcaa 2032 v.1 2001
GGGACAGGCCCCACTGTCGGTGTGCTTGGCCCATCCACTGGCACCTACCA 2050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2033
gggacaggccccactgtcggtgtgcttggcccatccactggcacctacca 2082 v.1 2051
CGGAGCTGAAGCCTGAGCCCCTCAGGAAGGTGCCTTAGGCCTGTTGGATT 2100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2083
cggagctgaagcctgagcccctcaggaaggtgccttaggcctgttggatt 2132 v.1 2101
CCTATTTATTGCCCACCTTTTCCTGGAGCCCAGGTCCAGGCCCGCCAGGA 2150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2133
cctatttattgcccaccttttcctggagcccaggtccaggcccgccagga 2182 v.1 2151
CTCTGCAGGTCACTGCTAGCTCCAGATGAGACCGTCCAGCGTTCCCCCTT 2200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2183
ctctgcaggtcactgctagctccagatgagaccgtccagcgttccccctt 2232 v.1 2201
CAAGAGAAACACTCATCCCGAACAGCCTAAAAAATTCCCATCCCTTCTCT 2250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2233
caagagaaacactcatcccgaacagcctaaaaaattcccatcccttctct 2282 v.1 2251
CTCACCCCTCCATATCTATCTCCCGAGTGGCTGGACAAAATGAGCTACGT 2300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2283
ctcacccctccatatctatctcccgagtggctggacaaaatgagctacgt 2332 v.1 2301
CTGGGTGCAGTAGTTATAGGTGGGGCAAGAGGTGGATGCCCACTTTCTGG 2350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2333
ctgggtgcagtagttataggtggggcaagaggtggatgcccactttctgg 2382 v.1 2351
TCAGACACCTTTAGGTTGCTCTGGGGAAGGCTGTCTTGCTAAATACCTCC 2400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2383
tcagacacctttaggttgctctggggaaggctgtcttgctaaatacctcc 2432 v.1 2401
AGGGTTCCCAGCAAGTGGCCACCAGGCCTTGTACAGGAAGACATTCAGTC 2450
11111111111111111111111111111111111111111111111111 v.2 2433
agggttcccagcaagtggccaccaggccttgtacaggaagacattcagtc 2482 v.1 2451
ACCGTGTAATTAGTAACACAGAAAGTCTGCCTGTCTGCATTGTACATAGT 2500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2483
accgtgtaattagtaacacagaaagtctgcctgtctgcattgtacatagt 2532 v.1 2501
GTTTATAATATTGTAATAATATATTTTACCTGTGGTATGTGGGCATGTTT 2550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2533
gtttataatattgtaataatatattttacctgtggtatgtgggcatgttt 2582 v.1 2551
ACTGCCACTGGCCTAGAGGAGACACAGACCTGGAGACCGTTTTAATGGGG 2600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2583
actgccactggcctagaggagacacagacctggagaccgttttaatgggg 2632 v.1 2601
GTTTTTGCCTCTGTGCCTGTTCAAGAGACTTGCAGGGCTAGGTAGAGGGC 2650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2633
gtttttgcctctgtgcctgttcaagagacttgcagggctaggtagagggc 2682 v.1 2651
CTTTGGGATGTTAAGGTGACTGCAGCTGATGCCAAGATGGACTCTGCAAT 2700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2683
ctttgggatgttaaggtgactgcagctgatgccaagatggactctgcaat 2732 v.1 2701
GGGCATACCTGGGGGCTCGTTCCCTGTCCCCAGAGGAAGCCCCCTCTCCT 2750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2733
gggcatacctgggggctcgttccctgtccccagaggaagccccctctcct 2782 v.1 2751
TCTCCATGGGCATGACTCTCCTTCGAGGCCACCACGTTTATCTCACAATG 2800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2783
tctccatgggcatgactctccttcgaggccaccacgtttatctcacaatg 2832 v.1 2801
ATGTGTTTTGCTTGACTTTCCCTTTGCGCTGTCTCGTGGGAAAGGTCATT 2850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2833
atgtgttttgcttgactttccctttgcgctgtctcgtgggaaaggtcatt 2882 v.1 2851
CTGTCTGAGACCCCAGCTCCTTCTCCAGCTTTGGCTGCGGGCATGGCCTG 2900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2883
ctgtctgagaccccagctccttctccagctttggctgcgggcatggcctg 2932 v.1 2901
AGCTTTCTGGAGAGCCTCTGCAGGGGGTTTGCCATCAGGGCCCTGTGGCT 2950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2933
agctttctggagagcctctgcagggggtttgccatcagggccctgtggct 2982 v.1 2951
GGGTCTGCTGCAGAGCTCCTTGGCTATCAGGAGAATCCTGGACACTGTAC 3000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 2983
gggtctgctgcagagctccttggctatcaggagaatcctggacactgtac 3032 v.1 3001
TGTGCCTCCCAGTTTACAAACACGCCCTTCATCTCAAGTGGCCCTTTAAA 3050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3033
tgtgcctcccagtttacaaacacgcccttcatctcaagtggccctttaaa 3082
v.1 3051 AGGCCTGCTGCCATGTGAGAGCTGTGAACAGCTCAGCTCTGAGTCGGCAG 3100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3083
aggcctgctgccatgtgagagctgtgaacagctcagctctgagtcggcag 3132 v.1 3101
GCTGGGGCTTCCTCCTGGGCCACCAGATGGAAAGGGGGTATTGTTTGCCT 3150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3133
gctggggcttcctcctgggccaccagatggaaagggggtattgtttgcct 3182 v.1 3151
CACTCCTGGATGCTGCGTTTTAAGGAAGTGAGTGAGAAAGAATGTGCCAA 3200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3183
cactcctggatgctgcgttttaaggaagtgagtgagaaagaatgtgccaa 3232 v.1 3201
GATACCTGGCTCCTGTGAAACCAGCCTCAGGAGGGAAACTGGGAGAGAGA 3250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3233
gatacctggctcctgtgaaaccagcctcaggagggaaactgggagagaga 3282 v.1 3251
AGCTGTGGTCTCCTGCTACATGCCCTGGGAGCTGGAAGAGAAAAACACTC 3300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3283
agctgtggtctcctgctacatgccctgggagctggaagagaaaaacactc 3332 v.1 3301
CCCTAAACAATCGCAAAATGATGAACCATCATGGGCCACTGTTCTCTTTG 3350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3333
ccctaaacaatcgcaaaatgatgaaccatcatgggccactgttctctttg 3382 v.1 3351
AGGGGACAGGTTTAGGGGTTTGCGTTCGCCCTTGTGGGCTGAAGCACTAG 3400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3383
aggggacaggtttaggggtttgcgttcgcccttgtgggctgaagcactag 3432 v.1 3401
CTTTTTGGTAGCTAGACACATCCTGCACCCAAAGGTTCTCTACAAAGGCC 3450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3433
ctttttggtagctagacacatcctgcacccaaaggttctctacaaaggcc 3482 v.1 3451
CAGATTTGTTTGTAAAGCACTTTGACTCTTACCTGGAGGCCCGCTCTCTA 3500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3483
cagatttgtttgtaaagcactttgactcttacctggaggcccgctctcta 3532 v.1 3501
AGGGCTTCCTGCGCTCCCACCTCATCTGTCCCTGAGATGCAGAGCAGGAT 3550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3533
agggcttcctgcgctcccacctcatctgtccctgagatgcagagcaggat 3582 v.1 3551
GGAGGGTCTGCTTCTAGCTCAGCTGTTTCTCCTTGAGGTTGCGGAGGAAT 3600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3583
ggagggtctgcttctagctcagctgtttctccttgaggttgcggaggaat 3632 v.1 3601
TGAATTGAATGGGACAGAGGGCAGGTGCTGTGGCCAAGAAGATCTCCGAG 3650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3633
tgaattgaatgggacagagggcaggtgctgtggccaagaagatctccgag 3682 v.1 3651
CAGCAGTGACGGGGCACCTTGCTGTGTGTCCTCTGGGCATGTTAACCCTT 3700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3683
cagcagtgacggggcaccttgctgtgtgtcctctgggcatgttaaccctt 3732 v.1 3701
CTGTGGGGCCAAAGGTTTGCATCGTGGATCCAGCTGTGCTCCAGTCTGTC 3750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3733
ctgtggggccaaaggtttgcatcgtggatccagctgtgctccagtctgtc 3782 v.1 3751
CCCTCCTCCTCCACTCTGACTGCCACGCCCCGGACCAGCAGCTTGGGGAC 3800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3783
ccctcctcctccactctgactgccacgccccggaccagcagcttggggac 3832 v.1 3801
CCTCCAGGGTACTAATGGGGCTCTGTTCTGAGATGGACAAATTCAGTGTT 3850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3833
cctccagggtactaatggggctctgttctgagatggacaaattcagtgtt 3882 v.1 3851
GGAAATACATGTTGTACTATGCACTTCCCATGCTCCTAGGGTTAGGAATA 3900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3883
ggaaatacatgttgtactatgcacttcccatgctcctagggttaggaata 3932 v.1 3901
GTTTCAAACATGATTGGCAGACATAACAACGGCAAATACTCGGACTGGGG 3950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3933
gtttcaaacatgattggcagacataacaacggcaaatactcggactgggg 3982 v.1 3951
CATAGGACTCCAGAGTAGGAAAAAGACAAAAGATTTGGCAGCCTGACACA 4000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 3983
cataggactccagagtaggaaaaagacaaaagatttggcagcctgacaca 4032 v.1 4001
GGCAACCTACCCCTCTCTCTCCAGCCTCTTTATGAAACTGTTTGTTTGCC 4050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4033
ggcaacctacccctctctctccagcctctttatgaaactgtttgtttgcc 4082 v.1 4051
AGTCCTGCCCTAAGGCAGAAGATGAATTGAAGATGCTGTGCATGTTTCCT 4100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4083
agtcctgccctaaggcagaagatgaattgaagatgctgtgcatgtttcct 4132 v.1 4101
AAGTCCTTGAGCAATCATGGTGGTGACAATTGCCACAAGGGATATGAGGC 4150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4133
aagtccttgagcaatcatggtggtgacaattgccacaagggatatgaggc 4182 v.1 4151
CAGTGCCACCAGAGGGTGGTGCCAAGTGCCACATCCCTTCCGATCCATTC 4200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4183
cagtgccaccagagggtggtgccaagtgccacatcccttccgatccattc 4232 v.1 4201
CCCTCTGCATCCTCGGAGCACCCCAGTTTGCCTTTGATGTGTCCGCTGTG 4250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4233
ccctctgcatcctcggagcaccccagtttgcctttgatgtgtccgctgtg 4282 v.1 4251
TATGTTAGCTGAACTTTGATGAGCAAAATTTCCTGAGCGAAACACTCCAA 4300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4283
tatgttagctgaactttgatgagcaaaatttcctgagcgaaacactccaa 4332 v.1 4301
AGAGATAGGAAAACTTGCCGCCTCTTCTTTTTTGTCCCTTAATCAAACTC 4350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4333
agagataggaaaacttgccgcctcttcttttttgtcccttaatcaaactc 4382 v.1 4351
AAATAAGCTTAAAAAAAATCCATGGAAGATCATGGACATGTGAAATGAGC 4400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4383
aaataagcttaaaaaaaatccatggaagatcatggacatgtgaaatgagc 4432 v.1 4401
ATTTTTTTCTTTTTTTTTTTTAACAAAGTCTGAACTGAACAGAACAAGAC 4450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4433
atttttttcttttttttttttaacaaagtctgaactgaacagaacaagac 4482 v.1 4451
TTTTTCCTCATACATCTCCAAATTGTTTAAACTTACTTTATGAGTGTTTG 4500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4483
tttttcctcatacatctccaaattgtttaaacttactttatgagtgtttg 4532 v.1 4501
TTTAGAAGTTCGGACCAACAGAAAAATGCAGTCAGATGTCATCTTGGAAT 4550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4533
tttagaagttcggaccaacagaaaaatgcagtcagatgtcatcttggaat 4582 v.1 4551
TGGTTTCTAAAAGAGTAAGGCATGTCCCTGCCCAGAAACTTAGGAAGCAT 4600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4583
tggtttctaaaagagtaaggcatgtccctgcccagaaacttaggaagcat 4632 v.1 4601
GAAATAAATCAAATGTTTATTTTCCTTCTTATTTAAAATCATGCAAATGC 4650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4633
gaaataaatcaaatgtttattttccttcttatttaaaatcatgcaaatgc 4682 v.1 4651
AACAGAAATAGAGGGTTTGTGCCAAATGCTATGAACGGCCCTTTCTTAAA 4700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 4683
aacagaaatagagggtttgtgccaaatgctatgaacggccctttcttaaa 4732 v.1 4701
GACAAGCAAGGGAGATTGATATATGTACAATTTGCTCTCATGTTTT 4746
|||||||||||||||||||||||||||||||||||||||||||||| v.2 4733
gacaagcaagggagattgatatatgtacaatttgctctcatgtttt 4778
TABLE-US-00066 TABLE LIV(a) Peptide sequences of protein coded by
202P5A05 v.2 (SEQ ID NO: 99) MSQESDNNKR LVALVPMPSD PPFNTRRAYT
SEDEAWKSYL ENPLTAATKA MMSINGDEDS 60 AAALGLLYDY YKVPRDKRLL
SVSKASDSQE DQEKRNCLGT SEAQSNLSGG ENRVQVLKTV 120 PVNLSLNQDH
LENSKREQYS ISFPESSAII PVSGITVVKA EDFTPVFMAP PVHYPRGDGE 180
EQRVVIFEQT QYDVPSLATH SAYLKDDQRS TPDSTYSESF KDAATEKFRS ASVGAEEYMY
240 DQTSSGTFQY TLEATKSLRQ KQGEGPMTYL NKGQFYAITL SETGDNKCFR
HPISKVRSVV 300 MVVFSEDKNR DEQLKYWKYW HSRQHTAKQR VLDIADYKES
FNTIGNIEEI AYNAVSFTWD 360 VNEEAKIFIT VNCLSTDFSS QKGVKGLPLM
IQIDTYSYNN RSNKPIHRAY CQIKVFCDKG 420 AERKIRDEER KQNRKKGKGQ
ASQTQCNSSS DGKLAAIPLQ KKSDITYFKT MPDLHSQPVL 480 FIPDVHFANL
QRTGQVYYNT DDEREGGSVL VKRMFRPMEE EFGPVPSKQM KEEGTKRVLL 540
YVRKETDDVF DALMLKSPTV KGLMEAISEK YGLPVEKIAK LYKKSKKGIL VNMDDNIIEH
600 YSNEDTFILN MESMVEGFKV TLMEI 625
TABLE-US-00067 TABLE LV(a) Amino acid sequence alignment of
202P5A05 v.2 (SEQ ID NO: 100) and 202P5A05 v.1 (SEQ ID NO: 101) v.1
1 MPSDPPFNTRRAYTSEDEAWKSYLENPLTAATKAMMSINGDEDSAAALGL 50
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 17
MPSDPPFNTRRAYTSEDEAWKSYLENPLTAATKAMMSINGDEDSAAALGL 66 v.1 51
LYDYYKVPRDKRLLSVSKASDSQEDQEKRNCLGTSEAQSNLSGGENRVQV 100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 67
LYDYYKVPRDKRLLSVSKASDSQEDQEKRNCLGTSEAQSNLSGGENRVQV 116 v.1 101
LKTVPVNLSLNQDHLENSKREQYSISFPESSAIIPVSGITVVKAEDFTPV 150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 117
LKTVPVNLSLNQDHLENSKREQYSISFPESSAIIPVSGITVVKAEDFTPV 166 v.1 151
FMAPPVHYPRGDGEEQRVVIFEQTQYDVPSLATHSAYLKDDQRSTPDSTY 200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 167
FMAPPVHYPRGDGEEQRVVIFEQTQYDVPSLATHSAYLKDDQRSTPDSTY 216 v.1 201
SESFKDAATEKFRSASVGAEEYMYDQTSSGTFQYTLEATKSLRQKQGEGP 250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 217
SESFKDAATEKFRSASVGAEEYMYDQTSSGTFQYTLEATKSLRQKQGEGP 266 v.1 251
MTYLNKGQFYAITLSETGDNKCFRHPISKVRSVVMVVFSEDKNRDEQLKY 300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 267
MTYLNKGQFYAITLSETGDNKCFRHPISKVRSVVMVVFSEDKNRDEQLKY 316 v.1 301
WKYWHSRQHTAKQRVLDIADYKESFNTIGNIEEIAYNAVSFTWDVNEEAK 350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 317
WKYWHSRQHTAKQRVLDIADYKESFNTIGNIEEIAYNAVSFTWDVNEEAK 366 v.1 351
IFITVNCLSTDFSSQKGVKGLPLMIQIDTYSYNNRSNKPIHRAYCQIKVF 400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 367
IFITVNCLSTDFSSQKGVKGLPLMIQIDTYSYNNRSNKPIHRAYCQIKVF 416 v.1 401
CDKGAERKIRDEERKQNRKKGKGQASQTQCNSSSDGKLAAIPLQKKSDIT 450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 417
CDKGAERKIRDEERKQNRKKGKGQASQTQCNSSSDGKLAAIPLQKKSDIT 466 v.1 451
YFKTMPDLHSQPVLFIPDVHFANLQRTGQVYYNTDDEREGGSVLVKRMFR 500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 467
YFKTMPDLHSQPVLFIPDVHFANLQRTGQVYYNTDDEREGGSVLVKRMFR 516 v.1 501
PMEEEFGPVPSKQMKEEGTKRVLLYVRKETDDVFDALMLKSPTVKGLMEA 550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 517
PMEEEFGPVPSKQMKEEGTKRVLLYVRKETDDVFDALMLKSPTVKGLMEA 566 v.1 551
ISEKYGLPVEKIAKLYKKSKKGILVNMDDNIIEHYSNEDTFILNMESMVE 600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.2 567
ISEKYGLPVEKIAKLYKKSKKGILVNMDDNIIEHYSNEDTFILNMESMVE 616 v.1 601
GFKVTLMEI 609 ||||||||| v.2 617 GFKVTLMEI 625
TABLE-US-00068 TABLE LII(b) Nucleotide sequence of transcript
variant 202P5A05 v.3 (SEQ ID NO: 102) attggatcaa acatgtcaca
agagtcggac aagtaagtgg atcacacgcg ccggctgctg 60 ctactactac
cactttgggc tgatggcaac tgtaataaaa gactagtggc cttagtgccc 120
atgcccagtg accctccatt caatacccga agagcctaca ccagtgagga tgaagcctgg
180 aagtcatact tggagaatcc cctgacagca gccaccaagg ccatgatgag
cattaatggt 240 gatgaggaca gtgctgctgc cctcggcctg ctctatgact
actacaaggt tcctcgagac 300 aagaggctgc tgtctgtaag caaagcaagt
gacagccaag aagaccagga gaaaagaaac 360 tgccttggca ccagtgaagc
ccagagtaat ttgagtggag gagaaaaccg agtgcaagtc 420 ctaaagactg
ttccagtgaa cctttcccta aatcaagatc acctggagaa ttccaagcgg 480
gaacagtaca gcatcagctt ccccgagagc tctgccatca tcccggtgtc gggaatcacg
540 gtggtgaaag ctgaagattt cacaccagtt ttcatggccc cacctgtgca
ctatccccgg 600 ggagatgggg aagagcaacg agtggttatc tttgaacaga
ctcagtatga cgtgccctcg 660 ctggccaccc acagcgccta tctcaaagac
gaccagcgca gcactccgga cagcacatac 720 agcgagagct tcaaggacgc
agccacagag aaatttcgga gtgcttcagt tggggctgag 780 gagtacatgt
atgatcagac atcaagtggc acatttcagt acaccctgga agccaccaaa 840
tctctccgtc agaagcaggg ggagggcccc atgacctacc tcaacaaagg acagttctat
900 gccataacac tcagcgagac cggagacaac aaatgcttcc gacaccccat
cagcaaagtc 960 aggagtgtgg tgatggtggt cttcagtgaa gacaaaaaca
gagatgaaca gctcaaatac 1020 tggaaatact ggcactctcg gcagcatacg
gcgaagcaga gggtccttga cattgccgat 1080 tacaaggaga gctttaatac
gattggaaac attgaagaga ttgcatataa tgctgtttcc 1140 tttacctggg
acgtgaatga agaggcgaag attttcatca ccgtgaattg cttgagcaca 1200
gatttctcct cccaaaaagg ggtgaaagga cttcctttga tgattcagat tgacacatac
1260 agttataaca atcgtagcaa taaacccatt catagagctt attgccagat
caaggtcttc 1320 tgtgacaaag gagcagaaag aaaaatccga gatgaagagc
ggaagcagaa caggaagaaa 1380 gggaaaggcc aggcctccca aactcaatgc
aacagctcct ctgatgggaa gttggctgcc 1440 atacctttac agaagaagag
tgacatcacc tacttcaaaa ccatgcctga tctccactca 1500 cagccagttc
tcttcatacc tgatgttcac tttgcaaacc tgcagaggac cggacaggtg 1560
tattacaaca cggatgatga acgagaaggt ggcagtgtcc ttgttaaacg gatgttccgg
1620 cccatggaag aggagtttgg tccagtgcct tcaaagcaga tgaaagaaga
agggacaaag 1680 cgagtgctct tgtacgtgag gaaggagact gacgatgtgt
tcgatgcatt gatgttgaag 1740 tctcccacag tgaagggcct gatggaagcg
atatctgaga aatatgggct gcccgtggag 1800 aagatagcaa agctttacaa
gaaaagcaaa aaaggcatct tggtgaacat ggatgacaac 1860 atcatcgagc
actactcgaa cgaggacacc ttcatcctca acatggagag catggtggag 1920
ggcttcaagg tcacgctcat ggaaatctag ccctgggttt ggcatccgct ttggctggag
1980 ctctcagtgc gttcctccct gagagagaca gaagccccag ccccagaacc
tggagaccca 2040 tctcccccat ctcacaactg ctgttacaag accgtgctgg
ggagtggggc aagggacagg 2100 ccccactgtc ggtgtgcttg gcccatccac
tggcacctac cacggagctg aagcctgagc 2160 ccctcaggaa ggtgccttag
gcctgttgga ttcctattta ttgcccacct tttcctggag 2220 cccaggtcca
ggcccgccag gactctgcag gtcactgcta gctccagatg agaccgtcca 2280
gcgttccccc ttcaagagaa acactcatcc cgaacagcct aaaaaattcc catcccttct
2340 ctctcacccc tccatatcta tctcccgagt ggctggacaa aatgagctac
gtctgggtgc 2400 agtagttata ggtggggcaa gaggtggatg cccactttct
ggtcagacac ctttaggttg 2460 ctctggggaa ggctgtcttg ctaaatacct
ccagggttcc cagcaagtgg ccaccaggcc 2520 ttgtacagga agacattcag
tcaccgtgta attagtaaca cagaaagtct gcctgtctgc 2580 attgtacata
gtgtttataa tattgtaata atatatttta cctgtggtat gtgggcatgt 2640
ttactgccac tggcctagag gagacacaga cctggagacc gttttaatgg gggtttttgc
2700 ctctgtgcct gttcaagaga cttgcagggc taggtagagg gcctttggga
tgttaaggtg 2760 actgcagctg atgccaagat ggactctgca atgggcatac
ctgggggctc gttccctgtc 2820 cccagaggaa gccccctctc cttctccatg
ggcatgactc tccttcgagg ccaccacgtt 2880 tatctcacaa tgatgtgttt
tgcttgactt tccctttgcg ctgtctcgtg ggaaaggtca 2940 ttctgtctga
gaccccagct ccttctccag ctttggctgc gggcatggcc tgagctttct 3000
ggagagcctc tgcagggggt ttgccatcag ggccctgtgg ctgggtctgc tgcagagctc
3060 cttggctatc aggagaatcc tggacactgt actgtgcctc ccagtttaca
aacacgccct 3120 tcatctcaag tggcccttta aaaggcctgc tgccatgtga
gagctgtgaa cagctcagct 3180 ctgagtcggc aggctggggc ttcctcctgg
gccaccagat ggaaaggggg tattgtttgc 3240 ctcactcctg gatgctgcgt
tttaaggaag tgagtgagaa agaatgtgcc aagatacctg 3300 gctcctgtga
aaccagcctc aggagggaaa ctgggagaga gaagctgtgg tctcctgcta 3360
catgccctgg gagctggaag agaaaaacac tcccctaaac aatcgcaaaa tgatgaacca
3420 tcatgggcca ctgttctctt tgaggggaca ggtttagggg tttgcgttcg
cccttgtggg 3480 ctgaagcact agctttttgg tagctagaca catcctgcac
ccaaaggttc tctacaaagg 3540 cccagatttg tttgtaaagc actttgactc
ttacctggag gcccgctctc taagggcttc 3600 ctgcgctccc acctcatctg
tccctgagat gcagagcagg atggagggtc tgcttctagc 3660 tcagctgttt
ctccttgagg ttgcggagga attgaattga atgggacaga gggcaggtgc 3720
tgtggccaag aagatctccg agcagcagtg acggggcacc ttgctgtgtg tcctctgggc
3780 atgttaaccc ttctgtgggg ccaaaggttt gcatcgtgga tccagctgtg
ctccagtctg 3840 tcccctcctc ctccactctg actgccacgc cccggaccag
cagcttgggg accctccagg 3900 gtactaatgg ggctctgttc tgagatggac
aaattcagtg ttggaaatac atgttgtact 3960 atgcacttcc catgctccta
gggttaggaa tagtttcaaa catgattggc agacataaca 4020 acggcaaata
ctcggactgg ggcataggac tccagagtag gaaaaagaca aaagatttgg 4080
cagcctgaca caggcaacct acccctctct ctccagcctc tttatgaaac tgtttgtttg
4140 ccagtcctgc cctaaggcag aagatgaatt gaagatgctg tgcatgtttc
ctaagtcctt 4200 gagcaatcat ggtggtgaca attgccacaa gggatatgag
gccagtgcca ccagagggtg 4260 gtgccaagtg ccacatccct tccgatccat
tcccctctgc atcctcggag caccccagtt 4320 tgcctttgat gtgtccgctg
tgtatgttag ctgaactttg atgagcaaaa tttcctgagc 4380 gaaacactcc
aaagagatag gaaaacttgc cgcctcttct tttttgtccc ttaatcaaac 4440
tcaaataagc ttaaaaaaaa tccatggaag atcatggaca tgtgaaatga gcattttttt
4500 cttttttttt tttaacaaag tctgaactga acagaacaag actttttcct
catacatctc 4560 caaattgttt aaacttactt tatgagtgtt tgtttagaag
ttcggaccaa cagaaaaatg 4620 cagtcagatg tcatcttgga attggtttct
aaaagagtaa ggcatgtccc tgcccagaaa 4680 cttaggaagc atgaaataaa
tcaaatgttt attttccttc ttatttaaaa tcatgcaaat 4740 gcaacagaaa
tagagggttt gtgccaaatg ctatgaacgg ccctttctta aagacaagca 4800
agggagattg atatatgtac aatttgctct catgttttaa aaaaaaaagg taaatgtaac
4860 ttaatagttt tgtaaatggg agagggggaa tctataaact ataaatacag
ttattttatt 4920 ttttgtacat ttttaaggag aaaaaaataa atattcataa
cataagagga aaa 4973
TABLE-US-00069 TABLE LIII(b) Nucleotide sequence alignment of
202P5A05 v.3 (SEQ ID NO: 103) and 202P5A05 v.1 (SEQ ID NO: 104) v.1
1 TAATAAAAGACTAGTGGCCTTAGTGCCCATGCCCAGTGACCCTCCATTCA 50
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 93
taataaaagactagtggccttagtgcccatgcccagtgaccctccattca 142 v.1 51
ATACCCGAAGAGCCTACACCAGTGAGGATGAAGCCTGGAAGTCATACTTG 100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 143
atacccgaagagcctacaccagtgaggatgaagcctggaagtcatacttg 192 v.1 101
GAGAATCCCCTGACAGCAGCCACCAAGGCCATGATGAGCATTAATGGTGA 150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 193
gagaatcccctgacagcagccaccaaggccatgatgagcattaatggtga 242 v.1 151
TGAGGACAGTGCTGCTGCCCTCGGCCTGCTCTATGACTACTACAAGGTTC 200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 243
tgaggacagtgctgctgccctcggcctgctctatgactactacaaggttc 292 v.1 201
CTCGAGACAAGAGGCTGCTGTCTGTAAGCAAAGCAAGTGACAGCCAAGAA 250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 293
ctcgagacaagaggctgctgtctgtaagcaaagcaagtgacagccaagaa 342 v.1 251
GACCAGGAGAAAAGAAACTGCCTTGGCACCAGTGAAGCCCAGAGTAATTT 300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 343
gaccaggagaaaagaaactgccttggcaccagtgaagcccagagtaattt 392 v.1 301
GAGTGGAGGAGAAAACCGAGTGCAAGTCCTAAAGACTGTTCCAGTGAACC 350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 393
gagtggaggagaaaaccgagtgcaagtcctaaagactgttccagtgaacc 442 v.1 351
TTTCCCTAAATCAAGATCACCTGGAGAATTCCAAGCGGGAACAGTACAGC 400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 443
tttccctaaatcaagatcacctggagaattccaagcgggaacagtacagc 492 v.1 401
ATCAGCTTCCCCGAGAGCTCTGCCATCATCCCGGTGTCGGGAATCACGGT 450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 493
atcagcttccccgagagctctgccatcatcccggtgtcgggaatcacggt 542 v.1 451
GGTGAAAGCTGAAGATTTCACACCAGTTTTCATGGCCCCACCTGTGCACT 500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 543
ggtgaaagctgaagatttcacaccagttttcatggccccacctgtgcact 592 v.1 501
ATCCCCGGGGAGATGGGGAAGAGCAACGAGTGGTTATCTTTGAACAGACT 550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 593
atccccggggagatggggaagagcaacgagtggttatctttgaacagact 642 v.1 551
CAGTATGACGTGCCCTCGCTGGCCACCCACAGCGCCTATCTCAAAGACGA 600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 643
cagtatgacgtgccctcgctggccacccacagcgcctatctcaaagacga 692 v.1 601
CCAGCGCAGCACTCCGGACAGCACATACAGCGAGAGCTTCAAGGACGCAG 650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 693
ccagcgcagcactccggacagcacatacagcgagagcttcaaggacgcag 742 v.1 651
CCACAGAGAAATTTCGGAGTGCTTCAGTTGGGGCTGAGGAGTACATGTAT 700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 743
ccacagagaaatttcggagtgcttcagttggggctgaggagtacatgtat 792 v.1 701
GATCAGACATCAAGTGGCACATTTCAGTACACCCTGGAAGCCACCAAATC 750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 793
gatcagacatcaagtggcacatttcagtacaccctggaagccaccaaatc 842 v.1 751
TCTCCGTCAGAAGCAGGGGGAGGGCCCCATGACCTACCTCAACAAAGGAC 800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 843
tctccgtcagaagcagggggagggccccatgacctacctcaacaaaggac 892 v.1 801
AGTTCTATGCCATAACACTCAGCGAGACCGGAGACAACAAATGCTTCCGA 850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 893
agttctatgccataacactcagcgagaccggagacaacaaatgcttccga 942 v.1 851
CACCCCATCAGCAAAGTCAGGAGTGTGGTGATGGTGGTCTTCAGTGAAGA 900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 943
caccccatcagcaaagtcaggagtgtggtgatggtggtcttcagtgaaga 992 v.1 901
CAAAAACAGAGATGAACAGCTCAAATACTGGAAATACTGGCACTCTCGGC 950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 993
caaaaacagagatgaacagctcaaatactggaaatactggcactctcggc 1042 v.1 951
AGCATACGGCGAAGCAGAGGGTCCTTGACATTGCCGATTACAAGGAGAGC 1000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1043
agcatacggcgaagcagagggtccttgacattgccgattacaaggagagc 1092 v.1 1001
TTTAATACGATTGGAAACATTGAAGAGATTGCATATAATGCTGTTTCCTT 1050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1093
tttaatacgattggaaacattgaagagattgcatataatgctgtttcctt 1142 v.1 1051
TACCTGGGACGTGAATGAAGAGGCGAAGATTTTCATCACCGTGAATTGCT 1100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1143
tacctgggacgtgaatgaagaggcgaagattttcatcaccgtgaattgct 1192 v.1 1101
TGAGCACAGATTTCTCCTCCCAAAAAGGGGTGAAAGGACTTCCTTTGATG 1150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1193
tgagcacagatttctcctcccaaaaaggggtgaaaggacttcctttgatg 1242 v.1 1151
ATTCAGATTGACACATACAGTTATAACAATCGTAGCAATAAACCCATTCA 1200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1243
attcagattgacacatacagttataacaatcgtagcaataaacccattca 1292 v.1 1201
TAGAGCTTATTGCCAGATCAAGGTCTTCTGTGACAAAGGAGCAGAAAGAA 1250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1293
tagagcttattgccagatcaaggtcttctgtgacaaaggagcagaaagaa 1342 v.1 1251
AAATCCGAGATGAAGAGCGGAAGCAGAACAGGAAGAAAGGGAAAGGCCAG 1300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1343
aaatccgagatgaagagcggaagcagaacaggaagaaagggaaaggccag 1392 v.1 1301
GCCTCCCAAACTCAATGCAACAGCTCCTCTGATGGGAAGTTGGCTGCCAT 1350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1393
gcctcccaaactcaatgcaacagctcctctgatgggaagttggctgccat 1442 v.1 1351
ACCTTTACAGAAGAAGAGTGACATCACCTACTTCAAAACCATGCCTGATC 1400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1443
acctttacagaagaagagtgacatcacctacttcaaaaccatgcctgatc 1492 v.1 1401
TCCACTCACAGCCAGTTCTCTTCATACCTGATGTTCACTTTGCAAACCTG 1450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1493
tccactcacagccagttctcttcatacctgatgttcactttgcaaacctg 1542 v.1 1451
CAGAGGACCGGACAGGTGTATTACAACACGGATGATGAACGAGAAGGTGG 1500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1543
cagaggaccggacaggtgtattacaacacggatgatgaacgagaaggtgg 1592 v.1 1501
CAGTGTCCTTGTTAAACGGATGTTCCGGCCCATGGAAGAGGAGTTTGGTC 1550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1593
cagtgtccttgttaaacggatgttccggcccatggaagaggagtttggtc 1642 v.1 1551
CAGTGCCTTCAAAGCAGATGAAAGAAGAAGGGACAAAGCGAGTGCTCTTG 1600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1643
cagtgccttcaaagcagatgaaagaagaagggacaaagcgagtgctcttg 1692 v.1 1601
TACGTGAGGAAGGAGACTGACGATGTGTTCGATGCATTGATGTTGAAGTC 1650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1693
tacgtgaggaaggagactgacgatgtgttcgatgcattgatgttgaagtc 1742 v.1 1651
TCCCACAGTGAAGGGCCTGATGGAAGCGATATCTGAGAAATATGGGCTGC 1700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1743
tcccacagtgaagggcctgatggaagcgatatctgagaaatatgggctgc 1792 v.1 1701
CCGTGGAGAAGATAGCAAAGCTTTACAAGAAAAGCAAAAAAGGCATCTTG 1750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1793
ccgtggagaagatagcaaagctttacaagaaaagcaaaaaaggcatcttg 1842 v.1 1751
GTGAACATGGATGACAACATCATCGAGCACTACTCGAACGAGGACACCTT 1800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1843
gtgaacatggatgacaacatcatcgagcactactcgaacgaggacacctt 1892 v.1 1801
CATCCTCAACATGGAGAGCATGGTGGAGGGCTTCAAGGTCACGCTCATGG 1850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1893
catcctcaacatggagagcatggtggagggcttcaaggtcacgctcatgg 1942 v.1 1851
AAATCTAGCCCTGGGTTTGGCATCCGCTTTGGCTGGAGCTCTCAGTGCGT 1900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1943
aaatctagccctgggtttggcatccgctttggctggagctctcagtgcgt 1992 v.1 1901
TCCTCCCTGAGAGAGACAGAAGCCCCAGCCCCAGAACCTGGAGACCCATC 1950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1993
tcctccctgagagagacagaagccccagccccagaacctggagacccatc 2042 v.1 1951
TCCCCCATCTCACAACTGCTGTTACAAGACCGTGCTGGGGAGTGGGGCAA 2000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2043
tcccccatctcacaactgctgttacaagaccgtgctggggagtggggcaa 2092 v.1 2001
GGGACAGGCCCCACTGTCGGTGTGCTTGGCCCATCCACTGGCACCTACCA 2050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2093
gggacaggccccactgtcggtgtgcttggcccatccactggcacctacca 2142 v.1 2051
CGGAGCTGAAGCCTGAGCCCCTCAGGAAGGTGCCTTAGGCCTGTTGGATT 2100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2143
cggagctgaagcctgagcccctcaggaaggtgccttaggcctgttggatt 2192 v.1 2101
CCTATTTATTGCCCACCTTTTCCTGGAGCCCAGGTCCAGGCCCGCCAGGA 2150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2193
cctatttattgcccaccttttcctggagcccaggtccaggcccgccagga 2242 v.1 2151
CTCTGCAGGTCACTGCTAGCTCCAGATGAGACCGTCCAGCGTTCCCCCTT 2200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2243
ctctgcaggtcactgctagctccagatgagaccgtccagcgttccccctt 2292 v.1 2201
CAAGAGAAACACTCATCCCGAACAGCCTAAAAAATTCCCATCCCTTCTCT 2250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2293
caagagaaacactcatcccgaacagcctaaaaaattcccatcccttctct 2342 v.1 2251
CTCACCCCTCCATATCTATCTCCCGAGTGGCTGGACAAAATGAGCTACGT 2300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2343
ctcacccctccatatctatctcccgagtggctggacaaaatgagctacgt 2392 v.1 2301
CTGGGTGCAGTAGTTATAGGTGGGGCAAGAGGTGGATGCCCACTTTCTGG 2350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2393
ctgggtgcagtagttataggtggggcaagaggtggatgcccactttctgg 2442 v.1 2351
TCAGACACCTTTAGGTTGCTCTGGGGAAGGCTGTCTTGCTAAATACCTCC 2400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2443
tcagacacctttaggttgctctggggaaggctgtcttgctaaatacctcc 2492 v.1 2401
AGGGTTCCCAGCAAGTGGCCACCAGGCCTTGTACAGGAAGACATTCAGTC 2450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2493
agggttcccagcaagtggccaccaggccttgtacaggaagacattcagtc 2542 v.1 2451
ACCGTGTAATTAGTAACACAGAAAGTCTGCCTGTCTGCATTGTACATAGT 2500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2543
accgtgtaattagtaacacagaaagtctgcctgtctgcattgtacatagt 2592 v.1 2501
GTTTATAATATTGTAATAATATATTTTACCTGTGGTATGTGGGCATGTTT 2550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2593
gtttataatattgtaataatatattttacctgtggtatgtgggcatgttt 2642 v.1 2551
ACTGCCACTGGCCTAGAGGAGACACAGACCTGGAGACCGTTTTAATGGGG 2600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2643
actgccactggcctagaggagacacagacctggagaccgttttaatgggg 2692 v.1 2601
GTTTTTGCCTCTGTGCCTGTTCAAGAGACTTGCAGGGCTAGGTAGAGGGC 2650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2693
gtttttgcctctgtgcctgttcaagagacttgcagggctaggtagagggc 2742 v.1 2651
CTTTGGGATGTTAAGGTGACTGCAGCTGATGCCAAGATGGACTCTGCAAT 2700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2743
ctttgggatgttaaggtgactgcagctgatgccaagatggactctgcaat 2792 v.1 2701
GGGCATACCTGGGGGCTCGTTCCCTGTCCCCAGAGGAAGCCCCCTCTCCT 2750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2793
gggcatacctgggggctcgttccctgtccccagaggaagccccctctcct 2842 v.1 2751
TCTCCATGGGCATGACTCTCCTTCGAGGCCACCACGTTTATCTCACAATG 2800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2843
tctccatgggcatgactctccttcgaggccaccacgtttatctcacaatg 2892 v.1 2801
ATGTGTTTTGCTTGACTTTCCCTTTGCGCTGTCTCGTGGGAAAGGTCATT 2850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2893
atgtgttttgcttgactttccctttgcgctgtctcgtgggaaaggtcatt 2942 v.1 2851
CTGTCTGAGACCCCAGCTCCTTCTCCAGCTTTGGCTGCGGGCATGGCCTG 2900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2943
ctgtctgagaccccagctccttctccagctttggctgcgggcatggcctg 2992 v.1 2901
AGCTTTCTGGAGAGCCTCTGCAGGGGGTTTGCCATCAGGGCCCTGTGGCT 2950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 2993
agctttctggagagcctctgcagggggtttgccatcagggccctgtggct 3042 v.1 2951
GGGTCTGCTGCAGAGCTCCTTGGCTATCAGGAGAATCCTGGACACTGTAC 3000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3043
gggtctgctgcagagctccttggctatcaggagaatcctggacactgtac 3092 v.1 3001
TGTGCCTCCCAGTTTACAAACACGCCCTTCATCTCAAGTGGCCCTTTAAA 3050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3093
tgtgcctcccagtttacaaacacgcccttcatctcaagtggccctttaaa 3142
v.1 3051 AGGCCTGCTGCCATGTGAGAGCTGTGAACAGCTCAGCTCTGAGTCGGCAG 3100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3143
aggcctgctgccatgtgagagctgtgaacagctcagctctgagtcggcag 3192 v.1 3101
GCTGGGGCTTCCTCCTGGGCCACCAGATGGAAAGGGGGTATTGTTTGCCT 3150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3193
gctggggcttcctcctgggccaccagatggaaagggggtattgtttgcct 3242 v.1 3151
CACTCCTGGATGCTGCGTTTTAAGGAAGTGAGTGAGAAAGAATGTGCCAA 3200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3243
cactcctggatgctgcgttttaaggaagtgagtgagaaagaatgtgccaa 3292 v.1 3201
GATACCTGGCTCCTGTGAAACCAGCCTCAGGAGGGAAACTGGGAGAGAGA 3250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3293
gatacctggctcctgtgaaaccagcctcaggagggaaactgggagagaga 3342 v.1 3251
AGCTGTGGTCTCCTGCTACATGCCCTGGGAGCTGGAAGAGAAAAACACTC 3300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3343
agctgtggtctcctgctacatgccctgggagctggaagagaaaaacactc 3392 v.1 3301
CCCTAAACAATCGCAAAATGATGAACCATCATGGGCCACTGTTCTCTTTG 3350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3393
ccctaaacaatcgcaaaatgatgaaccatcatgggccactgttctctttg 3442 v.1 3351
AGGGGACAGGTTTAGGGGTTTGCGTTCGCCCTTGTGGGCTGAAGCACTAG 3400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3443
aggggacaggtttaggggtttgcgttcgcccttgtgggctgaagcactag 3492 v.1 3401
CTTTTTGGTAGCTAGACACATCCTGCACCCAAAGGTTCTCTACAAAGGCC 3450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3493
ctttttggtagctagacacatcctgcacccaaaggttctctacaaaggcc 3542 v.1 3451
CAGATTTGTTTGTAAAGCACTTTGACTCTTACCTGGAGGCCCGCTCTCTA 3500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3543
cagatttgtttgtaaagcactttgactcttacctggaggcccgctctcta 3592 v.1 3501
AGGGCTTCCTGCGCTCCCACCTCATCTGTCCCTGAGATGCAGAGCAGGAT 3550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3593
agggcttcctgcgctcccacctcatctgtccctgagatgcagagcaggat 3642 v.1 3551
GGAGGGTCTGCTTCTAGCTCAGCTGTTTCTCCTTGAGGTTGCGGAGGAAT 3600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3643
ggagggtctgcttctagctcagctgtttctccttgaggttgcggaggaat 3692 v.1 3601
TGAATTGAATGGGACAGAGGGCAGGTGCTGTGGCCAAGAAGATCTCCGAG 3650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3693
tgaattgaatgggacagagggcaggtgctgtggccaagaagatctccgag 3742 v.1 3651
CAGCAGTGACGGGGCACCTTGCTGTGTGTCCTCTGGGCATGTTAACCCTT 3700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3743
cagcagtgacggggcaccttgctgtgtgtcctctgggcatgttaaccctt 3792 v.1 3701
CTGTGGGGCCAAAGGTTTGCATCGTGGATCCAGCTGTGCTCCAGTCTGTC 3750
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3793
ctgtggggccaaaggtttgcatcgtggatccagctgtgctccagtctgtc 3842 v.1 3751
CCCTCCTCCTCCACTCTGACTGCCACGCCCCGGACCAGCAGCTTGGGGAC 3800
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3843
ccctcctcctccactctgactgccacgccccggaccagcagcttggggac 3892 v.1 3801
CCTCCAGGGTACTAATGGGGCTCTGTTCTGAGATGGACAAATTCAGTGTT 3850
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3893
cctccagggtactaatggggctctgttctgagatggacaaattcagtgtt 3942 v.1 3851
GGAAATACATGTTGTACTATGCACTTCCCATGCTCCTAGGGTTAGGAATA 3900
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3943
ggaaatacatgttgtactatgcacttcccatgctcctagggttaggaata 3992 v.1 3901
GTTTCAAACATGATTGGCAGACATAACAACGGCAAATACTCGGACTGGGG 3950
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 3993
gtttcaaacatgattggcagacataacaacggcaaatactcggactgggg 4042 v.1 3951
CATAGGACTCCAGAGTAGGAAAAAGACAAAAGATTTGGCAGCCTGACACA 4000
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4043
cataggactccagagtaggaaaaagacaaaagatttggcagcctgacaca 4092 v.1 4001
GGCAACCTACCCCTCTCTCTCCAGCCTCTTTATGAAACTGTTTGTTTGCC 4050
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4093
ggcaacctacccctctctctccagcctctttatgaaactgtttgtttgcc 4142 v.1 4051
AGTCCTGCCCTAAGGCAGAAGATGAATTGAAGATGCTGTGCATGTTTCCT 4100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4143
agtcctgccctaaggcagaagatgaattgaagatgctgtgcatgtttcct 4192 v.1 4101
AAGTCCTTGAGCAATCATGGTGGTGACAATTGCCACAAGGGATATGAGGC 4150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4193
aagtccttgagcaatcatggtggtgacaattgccacaagggatatgaggc 4242 v.1 4151
CAGTGCCACCAGAGGGTGGTGCCAAGTGCCACATCCCTTCCGATCCATTC 4200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4243
cagtgccaccagagggtggtgccaagtgccacatcccttccgatccattc 4292 v.1 4201
CCCTCTGCATCCTCGGAGCACCCCAGTTTGCCTTTGATGTGTCCGCTGTG 4250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4293
ccctctgcatcctcggagcaccccagtttgcctttgatgtgtccgctgtg 4342 v.1 4251
TATGTTAGCTGAACTTTGATGAGCAAAATTTCCTGAGCGAAACACTCCAA 4300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4343
tatgttagctgaactttgatgagcaaaatttcctgagcgaaacactccaa 4392 v.1 4301
AGAGATAGGAAAACTTGCCGCCTCTTCTTTTTTGTCCCTTAATCAAACTC 4350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4393
agagataggaaaacttgccgcctcttcttttttgtcccttaatcaaactc 4442 v.1 4351
AAATAAGCTTAAAAAAAATCCATGGAAGATCATGGACATGTGAAATGAGC 4400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4443
aaataagcttaaaaaaaatccatggaagatcatggacatgtgaaatgagc 4492 v.1 4401
ATTTTTTTCTTTTTTTTTTTTAACAAAGTCTGAACTGAACAGAACAAGAC 4450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4493
atttttttcttttttttttttaacaaagtctgaactgaacagaacaagac 4542 v.1 4451
TTTTTCCTCATACATCTCCAAATTGTTTAAACTTACTTTATGAGTGTTTG 4500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4543
tttttcctcatacatctccaaattgtttaaacttactttatgagtgtttg 4592 v.1 4501
TTTAGAAGTTCGGACCAACAGAAAAATGCAGTCAGATGTCATCTTGGAAT 4550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4593
tttagaagttcggaccaacagaaaaatgcagtcagatgtcatcttggaat 4642 v.1 4551
TGGTTTCTAAAAGAGTAAGGCATGTCCCTGCCCAGAAACTTAGGAAGCAT 4600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4643
tggtttctaaaagagtaaggcatgtccctgcccagaaacttaggaagcat 4692 v.1 4601
GAAATAAATCAAATGTTTATTTTCCTTCTTATTTAAAATCATGCAAATGC 4650
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4693
gaaataaatcaaatgtttattttccttcttatttaaaatcatgcaaatgc 4742 v.1 4651
AACAGAAATAGAGGGTTTGTGCCAAATGCTATGAACGGCCCTTTCTTAAA 4700
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 4743
aacagaaatagagggtttgtgccaaatgctatgaacggccctttcttaaa 4792 v.1 4701
GACAAGCAAGGGAGATTGATATATGTACAATTTGCTCTCATGTTTT 4746
|||||||||||||||||||||||||||||||||||||||||||||| v.3 4793
gacaagcaagggagattgatatatgtacaatttgctctcatgtttt 4838
TABLE-US-00070 TABLE LIV(b) Peptide sequences of protein coded by
202P5A05 v.3 (SEQ ID NO: 105) MPSDPPFNTR RAYTSEDEAW KSYLENPLTA
ATKAMMSING DEDSAAALGL LYDYYKVPRD 60 KRLLSVSKAS DSQEDQEKRN
CLGTSEAQSN LSGGENRVQV LKTVPVNLSL NQDHLENSKR 120 EQYSISFPES
SAIIPVSGIT VVKAEDFTPV FMAPPVHYPR GDGEEQRVVI FEQTQYDVPS 180
LATHSAYLKD DQRSTPDSTY SESFKDAATE KFRSASVGAE EYMYDQTSSG TFQYTLEATK
240 SLRQKQGEGP MTYLNKGQFY AITLSETGDN KCFRHPISKV RSVVMVVFSE
DKNRDEQLKY 300 WKYWHSRQHT AKQRVLDIAD YKESFNTIGN IEEIAYNAVS
FTWDVNEEAK IFITVNCLST 360 DFSSQKGVKG LPLMIQIDTY SYNNRSNKPI
HRAYCQIKVF CDKGAERKIR DEERKQNRKK 420 GKGQASQTQC NSSSDGKLAA
IPLQKKSDIT YFKTMPDLHS QPVLFIPDVH FANLQRTGQV 480 YYNTDDEREG
GSVLVKRMFR PMEEEFGPVP SKQMKEEGTK RVLLYVRKET DDVFDALMLK 540
SPTVKGLMEA ISEKYGLPVE KIAKLYKKSK KGILVNMDDN IIEHYSNEDT FILNMESMVE
600 GFKVTLMEI 609
TABLE-US-00071 TABLE LV(b) Amino acid sequence alignment of
202P5A05 v.3 (SEQ ID NO: 106) and 202P5A05 v.1 (SEQ ID NO: 107) v.1
1 MPSDPPFNTRRAYTSEDEAWKSYLENPLTAATKAMMSINGDEDSAAALGL 50
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 1
MPSDPPFNTRRAYTSEDEAWKSYLENPLTAATKAMMSINGDEDSAAALGL 50 v.1 51
LYDYYKVPRDKRLLSVSKASDSQEDQEKRNCLGTSEAQSNLSGGENRVQV 100
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 51
LYDYYKVPRDKRLLSVSKASDSQEDQEKRNCLGTSEAQSNLSGGENRVQV 100 v.1 101
LKTVPVNLSLNQDHLENSKREQYSISFPESSAIIPVSGITVVKAEDFTPV 150
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 101
LKTVPVNLSLNQDHLENSKREQYSISFPESSAIIPVSGITVVKAEDFTPV 150 v.1 151
FMAPPVHYPRGDGEEQRVVIFEQTQYDVPSLATHSAYLKDDQRSTPDSTY 200
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 151
FMAPPVHYPRGDGEEQRVVIFEQTQYDVPSLATHSAYLKDDQRSTPDSTY 200 v.1 201
SESFKDAATEKFRSASVGAEEYMYDQTSSGTFQYTLEATKSLRQKQGEGP 250
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 201
SESFKDAATEKFRSASVGAEEYMYDQTSSGTFQYTLEATKSLRQKQGEGP 250 v.1 251
MTYLNKGQFYAITLSETGDNKCFRHPISKVRSVVMVVFSEDKNRDEQLKY 300
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 251
MTYLNKGQFYAITLSETGDNKCFRHPISKVRSVVMVVFSEDKNRDEQLKY 300 v.1 301
WKYWHSRQHTAKQRVLDIADYKESFNTIGNIEEIAYNAVSFTWDVNEEAK 350
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 301
WKYWHSRQHTAKQRVLDIADYKESFNTIGNIEEIAYNAVSFTWDVNEEAK 350 v.1 351
IFITVNCLSTDFSSQKGVKGLPLMIQIDTYSYNNRSNKPIHRAYCQIKVF 400
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 351
IFITVNCLSTDFSSQKGVKGLPLMIQIDTYSYNNRSNKPIHRAYCQIKVF 400 v.1 401
CDKGAERKIRDEERKQNRKKGKGQASQTQCNSSSDGKLAAIPLQKKSDIT 450
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 401
CDKGAERKIRDEERKQNRKKGKGQASQTQCNSSSDGKLAAIPLQKKSDIT 450 v.1 451
YFKTMPDLHSQPVLFIPDVHFANLQRTGQVYYNTDDEREGGSVLVKRMFR 500
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 451
YFKTMPDLHSQPVLFIPDVHFANLQRTGQVYYNTDDEREGGSVLVKRMFR 500 v.1 501
PMEEEFGPVPSKQMKEEGTKRVLLYVRKETDDVFDALMLKSPTVKGLMEA 550
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 501
PMEEEFGPVPSKQMKEEGTKRVLLYVRKETDDVFDALMLKSPTVKGLMEA 550 v.1 551
ISEKYGLPVEKIAKLYKKSKKGILVNMDDNIIEHYSNEDTFILNMESMVE 600
|||||||||||||||||||||||||||||||||||||||||||||||||| v.3 551
ISEKYGLPVEKIAKLYKKSKKGILVNMDDNIIEHYSNEDTFILNMESMVE 600 v.1 601
GFKVTLMEI 609 ||||||||| v.3 601 GFKVTLMEI 609
TABLE-US-00072 TABLE LVI SNP and codon changes in 202P5A5 v.1, v.2,
and v.3. v.1 Variant v.2 v.3 AA* AA relative AA AA AA AA SNP
Position change position to v.1 Position change position Position
change position g/t 138 S/I 37 v.4 170 S/I 53 230 S/I 37 g/a 1269
R/Q 414 v.5 1301 R/Q 430 1361 R/Q 414 a/c 1288 K/N 420 v.6 1320 K/N
436 1380 K/N 420 a/g 1552 P/P 508 v.7 1584 P/P 524 1644 P/P 508 a/t
1662 K/M 545 v.8 1694 K/M 561 1754 K/M 545 g/a 1899 Outside v.9
1931 Outside 1991 Outside ORF ORF ORF t/c 2057 Outside v.10 2089
Outside 2149 Outside ORF ORF ORF c/t 2143 Outside v.11 2175 Outside
2235 Outside ORF ORF ORF g/a 2144 Outside v.12 2176 Outside 2236
Outside ORF ORF ORF c/t 2249 Outside v.13 2281 Outside 2341 Outside
ORF ORF ORF --/at** 2269-70 Outside v.14 2301-2 Outside 2361-2
Outside ORF ORF ORF a/g 2576 Outside v.15 2608 Outside 2668 Outside
ORF ORF ORF t/c 2812 Outside v.16 2848 Outside 2908 Outside ORF ORF
ORF g/a 2836 Outside v.17 2868 Outside 2928 Outside ORF ORF ORF t/c
3059 Outside v.18 3091 Outside 3151 Outside ORF ORF ORF g/a 3101
Outside v.19 3133 Outside 3193 Outside ORF ORF ORF a/c 3309 Outside
v.20 3341 Outside 3401 Outside ORF ORF ORF t/c 3332 Outside v.21
3364 Outside 3424 Outside ORF ORF ORF a/-- 3427 Outside v.22 3459
Outside 3459 Outside ORF ORF ORF c/t 4208 Outside v.23 4240 Outside
4300 Outside ORF ORF ORF c/t 4264 Outside v.24 4296 Outside 4356
Outside ORF ORF ORF c/t 4409 Outside v.25 4441 Outside 4501 Outside
ORF ORF ORF a/t 4645 Outside v.26 4677 Outside 4737 Outside ORF ORF
ORF *AA: amino acid; **deletion of the corresponding base.
Sequence CWU 1
1
1081186DNAHomo sapiens 1gatcatggat tttttttaag cttatttgag tttgattaag
ggacaaaaaa gaagaggcgg 60caagttttcc tatctctttg gagtgtttcg ctcaggaaat
tttgctcatc aaaattcagc 120taacatacac agcggacaca tcaaaggcaa
actggggtgc tccgaggatg cagaggggaa 180tggatc 18624746DNAHomo
sapiensCDS(29)...(1858)misc_feature138v.1/v.4 K = G or T
2taataaaaga ctagtggcct tagtgccc atg ccc agt gac cct cca ttc aat 52
Met Pro Ser Asp Pro Pro Phe Asn 1 5acc cga aga gcc tac acc agt gag
gat gaa gcc tgg aag tca tac ttg 100Thr Arg Arg Ala Tyr Thr Ser Glu
Asp Glu Ala Trp Lys Ser Tyr Leu 10 15 20gag aat ccc ctg aca gca gcc
acc aag gcc atg atg akc att aat ggt 148Glu Asn Pro Leu Thr Ala Ala
Thr Lys Ala Met Met Ser Ile Asn Gly25 30 35 40gat gag gac agt gct
gct gcc ctc ggc ctg ctc tat gac tac tac aag 196Asp Glu Asp Ser Ala
Ala Ala Leu Gly Leu Leu Tyr Asp Tyr Tyr Lys 45 50 55gtt cct cga gac
aag agg ctg ctg tct gta agc aaa gca agt gac agc 244Val Pro Arg Asp
Lys Arg Leu Leu Ser Val Ser Lys Ala Ser Asp Ser 60 65 70caa gaa gac
cag gag aaa aga aac tgc ctt ggc acc agt gaa gcc cag 292Gln Glu Asp
Gln Glu Lys Arg Asn Cys Leu Gly Thr Ser Glu Ala Gln 75 80 85agt aat
ttg agt gga gga gaa aac cga gtg caa gtc cta aag act gtt 340Ser Asn
Leu Ser Gly Gly Glu Asn Arg Val Gln Val Leu Lys Thr Val 90 95
100cca gtg aac ctt tcc cta aat caa gat cac ctg gag aat tcc aag cgg
388Pro Val Asn Leu Ser Leu Asn Gln Asp His Leu Glu Asn Ser Lys
Arg105 110 115 120gaa cag tac agc atc agc ttc ccc gag agc tct gcc
atc atc ccg gtg 436Glu Gln Tyr Ser Ile Ser Phe Pro Glu Ser Ser Ala
Ile Ile Pro Val 125 130 135tcg gga atc acg gtg gtg aaa gct gaa gat
ttc aca cca gtt ttc atg 484Ser Gly Ile Thr Val Val Lys Ala Glu Asp
Phe Thr Pro Val Phe Met 140 145 150gcc cca cct gtg cac tat ccc cgg
gga gat ggg gaa gag caa cga gtg 532Ala Pro Pro Val His Tyr Pro Arg
Gly Asp Gly Glu Glu Gln Arg Val 155 160 165gtt atc ttt gaa cag act
cag tat gac gtg ccc tcg ctg gcc acc cac 580Val Ile Phe Glu Gln Thr
Gln Tyr Asp Val Pro Ser Leu Ala Thr His 170 175 180agc gcc tat ctc
aaa gac gac cag cgc agc act ccg gac agc aca tac 628Ser Ala Tyr Leu
Lys Asp Asp Gln Arg Ser Thr Pro Asp Ser Thr Tyr185 190 195 200agc
gag agc ttc aag gac gca gcc aca gag aaa ttt cgg agt gct tca 676Ser
Glu Ser Phe Lys Asp Ala Ala Thr Glu Lys Phe Arg Ser Ala Ser 205 210
215gtt ggg gct gag gag tac atg tat gat cag aca tca agt ggc aca ttt
724Val Gly Ala Glu Glu Tyr Met Tyr Asp Gln Thr Ser Ser Gly Thr Phe
220 225 230cag tac acc ctg gaa gcc acc aaa tct ctc cgt cag aag cag
ggg gag 772Gln Tyr Thr Leu Glu Ala Thr Lys Ser Leu Arg Gln Lys Gln
Gly Glu 235 240 245ggc ccc atg acc tac ctc aac aaa gga cag ttc tat
gcc ata aca ctc 820Gly Pro Met Thr Tyr Leu Asn Lys Gly Gln Phe Tyr
Ala Ile Thr Leu 250 255 260agc gag acc gga gac aac aaa tgc ttc cga
cac ccc atc agc aaa gtc 868Ser Glu Thr Gly Asp Asn Lys Cys Phe Arg
His Pro Ile Ser Lys Val265 270 275 280agg agt gtg gtg atg gtg gtc
ttc agt gaa gac aaa aac aga gat gaa 916Arg Ser Val Val Met Val Val
Phe Ser Glu Asp Lys Asn Arg Asp Glu 285 290 295cag ctc aaa tac tgg
aaa tac tgg cac tct cgg cag cat acg gcg aag 964Gln Leu Lys Tyr Trp
Lys Tyr Trp His Ser Arg Gln His Thr Ala Lys 300 305 310cag agg gtc
ctt gac att gcc gat tac aag gag agc ttt aat acg att 1012Gln Arg Val
Leu Asp Ile Ala Asp Tyr Lys Glu Ser Phe Asn Thr Ile 315 320 325gga
aac att gaa gag att gca tat aat gct gtt tcc ttt acc tgg gac 1060Gly
Asn Ile Glu Glu Ile Ala Tyr Asn Ala Val Ser Phe Thr Trp Asp 330 335
340gtg aat gaa gag gcg aag att ttc atc acc gtg aat tgc ttg agc aca
1108Val Asn Glu Glu Ala Lys Ile Phe Ile Thr Val Asn Cys Leu Ser
Thr345 350 355 360gat ttc tcc tcc caa aaa ggg gtg aaa gga ctt cct
ttg atg att cag 1156Asp Phe Ser Ser Gln Lys Gly Val Lys Gly Leu Pro
Leu Met Ile Gln 365 370 375att gac aca tac agt tat aac aat cgt agc
aat aaa ccc att cat aga 1204Ile Asp Thr Tyr Ser Tyr Asn Asn Arg Ser
Asn Lys Pro Ile His Arg 380 385 390gct tat tgc cag atc aag gtc ttc
tgt gac aaa gga gca gaa aga aaa 1252Ala Tyr Cys Gln Ile Lys Val Phe
Cys Asp Lys Gly Ala Glu Arg Lys 395 400 405atc cga gat gaa gag crg
aag cag aac agg aag aam ggg aaa ggc cag 1300Ile Arg Asp Glu Glu Arg
Lys Gln Asn Arg Lys Lys Gly Lys Gly Gln 410 415 420gcc tcc caa act
caa tgc aac agc tcc tct gat ggg aag ttg gct gcc 1348Ala Ser Gln Thr
Gln Cys Asn Ser Ser Ser Asp Gly Lys Leu Ala Ala425 430 435 440ata
cct tta cag aag aag agt gac atc acc tac ttc aaa acc atg cct 1396Ile
Pro Leu Gln Lys Lys Ser Asp Ile Thr Tyr Phe Lys Thr Met Pro 445 450
455gat ctc cac tca cag cca gtt ctc ttc ata cct gat gtt cac ttt gca
1444Asp Leu His Ser Gln Pro Val Leu Phe Ile Pro Asp Val His Phe Ala
460 465 470aac ctg cag agg acc gga cag gtg tat tac aac acg gat gat
gaa cga 1492Asn Leu Gln Arg Thr Gly Gln Val Tyr Tyr Asn Thr Asp Asp
Glu Arg 475 480 485gaa ggt ggc agt gtc ctt gtt aaa cgg atg ttc cgg
ccc atg gaa gag 1540Glu Gly Gly Ser Val Leu Val Lys Arg Met Phe Arg
Pro Met Glu Glu 490 495 500gag ttt ggt ccr gtg cct tca aag cag atg
aaa gaa gaa ggg aca aag 1588Glu Phe Gly Pro Val Pro Ser Lys Gln Met
Lys Glu Glu Gly Thr Lys505 510 515 520cga gtg ctc ttg tac gtg agg
aag gag act gac gat gtg ttc gat gca 1636Arg Val Leu Leu Tyr Val Arg
Lys Glu Thr Asp Asp Val Phe Asp Ala 525 530 535ttg atg ttg aag tct
ccc aca gtg awg ggc ctg atg gaa gcg ata tct 1684Leu Met Leu Lys Ser
Pro Thr Val Lys Gly Leu Met Glu Ala Ile Ser 540 545 550gag aaa tat
ggg ctg ccc gtg gag aag ata gca aag ctt tac aag aaa 1732Glu Lys Tyr
Gly Leu Pro Val Glu Lys Ile Ala Lys Leu Tyr Lys Lys 555 560 565agc
aaa aaa ggc atc ttg gtg aac atg gat gac aac atc atc gag cac 1780Ser
Lys Lys Gly Ile Leu Val Asn Met Asp Asp Asn Ile Ile Glu His 570 575
580tac tcg aac gag gac acc ttc atc ctc aac atg gag agc atg gtg gag
1828Tyr Ser Asn Glu Asp Thr Phe Ile Leu Asn Met Glu Ser Met Val
Glu585 590 595 600ggc ttc aag gtc acg ctc atg gaa atc tag
ccctgggttt ggcatccgct 1878Gly Phe Lys Val Thr Leu Met Glu Ile
605ttggctggag ctctcagtgc rttcctccct gagagagaca gaagccccag
ccccagaacc 1938tggagaccca tctcccccat ctcacaactg ctgttacaag
accgtgctgg ggagtggggc 1998aagggacagg ccccactgtc ggtgtgcttg
gcccatccac tggcacctac cacggagcyg 2058aagcctgagc ccctcaggaa
ggtgccttag gcctgttgga ttcctattta ttgcccacct 2118tttcctggag
cccaggtcca ggccyrccag gactctgcag gtcactgcta gctccagatg
2178agaccgtcca gcgttccccc ttcaagagaa acactcatcc cgaacagcct
aaaaaattcc 2238catcccttct ytctcacccc tccatatcta tctcccgagt
ggctggacaa aatgagctac 2298gtctgggtgc agtagttata ggtggggcaa
gaggtggatg cccactttct ggtcagacac 2358ctttaggttg ctctggggaa
ggctgtcttg ctaaatacct ccagggttcc cagcaagtgg 2418ccaccaggcc
ttgtacagga agacattcag tcaccgtgta attagtaaca cagaaagtct
2478gcctgtctgc attgtacata gtgtttataa tattgtaata atatatttta
cctgtggtat 2538gtgggcatgt ttactgccac tggcctagag gagacacrga
cctggagacc gttttaatgg 2598gggtttttgc ctctgtgcct gttcaagaga
cttgcagggc taggtagagg gcctttggga 2658tgttaaggtg actgcagctg
atgccaagat ggactctgca atgggcatac ctgggggctc 2718gttccctgtc
cccagaggaa gccccctctc cttctccatg ggcatgactc tccttcgagg
2778ccaccacgtt tatctcacaa tgatgtgttt tgcytgactt tccctttgcg
ctgtctcrtg 2838ggaaaggtca ttctgtctga gaccccagct ccttctccag
ctttggctgc gggcatggcc 2898tgagctttct ggagagcctc tgcagggggt
ttgccatcag ggccctgtgg ctgggtctgc 2958tgcagagctc cttggctatc
aggagaatcc tggacactgt actgtgcctc ccagtttaca 3018aacacgccct
tcatctcaag tggcccttta aaaggcctgc ygccatgtga gagctgtgaa
3078cagctcagct ctgagtcggc agrctggggc ttcctcctgg gccaccagat
ggaaaggggg 3138tattgtttgc ctcactcctg gatgctgcgt tttaaggaag
tgagtgagaa agaatgtgcc 3198aagatacctg gctcctgtga aaccagcctc
aggagggaaa ctgggagaga gaagctgtgg 3258tctcctgcta catgccctgg
gagctggaag agaaaaacac tcccctaaac matcgcaaaa 3318tgatgaacca
tcaygggcca ctgttctctt tgaggggaca ggtttagggg tttgcgttcg
3378cccttgtggg ctgaagcact agctttttgg tagctagaca catcctgcac
ccaaaggttc 3438tctacaaagg cccagatttg tttgtaaagc actttgactc
ttacctggag gcccgctctc 3498taagggcttc ctgcgctccc acctcatctg
tccctgagat gcagagcagg atggagggtc 3558tgcttctagc tcagctgttt
ctccttgagg ttgcggagga attgaattga atgggacaga 3618gggcaggtgc
tgtggccaag aagatctccg agcagcagtg acggggcacc ttgctgtgtg
3678tcctctgggc atgttaaccc ttctgtgggg ccaaaggttt gcatcgtgga
tccagctgtg 3738ctccagtctg tcccctcctc ctccactctg actgccacgc
cccggaccag cagcttgggg 3798accctccagg gtactaatgg ggctctgttc
tgagatggac aaattcagtg ttggaaatac 3858atgttgtact atgcacttcc
catgctccta gggttaggaa tagtttcaaa catgattggc 3918agacataaca
acggcaaata ctcggactgg ggcataggac tccagagtag gaaaaagaca
3978aaagatttgg cagcctgaca caggcaacct acccctctct ctccagcctc
tttatgaaac 4038tgtttgtttg ccagtcctgc cctaaggcag aagatgaatt
gaagatgctg tgcatgtttc 4098ctaagtcctt gagcaatcat ggtggtgaca
attgccacaa gggatatgag gccagtgcca 4158ccagagggtg gtgccaagtg
ccacatccct tccgatccat tcccctctgy atcctcggag 4218caccccagtt
tgcctttgat gtgtccgctg tgtatgttag ctgaaytttg atgagcaaaa
4278tttcctgagc gaaacactcc aaagagatag gaaaacttgc cgcctcttct
tttttgtccc 4338ttaatcaaac tcaaataagc ttaaaaaaaa tccatggaag
atcatggaca tgtgaaatga 4398gcattttttt yttttttttt tttaacaaag
tctgaactga acagaacaag actttttcct 4458catacatctc caaattgttt
aaacttactt tatgagtgtt tgtttagaag ttcggaccaa 4518cagaaaaatg
cagtcagatg tcatcttgga attggtttct aaaagagtaa ggcatgtccc
4578tgcccagaaa cttaggaagc atgaaataaa tcaaatgttt attttccttc
ttatttaaaa 4638tcatgcwaat gcaacagaaa tagagggttt gtgccaaatg
ctatgaacgg ccctttctta 4698aagacaagca agggagattg atatatgtac
aatttgctct catgtttt 47463609PRTHomo sapiensVARIANT37v.4 S=I 3Met
Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10
15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr
20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala
Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp
Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu
Asp Gln Glu Lys Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln
Ser Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys
Thr Val Pro Val Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu
Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser
Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140
Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145
150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr
Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu
Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu
Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser
Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly
Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg
Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly
Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265
270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe
275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys
Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu
Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly
Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp
Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys
Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu
Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg
Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390
395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys
Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln
Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu
Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp
Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe
Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr
Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met
Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510
Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515
520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr
Val 530 535 540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu
Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys
Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His
Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met
Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile
44913DNAHomo sapiensCDS(13)...(1890) 4attggatcaa ac atg tca caa gag
tcg gac aat aat aaa aga cta gtg gcc 51 Met Ser Gln Glu Ser Asp Asn
Asn Lys Arg Leu Val Ala 1 5 10tta gtg ccc atg ccc agt gac cct cca
ttc aat acc cga aga gcc tac 99Leu Val Pro Met Pro Ser Asp Pro Pro
Phe Asn Thr Arg Arg Ala Tyr 15 20 25acc agt gag gat gaa gcc tgg aag
tca tac ttg gag aat ccc ctg aca 147Thr Ser Glu Asp Glu Ala Trp Lys
Ser Tyr Leu Glu Asn Pro Leu Thr30 35 40 45gca gcc acc aag gcc atg
atg agc att aat ggt gat gag gac agt gct 195Ala Ala Thr Lys Ala Met
Met Ser Ile Asn Gly Asp Glu Asp Ser Ala 50 55 60gct gcc ctc ggc ctg
ctc tat gac tac tac aag gtt cct cga gac aag 243Ala Ala Leu Gly Leu
Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys 65 70 75agg ctg ctg tct
gta agc aaa gca agt gac agc caa gaa gac cag gag 291Arg Leu Leu Ser
Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu 80 85 90aaa aga aac
tgc ctt ggc acc agt gaa gcc cag agt aat ttg agt gga 339Lys Arg Asn
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly 95 100 105gga
gaa aac cga gtg caa gtc cta aag act gtt cca gtg aac ctt tcc 387Gly
Glu Asn Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser110 115
120 125cta aat caa gat cac ctg gag aat tcc aag cgg gaa cag tac agc
atc 435Leu Asn Gln Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser
Ile 130 135 140agc ttc ccc gag agc tct gcc atc atc ccg gtg tcg gga
atc acg gtg 483Ser Phe Pro Glu Ser Ser Ala Ile Ile Pro Val Ser Gly
Ile Thr Val 145 150 155gtg aaa gct gaa gat ttc aca cca gtt ttc atg
gcc cca cct gtg cac 531Val Lys Ala Glu Asp Phe Thr Pro Val Phe Met
Ala Pro Pro Val His 160 165 170tat ccc cgg gga gat ggg gaa gag caa
cga gtg gtt atc ttt
gaa cag 579Tyr Pro Arg Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe
Glu Gln 175 180 185act cag tat gac gtg ccc tcg ctg gcc acc cac agc
gcc tat ctc aaa 627Thr Gln Tyr Asp Val Pro Ser Leu Ala Thr His Ser
Ala Tyr Leu Lys190 195 200 205gac gac cag cgc agc act ccg gac agc
aca tac agc gag agc ttc aag 675Asp Asp Gln Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys 210 215 220gac gca gcc aca gag aaa ttt
cgg agt gct tca gtt ggg gct gag gag 723Asp Ala Ala Thr Glu Lys Phe
Arg Ser Ala Ser Val Gly Ala Glu Glu 225 230 235tac atg tat gat cag
aca tca agt ggc aca ttt cag tac acc ctg gaa 771Tyr Met Tyr Asp Gln
Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu 240 245 250gcc acc aaa
tct ctc cgt cag aag cag ggg gag ggc ccc atg acc tac 819Ala Thr Lys
Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr 255 260 265ctc
aac aaa gga cag ttc tat gcc ata aca ctc agc gag acc gga gac 867Leu
Asn Lys Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp270 275
280 285aac aaa tgc ttc cga cac ccc atc agc aaa gtc agg agt gtg gtg
atg 915Asn Lys Cys Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met 290 295 300gtg gtc ttc agt gaa gac aaa aac aga gat gaa cag ctc
aaa tac tgg 963Val Val Phe Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp 305 310 315aaa tac tgg cac tct cgg cag cat acg gcg aag
cag agg gtc ctt gac 1011Lys Tyr Trp His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp 320 325 330att gcc gat tac aag gag agc ttt aat
acg att gga aac att gaa gag 1059Ile Ala Asp Tyr Lys Glu Ser Phe Asn
Thr Ile Gly Asn Ile Glu Glu 335 340 345att gca tat aat gct gtt tcc
ttt acc tgg gac gtg aat gaa gag gcg 1107Ile Ala Tyr Asn Ala Val Ser
Phe Thr Trp Asp Val Asn Glu Glu Ala350 355 360 365aag att ttc atc
acc gtg aat tgc ttg agc aca gat ttc tcc tcc caa 1155Lys Ile Phe Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln 370 375 380aaa ggg
gtg aaa gga ctt cct ttg atg att cag att gac aca tac agt 1203Lys Gly
Val Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser 385 390
395tat aac aat cgt agc aat aaa ccc att cat aga gct tat tgc cag atc
1251Tyr Asn Asn Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile
400 405 410aag gtc ttc tgt gac aaa gga gca gaa aga aaa atc cga gat
gaa gag 1299Lys Val Phe Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu 415 420 425cgg aag cag aac agg aag aaa ggg aaa ggc cag gcc
tcc caa act caa 1347Arg Lys Gln Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Thr Gln430 435 440 445tgc aac agc tcc tct gat ggg aag ttg
gct gcc ata cct tta cag aag 1395Cys Asn Ser Ser Ser Asp Gly Lys Leu
Ala Ala Ile Pro Leu Gln Lys 450 455 460aag agt gac atc acc tac ttc
aaa acc atg cct gat ctc cac tca cag 1443Lys Ser Asp Ile Thr Tyr Phe
Lys Thr Met Pro Asp Leu His Ser Gln 465 470 475cca gtt ctc ttc ata
cct gat gtt cac ttt gca aac ctg cag agg acc 1491Pro Val Leu Phe Ile
Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr 480 485 490gga cag gtg
tat tac aac acg gat gat gaa cga gaa ggt ggc agt gtc 1539Gly Gln Val
Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val 495 500 505ctt
gtt aaa cgg atg ttc cgg ccc atg gaa gag gag ttt ggt cca gtg 1587Leu
Val Lys Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val510 515
520 525cct tca aag cag atg aaa gaa gaa ggg aca aag cga gtg ctc ttg
tac 1635Pro Ser Lys Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu
Tyr 530 535 540gtg agg aag gag act gac gat gtg ttc gat gca ttg atg
ttg aag tct 1683Val Arg Lys Glu Thr Asp Asp Val Phe Asp Ala Leu Met
Leu Lys Ser 545 550 555ccc aca gtg aag ggc ctg atg gaa gcg ata tct
gag aaa tat ggg ctg 1731Pro Thr Val Lys Gly Leu Met Glu Ala Ile Ser
Glu Lys Tyr Gly Leu 560 565 570ccc gtg gag aag ata gca aag ctt tac
aag aaa agc aaa aaa ggc atc 1779Pro Val Glu Lys Ile Ala Lys Leu Tyr
Lys Lys Ser Lys Lys Gly Ile 575 580 585ttg gtg aac atg gat gac aac
atc atc gag cac tac tcg aac gag gac 1827Leu Val Asn Met Asp Asp Asn
Ile Ile Glu His Tyr Ser Asn Glu Asp590 595 600 605acc ttc atc ctc
aac atg gag agc atg gtg gag ggc ttc aag gtc acg 1875Thr Phe Ile Leu
Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr 610 615 620ctc atg
gaa atc tag ccctgggttt ggcatccgct ttggctggag ctctcagtgc 1930Leu Met
Glu Ile 625gttcctccct gagagagaca gaagccccag ccccagaacc tggagaccca
tctcccccat 1990ctcacaactg ctgttacaag accgtgctgg ggagtggggc
aagggacagg ccccactgtc 2050ggtgtgcttg gcccatccac tggcacctac
cacggagctg aagcctgagc ccctcaggaa 2110ggtgccttag gcctgttgga
ttcctattta ttgcccacct tttcctggag cccaggtcca 2170ggcccgccag
gactctgcag gtcactgcta gctccagatg agaccgtcca gcgttccccc
2230ttcaagagaa acactcatcc cgaacagcct aaaaaattcc catcccttct
ctctcacccc 2290tccatatcta tctcccgagt ggctggacaa aatgagctac
gtctgggtgc agtagttata 2350ggtggggcaa gaggtggatg cccactttct
ggtcagacac ctttaggttg ctctggggaa 2410ggctgtcttg ctaaatacct
ccagggttcc cagcaagtgg ccaccaggcc ttgtacagga 2470agacattcag
tcaccgtgta attagtaaca cagaaagtct gcctgtctgc attgtacata
2530gtgtttataa tattgtaata atatatttta cctgtggtat gtgggcatgt
ttactgccac 2590tggcctagag gagacacaga cctggagacc gttttaatgg
gggtttttgc ctctgtgcct 2650gttcaagaga cttgcagggc taggtagagg
gcctttggga tgttaaggtg actgcagctg 2710atgccaagat ggactctgca
atgggcatac ctgggggctc gttccctgtc cccagaggaa 2770gccccctctc
cttctccatg ggcatgactc tccttcgagg ccaccacgtt tatctcacaa
2830tgatgtgttt tgcttgactt tccctttgcg ctgtctcgtg ggaaaggtca
ttctgtctga 2890gaccccagct ccttctccag ctttggctgc gggcatggcc
tgagctttct ggagagcctc 2950tgcagggggt ttgccatcag ggccctgtgg
ctgggtctgc tgcagagctc cttggctatc 3010aggagaatcc tggacactgt
actgtgcctc ccagtttaca aacacgccct tcatctcaag 3070tggcccttta
aaaggcctgc tgccatgtga gagctgtgaa cagctcagct ctgagtcggc
3130aggctggggc ttcctcctgg gccaccagat ggaaaggggg tattgtttgc
ctcactcctg 3190gatgctgcgt tttaaggaag tgagtgagaa agaatgtgcc
aagatacctg gctcctgtga 3250aaccagcctc aggagggaaa ctgggagaga
gaagctgtgg tctcctgcta catgccctgg 3310gagctggaag agaaaaacac
tcccctaaac aatcgcaaaa tgatgaacca tcatgggcca 3370ctgttctctt
tgaggggaca ggtttagggg tttgcgttcg cccttgtggg ctgaagcact
3430agctttttgg tagctagaca catcctgcac ccaaaggttc tctacaaagg
cccagatttg 3490tttgtaaagc actttgactc ttacctggag gcccgctctc
taagggcttc ctgcgctccc 3550acctcatctg tccctgagat gcagagcagg
atggagggtc tgcttctagc tcagctgttt 3610ctccttgagg ttgcggagga
attgaattga atgggacaga gggcaggtgc tgtggccaag 3670aagatctccg
agcagcagtg acggggcacc ttgctgtgtg tcctctgggc atgttaaccc
3730ttctgtgggg ccaaaggttt gcatcgtgga tccagctgtg ctccagtctg
tcccctcctc 3790ctccactctg actgccacgc cccggaccag cagcttgggg
accctccagg gtactaatgg 3850ggctctgttc tgagatggac aaattcagtg
ttggaaatac atgttgtact atgcacttcc 3910catgctccta gggttaggaa
tagtttcaaa catgattggc agacataaca acggcaaata 3970ctcggactgg
ggcataggac tccagagtag gaaaaagaca aaagatttgg cagcctgaca
4030caggcaacct acccctctct ctccagcctc tttatgaaac tgtttgtttg
ccagtcctgc 4090cctaaggcag aagatgaatt gaagatgctg tgcatgtttc
ctaagtcctt gagcaatcat 4150ggtggtgaca attgccacaa gggatatgag
gccagtgcca ccagagggtg gtgccaagtg 4210ccacatccct tccgatccat
tcccctctgc atcctcggag caccccagtt tgcctttgat 4270gtgtccgctg
tgtatgttag ctgaactttg atgagcaaaa tttcctgagc gaaacactcc
4330aaagagatag gaaaacttgc cgcctcttct tttttgtccc ttaatcaaac
tcaaataagc 4390ttaaaaaaaa tccatggaag atcatggaca tgtgaaatga
gcattttttt cttttttttt 4450tttaacaaag tctgaactga acagaacaag
actttttcct catacatctc caaattgttt 4510aaacttactt tatgagtgtt
tgtttagaag ttcggaccaa cagaaaaatg cagtcagatg 4570tcatcttgga
attggtttct aaaagagtaa ggcatgtccc tgcccagaaa cttaggaagc
4630atgaaataaa tcaaatgttt attttccttc ttatttaaaa tcatgcaaat
gcaacagaaa 4690tagagggttt gtgccaaatg ctatgaacgg ccctttctta
aagacaagca agggagattg 4750atatatgtac aatttgctct catgttttaa
aaaaaaaagg taaatgtaac ttaatagttt 4810tgtaaatggg agagggggaa
tctataaact ataaatacag ttattttatt ttttgtacat 4870ttttaaggag
aaaaaaataa atattcataa cataagagga aaa 49135625PRTHomo sapiens 5Met
Ser Gln Glu Ser Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro1 5 10
15 Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu
20 25 30 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala
Ala Thr 35 40 45 Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser
Ala Ala Ala Leu 50 55 60 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro
Arg Asp Lys Arg Leu Leu65 70 75 80 Ser Val Ser Lys Ala Ser Asp Ser
Gln Glu Asp Gln Glu Lys Arg Asn 85 90 95 Cys Leu Gly Thr Ser Glu
Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 100 105 110 Arg Val Gln Val
Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln 115 120 125 Asp His
Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 130 135 140
Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala145
150 155 160 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His Tyr
Pro Arg 165 170 175 Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu
Gln Thr Gln Tyr 180 185 190 Asp Val Pro Ser Leu Ala Thr His Ser Ala
Tyr Leu Lys Asp Asp Gln 195 200 205 Arg Ser Thr Pro Asp Ser Thr Tyr
Ser Glu Ser Phe Lys Asp Ala Ala 210 215 220 Thr Glu Lys Phe Arg Ser
Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr225 230 235 240 Asp Gln Thr
Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys 245 250 255 Ser
Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys 260 265
270 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys
275 280 285 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val
Val Phe 290 295 300 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr
Trp Lys Tyr Trp305 310 315 320 His Ser Arg Gln His Thr Ala Lys Gln
Arg Val Leu Asp Ile Ala Asp 325 330 335 Tyr Lys Glu Ser Phe Asn Thr
Ile Gly Asn Ile Glu Glu Ile Ala Tyr 340 345 350 Asn Ala Val Ser Phe
Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 355 360 365 Ile Thr Val
Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 370 375 380 Lys
Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn385 390
395 400 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val
Phe 405 410 415 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu
Arg Lys Gln 420 425 430 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln
Thr Gln Cys Asn Ser 435 440 445 Ser Ser Asp Gly Lys Leu Ala Ala Ile
Pro Leu Gln Lys Lys Ser Asp 450 455 460 Ile Thr Tyr Phe Lys Thr Met
Pro Asp Leu His Ser Gln Pro Val Leu465 470 475 480 Phe Ile Pro Asp
Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val 485 490 495 Tyr Tyr
Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 500 505 510
Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 515
520 525 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg
Lys 530 535 540 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser
Pro Thr Val545 550 555 560 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys
Tyr Gly Leu Pro Val Glu 565 570 575 Lys Ile Ala Lys Leu Tyr Lys Lys
Ser Lys Lys Gly Ile Leu Val Asn 580 585 590 Met Asp Asp Asn Ile Ile
Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 595 600 605 Leu Asn Met Glu
Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 610 615 620 Ile625
64973DNAHomo sapiensCDS(121)...(1950) 6attggatcaa acatgtcaca
agagtcggac aagtaagtgg atcacacgcg ccggctgctg 60ctactactac cactttgggc
tgatggcaac tgtaataaaa gactagtggc cttagtgccc 120atg ccc agt gac cct
cca ttc aat acc cga aga gcc tac acc agt gag 168Met Pro Ser Asp Pro
Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15gat gaa gcc tgg
aag tca tac ttg gag aat ccc ctg aca gca gcc acc 216Asp Glu Ala Trp
Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30aag gcc atg
atg agc att aat ggt gat gag gac agt gct gct gcc ctc 264Lys Ala Met
Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45ggc ctg
ctc tat gac tac tac aag gtt cct cga gac aag agg ctg ctg 312Gly Leu
Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60tct
gta agc aaa gca agt gac agc caa gaa gac cag gag aaa aga aac 360Ser
Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75
80tgc ctt ggc acc agt gaa gcc cag agt aat ttg agt gga gga gaa aac
408Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn
85 90 95cga gtg caa gtc cta aag act gtt cca gtg aac ctt tcc cta aat
caa 456Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110gat cac ctg gag aat tcc aag cgg gaa cag tac agc atc
agc ttc ccc 504Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125gag agc tct gcc atc atc ccg gtg tcg gga atc
acg gtg gtg aaa gct 552Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140gaa gat ttc aca cca gtt ttc atg gcc
cca cct gtg cac tat ccc cgg 600Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160gga gat ggg gaa gag caa
cga gtg gtt atc ttt gaa cag act cag tat 648Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175gac gtg ccc tcg
ctg gcc acc cac agc gcc tat ctc aaa gac gac cag 696Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190cgc agc
act ccg gac agc aca tac agc gag agc ttc aag gac gca gcc 744Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200
205aca gag aaa ttt cgg agt gct tca gtt ggg gct gag gag tac atg tat
792Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr
210 215 220gat cag aca tca agt ggc aca ttt cag tac acc ctg gaa gcc
acc aaa 840Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala
Thr Lys225 230 235 240tct ctc cgt cag aag cag ggg gag ggc ccc atg
acc tac ctc aac aaa 888Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met
Thr Tyr Leu Asn Lys 245 250 255gga cag ttc tat gcc ata aca ctc agc
gag acc gga gac aac aaa tgc 936Gly Gln Phe Tyr Ala Ile Thr Leu Ser
Glu Thr Gly Asp Asn Lys Cys 260 265 270ttc cga cac ccc atc agc aaa
gtc agg agt gtg gtg atg gtg gtc ttc 984Phe Arg His Pro Ile Ser Lys
Val Arg Ser Val Val Met Val Val Phe 275 280 285agt gaa gac aaa aac
aga gat gaa cag ctc aaa tac tgg aaa tac tgg 1032Ser Glu Asp Lys Asn
Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300cac tct cgg
cag cat acg gcg aag cag agg gtc ctt gac att gcc gat 1080His Ser Arg
Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315
320tac aag gag agc ttt aat acg att gga aac att gaa gag att gca tat
1128Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr
325 330 335aat gct gtt tcc ttt acc tgg gac gtg aat gaa gag gcg aag
att ttc 1176Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys
Ile Phe 340
345 350atc acc gtg aat tgc ttg agc aca gat ttc tcc tcc caa aaa ggg
gtg 1224Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly
Val 355 360 365aaa gga ctt cct ttg atg att cag att gac aca tac agt
tat aac aat 1272Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser
Tyr Asn Asn 370 375 380cgt agc aat aaa ccc att cat aga gct tat tgc
cag atc aag gtc ttc 1320Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys
Gln Ile Lys Val Phe385 390 395 400tgt gac aaa gga gca gaa aga aaa
atc cga gat gaa gag cgg aag cag 1368Cys Asp Lys Gly Ala Glu Arg Lys
Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415aac agg aag aaa ggg aaa
ggc cag gcc tcc caa act caa tgc aac agc 1416Asn Arg Lys Lys Gly Lys
Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430tcc tct gat ggg
aag ttg gct gcc ata cct tta cag aag aag agt gac 1464Ser Ser Asp Gly
Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445atc acc
tac ttc aaa acc atg cct gat ctc cac tca cag cca gtt ctc 1512Ile Thr
Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460ttc ata cct gat gtt cac ttt gca aac ctg cag agg acc gga cag gtg
1560Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480tat tac aac acg gat gat gaa cga gaa ggt ggc agt
gtc ctt gtt aaa 1608Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495cgg atg ttc cgg ccc atg gaa gag gag ttt
ggt cca gtg cct tca aag 1656Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510cag atg aaa gaa gaa ggg aca aag
cga gtg ctc ttg tac gtg agg aag 1704Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525gag act gac gat gtg ttc
gat gca ttg atg ttg aag tct ccc aca gtg 1752Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540aag ggc ctg atg
gaa gcg ata tct gag aaa tat ggg ctg ccc gtg gag 1800Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560aag
ata gca aag ctt tac aag aaa agc aaa aaa ggc atc ttg gtg aac 1848Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575atg gat gac aac atc atc gag cac tac tcg aac gag gac acc ttc atc
1896Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590ctc aac atg gag agc atg gtg gag ggc ttc aag gtc acg ctc
atg gaa 1944Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605atc tag ccctgggttt ggcatccgct ttggctggag
ctctcagtgc gttcctccct 2000Ile gagagagaca gaagccccag ccccagaacc
tggagaccca tctcccccat ctcacaactg 2060ctgttacaag accgtgctgg
ggagtggggc aagggacagg ccccactgtc ggtgtgcttg 2120gcccatccac
tggcacctac cacggagctg aagcctgagc ccctcaggaa ggtgccttag
2180gcctgttgga ttcctattta ttgcccacct tttcctggag cccaggtcca
ggcccgccag 2240gactctgcag gtcactgcta gctccagatg agaccgtcca
gcgttccccc ttcaagagaa 2300acactcatcc cgaacagcct aaaaaattcc
catcccttct ctctcacccc tccatatcta 2360tctcccgagt ggctggacaa
aatgagctac gtctgggtgc agtagttata ggtggggcaa 2420gaggtggatg
cccactttct ggtcagacac ctttaggttg ctctggggaa ggctgtcttg
2480ctaaatacct ccagggttcc cagcaagtgg ccaccaggcc ttgtacagga
agacattcag 2540tcaccgtgta attagtaaca cagaaagtct gcctgtctgc
attgtacata gtgtttataa 2600tattgtaata atatatttta cctgtggtat
gtgggcatgt ttactgccac tggcctagag 2660gagacacaga cctggagacc
gttttaatgg gggtttttgc ctctgtgcct gttcaagaga 2720cttgcagggc
taggtagagg gcctttggga tgttaaggtg actgcagctg atgccaagat
2780ggactctgca atgggcatac ctgggggctc gttccctgtc cccagaggaa
gccccctctc 2840cttctccatg ggcatgactc tccttcgagg ccaccacgtt
tatctcacaa tgatgtgttt 2900tgcttgactt tccctttgcg ctgtctcgtg
ggaaaggtca ttctgtctga gaccccagct 2960ccttctccag ctttggctgc
gggcatggcc tgagctttct ggagagcctc tgcagggggt 3020ttgccatcag
ggccctgtgg ctgggtctgc tgcagagctc cttggctatc aggagaatcc
3080tggacactgt actgtgcctc ccagtttaca aacacgccct tcatctcaag
tggcccttta 3140aaaggcctgc tgccatgtga gagctgtgaa cagctcagct
ctgagtcggc aggctggggc 3200ttcctcctgg gccaccagat ggaaaggggg
tattgtttgc ctcactcctg gatgctgcgt 3260tttaaggaag tgagtgagaa
agaatgtgcc aagatacctg gctcctgtga aaccagcctc 3320aggagggaaa
ctgggagaga gaagctgtgg tctcctgcta catgccctgg gagctggaag
3380agaaaaacac tcccctaaac aatcgcaaaa tgatgaacca tcatgggcca
ctgttctctt 3440tgaggggaca ggtttagggg tttgcgttcg cccttgtggg
ctgaagcact agctttttgg 3500tagctagaca catcctgcac ccaaaggttc
tctacaaagg cccagatttg tttgtaaagc 3560actttgactc ttacctggag
gcccgctctc taagggcttc ctgcgctccc acctcatctg 3620tccctgagat
gcagagcagg atggagggtc tgcttctagc tcagctgttt ctccttgagg
3680ttgcggagga attgaattga atgggacaga gggcaggtgc tgtggccaag
aagatctccg 3740agcagcagtg acggggcacc ttgctgtgtg tcctctgggc
atgttaaccc ttctgtgggg 3800ccaaaggttt gcatcgtgga tccagctgtg
ctccagtctg tcccctcctc ctccactctg 3860actgccacgc cccggaccag
cagcttgggg accctccagg gtactaatgg ggctctgttc 3920tgagatggac
aaattcagtg ttggaaatac atgttgtact atgcacttcc catgctccta
3980gggttaggaa tagtttcaaa catgattggc agacataaca acggcaaata
ctcggactgg 4040ggcataggac tccagagtag gaaaaagaca aaagatttgg
cagcctgaca caggcaacct 4100acccctctct ctccagcctc tttatgaaac
tgtttgtttg ccagtcctgc cctaaggcag 4160aagatgaatt gaagatgctg
tgcatgtttc ctaagtcctt gagcaatcat ggtggtgaca 4220attgccacaa
gggatatgag gccagtgcca ccagagggtg gtgccaagtg ccacatccct
4280tccgatccat tcccctctgc atcctcggag caccccagtt tgcctttgat
gtgtccgctg 4340tgtatgttag ctgaactttg atgagcaaaa tttcctgagc
gaaacactcc aaagagatag 4400gaaaacttgc cgcctcttct tttttgtccc
ttaatcaaac tcaaataagc ttaaaaaaaa 4460tccatggaag atcatggaca
tgtgaaatga gcattttttt cttttttttt tttaacaaag 4520tctgaactga
acagaacaag actttttcct catacatctc caaattgttt aaacttactt
4580tatgagtgtt tgtttagaag ttcggaccaa cagaaaaatg cagtcagatg
tcatcttgga 4640attggtttct aaaagagtaa ggcatgtccc tgcccagaaa
cttaggaagc atgaaataaa 4700tcaaatgttt attttccttc ttatttaaaa
tcatgcaaat gcaacagaaa tagagggttt 4760gtgccaaatg ctatgaacgg
ccctttctta aagacaagca agggagattg atatatgtac 4820aatttgctct
catgttttaa aaaaaaaagg taaatgtaac ttaatagttt tgtaaatggg
4880agagggggaa tctataaact ataaatacag ttattttatt ttttgtacat
ttttaaggag 4940aaaaaaataa atattcataa cataagagga aaa 49737609PRTHomo
sapiens 7Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr
Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu
Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu
Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys
Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser
Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80 Cys Leu Gly Thr
Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val
Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln 100 105 110
Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115
120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys
Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His
Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile
Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His
Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe
Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln
Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235
240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys
245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn
Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe
Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val
Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360
365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn
370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys
Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala
Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys
Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro
Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480
Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485
490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser
Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr
Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu
Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met Glu Ala Ile Ser Glu
Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr
Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn
Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn
Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605
Ile 84748DNAHomo sapiensCDS(29)...(1858) 8taataaaaga ctagtggcct
tagtgccc atg ccc agt gac cct cca ttc aat 52 Met Pro Ser Asp Pro Pro
Phe Asn 1 5acc cga aga gcc tac acc agt gag gat gaa gcc tgg aag tca
tac ttg 100Thr Arg Arg Ala Tyr Thr Ser Glu Asp Glu Ala Trp Lys Ser
Tyr Leu 10 15 20gag aat ccc ctg aca gca gcc acc aag gcc atg atg agc
att aat ggt 148Glu Asn Pro Leu Thr Ala Ala Thr Lys Ala Met Met Ser
Ile Asn Gly25 30 35 40gat gag gac agt gct gct gcc ctc ggc ctg ctc
tat gac tac tac aag 196Asp Glu Asp Ser Ala Ala Ala Leu Gly Leu Leu
Tyr Asp Tyr Tyr Lys 45 50 55gtt cct cga gac aag agg ctg ctg tct gta
agc aaa gca agt gac agc 244Val Pro Arg Asp Lys Arg Leu Leu Ser Val
Ser Lys Ala Ser Asp Ser 60 65 70caa gaa gac cag gag aaa aga aac tgc
ctt ggc acc agt gaa gcc cag 292Gln Glu Asp Gln Glu Lys Arg Asn Cys
Leu Gly Thr Ser Glu Ala Gln 75 80 85agt aat ttg agt gga gga gaa aac
cga gtg caa gtc cta aag act gtt 340Ser Asn Leu Ser Gly Gly Glu Asn
Arg Val Gln Val Leu Lys Thr Val 90 95 100cca gtg aac ctt tcc cta
aat caa gat cac ctg gag aat tcc aag cgg 388Pro Val Asn Leu Ser Leu
Asn Gln Asp His Leu Glu Asn Ser Lys Arg105 110 115 120gaa cag tac
agc atc agc ttc ccc gag agc tct gcc atc atc ccg gtg 436Glu Gln Tyr
Ser Ile Ser Phe Pro Glu Ser Ser Ala Ile Ile Pro Val 125 130 135tcg
gga atc acg gtg gtg aaa gct gaa gat ttc aca cca gtt ttc atg 484Ser
Gly Ile Thr Val Val Lys Ala Glu Asp Phe Thr Pro Val Phe Met 140 145
150gcc cca cct gtg cac tat ccc cgg gga gat ggg gaa gag caa cga gtg
532Ala Pro Pro Val His Tyr Pro Arg Gly Asp Gly Glu Glu Gln Arg Val
155 160 165gtt atc ttt gaa cag act cag tat gac gtg ccc tcg ctg gcc
acc cac 580Val Ile Phe Glu Gln Thr Gln Tyr Asp Val Pro Ser Leu Ala
Thr His 170 175 180agc gcc tat ctc aaa gac gac cag cgc agc act ccg
gac agc aca tac 628Ser Ala Tyr Leu Lys Asp Asp Gln Arg Ser Thr Pro
Asp Ser Thr Tyr185 190 195 200agc gag agc ttc aag gac gca gcc aca
gag aaa ttt cgg agt gct tca 676Ser Glu Ser Phe Lys Asp Ala Ala Thr
Glu Lys Phe Arg Ser Ala Ser 205 210 215gtt ggg gct gag gag tac atg
tat gat cag aca tca agt ggc aca ttt 724Val Gly Ala Glu Glu Tyr Met
Tyr Asp Gln Thr Ser Ser Gly Thr Phe 220 225 230cag tac acc ctg gaa
gcc acc aaa tct ctc cgt cag aag cag ggg gag 772Gln Tyr Thr Leu Glu
Ala Thr Lys Ser Leu Arg Gln Lys Gln Gly Glu 235 240 245ggc ccc atg
acc tac ctc aac aaa gga cag ttc tat gcc ata aca ctc 820Gly Pro Met
Thr Tyr Leu Asn Lys Gly Gln Phe Tyr Ala Ile Thr Leu 250 255 260agc
gag acc gga gac aac aaa tgc ttc cga cac ccc atc agc aaa gtc 868Ser
Glu Thr Gly Asp Asn Lys Cys Phe Arg His Pro Ile Ser Lys Val265 270
275 280agg agt gtg gtg atg gtg gtc ttc agt gaa gac aaa aac aga gat
gaa 916Arg Ser Val Val Met Val Val Phe Ser Glu Asp Lys Asn Arg Asp
Glu 285 290 295cag ctc aaa tac tgg aaa tac tgg cac tct cgg cag cat
acg gcg aag 964Gln Leu Lys Tyr Trp Lys Tyr Trp His Ser Arg Gln His
Thr Ala Lys 300 305 310cag agg gtc ctt gac att gcc gat tac aag gag
agc ttt aat acg att 1012Gln Arg Val Leu Asp Ile Ala Asp Tyr Lys Glu
Ser Phe Asn Thr Ile 315 320 325gga aac att gaa gag att gca tat aat
gct gtt tcc ttt acc tgg gac 1060Gly Asn Ile Glu Glu Ile Ala Tyr Asn
Ala Val Ser Phe Thr Trp Asp 330 335 340gtg aat gaa gag gcg aag att
ttc atc acc gtg aat tgc ttg agc aca 1108Val Asn Glu Glu Ala Lys Ile
Phe Ile Thr Val Asn Cys Leu Ser Thr345 350 355 360gat ttc tcc tcc
caa aaa ggg gtg aaa gga ctt cct ttg atg att cag 1156Asp Phe Ser Ser
Gln Lys Gly Val Lys Gly Leu Pro Leu Met Ile Gln 365 370 375att gac
aca tac agt tat aac aat cgt agc aat aaa ccc att cat aga 1204Ile Asp
Thr Tyr Ser Tyr Asn Asn Arg Ser Asn Lys Pro Ile His Arg 380 385
390gct tat tgc cag atc aag gtc ttc tgt gac aaa gga gca gaa aga aaa
1252Ala Tyr Cys Gln Ile Lys Val Phe Cys Asp Lys Gly Ala Glu Arg Lys
395 400 405atc cga gat gaa gag cgg aag cag aac agg aag aaa ggg aaa
ggc cag 1300Ile Arg Asp Glu Glu Arg Lys Gln Asn Arg Lys Lys Gly Lys
Gly Gln 410 415 420gcc tcc caa act caa tgc aac agc tcc tct gat ggg
aag ttg gct gcc 1348Ala Ser Gln Thr Gln Cys Asn Ser Ser Ser Asp Gly
Lys Leu Ala Ala425 430 435 440ata cct tta cag aag aag agt gac atc
acc tac ttc aaa acc atg cct 1396Ile Pro Leu Gln Lys Lys Ser Asp Ile
Thr Tyr Phe Lys Thr Met Pro 445 450 455gat ctc cac tca cag cca gtt
ctc ttc ata cct gat gtt cac ttt gca 1444Asp Leu His Ser Gln Pro Val
Leu Phe Ile Pro Asp Val His Phe Ala 460 465 470aac ctg cag agg acc
gga cag gtg tat tac aac acg gat gat gaa cga 1492Asn Leu Gln Arg Thr
Gly Gln Val Tyr Tyr Asn Thr Asp Asp Glu Arg 475 480 485gaa ggt ggc
agt gtc ctt gtt aaa cgg atg ttc cgg ccc atg gaa gag 1540Glu Gly Gly
Ser Val Leu Val Lys Arg Met Phe Arg Pro Met Glu Glu 490 495 500gag
ttt ggt cca gtg cct tca aag cag atg aaa gaa gaa ggg aca aag 1588Glu
Phe Gly Pro Val Pro Ser Lys Gln Met Lys Glu Glu Gly Thr Lys505 510
515 520cga gtg ctc ttg tac gtg agg aag gag act gac gat gtg ttc gat
gca 1636Arg Val Leu Leu Tyr Val Arg Lys Glu Thr Asp Asp Val Phe Asp
Ala 525 530 535ttg atg ttg aag tct ccc aca gtg aag ggc ctg atg gaa
gcg ata tct 1684Leu Met Leu Lys Ser Pro Thr Val Lys Gly Leu Met Glu
Ala Ile Ser 540 545
550gag aaa tat ggg ctg ccc gtg gag aag ata gca aag ctt tac aag aaa
1732Glu Lys Tyr Gly Leu Pro Val Glu Lys Ile Ala Lys Leu Tyr Lys Lys
555 560 565agc aaa aaa ggc atc ttg gtg aac atg gat gac aac atc atc
gag cac 1780Ser Lys Lys Gly Ile Leu Val Asn Met Asp Asp Asn Ile Ile
Glu His 570 575 580tac tcg aac gag gac acc ttc atc ctc aac atg gag
agc atg gtg gag 1828Tyr Ser Asn Glu Asp Thr Phe Ile Leu Asn Met Glu
Ser Met Val Glu585 590 595 600ggc ttc aag gtc acg ctc atg gaa atc
tag ccctgggttt ggcatccgct 1878Gly Phe Lys Val Thr Leu Met Glu Ile
605ttggctggag ctctcagtgc gttcctccct gagagagaca gaagccccag
ccccagaacc 1938tggagaccca tctcccccat ctcacaactg ctgttacaag
accgtgctgg ggagtggggc 1998aagggacagg ccccactgtc ggtgtgcttg
gcccatccac tggcacctac cacggagctg 2058aagcctgagc ccctcaggaa
ggtgccttag gcctgttgga ttcctattta ttgcccacct 2118tttcctggag
cccaggtcca ggcccgccag gactctgcag gtcactgcta gctccagatg
2178agaccgtcca gcgttccccc ttcaagagaa acactcatcc cgaacagcct
aaaaaattcc 2238catcccttct ctctcacccc tccatatcta tatctcccga
gtggctggac aaaatgagct 2298acgtctgggt gcagtagtta taggtggggc
aagaggtgga tgcccacttt ctggtcagac 2358acctttaggt tgctctgggg
aaggctgtct tgctaaatac ctccagggtt cccagcaagt 2418ggccaccagg
ccttgtacag gaagacattc agtcaccgtg taattagtaa cacagaaagt
2478ctgcctgtct gcattgtaca tagtgtttat aatattgtaa taatatattt
tacctgtggt 2538atgtgggcat gtttactgcc actggcctag aggagacaca
gacctggaga ccgttttaat 2598gggggttttt gcctctgtgc ctgttcaaga
gacttgcagg gctaggtaga gggcctttgg 2658gatgttaagg tgactgcagc
tgatgccaag atggactctg caatgggcat acctgggggc 2718tcgttccctg
tccccagagg aagccccctc tccttctcca tgggcatgac tctccttcga
2778ggccaccacg tttatctcac aatgatgtgt tttgcttgac tttccctttg
cgctgtctcg 2838tgggaaaggt cattctgtct gagaccccag ctccttctcc
agctttggct gcgggcatgg 2898cctgagcttt ctggagagcc tctgcagggg
gtttgccatc agggccctgt ggctgggtct 2958gctgcagagc tccttggcta
tcaggagaat cctggacact gtactgtgcc tcccagttta 3018caaacacgcc
cttcatctca agtggccctt taaaaggcct gctgccatgt gagagctgtg
3078aacagctcag ctctgagtcg gcaggctggg gcttcctcct gggccaccag
atggaaaggg 3138ggtattgttt gcctcactcc tggatgctgc gttttaagga
agtgagtgag aaagaatgtg 3198ccaagatacc tggctcctgt gaaaccagcc
tcaggaggga aactgggaga gagaagctgt 3258ggtctcctgc tacatgccct
gggagctgga agagaaaaac actcccctaa acaatcgcaa 3318aatgatgaac
catcatgggc cactgttctc tttgagggga caggtttagg ggtttgcgtt
3378cgcccttgtg ggctgaagca ctagcttttt ggtagctaga cacatcctgc
acccaaaggt 3438tctctacaaa ggcccagatt tgtttgtaaa gcactttgac
tcttacctgg aggcccgctc 3498tctaagggct tcctgcgctc ccacctcatc
tgtccctgag atgcagagca ggatggaggg 3558tctgcttcta gctcagctgt
ttctccttga ggttgcggag gaattgaatt gaatgggaca 3618gagggcaggt
gctgtggcca agaagatctc cgagcagcag tgacggggca ccttgctgtg
3678tgtcctctgg gcatgttaac ccttctgtgg ggccaaaggt ttgcatcgtg
gatccagctg 3738tgctccagtc tgtcccctcc tcctccactc tgactgccac
gccccggacc agcagcttgg 3798ggaccctcca gggtactaat ggggctctgt
tctgagatgg acaaattcag tgttggaaat 3858acatgttgta ctatgcactt
cccatgctcc tagggttagg aatagtttca aacatgattg 3918gcagacataa
caacggcaaa tactcggact ggggcatagg actccagagt aggaaaaaga
3978caaaagattt ggcagcctga cacaggcaac ctacccctct ctctccagcc
tctttatgaa 4038actgtttgtt tgccagtcct gccctaaggc agaagatgaa
ttgaagatgc tgtgcatgtt 4098tcctaagtcc ttgagcaatc atggtggtga
caattgccac aagggatatg aggccagtgc 4158caccagaggg tggtgccaag
tgccacatcc cttccgatcc attcccctct gcatcctcgg 4218agcaccccag
tttgcctttg atgtgtccgc tgtgtatgtt agctgaactt tgatgagcaa
4278aatttcctga gcgaaacact ccaaagagat aggaaaactt gccgcctctt
cttttttgtc 4338ccttaatcaa actcaaataa gcttaaaaaa aatccatgga
agatcatgga catgtgaaat 4398gagcattttt ttcttttttt tttttaacaa
agtctgaact gaacagaaca agactttttc 4458ctcatacatc tccaaattgt
ttaaacttac tttatgagtg tttgtttaga agttcggacc 4518aacagaaaaa
tgcagtcaga tgtcatcttg gaattggttt ctaaaagagt aaggcatgtc
4578cctgcccaga aacttaggaa gcatgaaata aatcaaatgt ttattttcct
tcttatttaa 4638aatcatgcaa atgcaacaga aatagagggt ttgtgccaaa
tgctatgaac ggccctttct 4698taaagacaag caagggagat tgatatatgt
acaatttgct ctcatgtttt 47489609PRTHomo sapiens 9Met Pro Ser Asp Pro
Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala
Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys
Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40
45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu
50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys
Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser
Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val
Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg
Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile
Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr
Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly
Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170
175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln
180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp
Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu
Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr
Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly
Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala
Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His
Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser
Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295
300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala
Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu
Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn
Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr
Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met
Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys
Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys
Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410
415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser
420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys
Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser
Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu
Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu
Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro
Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys
Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu
Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535
540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val
Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly
Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser
Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu
Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile 104745DNAHomo
sapiensCDS(29)...(1858) 10taataaaaga ctagtggcct tagtgccc atg ccc
agt gac cct cca ttc aat 52 Met Pro Ser Asp Pro Pro Phe Asn 1 5acc
cga aga gcc tac acc agt gag gat gaa gcc tgg aag tca tac ttg 100Thr
Arg Arg Ala Tyr Thr Ser Glu Asp Glu Ala Trp Lys Ser Tyr Leu 10 15
20gag aat ccc ctg aca gca gcc acc aag gcc atg atg agc att aat ggt
148Glu Asn Pro Leu Thr Ala Ala Thr Lys Ala Met Met Ser Ile Asn
Gly25 30 35 40gat gag gac agt gct gct gcc ctc ggc ctg ctc tat gac
tac tac aag 196Asp Glu Asp Ser Ala Ala Ala Leu Gly Leu Leu Tyr Asp
Tyr Tyr Lys 45 50 55gtt cct cga gac aag agg ctg ctg tct gta agc aaa
gca agt gac agc 244Val Pro Arg Asp Lys Arg Leu Leu Ser Val Ser Lys
Ala Ser Asp Ser 60 65 70caa gaa gac cag gag aaa aga aac tgc ctt ggc
acc agt gaa gcc cag 292Gln Glu Asp Gln Glu Lys Arg Asn Cys Leu Gly
Thr Ser Glu Ala Gln 75 80 85agt aat ttg agt gga gga gaa aac cga gtg
caa gtc cta aag act gtt 340Ser Asn Leu Ser Gly Gly Glu Asn Arg Val
Gln Val Leu Lys Thr Val 90 95 100cca gtg aac ctt tcc cta aat caa
gat cac ctg gag aat tcc aag cgg 388Pro Val Asn Leu Ser Leu Asn Gln
Asp His Leu Glu Asn Ser Lys Arg105 110 115 120gaa cag tac agc atc
agc ttc ccc gag agc tct gcc atc atc ccg gtg 436Glu Gln Tyr Ser Ile
Ser Phe Pro Glu Ser Ser Ala Ile Ile Pro Val 125 130 135tcg gga atc
acg gtg gtg aaa gct gaa gat ttc aca cca gtt ttc atg 484Ser Gly Ile
Thr Val Val Lys Ala Glu Asp Phe Thr Pro Val Phe Met 140 145 150gcc
cca cct gtg cac tat ccc cgg gga gat ggg gaa gag caa cga gtg 532Ala
Pro Pro Val His Tyr Pro Arg Gly Asp Gly Glu Glu Gln Arg Val 155 160
165gtt atc ttt gaa cag act cag tat gac gtg ccc tcg ctg gcc acc cac
580Val Ile Phe Glu Gln Thr Gln Tyr Asp Val Pro Ser Leu Ala Thr His
170 175 180agc gcc tat ctc aaa gac gac cag cgc agc act ccg gac agc
aca tac 628Ser Ala Tyr Leu Lys Asp Asp Gln Arg Ser Thr Pro Asp Ser
Thr Tyr185 190 195 200agc gag agc ttc aag gac gca gcc aca gag aaa
ttt cgg agt gct tca 676Ser Glu Ser Phe Lys Asp Ala Ala Thr Glu Lys
Phe Arg Ser Ala Ser 205 210 215gtt ggg gct gag gag tac atg tat gat
cag aca tca agt ggc aca ttt 724Val Gly Ala Glu Glu Tyr Met Tyr Asp
Gln Thr Ser Ser Gly Thr Phe 220 225 230cag tac acc ctg gaa gcc acc
aaa tct ctc cgt cag aag cag ggg gag 772Gln Tyr Thr Leu Glu Ala Thr
Lys Ser Leu Arg Gln Lys Gln Gly Glu 235 240 245ggc ccc atg acc tac
ctc aac aaa gga cag ttc tat gcc ata aca ctc 820Gly Pro Met Thr Tyr
Leu Asn Lys Gly Gln Phe Tyr Ala Ile Thr Leu 250 255 260agc gag acc
gga gac aac aaa tgc ttc cga cac ccc atc agc aaa gtc 868Ser Glu Thr
Gly Asp Asn Lys Cys Phe Arg His Pro Ile Ser Lys Val265 270 275
280agg agt gtg gtg atg gtg gtc ttc agt gaa gac aaa aac aga gat gaa
916Arg Ser Val Val Met Val Val Phe Ser Glu Asp Lys Asn Arg Asp Glu
285 290 295cag ctc aaa tac tgg aaa tac tgg cac tct cgg cag cat acg
gcg aag 964Gln Leu Lys Tyr Trp Lys Tyr Trp His Ser Arg Gln His Thr
Ala Lys 300 305 310cag agg gtc ctt gac att gcc gat tac aag gag agc
ttt aat acg att 1012Gln Arg Val Leu Asp Ile Ala Asp Tyr Lys Glu Ser
Phe Asn Thr Ile 315 320 325gga aac att gaa gag att gca tat aat gct
gtt tcc ttt acc tgg gac 1060Gly Asn Ile Glu Glu Ile Ala Tyr Asn Ala
Val Ser Phe Thr Trp Asp 330 335 340gtg aat gaa gag gcg aag att ttc
atc acc gtg aat tgc ttg agc aca 1108Val Asn Glu Glu Ala Lys Ile Phe
Ile Thr Val Asn Cys Leu Ser Thr345 350 355 360gat ttc tcc tcc caa
aaa ggg gtg aaa gga ctt cct ttg atg att cag 1156Asp Phe Ser Ser Gln
Lys Gly Val Lys Gly Leu Pro Leu Met Ile Gln 365 370 375att gac aca
tac agt tat aac aat cgt agc aat aaa ccc att cat aga 1204Ile Asp Thr
Tyr Ser Tyr Asn Asn Arg Ser Asn Lys Pro Ile His Arg 380 385 390gct
tat tgc cag atc aag gtc ttc tgt gac aaa gga gca gaa aga aaa 1252Ala
Tyr Cys Gln Ile Lys Val Phe Cys Asp Lys Gly Ala Glu Arg Lys 395 400
405atc cga gat gaa gag cgg aag cag aac agg aag aaa ggg aaa ggc cag
1300Ile Arg Asp Glu Glu Arg Lys Gln Asn Arg Lys Lys Gly Lys Gly Gln
410 415 420gcc tcc caa act caa tgc aac agc tcc tct gat ggg aag ttg
gct gcc 1348Ala Ser Gln Thr Gln Cys Asn Ser Ser Ser Asp Gly Lys Leu
Ala Ala425 430 435 440ata cct tta cag aag aag agt gac atc acc tac
ttc aaa acc atg cct 1396Ile Pro Leu Gln Lys Lys Ser Asp Ile Thr Tyr
Phe Lys Thr Met Pro 445 450 455gat ctc cac tca cag cca gtt ctc ttc
ata cct gat gtt cac ttt gca 1444Asp Leu His Ser Gln Pro Val Leu Phe
Ile Pro Asp Val His Phe Ala 460 465 470aac ctg cag agg acc gga cag
gtg tat tac aac acg gat gat gaa cga 1492Asn Leu Gln Arg Thr Gly Gln
Val Tyr Tyr Asn Thr Asp Asp Glu Arg 475 480 485gaa ggt ggc agt gtc
ctt gtt aaa cgg atg ttc cgg ccc atg gaa gag 1540Glu Gly Gly Ser Val
Leu Val Lys Arg Met Phe Arg Pro Met Glu Glu 490 495 500gag ttt ggt
cca gtg cct tca aag cag atg aaa gaa gaa ggg aca aag 1588Glu Phe Gly
Pro Val Pro Ser Lys Gln Met Lys Glu Glu Gly Thr Lys505 510 515
520cga gtg ctc ttg tac gtg agg aag gag act gac gat gtg ttc gat gca
1636Arg Val Leu Leu Tyr Val Arg Lys Glu Thr Asp Asp Val Phe Asp Ala
525 530 535ttg atg ttg aag tct ccc aca gtg aag ggc ctg atg gaa gcg
ata tct 1684Leu Met Leu Lys Ser Pro Thr Val Lys Gly Leu Met Glu Ala
Ile Ser 540 545 550gag aaa tat ggg ctg ccc gtg gag aag ata gca aag
ctt tac aag aaa 1732Glu Lys Tyr Gly Leu Pro Val Glu Lys Ile Ala Lys
Leu Tyr Lys Lys 555 560 565agc aaa aaa ggc atc ttg gtg aac atg gat
gac aac atc atc gag cac 1780Ser Lys Lys Gly Ile Leu Val Asn Met Asp
Asp Asn Ile Ile Glu His 570 575 580tac tcg aac gag gac acc ttc atc
ctc aac atg gag agc atg gtg gag 1828Tyr Ser Asn Glu Asp Thr Phe Ile
Leu Asn Met Glu Ser Met Val Glu585 590 595 600ggc ttc aag gtc acg
ctc atg gaa atc tag ccctgggttt ggcatccgct 1878Gly Phe Lys Val Thr
Leu Met Glu Ile 605ttggctggag ctctcagtgc gttcctccct gagagagaca
gaagccccag ccccagaacc 1938tggagaccca tctcccccat ctcacaactg
ctgttacaag accgtgctgg ggagtggggc 1998aagggacagg ccccactgtc
ggtgtgcttg gcccatccac tggcacctac cacggagctg 2058aagcctgagc
ccctcaggaa ggtgccttag gcctgttgga ttcctattta ttgcccacct
2118tttcctggag cccaggtcca ggcccgccag gactctgcag gtcactgcta
gctccagatg 2178agaccgtcca gcgttccccc ttcaagagaa acactcatcc
cgaacagcct aaaaaattcc 2238catcccttct ctctcacccc tccatatcta
tctcccgagt ggctggacaa aatgagctac 2298gtctgggtgc agtagttata
ggtggggcaa gaggtggatg cccactttct ggtcagacac 2358ctttaggttg
ctctggggaa ggctgtcttg ctaaatacct ccagggttcc cagcaagtgg
2418ccaccaggcc ttgtacagga agacattcag tcaccgtgta attagtaaca
cagaaagtct 2478gcctgtctgc attgtacata gtgtttataa tattgtaata
atatatttta cctgtggtat 2538gtgggcatgt ttactgccac tggcctagag
gagacacaga cctggagacc gttttaatgg 2598gggtttttgc ctctgtgcct
gttcaagaga cttgcagggc taggtagagg gcctttggga 2658tgttaaggtg
actgcagctg atgccaagat ggactctgca atgggcatac ctgggggctc
2718gttccctgtc cccagaggaa gccccctctc cttctccatg ggcatgactc
tccttcgagg 2778ccaccacgtt tatctcacaa tgatgtgttt tgcttgactt
tccctttgcg ctgtctcgtg 2838ggaaaggtca ttctgtctga gaccccagct
ccttctccag ctttggctgc gggcatggcc 2898tgagctttct ggagagcctc
tgcagggggt ttgccatcag ggccctgtgg ctgggtctgc 2958tgcagagctc
cttggctatc aggagaatcc tggacactgt actgtgcctc ccagtttaca
3018aacacgccct tcatctcaag tggcccttta aaaggcctgc tgccatgtga
gagctgtgaa 3078cagctcagct ctgagtcggc aggctggggc ttcctcctgg
gccaccagat ggaaaggggg 3138tattgtttgc ctcactcctg gatgctgcgt
tttaaggaag tgagtgagaa agaatgtgcc 3198aagatacctg gctcctgtga
aaccagcctc aggagggaaa ctgggagaga gaagctgtgg 3258tctcctgcta
catgccctgg gagctggaag agaaaaacac tcccctaaac aatcgcaaaa
3318tgatgaacca tcatgggcca ctgttctctt tgaggggaca ggtttagggg
tttgcgttcg 3378cccttgtggg ctgaagcact agctttttgg tagctagaca
catcctgcac ccaaaggttc 3438tctacaaagg
cccagatttg tttgtaaagc actttgactc ttacctggag gcccgctctc
3498taagggcttc ctgcgctccc acctcatcgt ccctgagatg cagagcagga
tggagggtct 3558gcttctagct cagctgtttc tccttgaggt tgcggaggaa
ttgaattgaa tgggacagag 3618ggcaggtgct gtggccaaga agatctccga
gcagcagtga cggggcacct tgctgtgtgt 3678cctctgggca tgttaaccct
tctgtggggc caaaggtttg catcgtggat ccagctgtgc 3738tccagtctgt
cccctcctcc tccactctga ctgccacgcc ccggaccagc agcttgggga
3798ccctccaggg tactaatggg gctctgttct gagatggaca aattcagtgt
tggaaataca 3858tgttgtacta tgcacttccc atgctcctag ggttaggaat
agtttcaaac atgattggca 3918gacataacaa cggcaaatac tcggactggg
gcataggact ccagagtagg aaaaagacaa 3978aagatttggc agcctgacac
aggcaaccta cccctctctc tccagcctct ttatgaaact 4038gtttgtttgc
cagtcctgcc ctaaggcaga agatgaattg aagatgctgt gcatgtttcc
4098taagtccttg agcaatcatg gtggtgacaa ttgccacaag ggatatgagg
ccagtgccac 4158cagagggtgg tgccaagtgc cacatccctt ccgatccatt
cccctctgca tcctcggagc 4218accccagttt gcctttgatg tgtccgctgt
gtatgttagc tgaactttga tgagcaaaat 4278ttcctgagcg aaacactcca
aagagatagg aaaacttgcc gcctcttctt ttttgtccct 4338taatcaaact
caaataagct taaaaaaaat ccatggaaga tcatggacat gtgaaatgag
4398catttttttc tttttttttt ttaacaaagt ctgaactgaa cagaacaaga
ctttttcctc 4458atacatctcc aaattgttta aacttacttt atgagtgttt
gtttagaagt tcggaccaac 4518agaaaaatgc agtcagatgt catcttggaa
ttggtttcta aaagagtaag gcatgtccct 4578gcccagaaac ttaggaagca
tgaaataaat caaatgttta ttttccttct tatttaaaat 4638catgcaaatg
caacagaaat agagggtttg tgccaaatgc tatgaacggc cctttcttaa
4698agacaagcaa gggagattga tatatgtaca atttgctctc atgtttt
474511609PRTHomo sapiens 11Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205
Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210
215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile 12609PRTHomo sapiens 12Met Pro Ser Asp Pro
Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala
Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys
Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40
45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu
50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys
Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser
Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val
Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg
Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile
Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr
Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly
Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170
175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln
180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp
Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu
Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr
Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly
Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala
Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His
Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser
Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295
300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala
Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu
Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn
Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr
Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met
Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys
Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys
Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410
415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser
420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys
Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser
Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu
Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu
Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro
Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys
Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu
Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535
540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val
Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly
Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser
Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu
Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile 13625PRTHomo
sapiens 13Met Ser Gln Glu Ser Asp Asn Asn Lys Arg Leu Val Ala Leu
Val Pro1 5 10 15 Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala
Tyr Thr Ser Glu 20 25 30 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn
Pro Leu Thr Ala Ala Thr 35 40 45 Lys Ala Met Met Ser Ile Asn Gly
Asp Glu Asp Ser Ala Ala Ala Leu 50 55 60 Gly Leu Leu Tyr Asp Tyr
Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu65 70 75 80 Ser Val Ser Lys
Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn 85 90 95 Cys Leu
Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 100 105 110
Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln 115
120 125 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe
Pro 130 135 140 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val
Val Lys Ala145 150 155 160 Glu Asp Phe Thr Pro Val Phe Met Ala Pro
Pro Val His Tyr Pro Arg 165 170 175 Gly Asp Gly Glu Glu Gln Arg Val
Val Ile Phe Glu Gln Thr Gln Tyr 180 185 190 Asp Val Pro Ser Leu Ala
Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 195 200 205 Arg Ser Thr Pro
Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 210 215 220 Thr Glu
Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr225 230 235
240 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys
245 250 255 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu
Asn Lys 260 265 270 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly
Asp Asn Lys Cys 275 280 285 Phe Arg His Pro Ile Ser Lys Val Arg Ser
Val Val Met Val Val Phe 290 295 300 Ser Glu Asp Lys Asn Arg Asp Glu
Gln Leu Lys Tyr Trp Lys Tyr Trp305 310 315 320 His Ser Arg Gln His
Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp 325 330 335 Tyr Lys Glu
Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 340 345 350 Asn
Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 355 360
365 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val
370 375 380 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr
Asn Asn385 390 395 400 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys
Gln Ile Lys Val Phe 405 410 415 Cys Asp Lys Gly Ala Glu Arg Lys Ile
Arg Asp Glu Glu Arg Lys Gln 420 425 430 Asn Arg Lys Lys Gly Lys Gly
Gln Ala Ser Gln Thr Gln Cys Asn Ser 435 440 445 Ser Ser Asp Gly Lys
Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 450 455 460 Ile Thr Tyr
Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu465 470 475 480
Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val 485
490 495 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val
Lys 500 505 510 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val
Pro Ser Lys 515 520 525 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu
Leu Tyr Val Arg Lys 530 535 540 Glu Thr Asp Asp Val Phe Asp Ala Leu
Met Leu Lys Ser Pro Thr Val545 550 555 560 Lys Gly Leu Met Glu Ala
Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu 565 570 575 Lys Ile Ala Lys
Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 580 585 590 Met Asp
Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 595 600 605
Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 610
615 620 Ile625 14609PRTHomo sapiens 14Met Pro Ser Asp Pro Pro Phe
Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys
Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met
Met Ile Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly
Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55
60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg
Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly
Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn
Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu
Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro
Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro
Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp
Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175
Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180
185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala
Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu
Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr
Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu
Gly Pro Met Thr Tyr Leu Asn Lys 245 250
255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys
260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val
Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr
Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys Gln Arg
Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr
Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe
Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val
Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys
Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375
380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val
Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu
Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser
Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala
Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr
Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp
Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480 Tyr
Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485 490
495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys
500 505 510 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val
Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys
Ser Pro Thr Val 530 535 540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys
Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys
Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile
Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met
Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile
15609PRTHomo sapiens 15Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg
Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu
Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile Asn
Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr Asp
Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val Ser
Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80 Cys
Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85 90
95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln
100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser
Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr
Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro
Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln Arg
Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser Leu
Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr
Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205 Thr
Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210 215
220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Gln Lys Gln 405 410 415 Asn Arg Lys Lys Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile 16609PRTHomo sapiens 16Met Pro Ser Asp Pro
Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala
Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys
Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40
45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu
50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys
Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser
Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val
Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg
Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile
Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr
Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly
Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170
175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln
180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp
Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu
Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr
Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly
Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala
Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His
Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser
Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295
300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala
Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu
Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn
Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr
Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met
Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys
Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys
Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410
415 Asn Arg Lys Asn Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser
420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys
Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser
Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu
Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu
Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro
Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys
Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu
Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535
540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val
Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly
Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser
Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu
Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile 17609PRTHomo
sapiens 17Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr
Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu
Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu
Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys
Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser
Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80 Cys Leu Gly Thr
Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val
Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln 100 105 110
Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115
120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys
Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His
Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile
Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His
Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe
Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln
Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235
240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys
245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn
Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe
Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val
Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360
365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn
370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys
Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala
Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys
Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro
Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480
Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485
490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser
Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr
Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu
Lys Ser Pro Thr Val 530 535 540 Met Gly Leu Met Glu Ala Ile Ser Glu
Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr
Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn
Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn
Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605
Ile 18609PRTHomo sapiens 18Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115
120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys
Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His
Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile
Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His
Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe
Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln
Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235
240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys
245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn
Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe
Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val
Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360
365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn
370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys
Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala
Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys
Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro
Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480
Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485
490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser
Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr
Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu
Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met Glu Ala Ile Ser Glu
Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr
Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn
Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn
Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605
Ile 19609PRTHomo sapiens 19Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ile Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205
Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210
215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Gln Lys Gln 405 410 415 Asn Arg Lys Asn Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Met Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile 20609PRTHomo sapiens 20Met Pro Ser Asp Pro
Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala
Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys
Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40
45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu
50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys
Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser
Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val
Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg
Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile
Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr
Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly
Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170
175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln
180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp
Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu
Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr
Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly
Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala
Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His
Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser
Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295
300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala
Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu
Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn
Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr
Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met
Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys
Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys
Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410
415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser
420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys
Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser
Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu
Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu
Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro
Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys
Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu
Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535
540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val
Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly
Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser
Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu
Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile 21609PRTHomo
sapiens 21Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr
Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu
Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu
Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys
Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser
Asp Ser Gln Glu Asp Gln Asp Lys Arg Asn65 70 75 80 Cys Leu Gly Thr
Ser Glu Ala Gln Ile Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val
Gln Val Leu Lys Thr Val Pro Val Asn Leu Cys Leu Ser Gln 100 105 110
Asp His Met Glu Asn Ser Lys Arg Glu Gln Tyr Ser Val Ser Ile Thr 115
120 125 Glu Ser Ser Ala Val Ile Pro Val Ser Gly Ile Thr Val Val Lys
Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His
Tyr Pro Arg145 150 155 160 Ala Asp Ser Glu Glu Gln Arg Val Val Ile
Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Leu Pro Ser Ile Ala Ser His
Ser Ser Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys Asp Gly Ala 195 200 205 Ser Glu Lys Phe
Arg Ser Thr Ser Val Gly Ala Asp Glu Tyr Thr Tyr 210 215 220 Asp Gln
Thr Gly Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235
240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys
245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn
Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe
Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val
Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360
365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn
370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys
Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Ala Gln Cys Asn Asn 420 425 430 Ser Ser Asp Gly Lys Met Ala
Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys
Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro
Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480
Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Ser Ser Val Leu Val Lys 485
490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Thr Pro Ser
Lys 500 505 510 Gln Ile Lys Glu Glu Asn Val Lys Arg Val Leu Leu Tyr
Val Arg Lys 515 520 525 Glu Asn Asp Asp Val Phe Asp Ala Leu Met Leu
Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met Glu Ala Leu Ser Glu
Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Thr Lys Leu Tyr
Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn
Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn
Met Glu Ser Met Val Glu Gly Phe Lys Ile Thr Leu Met Glu 595
600 605 Ile 22403PRTHomo sapiens 22Met Pro Ser Asp Pro Pro Phe Asn
Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser
Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met
Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu
Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60
Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65
70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly
Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu
Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln
Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val
Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val
Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly
Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp
Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185
190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala
195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr
Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu
Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly
Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr
Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile
Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp
Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His
Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310
315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala
Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala
Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser
Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile
Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His
Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp
Lys23403PRTHomo sapiens 23Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Asp Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ile Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Cys Leu Ser
Gln 100 105 110 Asp His Met Glu Asn Ser Lys Arg Glu Gln Tyr Ser Val
Ser Ile Thr 115 120 125 Glu Ser Ser Ala Val Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Ala Asp Ser Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Leu Pro Ser
Ile Ala Ser His Ser Ser Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Gly Ala 195 200 205
Ser Glu Lys Phe Arg Ser Thr Ser Val Gly Ala Asp Glu Tyr Thr Tyr 210
215 220 Asp Gln Thr Gly Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp
Lys2414PRTClostridium tetanitetanus toxoid 24Gln Tyr Ile Lys Ala
Asn Ser Lys Phe Ile Gly Ile Thr Glu1 5 10 2521PRTPlasmodium
falciparum 25Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser
Ser Val Phe1 5 10 15 Asn Val Val Asn Ser 20 2616PRTStreptococcus
26Gly Ala Val Asp Ser Ile Leu Gly Gly Val Ala Thr Tyr Gly Ala Ala1
5 10 15 2712PRTArtificial SequencePan-DR-binding epitope 27Xaa Lys
Xaa Val Ala Ala Trp Thr Leu Lys Ala Xaa1 5 10 2814DNAArtificial
SequencePrimer 28ttttgatcaa gctt 142942DNAArtificial SequencePrimer
29ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag
423012DNAArtificial SequencePrimer 30gatcctgccc gg
123140DNAArtificial SequencePrimer 31gtaatacgac tcactatagg
gcagcgtggt cgcggccgag 403210DNAArtificial SequencePrimer
32gatcctcggc 103322DNAArtificial SequencePrimer 33ctaatacgac
tcactatagg gc 223422DNAArtificial SequencePrimer 34tcgagcggcc
gcccgggcag ga 223520DNAArtificial SequencePrimer 35agcgtggtcg
cggccgagga 203625DNAArtificial SequencePrimer 36atatcgccgc
gctcgtcgtc gacaa 253726DNAArtificial SequencePrimer 37agccacacgc
agctcattgt agaagg 263824DNAArtificial SequencePrimer 38catttcacat
gtccatgatc ttcc 243924DNAArtificial SequencePrimer 39ctttgatgtg
tccgctgtgt atgt 244024DNAArtificial SequenceFLAG Epitope tag
40gattacaagg atgacgacga taag 24414PRTHomo sapiens 41Asn Leu Ser
Gly1 424PRTHomo sapiens 42Asn Leu Ser Leu1 434PRTHomo sapiens 43Asn
Arg Ser Asn1 444PRTHomo sapiens 44Asn Ser Ser Ser1 4515PRTHomo
sapiens 45Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr Asp Gln Thr Ser
Ser1 5 10 15 4615PRTHomo sapiens 46Val Gly Ala Glu Glu Tyr Met Tyr
Asp Gln Thr Ser Ser Gly Thr1 5 10 15 4715PRTHomo sapiens 47Arg Val
Leu Asp Ile Ala Asp Tyr Lys Glu Ser Phe Asn Thr Ile1 5 10 15
4815PRTHomo sapiens 48Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu
Asp Thr Phe Ile1 5 10 15 494PRTHomo sapiens 49Arg Lys Glu Thr1
504PRTHomo sapiens 50Thr Ser Glu Asp1 514PRTHomo sapiens 51Ser Glu
Asp Glu1 524PRTHomo sapiens 52Ser Tyr Leu Glu1 534PRTHomo sapiens
53Ser Gln Glu Asp1 544PRTHomo sapiens 54Ser Gly Gly Glu1 554PRTHomo
sapiens 55Ser Lys Arg Glu1 564PRTHomo sapiens 56Ser Phe Pro Glu1
574PRTHomo sapiens 57Thr Gln Tyr Asp1 584PRTHomo sapiens 58Ser Thr
Pro Asp1 594PRTHomo sapiens 59Thr Tyr Ser Glu1 604PRTHomo sapiens
60Ser Phe Lys Asp1 614PRTHomo sapiens 61Thr Leu Ser Glu1 624PRTHomo
sapiens 62Ser Ser Ser Asp1 634PRTHomo sapiens 63Thr Met Pro Asp1
644PRTHomo sapiens 64Thr Asp Asp Glu1 654PRTHomo sapiens 65Ser Asn
Glu Asp1 664PRTHomo sapiens 66Ser Met Val Glu1 674PRTHomo sapiens
67Thr Leu Met Glu1 688PRTHomo sapiens 68Arg Ser Thr Pro Asp Ser Thr
Tyr1 5 699PRTHomo sapiens 69Lys Asn Arg Asp Glu Gln Leu Lys Tyr1 5
708PRTHomo sapiens 70Arg Val Leu Asp Ile Ala Asp Tyr1 5 717PRTHomo
sapiens 71Lys Lys Ser Asp Ile Thr Tyr1 5 726PRTHomo sapiens 72Gly
Thr Ser Glu Ala Gln1 5 736PRTHomo sapiens 73Gly Gln Phe Tyr Ala
Ile1 5 746PRTHomo sapiens 74Gly Leu Met Glu Ala Ile1 5 756PRTHomo
sapiens 75Gly Ile Leu Val Asn Met1 5 7617PRTHomo sapiens 76Arg Lys
Ile Arg Asp Glu Glu Arg Lys Gln Asn Arg Lys Lys Gly Lys1 5 10 15
Gly77609PRTHomo sapiens 77Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205
Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210
215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile 7824PRTHomo sapiens 78Met Ser Gln Glu Ser
Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro1 5 10 15 Met Pro Ser
Asp Pro Pro Phe Asn 20 7925PRTHomo sapiens 79Met Ser Gln Glu Ser
Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro1 5 10 15 Met Pro Ser
Asp Pro Pro Phe Asn Thr 20 25 8030PRTHomo sapiens 80Met Ser Gln Glu
Ser Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro1 5 10 15 Met Pro
Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr 20 25 30
8117PRTHomo sapiens 81Thr Ala Ala Thr Lys Ala Met Met Ile Ile Asn
Gly Asp Glu Asp Ser1 5 10 15 Ala8219PRTHomo sapiens 82Leu Thr Ala
Ala Thr Lys Ala Met Met Ile Ile Asn Gly Asp Glu Asp1 5 10 15 Ser
Ala Ala8329PRTHomo sapiens 83Tyr Leu Glu Asn Pro Leu Thr Ala Ala
Thr Lys Ala Met Met Ile Ile1 5 10 15 Asn Gly Asp Glu Asp Ser Ala
Ala Ala Leu Gly Leu Leu 20 25 8417PRTHomo sapiens 84Glu Arg Lys Ile
Arg Asp Glu Glu Gln Lys Gln Asn Arg Lys Lys Gly1 5 10 15
Lys8519PRTHomo sapiens 85Ala Glu Arg Lys Ile Arg Asp Glu Glu Gln
Lys Gln Asn Arg Lys Lys1 5 10 15 Gly Lys Gly8629PRTHomo sapiens
86Phe Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Gln Lys1
5 10 15 Gln Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr 20 25
8717PRTHomo sapiens 87Glu Glu Arg Lys Gln Asn Arg Lys Asn Gly Lys
Gly Gln Ala Ser Gln1 5 10 15 Thr8819PRTHomo sapiens 88Asp Glu Glu
Arg Lys Gln Asn Arg Lys Asn Gly Lys Gly Gln Ala Ser1 5 10 15 Gln
Thr Gln8929PRTHomo sapiens 89Glu Arg Lys Ile Arg Asp Glu Glu Arg
Lys Gln Asn Arg Lys Asn Gly1 5 10 15 Lys Gly Gln Ala Ser Gln Thr
Gln Cys Asn Ser Ser Ser 20 25
9011PRTHomo sapiens 90Glu Glu Gln Lys Gln Asn Arg Lys Asn Gly Lys1
5 10 9113PRTHomo sapiens 91Asp Glu Glu Gln Lys Gln Asn Arg Lys Asn
Gly Lys Gly1 5 10 9223PRTHomo sapiens 92Glu Arg Lys Ile Arg Asp Glu
Glu Gln Lys Gln Asn Arg Lys Asn Gly1 5 10 15 Lys Gly Gln Ala Ser
Gln Thr 20 9317PRTHomo sapiens 93Leu Met Leu Lys Ser Pro Thr Val
Met Gly Leu Met Glu Ala Ile Ser1 5 10 15 Glu9419PRTHomo sapiens
94Ala Leu Met Leu Lys Ser Pro Thr Val Met Gly Leu Met Glu Ala Ile1
5 10 15 Ser Glu Lys9526PRTHomo sapiens 95Phe Asp Ala Leu Met Leu
Lys Ser Pro Thr Val Met Gly Leu Met Glu1 5 10 15 Ala Ile Ser Glu
Lys Tyr Gly Leu Pro Val 20 25 964913DNAHomo sapiens 96attggatcaa
acatgtcaca agagtcggac aataataaaa gactagtggc cttagtgccc 60atgcccagtg
accctccatt caatacccga agagcctaca ccagtgagga tgaagcctgg
120aagtcatact tggagaatcc cctgacagca gccaccaagg ccatgatgag
cattaatggt 180gatgaggaca gtgctgctgc cctcggcctg ctctatgact
actacaaggt tcctcgagac 240aagaggctgc tgtctgtaag caaagcaagt
gacagccaag aagaccagga gaaaagaaac 300tgccttggca ccagtgaagc
ccagagtaat ttgagtggag gagaaaaccg agtgcaagtc 360ctaaagactg
ttccagtgaa cctttcccta aatcaagatc acctggagaa ttccaagcgg
420gaacagtaca gcatcagctt ccccgagagc tctgccatca tcccggtgtc
gggaatcacg 480gtggtgaaag ctgaagattt cacaccagtt ttcatggccc
cacctgtgca ctatccccgg 540ggagatgggg aagagcaacg agtggttatc
tttgaacaga ctcagtatga cgtgccctcg 600ctggccaccc acagcgccta
tctcaaagac gaccagcgca gcactccgga cagcacatac 660agcgagagct
tcaaggacgc agccacagag aaatttcgga gtgcttcagt tggggctgag
720gagtacatgt atgatcagac atcaagtggc acatttcagt acaccctgga
agccaccaaa 780tctctccgtc agaagcaggg ggagggcccc atgacctacc
tcaacaaagg acagttctat 840gccataacac tcagcgagac cggagacaac
aaatgcttcc gacaccccat cagcaaagtc 900aggagtgtgg tgatggtggt
cttcagtgaa gacaaaaaca gagatgaaca gctcaaatac 960tggaaatact
ggcactctcg gcagcatacg gcgaagcaga gggtccttga cattgccgat
1020tacaaggaga gctttaatac gattggaaac attgaagaga ttgcatataa
tgctgtttcc 1080tttacctggg acgtgaatga agaggcgaag attttcatca
ccgtgaattg cttgagcaca 1140gatttctcct cccaaaaagg ggtgaaagga
cttcctttga tgattcagat tgacacatac 1200agttataaca atcgtagcaa
taaacccatt catagagctt attgccagat caaggtcttc 1260tgtgacaaag
gagcagaaag aaaaatccga gatgaagagc ggaagcagaa caggaagaaa
1320gggaaaggcc aggcctccca aactcaatgc aacagctcct ctgatgggaa
gttggctgcc 1380atacctttac agaagaagag tgacatcacc tacttcaaaa
ccatgcctga tctccactca 1440cagccagttc tcttcatacc tgatgttcac
tttgcaaacc tgcagaggac cggacaggtg 1500tattacaaca cggatgatga
acgagaaggt ggcagtgtcc ttgttaaacg gatgttccgg 1560cccatggaag
aggagtttgg tccagtgcct tcaaagcaga tgaaagaaga agggacaaag
1620cgagtgctct tgtacgtgag gaaggagact gacgatgtgt tcgatgcatt
gatgttgaag 1680tctcccacag tgaagggcct gatggaagcg atatctgaga
aatatgggct gcccgtggag 1740aagatagcaa agctttacaa gaaaagcaaa
aaaggcatct tggtgaacat ggatgacaac 1800atcatcgagc actactcgaa
cgaggacacc ttcatcctca acatggagag catggtggag 1860ggcttcaagg
tcacgctcat ggaaatctag ccctgggttt ggcatccgct ttggctggag
1920ctctcagtgc gttcctccct gagagagaca gaagccccag ccccagaacc
tggagaccca 1980tctcccccat ctcacaactg ctgttacaag accgtgctgg
ggagtggggc aagggacagg 2040ccccactgtc ggtgtgcttg gcccatccac
tggcacctac cacggagctg aagcctgagc 2100ccctcaggaa ggtgccttag
gcctgttgga ttcctattta ttgcccacct tttcctggag 2160cccaggtcca
ggcccgccag gactctgcag gtcactgcta gctccagatg agaccgtcca
2220gcgttccccc ttcaagagaa acactcatcc cgaacagcct aaaaaattcc
catcccttct 2280ctctcacccc tccatatcta tctcccgagt ggctggacaa
aatgagctac gtctgggtgc 2340agtagttata ggtggggcaa gaggtggatg
cccactttct ggtcagacac ctttaggttg 2400ctctggggaa ggctgtcttg
ctaaatacct ccagggttcc cagcaagtgg ccaccaggcc 2460ttgtacagga
agacattcag tcaccgtgta attagtaaca cagaaagtct gcctgtctgc
2520attgtacata gtgtttataa tattgtaata atatatttta cctgtggtat
gtgggcatgt 2580ttactgccac tggcctagag gagacacaga cctggagacc
gttttaatgg gggtttttgc 2640ctctgtgcct gttcaagaga cttgcagggc
taggtagagg gcctttggga tgttaaggtg 2700actgcagctg atgccaagat
ggactctgca atgggcatac ctgggggctc gttccctgtc 2760cccagaggaa
gccccctctc cttctccatg ggcatgactc tccttcgagg ccaccacgtt
2820tatctcacaa tgatgtgttt tgcttgactt tccctttgcg ctgtctcgtg
ggaaaggtca 2880ttctgtctga gaccccagct ccttctccag ctttggctgc
gggcatggcc tgagctttct 2940ggagagcctc tgcagggggt ttgccatcag
ggccctgtgg ctgggtctgc tgcagagctc 3000cttggctatc aggagaatcc
tggacactgt actgtgcctc ccagtttaca aacacgccct 3060tcatctcaag
tggcccttta aaaggcctgc tgccatgtga gagctgtgaa cagctcagct
3120ctgagtcggc aggctggggc ttcctcctgg gccaccagat ggaaaggggg
tattgtttgc 3180ctcactcctg gatgctgcgt tttaaggaag tgagtgagaa
agaatgtgcc aagatacctg 3240gctcctgtga aaccagcctc aggagggaaa
ctgggagaga gaagctgtgg tctcctgcta 3300catgccctgg gagctggaag
agaaaaacac tcccctaaac aatcgcaaaa tgatgaacca 3360tcatgggcca
ctgttctctt tgaggggaca ggtttagggg tttgcgttcg cccttgtggg
3420ctgaagcact agctttttgg tagctagaca catcctgcac ccaaaggttc
tctacaaagg 3480cccagatttg tttgtaaagc actttgactc ttacctggag
gcccgctctc taagggcttc 3540ctgcgctccc acctcatctg tccctgagat
gcagagcagg atggagggtc tgcttctagc 3600tcagctgttt ctccttgagg
ttgcggagga attgaattga atgggacaga gggcaggtgc 3660tgtggccaag
aagatctccg agcagcagtg acggggcacc ttgctgtgtg tcctctgggc
3720atgttaaccc ttctgtgggg ccaaaggttt gcatcgtgga tccagctgtg
ctccagtctg 3780tcccctcctc ctccactctg actgccacgc cccggaccag
cagcttgggg accctccagg 3840gtactaatgg ggctctgttc tgagatggac
aaattcagtg ttggaaatac atgttgtact 3900atgcacttcc catgctccta
gggttaggaa tagtttcaaa catgattggc agacataaca 3960acggcaaata
ctcggactgg ggcataggac tccagagtag gaaaaagaca aaagatttgg
4020cagcctgaca caggcaacct acccctctct ctccagcctc tttatgaaac
tgtttgtttg 4080ccagtcctgc cctaaggcag aagatgaatt gaagatgctg
tgcatgtttc ctaagtcctt 4140gagcaatcat ggtggtgaca attgccacaa
gggatatgag gccagtgcca ccagagggtg 4200gtgccaagtg ccacatccct
tccgatccat tcccctctgc atcctcggag caccccagtt 4260tgcctttgat
gtgtccgctg tgtatgttag ctgaactttg atgagcaaaa tttcctgagc
4320gaaacactcc aaagagatag gaaaacttgc cgcctcttct tttttgtccc
ttaatcaaac 4380tcaaataagc ttaaaaaaaa tccatggaag atcatggaca
tgtgaaatga gcattttttt 4440cttttttttt tttaacaaag tctgaactga
acagaacaag actttttcct catacatctc 4500caaattgttt aaacttactt
tatgagtgtt tgtttagaag ttcggaccaa cagaaaaatg 4560cagtcagatg
tcatcttgga attggtttct aaaagagtaa ggcatgtccc tgcccagaaa
4620cttaggaagc atgaaataaa tcaaatgttt attttccttc ttatttaaaa
tcatgcaaat 4680gcaacagaaa tagagggttt gtgccaaatg ctatgaacgg
ccctttctta aagacaagca 4740agggagattg atatatgtac aatttgctct
catgttttaa aaaaaaaagg taaatgtaac 4800ttaatagttt tgtaaatggg
agagggggaa tctataaact ataaatacag ttattttatt 4860ttttgtacat
ttttaaggag aaaaaaataa atattcataa cataagagga aaa 4913974746DNAHomo
sapiens 97taataaaaga ctagtggcct tagtgcccat gcccagtgac cctccattca
atacccgaag 60agcctacacc agtgaggatg aagcctggaa gtcatacttg gagaatcccc
tgacagcagc 120caccaaggcc atgatgagca ttaatggtga tgaggacagt
gctgctgccc tcggcctgct 180ctatgactac tacaaggttc ctcgagacaa
gaggctgctg tctgtaagca aagcaagtga 240cagccaagaa gaccaggaga
aaagaaactg ccttggcacc agtgaagccc agagtaattt 300gagtggagga
gaaaaccgag tgcaagtcct aaagactgtt ccagtgaacc tttccctaaa
360tcaagatcac ctggagaatt ccaagcggga acagtacagc atcagcttcc
ccgagagctc 420tgccatcatc ccggtgtcgg gaatcacggt ggtgaaagct
gaagatttca caccagtttt 480catggcccca cctgtgcact atccccgggg
agatggggaa gagcaacgag tggttatctt 540tgaacagact cagtatgacg
tgccctcgct ggccacccac agcgcctatc tcaaagacga 600ccagcgcagc
actccggaca gcacatacag cgagagcttc aaggacgcag ccacagagaa
660atttcggagt gcttcagttg gggctgagga gtacatgtat gatcagacat
caagtggcac 720atttcagtac accctggaag ccaccaaatc tctccgtcag
aagcaggggg agggccccat 780gacctacctc aacaaaggac agttctatgc
cataacactc agcgagaccg gagacaacaa 840atgcttccga caccccatca
gcaaagtcag gagtgtggtg atggtggtct tcagtgaaga 900caaaaacaga
gatgaacagc tcaaatactg gaaatactgg cactctcggc agcatacggc
960gaagcagagg gtccttgaca ttgccgatta caaggagagc tttaatacga
ttggaaacat 1020tgaagagatt gcatataatg ctgtttcctt tacctgggac
gtgaatgaag aggcgaagat 1080tttcatcacc gtgaattgct tgagcacaga
tttctcctcc caaaaagggg tgaaaggact 1140tcctttgatg attcagattg
acacatacag ttataacaat cgtagcaata aacccattca 1200tagagcttat
tgccagatca aggtcttctg tgacaaagga gcagaaagaa aaatccgaga
1260tgaagagcgg aagcagaaca ggaagaaagg gaaaggccag gcctcccaaa
ctcaatgcaa 1320cagctcctct gatgggaagt tggctgccat acctttacag
aagaagagtg acatcaccta 1380cttcaaaacc atgcctgatc tccactcaca
gccagttctc ttcatacctg atgttcactt 1440tgcaaacctg cagaggaccg
gacaggtgta ttacaacacg gatgatgaac gagaaggtgg 1500cagtgtcctt
gttaaacgga tgttccggcc catggaagag gagtttggtc cagtgccttc
1560aaagcagatg aaagaagaag ggacaaagcg agtgctcttg tacgtgagga
aggagactga 1620cgatgtgttc gatgcattga tgttgaagtc tcccacagtg
aagggcctga tggaagcgat 1680atctgagaaa tatgggctgc ccgtggagaa
gatagcaaag ctttacaaga aaagcaaaaa 1740aggcatcttg gtgaacatgg
atgacaacat catcgagcac tactcgaacg aggacacctt 1800catcctcaac
atggagagca tggtggaggg cttcaaggtc acgctcatgg aaatctagcc
1860ctgggtttgg catccgcttt ggctggagct ctcagtgcgt tcctccctga
gagagacaga 1920agccccagcc ccagaacctg gagacccatc tcccccatct
cacaactgct gttacaagac 1980cgtgctgggg agtggggcaa gggacaggcc
ccactgtcgg tgtgcttggc ccatccactg 2040gcacctacca cggagctgaa
gcctgagccc ctcaggaagg tgccttaggc ctgttggatt 2100cctatttatt
gcccaccttt tcctggagcc caggtccagg cccgccagga ctctgcaggt
2160cactgctagc tccagatgag accgtccagc gttccccctt caagagaaac
actcatcccg 2220aacagcctaa aaaattccca tcccttctct ctcacccctc
catatctatc tcccgagtgg 2280ctggacaaaa tgagctacgt ctgggtgcag
tagttatagg tggggcaaga ggtggatgcc 2340cactttctgg tcagacacct
ttaggttgct ctggggaagg ctgtcttgct aaatacctcc 2400agggttccca
gcaagtggcc accaggcctt gtacaggaag acattcagtc accgtgtaat
2460tagtaacaca gaaagtctgc ctgtctgcat tgtacatagt gtttataata
ttgtaataat 2520atattttacc tgtggtatgt gggcatgttt actgccactg
gcctagagga gacacagacc 2580tggagaccgt tttaatgggg gtttttgcct
ctgtgcctgt tcaagagact tgcagggcta 2640ggtagagggc ctttgggatg
ttaaggtgac tgcagctgat gccaagatgg actctgcaat 2700gggcatacct
gggggctcgt tccctgtccc cagaggaagc cccctctcct tctccatggg
2760catgactctc cttcgaggcc accacgttta tctcacaatg atgtgttttg
cttgactttc 2820cctttgcgct gtctcgtggg aaaggtcatt ctgtctgaga
ccccagctcc ttctccagct 2880ttggctgcgg gcatggcctg agctttctgg
agagcctctg cagggggttt gccatcaggg 2940ccctgtggct gggtctgctg
cagagctcct tggctatcag gagaatcctg gacactgtac 3000tgtgcctccc
agtttacaaa cacgcccttc atctcaagtg gccctttaaa aggcctgctg
3060ccatgtgaga gctgtgaaca gctcagctct gagtcggcag gctggggctt
cctcctgggc 3120caccagatgg aaagggggta ttgtttgcct cactcctgga
tgctgcgttt taaggaagtg 3180agtgagaaag aatgtgccaa gatacctggc
tcctgtgaaa ccagcctcag gagggaaact 3240gggagagaga agctgtggtc
tcctgctaca tgccctggga gctggaagag aaaaacactc 3300ccctaaacaa
tcgcaaaatg atgaaccatc atgggccact gttctctttg aggggacagg
3360tttaggggtt tgcgttcgcc cttgtgggct gaagcactag ctttttggta
gctagacaca 3420tcctgcaccc aaaggttctc tacaaaggcc cagatttgtt
tgtaaagcac tttgactctt 3480acctggaggc ccgctctcta agggcttcct
gcgctcccac ctcatctgtc cctgagatgc 3540agagcaggat ggagggtctg
cttctagctc agctgtttct ccttgaggtt gcggaggaat 3600tgaattgaat
gggacagagg gcaggtgctg tggccaagaa gatctccgag cagcagtgac
3660ggggcacctt gctgtgtgtc ctctgggcat gttaaccctt ctgtggggcc
aaaggtttgc 3720atcgtggatc cagctgtgct ccagtctgtc ccctcctcct
ccactctgac tgccacgccc 3780cggaccagca gcttggggac cctccagggt
actaatgggg ctctgttctg agatggacaa 3840attcagtgtt ggaaatacat
gttgtactat gcacttccca tgctcctagg gttaggaata 3900gtttcaaaca
tgattggcag acataacaac ggcaaatact cggactgggg cataggactc
3960cagagtagga aaaagacaaa agatttggca gcctgacaca ggcaacctac
ccctctctct 4020ccagcctctt tatgaaactg tttgtttgcc agtcctgccc
taaggcagaa gatgaattga 4080agatgctgtg catgtttcct aagtccttga
gcaatcatgg tggtgacaat tgccacaagg 4140gatatgaggc cagtgccacc
agagggtggt gccaagtgcc acatcccttc cgatccattc 4200ccctctgcat
cctcggagca ccccagtttg cctttgatgt gtccgctgtg tatgttagct
4260gaactttgat gagcaaaatt tcctgagcga aacactccaa agagatagga
aaacttgccg 4320cctcttcttt tttgtccctt aatcaaactc aaataagctt
aaaaaaaatc catggaagat 4380catggacatg tgaaatgagc atttttttct
tttttttttt taacaaagtc tgaactgaac 4440agaacaagac tttttcctca
tacatctcca aattgtttaa acttacttta tgagtgtttg 4500tttagaagtt
cggaccaaca gaaaaatgca gtcagatgtc atcttggaat tggtttctaa
4560aagagtaagg catgtccctg cccagaaact taggaagcat gaaataaatc
aaatgtttat 4620tttccttctt atttaaaatc atgcaaatgc aacagaaata
gagggtttgt gccaaatgct 4680atgaacggcc ctttcttaaa gacaagcaag
ggagattgat atatgtacaa tttgctctca 4740tgtttt 4746984746DNAHomo
sapiens 98taataaaaga ctagtggcct tagtgcccat gcccagtgac cctccattca
atacccgaag 60agcctacacc agtgaggatg aagcctggaa gtcatacttg gagaatcccc
tgacagcagc 120caccaaggcc atgatgagca ttaatggtga tgaggacagt
gctgctgccc tcggcctgct 180ctatgactac tacaaggttc ctcgagacaa
gaggctgctg tctgtaagca aagcaagtga 240cagccaagaa gaccaggaga
aaagaaactg ccttggcacc agtgaagccc agagtaattt 300gagtggagga
gaaaaccgag tgcaagtcct aaagactgtt ccagtgaacc tttccctaaa
360tcaagatcac ctggagaatt ccaagcggga acagtacagc atcagcttcc
ccgagagctc 420tgccatcatc ccggtgtcgg gaatcacggt ggtgaaagct
gaagatttca caccagtttt 480catggcccca cctgtgcact atccccgggg
agatggggaa gagcaacgag tggttatctt 540tgaacagact cagtatgacg
tgccctcgct ggccacccac agcgcctatc tcaaagacga 600ccagcgcagc
actccggaca gcacatacag cgagagcttc aaggacgcag ccacagagaa
660atttcggagt gcttcagttg gggctgagga gtacatgtat gatcagacat
caagtggcac 720atttcagtac accctggaag ccaccaaatc tctccgtcag
aagcaggggg agggccccat 780gacctacctc aacaaaggac agttctatgc
cataacactc agcgagaccg gagacaacaa 840atgcttccga caccccatca
gcaaagtcag gagtgtggtg atggtggtct tcagtgaaga 900caaaaacaga
gatgaacagc tcaaatactg gaaatactgg cactctcggc agcatacggc
960gaagcagagg gtccttgaca ttgccgatta caaggagagc tttaatacga
ttggaaacat 1020tgaagagatt gcatataatg ctgtttcctt tacctgggac
gtgaatgaag aggcgaagat 1080tttcatcacc gtgaattgct tgagcacaga
tttctcctcc caaaaagggg tgaaaggact 1140tcctttgatg attcagattg
acacatacag ttataacaat cgtagcaata aacccattca 1200tagagcttat
tgccagatca aggtcttctg tgacaaagga gcagaaagaa aaatccgaga
1260tgaagagcgg aagcagaaca ggaagaaagg gaaaggccag gcctcccaaa
ctcaatgcaa 1320cagctcctct gatgggaagt tggctgccat acctttacag
aagaagagtg acatcaccta 1380cttcaaaacc atgcctgatc tccactcaca
gccagttctc ttcatacctg atgttcactt 1440tgcaaacctg cagaggaccg
gacaggtgta ttacaacacg gatgatgaac gagaaggtgg 1500cagtgtcctt
gttaaacgga tgttccggcc catggaagag gagtttggtc cagtgccttc
1560aaagcagatg aaagaagaag ggacaaagcg agtgctcttg tacgtgagga
aggagactga 1620cgatgtgttc gatgcattga tgttgaagtc tcccacagtg
aagggcctga tggaagcgat 1680atctgagaaa tatgggctgc ccgtggagaa
gatagcaaag ctttacaaga aaagcaaaaa 1740aggcatcttg gtgaacatgg
atgacaacat catcgagcac tactcgaacg aggacacctt 1800catcctcaac
atggagagca tggtggaggg cttcaaggtc acgctcatgg aaatctagcc
1860ctgggtttgg catccgcttt ggctggagct ctcagtgcgt tcctccctga
gagagacaga 1920agccccagcc ccagaacctg gagacccatc tcccccatct
cacaactgct gttacaagac 1980cgtgctgggg agtggggcaa gggacaggcc
ccactgtcgg tgtgcttggc ccatccactg 2040gcacctacca cggagctgaa
gcctgagccc ctcaggaagg tgccttaggc ctgttggatt 2100cctatttatt
gcccaccttt tcctggagcc caggtccagg cccgccagga ctctgcaggt
2160cactgctagc tccagatgag accgtccagc gttccccctt caagagaaac
actcatcccg 2220aacagcctaa aaaattccca tcccttctct ctcacccctc
catatctatc tcccgagtgg 2280ctggacaaaa tgagctacgt ctgggtgcag
tagttatagg tggggcaaga ggtggatgcc 2340cactttctgg tcagacacct
ttaggttgct ctggggaagg ctgtcttgct aaatacctcc 2400agggttccca
gcaagtggcc accaggcctt gtacaggaag acattcagtc accgtgtaat
2460tagtaacaca gaaagtctgc ctgtctgcat tgtacatagt gtttataata
ttgtaataat 2520atattttacc tgtggtatgt gggcatgttt actgccactg
gcctagagga gacacagacc 2580tggagaccgt tttaatgggg gtttttgcct
ctgtgcctgt tcaagagact tgcagggcta 2640ggtagagggc ctttgggatg
ttaaggtgac tgcagctgat gccaagatgg actctgcaat 2700gggcatacct
gggggctcgt tccctgtccc cagaggaagc cccctctcct tctccatggg
2760catgactctc cttcgaggcc accacgttta tctcacaatg atgtgttttg
cttgactttc 2820cctttgcgct gtctcgtggg aaaggtcatt ctgtctgaga
ccccagctcc ttctccagct 2880ttggctgcgg gcatggcctg agctttctgg
agagcctctg cagggggttt gccatcaggg 2940ccctgtggct gggtctgctg
cagagctcct tggctatcag gagaatcctg gacactgtac 3000tgtgcctccc
agtttacaaa cacgcccttc atctcaagtg gccctttaaa aggcctgctg
3060ccatgtgaga gctgtgaaca gctcagctct gagtcggcag gctggggctt
cctcctgggc 3120caccagatgg aaagggggta ttgtttgcct cactcctgga
tgctgcgttt taaggaagtg 3180agtgagaaag aatgtgccaa gatacctggc
tcctgtgaaa ccagcctcag gagggaaact 3240gggagagaga agctgtggtc
tcctgctaca tgccctggga gctggaagag aaaaacactc 3300ccctaaacaa
tcgcaaaatg atgaaccatc atgggccact gttctctttg aggggacagg
3360tttaggggtt tgcgttcgcc cttgtgggct gaagcactag ctttttggta
gctagacaca 3420tcctgcaccc aaaggttctc tacaaaggcc cagatttgtt
tgtaaagcac tttgactctt 3480acctggaggc ccgctctcta agggcttcct
gcgctcccac ctcatctgtc cctgagatgc 3540agagcaggat ggagggtctg
cttctagctc agctgtttct ccttgaggtt gcggaggaat 3600tgaattgaat
gggacagagg gcaggtgctg tggccaagaa gatctccgag cagcagtgac
3660ggggcacctt gctgtgtgtc ctctgggcat gttaaccctt ctgtggggcc
aaaggtttgc 3720atcgtggatc cagctgtgct ccagtctgtc ccctcctcct
ccactctgac tgccacgccc 3780cggaccagca gcttggggac cctccagggt
actaatgggg ctctgttctg agatggacaa 3840attcagtgtt ggaaatacat
gttgtactat gcacttccca tgctcctagg gttaggaata 3900gtttcaaaca
tgattggcag acataacaac ggcaaatact cggactgggg cataggactc
3960cagagtagga aaaagacaaa agatttggca gcctgacaca ggcaacctac
ccctctctct 4020ccagcctctt tatgaaactg tttgtttgcc agtcctgccc
taaggcagaa gatgaattga 4080agatgctgtg catgtttcct aagtccttga
gcaatcatgg tggtgacaat tgccacaagg 4140gatatgaggc cagtgccacc
agagggtggt gccaagtgcc acatcccttc cgatccattc 4200ccctctgcat
cctcggagca ccccagtttg cctttgatgt gtccgctgtg tatgttagct
4260gaactttgat gagcaaaatt tcctgagcga aacactccaa agagatagga
aaacttgccg 4320cctcttcttt tttgtccctt aatcaaactc aaataagctt
aaaaaaaatc catggaagat 4380catggacatg tgaaatgagc atttttttct
tttttttttt taacaaagtc tgaactgaac 4440agaacaagac tttttcctca
tacatctcca aattgtttaa
acttacttta tgagtgtttg 4500tttagaagtt cggaccaaca gaaaaatgca
gtcagatgtc atcttggaat tggtttctaa 4560aagagtaagg catgtccctg
cccagaaact taggaagcat gaaataaatc aaatgtttat 4620tttccttctt
atttaaaatc atgcaaatgc aacagaaata gagggtttgt gccaaatgct
4680atgaacggcc ctttcttaaa gacaagcaag ggagattgat atatgtacaa
tttgctctca 4740tgtttt 474699625PRTHomo sapiens 99Met Ser Gln Glu
Ser Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro1 5 10 15 Met Pro
Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu 20 25 30
Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 35
40 45 Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala
Leu 50 55 60 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys
Arg Leu Leu65 70 75 80 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp
Gln Glu Lys Arg Asn 85 90 95 Cys Leu Gly Thr Ser Glu Ala Gln Ser
Asn Leu Ser Gly Gly Glu Asn 100 105 110 Arg Val Gln Val Leu Lys Thr
Val Pro Val Asn Leu Ser Leu Asn Gln 115 120 125 Asp His Leu Glu Asn
Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 130 135 140 Glu Ser Ser
Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala145 150 155 160
Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg 165
170 175 Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln
Tyr 180 185 190 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys
Asp Asp Gln 195 200 205 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser
Phe Lys Asp Ala Ala 210 215 220 Thr Glu Lys Phe Arg Ser Ala Ser Val
Gly Ala Glu Glu Tyr Met Tyr225 230 235 240 Asp Gln Thr Ser Ser Gly
Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys 245 250 255 Ser Leu Arg Gln
Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys 260 265 270 Gly Gln
Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 275 280 285
Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 290
295 300 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr
Trp305 310 315 320 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu
Asp Ile Ala Asp 325 330 335 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn
Ile Glu Glu Ile Ala Tyr 340 345 350 Asn Ala Val Ser Phe Thr Trp Asp
Val Asn Glu Glu Ala Lys Ile Phe 355 360 365 Ile Thr Val Asn Cys Leu
Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 370 375 380 Lys Gly Leu Pro
Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn385 390 395 400 Arg
Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe 405 410
415 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln
420 425 430 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys
Asn Ser 435 440 445 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln
Lys Lys Ser Asp 450 455 460 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu
His Ser Gln Pro Val Leu465 470 475 480 Phe Ile Pro Asp Val His Phe
Ala Asn Leu Gln Arg Thr Gly Gln Val 485 490 495 Tyr Tyr Asn Thr Asp
Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 500 505 510 Arg Met Phe
Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 515 520 525 Gln
Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 530 535
540 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr
Val545 550 555 560 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly
Leu Pro Val Glu 565 570 575 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys
Lys Gly Ile Leu Val Asn 580 585 590 Met Asp Asp Asn Ile Ile Glu His
Tyr Ser Asn Glu Asp Thr Phe Ile 595 600 605 Leu Asn Met Glu Ser Met
Val Glu Gly Phe Lys Val Thr Leu Met Glu 610 615 620 Ile625
100609PRTHomo sapiens 100Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205
Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210
215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile 101609PRTHomo sapiens 101Met Pro Ser Asp
Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu
Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30
Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35
40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu
Leu 50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu
Lys Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu
Ser Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro
Val Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys
Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile
Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe
Thr Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160
Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165
170 175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp
Gln 180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys
Asp Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala
Glu Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln
Tyr Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln
Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr
Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg
His Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285
Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290
295 300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala
Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu
Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn
Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr
Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met
Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys
Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys
Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410
415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser
420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys
Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser
Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu
Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu
Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro
Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys
Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu
Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535
540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val
Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly
Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser
Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu
Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile 1024973DNAHomo
sapiens 102attggatcaa acatgtcaca agagtcggac aagtaagtgg atcacacgcg
ccggctgctg 60ctactactac cactttgggc tgatggcaac tgtaataaaa gactagtggc
cttagtgccc 120atgcccagtg accctccatt caatacccga agagcctaca
ccagtgagga tgaagcctgg 180aagtcatact tggagaatcc cctgacagca
gccaccaagg ccatgatgag cattaatggt 240gatgaggaca gtgctgctgc
cctcggcctg ctctatgact actacaaggt tcctcgagac 300aagaggctgc
tgtctgtaag caaagcaagt gacagccaag aagaccagga gaaaagaaac
360tgccttggca ccagtgaagc ccagagtaat ttgagtggag gagaaaaccg
agtgcaagtc 420ctaaagactg ttccagtgaa cctttcccta aatcaagatc
acctggagaa ttccaagcgg 480gaacagtaca gcatcagctt ccccgagagc
tctgccatca tcccggtgtc gggaatcacg 540gtggtgaaag ctgaagattt
cacaccagtt ttcatggccc cacctgtgca ctatccccgg 600ggagatgggg
aagagcaacg agtggttatc tttgaacaga ctcagtatga cgtgccctcg
660ctggccaccc acagcgccta tctcaaagac gaccagcgca gcactccgga
cagcacatac 720agcgagagct tcaaggacgc agccacagag aaatttcgga
gtgcttcagt tggggctgag 780gagtacatgt atgatcagac atcaagtggc
acatttcagt acaccctgga agccaccaaa 840tctctccgtc agaagcaggg
ggagggcccc atgacctacc tcaacaaagg acagttctat 900gccataacac
tcagcgagac cggagacaac aaatgcttcc gacaccccat cagcaaagtc
960aggagtgtgg tgatggtggt cttcagtgaa gacaaaaaca gagatgaaca
gctcaaatac 1020tggaaatact ggcactctcg gcagcatacg gcgaagcaga
gggtccttga cattgccgat 1080tacaaggaga gctttaatac gattggaaac
attgaagaga ttgcatataa tgctgtttcc 1140tttacctggg acgtgaatga
agaggcgaag attttcatca ccgtgaattg cttgagcaca 1200gatttctcct
cccaaaaagg ggtgaaagga cttcctttga tgattcagat tgacacatac
1260agttataaca atcgtagcaa taaacccatt catagagctt attgccagat
caaggtcttc 1320tgtgacaaag gagcagaaag aaaaatccga gatgaagagc
ggaagcagaa caggaagaaa 1380gggaaaggcc aggcctccca aactcaatgc
aacagctcct ctgatgggaa gttggctgcc 1440atacctttac agaagaagag
tgacatcacc tacttcaaaa ccatgcctga tctccactca 1500cagccagttc
tcttcatacc tgatgttcac tttgcaaacc tgcagaggac cggacaggtg
1560tattacaaca cggatgatga acgagaaggt ggcagtgtcc ttgttaaacg
gatgttccgg 1620cccatggaag aggagtttgg tccagtgcct tcaaagcaga
tgaaagaaga agggacaaag 1680cgagtgctct tgtacgtgag gaaggagact
gacgatgtgt tcgatgcatt gatgttgaag 1740tctcccacag tgaagggcct
gatggaagcg atatctgaga aatatgggct gcccgtggag 1800aagatagcaa
agctttacaa gaaaagcaaa aaaggcatct tggtgaacat ggatgacaac
1860atcatcgagc actactcgaa cgaggacacc ttcatcctca acatggagag
catggtggag 1920ggcttcaagg tcacgctcat ggaaatctag ccctgggttt
ggcatccgct ttggctggag 1980ctctcagtgc gttcctccct gagagagaca
gaagccccag ccccagaacc tggagaccca 2040tctcccccat ctcacaactg
ctgttacaag accgtgctgg ggagtggggc aagggacagg 2100ccccactgtc
ggtgtgcttg gcccatccac tggcacctac cacggagctg aagcctgagc
2160ccctcaggaa ggtgccttag gcctgttgga ttcctattta ttgcccacct
tttcctggag 2220cccaggtcca ggcccgccag gactctgcag gtcactgcta
gctccagatg agaccgtcca 2280gcgttccccc ttcaagagaa acactcatcc
cgaacagcct aaaaaattcc catcccttct 2340ctctcacccc tccatatcta
tctcccgagt ggctggacaa aatgagctac gtctgggtgc 2400agtagttata
ggtggggcaa gaggtggatg cccactttct ggtcagacac ctttaggttg
2460ctctggggaa ggctgtcttg ctaaatacct ccagggttcc cagcaagtgg
ccaccaggcc 2520ttgtacagga agacattcag tcaccgtgta attagtaaca
cagaaagtct gcctgtctgc 2580attgtacata gtgtttataa tattgtaata
atatatttta cctgtggtat gtgggcatgt 2640ttactgccac tggcctagag
gagacacaga cctggagacc
gttttaatgg gggtttttgc 2700ctctgtgcct gttcaagaga cttgcagggc
taggtagagg gcctttggga tgttaaggtg 2760actgcagctg atgccaagat
ggactctgca atgggcatac ctgggggctc gttccctgtc 2820cccagaggaa
gccccctctc cttctccatg ggcatgactc tccttcgagg ccaccacgtt
2880tatctcacaa tgatgtgttt tgcttgactt tccctttgcg ctgtctcgtg
ggaaaggtca 2940ttctgtctga gaccccagct ccttctccag ctttggctgc
gggcatggcc tgagctttct 3000ggagagcctc tgcagggggt ttgccatcag
ggccctgtgg ctgggtctgc tgcagagctc 3060cttggctatc aggagaatcc
tggacactgt actgtgcctc ccagtttaca aacacgccct 3120tcatctcaag
tggcccttta aaaggcctgc tgccatgtga gagctgtgaa cagctcagct
3180ctgagtcggc aggctggggc ttcctcctgg gccaccagat ggaaaggggg
tattgtttgc 3240ctcactcctg gatgctgcgt tttaaggaag tgagtgagaa
agaatgtgcc aagatacctg 3300gctcctgtga aaccagcctc aggagggaaa
ctgggagaga gaagctgtgg tctcctgcta 3360catgccctgg gagctggaag
agaaaaacac tcccctaaac aatcgcaaaa tgatgaacca 3420tcatgggcca
ctgttctctt tgaggggaca ggtttagggg tttgcgttcg cccttgtggg
3480ctgaagcact agctttttgg tagctagaca catcctgcac ccaaaggttc
tctacaaagg 3540cccagatttg tttgtaaagc actttgactc ttacctggag
gcccgctctc taagggcttc 3600ctgcgctccc acctcatctg tccctgagat
gcagagcagg atggagggtc tgcttctagc 3660tcagctgttt ctccttgagg
ttgcggagga attgaattga atgggacaga gggcaggtgc 3720tgtggccaag
aagatctccg agcagcagtg acggggcacc ttgctgtgtg tcctctgggc
3780atgttaaccc ttctgtgggg ccaaaggttt gcatcgtgga tccagctgtg
ctccagtctg 3840tcccctcctc ctccactctg actgccacgc cccggaccag
cagcttgggg accctccagg 3900gtactaatgg ggctctgttc tgagatggac
aaattcagtg ttggaaatac atgttgtact 3960atgcacttcc catgctccta
gggttaggaa tagtttcaaa catgattggc agacataaca 4020acggcaaata
ctcggactgg ggcataggac tccagagtag gaaaaagaca aaagatttgg
4080cagcctgaca caggcaacct acccctctct ctccagcctc tttatgaaac
tgtttgtttg 4140ccagtcctgc cctaaggcag aagatgaatt gaagatgctg
tgcatgtttc ctaagtcctt 4200gagcaatcat ggtggtgaca attgccacaa
gggatatgag gccagtgcca ccagagggtg 4260gtgccaagtg ccacatccct
tccgatccat tcccctctgc atcctcggag caccccagtt 4320tgcctttgat
gtgtccgctg tgtatgttag ctgaactttg atgagcaaaa tttcctgagc
4380gaaacactcc aaagagatag gaaaacttgc cgcctcttct tttttgtccc
ttaatcaaac 4440tcaaataagc ttaaaaaaaa tccatggaag atcatggaca
tgtgaaatga gcattttttt 4500cttttttttt tttaacaaag tctgaactga
acagaacaag actttttcct catacatctc 4560caaattgttt aaacttactt
tatgagtgtt tgtttagaag ttcggaccaa cagaaaaatg 4620cagtcagatg
tcatcttgga attggtttct aaaagagtaa ggcatgtccc tgcccagaaa
4680cttaggaagc atgaaataaa tcaaatgttt attttccttc ttatttaaaa
tcatgcaaat 4740gcaacagaaa tagagggttt gtgccaaatg ctatgaacgg
ccctttctta aagacaagca 4800agggagattg atatatgtac aatttgctct
catgttttaa aaaaaaaagg taaatgtaac 4860ttaatagttt tgtaaatggg
agagggggaa tctataaact ataaatacag ttattttatt 4920ttttgtacat
ttttaaggag aaaaaaataa atattcataa cataagagga aaa 49731034746DNAHomo
sapiens 103taataaaaga ctagtggcct tagtgcccat gcccagtgac cctccattca
atacccgaag 60agcctacacc agtgaggatg aagcctggaa gtcatacttg gagaatcccc
tgacagcagc 120caccaaggcc atgatgagca ttaatggtga tgaggacagt
gctgctgccc tcggcctgct 180ctatgactac tacaaggttc ctcgagacaa
gaggctgctg tctgtaagca aagcaagtga 240cagccaagaa gaccaggaga
aaagaaactg ccttggcacc agtgaagccc agagtaattt 300gagtggagga
gaaaaccgag tgcaagtcct aaagactgtt ccagtgaacc tttccctaaa
360tcaagatcac ctggagaatt ccaagcggga acagtacagc atcagcttcc
ccgagagctc 420tgccatcatc ccggtgtcgg gaatcacggt ggtgaaagct
gaagatttca caccagtttt 480catggcccca cctgtgcact atccccgggg
agatggggaa gagcaacgag tggttatctt 540tgaacagact cagtatgacg
tgccctcgct ggccacccac agcgcctatc tcaaagacga 600ccagcgcagc
actccggaca gcacatacag cgagagcttc aaggacgcag ccacagagaa
660atttcggagt gcttcagttg gggctgagga gtacatgtat gatcagacat
caagtggcac 720atttcagtac accctggaag ccaccaaatc tctccgtcag
aagcaggggg agggccccat 780gacctacctc aacaaaggac agttctatgc
cataacactc agcgagaccg gagacaacaa 840atgcttccga caccccatca
gcaaagtcag gagtgtggtg atggtggtct tcagtgaaga 900caaaaacaga
gatgaacagc tcaaatactg gaaatactgg cactctcggc agcatacggc
960gaagcagagg gtccttgaca ttgccgatta caaggagagc tttaatacga
ttggaaacat 1020tgaagagatt gcatataatg ctgtttcctt tacctgggac
gtgaatgaag aggcgaagat 1080tttcatcacc gtgaattgct tgagcacaga
tttctcctcc caaaaagggg tgaaaggact 1140tcctttgatg attcagattg
acacatacag ttataacaat cgtagcaata aacccattca 1200tagagcttat
tgccagatca aggtcttctg tgacaaagga gcagaaagaa aaatccgaga
1260tgaagagcgg aagcagaaca ggaagaaagg gaaaggccag gcctcccaaa
ctcaatgcaa 1320cagctcctct gatgggaagt tggctgccat acctttacag
aagaagagtg acatcaccta 1380cttcaaaacc atgcctgatc tccactcaca
gccagttctc ttcatacctg atgttcactt 1440tgcaaacctg cagaggaccg
gacaggtgta ttacaacacg gatgatgaac gagaaggtgg 1500cagtgtcctt
gttaaacgga tgttccggcc catggaagag gagtttggtc cagtgccttc
1560aaagcagatg aaagaagaag ggacaaagcg agtgctcttg tacgtgagga
aggagactga 1620cgatgtgttc gatgcattga tgttgaagtc tcccacagtg
aagggcctga tggaagcgat 1680atctgagaaa tatgggctgc ccgtggagaa
gatagcaaag ctttacaaga aaagcaaaaa 1740aggcatcttg gtgaacatgg
atgacaacat catcgagcac tactcgaacg aggacacctt 1800catcctcaac
atggagagca tggtggaggg cttcaaggtc acgctcatgg aaatctagcc
1860ctgggtttgg catccgcttt ggctggagct ctcagtgcgt tcctccctga
gagagacaga 1920agccccagcc ccagaacctg gagacccatc tcccccatct
cacaactgct gttacaagac 1980cgtgctgggg agtggggcaa gggacaggcc
ccactgtcgg tgtgcttggc ccatccactg 2040gcacctacca cggagctgaa
gcctgagccc ctcaggaagg tgccttaggc ctgttggatt 2100cctatttatt
gcccaccttt tcctggagcc caggtccagg cccgccagga ctctgcaggt
2160cactgctagc tccagatgag accgtccagc gttccccctt caagagaaac
actcatcccg 2220aacagcctaa aaaattccca tcccttctct ctcacccctc
catatctatc tcccgagtgg 2280ctggacaaaa tgagctacgt ctgggtgcag
tagttatagg tggggcaaga ggtggatgcc 2340cactttctgg tcagacacct
ttaggttgct ctggggaagg ctgtcttgct aaatacctcc 2400agggttccca
gcaagtggcc accaggcctt gtacaggaag acattcagtc accgtgtaat
2460tagtaacaca gaaagtctgc ctgtctgcat tgtacatagt gtttataata
ttgtaataat 2520atattttacc tgtggtatgt gggcatgttt actgccactg
gcctagagga gacacagacc 2580tggagaccgt tttaatgggg gtttttgcct
ctgtgcctgt tcaagagact tgcagggcta 2640ggtagagggc ctttgggatg
ttaaggtgac tgcagctgat gccaagatgg actctgcaat 2700gggcatacct
gggggctcgt tccctgtccc cagaggaagc cccctctcct tctccatggg
2760catgactctc cttcgaggcc accacgttta tctcacaatg atgtgttttg
cttgactttc 2820cctttgcgct gtctcgtggg aaaggtcatt ctgtctgaga
ccccagctcc ttctccagct 2880ttggctgcgg gcatggcctg agctttctgg
agagcctctg cagggggttt gccatcaggg 2940ccctgtggct gggtctgctg
cagagctcct tggctatcag gagaatcctg gacactgtac 3000tgtgcctccc
agtttacaaa cacgcccttc atctcaagtg gccctttaaa aggcctgctg
3060ccatgtgaga gctgtgaaca gctcagctct gagtcggcag gctggggctt
cctcctgggc 3120caccagatgg aaagggggta ttgtttgcct cactcctgga
tgctgcgttt taaggaagtg 3180agtgagaaag aatgtgccaa gatacctggc
tcctgtgaaa ccagcctcag gagggaaact 3240gggagagaga agctgtggtc
tcctgctaca tgccctggga gctggaagag aaaaacactc 3300ccctaaacaa
tcgcaaaatg atgaaccatc atgggccact gttctctttg aggggacagg
3360tttaggggtt tgcgttcgcc cttgtgggct gaagcactag ctttttggta
gctagacaca 3420tcctgcaccc aaaggttctc tacaaaggcc cagatttgtt
tgtaaagcac tttgactctt 3480acctggaggc ccgctctcta agggcttcct
gcgctcccac ctcatctgtc cctgagatgc 3540agagcaggat ggagggtctg
cttctagctc agctgtttct ccttgaggtt gcggaggaat 3600tgaattgaat
gggacagagg gcaggtgctg tggccaagaa gatctccgag cagcagtgac
3660ggggcacctt gctgtgtgtc ctctgggcat gttaaccctt ctgtggggcc
aaaggtttgc 3720atcgtggatc cagctgtgct ccagtctgtc ccctcctcct
ccactctgac tgccacgccc 3780cggaccagca gcttggggac cctccagggt
actaatgggg ctctgttctg agatggacaa 3840attcagtgtt ggaaatacat
gttgtactat gcacttccca tgctcctagg gttaggaata 3900gtttcaaaca
tgattggcag acataacaac ggcaaatact cggactgggg cataggactc
3960cagagtagga aaaagacaaa agatttggca gcctgacaca ggcaacctac
ccctctctct 4020ccagcctctt tatgaaactg tttgtttgcc agtcctgccc
taaggcagaa gatgaattga 4080agatgctgtg catgtttcct aagtccttga
gcaatcatgg tggtgacaat tgccacaagg 4140gatatgaggc cagtgccacc
agagggtggt gccaagtgcc acatcccttc cgatccattc 4200ccctctgcat
cctcggagca ccccagtttg cctttgatgt gtccgctgtg tatgttagct
4260gaactttgat gagcaaaatt tcctgagcga aacactccaa agagatagga
aaacttgccg 4320cctcttcttt tttgtccctt aatcaaactc aaataagctt
aaaaaaaatc catggaagat 4380catggacatg tgaaatgagc atttttttct
tttttttttt taacaaagtc tgaactgaac 4440agaacaagac tttttcctca
tacatctcca aattgtttaa acttacttta tgagtgtttg 4500tttagaagtt
cggaccaaca gaaaaatgca gtcagatgtc atcttggaat tggtttctaa
4560aagagtaagg catgtccctg cccagaaact taggaagcat gaaataaatc
aaatgtttat 4620tttccttctt atttaaaatc atgcaaatgc aacagaaata
gagggtttgt gccaaatgct 4680atgaacggcc ctttcttaaa gacaagcaag
ggagattgat atatgtacaa tttgctctca 4740tgtttt 47461044746DNAHomo
sapiens 104taataaaaga ctagtggcct tagtgcccat gcccagtgac cctccattca
atacccgaag 60agcctacacc agtgaggatg aagcctggaa gtcatacttg gagaatcccc
tgacagcagc 120caccaaggcc atgatgagca ttaatggtga tgaggacagt
gctgctgccc tcggcctgct 180ctatgactac tacaaggttc ctcgagacaa
gaggctgctg tctgtaagca aagcaagtga 240cagccaagaa gaccaggaga
aaagaaactg ccttggcacc agtgaagccc agagtaattt 300gagtggagga
gaaaaccgag tgcaagtcct aaagactgtt ccagtgaacc tttccctaaa
360tcaagatcac ctggagaatt ccaagcggga acagtacagc atcagcttcc
ccgagagctc 420tgccatcatc ccggtgtcgg gaatcacggt ggtgaaagct
gaagatttca caccagtttt 480catggcccca cctgtgcact atccccgggg
agatggggaa gagcaacgag tggttatctt 540tgaacagact cagtatgacg
tgccctcgct ggccacccac agcgcctatc tcaaagacga 600ccagcgcagc
actccggaca gcacatacag cgagagcttc aaggacgcag ccacagagaa
660atttcggagt gcttcagttg gggctgagga gtacatgtat gatcagacat
caagtggcac 720atttcagtac accctggaag ccaccaaatc tctccgtcag
aagcaggggg agggccccat 780gacctacctc aacaaaggac agttctatgc
cataacactc agcgagaccg gagacaacaa 840atgcttccga caccccatca
gcaaagtcag gagtgtggtg atggtggtct tcagtgaaga 900caaaaacaga
gatgaacagc tcaaatactg gaaatactgg cactctcggc agcatacggc
960gaagcagagg gtccttgaca ttgccgatta caaggagagc tttaatacga
ttggaaacat 1020tgaagagatt gcatataatg ctgtttcctt tacctgggac
gtgaatgaag aggcgaagat 1080tttcatcacc gtgaattgct tgagcacaga
tttctcctcc caaaaagggg tgaaaggact 1140tcctttgatg attcagattg
acacatacag ttataacaat cgtagcaata aacccattca 1200tagagcttat
tgccagatca aggtcttctg tgacaaagga gcagaaagaa aaatccgaga
1260tgaagagcgg aagcagaaca ggaagaaagg gaaaggccag gcctcccaaa
ctcaatgcaa 1320cagctcctct gatgggaagt tggctgccat acctttacag
aagaagagtg acatcaccta 1380cttcaaaacc atgcctgatc tccactcaca
gccagttctc ttcatacctg atgttcactt 1440tgcaaacctg cagaggaccg
gacaggtgta ttacaacacg gatgatgaac gagaaggtgg 1500cagtgtcctt
gttaaacgga tgttccggcc catggaagag gagtttggtc cagtgccttc
1560aaagcagatg aaagaagaag ggacaaagcg agtgctcttg tacgtgagga
aggagactga 1620cgatgtgttc gatgcattga tgttgaagtc tcccacagtg
aagggcctga tggaagcgat 1680atctgagaaa tatgggctgc ccgtggagaa
gatagcaaag ctttacaaga aaagcaaaaa 1740aggcatcttg gtgaacatgg
atgacaacat catcgagcac tactcgaacg aggacacctt 1800catcctcaac
atggagagca tggtggaggg cttcaaggtc acgctcatgg aaatctagcc
1860ctgggtttgg catccgcttt ggctggagct ctcagtgcgt tcctccctga
gagagacaga 1920agccccagcc ccagaacctg gagacccatc tcccccatct
cacaactgct gttacaagac 1980cgtgctgggg agtggggcaa gggacaggcc
ccactgtcgg tgtgcttggc ccatccactg 2040gcacctacca cggagctgaa
gcctgagccc ctcaggaagg tgccttaggc ctgttggatt 2100cctatttatt
gcccaccttt tcctggagcc caggtccagg cccgccagga ctctgcaggt
2160cactgctagc tccagatgag accgtccagc gttccccctt caagagaaac
actcatcccg 2220aacagcctaa aaaattccca tcccttctct ctcacccctc
catatctatc tcccgagtgg 2280ctggacaaaa tgagctacgt ctgggtgcag
tagttatagg tggggcaaga ggtggatgcc 2340cactttctgg tcagacacct
ttaggttgct ctggggaagg ctgtcttgct aaatacctcc 2400agggttccca
gcaagtggcc accaggcctt gtacaggaag acattcagtc accgtgtaat
2460tagtaacaca gaaagtctgc ctgtctgcat tgtacatagt gtttataata
ttgtaataat 2520atattttacc tgtggtatgt gggcatgttt actgccactg
gcctagagga gacacagacc 2580tggagaccgt tttaatgggg gtttttgcct
ctgtgcctgt tcaagagact tgcagggcta 2640ggtagagggc ctttgggatg
ttaaggtgac tgcagctgat gccaagatgg actctgcaat 2700gggcatacct
gggggctcgt tccctgtccc cagaggaagc cccctctcct tctccatggg
2760catgactctc cttcgaggcc accacgttta tctcacaatg atgtgttttg
cttgactttc 2820cctttgcgct gtctcgtggg aaaggtcatt ctgtctgaga
ccccagctcc ttctccagct 2880ttggctgcgg gcatggcctg agctttctgg
agagcctctg cagggggttt gccatcaggg 2940ccctgtggct gggtctgctg
cagagctcct tggctatcag gagaatcctg gacactgtac 3000tgtgcctccc
agtttacaaa cacgcccttc atctcaagtg gccctttaaa aggcctgctg
3060ccatgtgaga gctgtgaaca gctcagctct gagtcggcag gctggggctt
cctcctgggc 3120caccagatgg aaagggggta ttgtttgcct cactcctgga
tgctgcgttt taaggaagtg 3180agtgagaaag aatgtgccaa gatacctggc
tcctgtgaaa ccagcctcag gagggaaact 3240gggagagaga agctgtggtc
tcctgctaca tgccctggga gctggaagag aaaaacactc 3300ccctaaacaa
tcgcaaaatg atgaaccatc atgggccact gttctctttg aggggacagg
3360tttaggggtt tgcgttcgcc cttgtgggct gaagcactag ctttttggta
gctagacaca 3420tcctgcaccc aaaggttctc tacaaaggcc cagatttgtt
tgtaaagcac tttgactctt 3480acctggaggc ccgctctcta agggcttcct
gcgctcccac ctcatctgtc cctgagatgc 3540agagcaggat ggagggtctg
cttctagctc agctgtttct ccttgaggtt gcggaggaat 3600tgaattgaat
gggacagagg gcaggtgctg tggccaagaa gatctccgag cagcagtgac
3660ggggcacctt gctgtgtgtc ctctgggcat gttaaccctt ctgtggggcc
aaaggtttgc 3720atcgtggatc cagctgtgct ccagtctgtc ccctcctcct
ccactctgac tgccacgccc 3780cggaccagca gcttggggac cctccagggt
actaatgggg ctctgttctg agatggacaa 3840attcagtgtt ggaaatacat
gttgtactat gcacttccca tgctcctagg gttaggaata 3900gtttcaaaca
tgattggcag acataacaac ggcaaatact cggactgggg cataggactc
3960cagagtagga aaaagacaaa agatttggca gcctgacaca ggcaacctac
ccctctctct 4020ccagcctctt tatgaaactg tttgtttgcc agtcctgccc
taaggcagaa gatgaattga 4080agatgctgtg catgtttcct aagtccttga
gcaatcatgg tggtgacaat tgccacaagg 4140gatatgaggc cagtgccacc
agagggtggt gccaagtgcc acatcccttc cgatccattc 4200ccctctgcat
cctcggagca ccccagtttg cctttgatgt gtccgctgtg tatgttagct
4260gaactttgat gagcaaaatt tcctgagcga aacactccaa agagatagga
aaacttgccg 4320cctcttcttt tttgtccctt aatcaaactc aaataagctt
aaaaaaaatc catggaagat 4380catggacatg tgaaatgagc atttttttct
tttttttttt taacaaagtc tgaactgaac 4440agaacaagac tttttcctca
tacatctcca aattgtttaa acttacttta tgagtgtttg 4500tttagaagtt
cggaccaaca gaaaaatgca gtcagatgtc atcttggaat tggtttctaa
4560aagagtaagg catgtccctg cccagaaact taggaagcat gaaataaatc
aaatgtttat 4620tttccttctt atttaaaatc atgcaaatgc aacagaaata
gagggtttgt gccaaatgct 4680atgaacggcc ctttcttaaa gacaagcaag
ggagattgat atatgtacaa tttgctctca 4740tgtttt 4746105609PRTHomo
sapiens 105Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr
Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu
Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu
Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys
Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser
Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80 Cys Leu Gly Thr
Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val
Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn Gln 100 105 110
Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115
120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys
Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His
Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile
Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His
Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser
Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe
Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln
Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235
240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys
245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn
Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val
Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu
Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys
Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe
Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val
Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile
Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360
365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn
370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys
Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp
Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala
Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala
Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys
Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro
Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val465
470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser Val Leu
Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro
Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys Arg Val
Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe Asp Ala
Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met Glu Ala
Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys Ile Ala
Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570 575 Met
Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile 580 585
590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu Met Glu
595 600 605 Ile 106609PRTHomo sapiens 106Met Pro Ser Asp Pro Pro
Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp
Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala
Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45
Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50
55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg
Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly
Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn
Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu
Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro
Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro
Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp
Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175
Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180
185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala
Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu
Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr
Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu
Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile
Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro
Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu
Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300
His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305
310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile
Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu
Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe
Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln
Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile
His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys
Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415 Asn
Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425
430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp
435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro
Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg
Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu
Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu
Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu
Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp
Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys
Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550
555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val
Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp
Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys
Val Thr Leu Met Glu 595 600 605 Ile 107609PRTHomo sapiens 107Met
Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu1 5 10
15 Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr
20 25 30 Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala
Ala Leu 35 40 45 Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp
Lys Arg Leu Leu 50 55 60 Ser Val Ser Lys Ala Ser Asp Ser Gln Glu
Asp Gln Glu Lys Arg Asn65 70 75 80 Cys Leu Gly Thr Ser Glu Ala Gln
Ser Asn Leu Ser Gly Gly Glu Asn 85 90 95 Arg Val Gln Val Leu Lys
Thr Val Pro Val Asn Leu Ser Leu Asn Gln 100 105 110 Asp His Leu Glu
Asn Ser Lys Arg Glu Gln Tyr Ser Ile Ser Phe Pro 115 120 125 Glu Ser
Ser Ala Ile Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala 130 135 140
Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg145
150 155 160 Gly Asp Gly Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr
Gln Tyr 165 170 175 Asp Val Pro Ser Leu Ala Thr His Ser Ala Tyr Leu
Lys Asp Asp Gln 180 185 190 Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu
Ser Phe Lys Asp Ala Ala 195 200 205 Thr Glu Lys Phe Arg Ser Ala Ser
Val Gly Ala Glu Glu Tyr Met Tyr 210 215 220 Asp Gln Thr Ser Ser Gly
Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys225 230 235 240 Ser Leu Arg
Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys 245 250 255 Gly
Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys 260 265
270 Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe
275 280 285 Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys
Tyr Trp 290 295 300 His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu
Asp Ile Ala Asp305 310 315 320 Tyr Lys Glu Ser Phe Asn Thr Ile Gly
Asn Ile Glu Glu Ile Ala Tyr 325 330 335 Asn Ala Val Ser Phe Thr Trp
Asp Val Asn Glu Glu Ala Lys Ile Phe 340 345 350 Ile Thr Val Asn Cys
Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val 355 360 365 Lys Gly Leu
Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn 370 375 380 Arg
Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe385 390
395 400 Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys
Gln 405 410 415 Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Thr Gln
Cys Asn Ser 420 425 430 Ser Ser Asp Gly Lys Leu Ala Ala Ile Pro Leu
Gln Lys Lys Ser Asp 435 440 445 Ile Thr Tyr Phe Lys Thr Met Pro Asp
Leu His Ser Gln Pro Val Leu 450 455 460 Phe Ile Pro Asp Val His Phe
Ala Asn Leu Gln Arg Thr Gly Gln Val465 470 475 480 Tyr Tyr Asn Thr
Asp Asp Glu Arg Glu Gly Gly Ser Val Leu Val Lys 485 490 495 Arg Met
Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Val Pro Ser Lys 500 505 510
Gln Met Lys Glu Glu Gly Thr Lys Arg Val Leu Leu Tyr Val Arg Lys 515
520 525 Glu Thr Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr
Val 530 535 540 Lys Gly Leu Met Glu Ala Ile Ser Glu Lys Tyr Gly Leu
Pro Val Glu545 550 555 560 Lys Ile Ala Lys Leu Tyr Lys Lys Ser Lys
Lys Gly Ile Leu Val Asn 565 570 575 Met Asp Asp Asn Ile Ile Glu His
Tyr Ser Asn Glu Asp Thr Phe Ile 580 585 590 Leu Asn Met Glu Ser Met
Val Glu Gly Phe Lys Val Thr Leu Met Glu 595 600 605 Ile
108609PRTHomo sapiens 108Met Pro Ser Asp Pro Pro Phe Asn Thr Arg
Arg Ala Tyr Thr Ser Glu1 5 10 15 Asp Glu Ala Trp Lys Ser Tyr Leu
Glu Asn Pro Leu Thr Ala Ala Thr 20 25 30 Lys Ala Met Met Ser Ile
Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu 35 40 45 Gly Leu Leu Tyr
Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu 50 55 60 Ser Val
Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Glu Lys Arg Asn65 70 75 80
Cys Leu Gly Thr Ser Glu Ala Gln Ser Asn Leu Ser Gly Gly Glu Asn 85
90 95 Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Ser Leu Asn
Gln 100 105 110 Asp His Leu Glu Asn Ser Lys Arg Glu Gln Tyr Ser Ile
Ser Phe Pro 115 120 125 Glu Ser Ser Ala Ile Ile Pro Val Ser Gly Ile
Thr Val Val Lys Ala 130 135 140 Glu Asp Phe Thr Pro Val Phe Met Ala
Pro Pro Val His Tyr Pro Arg145 150 155 160 Gly Asp Gly Glu Glu Gln
Arg Val Val Ile Phe Glu Gln Thr Gln Tyr 165 170 175 Asp Val Pro Ser
Leu Ala Thr His Ser Ala Tyr Leu Lys Asp Asp Gln 180 185 190 Arg Ser
Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Ala Ala 195 200 205
Thr Glu Lys Phe Arg Ser Ala Ser Val Gly Ala Glu Glu Tyr Met Tyr 210
215 220 Asp Gln Thr Ser Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr
Lys225 230 235 240 Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr
Tyr Leu Asn Lys 245 250 255 Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu
Thr Gly Asp Asn Lys Cys 260 265 270 Phe Arg His Pro Ile Ser Lys Val
Arg Ser Val Val Met Val Val Phe 275 280 285 Ser Glu Asp Lys Asn Arg
Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp 290 295 300 His Ser Arg Gln
His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp305 310 315 320 Tyr
Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr 325 330
335 Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe
340 345 350 Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys
Gly Val 355 360 365 Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr
Ser Tyr Asn Asn 370 375 380 Arg Ser Asn Lys Pro Ile His Arg Ala Tyr
Cys Gln Ile Lys Val Phe385 390 395 400 Cys Asp Lys Gly Ala Glu Arg
Lys Ile Arg Asp Glu Glu Arg Lys Gln 405 410 415 Asn Arg Lys Lys Gly
Lys Gly Gln Ala Ser Gln Thr Gln Cys Asn Ser 420 425 430 Ser Ser Asp
Gly Lys Leu Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp 435 440 445 Ile
Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu 450 455
460 Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln
Val465 470 475 480 Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Gly Ser
Val Leu Val Lys 485 490 495 Arg Met Phe Arg Pro Met Glu Glu Glu Phe
Gly Pro Val Pro Ser Lys 500 505 510 Gln Met Lys Glu Glu Gly Thr Lys
Arg Val Leu Leu Tyr Val Arg Lys 515 520 525 Glu Thr Asp Asp Val Phe
Asp Ala Leu Met Leu Lys Ser Pro Thr Val 530 535 540 Lys Gly Leu Met
Glu Ala Ile Ser Glu Lys Tyr Gly Leu Pro Val Glu545 550 555 560 Lys
Ile Ala Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn 565 570
575 Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile
580 585 590 Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Val Thr Leu
Met Glu 595 600 605 Ile
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