U.S. patent application number 13/450405 was filed with the patent office on 2012-08-09 for nucleic acid and corresponding protein entitled 193p1e1b useful in treatment and detection of cancer.
This patent application is currently assigned to AGENSYS, INC.. Invention is credited to Pia M. Challita-Eid, Mary Faris, Wangmao Ge, Rene S. Hubert, Aya Jakobovits, Arthur B. RAITANO.
Application Number | 20120202208 13/450405 |
Document ID | / |
Family ID | 21759312 |
Filed Date | 2012-08-09 |
United States Patent
Application |
20120202208 |
Kind Code |
A1 |
RAITANO; Arthur B. ; et
al. |
August 9, 2012 |
NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 193P1E1B USEFUL IN
TREATMENT AND DETECTION OF CANCER
Abstract
A novel gene 0193P1E1B (also designated 193P1E1B) and its
encoded protein, and variants thereof, are described wherein
193P1E1B exhibits tissue specific expression in normal adult
tissue, and is aberrantly expressed in the cancers listed in Table
I. Consequently, 193P1E1B provides a diagnostic, prognostic,
prophylactic and/or therapeutic target for cancer. The 193P1E1B
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
193P1E1B can be used in active or passive immunization.
Inventors: |
RAITANO; Arthur B.; (Los
Alamitos, CA) ; Challita-Eid; Pia M.; (Encino,
CA) ; Faris; Mary; (Los Angeles, CA) ; Hubert;
Rene S.; (Los Angeles, CA) ; Ge; Wangmao;
(Tampa, FL) ; Jakobovits; Aya; (Beverly Hills,
CA) |
Assignee: |
AGENSYS, INC.
Santa Monica
CA
|
Family ID: |
21759312 |
Appl. No.: |
13/450405 |
Filed: |
April 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12763982 |
Apr 20, 2010 |
8188228 |
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13450405 |
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12434526 |
May 1, 2009 |
7732584 |
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12763982 |
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10313972 |
Dec 6, 2002 |
7615379 |
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12434526 |
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10013312 |
Dec 7, 2001 |
7449548 |
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10313972 |
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Current U.S.
Class: |
435/6.11 ;
435/7.23 |
Current CPC
Class: |
A61P 35/02 20180101;
Y02A 50/466 20180101; A61P 35/04 20180101; A61P 35/00 20180101;
C07K 2317/34 20130101; C12Q 2600/158 20130101; C12Q 1/6886
20130101; C07K 16/30 20130101; C12Q 2600/136 20130101; A61K
2039/505 20130101; Y02A 50/30 20180101; A61P 43/00 20180101 |
Class at
Publication: |
435/6.11 ;
435/7.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method for detecting the presence of a 193P1E1B protein in a
sample, comprising: contacting the sample with an antibody or
fragment thereof that specifically binds to the 193P1E1B protein,
wherein the 193P1E1B protein comprises an amino acid sequence
selected from the group consisting of residues 1 to 412 of SEQ ID
NOS: 9, 11 and 13; and, detecting the formation and/or presence of
a complex comprising the antibody or fragment thereof and the
193P1E1B protein.
2. The method of claim 1, wherein the protein comprises the amino
acid sequence of SEQ ID NOS:9, 11, or 13.
3. The method of claim 1, wherein the protein comprises the amino
acid sequence of SEQ ID NO:9.
4. The method of claim 1, wherein the protein comprises the amino
acid sequence of SEQ ID NO:11.
5. The method of claim 1, wherein the protein comprises the amino
acid sequence of SEQ ID NO:13.
6. A method for detecting the presence of a 193P1E1B mRNA in a
sample, comprising: contacting the sample with a nucleotide probe
that specifically binds to the mRNA which encodes a protein
comprises an amino acid sequence selected from the group consisting
of residues 1 to 412 of SEQ ID NOS: 9, 11 and 13, and detecting the
formation and/or presence of a complex comprising the probe and the
mRNA.
7. The method of claim 6, wherein the protein comprises the amino
acid sequence of SEQ ID NOS:9, 11, or 13.
8. The method of claim 6, wherein the protein comprises the amino
acid sequence of SEQ ID NO:9.
9. The method of claim 6, wherein the protein comprises the amino
acid sequence of SEQ ID NO:11.
10. The method of claim 6, wherein the protein comprises the amino
acid sequence of SEQ ID NO:13.
11. The method of claim 6, further comprising: producing a cDNA
from the sample by reverse transcription using at least one primer;
amplifying the cDNA so produced using 193P1E1B polynucleotides as
sense and antisense primers, wherein the 193P1E1B polynucleotides
used as the sense and antisense primers serve to amplify a 193P1E1B
cDNA; and, detecting the presence of the amplified 193P1E1B cDNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 12/763,982, filed 20 Apr. 2010, now allowed,
which is a continuation of U.S. patent application Ser. No.
12/434,526, filed 1 May 2009, now U.S. Pat. No. 7,732,584, issued 8
Jun. 2010, which is a continuation of U.S. patent application Ser.
No. 10/313,972, filed 6 Dec. 2002, now U.S. Pat. No. 7,615,379,
issued 10 Nov. 2009, which is a continuation-in-part of U.S. patent
application Ser. No. 10/013,312, filed 7 Dec. 2001, now U.S. Pat.
No. 7,449,548, issued 11 Nov. 2008. The contents of each
application listed in this paragraph are fully incorporated by
reference herein.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] The entire content of the following electronic submission of
the sequence listing via the USPTO EFS-WEB server, as authorized
and set forth in MPEP .sctn.1730 II.B.2(a)(C), is incorporated
herein by reference in its entirety for all purposes. The sequence
listing is identified on the electronically filed text file as
follows:
TABLE-US-00001 File Name Date of Creation Size (bytes)
511582006312Seqlist April 18, 2012 294,471 bytes
FIELD OF THE INVENTION
[0003] The invention described herein relates to a gene and its
encoded protein, termed 193P1E1B, expressed in certain cancers, and
to diagnostic and therapeutic methods and compositions useful in
the management of cancers that express 193P1E1B.
BACKGROUND OF THE INVENTION
[0004] Cancer is the second leading cause of human death next to
coronary disease. Worldwide, millions of people die from cancer
every year. In the United States alone, as reported by the American
Cancer Society, cancer causes the death of well over a half-million
people annually, with over 1.2 million new cases diagnosed per
year. While deaths from heart disease have been declining
significantly, those resulting from cancer generally are on the
rise. In the early part of the next century, cancer is predicted to
become the leading cause of death.
[0005] Worldwide, several cancers stand out as the leading killers.
In particular, carcinomas of the lung, prostate, breast, colon,
pancreas, and ovary represent the primary causes of cancer death.
These and virtually all other carcinomas share a common lethal
feature. With very few exceptions, metastatic disease from a
carcinoma is fatal. Moreover, even for those cancer patients who
initially survive their primary cancers, common experience has
shown that their lives are dramatically altered. Many cancer
patients experience strong anxieties driven by the awareness of the
potential for recurrence or treatment failure. Many cancer patients
experience physical debilitations following treatment. Furthermore,
many cancer patients experience a recurrence.
[0006] Worldwide, prostate cancer is the fourth most prevalent
cancer in men. In North America and Northern Europe, it is by far
the most common cancer in males and is the second leading cause of
cancer death in men. In the United States alone, well over 30,000
men die annually of this disease--second only to lung cancer.
Despite the magnitude of these figures, there is still no effective
treatment for metastatic prostate cancer. Surgical prostatectomy,
radiation therapy, hormone ablation therapy, surgical castration
and chemotherapy continue to be the main treatment modalities.
Unfortunately, these treatments are ineffective for many and are
often associated with undesirable consequences.
[0007] On the diagnostic front, the lack of a prostate tumor marker
that can accurately detect early-stage, localized tumors remains a
significant limitation in the diagnosis and management of this
disease. Although the serum prostate specific antigen (PSA) assay
has been a very useful tool, however its specificity and general
utility is widely regarded as lacking in several important
respects.
[0008] Progress in identifying additional specific markers for
prostate cancer has been improved by the generation of prostate
cancer xenografts that can recapitulate different stages of the
disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts
are prostate cancer xenografts that have survived passage in severe
combined immune deficient (SCID) mice and have exhibited the
capacity to mimic the transition from androgen dependence to
androgen independence (Klein et al., 1997, Nat. Med. 3:402). More
recently identified prostate cancer markers include PCTA-1 (Su et
al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific
membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996
September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad
Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell
antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:
1735).
[0009] While previously identified markers such as PSA, PSM, PCTA
and PSCA have facilitated efforts to diagnose and treat prostate
cancer, there is need for the identification of additional markers
and therapeutic targets for prostate and related cancers in order
to further improve diagnosis and therapy.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 sequalae to these treatments. There is,
therefore, a need for efficacious breast cancer treatments.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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
[0027] The present invention relates to a gene, designated
193P1E1B, that has now been found to be over-expressed in the
cancer(s) listed in Table I. Northern blot expression analysis of
193P1E1B 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 193P1E1B are provided.
The tissue-related profile of 193P1E1B in normal adult tissues,
combined with the over-expression observed in the tissues listed in
Table I, shows that 193P1E1B 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.
[0028] The invention provides polynucleotides corresponding or
complementary to all or part of the 193P1E1B genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding 193P1E1B-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 193P1E1B-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 193P1E1B
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the 193P1E1B genes, mRNAs, or to
193P1E1B-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding 193P1E1B. Recombinant DNA
molecules containing 193P1E1B polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of 193P1E1B gene products are also provided. The
invention further provides antibodies that bind to 193P1E1B
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.
[0029] The invention further provides methods for detecting the
presence and status of 193P1E1B polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express 193P1E1B. A typical embodiment of this invention
provides methods for monitoring 193P1E1B gene products in a tissue
or hematology sample having or suspected of having some form of
growth dysregulation such as cancer.
[0030] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 193P1E1B such as cancers of tissues listed in Table I,
including therapies aimed at inhibiting the transcription,
translation, processing or function of 193P1E1B as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
193P1E1B 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
193P1E1B. Preferably, the carrier is a uniquely human carrier. In
another aspect of the invention, the agent is a moiety that is
immunoreactive with 193P1E1B 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.
[0031] 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 193P1E1B 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 193P1E1B as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
193P1E1B. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of 193P1E1B (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for 193P1E1B production) or a ribozyme effective to lyse
193P1E1B mRNA.
[0032] 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.
[0033] 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.
[0034] 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:
[0035] 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;
[0036] 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; iii) a peptide region of at least 5 amino acids
of a particular peptide of FIG. 3, in any whole number increment up
to the full length of that protein in FIG. 3, that includes an
amino acid position having a value equal to or greater than 0.5,
0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent
Accessible Residues profile of FIG. 7;
[0037] iv) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average
Flexibility profile of FIG. 8; or
[0038] v) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn
profile of FIG. 9.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1. The 193P1E1B SSH sequence of 227 nucleotides.
[0040] FIG. 2. A) The cDNA and amino acid sequence of 193P1E1B
variant 1 (also called "193P1E1B v.1" or "193P1E1B variant 1") is
shown in FIG. 2A. The start methionine is underlined. The open
reading frame extends from nucleic acid 805-2043 including the stop
codon.
[0041] B) The cDNA and amino acid sequence of 193P1E1B variant 2
(also called "193P1E1B v.2") is shown in FIG. 2B. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0042] C) The cDNA and amino acid sequence of 193P1E1B variant 3
(also called "193P1E1B v.3") is shown in FIG. 2C. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0043] D) The cDNA and amino acid sequence of 193P1E1B variant 4
(also called "193P1E1B v.4") is shown in FIG. 2D. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0044] E) The cDNA and amino acid sequence of 193P1E1B variant 5
(also called "193P1E1B v.5") is shown in FIG. 2E. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0045] F) The cDNA and amino acid sequence of 193P1E1B variant 6
(also called "193P1E1B v.6") is shown in FIG. 2F. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0046] G) The cDNA and amino acid sequence of 193P1E1B variant 7
(also called "193P1E1B v.7") is shown in FIG. 2G. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0047] H) The cDNA and amino acid sequence of 193P1E1B variant 8
(also called "193P1E1B v.8") is shown in FIG. 2H. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 805-2043 including the stop codon.
[0048] I) The cDNA and amino acid sequence of 193P1E1B variant 9
(also called "193P1E1B v.9") is shown in FIG. 2I. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 989-1981 including the stop codon.
[0049] J) The cDNA and amino acid sequence of 193P1E1B variant 10
(also called "193P1E1B v.10") is shown in FIG. 2J. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 805-1971 including the stop codon.
[0050] K) The cDNA and amino acid sequence of 193P1E1B variant 11
(also called "193P1E1B v.11") is shown in FIG. 2K. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 989-1909 including the stop codon.
[0051] L) The cDNA and amino acid sequence of 193P1E1B variant 12
(also called "193P1E1B v.12") is shown in FIG. 2L. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 805-1026 including the stop codon.
[0052] M) The cDNA and amino acid sequence of 193P1E1B variant 13
(also called "193P1E1B v.13") is shown in FIG. 2M. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 952-2070 including the stop codon.
[0053] FIG. 3.
[0054] A) Amino acid sequence of 193P1E1B v.1 is shown in FIG. 3A;
it has 412 amino acids.
[0055] B) The amino acid sequence of 193P1E1B v.5 is shown in FIG.
3B; it has 412 amino acids.
[0056] C) The amino acid sequence of 193P1E1B v.6 is shown in FIG.
3C; it has 412 amino acids.
[0057] D) The amino acid sequence of 193P1E1B v.9 is shown in FIG.
3D; it has 330 amino acids.
[0058] E) The amino acid sequence of 193P1E1B v.10 is shown in FIG.
3E; it has 388 amino acids.
[0059] F) The amino acid sequence of 193P1E1B v.11 is shown in FIG.
3F; it has 308 amino acids.
[0060] G) The amino acid sequence of 193P1E1B v.12 is shown in FIG.
3G; it has 73 amino acids.
[0061] H) The amino acid sequence of 193P1E1B v.13 is shown in FIG.
3H; it has 372 amino acids. As used herein, a reference to 193P1E1B
includes all variants thereof, including those shown in FIGS. 2, 3,
10, and 11, unless the context clearly indicates otherwise.
[0062] FIG. 4. FIG. 4A shows the alignment of 193P1E1B v.1 with gi
2178775. FIG. 4B shows the alignment of 193P1E1B v.5 with gi
2178775. FIG. 4C shows the alignment of 193P1E1B v.11 with gi
2178775. FIG. 4D shows the alignment of 193P1E1B v.12 with gi
2178775. FIG. 4E shows the alignment of 193P1E1B v.1 with E. coli
arginine repressor. FIG. 4F shows the Alignment of 193P1E1B v.1
with human adenosine deaminase.
[0063] FIG. 4G shows the Clustal alignment of 193P1E1B protein
variants.
[0064] FIG. 5. Hydrophilicity amino acid profile of 193P1E1B
determined by computer algorithm sequence analysis using the method
of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad.
Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located
on the World Wide Web through the ExPasy molecular biology
server.
[0065] FIG. 6. Hydropathicity amino acid profile of 193P1E1B
determined by computer algorithm sequence analysis using the method
of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol.
Biol. 157:105-132) accessed on the ProtScale website located on the
World Wide Web through the ExPasy molecular biology server.
[0066] FIG. 7. Percent accessible residues amino acid profile of
193P1E1B determined by computer algorithm sequence analysis using
the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on
the ProtScale website located on the World Wide Web through the
ExPasy molecular biology server.
[0067] FIG. 8. Average flexibility amino acid profile of 193P1E1B
determined by computer algorithm sequence analysis using the method
of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K.,
1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the
ProtScale website located on the World Wide Web through the ExPasy
molecular biology server.
[0068] FIG. 9. Beta-turn amino acid profile of 193P1E1B determined
by computer algorithm sequence analysis using the method of Deleage
and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294)
accessed on the ProtScale website located on the World Wide Web
through the ExPasy molecular biology server.
[0069] FIG. 10. Schematic alignment of SNP variants of 193P1E1B.
Variants 193P1E1B v.2 through v.8 are variants with single
nucleotide differences. Though these SNP variants are shown
separately, they could also occur in any combinations and in any
transcript variants that contains the base pairs. Numbers
correspond to those of 193P1E1B v.1. Black box shows the same
sequence as 193P1E1B v.1. SNPs are indicated above the box.
[0070] FIG. 11. Schematic alignment of protein variants of
193P1E1B. Protein variants correspond to nucleotide variants.
Nucleotide variants 193P1E1B v.2, v.3, v.4, v.7, and v.8 in FIG. 10
code for the same protein as 193P1E1B v.1. Nucleotide variants
193P1E1B v.9 through v.13 are splice variants of v.1. Single amino
acid differences were indicated above the boxes. Black boxes
represent the same sequence as 193P1E1B v.1. Numbers underneath the
box correspond to amino acid positions in 193P1E1B v.1.
[0071] FIG. 12. Intentionally Omitted.
[0072] FIG. 13. Secondary structure prediction for 193P1E1B (SEQ ID
NO:123). The secondary structure of 193P1E1B protein was predicted
using the HNN--Hierarchical Neural Network method, accessed from
the ExPasy molecular biology server. 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 as follows: h: Alpha
helix 29.13%; c: Random coil 60.92%; e: Extended strand 9.95%.
[0073] FIG. 14. Expression of 193P1E1B by RT-PCR. (A) The schematic
diagram depicts the location of PCR primers Set A and set B on the
sequences of the 3 variants of 193P1E1B. (B and C) First strand
cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney),
vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft
pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus,
prostate cancer pool, bladder cancer pool, kidney cancer pool,
colon cancer pool, lung cancer pool, ovary cancer pool, breast
cancer pool, metastasis cancer pool, pancreas cancer pool, and from
prostate cancer metastasis to lymph node from 2 different patients.
Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using primer Set A (B) or primer Set
B (C) to 193P1E1B, was performed at 30 cycles of amplification.
Strong expression of 193P1E1B was observed in prostate cancer
xenograft pool, bladder cancer pool, kidney cancer pool, colon
cancer pool, lung cancer pool, ovary cancer pool, breast cancer
pool, metastasis pool, pancreas cancer pool, and in the 2 different
prostate cancer metastasis to lymph node. Low expression was
observed in prostate cancer pool, but no expression was detected in
VP1 and VP2. FIG. 14C shows that the transcript encoding 193P1E1B
v.1 through v.8, is expressed at higher levels that the transcript
encoding 193P1E1B v.9. But both transcripts are expressed at
similar proportion in all tissues tested.
[0074] FIG. 15. Expression of 193P1E1B in normal human tissues. Two
multiple tissue northern blots, with 2 .mu.g of mRNA/lane, were
probed with 193P1E1B sequence. Size standards in kilobases (kb) are
indicated on the side. The results show expression of two 193P1E1B
transcripts, approximately 3.5 kb and 2 kb, in testis and
thymus.
[0075] FIG. 16. Expression of 193P1E1B in prostate cancer
xenografts. RNA was extracted from normal prostate, and from
prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and
LAPC-9AI. Northern blot with 10 .mu.g of total RNA/lane was probed
with 193P1E1B sequence. Size standards in kilobases (kb) are
indicated on the side. The results show expression of 193P1E1B in
all 4 xenografts but not in normal prostate.
[0076] FIG. 17. Expression of 193P1E1B in patient cancer specimens.
RNA was extracted from a pool of three patients for each of the
following, bladder cancer, colon cancer, ovary cancer and
metastasis cancer, as well as from normal prostate (NP), normal
bladder (NB), normal kidney (NK), normal colon (NC). Northern blots
with 10 .mu.g of total RNA/lane were probed with 193P1E1B sequence.
Size standards in kilobases (kb) are indicated on the side. The
results show expression of 193P1E1B in bladder cancer pool, colon
cancer pool, ovary cancer pool and metastasis cancer pool, but not
in any of the normal tissues tested.
[0077] FIG. 18. Expression of 193P1E1B in bladder cancer patient
specimens. RNA was extracted from bladder cancer cell lines (CL),
normal bladder (N), bladder tumors (T) and matched normal adjacent
tissue (NAT) isolated from bladder cancer patients. Northern blots
with 10 .mu.g of total RNA/lane were probed with 193P1E1B sequence.
Size standards in kilobases (kb) are indicated on the side. The
results show expression of 193P1E1B in the two bladder cancer cell
lines, and in 3 patient bladder tumors tested but not in normal
bladder tissues.
[0078] FIG. 19. Expression of 193P1E1B in cancer metastasis patient
specimens. RNA was extracted from the following cancer metastasis
tissues, colon metastasis to lung, lung metastasis to lymph node,
lung metastasis to skin, and breast metastasis to lymph node, as
well as from normal bladder (NB), normal lung (NL), normal breast
(NBr), and normal ovary (NO). Northern blots with 10 .mu.g of total
RNA/lane were probed with 193P1E1B sequence. Size standards in
kilobases (kb) are indicated on the side. The results show
expression of 193P1E1B in all four different cancer metastasis
samples but not in normal tissues.
[0079] FIG. 20. Expression of 193P1E1B in pancreas, ovary and
testis cancer patient specimens. RNA was extracted from pancreatic
cancer (P1), ovarian cancer (P2, P3), and testicular cancer (P4,
P5) isolated from cancer patients, as well as from normal pancreas
(NPa). Northern blots with 10 .mu.g of total RNA/lane were probed
with 193P1E1B sequence. Size standards in kilobases (kb) are
indicated on the side. The results show expression of 193P1E1B in
pancreatic, ovarian and testicular cancer specimens but not in
normal pancreas.
[0080] FIG. 21. Expression of 193P1E1B in Normal versus Patient
Cancer Specimens. First strand cDNA was prepared from a panel of
normal tissues (stomach, brain, heart, liver, spleen, skeletal
muscle, testis prostate, bladder, kidney, colon, lung and pancreas)
and from a panel of patient cancer pools (prostate cancer pool,
bladder cancer pool, kidney cancer pool, colon cancer pool, lung
cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer
pool, metastasis cancer pool, LAPC prostate xenograft pool (XP),
and from prostate cancer metastasis to lymph node from 2 different
patients (PMLN2). Normalization was performed by PCR using primers
to actin. Semi-quantitative PCR, using primer Set A as described in
FIG. 14, was performed 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. Relative
expression was calculated by normalizing to signal obtained using
actin primers. Results show restricted 193P1E1B expression in
normal testis amongst all normal tissues tested. 193P1E1B
expression was strongly upregulated in cancers of the bladder,
colon, lung, pancreas, ovary, breast, and to a lesser extent in
prostate and kidney cancers.
[0081] FIG. 22. Expression of 193P1E1B in Normal versus Patient
Cancer Specimens. First strand cDNA was prepared from a panel of
normal tissues (stomach, brain, heart, liver, spleen, skeletal
muscle, testis prostate, bladder, kidney, colon, lung and pancreas)
and from a panel of patient cancer pools (prostate cancer pool,
bladder cancer pool, kidney cancer pool, colon cancer pool, lung
cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer
pool, metastasis cancer pool, LAPC prostate xenograft pool (XP),
and from prostate cancer metastasis to lymph node from 2 different
patients (PMLN2). Normalization was performed by PCR using primers
to actin. Semi-quantitative PCR, using primer Set A as described in
FIG. 14, was performed 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. Relative
expression was calculated by normalizing to signal obtained using
actin primers. Results show restricted 193P1E1B expression in
normal testis amongst all normal tissues tested. 193P1E1B
expression was strongly upregulated in cancers of the bladder,
colon, lung, pancreas, ovary, breast, and to a lesser extent in
prostate and kidney cancers.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
[0082] I.) Definitions
[0083] II.) 193P1E1B Polynucleotides
[0084] II.A.) Uses of 193P1E1B Polynucleotides
[0085] II.A.1.) Monitoring of Genetic Abnormalities
[0086] II.A.2.) Antisense Embodiments
[0087] II.A.3.) Primers and Primer Pairs [0088] II.A.4.) Isolation
of 193P1E1B-Encoding Nucleic Acid Molecules
[0089] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0090] III.) 193P1E1B-related Proteins [0091] III.A.) Motif-bearing
Protein Embodiments [0092] III.B.) Expression of 193P1E1B-related
Proteins [0093] III.C.) Modifications of 193P1E1B-related Proteins
[0094] III.D.) Uses of 193P1E1B-related Proteins
[0095] IV.) 193P1E1B Antibodies
[0096] V.) 193P1E1B Cellular Immune Responses
[0097] VI.) 193P1E1B Transgenic Animals
[0098] VII.) Methods for the Detection of 193P1E1B
[0099] VIII.) Methods for Monitoring the Status of 193P1E1B-related
Genes and Their
Products
[0100] IX.) Identification of Molecules That Interact With
193P1E1B
[0101] X.) Therapeutic Methods and Compositions [0102] X.A.)
Anti-Cancer Vaccines
[0103] X.B.) 193P1E1B as a Target for Antibody-Based Therapy
[0104] X.C.) 193P1E1B as a Target for Cellular Immune Responses
[0105] X.C.1. Minigene Vaccines [0106] X.C.2. Combinations of CTL
Peptides with Helper Peptides [0107] X.C.3. Combinations of CTL
Peptides with T Cell Priming Agents [0108] X.C.4. Vaccine
Compositions Comprising DC Pulsed with CTL and/or
HTL Peptides
[0108] [0109] X.D.) Adoptive Immunotherapy
[0110] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0111] XI.) Diagnostic and Prognostic Embodiments of 193P1E1B.
[0112] XII.) Inhibition of 193P1E1B Protein Function [0113] XII.A.)
Inhibition of 193P1E1B With Intracellular Antibodies [0114] XII.B.)
Inhibition of 193P1E1B with Recombinant Proteins [0115] XII.C.)
Inhibition of 193P1E1B Transcription or Translation [0116] XII.D.)
General Considerations for Therapeutic Strategies
[0117] XIII.) Identification, Characterization and Use of
Modulators of 193P1E1B
[0118] XIV.) KITS/Articles of Manufacture
I.) DEFINITIONS
[0119] 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.
[0120] 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.
[0121] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 193P1E1B (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
193P1E1B. 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.
[0122] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 193P1E1B-related protein). For example, an analog
of a 193P1E1B protein can be specifically bound by an antibody or T
cell that specifically binds to 193P1E1B.
[0123] 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-193P1E1B 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.
[0124] 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-193P1E1B antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-193P1E1B antibody compositions with polyepitopic
specificity.
[0125] 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."
[0126] 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)).
[0127] 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).
[0128] 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.).
[0129] 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,
restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,
Sapaonaria officinalis inhibitor, and glucocorticoid and other
chemotherapeutic agents, as well as radioisotopes such as
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212 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.
[0130] 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.
[0131] "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.
[0132] 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.
[0133] 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.
[0134] "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).
[0135] 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.
[0136] 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 193P1E1B genes or that encode
polypeptides other than 193P1E1B gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 193P1E1B polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 193P1E1B proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 193P1E1B protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0137] 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.
[0138] 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 humorous. 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] A "motif", as in biological motif of a 193P1E1B-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.
[0144] 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.
[0145] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0146] 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).
[0147] 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".
[0148] 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.
[0149] "Radioisotopes" include, but are not limited to the
following (non-limiting exemplary uses are also set forth):
Examples of Medical Isotopes:
Isotope
Description of Use
Actinium-225
(AC-225)
See Thorium-229 (Th-229)
Actinium-227
(AC-227)
[0150] Parent of Radium-223 (Ra-223) which is an alpha emitter used
to treat metastases in the skeleton resulting from cancer (i.e.,
breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212
(Bi-212)
See Thorium-228 (Th-228)
Bismuth-213
(Bi-213)
See Thorium-229 (Th-229)
Cadmium-109
(Cd-109)
[0151] Cancer detection
Cobalt-60
(Co-60)
[0152] Radiation source for radiotherapy of cancer, for food
irradiators, and for sterilization of medical supplies
Copper-64
(Cu-64)
[0153] A positron emitter used for cancer therapy and SPECT
imaging
Copper-67
(Cu-67)
[0154] Beta/gamma emitter used in cancer radioimmunotherapy and
diagnostic studies (i.e., breast and colon cancers, and
lymphoma)
Examples of Medical Isotopes:
Isotope
Description of Use
Dysprosium-166
(Dy-166)
[0155] Cancer radioimmunotherapy
Erbium-169
(Er-169)
[0156] Rheumatoid arthritis treatment, particularly for the small
joints associated with fingers and toes
Europium-152
(Eu-152)
[0157] Radiation source for food irradiation and for sterilization
of medical supplies
Europium-154
(Eu-154)
[0158] Radiation source for food irradiation and for sterilization
of medical supplies
Gadolinium-153
(Gd-153)
[0159] Osteoporosis detection and nuclear medical quality assurance
devices
Gold-198
(Au-198)
[0160] Implant and intracavity therapy of ovarian, prostate, and
brain cancers
Holmium-166
(Ho-166)
[0161] Multiple myeloma treatment in targeted skeletal therapy,
cancer radioimmunotherapy, bone marrow ablation, and rheumatoid
arthritis treatment
Iodine-125
[0162] (1-125) Osteoporosis detection, diagnostic imaging, tracer
drugs, brain cancer treatment, radiolabeling, tumor imaging,
mapping of receptors in the brain, interstitial radiation therapy,
brachytherapy for treatment of prostate cancer, determination of
glomerular filtration rate (GFR), determination of plasma volume,
detection of deep vein thrombosis of the legs
Iodine-131
[0163] (1-131) Thyroid function evaluation, thyroid disease
detection, treatment of thyroid cancer as well as other
non-malignant thyroid diseases (i.e., Graves disease, goiters, and
hyperthyroidism), treatment of leukemia, lymphoma, and other forms
of cancer (e.g., breast cancer) using radioimmunotherapy
Examples of Medical Isotopes:
Isotope
Description of Use
Iridium-192
(Ir-192)
[0164] Brachytherapy, brain and spinal cord tumor treatment,
treatment of blocked arteries (i.e., arteriosclerosis and
restenosis), and implants for breast and prostate tumors
Lutetium-177
(Lu-177)
[0165] Cancer radioimmunotherapy and treatment of blocked arteries
(i.e., arteriosclerosis and restenosis)
Molybdenum-99
(Mo-99)
[0166] Parent of Technetium-99m (Tc-99m) which is used for imaging
the brain, liver, lungs, heart, and other organs. Currently, Tc-99m
is the most widely used radioisotope used for diagnostic imaging of
various cancers and diseases involving the brain, heart, liver,
lungs; also used in detection of deep vein thrombosis of the
legs
Osmium-194
(Os-194)
[0167] Cancer radioimmunotherapy
Palladium-103
(Pd-103)
[0168] Prostate cancer treatment
Platinum-195m
(Pt-195m)
[0169] Studies on biodistribution and metabolism of cisplatin, a
chemotherapeutic drug
Phosphorus-32
(P-32)
[0170] Polycythemia rubra vera (blood cell disease) and leukemia
treatment, bone cancer diagnosis/treatment; colon, pancreatic, and
liver cancer treatment; radiolabeling nucleic acids for in vitro
research, diagnosis of superficial tumors, treatment of blocked
arteries (i.e., arteriosclerosis and restenosis), and intracavity
therapy
Phosphorus-33
(P-33)
[0171] Leukemia treatment, bone disease diagnosis/treatment,
radiolabeling, and treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis)
Radium-223
(Ra-223)
See Actinium-227 (Ac-227)
Examples of Medical Isotopes:
Isotope
Description of Use
Rhenium-186
(Re-186)
[0172] Bone cancer pain relief, rheumatoid arthritis treatment, and
diagnosis and treatment of lymphoma and bone, breast, colon, and
liver cancers using radioimmunotherapy
Rhenium-188
(Re-188)
[0173] Cancer diagnosis and treatment using radioimmunotherapy,
bone cancer pain relief, treatment of rheumatoid arthritis, and
treatment of prostate cancer
Rhodium-105
(Rh-105)
[0174] Cancer radioimmunotherapy
Samarium-145
(Sm-145)
[0175] Ocular cancer treatment
Samarium-153
(Sm-153)
[0176] Cancer radioimmunotherapy and bone cancer pain relief
Scandium-47
(Sc-47)
[0177] Cancer radioimmunotherapy and bone cancer pain relief
Selenium-75
(Se-75)
[0178] Radiotracer used in brain studies, imaging of adrenal cortex
by gamma-scintigraphy, lateral locations of steroid secreting
tumors, pancreatic scanning, detection of hyperactive parathyroid
glands, measure rate of bile acid loss from the endogenous pool
Strontium-85
(Sr-85)
[0179] Bone cancer detection and brain scans
Strontium-89
(Sr-89)
[0180] Bone cancer pain relief, multiple myeloma treatment, and
osteoblastic therapy
Technetium-99m
(Tc-99m)
See Molybdenum-99 (Mo-99)
Examples of Medical Isotopes:
Isotope
Description of Use
Thorium-228
(Th-228)
[0181] Parent of Bismuth-212 (Bi-212) which is an alpha emitter
used in cancer radioimmunotherapy
Thorium-229
(Th-229)
[0182] Parent of Actinium-225 (Ac-225) and grandparent of
Bismuth-213 (Bi-213) which are alpha emitters used in cancer
radioimmunotherapy
Thulium-170
(Tm-170)
[0183] Gamma source for blood irradiators, energy source for
implanted medical devices
Tin-117m
(Sn-117m)
[0184] Cancer immunotherapy and bone cancer pain relief
Tungsten-188
(W-188)
[0185] Parent for Rhenium-188 (Re-188) which is used for cancer
diagnostics/treatment, bone cancer pain relief, rheumatoid
arthritis treatment, and treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis)
Xenon-127
(Xe-127)
[0186] Neuroimaging of brain disorders, high resolution SPECT
studies, pulmonary function tests, and cerebral blood flow
studies
Ytterbium-175
(Yb-175)
[0187] Cancer radioimmunotherapy
Yttrium-90
(Y-90)
[0188] Microseeds obtained from irradiating Yttrium-89 (Y-89) for
liver cancer treatment
Yttrium-91
(Y-91)
[0189] A gamma-emitting label for Yttrium-90 (Y-90) which is used
for cancer radioimmunotherapy (i.e., lymphoma, breast, colon,
kidney, lung, ovarian, prostate, pancreatic, and inoperable liver
cancers)
[0190] 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.
[0191] 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.
[0192] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0193] Non-limiting examples of small molecules include compounds
that bind or interact with 193P1E1B, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit 193P1E1B
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, 193P1E1B protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions
[0194] "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).
[0195] "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.
[0196] 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 supertypes are as follows: [0197] A2:
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901,
A*0207 [0198] A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101,
A*3101 [0199] 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 [0200] B44:
B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006) [0201] A1:
A*0102, A*2604, A*3601, A*4301, A*8001 [0202] A24: A*24, A*30,
A*2403, A*2404, A*3002, A*3003 [0203] 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 [0204] B58: B*1516, B*1517, B*5701,
B*5702, B58 [0205] B62: B*4601, B52, B*1501 (B62), B*1502 (B75),
B*1513 (B77)
[0206] Calculated population coverage afforded by different
HLA-supertype combinations are set forth in Table IV (G).
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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 193P1E1B
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.
[0211] The "193P1E1B-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 193P1E1B proteins or fragments thereof, as well as fusion
proteins of a 193P1E1B protein and a heterologous polypeptide are
also included. Such 193P1E1B proteins are collectively referred to
as the 193P1E1B-related proteins, the proteins of the invention, or
193P1E1B. The term "193P1E1B-related protein" refers to a
polypeptide fragment or a 193P1E1B 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, 600, 625, 650, or 664 or more amino acids.
II.) 193P1E1B POLYNUCLEOTIDES
[0212] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of a 193P1E1B gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding a 193P1E1B-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to a 193P1E1B
gene or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to a 193P1E1B gene, mRNA, or to a
193P1E1B encoding polynucleotide (collectively, "193P1E1B
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0213] Embodiments of a 193P1E1B polynucleotide include: a 193P1E1B
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 193P1E1B 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 193P1E1B nucleotides comprise, without
limitation: [0214] (I) a polynucleotide comprising, consisting
essentially of, or consisting of a sequence as shown in FIG. 2,
wherein T can also be U; [0215] (II) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2A, from nucleotide residue number 805 through nucleotide
residue number 2043, including the stop codon, wherein T can also
be U; [0216] (III) a polynucleotide comprising, consisting
essentially of, or consisting of the sequence as shown in FIG. 2B,
from nucleotide residue number 805 through nucleotide residue
number 2043, including the stop codon, wherein T can also be U;
[0217] (IV) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2C, from nucleotide
residue number 805 through nucleotide residue number 2043,
including the a stop codon, wherein T can also be U; [0218] (V) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2D, from nucleotide residue number
805 through nucleotide residue number 2043, including the stop
codon, wherein T can also be U; [0219] (VI) a polynucleotide
comprising, consisting essentially of, or consisting of the
sequence as shown in FIG. 2E, from nucleotide residue number 805
through nucleotide residue number 2043, including the stop codon,
wherein T can also be U; [0220] (VII) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2F, from nucleotide residue number 805 through nucleotide
residue number 2043, including the stop codon, wherein T can also
be U; [0221] (VIII) a polynucleotide comprising, consisting
essentially of, or consisting of the sequence as shown in FIG. 2G,
from nucleotide residue number 805 through nucleotide residue
number 2043, including the stop codon, wherein T can also be U;
[0222] (IX) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2H, from nucleotide
residue number 805 through nucleotide residue number 2043,
including the stop codon, wherein T can also be U; [0223] (X) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2I, from nucleotide residue number
989 through nucleotide residue number 1981, including the stop
codon, wherein T can also be U; [0224] (XI) a polynucleotide
comprising, consisting essentially of, or consisting of the
sequence as shown in FIG. 2J, from nucleotide residue number 805
through nucleotide residue number 1971, including the stop codon,
wherein T can also be U; [0225] (XII) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2K, from nucleotide residue number 989 through nucleotide
residue number 1909, including the stop codon, wherein T can also
be U; [0226] (XIII) a polynucleotide comprising, consisting
essentially of, or consisting of the sequence as shown in FIG. 2L,
from nucleotide residue number 805 through nucleotide residue
number 1026, including the stop codon, wherein T can also be U;
[0227] (XIV) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2M, from
nucleotide residue number 952 through nucleotide residue number
2070, including the stop codon, wherein T can also be U; [0228]
(XV) a polynucleotide that encodes a 193P1E1B-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 FIGS. 2A-M;
[0229] (XVI) a polynucleotide that encodes a 193P1E1B-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 FIGS.
2A-M; [0230] (XVII) a polynucleotide that encodes at least one
peptide set forth in Tables VIII-XXI and XXII-XLIX; [0231] (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. 3A-C in any whole number increment up to 412 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; [0232] (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. 3A-C in
any whole number increment up to 412 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; [0233] (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. 3A-C in any whole
number increment up to 412 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; [0234] (XXI) 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. 3A-C in any whole number
increment up to 412 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; [0235] (XXII) 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. 3A-C in any whole number increment up to
412 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; [0236] (XXIII) 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. 3D in any whole number increment up to 330 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; [0237] (XXIV) 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. 3D in
any whole number increment up to 330 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; [0238] (XXV) 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. 3D in any whole number
increment up to 330 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; [0239] (XXVI) 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. 3D in any whole number
increment up to 330 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; [0240] (XXVII) 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. 3D in any whole number increment up to
330 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 [0241] (XXVIII) 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. 3E in any whole number increment up to 388 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; [0242] (XXIX) 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. 3E in
any whole number increment up to 388 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; [0243] (XXX) 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. 3E in any whole number
increment up to 388 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; [0244] (XXXI) 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. 3E in any whole number
increment up to 388 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; [0245] (XXXII) 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. 3E in any whole number increment up to
388 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 [0246] (XXXIII) 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. 3F in any whole number increment up to 308 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; [0247] (XXXIV) 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. 3F in
any whole number increment up to 308 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; [0248] (XXXV) 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. 3F in any whole number
increment up to 308 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; [0249] (XXXVI) 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. 3F in any whole number
increment up to 308 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; [0250] (XXXVII) 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. 3F in any whole number increment
up to 308 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 [0251] (XXXVIII) 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. 3G in any whole number increment up to 73 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; [0252] (XXXIX) 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. 3G in any whole number
increment up to 73 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; [0253] (XL)
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. 3G in any whole number increment up to 73 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; [0254] (XLI) 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. 3G in any whole number increment up to 73 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; [0255] (XLII) 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. 3G in any whole
number increment up to 73 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 [0256]
(XLIII) 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. 3H in any whole number increment up to 372 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; [0257] (XLIV) 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. 3H in
any whole number increment up to 372 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; [0258] (XLV) 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. 3H in any whole number
increment up to 372 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; [0259] (XLVI) 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. 3H in any whole number
increment up to 372 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; [0260] (XLVII) 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. 3H in any whole number increment up to
372 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; [0261] (XLVIII) a
polynucleotide that is fully complementary to a polynucleotide of
any one of (I)-(XLVII); [0262] (XLIX) a peptide that is encoded by
any of (I) to (XLVIII); and [0263] (L) a composition comprising a
polynucleotide of any of (I)-(XLVIII) or peptide of (XLIX) together
with a pharmaceutical excipient and/or in a human unit dose form;
[0264] (LI) a method of using a polynucleotide of any (I)-(XLVIII)
or peptide of (XLIX) or a composition of (L) in a method to
modulate a cell expressing 193P1E1B; [0265] (LII) a method of using
a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a
composition of (L) in a method to diagnose, prophylax, prognose, or
treat an individual who bears a cell expressing 193P1E1B; [0266]
(LIII) a method of using a polynucleotide of any (I)-(XLVIII) or
peptide of (XLIX) or a composition of (L) in a method to diagnose,
prophylax, prognose, or treat an individual who bears a cell
expressing 193P1E1B, said cell from a cancer of a tissue listed in
Table I; [0267] (LIV) a method of using a polynucleotide of any
(I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a
method to diagnose, prophylax, prognose, or treat a cancer; [0268]
(LV) a method of using a polynucleotide of any (I)-(XLVIII) or
peptide of (XLIX) or a composition of (L) in a method to diagnose,
prophylax, prognose, or treat a cancer of a tissue listed in Table
I; and, [0269] (LVI) a method of using a polynucleotide of any
(I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a
method to identify or characterize a modulator of a cell expressing
193P1E1B.
[0270] As used herein, a range is understood to disclose
specifically all whole unit positions thereof.
[0271] Typical embodiments of the invention disclosed herein
include 193P1E1B polynucleotides that encode specific portions of
193P1E1B mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0272] (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, 410, 412 or more contiguous amino
acids of 193P1E1B variant 1; the maximal lengths relevant for other
variants are: variant 5, 412 amino acids; variant 6, 412 amino
acids, variant 9, 330 amino acids, variant 10, 388 amino acids,
variant 11, 308 amino acids, variant 12, 73 amino acids, and
variant 13, 372 amino acids.
[0273] 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 193P1E1B protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 193P1E1B
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 193P1E1B protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 193P1E1B protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 193P1E1B protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 193P1E1B
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 193P1E1B protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 193P1E1B 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 193P1E1B 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.
[0274] Polynucleotides encoding relatively long portions of a
193P1E1B 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 193P1E1B 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 193P1E1B
sequence as shown in FIG. 2.
[0275] Additional illustrative embodiments of the invention
disclosed herein include 193P1E1B polynucleotide fragments encoding
one or more of the biological motifs contained within a 193P1E1B
protein "or variant" sequence, including one or more of the
motif-bearing subsequences of a 193P1E1B 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 193P1E1B 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 193P1E1B 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.
[0276] 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 LVII. 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.
[0277] II.A.) Uses of 193P1E1B Polynucleotides
[0278] II.A.1.) Monitoring of Genetic Abnormalities
[0279] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 193P1E1B gene maps to
the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 193P1E1B." For example, because the
193P1E1B gene maps to this chromosome, polynucleotides that encode
different regions of the 193P1E1B 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 193P1E1B proteins provide new tools that
can be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 193P1E1B 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)).
[0280] Furthermore, as 193P1E1B was shown to be highly expressed in
bladder and other cancers, 193P1E1B polynucleotides are used in
methods assessing the status of 193P1E1B gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 193P1E1B 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 193P1E1B 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.
[0281] II.A.2.) Antisense Embodiments
[0282] 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 193P1E1B. 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 193P1E1B
polynucleotides and polynucleotide sequences disclosed herein.
[0283] 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., 193P1E1B. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 193P1E1B 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 193P1E1B 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).
[0284] The 193P1E1B 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 193P1E1B 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 193P1E1B mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 193P1E1B antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 193P1E1B mRNA. Optionally, 193P1E1B 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
193P1E1B. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 193P1E1B expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet. 12:
510-515 (1996).
[0285] II.A.3.) Primers and Primer Pairs
[0286] 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 193P1E1B polynucleotide in a sample and as a means
for detecting a cell expressing a 193P1E1B protein.
[0287] Examples of such probes include polypeptides comprising all
or part of the human 193P1E1B cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying
193P1E1B 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 193P1E1B mRNA.
[0288] The 193P1E1B 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
193P1E1B 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
193P1E1B polypeptides; as tools for modulating or inhibiting the
expression of the 193P1E1B gene(s) and/or translation of the
193P1E1B transcript(s); and as therapeutic agents.
[0289] The present invention includes the use of any probe as
described herein to identify and isolate a 193P1E1B or 193P1E1B
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.
[0290] II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid
Molecules
[0291] The 193P1E1B cDNA sequences described herein enable the
isolation of other polynucleotides encoding 193P1E1B gene
product(s), as well as the isolation of polynucleotides encoding
193P1E1B gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 193P1E1B gene product as
well as polynucleotides that encode analogs of 193P1E1B-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding a 193P1E1B 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 193P1E1B gene cDNAs can be identified by
probing with a labeled 193P1E1B cDNA or a fragment thereof. For
example, in one embodiment, a 193P1E1B 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 193P1E1B gene.
A 193P1E1B gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 193P1E1B
DNA probes or primers.
[0292] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0293] The invention also provides recombinant DNA or RNA molecules
containing a 193P1E1B 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).
[0294] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a 193P1E1B
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
[0295] (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 193P1E1B or a fragment, analog or homolog
thereof can be used to generate 193P1E1B proteins or fragments
thereof using any number of host-vector systems routinely used and
widely known in the art.
[0296] A wide range of host-vector systems suitable for the
expression of 193P1E1B 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, 193P1E1B
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 193P1E1B protein or fragment thereof. Such
host-vector systems can be employed to study the functional
properties of 193P1E1B and 193P1E1B mutations or analogs.
[0297] Recombinant human 193P1E1B protein or an analog or homolog
or fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 193P1E1B-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 193P1E1B or fragment, analog or homolog thereof, a
193P1E1B-related protein is expressed in the 293T cells, and the
recombinant 193P1E1B protein is isolated using standard
purification methods (e.g., affinity purification using
anti-193P1E1B antibodies). In another embodiment, a 193P1E1B 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 193P1E1B 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 193P1E1B coding sequence can be used
for the generation of a secreted form of recombinant 193P1E1B
protein.
[0298] As discussed herein, redundancy in the genetic code permits
variation in 193P1E1B 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.
[0299] Additional sequence modifications are known to enhance
protein expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon/intron
splice site signals, transposon-like repeats, and/or other such
well-characterized sequences that are deleterious to gene
expression. The GC content of the sequence is adjusted to levels
average for a given cellular host, as calculated by reference to
known genes expressed in the host cell. Where possible, the
sequence is modified to avoid predicted hairpin secondary mRNA
structures. Other useful modifications include the addition of a
translational initiation consensus sequence at the start of the
open reading frame, as described in Kozak, Mol. Cell. Biol.,
9:5073-5080 (1989). Skilled artisans understand that the general
rule that eukaryotic ribosomes initiate translation exclusively at
the 5' proximal AUG codon is abrogated only under rare conditions
(see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR
15(20): 8125-8148 (1987)).
III.) 193P1E1B-RELATED PROTEINS
[0300] Another aspect of the present invention provides
193P1E1B-related proteins. Specific embodiments of 193P1E1B
proteins comprise a polypeptide having all or part of the amino
acid sequence of human 193P1E1B as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 193P1E1B proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 193P1E1B shown in FIG. 2 or FIG. 3.
[0301] Embodiments of a 193P1E1B polypeptide include: a 193P1E1B
polypeptide having a sequence shown in FIG. 2, a peptide sequence
of a 193P1E1B 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 193P1E1B peptides comprise, without
limitation: [0302] (I) a protein comprising, consisting essentially
of, or consisting of an amino acid sequence as shown in FIG. 2A-M
or FIGS. 3A-H; [0303] (II) a 193P1E1B-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 FIGS. 2A-M; [0304] (III) a
193P1E1B-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-M or 3A-H; [0305] (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; [0306] (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; [0307] (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; [0308] (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;
[0309] (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; [0310] (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, 3B, 3C, 3D, 3E,
3F, 3G, or 3H in any whole number increment up to 412, 412, 412,
330, 388, 308, 73, or 372 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; [0311] (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, 3B, 3C, 3D, 3E, 3F, 3G, or
3H in any whole number increment up to 412, 412, 412, 330, 388,
308, 73, or 372 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 less than 0.5 in the Hydropathicity
profile of FIG. 6; [0312] (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, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole
number increment up to 412, 412, 412, 330, 388, 308, 73, or 372
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 Percent Accessible Residues profile
of FIG. 7; [0313] (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, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number
increment up to 412, 412, 412, 330, 388, 308, 73, or 372
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 Average Flexibility profile of FIG.
8; [0314] (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, 35 amino acids of a protein of FIG.
3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up
to 412, 412, 412, 330, 388, 308, 73, or 372 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 Beta-turn profile of FIG. 9; [0315] (XIV) a peptide that
occurs at least twice in Tables VIII-XXI and XXII to XLIX,
collectively; [0316] (XV) a peptide that occurs at least three
times in Tables VIII-XXI and XXII to XLIX, collectively; [0317]
(XVI) a peptide that occurs at least four times in Tables VIII-XXI
and XXII to XLIX, collectively; [0318] (XVII) a peptide that occurs
at least five times in Tables VIII-XXI and XXII to XLIX,
collectively; [0319] (XVIII) a peptide that occurs at least once in
Tables VIII-XXI, and at least once in tables XXII to XLIX; [0320]
(XIX) a peptide that occurs at least once in Tables VIII-XXI, and
at least twice in tables XXII to XLIX; [0321] (XX) a peptide that
occurs at least twice in Tables VIII-XXI, and at least once in
tables XXII to XLIX; [0322] (XXI) a peptide that occurs at least
twice in Tables VIII-XXI, and at least twice in tables XXII to
XLIX; [0323] (XXII) a peptide which comprises one two, three, four,
or five of the following characteristics, or an oligonucleotide
encoding such peptide: [0324] 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; [0325] 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; [0326] 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; [0327]
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, [0328] 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; [0329] (XXIII) a composition comprising a peptide of
(I)-(XXII) or an antibody or binding region thereof together with a
pharmaceutical excipient and/or in a human unit dose form; [0330]
(XXIV) a method of using a peptide of (I)-(XXII), or an antibody or
binding region thereof or a composition of (XXIII) in a method to
modulate a cell expressing 193P1E1B; [0331] (XXV) a method of using
a peptide of (I)-(XXII) or an antibody or binding region thereof or
a composition of (XXIII) in a method to diagnose, prophylax,
prognose, or treat an individual who bears a cell expressing
193P1E1B; [0332] (XXVI) a method of using a peptide of (I)-(XXII)
or an antibody or binding region thereof or a composition (XXIII)
in a method to diagnose, prophylax, prognose, or treat an
individual who bears a cell expressing 193P1E1B, said cell from a
cancer of a tissue listed in Table I; [0333] (XXVII) a method of
using a peptide of (I)-(XXII) or an antibody or binding region
thereof or a composition of (XXIII) in a method to diagnose,
prophylax, prognose, or treat a cancer; [0334] (XXVIII) a method of
using a peptide of (I)-(XXII) or an antibody or binding region
thereof or a composition of (XXIII) in a method to diagnose,
prophylax, prognose, or treat a cancer of a tissue listed in Table
I; and, [0335] (XXIX) a method of using a a peptide of (I)-(XXII)
or an antibody or binding region thereof or a composition (XXIII)
in a method to identify or characterize a modulator of a cell
expressing 193P1E1B.
[0336] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0337] Typical embodiments of the invention disclosed herein
include 193P1E1B polynucleotides that encode specific portions of
193P1E1B mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0338] (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, 410, 412 or more contiguous amino
acids of 193P1E1B variant 1; the maximal lengths relevant for other
variants are: variant 5, 412 amino acids; variant 6, 412 amino
acids, variant 9, 330, variant 10, 388 amino acids, variant 11, 308
amino acids, variant 12, 73, and variant 13, 372 amino acids.
[0339] In general, naturally occurring allelic variants of human
193P1E1B share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
193P1E1B protein contain conservative amino acid substitutions
within the 193P1E1B sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 193P1E1B. One class of 193P1E1B allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 193P1E1B 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.
[0340] 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).
[0341] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 193P1E1B proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 193P1E1B 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
193P1E1B variant DNA.
[0342] 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.
[0343] As defined herein, 193P1E1B variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 193P1E1B 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
193P1E1B variant also specifically binds to a 193P1E1B 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 193P1E1B
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.
[0344] Other classes of 193P1E1B-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 193P1E1B protein variants or analogs comprises one or more of
the 193P1E1B biological motifs described herein or presently known
in the art. Thus, encompassed by the present invention are analogs
of 193P1E1B 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.
[0345] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a 193P1E1B 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 193P1E1B protein shown in
FIG. 2 or FIG. 3.
[0346] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of a 193P1E1B protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a 193P1E1B protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of a
193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of a 193P1E1B protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 193P1E1B protein shown in FIG.
2 or FIG. 3, polypeptides consisting of about amino acid 60 to
about amino acid 70 of a 193P1E1B protein shown in FIG. 2 or FIG.
3, polypeptides consisting of about amino acid 70 to about amino
acid 80 of a 193P1E1B protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 80 to about amino acid
90 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 90 to about amino acid 100 of a
193P1E1B protein shown in FIG. 2 or FIG. 3, etc. throughout the
entirety of a 193P1E1B 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 193P1E1B 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.
[0347] 193P1E1B-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
193P1E1B-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a 193P1E1B protein
(or variants, homologs or analogs thereof).
[0348] III.A.) Motif-Bearing Protein Embodiments
[0349] Additional illustrative embodiments of the invention
disclosed herein include 193P1E1B polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a 193P1E1B 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.
[0350] Motif bearing subsequences of all 193P1E1B variant proteins
are set forth and identified in Tables VIII-XXI and XXII-XLIX.
[0351] Table V sets forth several frequently occurring motifs based
on pfam searches. 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.
[0352] Polypeptides comprising one or more of the 193P1E1B motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 193P1E1B motifs discussed above are associated with growth
dysregulation and because 193P1E1B 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)).
[0353] 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 193P1E1B protein
that are capable of optimally binding to specified HLA alleles.
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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 193P1E1B-related proteins are embodied in many forms,
preferably in isolated form. A purified 193P1E1B protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 193P1E1B to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 193P1E1B-related proteins
include purified 193P1E1B-related proteins and functional, soluble
193P1E1B-related proteins. In one embodiment, a functional, soluble
193P1E1B protein or fragment thereof retains the ability to be
bound by antibody, T cell or other ligand.
[0358] The invention also provides 193P1E1B proteins comprising
biologically active fragments of a 193P1E1B amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting 193P1E1B protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting 193P1E1B 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.
[0359] 193P1E1B-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-193P1E1B antibodies or T cells or in identifying cellular
factors that bind to 193P1E1B. 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.
[0360] CTL epitopes can be determined using specific algorithms to
identify peptides within a 193P1E1B protein that are capable of
optimally binding to specified HLA alleles (e.g., by using the
SYFPEITHI site at World Wide Web; Epimatrix.TM. and Epimer.TM.,
Brown University; and BIMAS). Illustrating this, peptide epitopes
from 193P1E1B that are presented in the context of human MHC Class
I molecules, e.g., HLA-A1, A2, A3, All, A24, B7 and B35 were
predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically,
the complete amino acid sequence of the 193P1E1B 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.
[0361] 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 193P1E1B 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.
[0362] 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.
[0363] 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
SYFPEITHI or BIMAS) are to be "applied" to a 193P1E1B protein in
accordance with the invention. As used in this context "applied"
means that a 193P1E1B 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 193P1E1B 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.
[0364] III.B.) Expression of 193P1E1B-Related Proteins
[0365] In an embodiment described in the examples that follow,
193P1E1B 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 193P1E1B 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 193P1E1B protein in transfected cells.
The secreted HIS-tagged 193P1E1B in the culture media can be
purified, e.g., using a nickel column using standard
techniques.
[0366] III.C.) Modifications of 193P1E1B-Related Proteins
[0367] Modifications of 193P1E1B-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 193P1E1B polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a 193P1E1B protein. Another type of
covalent modification of a 193P1E1B 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 193P1E1B comprises linking a 193P1E1B polypeptide
to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0368] The 193P1E1B-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 193P1E1B
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 193P1E1B 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 193P1E1B. A chimeric
molecule can comprise a fusion of a 193P1E1B-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 193P1E1B protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
193P1E1B-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 193P1E1B 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, CHI, 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.
[0369] III.D.) Uses of 193P1E1B-Related Proteins
[0370] The proteins of the invention have a number of different
specific uses. As 193P1E1B is highly expressed in prostate and
other cancers, 193P1E1B-related proteins are used in methods that
assess the status of 193P1E1B gene products in normal versus
cancerous tissues, thereby elucidating the malignant phenotype.
Typically, polypeptides from specific regions of a 193P1E1B 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 193P1E1B-related proteins
comprising the amino acid residues of one or more of the biological
motifs contained within a 193P1E1B 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, 193P1E1B-related proteins that contain the amino
acid residues of one or more of the biological motifs in a 193P1E1B
protein are used to screen for factors that interact with that
region of 193P1E1B.
[0371] 193P1E1B protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of a 193P1E1B protein), for identifying agents or cellular
factors that bind to 193P1E1B 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.
[0372] Proteins encoded by the 193P1E1B 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 193P1E1B gene product. Antibodies raised against a
193P1E1B protein or fragment thereof are useful in diagnostic and
prognostic assays, and imaging methodologies in the management of
human cancers characterized by expression of 193P1E1B protein, such
as those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 193P1E1B-related nucleic acids or proteins are also used
in generating HTL or CTL responses.
[0373] Various immunological assays useful for the detection of
193P1E1B 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
193P1E1B-expressing cells (e.g., in radioscintigraphic imaging
methods). 193P1E1B proteins are also particularly useful in
generating cancer vaccines, as further described herein.
IV.) 193P1E1B ANTIBODIES
[0374] Another aspect of the invention provides antibodies that
bind to 193P1E1B-related proteins. Preferred antibodies
specifically bind to a 193P1E1B-related protein and do not bind (or
bind weakly) to peptides or proteins that are not 193P1E1B-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
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 193P1E1B can bind
193P1E1B-related proteins such as the homologs or analogs
thereof.
[0375] 193P1E1B 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 193P1E1B 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 193P1E1B is involved, such as
advanced or metastatic prostate cancers.
[0376] The invention also provides various immunological assays
useful for the detection and quantification of 193P1E1B and mutant
193P1E1B-related proteins. Such assays can comprise one or more
193P1E1B antibodies capable of recognizing and binding a
193P1E1B-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.
[0377] 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.
[0378] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing 193P1E1B are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 193P1E1B
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 193P1E1B expressing cancers such as
prostate cancer.
[0379] 193P1E1B antibodies are also used in methods for purifying a
193P1E1B-related protein and for isolating 193P1E1B homologues and
related molecules. For example, a method of purifying a
193P1E1B-related protein comprises incubating a 193P1E1B antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 193P1E1B-related protein under conditions
that permit the 193P1E1B antibody to bind to the 193P1E1B-related
protein; washing the solid matrix to eliminate impurities; and
eluting the 193P1E1B-related protein from the coupled antibody.
Other uses of 193P1E1B antibodies in accordance with the invention
include generating anti-idiotypic antibodies that mimic a 193P1E1B
protein.
[0380] 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 193P1E1B-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 193P1E1B can also be used, such as
a 193P1E1B 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 193P1E1B-related
protein is synthesized and used as an immunogen.
[0381] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 193P1E1B-related protein or
193P1E1B expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann Rev.
Immunol. 15: 617-648).
[0382] The amino acid sequence of a 193P1E1B protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
193P1E1B protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 193P1E1B amino acid
sequence are used to identify hydrophilic regions in the 193P1E1B
structure. Regions of a 193P1E1B 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 193P1E1B 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
193P1E1B 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.
[0383] 193P1E1B 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
193P1E1B-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.
[0384] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a 193P1E1B protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
193P1E1B 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.
[0385] 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 193P1E1B 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 193P1E1B
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued
19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and, 6,114,598 issued
5 Sep. 2000). This method avoids the in vitro manipulation required
with phage display technology and efficiently produces high
affinity authentic human antibodies.
[0386] Reactivity of 193P1E1B antibodies with a 193P1E1B-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 193P1E1B-related proteins,
193P1E1B-expressing cells or extracts thereof. A 193P1E1B 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 193P1E1B epitopes are generated using
methods generally known in the art. Homodimeric antibodies can also
be generated by cross-linking techniques known in the art (e.g.,
Wolff et al., Cancer Res. 53: 2560-2565).
V.) 193P1E1B CELLULAR IMMUNE RESPONSES
[0387] 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.
[0388] 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; 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).
[0389] 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.)
[0390] 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).
[0391] 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.
[0392] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0393] 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.
[0394] 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.
[0395] 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.
VI.) 193P1E1B TRANSGENIC ANIMALS
[0396] Nucleic acids that encode a 193P1E1B-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 193P1E1B can be used to clone genomic DNA
that encodes 193P1E1B. The cloned genomic sequences can then be
used to generate transgenic animals containing cells that express
DNA that encode 193P1E1B. 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
September 1989. Typically, particular cells would be targeted for
193P1E1B transgene incorporation with tissue-specific
enhancers.
[0397] Transgenic animals that include a copy of a transgene
encoding 193P1E1B can be used to examine the effect of increased
expression of DNA that encodes 193P1E1B. 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.
[0398] Alternatively, non-human homologues of 193P1E1B can be used
to construct a 193P1E1B "knock out" animal that has a defective or
altered gene encoding 193P1E1B as a result of homologous
recombination between the endogenous gene encoding 193P1E1B and
altered genomic DNA encoding 193P1E1B introduced into an embryonic
cell of the animal. For example, cDNA that encodes 193P1E1B can be
used to clone genomic DNA encoding 193P1E1B in accordance with
established techniques. A portion of the genomic DNA encoding
193P1E1B 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 193P1E1B
polypeptide.
VII.) METHODS FOR THE DETECTION OF 193P1E1B
[0399] Another aspect of the present invention relates to methods
for detecting 193P1E1B polynucleotides and 193P1E1B-related
proteins, as well as methods for identifying a cell that expresses
193P1E1B. The expression profile of 193P1E1B makes it a diagnostic
marker for metastasized disease. Accordingly, the status of
193P1E1B 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 193P1E1B 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.
[0400] More particularly, the invention provides assays for the
detection of 193P1E1B polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 193P1E1B polynucleotides
include, for example, a 193P1E1B gene or fragment thereof, 193P1E1B
mRNA, alternative splice variant 193P1E1B mRNAs, and recombinant
DNA or RNA molecules that contain a 193P1E1B polynucleotide. A
number of methods for amplifying and/or detecting the presence of
193P1E1B polynucleotides are well known in the art and can be
employed in the practice of this aspect of the invention.
[0401] In one embodiment, a method for detecting a 193P1E1B 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 193P1E1B polynucleotides as sense and
antisense primers to amplify 193P1E1B cDNAs therein; and detecting
the presence of the amplified 193P1E1B cDNA. Optionally, the
sequence of the amplified 193P1E1B cDNA can be determined.
[0402] In another embodiment, a method of detecting a 193P1E1B gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 193P1E1B
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 193P1E1B gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
193P1E1B nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0403] The invention also provides assays for detecting the
presence of a 193P1E1B protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a
193P1E1B-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
193P1E1B-related protein in a biological sample comprises first
contacting the sample with a 193P1E1B antibody, a 193P1E1B-reactive
fragment thereof, or a recombinant protein containing an
antigen-binding region of a 193P1E1B antibody; and then detecting
the binding of 193P1E1B-related protein in the sample.
[0404] Methods for identifying a cell that expresses 193P1E1B are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 193P1E1B gene comprises
detecting the presence of 193P1E1B 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 193P1E1B
riboprobes, Northern blot and related techniques) and various
nucleic acid amplification assays (such as RT-PCR using
complementary primers specific for 193P1E1B, 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 193P1E1B gene comprises
detecting the presence of 193P1E1B-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
193P1E1B-related proteins and cells that express 193P1E1B-related
proteins.
[0405] 193P1E1B expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 193P1E1B gene
expression. For example, 193P1E1B 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 193P1E1B 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 193P1E1B expression by RT-PCR, nucleic acid
hybridization or antibody binding.
VIII.) METHODS FOR MONITORING THE STATUS OF 193P1E1B-RELATED GENES
AND THEIR PRODUCTS
[0406] 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 193P1E1B 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 193P1E1B in a biological
sample of interest can be compared, for example, to the status of
193P1E1B 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 193P1E1B 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 193P1E1B status in a sample.
[0407] 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 193P1E1B
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 193P1E1B mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 193P1E1B
comprises a change in the location of 193P1E1B and/or 193P1E1B
expressing cells and/or an increase in 193P1E1B mRNA and/or protein
expression.
[0408] 193P1E1B 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 193P1E1B 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 193P1E1B 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 193P1E1B gene), Northern analysis and/or PCR analysis of 193P1E1B
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 193P1E1B 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
193P1E1B proteins and/or associations of 193P1E1B proteins with
polypeptide binding partners). Detectable 193P1E1B polynucleotides
include, for example, a 193P1E1B gene or fragment thereof, 193P1E1B
mRNA, alternative splice variants, 193P1E1B mRNAs, and recombinant
DNA or RNA molecules containing a 193P1E1B polynucleotide.
[0409] The expression profile of 193P1E1B 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 193P1E1B provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 193P1E1B status and diagnosing
cancers that express 193P1E1B, such as cancers of the tissues
listed in Table I. For example, because 193P1E1B mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of 193P1E1B mRNA
transcripts or proteins in a biological sample can be used to
diagnose a disease associated with 193P1E1B dysregulation, and can
provide prognostic information useful in defining appropriate
therapeutic options.
[0410] The expression status of 193P1E1B 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 193P1E1B 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.
[0411] As described above, the status of 193P1E1B in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 193P1E1B 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 193P1E1B
expressing cells (e.g. those that express 193P1E1B mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 193P1E1B-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 193P1E1B 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 Aug. 154(2 Pt 1):474-8).
[0412] In one aspect, the invention provides methods for monitoring
193P1E1B gene products by determining the status of 193P1E1B 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 193P1E1B gene products in a corresponding normal
sample. The presence of aberrant 193P1E1B 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.
[0413] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 193P1E1B 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
193P1E1B mRNA can, for example, be evaluated in tissues including
but not limited to those listed in Table I. The presence of
significant 193P1E1B 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 193P1E1B mRNA
or express it at lower levels.
[0414] In a related embodiment, 193P1E1B status is determined at
the protein level rather than at the nucleic acid level. For
example, such a method comprises determining the level of 193P1E1B
protein expressed by cells in a test tissue sample and comparing
the level so determined to the level of 193P1E1B expressed in a
corresponding normal sample. In one embodiment, the presence of
193P1E1B protein is evaluated, for example, using
immunohistochemical methods. 193P1E1B antibodies or binding
partners capable of detecting 193P1E1B protein expression are used
in a variety of assay formats well known in the art for this
purpose.
[0415] In a further embodiment, one can evaluate the status of
193P1E1B 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
193P1E1B may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 193P1E1B indicates a potential loss of function or
increase in tumor growth.
[0416] 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 193P1E1B 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).
[0417] Additionally, one can examine the methylation status of a
193P1E1B 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.
[0418] Gene amplification is an additional method for assessing the
status of 193P1E1B. 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.
[0419] 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 193P1E1B
expression. The presence of RT-PCR amplifiable 193P1E1B 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).
[0420] 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 193P1E1B mRNA or 193P1E1B protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 193P1E1B mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 193P1E1B
in prostate or other tissue is examined, with the presence of
193P1E1B 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 193P1E1B 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 193P1E1B gene products in the sample
is an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0421] 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
193P1E1B mRNA or 193P1E1B protein expressed by tumor cells,
comparing the level so determined to the level of 193P1E1B mRNA or
193P1E1B protein expressed in a corresponding normal tissue taken
from the same individual or a normal tissue reference sample,
wherein the degree of 193P1E1B mRNA or 193P1E1B 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 193P1E1B
is expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 193P1E1B 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.
[0422] 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 193P1E1B mRNA or 193P1E1B protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 193P1E1B mRNA or 193P1E1B protein expressed in an equivalent
tissue sample taken from the same individual at a different
time,
[0423] wherein the degree of 193P1E1B mRNA or 193P1E1B 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 193P1E1B
expression in the tumor cells over time, where increased expression
over time indicates a progression of the cancer. Also, one can
evaluate the integrity 193P1E1B 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.
[0424] 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 193P1E1B gene and 193P1E1B gene products (or
perturbations in 193P1E1B gene and 193P1E1B 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 193P1E1B gene
and 193P1E1B gene products (or perturbations in 193P1E1B gene and
193P1E1B 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.
[0425] In one embodiment, methods for observing a coincidence
between the expression of 193P1E1B gene and 193P1E1B gene products
(or perturbations in 193P1E1B gene and 193P1E1B gene products) and
another factor associated with malignancy entails detecting the
overexpression of 193P1E1B 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
193P1E1B mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of 193P1E1B and PSA mRNA in prostate tissue is examined, where the
coincidence of 193P1E1B 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.
[0426] Methods for detecting and quantifying the expression of
193P1E1B 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 193P1E1B mRNA include in situ hybridization using
labeled 193P1E1B riboprobes, Northern blot and related techniques
using 193P1E1B polynucleotide probes, RT-PCR analysis using primers
specific for 193P1E1B, 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 193P1E1B mRNA expression. Any number of primers
capable of amplifying 193P1E1B 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
193P1E1B protein can be used in an immunohistochemical assay of
biopsied tissue.
IX.) IDENTIFICATION OF MOLECULES THAT INTERACT WITH 193P1E1B
[0427] The 193P1E1B protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 193P1E1B, as well as
pathways activated by 193P1E1B 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).
[0428] Alternatively one can screen peptide libraries to identify
molecules that interact with 193P1E1B protein sequences. In such
methods, peptides that bind to 193P1E1B 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 193P1E1B protein(s).
[0429] 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 193P1E1B 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.
[0430] Alternatively, cell lines that express 193P1E1B are used to
identify protein-protein interactions mediated by 193P1E1B. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 193P1E1B protein can be immunoprecipitated from
193P1E1B-expressing cell lines using anti-193P1E1B antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 193P1E1B 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.
[0431] Small molecules and ligands that interact with 193P1E1B 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
193P1E1B'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
193P1E1B-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses 193P1E1B (see, e.g., Hille, B., Ionic
Channels of Excitable Membranes 2.sup.nd Ed., Sinauer Assoc.,
Sunderland, Mass., 1992). Moreover, ligands that regulate 193P1E1B
function can be identified based on their ability to bind 193P1E1B
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 193P1E1B 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 193P1E1B.
[0432] An embodiment of this invention comprises a method of
screening for a molecule that interacts with a 193P1E1B amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a 193P1E1B amino acid
sequence, allowing the population of molecules and the 193P1E1B
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 193P1E1B amino acid sequence, and then separating
molecules that do not interact with the 193P1E1B 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 193P1E1B amino acid sequence.
The identified molecule can be used to modulate a function
performed by 193P1E1B. In a preferred embodiment, the 193P1E1B
amino acid sequence is contacted with a library of peptides.
X.) THERAPEUTIC METHODS AND COMPOSITIONS
[0433] The identification of 193P1E1B as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in prostate and other cancers, opens a number of
therapeutic approaches to the treatment of such cancers. As
contemplated herein, 193P1E1B functions as a transcription factor
involved in activating tumor-promoting genes or repressing genes
that block tumorigenesis.
[0434] Accordingly, therapeutic approaches that inhibit the
activity of a 193P1E1B protein are useful for patients suffering
from a cancer that expresses 193P1E1B. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a 193P1E1B
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a 193P1E1B gene or translation of 193P1E1B
mRNA.
[0435] X.A.) Anti-Cancer Vaccines
[0436] The invention provides cancer vaccines comprising a
193P1E1B-related protein or 193P1E1B-related nucleic acid. In view
of the expression of 193P1E1B, cancer vaccines prevent and/or treat
193P1E1B-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).
[0437] Such methods can be readily practiced by employing a
193P1E1B-related protein, or a 193P1E1B-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 193P1E1B immunogen (which typically comprises a
number of antibody or T cell epitopes). Skilled artisans understand
that a wide variety of vaccine systems for delivery of
immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer
Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of
generating an immune response (e.g. humoral and/or cell-mediated)
in a mammal, comprise the steps of: exposing the mammal's immune
system to an immunoreactive epitope (e.g. an epitope present in a
193P1E1B 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 193P1E1B immunogen contains
a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a
peptide of a size range from 193P1E1B indicated in FIG. 5, FIG. 6,
FIG. 7, FIG. 8, and FIG. 9.
[0438] The entire 193P1E1B 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.
[0439] In patients with 193P1E1B-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.
[0440] Cellular Vaccines:
[0441] CTL epitopes can be determined using specific algorithms to
identify peptides within 193P1E1B protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University, BIMAS, and SYFPEITHI. In a preferred embodiment,
a 193P1E1B 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.
[0442] Antibody-Based Vaccines
[0443] 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 193P1E1B protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to 193P1E1B in a
host, by contacting the host with a sufficient amount of at least
one 193P1E1B B cell or cytotoxic T-cell epitope or analog thereof;
and at least one periodic interval thereafter re-contacting the
host with the 193P1E1B B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a 193P1E1B-related protein or a man-made
multiepitopic peptide comprising: administering 193P1E1B immunogen
(e.g. a 193P1E1B protein or a peptide fragment thereof, a 193P1E1B
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 193P1E1B 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 193P1E1B
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 193P1E1B, in
order to generate a response to the target antigen.
[0444] Nucleic Acid Vaccines:
[0445] 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 193P1E1B. Constructs comprising DNA encoding a
193P1E1B-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 193P1E1B protein/immunogen.
Alternatively, a vaccine comprises a 193P1E1B-related protein.
Expression of the 193P1E1B-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a 193P1E1B 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
(bupivacaine, 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).
[0446] 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
193P1E1B-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0447] 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.
[0448] Thus, gene delivery systems are used to deliver a
193P1E1B-related nucleic acid molecule. In one embodiment, the
full-length human 193P1E1B cDNA is employed. In another embodiment,
193P1E1B nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0449] Ex Vivo Vaccines
[0450] 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
193P1E1B 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 193P1E1B peptides to T cells in the context of MHC class
I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with 193P1E1B peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete 193P1E1B protein. Yet another
embodiment involves engineering the overexpression of a 193P1E1B
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 193P1E1B can also be engineered to express
immune modulators, such as GM-CSF, and used as immunizing
agents.
[0451] X.B.) 193P1E1B as a Target for Antibody-Based Therapy
[0452] 193P1E1B 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 193P1E1B is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 193P1E1B-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 193P1E1B are
useful to treat 193P1E1B-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0453] 193P1E1B antibodies can be introduced into a patient such
that the antibody binds to 193P1E1B 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 193P1E1B, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0454] 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 193P1E1B 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., Sievers 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. 193P1E1B), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0455] 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-193P1E1B antibody) that binds to a marker (e.g. 193P1E1B)
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 193P1E1B, comprising
conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 193P1E1B 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.
[0456] Cancer immunotherapy using anti-193P1E1B 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, 193P1E1B 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).
[0457] Although 193P1E1B 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.
[0458] Although 193P1E1B 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.
[0459] Cancer patients can be evaluated for the presence and level
of 193P1E1B expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 193P1E1B imaging, or
other techniques that reliably indicate the presence and degree of
193P1E1B 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.
[0460] Anti-193P1E1B 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-193P1E1B 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-193P1E1B mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 193P1E1B.
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-193P1E1B 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.
[0461] 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 193P1E1B antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0462] Therapeutic methods of the invention contemplate the
administration of single anti-193P1E1B 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-193P1E1B 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-193P1E1B mAbs are administered in
their "naked" or unconjugated form, or can have a therapeutic
agent(s) conjugated to them.
[0463] Anti-193P1E1B 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-193P1E1B 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.
[0464] 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-193P1E1B 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 193P1E1B expression in the patient, the
extent of circulating shed 193P1E1B 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.
[0465] Optionally, patients should be evaluated for the levels of
193P1E1B in a given sample (e.g. the levels of circulating 193P1E1B
antigen and/or 193P1E1B 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).
[0466] Anti-idiotypic anti-193P1E1B antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 193P1E1B-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-193P1E1B antibodies that mimic an epitope on a
193P1E1B-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.
[0467] X.C.) 193P1E1B as a Target for Cellular Immune Responses
[0468] 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.
[0469] 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-phosphorothioated-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))
[0470] 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 193P1E1B antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0471] 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).
[0472] 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.
[0473] 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.
[0474] 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.
[0475] 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.
[0476] 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.
[0477] 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.
[0478] 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.
[0479] 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.
[0480] 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.
[0481] X.C.1. Minigene Vaccines
[0482] 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.
[0483] 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 193P1E1B, the PADRE.RTM. universal helper T cell
epitope or multiple HTL epitopes from 193P1E1B (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.
[0484] 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.
[0485] 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.
[0486] 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.
[0487] 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.
[0488] 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.
[0489] 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.
[0490] 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.
[0491] 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.
[0492] 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.
[0493] 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 Feigner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0494] 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.
[0495] 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.
[0496] 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.
[0497] 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.
[0498] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0499] 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.
[0500] 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.
[0501] 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:48), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:49), and Streptococcus 18 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO:50).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0502] 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: AKXVAAWTLKAAA
(SEQ ID NO:51), 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.
[0503] 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.
[0504] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0505] 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.
[0506] 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.
[0507] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0508] 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.
[0509] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 193P1E1B. 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 193P1E1B.
[0510] X.D. Adoptive Immunotherapy
[0511] Antigenic 193P1E1B-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.
[0512] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0513] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 193P1E1B. 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.
[0514] 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 193P1E1B. 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.
[0515] For therapeutic use, administration should generally begin
at the first diagnosis of 193P1E1B-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 193P1E1B, a vaccine comprising
193P1E1B-specific CTL may be more efficacious in killing tumor
cells in patient with advanced disease than alternative
embodiments.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] 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.
[0520] 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.
[0521] 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.
[0522] 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.
[0523] 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.
[0524] 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.
[0525] For antibodies, a treatment generally involves repeated
administration of the anti-193P1E1B 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-193P1E1B
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
193P1E1B expression in the patient, the extent of circulating shed
193P1E1B 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.
[0526] 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.
[0527] 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.
[0528] 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.
[0529] 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.
[0530] 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%.
[0531] For aerosol administration, immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are about 0.01%-20%
by weight, preferably about 1%-10%. The surfactant must, of course,
be nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters of
fatty acids containing from about 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids with an aliphatic polyhydric alcohol or
its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be employed. The surfactant may constitute about
0.1%-20% by weight of the composition, preferably about 0.25-5%.
The balance of the composition is ordinarily propellant. A carrier
can also be included, as desired, as with, e.g., lecithin for
intranasal delivery.
XI.) DIAGNOSTIC AND PROGNOSTIC EMBODIMENTS OF 193P1E1B
[0532] As disclosed herein, 193P1E1B 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 193P1E1B in normal tissues, and patient
specimens").
[0533] 193P1E1B 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
193P1E1B polynucleotides and polypeptides (as well as 193P1E1B
polynucleotide probes and anti-193P1E1B 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.
[0534] Typical embodiments of diagnostic methods which utilize the
193P1E1B 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
193P1E1B polynucleotides described herein can be utilized in the
same way to detect 193P1E1B 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 193P1E1B
polypeptides described herein can be utilized to generate
antibodies for use in detecting 193P1E1B overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0535] 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 193P1E1B 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
193P1E1B-expressing cells (lymph node) is found to contain
193P1E1B-expressing cells such as the 193P1E1B expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0536] Alternatively 193P1E1B 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 193P1E1B or
express 193P1E1B at a different level are found to express 193P1E1B
or have an increased expression of 193P1E1B (see, e.g., the
193P1E1B 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 193P1E1B) such as
PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract.
192(3): 233-237 (1996)).
[0537] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, 193P1E1B 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 193P1E1B in normal
tissues, and patient specimens," where a 193P1E1B polynucleotide
fragment is used as a probe to show the expression of 193P1E1B 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 193P1E1B polynucleotide shown in
FIG. 2 or variant thereof) under conditions of high stringency.
[0538] 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. 193P1E1B
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
193P1E1B 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 193P1E1B
polypeptide shown in FIG. 3).
[0539] As shown herein, the 193P1E1B polynucleotides and
polypeptides (as well as the 193P1E1B polynucleotide probes and
anti-193P1E1B 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 193P1E1B 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 193P1E1B polynucleotides and
polypeptides (as well as the 193P1E1B polynucleotide probes and
anti-193P1E1B antibodies used to identify the presence of these
molecules) need to be employed to confirm a metastases of prostatic
origin.
[0540] Finally, in addition to their use in diagnostic assays, the
193P1E1B 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 193P1E1B gene maps (see the Example entitled "Chromosomal
Mapping of 193P1E1B" below). Moreover, in addition to their use in
diagnostic assays, the 193P1E1B-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).
[0541] Additionally, 193P1E1B-related proteins or polynucleotides
of the invention can be used to treat a pathologic condition
characterized by the over-expression of 193P1E1B. 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 193P1E1B antigen. Antibodies or other molecules that react with
193P1E1B can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
XII.) INHIBITION Of 193P1E1B PROTEIN FUNCTION
[0542] The invention includes various methods and compositions for
inhibiting the binding of 193P1E1B to its binding partner or its
association with other protein(s) as well as methods for inhibiting
193P1E1B function.
[0543] XII.A.) Inhibition of 193P1E1B with Intracellular
Antibodies
[0544] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 193P1E1B are introduced
into 193P1E1B expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-193P1E1B antibody is
expressed intracellularly, binds to 193P1E1B 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).
[0545] 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.
[0546] In one embodiment, intrabodies are used to capture 193P1E1B
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 193P1E1B
intrabodies in order to achieve the desired targeting. Such
193P1E1B intrabodies are designed to bind specifically to a
particular 193P1E1B domain. In another embodiment, cytosolic
intrabodies that specifically bind to a 193P1E1B protein are used
to prevent 193P1E1B from gaining access to the nucleus, thereby
preventing it from exerting any biological activity within the
nucleus (e.g., preventing 193P1E1B from forming transcription
complexes with other factors).
[0547] 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).
[0548] XII.B.) Inhibition of 193P1E1B with Recombinant Proteins
[0549] In another approach, recombinant molecules bind to 193P1E1B
and thereby inhibit 193P1E1B function. For example, these
recombinant molecules prevent or inhibit 193P1E1B 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 193P1E1B specific antibody
molecule. In a particular embodiment, the 193P1E1B binding domain
of a 193P1E1B binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 193P1E1B 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 193P1E1B, whereby the dimeric fusion protein
specifically binds to 193P1E1B and blocks 193P1E1B interaction with
a binding partner. Such dimeric fusion proteins are further
combined into multimeric proteins using known antibody linking
technologies.
[0550] XII.C.) Inhibition of 193P1E1B Transcription or
Translation
[0551] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 193P1E1B gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 193P1E1B mRNA into protein.
[0552] In one approach, a method of inhibiting the transcription of
the 193P1E1B gene comprises contacting the 193P1E1B gene with a
193P1E1B antisense polynucleotide. In another approach, a method of
inhibiting 193P1E1B mRNA translation comprises contacting a
193P1E1B mRNA with an antisense polynucleotide. In another
approach, a 193P1E1B specific ribozyme is used to cleave a 193P1E1B
message, thereby inhibiting translation. Such antisense and
ribozyme based methods can also be directed to the regulatory
regions of the 193P1E1B gene, such as 193P1E1B promoter and/or
enhancer elements. Similarly, proteins capable of inhibiting a
193P1E1B gene transcription factor are used to inhibit 193P1E1B
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.
[0553] Other factors that inhibit the transcription of 193P1E1B by
interfering with 193P1E1B transcriptional activation are also
useful to treat cancers expressing 193P1E1B. Similarly, factors
that interfere with 193P1E1B processing are useful to treat cancers
that express 193P1E1B. Cancer treatment methods utilizing such
factors are also within the scope of the invention.
[0554] XII.D.) General Considerations for Therapeutic
Strategies
[0555] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 193P1E1B (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 193P1E1B inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 193P1E1B antisense polynucleotides, ribozymes,
factors capable of interfering with 193P1E1B transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0556] 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.
[0557] 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 193P1E1B to a binding partner, etc.
[0558] In vivo, the effect of a 193P1E1B therapeutic composition
can be evaluated in a suitable animal model. For example,
xenogeneic 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.
[0559] 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.
[0560] 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).
[0561] 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.
[0562] Dosages and administration protocols for the treatment of
cancers using the foregoing methods will vary with the method and
the target cancer, and will generally depend on a number of other
factors appreciated in the art.
XIII.) IDENTIFICATION, CHARACTERIZATION AND USE OF MODULATORS OF
193P1E1B
[0563] Methods to Identify and Use Modulators
[0564] 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.
[0565] 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.
[0566] Modulator-Related Identification and Screening Assays:
[0567] Gene Expression-Related Assays
[0568] 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).
[0569] 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.
[0570] 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.
[0571] 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.
[0572] Expression Monitoring to Identify Compounds that Modify Gene
Expression
[0573] 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.
[0574] 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.
[0575] 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.
[0576] 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.
[0577] 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.
[0578] 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.
[0579] 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.
[0580] 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.
[0581] 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.
[0582] 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.
[0583] Biological Activity-Related Assays
[0584] 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.
[0585] 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.
[0586] 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.
[0587] 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.
[0588] High Throughput Screening to Identify Modulators
[0589] 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.
[0590] 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.
[0591] Use of Soft Agar Growth and Colony Formation to Identify and
Characterize Modulators
[0592] 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.
[0593] 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.
[0594] Evaluation of Contact Inhibition and Growth Density
Limitation to Identify and Characterize Modulators
[0595] 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.
[0596] 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.
[0597] 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.
[0598] Evaluation of Growth Factor or Serum Dependence to Identify
and Characterize Modulators
[0599] 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.
[0600] Use of Tumor-Specific Marker Levels to Identify and
Characterize Modulators
[0601] 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).
[0602] 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.
[0603] Invasiveness into Matrigel to Identify and Characterize
Modulators
[0604] 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.
[0605] Evaluation of Tumor Growth In Vivo to Identify and
Characterize Modulators
[0606] 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.
[0607] 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).
[0608] 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.
[0609] In Vitro Assays to Identify and Characterize Modulators
[0610] 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.
[0611] Alternatively, a reporter gene system can be devised using a
cancer protein promoter operably linked to a reporter gene such as
luciferase, green fluorescent protein, CAT, or P-gal. The reporter
construct is typically transfected into a cell. After treatment
with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured according to
standard techniques known to those of skill in the art (Davis G F,
supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol.
1998: 9:624).
[0612] 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.
[0613] 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.
[0614] Binding Assays to Identify and Characterize Modulators
[0615] 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.
[0616] 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.
[0617] 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.
[0618] 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.
[0619] 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.
[0620] 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.
[0621] 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.
[0622] 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.
[0623] Competitive Binding to Identify and Characterize
Modulators
[0624] 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.
[0625] 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.
[0626] 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.
[0627] 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.
[0628] 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.
[0629] 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.
[0630] 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.
[0631] Use of Polynucleotides to Down-Regulate or Inhibit a Protein
of the Invention.
[0632] 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.
[0633] Inhibitory and Antisense Nucleotides
[0634] 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.
[0635] 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.
[0636] 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.
[0637] 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)).
[0638] Ribozymes
[0639] 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).
[0640] 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)).
[0641] Use of Modulators in Phenotypic Screening
[0642] 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
[0643] 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.
[0644] Use of Modulators to Affect Peptides of the Invention
[0645] 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.
[0646] Methods of Identifying Characterizing Cancer-Associated
Sequences
[0647] 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.
[0648] 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
[0649] For use in the diagnostic and therapeutic applications
described herein, kits are also 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. 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 FIG. 2--related protein or a FIG. 2 gene or message,
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 and/or a container comprising a reporter-means, such
as a biotin-binding protein, such as avidin or streptavidin, bound
to a reporter molecule, such as an enzymatic, florescent, or
radioisotope label. 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 molecules that encodes such amino acid sequences.
[0650] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising 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.
[0651] A label can be present on the container to indicate that the
composition is used for a specific therapy or non-therapeutic
application, such as a 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.
[0652] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0653] 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 or plastic. The container can hold amino
acid sequence(s), small molecule(s), nucleic acid sequence(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.
[0654] The container can alternatively hold a composition which 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 193P1E1B and modulating the function of
193P1E1B.
[0655] 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. The article of manufacture can further comprise a
second container comprising a pharmaceutically-acceptable buffer,
such as phosphate-buffered saline, Ringer's solution and/ordextrose
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
[0656] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the 193P1E1B Gene
[0657] To isolate genes that are over-expressed in prostate cancer
we used the Suppression Subtractive Hybridization (SSH) procedure
using cDNA derived from prostate cancer xenograft tissues. LAPC-9AD
xenograft was obtained from Dr. Charles Sawyers (UCLA) and was
generated as described (Klein et al., 1997, Nature Med. 3:402-408;
Craft et al., 1999, Cancer Res. 59:5030-5036). LAPC-9AD.sup.2 was
generated from LAPC-9AD xenograft by growing LAPC-9AD xenograft
tissues within a piece of human bone implanted in SCID mice. Tumors
were then harvested and subsequently passaged subcutaneously into
other SCID animals to generate LAPC-9AD.sup.2.
[0658] The 193P1E1B SSH cDNA sequence was derived from a
subtraction consisting of a prostate cancer xenograft
LAPC-9AD.sup.2 minus prostate cancer xenograft LAPC-9AD. By RT-PCR,
the 193P1E1B cDNA was identified as highly expressed in the
prostate cancer xenograft pool (LAPC4-AD, LAPC4-AI, LAPC9-AD,
LAPC9-AI), bladder cancer pool, kidney cancer pool, colon cancer
pool, lung cancer pool, ovary cancer pool, breast cancer pool,
metastasis cancer pool, pancreas cancer pool, with low expression
observed in the prostate cancer pool, and no expression observed in
vital pool 1 (kidney, liver, lung), and in vital pool 2 (stomach,
colon, pancreas) (FIG. 14).
[0659] The 193P1E1B SSH cDNA of 227 bp is listed in FIG. 1. The
full length 193P1E1B cDNAs and ORFs are described in FIG. 2 with
the protein sequences listed in FIG. 3. 193P1E1B v.1, v.2, v.3,
v.4, v.5, v.6, v.7, v.8, v.11, v.12 and v.13 are novel proteins and
have not been previously described. 193P1E1B v.9 shows 99% identity
to a hypothetical protein, MGC4832. 193P1E1B v.10 shows 100%
identity to a novel unnamed protein BAC03484.1.
[0660] Materials and Methods
[0661] RNA Isolation:
[0662] Tumor tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10.sup.8
cells 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.
[0663] Oligonucleotides:
[0664] The following HPLC purified oligonucleotides were used.
TABLE-US-00002 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 52)
5'TTTTGATCAAGCTT.sub.303' Adaptor 1: (SEQ ID NO: 53)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 54)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 55)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 56)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 57)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO: 58)
5'TCGAGCGGCCGCCCGGGCAGGA3' Nested primer (NP)2: (SEQ ID NO: 59)
5'AGCGTGGTCGCGGCCGAGGA3'
[0665] Suppression Subtractive Hybridization:
[0666] 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 xenograft LAPC-9AD.sup.2. The gene 193P1E1B was
derived from a prostate cancer xenograft LAPC-9AD.sup.2 minus
prostate cancer xenograft LAPC-9AD tissues. The SSH DNA sequence
(FIG. 1) was identified.
[0667] The cDNA derived from prostate cancer xenograft LAPC-9AD
tissue was used as the source of the "driver" cDNA, while the cDNA
from prostate cancer xenograft LAPC-9AD.sup.2 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 tissue, as described
above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of
oligonucleotide DPNCDN as primer. First- and second-strand
synthesis were carried out as described in the Kit's user manual
protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The
resulting cDNA was digested with Dpn II for 3 hrs at 37.degree. C.
Digested cDNA was extracted with phenol/chloroform (1:1) and
ethanol precipitated.
[0668] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH).
Ligation was terminated with 1 .mu.l of 0.2 M EDTA and heating at
72.degree. C. for 5 min
[0669] The first hybridization was performed by adding 1.5 it (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.
[0670] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0671] 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.l ), 2.5
.mu.l 10.times. reaction buffer (CLONTECH) and 0.5 .mu.l
50.times.Advantage cDNA polymerase Mix (CLONTECH) in a final volume
of 25 .mu.l. PCR 1 was conducted using the following conditions:
75.degree. C. for 5 min, 94.degree. C. for 25 sec., then 27 cycles
of 94.degree. C. for 10 sec, 66.degree. C. for 30 sec, 72.degree.
C. for 1.5 min Five separate primary PCR reactions were performed
for each experiment. The products were pooled and diluted 1:10 with
water. For the secondary PCR reaction, 1 .mu.l from the pooled and
diluted primary PCR reaction was added to the same reaction mix as
used for PCR 1, except that primers NP1 and NP2 (10 .mu.M) were
used instead of PCR primer 1. PCR 2 was performed using 10-12
cycles of 94.degree. C. for 10 sec, 68.degree. C. for 30 sec, and
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0672] The PCR products were inserted into pCR2.1 using the T/A
vector cloning kit (Invitrogen). Transformed E. coli were subjected
to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in liquid culture
overnight. To identify inserts, PCR amplification was performed on
1 ml of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0673] 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.
[0674] RT-PCR Expression Analysis:
[0675] 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.
[0676] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers 5'
atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO:60) and 5'
agccacacgcagctcattgtagaagg 3' (SEQ ID NO:61) 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 bp .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.
[0677] To determine expression levels of the 193P1E1B 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.
[0678] A typical RT-PCR expression analysis is shown in FIG. 14.
RT-PCR expression analysis was performed on first strand cDNA
generated using pools of tissues from multiple samples. The cDNA
samples were shown to be normalized using beta-actin PCR. Strong
expression of 193P1E1B was observed in prostate cancer xenograft
pool, bladder cancer pool, kidney cancer pool, colon cancer pool,
lung cancer pool, ovary cancer pool, breast cancer pool, and
metastasis pool. Low expression was observed in prostate cancer
pool, but no expression was detected in VP1 and VP2.
Example 2
Isolation of Full Length 193P1E1B Encoding cDNA
[0679] To isolate genes that are involved in prostate cancer, an
experiment was conducted using the prostate cancer xenograft
LAPC-9AD.sup.2. The gene 193P1E1B was derived from a subtraction
consisting of a prostate cancer xenograft LAPC-9AD.sup.2 minus
prostate cancer xenograft LAPC-9AD. The SSH DNA sequence (FIG. 1)
was designated 193P1E1B. Thirteen variants of 193P1E1B were
identified (FIGS. 2 and 3). cDNA clone 193P1E1B v.1 and 193P1E1B
v.5 were cloned from bladder cancer pool cDNA. 193P1E1B v.9 was
cloned from LAPC-4AD cDNA library. All other variants were
identified by bioinformatic analysis.
[0680] 193P1E1B v.1 through v.8 differ from each other by one
nucleic acid substitution. 193P1E1B v.1, v.2, v.4 v.7 and v.8 code
for the same protein, whereas 193P1E1B v.5 and v.6 contain one
amino acid substitution as shown in FIG. 12.
[0681] Absence of a 62-nucleotide sequence was identified in
193P1E1B v.9, nucleic acid positions 907-969 of 193P1E1B v.1. This
resulted in an 82-amino acid truncation at the amino terminus of
193P1E1B v.9. Other splice variants were identified and referred to
as 193P1E1B v.10, v.11, v.12 and v.13.
[0682] 193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11, v.12
and v.13 are novel proteins and have not been previously described.
193P1E1B v.9 shows 99% identity to a hypothetical protein, MGC4832.
193P1E1B v.10 shows 100% identity to a novel unnamed protein
BAC03484.1.
Example 3
Chromosomal Mappine of 193P1E1B
[0683] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are available
including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics
7:22; Research Genetics, Huntsville Ala.), human-rodent somatic
cell hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0684] 193P1E1B maps to chromosome 13q11, using 193P1E1B sequence
and the NCBI BLAST tool. This 13q11 region has been previously
implicated in bladder cancer (Wada T, Louhelainen J, Hemminki K,
Adolfsson J, Wijkstrom H, Norming U, Borgstrom E, Hansson J,
Sandstedt B, Steineck G. Bladder cancer: allelic deletions at and
around the retinoblastoma tumor suppressor gene in relation to
stage and grade. Clin Cancer Res. 2000 February; 6(2):610-5).
Example 4
Expression Analysis of 193P1E1B
[0685] Expression of 193P1E1B was analyzed using 2 sets of primers
as illustrated in FIG. 14A. First strand cDNA was prepared from
vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2,
pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,
LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool,
bladder cancer pool, kidney cancer pool, colon cancer pool, lung
cancer pool, ovary cancer pool, breast cancer pool, metastasis
cancer pool, pancreas cancer pool, and from prostate cancer
metastasis to lymph node from 2 different patients. Normalization
was performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using Primer Set A (B) or Primer Set B (C)
to 193P1E1B, was performed at 30 cycles of amplification. A
schematic diagram depicting the location of the 2 primer sets A and
B is shown in FIG. 14A. Primer Set A detected a PCR product of 190
bp which is identical in all variants of 193P1E1B (FIG. 14B).
Expression of 193P1E1B was observed in prostate cancer xenograft
pool, prostate cancer pool, bladder cancer pool, kidney cancer
pool, colon cancer pool, lung cancer pool, ovary cancer pool,
breast cancer pool, metastasis cancer pool, pancreas cancer pool,
as well as the 2 prostate metastasis to lymph node, but not in VP1
and VP2 (FIG. 14B). In order to test abundance of expression of
193P1E1B v.1 through v.8 compared to 193P1E1B v.9, an experiment
was conducted in which RT-PCR was performed using Primer Set B
(FIG. 14C). Primer Set B detected a PCR product of 239 bp from
193P1E1B v.1 through v.8, and of 177 bp from 193P1E1B v.9 (FIG.
14C). FIG. 14C shows that the transcript encoding 193P1E1B v.1
through v.8, is expressed ate higher levels that the transcript
encoding 193P1E1B v.9. But both transcripts are expressed at
similar proportion in all tissues tested.
[0686] Extensive northern blot analysis of 193P1E1B in 16 human
normal tissues confirms the expression observed by RT-PCR (FIG.
15). Two transcripts of approximately 3.5 kb and 2 kb are only
detected in testis and thymus, but not in any other normal tissue
tested.
[0687] FIG. 16 shows expression of 193P1E1B in prostate cancer
xenografts. RNA was extracted from normal prostate, and from
prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and
LAPC-9AI. Northern blot with 10 ug of total RNA/lane was probed
with 193P1E1B SSH sequence. Northern blot analysis shows expression
of 193P1E1B in all 4 tissues, LAPC-4AD, LAPC-4AI, LAPC-9AD,
LAPC-9AI, with the lowest expression detected in the LAPC-9AD
tissue, but not in normal prostate.
[0688] To test expression of 193P1E1B in patient cancer specimens,
RNA was extracted from a pool of three patients for each of the
following, bladder cancer, colon cancer, ovary cancer and
metastasis cancer, as well as from normal prostate (NP), normal
bladder (NB), normal kidney (NK), normal colon (NC). Northern blots
with 10 ug of total RNA/lane were probed with 193P1E1B SSH sequence
(FIG. 17). Results show expression of 193P1E1B in bladder cancer
pool, colon cancer pool, ovary cancer pool and metastasis cancer
pool, but not in any of the normal tissues tested.
[0689] Analysis of individual bladder cancer tissues by northern
blot shows expression of 193P1E1B in the 2 bladder cancer cell
lines and in the 3 bladder cancer patient specimens tested, but not
in normal bladder tissues (FIG. 18).
[0690] FIG. 19 shows expression of 193P1E1B in cancer metastasis
patient specimens. RNA was extracted from the following cancer
metastasis tissues, colon metastasis to lung, lung metastasis to
lymph node, lung metastasis to skin, and breast metastasis to lymph
node, as well as from normal bladder (NB), normal lung (NL), normal
breast (NBr), and normal ovary (NO). Northern blots with 10 ug of
total RNA/lane were probed with 193P1E1B sequence. Size standards
in kilobases (kb) are indicated on the side. The results show
expression of 193P1E1B in all four different cancer metastasis
samples but not in the normal tissues tested.
[0691] FIG. 20 shows expression of 193P1E1B in pancreatic, ovarian
and testicular cancer patient specimens. RNA was extracted from
pancreatic cancer (P1), ovarian cancer (P2, P3), and testicular
cancer (P4, P5) isolated from cancer patients, as well as from
normal pancreas (NPa). Northern blots with 10 ug of total RNA/lane
were probed with 193P1E1B sequence. Size standards in kilobases
(kb) are indicated on the side. The results show expression of
193P1E1B in pancreatic, ovarian and testicular cancer specimens but
not in normal pancreas.
[0692] FIG. 21 shows expression of 193P1E1B in normal compared to
patient cancer specimens. First strand cDNA was prepared from a
panel of normal tissues (stomach, brain, heart, liver, spleen,
skeletal muscle, testis prostate, bladder, kidney, colon, lung and
pancreas) and from a panel of patient cancer pools (prostate cancer
pool, bladder cancer pool, kidney cancer pool, colon cancer pool,
lung cancer pool, pancreas cancer pool, ovary cancer pool, breast
cancer pool, metastasis cancer pool, LAPC prostate xenograft pool
(XP), and from prostate cancer metastasis to lymph node from 2
different patients (PMLN2). Normalization was performed by PCR
using primers to actin. Semi-quantitative PCR, using primer Set A
as described in FIG. 14, was performed 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.
Relative expression was calculated by normalizing to signal
obtained using actin primers. Results show restricted 193P1E1B
expression in normal testis amongst all normal tissues tested.
193P1E1B expression was strongly upregulated in cancers of the
bladder, colon, lung, pancreas, ovary, breast, and to a lesser
extent in prostate and kidney cancers.
[0693] 193P1E1B was also shown to be expressed in uterus, melanoma
and bone cancer patient specimens. First strand cDNA was prepared
from a panel of uterus patient cancer specimens (A), melanoma and
bone cancer specimens (B). Semi-quantitative PCR, using primers to
193P1E1B, 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, or strong. Results show expression of 193P1E1B in
the majority of uterus patient cancer specimens tested, as well as
in the 2 melanoma specimens and in the bone tumor tested.
[0694] 193P1E1B expression is reminiscent of a cancer-testis gene.
Its restricted normal tissue expression to normal testis, and the
upregulation detected in prostate cancer, bladder cancer, kidney
cancer, colon cancer, lung cancer, ovary cancer, breast cancer and
pancreatic cancer suggest that 193P1E1B is a potential therapeutic
target and a diagnostic marker for human cancers.
Example 5
Transcript Variants of 193P1E1B
[0695] 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.
[0696] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified by full-length cloning experiment,
or by use of full-length transcript and EST sequences. First, all
human ESTs were grouped into clusters which show direct or indirect
identity with each other. Second, ESTs in the same cluster were
further grouped into sub-clusters and assembled into a consensus
sequence. The original gene sequence is compared to the consensus
sequence(s) or other full-length sequences. Each consensus sequence
is a potential splice variant for that gene. Even when a variant is
identified that is not a full-length clone, that portion of the
variant is very useful for antigen generation and for further
cloning of the full-length splice variant, using techniques known
in the art.
[0697] Moreover, computer programs are available in the art that
identify transcript variants based on genomic sequences.
Genomic-based transcript variant identification programs include
FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in
Drosophila genomic DNA," Genome Research. 2000 April;
10(4):516-22); Grail and GenScan. 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.
[0698] 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).
[0699] 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 193P1E1B has a particular expression profile related to
cancer. Alternative transcripts and splice variants of 193P1E1B may
also be involved in cancers in the same or different tissues, thus
serving as tumor-associated markers/antigens.
[0700] Using the full-length gene and EST sequences, three
transcript variants were identified, designated as 193P1E1B v.7,
v.8 and v.9. Compared with 193P1E1B v.1, transcript variant
193P1E1B v.7 has spliced out exons 10 and 11 from variant 193P1E1B
v.1, as shown in FIG. 12. Variant 193P1E1B v.8 inserted 36 bp in
between 1931 and 1932 of variant 193P1E1B v.1 and variant 193P1E1B
v.9 replaced with 36 bp the segment 1136-1163 of variant 193P1E1B
v.1. Theoretically, each different combination of exons in spatial
order, e.g. exons 2 and 3, is a potential splice variant.
[0701] Tables LI through LXX are set forth on a variant-by-variant
bases. Tables LI, LV, LIX, LXIII, and LXVII show the nucleotide
sequence of the transcript variant. Tables LII, LVI, LX, LXIV, and
LXVIII show the alignment of the transcript variant with nucleic
acid sequence of 193P1E1B v.1. Tables LIB, LVII, LXI, LXV, and LXIX
show the amino acid translation of the transcript variant for the
identified reading frame orientation. Tables LIV, LVIII, LXII,
LXVI, and LXX display alignments of the amino acid sequence encoded
by the splice variant with that of 193P1E1B v.1.
Example 6
Single Nucleotide Polymorphisms of 193P1E1B
[0702] 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 cSNPs. These cSNPs may change amino acids of the protein
encoded by the gene and thus change the functions of the protein.
Some SNPs cause inherited diseases; others contribute to
quantitative variations in phenotype and reactions to environmental
factors including diet and drugs among individuals. Therefore, SNPs
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).
[0703] 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 are 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).
[0704] Using the methods described above, seven SNPs were
identified in the original transcript, 193P1E1B v.1, at positions
57 (A/G), 792 (C/G), 804 (C/A), 1253 (G/A), 1564 (A/G), 2268 (C/T)
and 2387 (C/T). The transcripts or proteins with alternative
alleles were designated as variants 193P1E1B v.2, v.3, v.4, v.5 and
v.6, respectively. FIG. 10 shows the schematic alignment of the SNP
variants. FIG. 11 shows the schematic alignment of protein
variants, corresponding to nucleotide variants. Nucleotide variants
that code for the same amino acid sequence as variant 1 are not
shown in FIG. 11. These alleles of the SNPs, though shown
separately here, can occur in different combinations (haplotypes)
and in any one of the transcript variants (such as 193P1E1B v.9)
that contains the sequence context of the SNPs.
Example 7
Production of Recombinant 193P1E1B in Prokaryotic Systems
[0705] To express recombinant 193P1E1B in prokaryotic cells, the
full or partial length 193P1E1B cDNA sequences can be cloned into
any one of a variety of expression vectors known in the art. One or
more of the following regions of 193P1E1B are expressed in these
constructs, amino acids 1 to 412 of variant 5 or variant 2; or
amino acids 1 to 388 of variant 10, 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 193P1E1B, variants, or analogs
thereof. In certain embodiments a region of 193P1E1B is expressed
that encodes an amino acid not shared amongst at least
variants.
[0706] A. In Vitro Transcription and Translation Constructs:
[0707] pCR11: To generate 193P1E1B sense and anti-sense RNA probes
for RNA in situ investigations, pCR11 constructs (Invitrogen,
Carlsbad Calif.) are generated encoding either all or fragments of
a 193P1E1B cDNA. The pCR11 vector has Sp6 and T7 promoters flanking
the insert to drive the transcription of 193P1E1B RNA for use as
probes in RNA in situ hybridization experiments. These probes are
used to analyze the cell and tissue expression of 193P1E1B at the
RNA level. Transcribed 193P1E1B RNA representing the cDNA amino
acid coding region of the 193P1E1B gene is used in in vitro
translation systems such as the TnT.TM. Coupled Reticulolysate
System (Promega, Corp., Madison, Wis.) to synthesize 193P1E1B
protein.
[0708] B. Bacterial Constructs:
[0709] pGEX Constructs: To generate recombinant 193P1E1B proteins
in bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of a 193P1E1B cDNA protein coding sequence
are fused to the GST gene by cloning into pGEX-6P-1 or any other
GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech,
Piscataway, N.J.). These constructs allow controlled expression of
recombinant 193P1E1B 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 193P1E1B-related protein. The ampicillin resistance gene
and pBR322 origin permits selection and maintenance of the pGEX
plasmids in E. coli.
[0710] pMAL Constructs: To generate, in bacteria, recombinant
193P1E1B proteins that are fused to maltose-binding protein (MBP),
all or parts of a 193P1E1B cDNA protein coding sequence are fused
to the MBP gene by cloning into the pMAL-c2.times. and
pMAL-p2.times. vectors (New England Biolabs, Beverly, Mass.). These
constructs allow controlled expression of recombinant 193P1E1B
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 193P1E1B. The
pMAL-c2.times. and pMAL-p2.times. vectors are optimized to express
the recombinant protein in the cytoplasm or periplasm respectively.
Periplasm expression enhances folding of proteins with disulfide
bonds.
[0711] pET Constructs: To express 193P1E1B in bacterial cells, all
or parts of a 193P1E1B cDNA protein coding sequence are cloned into
the pET family of vectors (Novagen, Madison, Wis.). These vectors
allow tightly controlled expression of recombinant 193P1E1B 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 a
193P1E1B protein are expressed as amino-terminal fusions to
NusA.
[0712] C. Yeast Constructs:
[0713] pESC Constructs: To express 193P1E1B in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of a 193P1E1B 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 193P1E1B. In addition, expression
in yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations, that are found when expressed
in eukaryotic cells.
[0714] pESP Constructs: To express 193P1E1B in the yeast species
Saccharomyces pombe, all or parts of a 193P1E1B cDNA protein coding
sequence are cloned into the pESP family of vectors. These vectors
allow controlled high level of expression of a 193P1E1B 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 193P1E1B in Higher Eukaryotic Systems
[0715] A. Mammalian Constructs:
[0716] To express recombinant 193P1E1B in eukaryotic cells, the
full or partial length 193P1E1B cDNA sequences can be cloned into
any one of a variety of expression vectors known in the art. One or
more of the following regions of 193P1E1B are expressed in these
constructs, amino acids 1 to 412; 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 193P1E1B, variants, or analogs
thereof.
[0717] 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-193P1E1B polyclonal serum,
described herein.
[0718] pcDNA4/HisMax Constructs: To express 193P1E1B in mammalian
cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B 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.
[0719] pcDNA3.1/MycHis Constructs: To express 193P1E1B in mammalian
cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B with a
consensus Kozak translation initiation site are 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.
[0720] pcDNA3.1/CT-GFP-TOPO Construct: To express 193P1E1B in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, the 193P1E1B ORF, or portions thereof, of
193P1E1B 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 the
193P1E1B proteins.
[0721] PAPtag: The 193P1E1B ORF, or portions thereof, of 193P1E1B
are cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This
construct generates an alkaline phosphatase fusion at the
carboxyl-terminus of the 193P1E1B proteins 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
193P1E1B proteins. The resulting recombinant 193P1E1B 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 the 193P1E1B 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.
[0722] ptag5: The 193P1E1B ORF, or portions thereof, of 193P1E1B
are cloned into pTag-5. This vector is similar to pAPtag but
without the alkaline phosphatase fusion. This construct generates
193P1E1B 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 193P1E1B 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 193P1E1B 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.
[0723] PsecFc: The 193P1E1B ORF, or portions thereof, of 193P1E1B
are 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
193P1E1B proteins, while fusing the IgGK signal sequence to
N-terminus 193P1E1B fusions utilizing the murine IgG1 Fc region are
also used. The resulting recombinant 193P1E1B 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 the 193P1E1B 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.
[0724] pSR.alpha. Constructs: To generate mammalian cell lines that
express 193P1E1B constitutively, 193P1E1B ORF, or portions thereof,
of 193P1E1B are cloned into pSR.alpha. constructs. Amphotropic and
ecotropic retroviruses are generated by transfection of pSR.alpha.
constructs into the 293T-10A1 packaging line or co-transfection of
pSR.alpha. and a helper plasmid (containing deleted packaging
sequences) into the 293 cells, respectively. The retrovirus is used
to infect a variety of mammalian cell lines, resulting in the
integration of the cloned gene, 193P1E1B, into the host cell-lines.
Protein expression is driven from a long terminal repeat (LTR). The
Neomycin resistance gene present in the vector allows for selection
of mammalian cells that express the protein, and the ampicillin
resistance gene and ColE1 origin permit selection and maintenance
of the plasmid in E. coli. The retroviral vectors can thereafter be
used for infection and generation of various cell lines using, for
example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
[0725] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
193P1E1B sequences to allow detection using anti-Flag antibodies.
For example, the FLAG.TM. sequence 5' gat tac aag gat gac gac gat
aag 3' (SEQ ID NO:62) is added to cloning primer at the 3' end of
the ORF. Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length 193P1E1B proteins.
[0726] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 193P1E1B. High virus
titer leading to high level expression of 193P1E1B is achieved in
viral delivery systems such as adenoviral vectors and herpes
amplicon vectors. The 193P1E1B 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, 193P1E1B 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.
[0727] Regulated Expression Systems: To control expression of
193P1E1B in mammalian cells, coding sequences of 193P1E1B, 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 193P1E1B. These
vectors are thereafter used to control expression of 193P1E1B in
various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0728] B. Baculovirus Expression Systems
[0729] To generate recombinant 193P1E1B proteins in a baculovirus
expression system, 193P1E1B 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-193P1E1B 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.
[0730] Recombinant 193P1E1B protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 193P1E1B protein can be detected using anti-193P1E1B or
anti-His-tag antibody. 193P1E1B protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 193P1E1B.
Example 9
Antigenicity Profiles and Secondary Structure
[0731] FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict
graphically five amino acid profiles of the 193P1E1B amino acid
sequence (variant 1), each assessment is available by accessing the
ProtScale website on the ExPasy molecular biology server.
[0732] 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 193P1E1B protein. Each of the above amino acid profiles
of 193P1E1B 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.
[0733] 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 profiles, 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.
[0734] 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.
[0735] Antigenic sequences of the full length 193P1E1B protein
(variant 1) 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-193P1E1B 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 193P1E1B protein. In particular, peptide
immunogens of the invention can comprise, a peptide region of at
least 5 amino acids of FIG. 2 in any whole number increment up to
412 that includes an amino acid position having a value greater
than 0.5 in the Hydrophilicity profile of FIG. 5; a peptide region
of at least 5 amino acids of FIG. 2 in any whole number increment
up to 412 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 FIG. 2 in any whole number increment
up to 412 that includes an amino acid position having a value
greater than 0.5 in the Percent Accessible Residues profile of FIG.
7; a peptide region of at least 5 amino acids of FIG. 2 in any
whole number increment up to 412 that includes an amino acid
position having a value greater than 0.5 in the Average Flexibility
profile on FIG. 8; and, a peptide region of at least 5 amino acids
of FIG. 2 in any whole number increment up to 412 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.
[0736] 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.
[0737] The secondary structure of 193P1E1B, namely the predicted
presence and location of alpha helices, extended strands, and
random coils, is predicted from the primary amino acid sequence of
193P1E1B variant 1 using the HNN--Hierarchical Neural Network
method, accessed from the ExPasy molecular biology server. The
analysis indicates that 193P1E1B is composed 29.13% alpha helix,
9.95% extended strand, and 60.92% random coil (FIG. 13).
[0738] Analysis of 193P1E1B using a variety of transmembrane
prediction algorithms accessed from the ExPasy molecular biology
server did not predict the presence of such domains, confirming
that 193P1E1B is a soluble protein.
Example 10
Generation of 193P1E1B Polyclonal Antibodies
[0739] 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 the full
length 193P1E1B protein, 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 Example 9 entitled
"Antigenicity Profiles and Secondary Structure"). Such regions
would be predicted to be hydrophilic, flexible, in beta-turn
conformations, and be exposed on the surface of the protein (see,
e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid
profiles that indicate such regions of 193P1E1B).
[0740] For example, 193P1E1B recombinant bacterial fusion proteins
or peptides containing hydrophilic, flexible, beta-turn regions of
193P1E1B are used as antigens to generate polyclonal antibodies in
New Zealand White rabbits. For example, such regions include, but
are not limited to, amino acids 20-43, amino acids 100-164, amino
acids 241-261, or amino acids 310-331. 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 241-261 of
193P1E1B is conjugated to KLH and used to immunize the rabbit.
Alternatively the immunizing agent may include all or portions of a
193P 1E1B protein, analogs or fusion proteins thereof. For example,
a 193P1E1B 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. Such fusion proteins are purified
from induced bacteria using the appropriate affinity matrix.
[0741] In one embodiment, a GST-fusion protein containing an entire
193P1E1B coding sequence is produced and purified and used as
immunogen. Other recombinant bacterial fusion proteins that can be
employed include maltose binding protein, LacZ, thioredoxin, NusA,
or an immunoglobulin constant region (see the section entitled
"Production of 193P1E1B 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., Limes, M.,
Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med.
174, 561-566).
[0742] 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 Example 8 entitled "Production of
Recombinant 193P1E1B in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, an entire 193P1E1B coding
sequence is cloned into the Tag5 mammalian secretion vector. The
recombinant protein is purified by metal chelate chromatography
from tissue culture supernatants of 293T cells stably expressing
the recombinant vector. The purified Tag5 193P1E1B protein is then
used as immunogen.
[0743] 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).
[0744] 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.
[0745] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with Tag5 193P1E1B
protein or KLH-coupled peptide encoding amino acids 241-261, the
full-length 193P1E1B cDNA is cloned into pCDNA 3.1 myc-his
expression vector (Invitrogen, see the Example entitled "Production
of Recombinant 193P1E1B in Eukaryotic Systems"). After transfection
of the constructs into 293T cells, cell lysates are probed with the
anti-193P1E1B serum and with anti-His antibody (Santa Cruz
Biotechnologies, Santa Cruz, Calif.) to determine specific
reactivity to denatured 193P1E1B protein using the Western blot
technique. Immunoprecipitation and flow cytometric analyses of 293T
and other recombinant 193P1E1B-expressing cells determine
recognition of native protein by the antiserum. In addition,
Western blot, immunoprecipitation, fluorescent microscopy, and flow
cytometric techniques using cells that endogenously express
193P1E1B are carried out to test specificity.
[0746] The anti-serum from the Tag5 193P1E1B immunized rabbit is
affinity purified by passage over a column composed of the Tag5
antigen covalently coupled to Affigel matrix (BioRad, Hercules,
Calif.). The serum is then further purified by protein G affinity
chromatography to isolate the IgG fraction. Serum from rabbits
immunized with 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. 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 193P1E1B Monoclonal Antibodies (mAbs)
[0747] In one embodiment, therapeutic mAbs to 193P1E1B comprise
those that react with epitopes of the protein that would disrupt or
modulate the biological function of 193P1E1B, for example those
that would disrupt its interaction with ligands, proteins, or
substrates that mediate its biological activity. Immunogens for
generation of such mAbs include those designed to encode or contain
an entire 193P1E1B protein or its variants or regions of a193P1E1B
protein 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 Example 9 entitled "Antigenicity Profiles and Secondary
Structure"). Immunogens include peptides, recombinant bacterial
proteins, and mammalian expressed Tag 5 proteins and human and
murine IgG FC fusion proteins. In addition, cells expressing high
levels of 193P1E1B, such as 293T-193P1E1B or 300.19-193P1E1B murine
Pre-B cells, are used to immunize mice.
[0748] To generate mAbs to 193P1E1B, mice are first immunized
intraperitoneally (IP) with, typically, 10-50 .mu.g of protein
immunogen or 10.sup.7 193P1E1B-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 193P1E1B sequence is used to
immunize mice by direct injection of the plasmid DNA. For example,
an entire coding sequence of 193P1E1B, e.g., amino acids 1-412 of
193P1E1B variant 1, is cloned into the Tag5 mammalian secretion
vector and the recombinant vector is used as immunogen. In another
example the amino acids are cloned into an Fc-fusion secretion
vector in which a 193P1E1B 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
193P1E1B.
[0749] 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).
[0750] In one embodiment, monoclonal antibodies are derived that
distinguish between, e.g., the various 193P1E1B variants, e.g., the
amino terminal truncated splice variant 3, encoding amino acids
83-412 and the full length protein encoding amino acids 1-412. In
one method, two different Fc-fusion proteins are derived, one
encoding amino acids 1-82, and the other encoding amino acids
83-412. These are expressed and purified from stably transfected
293T cells. Balb C mice are initially immunized intraperitoneally
with 25 .mu.g of the Tag5-193P1E1B 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 Tag5 antigen
determines the titer of serum from immunized mice. Reactivity and
specificity of serum to the full length 193P1E1B protein and to
amino terminal truncated variant 3 is monitored by Western
blotting, immunoprecipitation and flow cytometry using 293T cells
transfected with an expression vector encoding each of the
respective 193P1E1B cDNAs (see e.g., the Example entitled
"Production of Recombinant 193P1E1B in Eukaryotic Systems"). Other
recombinant 193P1E1B-expressing cells or cells endogenously
expressing 193P1E1B 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 (see, e.g., Harlow and Lane, 1988).
Supernatants from HAT selected growth wells are screened by ELISA,
Western blot, immunoprecipitation, fluorescent microscopy, and flow
cytometry to identify 193P1E1B specific antibody-producing
clones.
[0751] The binding affinity of a 193P1E1B monoclonal antibody is
determined using standard technologies. Affinity measurements
quantify the strength of antibody to epitope binding and are used
to help define which 193P1E1B monoclonal antibodies preferred,
e.g., 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
[0752] 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.
[0753] 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.
[0754] 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
[0755] 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.
[0756] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0757] 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 193P1E1B set forth in FIGS. 2 and 3, the
specific search peptides used to generate the tables are listed in
Table VII.
[0758] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
193P1E1B 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.
[0759] 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
[0760] where a.sub.ji is a coefficient which represents the effect
of the presence of a given amino acid (j) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial
assumption of this method is that the effects at each position are
essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at position i in the
peptide, it is assumed to contribute a constant amount j.sub.i to
the free energy of binding of the peptide irrespective of the
sequence of the rest of the peptide.
[0761] 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.
[0762] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0763] Protein sequences from 193P1E1B 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).
[0764] 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.
[0765] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0766] The 193P1E1B 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.
[0767] Selection of HLA-B7 Supermotif Bearing Epitopes
[0768] The 193P1E1B 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 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.
[0769] Selection of A1 and A24 Motif-Bearing Epitopes
[0770] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 193P1E1B protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0771] 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
[0772] 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:
[0773] Target Cell Lines for Cellular Screening:
[0774] 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.
[0775] Primary CTL Induction Cultures:
[0776] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI
with 30 .mu.g/ml DNAse, washed twice and resuspended in complete
medium (RPMI-1640 plus 5% AB human serum, non-essential amino
acids, sodium pyruvate, L-glutamine and penicillin/streptomycin).
The monocytes are purified by plating 10.times.10.sup.6 PBMC/well
in a 6-well plate. After 2 hours at 37.degree. C., the non-adherent
cells are removed by gently shaking the plates and aspirating the
supernatants. The wells are washed a total of three times with 3 ml
RPMI to remove most of the non-adherent and loosely adherent cells.
Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000
U/ml of IL-4 are then added to each well. TNF.alpha. is added to
the DCs on day 6 at 75 ng/ml and the cells are used for CTL
induction cultures on day 7.
[0777] Induction of CTL with DC and Peptide: 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 (140 .mu.l
beads/20.times.10.sup.6 cells) and incubated for 1 hour at
4.degree. C. with continuous mixing. The beads and cells are washed
4.times. with PBS/AB serum to remove the nonadherent cells and
resuspended at 100.times.10.sup.6 cells/ml (based on the original
cell number) in PBS/AB serum containing 100 .mu.l/ml
Detacha-Bead.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.
[0778] Setting up induction cultures: 0.25 ml cytokine-generated DC
(at 1.times.10.sup.5 cells/ml) are co-cultured with 0.25 ml of CD8+
T-cells (at 2.times.10.sup.6 cell/ml) in each well of a 48-well
plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10
is added the next day at a final concentration of 10 ng/ml and
rhuman IL-2 is added 48 hours later at 10 IU/ml.
[0779] Restimulation of the induction cultures with peptide-pulsed
adherent cells: Seven and fourteen days after the primary
induction, the cells are restimulated with peptide-pulsed adherent
cells. The PBMCs are thawed and washed twice with RPMI and DNAse.
The cells are resuspended at 5.times.10.sup.6 cells/ml and
irradiated at .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.
[0780] Measurement of CTL Lytic Activity by .sup.51Cr Release.
[0781] 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.
[0782] Adherent target cells are removed from culture flasks with
trypsin-EDTA. Target cells are labeled with 200 .mu.Ci of .sup.51Cr
sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37.degree.
C. Labeled target cells are resuspended at 10.sup.6 per ml and
diluted 1:10 with K562 cells at a concentration of
3.3.times.10.sup.6/ml (an NK-sensitive erythroblastoma cell line
used to reduce non-specific lysis). Target cells (100 .mu.l) and
effectors (100 .mu.l) are plated in 96 well round-bottom plates and
incubated for 5 hours at 37.degree. C. At that time, 100 .mu.l of
supernatant are collected from each well and percent lysis is
determined according to the formula:
[(cpm of the test sample-cpm of the spontaneous .sup.51Cr release
sample)/(cpm of the maximal .sup.51Cr release sample-cpm of the
spontaneous .sup.51Cr release sample)].times.100.
[0783] 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.
[0784] In Situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-Specific and Endogenous Recognition
[0785] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1 M 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.
[0786] Recombinant human IFN-gamma is added to the standard wells
starting at 400 pg or 1200 pg/100 microliter/well and the plate
incubated for two hours at 37.degree. C. The plates are washed and
100 .mu.l of biotinylated mouse anti-human IFN-gamma monoclonal
antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added
and incubated for 2 hours at room temperature. After washing again,
100 microliter HRP-streptavidin (1:4000) are added and the plates
incubated for one hour at room temperature. The plates are then
washed 6.times. with wash buffer, 100 microliter/well developing
solution (TMB 1:1) are added, and the plates allowed to develop for
5-15 minutes. The reaction is stopped with 50 microliter/well 1M
H.sub.3PO.sub.4 and read at OD450. A culture is considered positive
if it measured at least 50 pg of IFN-gamma/well above background
and is twice the background level of expression.
[0787] CTL Expansion.
[0788] 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.
[0789] 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.
[0790] Immunogenicity of A2 Supermotif-Bearing Peptides
[0791] 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.
[0792] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 193P1E1B. 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.
[0793] Evaluation of A*03/A11 Immunogenicity
[0794] 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.
[0795] Evaluation of B7 Immunogenicity
[0796] 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.
[0797] 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
[0798] 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.
[0799] Analoging at Primary Anchor Residues
[0800] 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
[0801] 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.
[0802] 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.
[0803] 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).
[0804] 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.
[0805] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0806] 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.
[0807] 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.
[0808] 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).
[0809] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0810] 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.
[0811] Analoging at Secondary Anchor Residues
[0812] 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.
[0813] 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 193P1E1B-expressing tumors.
[0814] Other Analoging Strategies
[0815] 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).
[0816] 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 193P1E1B-Derived Sequences with
HLA-DR Binding Motifs
[0817] 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.
[0818] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0819] To identify 193P1E1B-derived, HLA class II HTL epitopes, a
193P1E1B 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).
[0820] 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.
[0821] The 193P1E1B-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. 193P1E1B-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0822] Selection of DR3 Motif Peptides
[0823] 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.
[0824] To efficiently identify peptides that bind DR3, target
193P1E1B 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.
[0825] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0826] 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 193P1E1B-Derived HTL Epitopes
[0827] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0828] 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 193P1E1B-expressing
tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0829] 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.
[0830] 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].
[0831] 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).
[0832] 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.
[0833] 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.
[0834] 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
[0835] 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.
[0836] 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 193P1E1B
expression vectors.
[0837] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
193P1E1B 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
[0838] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 193P1E1B-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 193P1E1B-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.
[0839] Immunization procedures: Immunization of transgenic mice is
performed as described (Alexander et al., J. Immunol.
159:4753-4761, 1997). For example, A2/K.sup.b mice, which are
transgenic for the human HLA A2.1 allele and are used to confirm
the immunogenicity of HLA-A*0201 motif- or HLA-A2
supermotif-bearing epitopes, and are primed subcutaneously (base of
the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant,
or if the peptide composition is a lipidated 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.
[0840] Cell lines: Target cells for peptide-specific cytotoxicity
assays are Jurkat cells transfected with the HLA-A2.1/K.sup.b
chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007,
1991)
[0841] In vitro CTL activation: One week after priming, spleen
cells (30.times.10.sup.6 cells/flask) are co-cultured at 37.degree.
C. with 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.
[0842] Assay for cytotoxic activity: 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.
[0843] 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
193P1E1B-Specific Vaccine
[0844] 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.
[0845] 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.
[0846] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 193P1E1B clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 193P1E1B. For example, if it has been observed
that patients who spontaneously clear 193P1E1B-expressing cells
generate an immune response to at least three (3) epitopes from
193P1E1B antigen, then at least three epitopes should be included
for HLA class I. A similar rationale is used to determine HLA class
II epitopes.
[0847] 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.
[0848] 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.
[0849] 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 193P1E1B, 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.
[0850] 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 193P1E1B.
Example 22
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0851] 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.
[0852] 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 193P1E1B, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 193P1E1B 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.
[0853] 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.
[0854] 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.
[0855] 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.
[0856] 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
[0857] 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
[0858] 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).
[0859] 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.
[0860] 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.
[0861] 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.
[0862] 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.
[0863] 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.
[0864] 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).
[0865] 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.
[0866] 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
[0867] Vaccine compositions of the present invention can be used to
prevent 193P1E1B 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
193P1E1B-associated tumor.
[0868] 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 193P1E1B-associated disease.
[0869] 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 193P1E1B
Sequences
[0870] A native 193P1E1B 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.
[0871] The vaccine composition will include, for example, multiple
CTL epitopes from 193P1E1B 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.
[0872] 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 193P1E1B,
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.
[0873] 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
[0874] The 193P1E1B 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
193P1E1B and such other antigens. For example, a vaccine
composition can be provided as a single polypeptide that
incorporates multiple epitopes from 193P1E1B as well as
tumor-associated antigens that are often expressed with a target
cancer associated with 193P1E1B 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
[0875] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 193P1E1B. 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.
[0876] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 193P1E1B HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising a 193P1E1B
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.
[0877] 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 193P1E1B epitope, and thus the
status of exposure to 193P1E1B, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
[0878] 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 193P1E1B-associated disease or who have been
vaccinated with a 193P1E1B vaccine.
[0879] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
193P1E1B 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.
[0880] 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.
[0881] 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).
[0882] 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).
[0883] 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.51 Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0884] 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.
[0885] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 193P1E1B or a 193P1E1B vaccine.
[0886] 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 193P1E1B
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
[0887] 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:
[0888] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0889] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0890] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0891] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0892] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0893] 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.
[0894] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0895] 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.
[0896] The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 193P1E1B
[0897] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 193P1E1B. The main objectives of the trial
are to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 193P1E1B, 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:
[0898] 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.
[0899] 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 193P1E1B.
[0900] 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 193P1E1B-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
[0901] 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.
[0902] 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.
[0903] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 193P1E1B is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0904] 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
193P1E1B protein from which the epitopes in the vaccine are
derived.
[0905] 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.
[0906] 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.
[0907] 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.
[0908] Ex Vivo Activation of CTL/HTL Responses
[0909] Alternatively, ex vivo CTL or HTL responses to 193P1E1B
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
[0910] 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. 193P1E1B.
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.
[0911] 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
193P1E1B to isolate peptides corresponding to 193P1E1B 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.
[0912] 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
[0913] Sequences complementary to the 193P1E1B-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 193P1E1B. 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 193P1E1B. 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 193P1E1B-encoding
transcript.
Example 35
Purification of Naturally-Occurring or Recombinant 193P1E1B Using
193P1E1B-Specific Antibodies
[0914] Naturally occurring or recombinant 193P1E1B is substantially
purified by immunoaffinity chromatography using antibodies specific
for 193P1E1B. An immunoaffinity column is constructed by covalently
coupling anti-193P1E1B 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.
[0915] Media containing 193P1E1B are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 193P1E1B (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/193P1E1B 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 193P1E1B
[0916] 193P1E1B, or biologically active fragments thereof, are
labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
193P1E1B, washed, and any wells with labeled 193P1E1B complex are
assayed. Data obtained using different concentrations of 193P1E1B
are used to calculate values for the number, affinity, and
association of 193P1E1B with the candidate molecules.
Example 37
In Vivo Assay for 193P1E1B Tumor Growth Promotion
[0917] The effect of a 193P1E1B protein on tumor cell growth can be
confirmed in vivo by gene overexpression in a variety of cancer
cells such as those in Table I. For example, SCID mice can be
injected SQ on each flank with 1.times.10.sup.6 prostate, kidney,
colon or bladder cancer cells (such as PC3, LNCaP, SCaBER, UM-UC-3,
HT1376, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839 cells)
containing tkNeo empty vector or 193P1E1B.
[0918] At least two strategies can be used:
[0919] (1) Constitutive 193P1E1B 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.
[0920] (2) Regulated expression under control of an inducible
vector system, such as ecdysone, tet, etc., can be used provided
such promoters are compatible with the host cell systems. Tumor
volume is then monitored at the appearance of palpable tumors or by
following serum markers such as PSA. Tumor development is followed
over time to validate that 193P1E1B-expressing cells grow at a
faster rate and/or that tumors produced by 193P1E1B-expressing
cells demonstrate characteristics of altered aggressiveness (e.g.,
enhanced metastasis, vascularization, reduced responsiveness to
chemotherapeutic drugs). Tumor volume is evaluated by caliper
measurements. Additionally, mice can be implanted with the same
cells orthotopically in the prostate, bladder, colon or kidney to
determine if 193P1E1B has an effect on local growth, e.g., in the
prostate, bladder, colon or kidney or on the ability of the cells
to metastasize, specifically to lungs or lymph nodes (Saffran et
al., Proc Natl Acad Sci USA. 2001, 98: 2658; Fu, X., et al., Int.
J. Cancer, 1991. 49: 938-939; Chang, S., et al., Anticancer Res.,
1997, 17: 3239-3242; Peralta, E. A., et al., J. Urol., 1999. 162:
1806-1811). For instance, the orthotopic growth of PC3 and
PC3-193P1E1B can be compared in the prostate of SCID mice. Such
experiments reveal the effect of 193P1E1B on orthotopic tumor
growth, metastasis and/or angiogenic potential.
[0921] Furthermore, this assay is useful to confirm the inhibitory
effect of candidate therapeutic compositions, such as 193P1E1B
antibodies or intrabodies, and 193P1E1B antisense molecules or
ribozymes, or 193P1E1B directed small molecules, on cells that
express a 193P1E1B protein.
Example 38
193P1E1B Monoclonal Antibody-mediated Inhibition of Prostate Tumors
In Vivo
[0922] The significant expression of 193P1E1B, in cancer tissues,
together with its restricted expression in normal tissues makes
193P1E1B an excellent target for antibody therapy. Similarly,
193P1E1B is a target for T cell-based immunotherapy. Thus, the
therapeutic efficacy of anti-193P1E1B mAbs is evaluated, e.g., in
human prostate cancer xenograft mouse models using
androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et
al. Cancer Res, 1999. 59(19): p. 5030-5036), kidney cancer
xenografts (AGS-K3, AGS-K6), kidney cancer metastases to lymph node
(AGS-K6 met) xenografts, and kidney cancer cell lines transfected
with 193P1E1B, such as 769P-193P1E1B, A498-193P1E1B.
[0923] Antibody efficacy on tumor growth and metastasis formation
is studied, e.g., in mouse orthotopic prostate cancer xenograft
models and mouse kidney 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-193P1E1B
mAbs inhibit formation of both the androgen-dependent LAPC-9 and
androgen-independent PC3-193P1E1B tumor xenografts. Anti-193P1E1B
mAbs also retard the growth of established orthotopic tumors and
prolonged survival of tumor-bearing mice. These results indicate
the utility of anti-193P1E1B mAbs in the treatment of local and
advanced stages of, e.g., prostate cancer. (See, e.g., Saffran, D.,
et al., PNAS 10:1073-1078.) Similarly, anti-193P1E1B mAbs inhibit
formation of AGS-K3 and AGS-K6 tumors in SCID mice, and prevent or
retard the growth A498-193P1E1B tumor xenografts. These results
indicate the use of anti-193P1E1B mAbs in the treatment of prostate
and/or kidney cancer.
[0924] Administration of the anti-193P1E1B mAbs leads 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 193P1E1B is an attractive target for immunotherapy
and demonstrate the therapeutic use of anti-193P1E1B mAbs for the
treatment of local and metastatic cancer. This example demonstrates
that unconjugated 193P1E1B monoclonal antibodies are effective to
inhibit the growth of human prostate tumor xenografts and human
kidney xenografts grown in SCID mice.
[0925] Tumor Inhibition Using Multiple Unconjugated 193P1E1B
mAbs
Materials and Methods
[0926] 193P1E1B Monoclonal Antibodies:
[0927] Monoclonal antibodies are obtained against 193P1E1B, as
described in Example 11 entitled: Generation of 193P1E1B Monoclonal
Antibodies (mAbs), or may be obtained commercially. The antibodies
are characterized by ELISA, Western blot, FACS, and
immunoprecipitation for their capacity to bind 193P1E1B. Epitope
mapping data for the anti-193P1E1B mAbs, as determined by ELISA and
Western analysis, recognize epitopes on a 193P1E1B protein
Immunohistochemical analysis of cancer tissues and cells is
performed with these antibodies.
[0928] 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, e.g., LAPC-9 prostate tumor xenografts.
[0929] Cancer Xenografts and Cell Lines
[0930] 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 subcutaneous (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. The prostate carcinoma cell
line PC3 (American Type Culture Collection) is maintained in RPMI
supplemented with L-glutamine and 10% FBS, and the kidney carcinoma
line A498 (American Type Culture Collection) is maintained in DMEM
supplemented with L-glutamine and 10% FBS.
[0931] PC3-193P1E1B and A498-193P1E1B cell populations are
generated by retroviral gene transfer as described in Hubert, R.
S., et al., STEAP: A Prostate-specific Cell-surface Antigen Highly
Expressed in Human Prostate Tumors, Proc Natl. Acad. Sci. USA,
1999. 96(25): p. 14523-14528. Anti-193P1E1B staining is detected by
using, e.g., an FITC-conjugated goat anti-mouse antibody (Southern
Biotechnology Associates) followed by analysis on a Coulter
Epics-XL f low cytometer.
[0932] Xenograft Mouse Models.
[0933] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10.sup.6 LAPC-9, AGS-K3, AGS-K6, PC3, PC3-193P1E1B, A498 or
A498-193P1E1B cells mixed at a 1:1 dilution with Matrigel
(Collaborative Research) in the right flank of male SCID mice. To
test antibody efficacy on tumor formation, i.p. antibody injections
are started on the same day as tumor-cell injections. As a control,
mice are injected with either purified mouse IgG (ICN) or PBS; or a
purified monoclonal antibody that recognizes an irrelevant antigen
not expressed in human cells. In preliminary studies, no difference
is found between mouse IgG or PBS on tumor growth. Tumor sizes are
determined by vernier caliper measurements, and the tumor volume is
calculated as length.times.width.times.height. Mice with s.c.
tumors greater than 1.5 cm in diameter are sacrificed. PSA levels
are determined by using a PSA ELISA kit (Anogen, Mississauga,
Ontario). Circulating levels of anti-193P1E1B mAbs are determined
by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.).
(See, e.g., (Saffran, D., et al., PNAS 10:1073-1078).
[0934] Orthotopic prostate 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.10.sup.5) 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 kidney
orthotopic models, an incision is made through the abdominal
muscles to expose the kidney. AGS-K3 or AGS-K6 cells mixed with
Matrigel are injected under the kidney capsule. The mice are
segregated into groups for appropriate treatments, with
anti-193P1E1B or control mAbs being injected i.p.
[0935] Anti-193P1E1B mAbs Inhibit Growth of 193P1E1B-Expressing
Xenograft-Cancer Tumors
[0936] The effect of anti-193P1E1B mAbs on tumor formation is
tested by using, e.g., LAPC-9 and/or AGS-K3 orthotopic models. As
compared with the s.c. tumor model, the orthotopic model, which
requires injection of tumor cells directly in the mouse prostate or
kidney, respectively, 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; Fu, X., et al.,
Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem,
1994. 56(1): p. 4-8). The features make the orthotopic model more
representative of human disease progression and allow for tracking
of the therapeutic effect of mAbs on clinically relevant end
points.
[0937] Accordingly, tumor cells are injected into the mouse
prostate or kidney, and the mice are segregated into two groups and
treated with either: a) 200-500 .mu.g, of anti-193P1E1B Ab, or b)
PBS for two to five weeks.
[0938] As noted, a major advantage of the orthotopic
prostate-cancer model is the ability to study the development of
metastases. Formation of metastasis in mice bearing established
orthotopic tumors is studied by IHC analysis on lung sections using
an antibody against a prostate-specific cell-surface protein STEAP
expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et
al., Proc Natl. Acad. Sci. USA, 1999. 96(25): p. 14523-14528) or
anti-G250 antibody for kidney cancer models. G250 is a clinically
relevant marker for renal clear cell carcinoma, which is
selectively expressed on tumor but not normal kidney cells
(Grabmaier K et al, Int J. Cancer. 2000, 85: 865).
[0939] Mice bearing established orthotopic LAPC-9 tumors are
administered 500-1000 .mu.g injections of either anti-193P1E1B mAb
or PBS over a 4-week period. Mice in both groups are allowed to
establish a high tumor burden (PSA levels greater than 300 ng/ml),
to ensure a high frequency of metastasis formation in mouse lungs.
Mice then are killed and their prostate/kidney and lungs are
analyzed for the presence of tumor cells by IHC analysis.
[0940] These studies demonstrate a broad anti-tumor efficacy of
anti-193P1E1B antibodies on initiation and/or progression of
prostate and kidney cancer in xenograft mouse models. Anti-193P1E1B
antibodies inhibit tumor formation of both androgen-dependent and
androgen-independent prostate tumors as well as retarding the
growth of already established tumors and prolong the survival of
treated mice. Moreover, anti-193P1E1B 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. Similar
therapeutic effects are seen in the kidney cancer model. Thus,
anti-193P1E1B mAbs are efficacious on major clinically relevant end
points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-193P1E1B Antibodies in
Humans
[0941] Anti-193P1E1B 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-193P1E1B mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 193P1E1B in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-193P1E1B antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0942] As determined by flow cytometry, anti-193P1E1B mAb
specifically binds to carcinoma cells. Thus, anti-193P1E1B
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 193P1E1B. Shedding or release of an extracellular
domain of 193P1E1B into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 193P1E1B by
anti-193P1E1B antibodies in serum and/or urine samples from suspect
patients.
[0943] Anti-193P1E1B antibodies that specifically bind 193P1E1B are
used in therapeutic applications for the treatment of cancers that
express 193P1E1B. Anti-193P1E1B 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-193P1E1B 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 "193P1E1B Monoclonal Antibody-mediated
Inhibition of Bladder and Lung Tumors In Vivo"). Either conjugated
and unconjugated anti-193P1E1B 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-193P1E1B Antibodies In
Vivo
[0944] Antibodies are used in accordance with the present invention
which recognize an epitope on 193P1E1B, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 193P1E1B 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.
[0945] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-193P1E1B 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-193P1E1B
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-193P1E1B 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).
[0946] II.) Monotherapy: In connection with the use of the
anti-193P1E1B 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.
[0947] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-193P1E1B
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 193P1E1B. In connection with the use of
the anti-193P1E1B antibodies as imaging agents, the antibodies are
used as an adjunct to surgical treatment of solid tumors, as both a
pre-surgical screen as well as a post-operative follow-up to
determine what tumor remains and/or returns. In one embodiment, a
(.sup.111In) 193P1E1B antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses 193P1E1B (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
[0948] Dose and Route of Administration
[0949] 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-193P1E1B
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-193P1E1B 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-193P1E1B antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-193P1E1B 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.
[0950] Three distinct delivery approaches are useful for delivery
of anti-193P1E1B 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.
[0951] Clinical Development Plan (CDP)
[0952] Overview: The CDP follows and develops treatments of
anti-193P1E1B 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-193P1E1B antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
193P1E1B expression levels in their tumors as determined by
biopsy.
[0953] 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 193P1E1B. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-193P1E1B antibodies are
found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-193P1E1B
Antibody and Chemotherapeutic Agent
[0954] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-193P1E1B 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-193P1E1B 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-193P1E1B
antibody with dosage of antibody escalating from approximately
about 25 mg/m.sup.2 to about 275 mg/m.sup.2 over the course of the
treatment in accordance with the following schedule:
TABLE-US-00003 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25
75 125 175 225 275 mg/ mg/ mg/ mg/ mg/ mg/ m.sup.2 m.sup.2 m.sup.2
m.sup.2 m.sup.2 m.sup.2 Chemotherapy + + + + + + (standard
dose)
[0955] 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 193P1E1B. 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.
[0956] The anti-193P1E1B 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-193P1E1B
Antibody
[0957] Anti-193P1E1B 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-193P1E1B
antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-193P1E1B
Antibody
[0958] 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-193P1E1B 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 193P1E1B to Known Sequences
[0959] The 193P1E1B protein has several forms, including 3 SNPs and
5 splice variants (FIG. 4G). Three variants, namely 193P1E1B v.1,
v.5 and v.6, consist of 412 amino acids each, with calculated
molecular weight of 46.25 kDa, and pI of 5.18, and differ from each
other by one amino acid. 193P1E1B v.10, v.9 and v.12 are
progressively smaller proteins, with 388, 330, and 73 amino acids
respectively. These variants differ with regards to their molecular
weights and isoelectic points, as shown in Table L. All variants of
193P1E1B are predicted to be nuclear proteins, with possible
localization to the mitochondria (193P1E3B v.1, v.5, v.6, v.9,
v.10, v.11 and v.13) or cytoplasm (193P1E3B v.12). Motif analysis
revealed no known motifs.
[0960] All protein variants of 193P1E1B show best homology to a
human un-named protein (gi 21748775) of unknown function, with
193P1E1B v.5 showing 100% identity with gi 21748775 over the entire
length of the protein, and 193P1E1B sharing 99% identity with the
same protein. Similarly, the other variants show highest homology
to the human un-named protein (gi 21748775). The variant with the
lowest homology to gi 21748775 is 193P1E1B v.12, with 89% identity
and 89% homology over the first 39 amino acids of the protein (FIG.
4A-D).
[0961] The 193P1E1B protein shows homology to a protein of known
function, namely the arginine repressor (gi 14349114) of E. coli,
also known as carbamate kinase. Variant 193P1E1B v.1 shows 30%
identity and 57% homology with that protein (FIG. 4E). This
homology indicates that 193P1E1B may regulate ATP synthesis and
metabolism (Marina A et al, Eur J Biochem 1998, 253:280; Alcantara
C et al, FEBS Lett. 2000, 484:261), a key factor in cell growth and
biological function.
[0962] In addition, 193P1E1B also exhibit some homology to human
double-stranded RNA-specific adenosine deaminase (ADAR-c isoform)
(gi 7669475). 193P1E1Bv.1 shares 26% identity and 40% homology with
ADAR-c (FIG. 4F). Similar results were obtained with 193P1E1Bv.5,
v.6, v.9, v.10 and v.13. This suggests that 193P1E1B has the
ability to bind specifically to double stranded RNA or DNA
(Schwartz T., et al., Nature Struc. Biol. 2001, 8:761). Adenosine
deaminases acting on RNA have been shown to be involved in RNA
editing (Raitskin, O., et al., Proc. Natl. Acad. Sci. 2001,
98:6571). Recent studies have associated adenosine deaminase with
cancer and cellular proliferation (Eroglu A, et al., Med Oncol.
2000, 17:319-24; Barry C. P., and, Lind, S. E., Cancer Res. 2000,
60:1887-94). In addition, adenosine deaminase is highly expressed
in tumor tissue relative to normal tissues in such cancers as
colon, leukemia and other lymphoid cancers (Blatt, J., et al., N
Engl J Med. 1980; 303:918; Eroglu, A., et al., Med Oncol. 2000,
17:319). Adenosine deaminase has been considered a potential marker
for lymphoid malignancies (Blatt J et al., N Engl J Med. 1980; 303:
918). In addition, inhibition of adenosine deaminase was found to
result in cell death of epithelial cells (Barry, C. P., and, Lind,
S. E., Cancer Res. 2000, 60:1887).
[0963] This information indicates that 193P1E1B plays a role in the
transformation of mammalian cells, supports cell survival and
proliferation, and regulates gene transcription by regulating
events in the nucleus.
[0964] Accordingly, when 193P1E1B functions as a regulator of cell
transformation, tumor formation, or as a modulator of transcription
involved in activating genes associated with inflammation,
tumorigenesis or proliferation, 193P1E1B is used for therapeutic,
diagnostic, prognostic and/or preventative purposes.
Example 45
Identification and Confirmation of Potential Signal Transduction
Pathways
[0965] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J Neurochem. 2001; 76:217-223). In particular, adenosine
deaminase has been found to associate with G-proteins, thereby
regulating several signaling pathways (Ciruela F et al, FEBS Lett.
1996, 380:219). Using immunoprecipitation and Western blotting
techniques, proteins are identified that associate with 193P1E1B
and mediate signaling events. Several pathways known to play a role
in cancer biology can be regulated by 193P1E1B, including
phospholipid pathways such as PI3K, AKT, etc, adhesion and
migration pathways, including FAK, Rho, Rac-1, etc, as well as
mitogenic/survival cascades such as ERK, p38, etc. (Cell Growth
Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,
19:3003, J. Cell Biol. 1997, 138:913).
[0966] To confirm that 193P1E1B 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.
[0967] 1 NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0968] 2SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0969] 3 AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0970] 4 ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0971] 5 p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0972] 6 CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0973] 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.
[0974] Signaling pathways activated by 193P1E1B are mapped and used
for the identification and validation of therapeutic targets. When
193P1E1B is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 46
Regulation of Transcription
[0975] The nuclear localization of 193P1E1B and its ability to
regulate adenosine deaminase indicate that it is effectively used
as a modulator of the transcriptional regulation of eukaryotic
genes. Regulation of gene expression is confirmed, e.g., by
studying gene expression in cells expressing or lacking 193P1E1B.
For this purpose, two types of experiments are performed.
[0976] In the first set of experiments, RNA from parental and
193P1E1B-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, pheromones, 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.).
[0977] 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.
[0978] Thus, 193P1E1B plays a role in gene regulation, and it is
used as a target for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 47
Involvement in Tumor Progression
[0979] The 193P1E1B gene can contribute to the growth of cancer
cells. The role of 193P1E1B in tumor growth is confirmed in a
variety of primary and transfected cell lines including prostate,
colon, bladder and kidney cell lines, as well as NIH 3T3 cells
engineered to stably express 193P1E1B. Parental cells lacking
193P1E1B and cells expressing 193P1E1B are evaluated for cell
growth using a well-documented proliferation assay (Fraser S P, et
al., Prostate 2000; 44:61, Johnson D E, Ochieng J, Evans S L.
Anticancer Drugs. 1996, 7:288). The effect of 193P1E1B can also be
observed on cell cycle progression. Control and 193P1E1B-expressing
cells are grown in low serum overnight, and treated with 10% FBS
for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide
incorporation by FACS analysis.
[0980] To confirm the role of 193P1E1B in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH-3T3 cells lacking 193P1E1B are compared to NIH-3T3
cells expressing 193P1E1B, using a soft agar assay under stringent
and more permissive conditions (Song Z. et al. Cancer Res. 2000;
60:6730).
[0981] To confirm the role of 193P1E1B in invasion and metastasis
of cancer cells, a well-established assay is used. A non-limiting
example is the use of an assay which provides a basement membrane
or an analog thereof used to detect whether cells are invasive
(e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer
Res. 1999; 59:6010)). Control cells, including prostate, and
bladder cell lines lacking 193P1E1B are compared to cells
expressing 193P1E1B. Cells are loaded with the fluorescent dye,
calcein, and plated in the top well of a support structure coated
with a basement membrane analog (e.g. the Transwell insert) and
used in the assay. Invasion is determined by fluorescence of cells
in the lower chamber relative to the fluorescence of the entire
cell population.
[0982] 193P1E1B can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 193P1E1B 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 193P1E1B, including normal and tumor prostate, and
kidney cells. Engineered and parental cells are treated with
various chemotherapeutic agents, such as etoposide, flutamide, 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 193P1E1B can play a
critical role in regulating tumor progression and tumor load.
[0983] When 193P1E1B 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
[0984] 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 phsophodieseterase inhibitors on endothelial cells,
193P1E1B plays a role in angiogenesis (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 193P1E1B in angiogenesis,
enhancement or inhibition, is confirmed. For example, endothelial
cells engineered to express 193P1E1B are evaluated using tube
formation and proliferation assays. The effect of 193P1E1B is also
confirmed in animal models in vivo. For example, cells either
expressing or lacking 193P1E1B are implanted subcutaneously in
immunocompromised mice. Endothelial cell migration and angiogenesis
are evaluated 5-15 days later using immunohistochemistry
techniques. 193P1E1B affects angiogenesis, and it is used as a
target for diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 49
Involvement in Cell Adhesion
[0985] Cell adhesion plays a critical role in tissue colonization
and metastasis. 193P1E1B can participate in cellular organization,
and as a consequence cell adhesion and motility. To confirm that
193P1E1B regulates cell adhesion, control cells lacking 193P1E1B
are compared to cells expressing 193P1E1B, using techniques
previously described (see, e.g., Haier et al, Br. J. Cancer. 1999,
80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118).
Briefly, in one embodiment, cells labeled with a fluorescent
indicator, such as calcein, are incubated on tissue culture wells
coated with media alone or with matrix proteins. Adherent cells are
detected by fluorimetric analysis and percent adhesion is
calculated. In another embodiment, cells lacking or expressing
193P1E1B are analyzed for their ability to mediate cell-cell
adhesion using similar experimental techniques as described above.
Both of these experimental systems are used to identify proteins,
antibodies and/or small molecules that modulate cell adhesion to
extracellular matrix and cell-cell interaction. Cell adhesion plays
a critical role in tumor growth, progression, and, colonization,
and 193P1E1B is involved in these processes. Thus, it serves as a
diagnostic, prognostic, preventative and/or therapeutic
modality.
Example 50
Protein-Protein Association
[0986] Several adenosine deaminasess have been shown to interact
with other proteins, thereby regulating gene transcription, protein
function, as well as cell growth (Raitskin et al above; Morimoto C,
and Schlossman S F, Immunol Rev. 1998, 161:55.). Using
immunoprecipitation techniques as well as two yeast hybrid systems,
proteins are identified that associate with 193P1E1B
Immunoprecipitates from cells expressing 193P1E1B and cells lacking
193P1E1B are compared for specific protein-protein
associations.
[0987] Studies are performed to confirm the extent of association
of 193P1E1B with effector molecules, such as nuclear proteins,
transcription factors, kinases, phosphates, etc. Studies comparing
193P1E1B positive and 193P1E1B 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.
[0988] 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 193P1E1B-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 193P1E1B, 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 193P1E1B.
[0989] Thus it is found that 193P1E1B associates with proteins and
small molecules. Accordingly, 193P1E1 Band these proteins and small
molecules are used for diagnostic, prognostic, preventative and/or
therapeutic purposes.
[0990] 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.
[0991] 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-00004 [0992] TABLE I Tissues that Express 193P1E1B: a.
Malignant Tissues Prostate Bladder Kidney Colon Lung Ovary Breast
Pancreas Testis Uterus Skin Bone
TABLE-US-00005 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 aspartic acid E Glu glutamic acid G Gly
glycine
TABLE-US-00006 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. 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-00007 [0993] TABLE IV (A) HLA Class I Supermotifs/Motifs
POSITION POSITION 3 (Primary POSITION 2 (Primary Anchor) Anchor) C
Terminus (Primary Anchor) SUPER- MOTIF A1 TILVMS (SEQ ID NO: 124)
FWY A2 LIVMATQ (SEQ ID NO: 125) IVMATL (SEQ ID NO: 126) A3 VSMATLI
(SEQ ID NO: 127) RK A24 YFWIVLMT (SEQ ID NO: 128) FIYWLM (SEQ ID
NO: 129) B7 P VILFMWYA (SEQ ID NO: 130) B27 RHK FYLWMIVA (SEQ ID
NO: 131) B44 ED FWYLIMVA (SEQ ID NO: 132) B58 ATS FWYLIVMA (SEQ ID
NO: 133) B62 QLIVMP (SEQ ID NO: 134) FWYMIVLA (SEQ ID NO: 135)
MOTIFS A1 TSM Y A1 DEAS Y (SEQ ID NO: 247) A2.1 LMVQIAT (SEQ ID NO:
136) VLIMAT (SEQ ID NO: 137) A3 LMVISATFCGD (SEQ ID NO: 138) KYRHFA
(SEQ ID NO: 139) A11 VTMLISAGNCDF (SEQ ID NO: 140) KRYH (SEQ ID NO:
141) A24 YFWM (SEQ ID NO: 142) FLIW (SEQ ID NO: 143) A*3101 MVTALIS
(SEQ ID NO: 144) RK A*3301 MVALFIST (SEQ ID NO: 145) RK A*6801
AVTMSLI (SEQ ID NO: 146) RK B*0702 P LMFWYAIV (SEQ ID NO: 147)
B*3501 P LMFWYIVA (SEQ ID NO: 148) B51 P LIVFWYAM (SEQ ID NO: 149)
B*5301 P IMFWYALV (SEQ ID NO: 150) B*5401 P ATIVLMFWY (SEQ ID NO:
151) 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-00008 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-00009 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 (SEQ ID NO: 152) (SEQ ID NO: 153) deleterious W R
WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM (SEQ ID NO: 154)
(SEQ ID NO: 155) (SEQ ID NO: 156) deleterious C CH FD CWD GDE D DR7
preferred MFLIVWY M W A IVMSACTPL M IV (SEQ ID NO: 157) (SEQ ID NO:
158) 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 LIVMFY D preferred
(SEQ ID NO: 159) Motif b LIVMFAY DNQEST KRH preferred (SEQ ID NO:
160) (SEQ ID NO: 161) DR MFLIVWY VMSTACPLI Supermotif (SEQ ID NO:
162) (SEQ ID NO: 163) Italicized residues indicate less preferred
or "tolerated" residues
TABLE-US-00010 TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS
POSITION 1 2 3 4 5 6 7 8 C-terminus A1 1.degree. Anchor 1.degree.
Anchor TILVMS FWY (SEQ ID NO: 164) A2 1.degree. Anchor 1.degree.
Anchor LIVMATQ LIVMAT (SEQ ID NO: 165) (SEQ ID NO: 166) A3
Preferred 1.degree. Anchor YFW YFW YFW P 1.degree. Anchor VSMATLI
(4/5) (3/5) (4/5) (4/5) RK (SEQ ID NO: 167) deleterious DE (3/5); P
(5/5) DE (4/5) A24 1.degree. Anchor 1.degree. Anchor YFWIVLMT
FIYWLM (SEQ ID NO: 168) (SEQ ID NO: 169) B7 Preferred FWY (5/5);
LIVM 1.degree. Anchor FWY FWY 1.degree. Anchor (3/5) P (4/5) (3/5)
VILFMWYA (SEQ ID NO: 170) (SEQ ID NO: 171) deleterious DE (3/5);
P(5/5); DE G QN DE G(4/5); A(3/5); (3/5) (4/5) (4/5) (4/5) QN(3/5)
B27 1.degree. Anchor 1.degree. Anchor RHK FYLWMIVA (SEQ ID NO: 172)
B44 1.degree. Anchor 1.degree. Anchor ED FWYLIMVA (SEQ ID NO: 173)
B58 1.degree. Anchor 1.degree. Anchor ATS FWYLIVMA (SEQ ID NO: 174)
B62 1.degree. Anchor 1.degree. Anchor QLIVMP FWYMIVLA (SEQ ID NO:
175) (SEQ ID NO: 176) Italicized residues indicate less preferred
or "tolerated" residues
TABLE-US-00011 TABLE IV (E) HLA Class I Motifs POSITION 1 2 3 4 5 6
A1 preferred GFYW 1.degree. Anchor DEA YFW P 9-mer (SEQ ID STM NO:
177) deleterious DE RHKLIVMP A G A (SEQ ID NO: 179) A1 preferred
GRHK ASTCLIVM 1.degree. Anchor GSTC ASTC 9-mer (SEQ ID (SEQ ID DEAS
(SEQ ID (SEQ ID NO: 180) NO: 181) (SEQ ID NO: 183) NO: 184) NO:
182) deleterious A RHKDEPYFW DE PQN RHK (SEQ ID NO: 186) A1
preferred YFW 1.degree. Anchor DEAQN A YFWQN 10-mer STM (SEQ ID
(SEQ ID NO: 187) NO: 188) deleterious GP RHKGLIVM DE RHK QNA (SEQ
ID NO: 190) A1 preferred YFW STCLIVM 1.degree. Anchor A YFW 10-mer
(SEQ ID DEAS NO: 192) (SEQ ID NO: 193) deleterious RHK RHKDEPYFW P
G (SEQ ID NO: 194) A2.1 preferred YFW 1.degree. Anchor YFW STC YFW
A 9-mer LMIVQAT (SEQ ID NO: 196) deleterious DEP DERKH RKH (SEQ ID
NO: 198) A2.1 preferred AYFW 1.degree. Anchor LVIM G G 10-mer
LMIVQAT (SEQ ID (SEQ ID NO: 201) NO: 200) deleterious DEP DE RKHA P
(SEQ ID NO: 204) A3 preferred RHK 1.degree. Anchor YFW PRHKYFW A
YFW LMVISATF (SEQ ID CGD NO: 207) (SEQ ID NO: 206) deleterious DEP
DE A11 preferred A 1.degree. Anchor YFW YFW A YFW VTLMISAGN CDF
(SEQ ID NO: 209) deleterious DEP A24 preferred YFWRHK 1.degree.
Anchor STC 9-mer (SEQ ID YFWM NO: 211) (SEQ ID NO: 212) deleterious
DEG DE G QNP DERHK (SEQ ID NO: 214) A24 preferred 1.degree. Anchor
P YFWP 10-mer YFWM (SEQ ID (SEQ ID NO: 216) NO: 215) deleterious
GDE QN RHK DE A3101 preferred RHK 1.degree. Anchor YFW P YFW
MVTALIS (SEQ ID NO: 218) deleterious DEP DE ADE DE A3301 preferred
1.degree. Anchor YFW MVALFIST (SEQ ID NO: 219) deleterious GP DE
A6801 preferred YFWSTC 1.degree. Anchor YFWLIVM (SEQ ID AVTMSLI
(SEQ ID NO: 221) (SEQ ID NO: 223) NO: 222) deleterious GP DEG RHK
B0702 preferred RHKFWY 1.degree. Anchor RHK RHK RHK (SEQ ID P NO:
224) deleterious DEQNP DEP DE DE GDE (SEQ ID NO: 226) B3501
preferred FWYLIVM 1.degree. Anchor FWY (SEQ ID P NO: 227)
deleterious AGP G G B51 preferred LIVMFWY 1.degree. Anchor FWY STC
FWY (SEQ ID P NO: 229) deleterious AGPDERH DE G KSTC (SEQ ID NO:
231) B5301 preferred LIVMFWY 1.degree. Anchor FWY STC FWY (SEQ ID P
NO: 233) deleterious AGPQN G (SEQ ID NO: 236) B5401 preferred FWY
1.degree. Anchor FWYLIVM LIVM P (SEQ ID (SEQ ID NO: 238) NO: 239)
deleterious GPQNDE GDESTC RHKDE DE (SEQ ID (SEQ ID (SEQ ID NO: 243)
NO: 244) NO: 245) POSITION 7 8 9 or C-terminus C-terminus A1
preferred DEQN YFW 1.degree. Anchor 9-mer (SEQ ID Y NO: 178)
deleterious A1 LIVM DE 1.degree. Anchor 9-mer (SEQ ID Y NO: 185)
deleterious PG GP A1 preferred PASTC GDE P 1.degree. Anchor 10-mer
(SEQ ID Y NO: 189) deleterious RHKYFW RHK A (SEQ ID NO: 191) A1
preferred PG G YFW 1.degree. Anchor 10-mer Y deleterious PRHK QN
(SEQ ID NO: 195) A2.1 preferred A P 1.degree. Anchor 9-mer VLIMAT
(SEQ ID NO: 197) deleterious DERKH (SEQ ID NO: 199) A2.1 preferred
FYWLVI 1.degree. Anchor 10-mer M VLIMAT (SEQ ID (SEQ ID NO: 202)
NO: 203) deleterious RKH DERKH RKH (SEQ ID NO: 205) A3 preferred P
1.degree. Anchor KYRHFA (SEQ ID NO: 208) deleterious A11 preferred
YFW P 1.degree. Anchor KRYH (SEQ ID NO: 210) deleterious A G A24
preferred YFW YFW 1.degree. Anchor 9-mer FLIW (SEQ ID NO: 213)
deleterious G AQN A24 preferred P 1.degree. Anchor 10-mer FLIW (SEQ
ID NO: 217) deleterious A QN DEA A3101 preferred YFW AP 1.degree.
Anchor RK deleterious DE DE A3301 preferred AYFW 1.degree. Anchor
(SEQ ID RK NO: 220) deleterious A6801 preferred YFW P 1.degree.
Anchor RK deleterious A B0702 preferred RHK PA 1.degree. Anchor
LMFWYAIV (SEQ ID NO: 225) deleterious QN DE B3501 preferred FWY
1.degree. Anchor LMFWYIVA (SEQ ID NO: 228) deleterious B51
preferred G FWY 1.degree. Anchor LIVFWYAM (SEQ ID NO: 230) DEQN GDE
deleterious (SEQ ID NO: 232) B5301 preferred LIVMFWY FWY 1.degree.
Anchor (SEQ ID IMFWYALV NO: 234) (SEQ ID NO: 235) deleterious RHKQN
DE (SEQ ID NO: 237) B5401 preferred ALIVM FWYAP 1.degree. Anchor
(SEQ ID (SEQ ID ATIVLMFWY NO: 240) NO: 241) (SEQ ID NO: 242)
deleterious QNDGE DE (SEQ ID NO: 246)
TABLE-US-00012 TABLE IV (F) Summary of HLA-supertypes Overall
phenotypic frequencies of HLA-supertypes in different ethnic
populations Specificity Phenotypic frequency N.A. Supertype
Position 2 C-Terminus Caucasian Black Japanese Chinese Hispanic
Average B7 P AILMVFWY (SEQ ID 43.2 55.1 57.1 43.0 49.3 49.5 NO:
248) A3 AILMVST (SEQ ID RK 37.5 42.1 45.8 52.7 43.1 44.2 NO: 249)
A2 AILMVT (SEQ ID AILMVT (SEQ ID 45.8 39.0 42.4 45.9 43.0 42.2 NO:
250) NO: 251) A24 YF (SEQ ID FI (YWLM) (SEQ ID 23.9 38.9 58.6 40.1
38.3 40.0 (WIVLMT) NO: 252) NO: 253) B44 E (D) FWYLIMVA (SEQ ID
43.0 21.2 42.9 39.1 39.0 37.0 NO: 254) A1 TI (LVMS) (SEQ ID FWY
47.1 16.1 21.8 14.7 26.3 25.2 NO: 255) B27 RHK FYL (WMI) (SEQ ID
28.4 26.1 13.3 13.9 35.3 23.4 NO: 256) B62 QL (IVMP) (SEQ ID FWY
(MIV) (SEQ ID 12.6 4.8 36.5 25.4 11.1 18.1 NO: 257) NO: 258) B58
ATS FWY (LIV) (SEQ ID 10.0 25.1 1.6 9.0 5.9 10.3 NO: 259)
TABLE-US-00013 TABLE IV (G): Calculated population coverage
afforded by different HLA-supertype combinations Phenotypic
frequency Cauca- N. A. Japa- Chi- His- Aver- HLA-supertypes sian
Blacks nese nese panic age 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
99.9 99.6 100.0 99.8 99.9 99.8 A2, A3, B7, A24, B44, A1, B27, B62,
and B 58 Motifs indicate the residues defining supertype
specificities. 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-00014 TABLE V Frequently Occurring Motifs avrg. % Name
identity Description Potential Function zf-C2H2 34% Zinc finger,
C2H2 type Nucleic acid-binding protein functions as transcription
factor, nuclear location probable cytochrome_b_N 68% Cytochrome
b(N- membrane bound oxidase, generate superoxide terminal)/b6/petB
Ig 19% Immunoglobulin domain domains are one hundred amino acids
long and include a conserved intradomain disulfide bond. WD40 18%
WD domain, G-beta repeat tandem repeats of about 40 residues, each
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
translocation Ubiquinone/plastoquinone across the membrane (complex
I), various chains Efhand 24% EF hand calcium-binding domain,
consists of a12 residue loop flanked on both sides by a 12 residue
alpha-helical domain Rvp 79% Retroviral aspartyl protease Aspartyl
or acid proteases, centered on a catalytic aspartyl residue
Collagen 42% Collagen triple helix repeat extracellular structural
proteins involved in formation of (20 copies) connective tissue.
The sequence consists of the G-X-Y and the polypeptide chains forms
a triple helix. Fn3 20% Fibronectin type III domain Located in the
extracellular ligand-binding 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 receptor seven
hydrophobic transmembrane regions, with the N- (rhodopsin family)
terminus located extracellularly while the C-terminus is
cytoplasmic. Signal through G proteins
TABLE-US-00015 TABLE VI Motifs and Post-translational Modifications
of 193P1E1B N-glycosylation site Number of matches: 3 1 246-249
NKSE (SEQ ID NO: 63) 2 316-319 NSSS (SEQ ID NO: 64) 3 340-343 NLTD
(SEQ ID NO: 65) cAMP- and cGMP-dependent protein kinase
phosphorylation site 107-110 KKNS (SEQ ID NO: 66) Protein kinase C
phosphorylation site Number of matches: 10 1 22-24 TAR 2 53-55 TLK
3 103-105 SPR 4 152-154 SPR 5 149-151 SEK 6 103-105 SPR 7 152-154
SPR 8 203-205 TPK 9 217-219 TPK 10 203-205 TPK Casein kinase II
phosphorylation site Number of matches: 12 1 16-19 STLD (SEQ ID NO:
67) 2 34-37 SDFE (SEQ ID NO: 68) 3 53-56 TLKD (SEQ ID NO: 69) 4
110-113 SVHE (SEQ ID NO: 70) 5 119-122 SDPE (SEQ ID NO: 71) 6
124-127 SNCE (SEQ ID NO: 72) 7 276-279 SDAE (SEQ ID NO: 73) 8
318-321 SSND (SEQ ID NO: 74) 9 336-339 TCFE (SEQ ID NO: 75) 10
350-353 SSYE (SEQ ID NO: 76) 11 360-363 TPPE (SEQ ID NO: 77) 12
408-411 SNKE (SEQ ID NO: 78) N-myristoylation site 239-244 GLKNAR
(SEQ ID NO: 79)
TABLE-US-00016 TABLE VII Search Peptides variant 1: 9-mers, 10-mers
and 15-mers (SEQ ID NO: 80)
MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI
PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN
LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD
NYKEEPVIVIPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL
KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK
NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT
PPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN variant 5:
9-mers PVASSCISEKSPRSPQL (SEQ ID NO: 81) 10-mers
PPVASSCISEKSPRSPQLS (SEQ ID NO: 82) 15-mers
DDLSDPPVASSCISEKSPRSPQLSDFGLE (SEQ ID NO: 83) Variant 6: 9-mers
NKSEEAIDAESRLND NV (SEQ ID NO: 84) 10-mers NNKSEEAIDAESRLND NVF
(SEQ ID NO: 85) 15-mers LKNARNNKSEEAIDAESRLND NVFATPSP (SEQ ID NO:
86) Variant 10: 9-mers KIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 87)
10-mers TKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 88) 15-mers
TPPEVTKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 89) Variant 12: 9-mers
RALDGEESLLSKYNSN (SEQ ID NO: 90) 10-mers QRALDGEESLLSKYNSNL (SEQ ID
NO: 91) 15-mers ETARLQRALDGEESLLSKYNSNLATPIA (SEQ ID NO: 92)
Tables VIII-XXI:
TABLE-US-00017 [0994] TABLE VIII-V1 HLA-A1-9mers-193P1EIB 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 19 DCETARLQR 45.000 98 LSDFGLERY 37.500 341
LTDPSSPTI 25.000 132 GIDFIKATK 20.000 78 LSDPPVASS 15.000 129
NQEGIDFIK 13.500 272 QLEKSDAEY 9.000 28 ALDGEESDF 5.000 253
DTESRLNDN 2.250 31 GEESDFEDY 2.250 33 ESDFEDYPM 1.500 306 VSTNYPLSK
1.500 63 LSNCENFQK 1.500 225 ISEYTMCLN 1.350 37 EDYPMRILY 1.250 228
YTMCLNEDY 1.250 319 SNDLEVEDR 1.250 71 KTDVKDDLS 1.250 232
LNEDYTMGL 1.125 358 TPTPPEVTK 1.000 215 CVTPKLEHF 1.000 389
KAVPPSKRF 1.000 17 TLDCETARL 1.000 277 DAEYTNSPL 0.900 321
DLEVEDRTS 0.900 323 EVEDRTSLV 0.900 344 PSSPTISSY 0.750 349
ISSYENLLR 0.750 333 NSDTCFENL 0.750 275 KSDAEYTNS 0.750 48
HSEVQTLKD 0.675 233 NEDYTMGLK 0.500 382 LATPIAIKA 0.500 281
TNSPLVPTF 0.500 251 AIDTESRLN 0.500 370 DILQLLSKY 0.500 263
FATPSPIIQ 0.500 302 SIALVSTNY 0.500 97 QLSDFGLER 0.500 219
KLEHFGISE 0.450 381 NLATPIAIK 0.400 236 YTMGLKNAR 0.250 16
STLDCETAR 0.250 391 VPPSKRFLK 0.250 267 SPIIQQLEK 0.250 209
KMDDFECVT 0.250 121 LLDKARLEN 0.250 189 VTPPTKQSL 0.250 60
IPELSNCEN 0.225 367 IPEDILQLL 0.225 171 VHEQEAINS 0.225 35
DFEDYPMRI 0.225 142 LMEKNSMDI 0.225 182 YKEEPVIVT 0.225 175
EAINSDNYK 0.200 201 LKTPKCALK 0.200 110 QVLPNPPQA 0.200 83
VASSCISGK 0.200 102 GLERYIVSQ 0.180 146 NSMDIMKIR 0.150 173
EQEAINSDN 0.135 247 KSEEAIDTE 0.135 290 CTPGLKIPS 0.125 147
SMDIMKIRE 0.125 264 ATPSPIIQQ 0.125 30 DGEESDFED 0.113 86 SCISGKSPR
0.100 330 LVLNSDTCF 0.100 188 IVTPPTKQS 0.100 118 AVNLLDKAR 0.100
205 KCALKMDDF 0.100 160 YGYSPRVKK 0.100 137 KATKVLMEK 0.100 390
AVPPSKRFL 0.100 126 RLENQEGID 0.090 183 KEEPVIVTP 0.090 65
NCENFQKTD 0.090 212 DFECVTPKL 0.090 12 RSLASTLDC 0.075 178
NSDNYKEEP 0.075 5 RSFCGKLRS 0.075 316 NSSSNDLEV 0.075 350 SSYENLLRT
0.075 195 QSLVKVLKT 0.075 194 KQSLVKVLK 0.060 287 PTFCTPGLK 0.050
57 DVNIPELSN 0.050 112 LPNPPQAVN 0.050 280 YTNSPLVPT 0.050 106
YIVSQVLPN 0.050 224 GISEYTMCL 0.050 154 REYFQKYGY 0.050 257
RLNDNVFAT 0.050 369 EDILQLLSK 0.050 55 KDDVNIPEL 0.050 152
KIREYFQKY 0.050 366 KIPEDILQL 0.050 67 ENFQKTDVK 0.050 75 KDDLSDPPV
0.050 214 ECVTPKLEH 0.050
TABLE-US-00018 TABLE VIII-V5 HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
VASSCISEK 0.200 5 SCISEKSPR 0.100 6 CISEKSPRS 0.020 3 ASSCISEKS
0.015 7 ISEKSPRSP 0.014 9 EKSPRSPQL 0.010 4 SSCISEKSP 0.002 1
PVASSCISE 0.001 8 SEKSPRSPQ 0.000
TABLE-US-00019 TABLE VIII-V6 HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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 8
DAESRLNDN 0.900 6 AIDAESRLN 0.500 2 KSEEAIDAE 0.135 3 SEEAIDAES
0.090 5 EAIDAESRL 0.010 4 EEAIDAESR 0.005 1 NKSEEAIDA 0.003 7
IDAESRLND 0.000 9 AESRLNDNV 0.000
TABLE-US-00020 TABLE VIII-V10 HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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
IPEDILQKF 2.250 1 KIPEDILQK 1.000 12 WIYPTQKLN 0.100 13 IYPTQKLNK
0.050 6 ILQKFQWIY 0.050 5 DILQKFQWI 0.010 10 FQWIYPTQK 0.003 3
PEDILQKFQ 0.003 15 PTQKLNKMR 0.003 4 EDILQKFQW 0.003 14 YPTQKLNKM
0.003 8 QKFQWIYPT 0.001 9 KFQWIYPTQ 0.001 11 QWIYPTQKL 0.001 7
LQKFQWIYP 0.000
TABLE-US-00021 TABLE VIII-V12 HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
DGEESLLSK 22.500 5 GEESLLSKY 2.250 2 ALDGEESLL 0.500 7 ESLLSKYNS
0.030 8 SLLSKYNSN 0.010 1 RALDGEESL 0.010 3 LDGEESLLS 0.003 6
EESLLSKYN 0.001
TABLE-US-00022 TABLE IX-V1 HLA-A1-10mers-193P1EIB 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
219 KLEHFGISEY 45.000 126 RLENQEGIDF 45.000 33 ESDFEDYPMR 15.000
341 LTDPSSPTIS 12.500 147 SMDIMKIREY 12.500 30 DGEESDFEDY 11.250
390 AVPPSKRFLK 10.000 78 LSDPPVASSC 7.500 173 EQEAINSDNY 6.750 36
FEDYPMRILY 6.250 323 EVEDRTSLVL 4.500 153 IREYFQKYGY 4.500 232
LNEDYTMGLK 4.500 60 IPELSNCENF 2.250 253 DTESRLNDNV 2.250 277
DAEYTNSPLV 1.800 247 KSEEAIDTES 1.350 367 IPEDILQLLS 1.125 357
RTPTPPEVTK 1.000 305 LVSTNYPLSK 1.000 62 ELSNCENFQK 1.000 19
DCETARLQRA 0.900 321 DLEVEDRTSL 0.900 65 NCENFQKTDV 0.900 301
NSIALVSTNY 0.750 333 NSDTCFENLT 0.750 178 NSDNYKEEPV 0.750 98
LSDFGLERYI 0.750 225 ISEYTMCLNE 0.675 129 NQEGIDFIKA 0.675 71
KTDVKDDLSD 0.625 17 TLDCETARLQ 0.500 263 FATPSPIIQQ 0.500 210
MDDFECVTPK 0.500 132 GIDFIKATKV 0.500 348 TISSYENLLR 0.500 289
FCTPGLKIPS 0.500 280 YTNSPLVPTF 0.500 97 QLSDFGLERY 0.500 248
SEEAIDTESR 0.450 368 PEDILQLLSK 0.250 190 TPPTKQSLVK 0.250 189
VTPPTKQSLV 0.250 128 ENQEGIDFIK 0.250 251 AIDTESRLND 0.250 46
DLHSEVQTLK 0.200 266 PSPIIQQLEK 0.150 85 SSCISGKSPR 0.150 15
ASTLDCETAR 0.150 318 SSNDLEVEDR 0.150 271 QQLEKSDAEY 0.150 48
HSEVQTLKDD 0.135 343 DPSSPTISSY 0.125 258 LNDNVFATPS 0.125 233
NEDYTMGLKN 0.125 319 SNDLEVEDRT 0.125 380 SNLATPIAIK 0.100 188
IVTPPTKQSL 0.100 185 EPVIVTPPTK 0.100 110 QVLPNPPQAV 0.100 214
ECVTPKLEHF 0.100 27 RALDGEESDF 0.100 131 EGIDFIKATK 0.100 382
LATPIAIKAV 0.100 329 SLVLNSDTCF 0.100 215 CVTPKLEHFG 0.100 117
QAVNLLDKAR 0.100 389 KAVPPSKRFL 0.100 102 GLERYIVSQV 0.090 272
QLEKSDAEYT 0.090 337 CFENLTDPSS 0.090 379 NSNLATPIAI 0.075 275
KSDAEYTNSP 0.075 94 RSPQLSDFGL 0.075 349 ISSYENLLRT 0.075 282
NSPLVPTFCT 0.075 291 TPGLKIPSTK 0.050 331 VLNSDTCFEN 0.050 54
LKDDVNIPEL 0.050 381 NLATPIAIKA 0.050 16 STLDCETARL 0.050 44
LYDLHSEVQT 0.050 236 YTMGLKNARN 0.050 28 ALDGEESDFE 0.050 112
LPNPPQAVNL 0.050 75 KDDLSDPPVA 0.050 286 VPTFCTPGLK 0.050 121
LLDKARLENQ 0.050 170 SVHEQEAINS 0.050 231 CLNEDYTMGL 0.050 141
VLMEKNSMDI 0.050 74 VKDDLSDPPV 0.050 150 IMKIREYFQK 0.050 120
NLLDKARLEN 0.050 209 KMDDFECVTP 0.050 290 CTPGLKIPST 0.050 136
IKATKVLMEK 0.050 183 KEEPVIVTPP 0.045 351 SYENLLRTPT 0.045 35
DFEDYPMRIL 0.045
TABLE-US-00023 TABLE IX-V5 HLA-A1-10mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 11; 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
SSCISEKSPR 0.150 8 ISEKSPRSPQ 0.135 6 SCISEKSPRS 0.020 2 PVASSCISEK
0.020 3 VASSCISEKS 0.010 10 EKSPRSPQLS 0.005 4 ASSCISEKSP 0.002 7
CISEKSPRSP 0.001 1 PPVASSCISE 0.000 9 SEKSPRSPQL 0.000
TABLE-US-00024 TABLE IX-V6 HLA-A1-10mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 13; 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
KSEEAIDAES 2.700 9 DAESRLNDNV 0.900 4 SEEAIDAESR 0.450 7 AIDAESRLND
0.250 6 EAIDAESRLN 0.010 1 NNKSEEAIDA 0.001 10 AESRLNDNVF 0.001 8
IDAESRLNDN 0.001 5 EEAIDAESRL 0.001 2 NKSEEAIDAE 0.000
TABLE-US-00025 TABLE IX-V10 HLA-A1-10mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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 13
WIYPTQKLNK 10.000 1 TKIPEDILQK 0.500 6 DILQKFQWIY 0.500 3
IPEDILQKFQ 0.225 2 KIPEDILQKF 0.100 15 YPTQKLNKMR 0.025 4
PEDILQKFQW 0.013 10 KFQWIYPTQK 0.010 9 QKFQWIYPTQ 0.001 7
ILQKFQWIYP 0.001 14 IYPTQKLNKM 0.001 12 QWIYPTQKLN 0.001 5
EDILQKFQWI 0.001 8 LQKFQWIYPT 0.000 11 FQWIYPTQKL 0.000
TABLE-US-00026 TABLE IX-V12 HLA-A1-10mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
DGEESLLSKY 11.250 3 ALDGEESLLS 2.500 4 LDGEESLLSK 0.050 6
GEESLLSKYN 0.045 8 ESLLSKYNSN 0.015 9 SLLSKYNSNL 0.010 2 RALDGEESLL
0.010 7 EESLLSKYNS 0.001 1 QRALDGEESL 0.001
TABLE-US-00027 TABLE X-V1 HLA-A0201-9mers-193P1E1B 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
43 ILYDLHSEV 1551.288 257 RLNDNVFAT 407.580 111 VLPNPPQAV 118.238
366 KIPEDILQL 96.947 209 KMDDFECVT 48.131 374 LLSKYNSNL 36.316 340
NLTDPSSPT 30.553 304 ALVSTNYPL 21.362 229 TMCLNEDYT 14.504 224
GISEYTMCL 12.043 17 TLDCETARL 8.545 10 KLRSLASTL 5.682 140
KVLMEKNSM 5.629 199 KVLKTPKCA 5.629 39 YPMRILYDL 5.459 295
KIPSTKNSI 5.021 278 AEYTNSPLV 4.328 46 DLHSEVQTL 3.685 261
NVFATPSPI 3.378 348 TISSYENLL 2.937 322 LEVEDRTSL 2.895 329
SLVLNSDTC 2.434 208 LKMDDFECV 2.319 383 ATPIAIKAV 2.222 390
AVPPSKRFL 2.056 110 QVLPNPPQA 1.608 350 SSYENLLRT 1.468 66
CENFQKTDV 1.352 200 VLKTPKCAL 1.271 280 YTNSPLVPT 1.095 283
SPLVPTFCT 1.044 314 KTNSSSNDL 1.038 207 ALKMDDFEC 1.009 119
VNLLDKARL 0.877 270 IQQLEKSDA 0.856 142 LMEKNSMDI 0.820 95
SPQLSDFGL 0.809 371 ILQLLSKYN 0.697 352 YENLLRTPT 0.667 254
TESRLNDNV 0.663 133 IDFIKATKV 0.608 145 KNSMDIMKI 0.548 52
QTLKDDVNI 0.536 189 VTPPTKQSL 0.504 231 CLNEDYTMG 0.458 316
NSSSNDLEV 0.454 190 TPPTKQSLV 0.454 195 QSLVKVLKT 0.414 373
QLLSKYNSN 0.414 103 LERYIVSQV 0.402 300 KNSIALVST 0.392 141
VLMEKNSMD 0.384 64 SNCENFQKT 0.379 114 NPPQAVNLL 0.321 265
TPSPIIQQL 0.321 378 YNSNLATPI 0.313 158 QKYGYSPRV 0.309 380
SNLATPIAI 0.252 202 KTPKCALKM 0.242 307 STNYPLSKT 0.238 286
VPTFCTPGL 0.237 97 QLSDFGLER 0.232 223 FGISEYTMC 0.224 50 EVQTLKDDV
0.224 20 CETARLQRA 0.222 230 MCLNEDYTM 0.204 130 QEGIDFIKA 0.184
328 TSLVLNSDT 0.180 282 NSPLVPTFC 0.178 45 YDLHSEVQT 0.176 14
LASTLDCET 0.176 7 FCGKLRSLA 0.149 36 FEDYPMRIL 0.144 367 IPEDILQLL
0.143 331 VLNSDTCFE 0.139 75 KDDLSDPPV 0.135 131 EGIDFIKAT 0.131
128 ENQEGIDFI 0.130 58 VNIPELSNC 0.127 12 RSLASTLDC 0.120 323
EVEDRTSLV 0.120 382 LATPIAIKA 0.117 324 VEDRTSLVL 0.116 168
KNSVHEQEA 0.114 291 TPGLKIPST 0.112 135 FIKATKVLM 0.110 106
YIVSQVLPN 0.108 341 LTDPSSPTI 0.099 55 KDDVNIPEL 0.096 250
EAIDTESRL 0.091 9 GKLRSLAST 0.088 399 KHGQNIRDV 0.078 117 QAVNLLDKA
0.078 298 STKNSIALV 0.078 320 NDLEVEDRT 0.077 232 LNEDYTMGL 0.062
70 QKTDVKDDL 0.060 99 SDFGLERYI 0.059 354 NLLRTPTPP 0.055 237
TMGLKNARN 0.054
TABLE-US-00028 TABLE X-V5 HLA-A0201-9mers-193P1EB Each peptide is a
portion of SEQ ID NO: 11; 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
CISEKSPRS 0.042 9 EKSPRSPQL 0.002 2 VASSCISEK 0.001 3 ASSCISEKS
0.000 5 SCISEKSPR 0.000 4 SSCISEKSP 0.000 1 PVASSCISE 0.000 8
SEKSPRSPQ 0.000 7 ISEKSPRSP 0.000
TABLE-US-00029 TABLE X-V6 HLA-A0201-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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
AESRLNDNV 0.663 5 EAIDAESRL 0.091 1 NKSEEAIDA 0.028 6 AIDAESRLN
0.001 7 IDAESRLND 0.000 2 KSEEAIDAE 0.000 3 SEEAIDAES 0.000 8
DAESRLNDN 0.000 4 EEAIDAESR 0.000
TABLE-US-00030 TABLE X-V10-HLA-A0201-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; each start position s specified the
length of peptide is 9 no acids, and the end position for each
peptide is the start position plus eight. Start Subsequence Score 5
DILQKFQWI 4.160 6 ILQKFQWIY 1.480 14 YPTQKLNKM 0.343 12 WIYPTQKLN
0.151 8 QKFQWIYPT 0.088 1 KIPEDILQK 0.068 10 FQWIYPTQK 0.058 11
QWIYPTQKL 0.003 7 LQKFQWIYP 0.001 2 IPEDILQKF 0.000 9 KFQWIYPTQ
0.000 4 EDILQKFQW 0.000 3 PEDILQKFQ 0.000 15 PTQKLNKMR 0.000 13
IYPTQKLNK 0.000
TABLE-US-00031 TABLE X-V12-HLA-A0201-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
ALDGEESLL 8.545 1 RALDGEESL 2.205 8 SLLSKYNSN 0.414 3 LDGEESLLS
0.001 6 EESLLSKYN 0.001 5 GEESLLSKY 0.000 7 ESLLSKYNS 0.000 4
DGEESLLSK 0.000
TABLE-US-00032 TABLE XI-V1-HLA-A0201-10-MERS-193P1E1B 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
141 VLMEKNSMDI 269.051 366 KIPEDILQLL 96.947 231 CLNEDYTMGL 87.586
373 QLLSKYNSNL 79.041 340 NLTDPSSPTI 42.774 42 RILYDLHSEV 35.385
199 KVLKTPKCAL 24.206 110 QVLPNPPQAV 22.517 102 GLERYIVSQV 10.238
322 LEVEDRTSLV 9.426 355 LRTPTPTPEV 8.986 374 LLSKYNSNLA 8.446 13
SLASTLDCET 7.452 194 KQSLVKVLKT 6.082 381 NLATPIAIKA 4.968 228
YTMCLNEDYI 4.747 16 STLDCETARL 4.501 132 GIDFIKATKV 3.825 382
LATPIAIKAV 3.777 229 TMCLNEDYTM 3.588 188 IVTPPTKQSL 3.178 285
LVPTFCTPGL 3.178 207 ALKMDDFECV 2.266 127 LENQEGIDFI 2.138 162
YSPRVKKNSV 2.088 118 AVNLLDKARL 1.869 303 IALVSTNYPL 1.866 51
VQTLKDDVNI 1.798 189 VTPPTKQSLA 1.642 206 CALKMDDFEC 1.481 216
VTPKLEHFGI 1.429 261 NVFATPSPII 1.385 272 QLEKSDAEYI 1.285 130
QEGIDFIKAT 1.266 45 YDLHSEVQTL 1.161 269 IIQQLEKSDA 1.161 389
KAVPPSKRFL 1.142 157 FQKYGYSPRA 1.135 120 NLLDKARLEN 1.130 295
KIPSTKNSIA 0.980 332 LNSDTCFENL 0.905 94 RSPQLSDFGL 0.809 331
VLNSDTCFEN 0.735 264 ATPSPIIQQL 0.682 220 LEHFGISEYT 0.664 49
SEVQTLKDDV 0.663 223 FGISEYTMCL 0.641 109 SQVLPNPPQN 0.504 315
TNSSSNDLEV 0.454 208 LKMDDFECVT 0.416 97 QLSDFGLERY 0.344 282
NSPLVPTFCT 0.282 74 VKDDLSDPPV 0.269 290 CTPGLKIPSV 0.238 5
RSFCGKLRSL 0.237 112 LPNPPQAVNL 0.237 296 IPSTKNSIAL 0.237 152
KIREYFQKYG 0.234 34 SDFEDYPMRI 0.220 54 LKDDVNIPEL 0.190 306
VSTNYPLSKT 0.190 349 ISSYENLLRT 0.190 142 LMEKNSMDIM 0.180 281
TNSPLVPTFC 0.178 69 FQKTDVKDDL 0.171 63 LSNCENFQKT 0.157 358
TPTPPEVTKI 0.157 99 SDFGLERYIV 0.147 371 ILQLLSKYNS 0.127 276
SDAEYTNSPL 0.122 168 KNSVHEQEAI 0.117 257 RLNDNVFATP 0.116 271
QQLEKSDAEY 0.115 304 ALVSTNYPLS 0.112 9 GKLRSLASTL 0.110 327
RTSLVLNSDT 0.104 321 DLEVEDRTSL 0.103 346 SPTISSYENL 0.102 224
GISEYTMCLN 0.097 178 NSDNYKEEPA 0.089 20 CETARLQRAL 0.083 133
IDFIKATKVL 0.077 398 LKHGQNIRDM 0.076 57 DVNIPELSNC 0.075 329
SLVLNSDTCF 0.075 333 NSDTCFENLD 0.074 372 LQLLSKYNSN 0.071 365
TKIPEDILQL 0.068 379 NSNLATPIAI 0.068 43 ILYDLHSEVQ 0.067 209
KMDDFECVTP 0.062 129 FQEGIDFIKA 0.061 378 YNSNLATPIA 0.061 328
TSLVLNSDTC 0.059 14 LASTLDCETA 0.057 354 NLLRTPTPPE 0.055 77
DLSDPPVASS 0.053 111 VLPNPPQAVN 0.052 256 SRLNDNVFAT 0.051 98
LSDFGLERYI 0.051
TABLE-US-00033 TABLE XI-V5-HLA-A0201-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 SEKSPRSPQL 0.015 7 CISEKSPRSP 0.002 3 VASSCISEKS 0.001 6
SCISEKSPRS 0.000 5 SSCISEKSPR 0.000 4 ASSCISEKSP 0.000 2 PVASSCISEK
0.000 8 ISEKSPRSPQ 0.000 10 EKSPRSPQLS 0.000 1 PPVASSCISE 0.000
TABLE-US-00034 TABLE XI-V6-HLA-A0201-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EEAIDAESRL 0.031 9 DAESRLNDNV 0.002 8 IDAESRLNDN 0.002 1
NNKSEEAIDA 0.001 7 AIDAESRLND 0.001 10 AESRLNDNVF 0.001 3
KSEEAIDAES 0.000 2 NKSEEAIDAE 0.000 6 EAIDAESRLN 0.000 4 SEEAIDAESR
0.000
TABLE-US-00035 TABLE XI-V10-HLA-A0201-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; each start 1position 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 11 FQWIYPTQKL 82.694 2 KIPEDILQKF 0.338 7 ILQKFQWIYP 0.237 8
LQKFQWIYPV 0.103 6 DILQKFQWIY 0.033 13 WIYPTQKLNK 0.030 5
EDILQKFQWI 0.011 14 IYPTQKLNKM 0.003 15 YPTQKLNKMR 0.000 3
IPEDILQKFQ 0.000 9 QKFQWIYPTQ 0.000 4 PEDILQKFQW 0.000 1 TKIPEDILQK
0.000 10 KFQWIYPTQK 0.000 12 QWIYPTQKLN 0.000
TABLE-US-00036 TABLE XI-V12-HLA-A0201-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 Subsectuence
Score 9 SLLSKYNSNL 79.041 2 RALDGEESLL 4.501 3 ALDGEESLLS 0.030 1
QRALDGEESL 0.001 6 GEESLLSKYN 0.001 4 LDGEESLLSK 0.000 8 ESLLSKYNSN
0.000 7 EESLLSKYNS 0.000 5 DGEESLLSKY 0.000
TABLE-US-00037 TABLE XII-V1-HLA-A3-9MERS-193P1E1B 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
239 GLKNARNNK 60.000 381 NLATPIAIK 45.000 97 QLSDFGLER 24.000 152
KIREYFQKY 16.200 132 GIDFIKATK 19.000 129 NQEGIDFIK 4.050 397
FLKHGQNIR 4.000 272 QLEKSDAEY 4.000 387 AIKAVPPSK 3.000 28
ALDGEESDF 3.000 194 KQSLVKVLK 2.700 137 KATKVLMEK 2.700 304
ALVSTNYPL 2.700 197 LVKVLKTPK 2.000 374 LLSKYNSNL 1.800 10
KLRSLASTL 1.800 224 GISEYTMCL 1.620 142 LMEKNSMDI 1.200 43
ILYDLHSEV 1.000 209 KMDDFECVT 0.900 200 VLKTPKCAL 0.900 257
RLNDNVFAT 0.900 366 KIPEDILQL 0.810 267 SPIIQQLEK 0.600 207
ALKMDDFEC 0.600 306 VSTNYPLSK 0.600 391 VPPSKRFLK 0.600 302
SIALVSTNY 0.600 17 TLDCETARL 0.600 46 DLHSEVQTL 0.540 358 TPTPPEVTK
0.450 236 YTMGLKNAR 0.450 215 CVTPKLEHF 0.450 219 KLEHFGISE 0.360
861 PVIVTPPTK 0.300 83 VASSCISGK 0.300 111 VLPNPPQAV 0.300 63
LSNCENFQK 0.300 330 LVLNSDTCF 0.300 16 STLDCETAR 0.300 261
NVFATPSPI 0.300 228 YTMCLNEDY 0.300 329 SLVLNSDTC 0.300 2 DPIRSFCGK
0.270 102 GLERYIVSQ 0.270 370 DILQLLSKY 0.270 118 AVNLLDKAR 0.200
116 PQAVNLLDK 0.180 402 QNIRDVSNK 0.107 295 KIPSTKNSI 0.180 348
TISSYENLL 0.180 154 REYFQKYGY 0.180 160 YGYSPRVKK 0.150 287
PTFCTPGLK 0.150 340 NLTDPSSPT 0.150 149 DIMKIREYF 0.135 211
DDFECVTPK 0.135 157 FQKYGYSPR 0.120 31 GEESDFEDY 0.108 389
KAVPPSKRF 0.101 229 TMCLNEDYT 0.100 151 MKIREYFQK 0.090 140
KVLMEKNSM 0.090 314 KTNSSSNDL 0.090 205 KCALKMDDF 0.090 293
GLKIPSTKN 0.090 175 EAINSDNYK 0.090 39 YPMRILYDL 0.061 67 ENFQKTDVK
0.060 202 KTPKCALKM 0.060 150 IMKIREYFQ 0.060 86 SCISGKSPR 0.060 53
TLKDDVNIP 0.060 47 LHSEVQTLK 0.045 199 KVLKTPKCA 0.045 110 QVLPNPPA
0.045 364 VTKIPEDIL 0.045 141 VLMEKNSMD 0.045 52 QTLKDDVNI 0.045
189 VTPPTKQSL 0.045 341 LTDPSSPTI 0.045 349 ISSYENLLR 0.040 121
LLDKARLEN 0.040 147 SMDIMKIRE 0.040 191 PPTKQSLVK 0.040 196
SLVKVLKTP 0.034 146 NSMDIMKIR 0.034 201 LKTPKCALK 0.030 231
CLNEDYTMG 0.030 373 QLLSKYNSN 0.030 34 SDFEDYPMR 0.030 24 RLQRALDGE
0.030 98 LSDFGLERY 0.030 354 NLLRTPTPP 0.030 355 LLRTPTPPE 0.030 13
SLASTLDCE 0.030 363 EVTKIPEDI 0.027 369 EDILQLLSK 0.027 233
NEDYTMGLK 0.027 319 SNDLEVEDR 0.024
TABLE-US-00038 TABLE XII-V5-HLA-A3-9MERS-193P1E1B Each peptide is a
portion of SEQ ID NO: 11; 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
VASSCISEK 0.300 5 SCISEKSPR 0.060 6 CISEKSPRS 0.006 1 PVASSCISE
0.000 3 ASSCISEKS 0.000 9 EKSPRSPQL 0.000 8 SEKSPRSPQ 0.000 4
SSCISEKSP 0.000 7 ISEKSPRSP 0.000
TABLE-US-00039 TABLE XII-V6-HLA-A3-9MERS-193P1E1B Each peptide is a
portion of SEQ ID NO: 13; 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 Subsectuence Score
4 EEAIDAESR 0.004 5 EAIDAESRL 0.003 2 KSEEAIDAE 0.001 1 NKSEEAIDA
0.001 9 AESRLNDNV 0.001 6 AIDAESRLN 0.000 3 SEEAIDAES 0.000 8
DAESRLNDN 0.000 7 IDAESRLND 0.000
TABLE-US-00040 TABLE XII-V10-HLA-A3-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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
ILQKFQWIY 36.000 1 KIPEDILQK 27.000 10 FQWIYPTQK 9.000 5 DILQKFQWI
0.081 2 IPEDILQKF 0.045 13 IYPTQKLNK 0.040 15 PTQKLNKMR 0.010 12
WIYPTQKLN 0.007 8 QKFQWIYPT 0.007 14 YPTQKLNKM 0.003 11 QWIYPTQKL
0.001 7 LQKFQWIYP 0.001 4 EDILQKFQW 0.000 9 KFQWIYPTQ 0.000 3
PEDILQKFQ 0.000
TABLE-US-00041 TABLE XII-V12-HLA-A3-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
ALDGEESLL 0.900 5 GEESLLSKY 0.054 8 SLLSKYNSN 0.030 4 DGEESLLSK
0.027 1 RALDGEESL 0.009 7 ESLLSKYNS 0.000 3 LDGEESLLS 0.000 6
EESLLSKYN 0.000
TABLE-US-00042 TABLE XIII-V1-HLA-A3-10MERS-193P1E1B 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
150 IMKIREYFQK 60.000 196 SLVKVLKTPK 30.000 200 VLKTPKCALK 20.000
219 KLEHFGISEY 18.000 62 ELSNCENFQK 18.000 305 LVSTNYPLSK 12.000 46
DLHSEVQTLM 9.000 390 AVPPSKRFLK 9.000 97 QLSDFGLERY 6.000 231
CLNEDYTMGL 5.400 401 GQNIRDVSNK 5.400 126 RLENQEGIDF 4.000 329
SLVLNSDTCF 3.000 102 GLERYIVSQV 2.700 373 QLLSKYNSNL 2.700 141
VLMEKNSMDI 2.700 357 RTPTPPEVTK 1.500 348 TISSYENLLR 0.800 366
KIPEDILQLL 0.608 207 ALKMDDFECV 0.600 147 SMDIMKIREY 0.600 381
NLATPIAIKA 0.600 387 AIKAVPPSKR 0.600 340 NLTDPSSPTI 0.600 229
TMCLNEDYTM 0.600 386 IAIKAVPPSK 0.450 261 NVFATPSPII 0.450 199
KVLKTPKCAL 0.405 190 TPPTKQSLVK 0.400 82 PVASSCISGK 0.300 291
TPGLKIPSTM 0.300 142 LMEKNSMDIM 0.300 280 YTNSPLVPTF 0.300 271
QQLEKSDAEY 0.270 355 LLRTPTPREV 0.200 374 LLSKYNSNLA 0.200 321
DLEVEDRTSL 0.180 380 SNLATPIAIK 0.135 143 MEKNSMDIMQ 0.120 371
ILQLLSKYNS 0.120 239 GLKNARNNKS 0.120 96 PQLSDFGLER 0.108 13
SLASTLDCET 0.100 43 ILYDLHSEVQ 0.100 272 QLEKSDAEYT 0.100 159
KYGYSPRVKK 0.090 136 IKATKVLMEK 0.090 216 VTPKLEHRGI 0.090 188
IVTPPTKQSL 0.090 257 RLNDNVFATP 0.090 185 EPVIVTPPTK 0.090 126
ENQEGIDFIM 0.081 264 ATPSPIIQQL 0.068 110 QVLPNRRQAV 0.068 1
MDPIRSFCGK 0.060 210 MDDFECVTPK 0.060 115 PPQAVNLLDK 0.060 304
ALVSTNYPLS 0.060 295 KIPSTKNSIN 0.060 293 GLKIPSTKNS 0.060 53
TLKDDVNIPE 0.060 118 AVNLLDKARL 0.060 132 GIDFIKATKV 0.060 331
VLNSDTCFEN 0.060 174 QEAINSDNYM 0.060 66 CENFQKTDVK 0.060 10
KLRSLASTLE 0.060 286 VPTFCTPGLK 0.060 285 LVPTFCTPGL 0.060 318
SSNDLEVEDR 0.060 209 KMDDFECVTP 0.060 120 NLLDKARLEN 0.060 77
DLSDPPVASS 0.054 194 KQSLVKVLKT 0.054 129 NQEGIDFIKA 0.054 151
MKIREYFQKY 0.054 301 NSIALVSTNY 0.045 354 NLLRIPTPPE 0.045 16
STLDCETARL 0.045 287 PTFCTPGLKI 0.045 397 FLKHGQNIRD 0.040 323
EVEDRTSLVL 0.036 173 EQEAINSDNY 0.036 193 TKQSLVKVLK 0.030 238
MGLKNARNNK 0.030 28 ALDGEESDFE 0.030 42 RILYDLHSEV 0.030 117
QAVNLLDKAR 0.030 27 RALDGEESDN 0.030 111 VLPNPPQAVN 0.030 92
SPRSPQLSDF 0.030 121 LLDKARLENQ 0.030 358 TPTPPEVTKI 0.027 303
IALVSTNYPL 0.027 34 DFEDYPMRI 0.027 363 EVTKIPEDIL 0.027 145
KNSMDIMKIR 0.027 69 FQKTDVEDDL 0.027 36 FEDYPMRILY 0.024 40
PMRILYDLHS 0.024
TABLE-US-00043 TABLE XIII-V5-HLA-A3-10MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
PVASSCISEK 0.300 5 SSCISEKSPR 0.020 9 SEKSPRSPQL 0.002 6 SCISEKSPRS
0.001 3 VASSCISEKS 0.001 7 CISEKSPRSP 0.000 8 ISEKSPRSPQ 0.000 1
PPVASSCISE 0.000 4 ASSCISEKSP 0.000 10 EKSPRSPQLS 0.000
TABLE-US-00044 TABLE XIII-V6-HLA-A3-10MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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
SEEAIDAESR 0.012 10 AESRLNDNVF 0.004 7 AIDAESRLND 0.001 3
KSEEAIDAES 0.001 1 NNKSEEAIDA 0.001 9 DAESRLNDNV 0.001 5 EEAIDAESRL
0.001 8 IDAESRLNDN 0.000 2 NKSEEAIDAE 0.000 6 EAIDAESRLN 0.000
TABLE-US-00045 TABLE XIII V10-HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 13 WIYPTQKLNK 30.000 2 KIPEDILQKF 2.025 6 DILQKFQWIY 1.620 10
KFQWIYPTQK 0.180 1 TKIPEDILQK 0.135 11 FQWIYPTQKL 0.135 8
LQKFQWIYPT 0.041 7 ILQKFQWIYP 0.040 15 YPTQKLNKMR 0.020 5
EDILQKFQWI 0.001 14 IYPTQKLNKM 0.000 4 PEDILQKFQW 0.000 9
QKFQWIYPTQ 0.000 3 IPEDILQKFQ 0.000 12 QWIYPTQKLN 0.000
TABLE-US-00046 TABLE XIII V12-HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 SLLSKYNSNL 2.700 3 ALDGEESLLS 0.120 4 LDGEESLLSK 0.090 2
RALDGEESLL 0.009 5 DGEESLLSKY 0.003 1 QRALDGEESL 0.001 7 EESLLSKYNS
0.000 6 GEESLLSKYN 0.000 8 ESLLSKYNSN 0.000
TABLE-US-00047 TABLE XIV V1-HLA-A1101-9mers-193P1E1B 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
197 LVKVLKTPK 2.000 194 KQSLVKVLK 1.800 129 NQEGIDFIK 1.800 239
GLKNARNNK 1.200 137 KATKVLMEK 1.200 132 GIDFIKATK 1.200 391
VPPSKRFLK 0.600 267 SPIIQQLEK 0.600 387 AIKAVPPSK 0.400 236
YTMGLKNAR 0.400 381 NLATPIAIK 0.400 186 PVIVTPPTK 0.300 16
STLDCETAR 0.300 118 AVNLLDKAR 0.200 287 PTFCTPGLK 0.200 358
TPTPPEVTK 0.200 83 VASSCISGK 0.200 97 QLSDFGLER 0.160 116 PQAVNLLDK
0.120 159 KYGYSPRVK 0.120 157 FQKYGYSPR 0.120 151 MKIREYFQK 0.090
140 KVLMEKNSM 0.090 175 EAINSDNYK 0.090 2 DPIRSFCGK 0.090 397
FLKHGQNIR 0.080 402 QNIRDVSNK 0.060 63 LSNCENFQK 0.060 202
KTPKCALKM 0.060 233 NEDYTMGLK 0.060 86 SCISGKSPR 0.060 199
KVLKTPKCA 0.045 160 YGYSPRVKK 0.040 191 PPTKQSLVK 0.040 306
VSTNYPLSK 0.040 261 NVFATPSPI 0.040 330 LVLNSDTCF 0.030 110
QVLPNPPQA 0.030 314 KTNSSSNDL 0.030 67 ENFQKTDVK 0.024 224
GISEYYMCL 0.024 19 DCETARLQR 0.024 366 KIPEDILQL 0.024 47 LHSEVQTLK
0.020 228 YTMCLNEDY 0.020 215 CVTPKLEHF 0.020 201 LKTPKCALK 0.020
369 EDILQLLSK 0.018 52 QTLKDDVNI 0.015 295 KIPSTKNSI 0.012 144
EKNSMDIMK 0.012 211 DDFECVTPK 0.012 304 ALVSTNYPL 0.012 10
KLRSLASTL 0.012 152 KIREYFQKY 0.012 298 STKNSIALV 0.010 189
VTPPTKQSL 0.010 341 LTDPSSPTI 0.010 364 VTKIPEDIL 0.010 396
RFLKHGQNI 0.009 142 LMEKNSMDI 0.008 349 ISSYENLLR 0.008 34
SDFEDYPMR 0.008 319 SNDLEVEDR 0.008 43 ILYDLHSEV 0.008 39 YPMRILYDL
0.008 154 REYFQKYGY 0.007 323 EVEDRTSLV 0.006 165 RVKKNSVHE 0.006
50 EVQTLKDDV 0.006 363 EVTKIPEDI 0.006 205 KCALKMDDF 0.006 217
TPKLEHFGI 0.006 95 SPQLSDFGL 0.006 270 IQQLEKSDA 0.006 230
MCLNEDYTM 0.006 383 ATPIAIKAV 0.005 389 KAVPPSKRF 0.005 200
VLKTPKCAL 0.004 146 NSMDIMKIR 0.004 382 LATPIAIKA 0.004 222
HFGISEYTM 0.004 272 QLEKSDAEY 0.004 28 ALDGEESDF 0.004 111
VLPNPPQAV 0.004 288 TFCTPGLKI 0.004 135 FIKATKVLM 0.004 388
IKAVPPSKR 0.004 17 TLDCETARL 0.004 374 LLSKYNSNL 0.004 348
TISSYENLL 0.004 181 NYKEEPVIV 0.004 302 SIALVSTNY 0.004 257
RLNDNVFAT 0.004 249 EEAIDTESR 0.004 292 PGLKIPSTK 0.003 71
KTDVKDDLS 0.003 357 RTPTPPEVT 0.003 117 QAVNLLDKA 0.003 327
RTSLVLNSD 0.003
TABLE-US-00048 TABLE XIV V5-HLA-A1101-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 VASSCISEK 0.200 5 SCISEKSPR 0.060 1 PVASSCISE 0.000 6
CISEKSPRS 0.000 9 EKSPRSPQL 0.000 8 SEKSPRSPQ 0.000 3 ASSCISEKS
0.000 4 SSCISEKSP 0.000 7 ISEKSPRSP 0.000
TABLE-US-00049 TABLE XIV V6-HLA-A1101-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EEAIDAESR 0.004 5 EAIDAESRL 0.001 9 AESRLNDNV 0.001 1
NKSEEAIDA 0.000 2 KSEEAIDAE 0.000 8 DAESRLNDN 0.000 3 SEEAIDAES
0.000 7 IDAESRLND 0.000 6 AIDAESRLN 0.000
TABLE-US-00050 TABLE XIV V10-HLA-A1101-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 KIPEDILQK 2.400 10 FQWIYPTQK 1.200 13 IYPTQKLNK 0.800 15
PTQKLNKMR 0.010 6 ILQKFQWIY 0.008 14 YPTQKLNKM 0.002 2 IPEDILQKF
0.002 5 DILQKFQWI 0.002 7 LQKFQWIYP 0.001 9 KFQWIYPTQ 0.001 12
WIYPTQKLN 0.000 11 QWIYPTQKL 0.000 4 EDILQKFQW 0.000 8 QKFQWIYPT
0.000 3 PEDILQKFQ 0.000
TABLE-US-00051 Table XIV V12-HLA-A1101-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 DGEESLLSK 0.012 1 RALDGEESL 0.009 2 ALDGEESLL 0.004 5
GEESLLSKY 0.002 8 SLLSKYNSN 0.001 3 LDGEESLLS 0.000 7 ESLLSKYNS
0.000 6 EESLLSKYN 0.000
TABLE-US-00052 TABLE XV V1-HLA-A11-10mers-193P1E1B 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
390 AVPPSKRFLK 6.000 305 LVSTNYPLSK 4.000 357 RTPTPPEVTK 3.000 401
GQNIRDVSNK 1.800 159 KYGYSPRVKK 1.200 150 IMKIREYFQK 1.200 196
SLVKVLKTPK 0.600 200 VLKTPKCALK 0.400 190 TPPTKQSLVK 0.400 62
ELSNCENFQK 0.360 386 IAIKAVPPSK 0.300 291 TPGLKIPSTK 0.200 286
VPTFCTPGLK 0.200 82 PVASSCISGK 0.200 396 RFLKHGQNIR 0.180 348
TISSYENLLR 0.160 46 DLHSEVQTLK 0.120 143 MEKNSMDIMK 0.120 199
KVLKTPKCAL 0.090 185 EPVIVTPPTK 0.090 387 AIKAVPPSKR 0.080 380
SNLATPIAIK 0.060 174 QEAINSDNYK 0.060 66 CENFQKTDVK 0.060 136
IKATKVLMEK 0.040 115 PPQAVNLLDK 0.040 156 YFQKYGYSPR 0.040 261
NVFATPSPII 0.040 232 LNEDYTMGLK 0.040 96 PQLSDFGLER 0.036 128
ENQEGIDFIK 0.036 238 MGLKNARNNK 0.030 110 QVLPNPPQAV 0.030 117
QAVNLLDKAR 0.030 216 VTPKLEHFGI 0.030 235 DYTMGLKNAR 0.024 126
RLENQEGIDF 0.024 188 IVTPPTKQSL 0.020 193 TKQSLVKVLK 0.020 1
MDPIRSFCGK 0.020 210 MDDFECVTPK 0.020 118 AVNLLDKARL 0.020 285
LVPTFCTPGL 0.018 42 RILYDLHSEV 0.018 141 VLMEKNSMDI 0.016 16
STLDCETARL 0.015 145 KNSMDIMKIR 0.012 129 NQEGIDFIKA 0.012 219
KLEHFGISEY 0.014 132 GIDFIKATKV 0.012 368 PEDILQLLSK 0.012 366
KIPEDILQLL 0.012 295 KIPSTKNSIA 0.012 323 EVEDRTSLVL 0.012 246
SEEAIDTESR 0.012 102 GLERYIVSQV 0.012 377 KYNSNLATPI 0.012 264
ATPSPIIQQL 0.012 280 YTNSPLVPTF 0.010 189 VTPPTKQSLV 0.010 27
RALDGEESDF 0.009 131 EGIDFIKATK 0.009 140 KVLMEKNSMD 0.009 109
SQVLPNPPQA 0.009 271 QQLEKSDAEY 0.009 231 CLNEDYTMGL 0.008 18
LDCETARLQR 0.008 381 NLATPIAIKA 0.008 229 TMCLNEDYTM 0.008 51
VQTLKDDVNI 0.006 373 QLLSKYNSNL 0.006 69 FQKTDVKDDL 0.006 303
IALVSTNYPL 0.006 329 SLVLNSDTCF 0.006 124 KARLENQEGI 0.006 363
EVTKIPEDIL 0.006 71 KTDVKDDLSD 0.006 157 FQKYGYSPRV 0.006 165
RVKKNSVHEQ 0.006 158 QKYGYSPRVK 0.004 266 PSPIIQQLEK 0.004 269
IIQQLEKSDA 0.004 287 PTFCTPGLKI 0.004 85 SSCISGKSPR 0.004 39
YPMRILYDLH 0.004 374 LLSKYNSNLA 0.004 355 LLRTPTPPEV 0.004 3
PIRSFCGKLR 0.004 296 IPSTKNSIAL 0.004 15 ASTLDCETAR 0.004 97
QLSDFGLERY 0.004 207 ALKMDDFECV 0.004 170 SVHEQEAINS 0.004 318
SSNDLEVEDR 0.004 142 LMEKNSMDIM 0.004 391 VPPSKRFLKH 0.004 340
NLTDPSSPTI 0.004 194 KQSLVKVLKT 0.004 105 RYIVSQVPLN 0.004 314
KTNSSSNDLE 0.003
TABLE-US-00053 TABLE XV V5-HLA-A1101-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 PVASSCISEK 0.200 5 SSCISEKSPR 0.004 9 SEKSPRSPQL 0.001 6
SCISEKSPRS 0.000 3 VASSCISEKS 0.000 1 PPVASSCISE 0.000 7 CISEKSPRSP
0.000 8 ISEKSPRSPQ 0.000 4 ASSCISEKSP 0.000 10 EKSPRSPQLS 0.000
TABLE-US-00054 TABLE XV V10-HLA-A1101-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 13 WIYPTQKLNK 1.600 10 KFQWIYPTQK 0.600 1 TKIPEDILQK 0.060 15
YPTQKLNKMR 0.020 2 KIPEDILQKF 0.012 11 FQWIYPTQKL 0.012 14
IYPTQKLNKM 0.004 6 DILQKFQWIY 0 004 8 LQKFQWIYPT 0.001 7 ILQKFQWIYP
0.001 4 PEDILQKFQW 0.000 3 IPEDILQKFQ 0.000 5 EDILQKFQWI 0.000 9
QKFQWIYPTQ 0.000 12 QWIYPTQKLN 0.000
TABLE-US-00055 TABLE XV V6-HLA-A1101-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 SEEAIDAESR 0.012 1 NNKSEEAIDA 0.001 7 AIDAESRLND 0.001 9
DAESRLNDNV 0.001 10 AESRLNDNVF 0.001 5 EEAIDAESRL 0.000 3
KSEEAIDAES 0.000 8 IDAESRLNDN 0.000 2 NKSEEAIDAE 0.000 6 EAIDAESRLN
0.000
TABLE-US-00056 TABLE XV V12-HLA-A1101-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 LDGEESLLSK 0.000 2 RALDGEESLL 0.009 9 SLLSKYNSNL 0.006 3
ALDGEESLLS 0.001 1 QRALDGEESL 0.000 6 GEESLLSKYN 0.000 5 DGEESLLSKY
0.000 7 EESLLSKYNS 0.000 8 ESLLSKYNSN 0.000
TABLE-US-00057 TABLE XVI V1-HLA-A24-9mers-193P1E1B 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
212 DFECVTPKL 46.200 134 DFIKATKVL 30.000 6 SFCGKLRSL 20.000 396
RFLKHGQNI 18.000 366 KIPEDILQL 14.400 314 KTNSSSNDL 14.400 367
IPEDILQLL 12.096 10 KLRSLASTL 9.600 35 DFEDYPMRI 9.000 189
VTPPTKQSL 8.640 39 YPMRILYDL 8.400 265 TPSPIIQQL 8.064 309
NYPLSKTNS 7.500 114 NPPQAVNLL 7.200 250 EAIDTESRL 7.200 389
KAVPPSKRF 7.200 390 AVPPSKRFL 7.200 232 LNEDYTMGL 7.200 161
GYSPRVKKN 6.600 95 SPQLSDFGL 6.000 119 VNLLDKARL 6.000 304
ALVSTNYPL 6.000 277 DAEYTNSPL 6.000 181 NYKEEPVIV 6.000 288
TFCTPGLKI 5.500 235 DYTMGLKNA 5.000 262 VFATPSPII 5.000 155
EYFQKYGYS 5.000 224 GISEYTMCL 4.800 46 DLHSEVQTL 4.800 21 ETARLQRAL
4.800 333 NSDTCFENL 4.800 348 TISSYENLL 4.800 149 DIMKIREYF 4.200
200 VLKTPKCAL 4.000 286 VPTFCTPGL 4.000 364 VTKIPEDIL 4.000 17
TLDCETARL 4.000 205 KCALKMDDF 4.000 374 LLSKYNSNL 4.000 295
KIPSTKNSI 3.600 330 LVLNSDTCF 3.000 244 RNNKSEEAI 3.000 281
TNSPLVPTF 2.880 222 HFGISEYTM 2.500 215 CVTPKLEHF 2.400 255
ESRLNDNVF 2.400 145 KNSMDIMKI 2.200 28 ALDGEESDF 2.000 128
ENQEGIDFI 1.800 140 KVLMEKNSM 1.800 202 KTPKCALKM 1.650 380
SNLATPIAI 1.500 105 RYIVSQVLP 1.500 142 LMEKNSMDI 1.500 169
NSVHEQEAI 1.500 80 DPPVASSCI 1.500 52 QTLKDDVNI 1.500 377 KYNSNLATP
1.500 363 EVTKIPEDI 1.400 341 LTDPSSPTI 1.200 378 YNSNLATPI 1.200
180 DNYKEEPVI 1.000 217 TPKLEHFGI 1.000 159 KYGYSPRVK 1.000 261
NVFATPSPI 1.000 337 CFENLTDPS 0.900 351 SYENLLRTP 0.900 55
KDDVNIPEL 0.880 230 MCLNEDYTM 0.750 38 DYPMRILYD 0.750 322
LEVEDRTSL 0.720 193 TKQSLVKVL 0.720 113 PNPPQAVNL 0.720 104
ERYIVSQVL 0.672 70 QKTDVKDDL 0.672 227 EYTMCLNED 0.660 347
PTISSYENL 0.600 33 ESDFEDYPM 0.500 44 LYDLHSEVQ 0.500 135 FIKATKVLM
0.500 279 EYTNSPLVP 0.500 100 DFGLERYIV 0.500 90 GKSPRSPQL 0.480 3
PIRSFCGKL 0.440 324 VEDRTSLVL 0.400 297 PSTKNSIAL 0.400 36
FEDYPMRIL 0.400 152 KIREYFQKY 0.380 91 KSPRSPQLS 0.360 257
RLNDNVFAT 0.360 199 KVLKTPKCA 0.300 357 RTPTPPEVT 0.300 12
RSLASTLDC 0.300 127 LENQEGIDF 0.300 168 KNSVHEQEA 0.264 209
KMDDFECVT 0.240 185 EPVIVTPPT 0.210 282 NSPLVPTFC 0.210 400
HGQNIRDVS 0.210
TABLE-US-00058 TABLE XVI V5-HLA-A24-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
EKSPRSPQL 0.480 3 ASSCISEKS 0.154 6 CISEKSPRS 0.120 7 ISEKSPRSP
0.015 5 SCISEKSPR 0.015 2 VASSCISEK 0.011 4 SSCISEKSP 0.010 8
SEKSPRSPQ 0.001 1 PVAAACISE 0.001
TABLE-US-00059 TABLE XVI V6-HLA-A24-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; each start position is specified, is
the length of peptide is 9 amino acids, and the end position for
each peptide is the start position plus eight. Start Subsequence
Score 5 EAIDAESRL 7.200 8 DAESRLNDN 0.180 6 AIDAESRLN 0.100 2
KSEEAIDAE 0.036 3 SEEAIDAES 0.023 9 AESRLNDNV 0.012 1 NKSEEAIDA
0.012 7 IDAESRLND 0.001 4 EEAIDAESR 0.001
TABLE-US-00060 TABLE XVI-V10-HLA-A24-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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 11
QWIYPTQKL 7.920 2 IPEDILQKF 6.653 5 DILQKFQWI 2.160 13 IYPTQKLNK
0.750 14 YPTQKLNKM 0.660 9 KFQWIYPTQ 0.210 6 ILQKFQWIY 0.150 12
WIYPTQKLN 0.120 1 KIPEDILQK 0.036 4 EDILQKFQW 0.015 7 LQKFQWIYP
0.010 8 QKFQWIYPT 0.010 10 FQWIYPTQK 0.010 15 PTQKLNKMR 0.002 3
PEDILQKFQ 0.000
TABLE-US-00061 TABLE XVI-V12-HLA-A24-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
RALDGEESL 14.400 2 ALDGEESLL 4.000 8 SLLSKYNSN 0.180 7 ESLLSKYNS
0.150 5 GEESLLSKY 0.020 4 DGEESLLSK 0.018 6 EESLLSKYN 0.012 3
LDGEESLLS 0.012
TABLE-US-00062 TABLE XVII-V1-HLA-A24-10MERS-193P1E1B 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 38
DYPMRILYDL 420.000 377 KYNSNLATPI 180.000 35 DFEDYPMRIL 36.000 366
KIPEDILQLL 24.192 105 RYIVSQVLPN 15.000 389 KAVPPSKRFL 14.400 94
RSPQLSDFGL 12.000 199 KVLKTPKCAL 12.000 264 ATPSPIIQQL 10.080 351
SYENLLRTPT 9.000 309 NYPLSKINSS 9.000 161 GYSPRVKKNS 8.400 5
RSFCGKLRSL 8.000 231 CLNEDYTMGL 7.200 16 STLDCETARL 7.200 112
LPNPPQAVNL 7.200 323 EVEDRTSLVL 7.200 27 RALDGEESDF 7.200 2
DPIRSFCGKL 6.600 227 EYTMCLNEDY 6.000 181 NYKEEPVIVT 6.000 321
DLEVEDRTSL 6.000 118 AVNLLDKARL 6.000 373 QLLSKYNSNL 6.000 126
RLENQEGIDF 6.000 285 LVPTFCTPGL 6.000 223 FGISEYTMCL 6.000 303
IALVSTNYPL 6.000 188 IVTPPTKQSL 5.760 332 LNSDTCFENL 5.760 69
FQKTDVKDDL 5.600 44 LYDLHSEVQT 5.000 279 EYTNSPLVPT 5.000 296
IPSTKNSIAL 4.000 363 EVTKIPEDIL 4.000 89 SGKSPRSPQL 4.000 346
SPTISSYENL 4.000 134 DFIKATKVLM 3.750 280 YTNSPLVPTF 3.600 60
IPELSNCENF 3.000 214 ECVTPKLEHF 3.000 329 SLVLNSDTCF 3.000 124
KARLENQEGI 2.000 92 SPRSPQLSDF 2.000 168 KNSVHEQEAI 2.000 141
VLMEKNSMDI 1.800 260 DNVFATPSPI 1.500 379 NSNLATPIAI 1.500 216
VTPKLEHFGI 1.500 358 TPTPPEVTKI 1.320 340 NLTDPSSPTI 1.200 98
LSDFGLERYI 1.200 159 KYGYSPRVKK 1.100 51 VQTLKDDVNI 1.000 261
NVFATPSPII 1.000 113 PNPPQAVNLL 0.864 337 CFENLTDPSS 0.750 142
LMEKNSMDIM 0.750 211 DDFECVTPKL 0.739 9 GKLRSLASTL 0.720 365
TKIPEDILQL 0.720 347 PTISSYENLL 0.720 45 YDLHSEVQTL 0.720 235
DYTMGLKNAR 0.720 103 LERYIVSQVL 0.672 6 SFCGKLRSLA 0.600 247
KSEEAIDTES 0.554 54 LKDDVNIPEL 0.528 155 EYFQKYGYSP 0.500 222
HFGISEYTMC 0.500 100 DFGLERYIVS 0.500 229 TMCLNEDYTM 0.500 276
SDAEYTNSPL 0.480 20 CETARLQRAL 0.480 192 PTKQSLVKVL 0.480 313
SKTNSSSNDL 0.480 148 MDIMKIREYF 0.420 133 IDFIKATKVL 0.400 249
EEAIDTESRL 0.400 42 RILYDLHSEV 0.396 219 KLEHFGISEY 0.330 295
KIPSTKNSIA 0.300 137 KATKVLMEKN 0.264 300 KNSIALVSTN 0.240 254
TESRLNDNVF 0.240 395 KRFLKHGQNI 0.240 327 RTSLVLNSDT 0.240 63
LSNCENFQKT 0.238 194 KQSLVKVLKT 0.220 294 LKIPSTKNSI 0.216 110
QVLPNPPQAV 0.216 367 IPEDILQLLS 0.216 30 DGEESDFEDY 0.216 102
GLERYIVSQV 0.210 301 NSIALVSTNY 0.210 388 IKAVPPSKRF 0.200 271
QQLEKSDAEY 0.198 59 NIPELSNCEN 0.198 129 NQEGIDFIKA 0.198 120
NLLDKARLEN 0.198
TABLE-US-00063 TABLE XVII-V5-HLA-A24-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 SEKSPRSPQL 0.400 3 VASSCISEKS 0.154 6 SCISEKSPRS 0.150 8
ISEKSPRSPQ 0.015 10 EKSPRSPQLS 0.014 7 CISEKSPRSP 0.012 4
ASSCISEKSP 0.010 5 SSCISEKSPR 0.010 1 PPVASSCISE 0.002 2 PVASSCISEK
0.001
TABLE-US-00064 TABLE XVII-V6-HLA-A24-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 KSEEAIDAES 0.554 5 EEAIDAESRL 0.400 10 AESRLNDNVF 0.240 6
EAIDAESRLN 0.180 9 DAESRLNDNA 0.180 1 NNKSEEAIDM 0.100 8 IDAESRLNDN
0.014 7 AIDAESRLND 0.010 4 SEEAIDAESR 0.002 2 NKSEEAIDAE 0.001
TABLE-US-00065 TABLE XVII-V10-HLA-A24-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 IYPTQKLNKM 49.500 2 KIPEDILQKF 13.306 11 FQWIYPTQKL 5.280
5 EDILQKFQWI 0.216 10 KFQWIYPTQK 0.150 6 DILQKFQWIY 0.150 12
QWIYPTQKLN 0.150 8 LQKFQWIYPT 0.100 3 IPEDILQKFQ 0.022 7 ILQKFQWIYP
0.015 15 YPTQKLNKMR 0.012 13 WIYPTQKLNK 0.012 1 TKIPEDILQK 0.002 9
QKFQWIYPTQ 0.001 4 PEDILQKFQW 0.001
TABLE-US-00066 TABLE XVII-V12-HLA-A24-10MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 RALDGEESLL 14.400 9 SLLSKYNSNL 6.000 1 QRALDGEESL 0.400 5
DGEESLLSKY 0.238 8 ESLLSKYNSN 0.180 3 ALDGEESLLS 0.100 6 GEESLLSKYN
0.018 7 EESLLSKYNS 0.010 4 LDGEESLLSK 0.001
TABLE-US-00067 TABLE XVIII-V1-HLA-B7-9MERS-193P1E1B 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
39 YPMRILYDL 240.000 265 TPSPIIQQL 80.000 114 NPPQAVNLL 80.000 95
SPQLSDFGL 80.000 286 VPTFCTPGL 80.000 390 AVPPSKRFL 60.000 10
KLRSLASTL 40.000 163 SPRVKKNSV 40.000 367 IPEDILQLL 24.000 304
ALVSTNYPL 12.000 250 EAIDTESRL 12.000 80 DPPVASSCI 8.000 217
TPKLEHFGI 8.000 200 VLKTPKCAL 6.000 364 VTKIPEDIL 6.000 140
KVLMEKNSM 5.000 189 VTPPTKQSL 4.000 3 PIRSFCGKL 4.000 314 KTNSSSNDL
4.000 374 LLSKYNSNL 4.000 224 GISEYTMCL 4.000 190 TPPTKQSLV 4.000
119 VNLLDKARL 4.000 366 KIPEDILQL 4.000 46 DLHSEVQTL 4.000 348
TISSYENLL 4.000 21 ETARLARAL 4.000 277 DAEYTNSPL 3.600 92 SPRSPQLSD
3.000 283 SPLVPTFCT 3.000 185 EPVIVTPPT 2.000 261 NVFATPSPI 2.000
296 TPGLKIPST 2.000 232 LNEDYTMGL 1.200 17 TLDCETARL 1.200 333
NSDTCFENL 1.200 50 EVQTLKDDV 1.000 135 FIKATKVLM 1.000 230
MCLNEDYTM 1.000 202 KTPKCALKM 1.000 383 ATPIAIKAV 0.600 343
DPSSPTISS 0.600 112 LPNPPQAVN 0.600 322 LEVEDRTSL 0.600 110
QVLPNPPQA 0.500 199 KVLKTPKCA 0.500 22 TARLQRALD 0.450 113
PNPPQAVNL 0.400 70 QKTDVKDDL 0.400 297 PSTKNSIAL 0.400 145
KNSMDIMKI 0.400 169 NSVHEQEAI 0.400 347 PTISSTENL 0.400 90
GKSPRSPQL 0.400 134 DFIKATKVL 0.400 310 YPLSKTNSS 0.400 346
SPTISSYEN 0.400 378 YNSNLATPI 0.400 6 SFCGKLRSL 0.400 193 TKQSLVKVL
0.400 180 DNYKEEPVI 0.400 295 KIPSTKNSI 0.400 244 RNNKSEEAI 0.400
104 ERYIVSQVL 0.400 128 ENQEGIDFI 0.400 380 SNLATPIAI 0.400 52
QTLKDDVNI 0.400 323 EVEDRTSLV 0.300 117 QAVNLLDKA 0.300 15
ASTLDCETA 0.300 33 ESDFEDYPM 0.300 382 LATPIAIKA 0.300 242
NARNNKSEE 0.300 207 ALKMDDFEC 0.300 111 VLPNPPQAV 0.300 391
VPPSKRFLK 0.300 124 KARLENQEG 0.300 358 TPTPPEVTK 0.300 14
LASTLDCET 0.300 384 TPIAIKAVP 0.200 360 TPPEVTKIP 0.200 2 DPIRSFCGK
0.200 298 STKNSIALV 0.200 152 KIREYFQKY 0.200 43 ILYDLHSEV 0.200
203 TPKCALKMD 0.200 316 NSSSNDLEV 0.200 267 SPIIQQLEK 0.200 103
LERYIVSQV 0.200 255 ESRLNDNVF 0.200 36 FEDYPMRIL 0.180 355
LLRTPTPPE 0.150 280 YTNSPLVPT 0.150 57 DVNIPELSN 0.150 340
NLTDPSSPT 0.150 7 FCGKLRSLA 0.150 118 AVNLLDKAR 0.150 307 STNYPLSKT
0.150
TABLE-US-00068 TABLE XVIII-V5-HLA-B7-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
EKSPRSPQL 0.400 3 ASSCISEKS 0.060 2 VASSCISEK 0.030 6 CISEKSPRS
0.020 5 SCISEKSPR 0.010 4 SSCISEKSP 0.010 1 PVASSCISE 0.005 7
ISEKSPRSP 0.003 8 SEKSPRSPQ 0.002
TABLE-US-00069 TABLE XVIII-V6-HLA-B7-9MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; each il 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 5 EAIDAESRL 12.000 9 AESRLNDNV 0.060 6 AIDAESRLN 0.018 8
DAESRLNDN 0.018 1 NKSEEAIDA 0.010 2 KSEEAIDAE 0.003 7 IDAESRLND
0.002 4 EEAIDAESR 0.001 3 SEEAIDAES 0.001
TABLE-US-00070 TABLE XVIII-V10-HLA-B7-9MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 14 YPTQKLNKM 20.000 11 QWIYPTQKL 0.600 5 DILQKFQWI 0.400 2
IPEDILQKF 0.120 12 WIYPTQKLN 0.020 6 ILQKFQWIY 0.020 8 QKFQWIYPT
0.010 7 LQKFQWIYP 0.010 10 FQWIYPTQK 0.010 1 KIPEDILQK 0.010 4
EDILQKFQW 0.002 13 IYPTQKLNK 0.001 15 PTQKLNKMR 0.001 9 KFQWIYPTQ
0.001 3 PEDILQKFQ 0.000
TABLE-US-00071 TABLE XVIII-V12-HLA-B7-9MERS-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 RALDGEESL 12.000 2 ALDGEESLL 5.600 7 ESLLSKYNS 0.020 8
SLLSKYNSN 0.020 4 DGEESLLSK 0.003 3 LDGEESLLS 0.002 6 EESLLSKYN
0.002 5 GEESLLSKY 0.001
TABLE-US-00072 TABLE XIX-V1-HLA-B7-10MERS-193P1E1B 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
296 IPSTKNSIAL 80.000 2 DPIRSFCGKL 80.000 112 LPNPPQAVNL 80.000 346
SPTISSYENL 80.000 118 AVNLLDKARL 60.000 363 EVTKIPEDIL 30.000 199
KVLKTPKCAL 30.000 188 IVTPPTKQSL 20.000 285 LVPTFCTPGL 20.000 303
IALVSTNYPL 12.000 389 KAVPPSKRFL 12.000 264 ATPSPIIQQL 12.000 124
KARLENQEGI 12.000 358 TPTPPEVTKI 8.000 323 EVEDRTSLVL 6.000 231
CLNEDYTMGL 4.000 366 KIPEDILQLL 4.000 103 LERYIVSQVL 4.000 69
FQKTDVKDDL 4.000 92 SPRSPQLSDF 4.000 94 RSPQLSDFGL 4.000 5
RSFCGKLRSL 4.000 223 FGISEYTMCL 4.000 332 LNSDTCFENL 4.000 16
STLDCETARL 4.000 89 SGKSPRSPQL 4.000 373 QLLSKYNSNL 4.000 261
NVFATPSPII 3.000 242 NARNNKSEEA 3.000 355 LLRTPTPPEV 2.000 163
SPRVKKNSVH 2.000 321 DLEVEDRTSL 1.800 110 QVLPNPPQAV 1.500 141
VLMEKNSMDI 1.200 255 ESRLNDNVFA 1.000 229 TMCLNEDYTM 1.000 207
ALKMDDFECV 0.600 382 LATPIAIKAV 0.600 39 YPMRILYDLH 0.600 57
DVNIPELSNC 0.500 197 LVKVLKTPKC 0.500 133 IDFIKATKVL 0.400 379
NSNLATPIAI 0.400 191 PPTKQSLVKA 0.400 365 TKIPEDILQL 0.400 343
DPSSPTISSI 0.400 9 GKLRSLASTL 0.400 168 KNSVHEQEAI 0.400 276
SDAEYTNSPL 0.400 38 DYPMRILYDL 0.400 216 VTPKLEHFGI 0.400 211
DDFECVTPKL 0.400 45 YDLHSEVQTL 0.400 51 VQTLKDDVNI 0.400 20
CETARLQRAL 0.400 260 DNVFATPSPI 0.400 192 PTKQSLVKVL 0.400 313
SKTNSSSNDL 0.400 340 NLTDPSSPTI 0.400 267 SPIIQQLEKS 0.400 113
PNPPQAVNLL 0.400 249 EEAIDTESRL 0.400 80 DPPVASSCIS 0.400 347
PTISSYENLL 0.400 310 YPLSKTNSSS 0.400 228 YTMCLNEDYT 0.300 14
LASTLDCETA 0.300 22 TARLQRALDG 0.300 142 LMEKNSMDIM 0.300 206
CALKMDDFEC 0.300 390 AVPPSKRFLK 0.225 283 SPLVPTFCTP 0.200 25
LQRALDGEES 0.200 291 TPGLKIPSTK 0.200 265 TPSPIIQQLE 0.200 185
EPVIVTPPTK 0.200 180 DNYKEEPVIV 0.200 203 TPKCALKMDD 0.200 286
VPTFCTPGLK 0.200 403 NIRDVSNKEN 0.200 391 VPPSKRFLKH 0.200 189
VTPPTKQSLV 0.200 114 NPPQAVNLLD 0.200 384 TPIAIKAVPP 0.200 42
RILYDLHSEV 0.200 95 SPQLSDFGLE 0.200 315 TNSSSNDLEV 0.200 190
TPPTKQSLVK 0.200 157 FQKYGYSPRV 0.200 360 TPPEVTKIPE 0.200 162
YSPRVKKNSV 0.200 35 DFEDYPMRIL 0.180 277 DAEYTNSPLV 0.180 306
VSTNYPLSKI 0.150 339 ENLTDPSSPT 0.150 282 NSPLVPTFCT 0.150 60
IPELSNCENF 0.120 243 ARNNKSEEAI 0.120 367 IPEDILQLLS 0.120
TABLE-US-00073 TABLE XIX-V5-HLA-B7-10MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
SEKSPRSPQL 0.400 3 VASSCISEKS 0.060 4 ASSCISEKSP 0.030 6 SCISEKSPRS
0.020 1 PPVASSCISE 0.020 5 SSCISEKSPR 0.010 7 CISEKSPRSP 0.010 8
ISEKSPRSPQ 0.007 2 PVASSCISEK 0.005 10 EKSPRSPQLS 0.002
TABLE-US-00074 TABLE XIX-V6-HLA-B7410MERS-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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
EEAIDAESRL 0.400 9 DAESRLNDNV 0.180 1 NNKSEEAIDA 0.100 6 EAIDAESRLN
0.060 7 AIDAESRLND 0.013 10 AESRLNDNVF 0.006 3 KSEEAIDAES 0.006 8
IDAESRLNDN 0.002 2 NKSEEAIDAE 0.001 4 SEEAIDAESR 0.000
TABLE-US-00075 TABLE XIX V10-HLA-B7-10mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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 11
FQWIYPTQKL 6.000 15 YPTQKLNKMR 0.200 14 IYPTQKLNKM 0.100 8
LQKFQWIYPT 0.100 3 IPEDILQKFQ 0.060 5 EDILQKFQWI 0.040 6 DILQKFQWIY
0.020 2 KIPEDILQKF 0.020 13 WIYPTQKLNK 0.010 7 ILQKFQWIYP 0.010 12
QWIYPTQKLN 0.002 9 QKFQWIYPTQ 0.001 10 KFQWIYPTQK 0.001 1
TKIPEDILQK 0.001 4 PEDILQKFQW 0.000
TABLE-US-00076 TABLE XIX V12-HLA-B7-10mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
RALDGEESLL 12.000 9 SLLSKYNSNL 4.000 1 QRALDGEESL 0.400 8
ESLLSKYNSN 0.020 3 ALDGEESLLS 0.018 5 DGEESLLSKY 0.006 7 EESLLSKYNS
0.002 4 LDGEESLLSK 0.001 6 GEESLLSKYN 0.001
TABLE-US-00077 TABLE XX V1-HLA-B3501-9mers-193P1E1B 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
217 TPKLEHFGI 36.000 152 KIREYFQKY 24.000 39 YPMRILYDL 20.000 95
SPQLSDFGL 20.000 114 NPPQAVNLL 20.000 286 VPTFCTPGL 20.000 265
TPSPIIQQL 20.000 255 ESRLNDNVF 15.000 163 SPRVKKNSV 12.000 367
IPEDILQLL 12.000 80 DPPVASSCI 8.000 250 EAIDTESRL 6.000 366
KIPEDILQL 6.000 140 KVLMEKNSM 6.000 135 FIKATKVLM 6.000 389
KAVPPSKRF 6.000 10 KLRSLASTL 6.000 33 ESDFEDYPM 4.500 202 KTPKCALKM
4.000 190 TPPTKQSLV 4.000 230 MCLNEDYTM 3.000 364 VTKIPEDIL 3.000
98 LSDFGLERY 3.000 200 VLKTPKCAL 3.000 169 NSVHEQEAI 3.000 302
SIALVSTNY 2.000 283 SPLVPTFCT 2.000 296 IPSTKNSIA 2.000 112
LPNPPQAVN 2.000 310 YPLSKTNSS 2.000 370 DILQLLSKY 2.000 224
GISEYTMCL 2.000 185 EPVIVTPPT 2.000 228 YTMCLNEDY 2.000 314
KTNSSSNDL 2.000 346 SPTISSYEN 2.000 205 KCALKMDDF 2.000 291
TPGLKIPST 2.000 343 DPSSPTISS 2.000 312 LSKTNSSSN 1.500 46
DLHSEVQTL 1.500 119 VNLLDKARL 1.500 333 NSDTCFENL 1.500 375
LSKYNSNLA 1.500 145 KNSMDIMKI 1.200 5 RSFCGKLRS 1.000 215 CVTPKLEHF
1.000 390 AVPPSKRFL 1.000 304 ALVSTNYPL 1.000 189 VTPPTKQSL 1.000
350 SSYENLLRT 1.000 149 DIMKIREYF 1.000 374 LLSKYNSNL 1.000 316
NSSSNDLEV 1.000 330 LVLNSDTCF 1.000 344 PSSPTISSY 1.000 348
TISSYENLL 1.000 91 KSPRSPQLS 1.000 12 RSLASTLDC 1.000 281 TNSPLVPTF
1.000 21 ETARLQRAL 1.000 277 DAEYTNSPL 0.900 244 RNNKSEEAI 0.800
128 ENQEGIDFI 0.800 295 KIPSTKNSI 0.800 15 ASTLDCETA 0.750 272
QLEKSDAEY 0.600 60 IPELSNCEN 0.600 143 MEKNSMDIM 0.600 180
DNYKEEPVI 0.600 52 QTLKDDVNI 0.600 298 STKNSIALV 0.600 92 SPRSPQLSD
0.600 232 LNEDYTMGL 0.600 203 TPKCALKMD 0.600 379 NSNLATPIA 0.500
162 YSPRVKKNS 0.500 328 TSLVLNSDT 0.500 297 PSTKNSIAL 0.500 301
NSIALVSTN 0.500 282 NSPLVPTFC 0.500 195 QSLVKVLKT 0.500 84
ASSCISGKS 0.500 17 TLDCETARL 0.450 28 ALDGEESDF 0.450 207 ALKMDDFEC
0.450 275 KSDAEYTNS 0.450 380 SNLATPIAI 0.400 261 NVFATPSPI 0.400
154 REYFQKYGY 0.400 363 EVTKIPEDI 0.400 360 TPPEVTKIP 0.400 43
ILYDLHSEV 0.400 378 YNSNLATPI 0.400 257 RLNDNVFAT 0.400 386
IAIKAVPPS 0.300 8 CGKLRSLAS 0.300 117 QAVNLLDKA 0.300 14 LASTLDCET
0.300 3 PIRSFCGKL 0.300
TABLE-US-00078 TABLE XX V5-HLA-B3501-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
ASSCISEKS 0.500 6 CISEKSPRS 0.200 9 EKSPRSPQL 0.100 4 SSCISEKSP
0.050 2 VASSCISEK 0.030 7 ISEKSPRSP 0.015 5 SCISEKSPR 0.015 8
SEKSPRSPQ 0.003 1 PVASSCISE 0.001
TABLE-US-00079 TABLE XX V6-HLA-B3501-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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 5
EAIDAESRL 6.000 8 DAESRLNDN 0.090 2 KSEEAIDAE 0.060 6 AIDAESRLN
0.045 1 NKSEEAIDA 0.030 9 AESRLNDNV 0.020 3 SEEAIDAES 0.003 7
IDAESRLND 0.002 4 EEAIDAESR 0.002
TABLE-US-00080 TABLE XX V10-HLA-B3501-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 14 YPTQKLNKM 40.000 2 IPEDILQKF 12.000 6 ILQKFQWIY 2.000 5
DILQKFQWI 0.400 11 QWIYPTQKL 0.100 12 WIYPTQKLN 0.100 1 KIPEDILQK
0.060 4 EDILQKFQW 0.050 7 LQKFQWIYP 0.030 10 FQWIYPTQK 0.010 8
QKFQWIYPT 0.010 9 KFQWIYPTQ 0.002 13 IYPTQKLNK 0.001 15 PTQKLNKMR
0.001 3 PEDILQKFQ 0.000
TABLE-US-00081 TABLE XX V12-HLA-B3501-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 RALDGEESL 12.000 7 ESLLSKYNS 0.500 2 ALDGEESLL 0.450 8
SLLSKYNSN 0.100 5 GEESLLSKY 0.060 3 LDGEESLLS 0.030 6 EESLLSKYN
0.010 4 DGEESLLSK 0.006
TABLE-US-00082 TABLE XXI V1-HLA-B3501-10mers-193P1E1B 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 92
SPRSPQLSDF 60.000 343 DPSSPTISSY 40.000 346 SPTISSYENL 20.000 2
DPIRSFCGKL 20.000 296 IPSTKNSIAL 20.000 112 LPNPPQAVNL 20.000 27
RALDGEESDF 18.000 358 TPTPPEVTKI 12.000 301 NSIALVSTNY 10.000 94
RSPQLSDFGL 10.000 5 RSFCGKLRSL 10.000 124 KARLENQEGI 7.200 389
KAVPPSKRFL 6.000 217 TPKLEHFGIS 6.000 60 IPELSNCENF 6.000 271
QQLEKSDAEY 4.000 366 KIPEDILQLL 4.000 97 QLSDFGLERY 4.000 303
IALVSTNYPL 3.000 89 SGKSPRSPQL 3.000 69 FQKTDVKDDL 3.000 229
TMCLNEDYTM 3.000 16 STLDCETARL 3.000 255 ESRLNDNVFA 2.250 310
YPLSKTNSSS 2.000 199 KVLKTPKCAL 2.000 231 CLNEDYTMGL 2.000 332
LNSDTCFENL 2.000 379 NSNLATPIAI 2.000 80 DPPVASSCIS 2.000 267
SPIIQQLEKS 2.000 30 DGEESDFEDY 1.800 375 LSKYNSNLAT 1.500 118
AVNLLDKARL 1.500 168 KNSVHEQEAI 1.200 367 IPEDILQLLS 1.200 219
KLEHFGISEY 1.200 188 IVTPPTKQSL 1.000 280 YTNSPLVPTF 1.000 162
YSPRVKKNSV 1.000 329 SLVLNSDTCF 1.000 363 EVTKIPEDIL 1.000 264
ATPSPIIQQL 1.000 223 FGISEYTMCL 1.000 373 QLLSKYNSNL 1.000 285
LVPTFCTPGL 1.000 214 ECVTPKLEHF 1.000 126 RLENQEGIDF 0.900 207
ALKMDDFECV 0.900 242 NARNNKSEEA 0.900 250 EAIDTESRLN 0.900 340
NLTDPSSPTI 0.800 141 VLMEKNSMDI 0.800 216 VTPKLEHFGI 0.600 323
EVEDRTSLVL 0.600 147 SMDIMKIREY 0.600 382 LATPIAIKAV 0.600 32
EESDFEDYPM 0.600 157 FQKYGYSPRV 0.600 203 TPKCALKMDD 0.600 51
VQTLKDDVNI 0.600 142 LMEKNSMDIM 0.600 137 KATKVLMEKN 0.600 403
NIRDVSNKEN 0.600 247 KSEEAIDTES 0.600 98 LSDFGLERYI 0.600 173
EQEAINSDNY 0.600 355 LLRTPTPPEV 0.600 163 SPRVKKNSVH 0.600 306
VSTNYPLSKT 0.500 282 NSPLVPTFCT 0.500 345 SSPTISSYEN 0.500 169
NSVHEQEAIN 0.500 328 TSLVLNSDTC 0.500 349 ISSYENLLRT 0.500 63
LSNCENFQKT 0.500 25 LQRALDGEES 0.450 245 NNKSEEAIDT 0.450 14
LASTLDCETA 0.450 206 CALKMDDFEC 0.450 321 DLEVEDRTSL 0.450 260
DNVFATPSPI 0.400 261 NVFATPSPII 0.400 42 RILYDLHSEV 0.400 191
PPTKQSLVKV 0.400 360 TPPEVTKIPE 0.400 138 ATKVLMEKNS 0.300 298
STKNSIALVS 0.300 180 DNYKEEPVIV 0.300 192 PTKQSLVKVL 0.300 170
SVHEQEAINS 0.300 197 LVKVLKTPKC 0.300 239 GLKNARNNKS 0.300 8
CGKLRSLAST 0.300 139 TKVLMEKNSM 0.300 103 LERYIVSQVL 0.300 178
NSDNYKEEPV 0.300 83 VASSCISGKS 0.300 95 SPQLSDFGLE 0.300 293
GLKIPSTKNS 0.300
TABLE-US-00083 TABLE XXI V5-HLA-B3501-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 SEKSPRSPQL 0.300 3 VASSCISEKS 0.300 6 SCISEKSPRS 0.100 5
SSCISEKSPR 0.075 4 ASSCISEKSP 0.050 7 CISEKSPRSP 0.020 1 PPVASSCISE
0.020 8 ISEKSPRSPQ 0.015 10 EKSPRSPQLS 0.010 2 PVASSCISEK 0.001
TABLE-US-00084 TABLE XXI V6-HLA-B3501-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRLN 0.900 3 KSEEAIDAES 0.600 1 NNKSEEAIDA 0.450 9
DAESRLNDNV 0.180 5 EEAIDAESRL 0.100 10 AESRLNDNVF 0.100 8
IDAESRLNDN 0.020 7 AIDAESRLND 0.003 2 NKSEEAIDAE 0.002 4 SEEAIDAESR
0.000
TABLE-US-00085 TABLE XXI V10-HLA-B3501-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 KIPEDILQKF 4.000 6 DILQKFQWIY 2.000 11 FQWIYPTQKL 1.000 8
LQKFQWIYPT 0.300 15 YPTQKLNKMR 0.200 14 IYPTQKLNKM 0.200 3
IPEDILQKFQ 0.120 5 EDILQKFQWI 0.040 7 ILQKFQWIYP 0.010 12
QWIYPTQKLN 0.010 13 WIYPTQKLNK 0.010 10 KFQWIYPTQK 0.002 4
PEDILQKFQW 0.002 1 TKIPEDILQK 0.002 9 QKFQWIYPTQ 0.001
TABLE-US-00086 TABLE XXI V12-HLA-B3501-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 RALDGEESLL 18.000 5 DGEESLLSKY 1.200 9 SLLSKYNSNL 1.000 8
ESLLSKYNSN 0.500 1 QRALDGEESL 0.100 3 ALDGEESLLS 0.045 7 EESLLSKYNS
0.010 6 GEESLLSKYN 0.003 4 LDGEESLLSK 0.002
Tables XXII-XLIX:
TABLE-US-00087 [0995] TABLE XII V1-HLA-A1-9mers-193P1E1B 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 98 LSDFGLERY 31 31 GEESDFEDY 28 37 EDYPMRILY 26 272
QLEKSDAEY 26 48 HSEVQTLKD 24 228 YTMCLNEDY 24 344 PSSPTISSY 23 152
KIREYFQKY 21 78 LSDPPVASS 20 341 LTDPSSPTI 20 182 YKEEPVIVT 19 302
SIALVSTNY 19 71 KTDVKDDLS 18 121 LLDKARLEN 18 324 VEDRTSLVL 18 368
PEDILQLLS 18 19 DCETARLQR 17 54 LKDDVNIPE 17 154 REYFQKYGY 17 220
LEHFGISEY 17 333 NSDTCFENL 17 370 DILQLLSKY 17 147 SMDIMKIRE 16 183
KEEPVIVTP 16 219 KLEHFGISE 16 225 ISEYTMCLN 16 253 DTESRLNDN 16 148
MDIMKIREY 15 171 VHEQEAINS 15 174 QEAINSDNY 15 178 NSDNYKEEP 15 258
LNDNVFATP 15
TABLE-US-00088 TABLE XXII V5-HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
ISEKSPRSP 14 3 ASSCISEKS 7 4 SSCISEKSP 6
TABLE-US-00089 TABLE XXII V6-HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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
KSEEAIDAE 14 6 AIDAESRLN 13 3 SEEAIDAES 12 8 DAESRLNDN 10 7
IDAESRLND 7
TABLE-US-00090 TABLE XXII V10-HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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
ILQKFQWIY 16 2 IPEDILQKF 12 3 PEDILQKFQ 10 13 IYPTQKLNK 8 15
PTQKLNKMR 8
TABLE-US-00091 TABLE XXII V12-HLA-A1-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 25; 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
GEESLLSKY 27 4 DGEESLLSK 16 2 ALDGEESLL 15
TABLE-US-00092 TABLE XXIII V1-HLA-A0201-9mers-193P1E1B 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 43
ILYDLHSEV 27 366 KIPEDILQL 26 46 DLHSEVQTL 25 10 KLRSLASTL 24 17
TLDCETARL 24 224 GISEYTMCL 24 111 VLPNPPQAV 23 304 ALVSTNYPL 23 374
LLSKYNSNL 23 200 VLKTPKCAL 22 6 SFCGKLRSL 21 257 RLNDNVFAT 21 298
STKNSIALV 21 295 KIPSTKNSI 20 348 TISSYENLL 20 383 ATPIAIKAV 20 102
GLERYIVSQ 19 189 VTPPTKQSL 19 341 LTDPSSPTI 19 381 NLATPIAIK 19 3
PIRSFCGKL 18 13 SLASTLDCE 18 39 YPMRILYDL 18 55 KDDVNIPEL 18 192
PTKQSLVKV 18 280 YTNSPLVPT 18 390 AVPPSKRFL 18 142 LMEKNSMDI 17 196
SLVKVLKTP 17 307 STNYPLSKT 17 314 KTNSSSNDL 17 359 PTPPEVTKI 17 42
RILYDLHSE 16 52 QTLKDDVNI 16 133 IDFIKATKV 16 163 SPRVKKNSV 16 232
LNEDYTMGL 16 265 TPSPIIQQL 16 322 LEVEDRTSL 16 355 LLRTPTPPE 16 356
LRTPTPPEV 16 367 IPEDILQLL 16 370 DILQLLSKY 16 373 QLLSKYNSN 16 9
GKLRSLAST 15 21 ETARLQRAL 15 24 RLQRALDGE 15 53 TLKDDVNIP 15 97
QLSDFGLER 15 103 LERYIVSQV 15 106 YIVSQVLPN 15 114 NPPQAVNLL 15 117
QAVNLLDKA 15 119 VNLLDKARL 15 121 LLDKARLEN 15 125 ARLENQEGI 15 141
VLMEKNSMD 15 145 KNSMDIMKI 15 158 QKYGYSPRV 15 209 KMDDFECVT 15 340
NLTDPSSPT 15 364 VTKIPEDIL 15 399 KHGQNIRDV 15 28 ALDGEESDF 14 77
DLSDPPVAS 14 90 GKSPRSPQL 14 99 SDFGLERYI 14 120 NLLDKARLE 14 135
FIKATKVLM 14 140 KVLMEKNSM 14 152 KIREYFQKY 14 176 AINSDNYKE 14 193
TKQSLVKVL 14 195 QSLVKVLKT 14 208 LKMDDFECV 14 250 EAIDTESRL 14 278
AEYTNSPLV 14 329 SLVLNSDTC 14 331 VLNSDTCFE 14 350 SSYENLLRT 14 14
LASTLDCET 13 78 LSDPPVASS 13 95 SPQLSDFGL 13 110 QVLPNPPQA 13 128
ENQEGIDFI 13 179 SDNYKEEPV 13 181 NYKEEPVIV 13 207 ALKMDDFEC 13 212
DFECVTPKL 13 219 KLEHFGISE 13 231 CLNEDYTMG 13 261 NVFATPSPI 13 262
VFATPSPII 13 268 PIIQQLEKS 13 272 QLEKSDAEY 13 286 VPTFCTPGL 13 293
GLKIPSTKN 13 300 KNSIALVST 13 316 NSSSNDLEV 13 323 EVEDRTSLV 13 347
PTISSYENL 13 380 SNLATPIAI 13 382 LATPIAIKA 13 386 IAIKAVPPS 13 397
FLKHGQNIR 13 50 EVQTLKDDV 12 59 NIPELSNCE 12 75 KDDLSDPPV 12 87
CISGKSPRS 12 132 GIDFIKATK 12 134 DFIKATKVL 12 182 YKEEPVIVT 12 187
VIVTPPTKQ 12 202 KTPKCALKM 12 229 TMCLNEDYT 12 236 YTMGLKNAR 12 269
IIQQLEKSD 12 276 SDAEYTNSP 12 288 TFCTPGLKI 12
291 TPGLKIPST 12 302 SIALVSTNY 12 324 VEDRTSLVL 12 354 NLLRTPTPP 12
371 ILQLLSKYN 12 387 AIKAVPPSK 12 403 NIRDVSNKE 12
TABLE-US-00093 TABLE XXIII V5-HLA-A0201-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 CISEKSPRS 12 2 VASSCISEK 11 9 EKSPRSPQL 10 1 PVASSCISE 5
TABLE-US-00094 TABLE XXIII V6-HLA-A0201-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRL 14 9 AESRLNDNV 13 6 AIDAESRLN 10 2 KSEEAIDAE 7 7
IDAESRLND 7 8 DAESRLNDN 7 1 NKSEEAIDA 6
TABLE-US-00095 TABLE XXIII V10-HLA-A0201-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 DILQKFQWI 18 1 KIPEDILQK 16 11 QWIYPTQKL 16 6
ILQKFQWIY 13 14 YPTQKLNKM 13 8 QKFQWIYPT 10 12 WIYPTQKLN 10 2
IPEDILQKF 8
TABLE-US-00096 TABLE XXIII V12-HLA-A0201-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 ALDGEESLL 24 1 RALDGEESL 21 8 SLLSKYNSN 18
TABLE-US-00097 TABLE XXIV-V1-HLA-A0203-9mers-193P1E1B Pos 123456789
score NoResultsFound.
TABLE-US-00098 TABLE XXIV-V5-HLA-A0203-9mers-193P1E1B Pos 123456789
score NoResultsFound.
TABLE-US-00099 TABLE XXIV-V6-HLA-A0203-9mers-193P1E1B Pos 123456789
score NoResultsFound.
TABLE-US-00100 TABLE XXIV-V10-HLA-A0203-9mers-193P1E1B Pos
123456789 score NoResultsFound.
TABLE-US-00101 TABLE XXIV-V12-HLA-A0203-9mers-193P1E1B Pos
123456789 score NoResultsFound.
TABLE-US-00102 TABLE XXV V1-HLA-A3-9mers-193P1E1B 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 186
PVIVTPPTK 28 387 AIKAVPPSK 28 10 KLRSLASTL 26 132 GIDFIKATK 25 381
NLATPIAIK 24 97 QLSDFGLER 23 239 GLKNARNNK 23 28 ALDGEESDF 22 197
LVKVLKTPK 22 110 QVLPNPPQA 21 152 KIREYFQKY 21 272 QLEKSDAEY 21 358
TPTPPEVTK 21 402 QNIRDVSNK 21 43 ILYDLHSEV 20 292 PGLKIPSTK 20 102
GLERYIVSQ 19 118 AVNLLDKAR 19 151 MKIREYFQK 19 160 YGYSPRVKK 19 165
RVKKNSVHE 19 194 KQSLVKVLK 19 302 SIALVSTNY 19 369 EDILQLLSK 19 370
DILQLLSKY 19 159 KYGYSPRVK 18 188 IVTPPTKQS 18 215 CVTPKLEHF 18 219
KLEHFGISE 18 267 SPIIQQLEK 18 330 LVLNSDTCF 18 366 KIPEDILQL 18 385
PIAIKAVPP 18 24 RLQRALDGE 17 77 DLSDPPVAS 17 120 NLLDKARLE 17 140
KVLMEKNSM 17 191 PPTKQSLVK 17 201 LKTPKCALK 17 284 PLVPTFCTP 17 306
VSTNYPLSK 17 354 NLLRTPTPP 17 373 QLLSKYNSN 17 397 FLKHGQNIR 17 2
DPIRSFCGK 16 42 RILYDLHSE 16 57 DVNIPELSN 16 126 RLENQEGID 16 141
VLMEKNSMD 16 196 SLVKVLKTP 16 199 KVLKTPKCA 16 257 RLNDNVFAT 16 261
NVFATPSPI 16 323 EVEDRTSLV 16 329 SLVLNSDTC 16 390 AVPPSKRFL 16 67
ENFQKTDVK 15 73 DVKDDLSDP 15 135 FIKATKVLM 15 137 KATKVLMEK 15 170
SVHEQEAIN 15 183 KEEPVIVTP 15 311 PLSKTNSSS 15 37 EDYPMRILY 14 46
DLHSEVQTL 14 116 PQAVNLLDK 14 121 LLDKARLEN 14 154 REYFQKYGY 14 175
EAINSDNYK 14 207 ALKMDDFEC 14 321 DLEVEDRTS 14 340 NLTDPSSPT 14 344
PSSPTISSY 14 17 TLDCETARL 13 23 ARLQRALDG 13 47 LHSEVQTLK 13 63
LSNCENFQK 13 79 SDPPVASSC 13 82 PVASSCISG 13 83 VASSCISGK 13 86
SCISGKSPR 13 107 IVSQVLPNP 13 111 VLPNPPQAV 13 149 DIMKIREYF 13 231
CLNEDYTMG 13 293 GLKIPSTKN 13 295 KIPSTKNSI 13 304 ALVSTNYPL 13 355
LLRTPTPPE 13 371 ILQLLSKYN 13 374 LLSKYNSNL 13 403 NIRDVSNKE 13
TABLE-US-00103 TABLE XXV V5-HLA-A3-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 11; 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
PVASSCISE 13 2 VASSCISEK 13 5 SCISEKSPR 11 6 CISEKSPRS 10 9
EKSPRSPQL 8 8 SEKSPRSPQ 6
TABLE-US-00104 TABLE XXV V6-HLA-A3-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 13; 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
AIDAESRLN 13 4 EEAIDAESR 11 7 IDAESRLND 8 5 EAIDAESRL 7 3 SEEAIDAES
6 9 AESRLNDNV 6
TABLE-US-00105 TABLE XXV V10-HLA-A3-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 21; 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
KIPEDILQK 28 6 ILQKFQWIY 18 10 FQWIYPTQK 16 12 WIYPTQKLN 15 13
IYPTQKLNK 15
TABLE-US-00106 TABLE XXV V12-HLA-A3-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO:25; 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
ALDGEESLL 18 4 DGEESLLSK 16 8 SLLSKYNSN 16 1 RALDGEESL 11 5
GEESLLSKY 9
TABLE-US-00107 TABLE XXVI V1-HLA-A26-9mers-193P1E1B 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 370
DILQLLSKY 29 21 ETARLQRAL 28 215 CVTPKLEHF 25 73 DVKDDLSDP 24 250
EAIDTESRL 24 363 EVTKIPEDI 23 37 EDYPMRILY 22 46 DLHSEVQTL 22 149
DIMKIREYF 22 323 EVEDRTSLV 22 50 EVQTLKDDV 21 253 DTESRLNDN 21 347
PTISSYENL 21 57 DVNIPELSN 20 131 EGIDFIKAT 20 134 DFIKATKVL 20 366
KIPEDILQL 20 369 EDILQLLSK 20 148 MDIMKIREY 19 228 YTMCLNEDY 19 255
ESRLNDNVF 19 390 AVPPSKRFL 19 104 ERYIVSQVL 18 189 VTPPTKQSL 18 211
DDFECVTPK 18 330 LVLNSDTCF 18 335 DTCFENLTD 18 152 KIREYFQKY 17 212
DFECVTPKL 17 265 TPSPIIQQL 17 277 DAEYTNSPL 17 314 KTNSSSNDL 17 344
PSSPTISSY 17 128 ENQEGIDFI 16 155 EYFQKYGYS 16 184 EEPVIVTPP 16 221
EHFGISEYT 16 281 TNSPLVPTF 16 326 DRTSLVLNS 16 364 VTKIPEDIL 16 3
PIRSFCGKL 15 6 SFCGKLRSL 15 67 ENFQKTDVK 15 186 PVIVTPPTK 15 193
TKQSLVKVL 15 214 ECVTPKLEH 15 220 LEHFGISEY 15 227 EYTMCLNED 15 261
NVFATPSPI 15 264 ATPSPIIQQ 15 302 SIALVSTNY 15 307 STNYPLSKT 15 322
LEVEDRTSL 15 367 IPEDILQLL 15 39 YPMRILYDL 14 56 DDVNIPELS 14 93
PRSPQLSDF 14 98 LSDFGLERY 14 106 YIVSQVLPN 14 107 IVSQVLPNP 14 175
EAINSDNYK 14 185 EPVIVTPPT 14 224 GISEYTMCL 14 325 EDRTSLVLN 14 348
TISSYENLL 14 359 PTPPEVTKI 14
TABLE-US-00108 TABLE XXVI V5-HLA-A26-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 11; 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
EKSPRSPQL 20 1 PVASSCISE 13
TABLE-US-00109 TABLE XXVI V6-HLA-A26-9mers-193P1E1B Each peptide is
a portion of SEQ ID NO: 13; 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
EAIDAESRL 24 8 DAESRLNDN 13 4 EEAIDAESR 11
TABLE-US-00110 TABLE XXVI V10-HLA-A26-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 IPEDILQKF 16 4 EDILQKFQW 15 5 DILQKFQWI 13 11 QWIYPTQKL 13 1
KIPEDILQK 12 6 ILQKFQWIY 10 15 PTQKLNKMR 10 8 QKFQWIYPT 8 14
YPTQKLNKM 7
TABLE-US-00111 TABLE XXVI V12-HLA-A26-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 GEESLLSKY 18 4 DGEESLLSK 16 6 EESLLSKYN 11 1 RALDGEESL 10 7
ESLLSKYNS 10 2 ALDGEESLL 9
TABLE-US-00112 TABLE XXVII V1-HLA-B0702-9mers-193P1E1B 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 265
TPSPIIQQL 23 286 VPTFCTPGL 22 39 YPMRILYDL 21 114 NPPQAVNLL 21 367
IPEDILQLL 21 95 SPQLSDFGL 20 185 EPVIVTPPT 20 283 SPLVPTFCT 20 296
IPSTKNSIA 19 163 SPRVKKNSV 18 291 TPGLKIPST 18 92 SPRSPQLSD 17 80
DPPVASSCI 16 112 LPNPPQAVN 16 190 TPPTKQSLV 16 217 TPKLEHFGI 16 343
DPSSPTISS 16 358 TPTPPEVTK 16 392 PPSKRFLKH 16 36 FEDYPMRIL 14 90
GKSPRSPQL 14 191 PPTKQSLVK 14 200 VLKTPKCAL 14 324 VEDRTSLVL 14 390
AVPPSKRFL 14 10 KLRSLASTL 13 17 TLDCETARL 13 21 ETARLQRAL 13 55
KDDVNIPEL 13 113 PNPPQAVNL 13 115 PPQAVNLLD 13 134 DFIKATKVL 13 224
GISEYTMCL 13 304 ALVSTNYPL 13 364 VTKIPEDIL 13 366 KIPEDILQL 13 374
LLSKYNSNL 13 384 TPIAIKAVP 13 3 PIRSFCGKL 12 6 SFCGKLRSL 12 104
ERYIVSQVL 12 193 TKQSLVKVL 12 212 DFECVTPKL 12 267 SPIIQQLEK 12 280
YTNSPLVPT 12 288 TFCTPGLKI 12 297 PSTKNSIAL 12 322 LEVEDRTSL 12 333
NSDTCFENL 12 348 TISSYENLL 12 2 DPIRSFCGK 11 28 ALDGEESDF 11 46
DLHSEVQTL 11 60 IPELSNCEN 11 81 PPVASSCIS 11 119 VNLLDKARL 11 182
YKEEPVIVT 11 189 VTPPTKQSL 11 232 LNEDYTMGL 11 250 EAIDTESRL 11 262
VFATPSPII 11 277 DAEYTNSPL 11 281 TNSPLVPTF 11 300 KNSIALVST 11 310
YPLSKTNSS 11 314 KTNSSSNDL 11 357 RTPTPPEVT 11 360 TPPEVTKIP 11 361
PPEVTKIPE 11 389 KAVPPSKRF 11 391 VPPSKRFLK 11
TABLE-US-00113 TABLE XXVII V5-HLA-B0702-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 EKSPRSPQL 15
TABLE-US-00114 TABLE XXVII V6-HLA-B0702-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRL 11 9 AESRLNDNV 10 1 NKSEEAIDA 8 7 IDAESRLND 5
TABLE-US-00115 TABLE XXVII V10-HLA-B0702-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 IPEDILQKF 17 14 YPTQKLNKM 16 11 QWIYPTQKL 14
TABLE-US-00116 TABLE XXVII V12-HLA-B0702-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 ALDGEESLL 15 1 RALDGEESL 11
TABLE-US-00117 TABLE XXVIII V1-HLA-B08-9mers-193P1E1B 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 200
VLKTPKCAL 28 163 SPRVKKNSV 25 217 TPKLEHFGI 23 6 SFCGKLRSL 22 10
KLRSLASTL 22 364 VTKIPEDIL 21 3 PIRSFCGKL 20 8 CGKLRSLAS 20 141
VLMEKNSMD 20 90 GKSPRSPQL 19 95 SPQLSDFGL 19 150 IMKIREYFQ 19 122
LDKARLENQ 18 291 TPGLKIPST 18 296 IPSTKNSIA 18 46 DLHSEVQTL 17 53
TLKDDVNIP 17 114 NPPQAVNLL 17 203 TPKCALKMD 17 205 KCALKMDDF 17 207
ALKMDDFEC 17 224 GISEYTMCL 17 239 GLKNARNNK 17 265 TPSPIIQQL 17 286
VPTFCTPGL 17 293 GLKIPSTKN 17 304 ALVSTNYPL 17 366 KIPEDILQL 17 367
IPEDILQLL 17 374 LLSKYNSNL 17 391 VPPSKRFLK 17 397 FLKHGQNIR 17 17
TLDCETARL 16 39 YPMRILYDL 16 120 NLLDKARLE 16 135 FIKATKVLM 16 190
TPPTKQSLV 16 215 CVTPKLEHF 16 250 EAIDTESRL 16 277 DAEYTNSPL 16 310
YPLSKTNSS 16 373 QLLSKYNSN 16 255 ESRLNDNVF 15 385 PIAIKAVPP 15 87
CISGKSPRS 14 92 SPRSPQLSD 14 348 TISSYENLL 14 387 AIKAVPPSK 14 392
PPSKRFLKH 14 21 ETARLQRAL 13 55 KDDVNIPEL 13 69 FQKTDVKDD 13 80
DPPVASSCI 13 124 KARLENQEG 13 166 VKKNSVHEQ 13 179 SDNYKEEPV 13 181
NYKEEPVIV 13 271 QQLEKSDAE 13 298 STKNSIALV 13
TABLE-US-00118 TABLE XXVIII V5-HLA-B08-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 EKSPRSPQL 20 6 CISEKSPRS 16 8 SEKSPRSPQ 12
TABLE-US-00119 TABLE XXVIII V6-HLA-B08-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRL 16 8 DAESRLNDN 12
TABLE-US-00120 TABLE XXVIII V10-HLA-B08-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 DILQKFQWI 20 14 YPTQKLNKM 16 2 IPEDILQKF 13 11 QWIYPTQKL 11 7
LQKFQWIYP 10
TABLE-US-00121 TABLE XXVIII V12-HLA-B08-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 SLLSKYNSN 18 2 ALDGEESLL 16 1 RALDGEESL 14
TABLE-US-00122 TABLE XXIX V1-HLA-B1510-9mers-193P1E1B 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
ETARLQRAL 15 55 KDDVNIPEL 15 90 GKSPRSPQL 15 265 TPSPIIQQL 15 390
AVPPSKRFL 15 399 KHGQNIRDV 15 36 FEDYPMRIL 14 113 PNPPQAVNL 14 193
TKQSLVKVL 14 250 EAIDTESRL 14 367 IPEDILQLL 14 6 SFCGKLRSL 13 17
TLDCETARL 13 104 ERYIVSQVL 13 119 VNLLDKARL 13 134 DFIKATKVL 13 189
VTPPTKQSL 13 200 VLKTPKCAL 13 224 GISEYTMCL 13 281 TNSPLVPTF 13 297
PSTKNSIAL 13 46 DLHSEVQTL 12 47 LHSEVQTLK 12 70 QKTDVKDDL 12 114
NPPQAVNLL 12 171 VHEQEAINS 12 212 DFECVTPKL 12 221 EHFGISEYT 12 232
LNEDYTMGL 12 322 LEVEDRTSL 12 324 VEDRTSLVL 12 348 TISSYENLL 12 366
KIPEDILQL 12 374 LLSKYNSNL 12 10 KLRSLASTL 11 39 YPMRILYDL 11 277
DAEYTNSPL 11 286 VPTFCTPGL 11 364 VTKIPEDIL 11 389 KAVPPSKRF 11 3
PIRSFCGKL 10 95 SPQLSDFGL 10 255 ESRLNDNVF 10 304 ALVSTNYPL 10 314
KTNSSSNDL 10 333 NSDTCFENL 10 347 PTISSYENL 10 93 PRSPQLSDF 9 135
FIKATKVLM 9 149 DIMKIREYF 8 205 KCALKMDDF 8 215 CVTPKLEHF 8 28
ALDGEESDF 7 33 ESDFEDYPM 7 61 PELSNCENF 7 77 DLSDPPVAS 7 127
LENQEGIDF 7 140 KVLMEKNSM 7 143 MEKNSMDIM 7 182 YKEEPVIVT 7 183
KEEPVIVTP 7 202 KTPKCALKM 7 222 HFGISEYTM 7 230 MCLNEDYTM 7 358
TPTPPEVTK 7
TABLE-US-00123 TABLE XXIX V5-HLA-B1510-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 EKSPRSPQL 15 7 ISEKSPRSP 7
TABLE-US-00124 TABLE XXIX V6-HLA-B1510-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO:13; 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
EAIDAESRL 14
TABLE-US-00125 TABLE XXIX V10-HLA-B1510-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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
11 QWIYPTQKL 11 2 IPEDILQKF 10 14 YPTQKLNKM 8
TABLE-US-00126 TABLE XXIX V12-HLA-B1510-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 RALDGEESL 12 2 ALDGEESLL 11
TABLE-US-00127 TABLE XXX V1-HLA-B2705-9mers-193P1E1B 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 104
ERYIVSQVL 25 164 PRVKKNSVH 25 93 PRSPQLSDF 23 125 ARLENQEGI 22 4
IRSFCGKLR 21 137 KATKVLMEK 18 366 KIPEDILQL 18 389 KAVPPSKRF 18 395
KRFLKHGQN 18 23 ARLQRALDG 17 55 KDDVNIPEL 17 67 ENFQKTDVK 17 90
GKSPRSPQL 17 119 VNLLDKARL 17 132 GIDFIKATK 17 211 DDFECVTPK 17 292
PGLKIPSTK 17 370 DILQLLSKY 17 10 KLRSLASTL 16 47 LHSEVQTLK 16 86
SCISGKSPR 16 140 KVLMEKNSM 16 154 REYFQKYGY 16 160 YGYSPRVKK 16 194
KQSLVKVLK 16 202 KTPKCALKM 16 239 GLKNARNNK 16 265 TPSPIIQQL 16 267
SPIIQQLEK 16 330 LVLNSDTCF 16 369 EDILQLLSK 16 374 LLSKYNSNL 16 396
RFLKHGQNI 16 402 QNIRDVSNK 16 6 SFCGKLRSL 15 28 ALDGEESDF 15 34
SDFEDYPMR 15 41 MRILYDLHS 15 113 PNPPQAVNL 15 134 DFIKATKVL 15 148
MDIMKIREY 15 175 EAINSDNYK 15 191 PPTKQSLVK 15 220 LEHFGISEY 15 224
GISEYTMCL 15 236 YTMGLKNAR 15 250 EAIDTESRL 15 281 TNSPLVPTF 15 302
SIALVSTNY 15 314 KTNSSSNDL 15 322 LEVEDRTSL 15 326 DRTSLVLNS 15 347
PTISSYENL 15 381 NLATPIAIK 15 388 IKAVPPSKR 15 397 FLKHGQNIR 15 11
LRSLASTLD 14 16 STLDCETAR 14 17 TLDCETARL 14 52 QTLKDDVNI 14 61
PELSNCENF 14 83 VASSCISGK 14 114 NPPQAVNLL 14 118 AVNLLDKAR 14 127
LENQEGIDF 14 129 NQEGIDFIK 14 145 KNSMDIMKI 14 151 MKIREYFQK 14 159
KYGYSPRVK 14 186 PVIVTPPTK 14 197 LVKVLKTPK 14 205 KCALKMDDF 14 212
DFECVTPKL 14 230 MCLNEDYTM 14 243 ARNNKSEEA 14 255 ESRLNDNVF 14 256
SRLNDNVFA 14 272 QLEKSDAEY 14 297 PSTKNSIAL 14 304 ALVSTNYPL 14 344
PSSPTISSY 14 349 ISSYENLLR 14 358 TPTPPEVTK 14 387 AIKAVPPSK 14 390
AVPPSKRFL 14 404 IRDVSNKEN 14 2 DPIRSFCGK 13 5 RSFCGKLRS 13 21
ETARLQRAL 13 39 YPMRILYDL 13 46 DLHSEVQTL 13 63 LSNCENFQK 13 95
SPQLSDFGL 13 98 LSDFGLERY 13 149 DIMKIREYF 13 152 KIREYFQKY 13 153
IREYFQKYG 13 157 FQKYGYSPR 13 180 DNYKEEPVI 13 189 VTPPTKQSL 13 193
TKQSLVKVL 13 201 LKTPKCALK 13 214 ECVTPKLEH 13 215 CVTPKLEHF 13 244
RNNKSEEAI 13 287 PTFCTPGLK 13 319 SNDLEVEDR 13 324 VEDRTSLVL 13 356
LRTPTPPEV 13 359 PTPPEVTKI 13 367 IPEDILQLL 13 392 PPSKRFLKH 13 3
PIRSFCGKL 12 19 DCETARLQR 12 26 QRALDGEES 12 37 EDYPMRILY 12 70
QKTDVKDDL 12 97 QLSDFGLER 12 99 SDFGLERYI 12
116 PQAVNLLDK 12 128 ENQEGIDFI 12 144 EKNSMDIMK 12 200 VLKTPKCAL 12
277 DAEYTNSPL 12 306 VSTNYPLSK 12 333 NSDTCFENL 12
TABLE-US-00128 TABLE XXX V5-B2705-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 11; 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
VASSCISEK 15 5 SCISEKSPR 15 9 EKSPRSPQL 14
TABLE-US-00129 TABLE XXX V6-B2705-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 13; 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
EAIDAESRL 15 4 EEAIDAESR 12
TABLE-US-00130 TABLE XXX V10-B2705-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 21; 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
KIPEDILQK 18 2 IPEDILQKF 15 11 QWIYPTQKL 15 13 IYPTQKLNK 15 14
YPTQKLNKM 15 6 ILQKFQWIY 14 15 PTQKLNKMR 14 10 FQWIYPTQK 13 5
DILQKFQWI 11 8 QKFQWIYPT 9
TABLE-US-00131 TABLE XXX V12-B2705-9mers-193P1E1B Each peptide is a
portion of SEQ ID NO: 25; 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
RALDGEESL 19 5 GEESLLSKY 16 2 ALDGEESLL 15 4 DGEESLLSK 15
TABLE-US-00132 TABLE XXXI V1-HLA-B2709-9mers-193P1E1B 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 104
ERYIVSQVL 22 125 ARLENQEGI 21 356 LRTPTPPEV 21 93 PRSPQLSDF 19 90
GKSPRSPQL 16 23 ARLQRALDG 15 326 DRTSLVLNS 15 366 KIPEDILQL 15 395
KRFLKHGQN 15 396 RFLKHGQNI 15 10 KLRSLASTL 14 113 PNPPQAVNL 14 119
VNLLDKARL 14 256 SRLNDNVFA 14 304 ALVSTNYPL 14 52 QTLKDDVNI 13 55
KDDVNIPEL 13 224 GISEYTMCL 13 265 TPSPIIQQL 13 314 KTNSSSNDL 13 347
PTISSYENL 13 389 KAVPPSKRF 13 39 YPMRILYDL 12 41 MRILYDLHS 12 46
DLHSEVQTL 12 61 PELSNCENF 12 133 IDFIKATKV 12 140 KVLMEKNSM 12 158
QKYGYSPRV 12 193 TKQSLVKVL 12 202 KTPKCALKM 12 244 RNNKSEEAI 12 250
EAIDTESRL 12 278 AEYTNSPLV 12 286 VPTFCTPGL 12 322 LEVEDRTSL 12 367
IPEDILQLL 12 390 AVPPSKRFL 12 3 PIRSFCGKL 11 4 IRSFCGKLR 11 17
TLDCETARL 11 26 QRALDGEES 11 43 ILYDLHSEV 11 70 QKTDVKDDL 11 75
KDDLSDPPV 11 103 LERYIVSQV 11 114 NPPQAVNLL 11 134 DFIKATKVL 11 145
KNSMDIMKI 11 153 IREYFQKYG 11 164 PRVKKNSVH 11 180 DNYKEEPVI 11 189
VTPPTKQSL 11 212 DFECVTPKL 11 230 MCLNEDYTM 11 243 ARNNKSEEA 11 281
TNSPLVPTF 11 295 KIPSTKNSI 11 297 PSTKNSIAL 11 324 VEDRTSLVL 11 333
NSDTCFENL 11 348 TISSYENLL 11 374 LLSKYNSNL 11 404 IRDVSNKEN 11 6
SFCGKLRSL 10 11 LRSLASTLD 10 21 ETARLQRAL 10 36 FEDYPMRIL 10 95
SPQLSDFGL 10 99 SDFGLERYI 10 200 VLKTPKCAL 10 205 KCALKMDDF 10 215
CVTPKLEHF 10 232 LNEDYTMGL 10 261 NVFATPSPI 10 277 DAEYTNSPL 10 316
NSSSNDLEV 10 330 LVLNSDTCF 10 341 LTDPSSPTI 10 359 PTPPEVTKI 10 363
EVTKIPEDI 10 364 VTKIPEDIL 10 380 SNLATPIAI 10 399 KHGQNIRDV 10
TABLE-US-00133 TABLE XXXI V5-HLA-B2709-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 EKSPRSPQL 13
TABLE-US-00134 TABLE XXXI V6-HLA-B2709-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRL 12 9 AESRLNDNV 10
TABLE-US-00135 TABLE XXXI V10-HLA-B2709-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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
11 QWIYPTQKL 11 2 IPEDILQKF 10 5 DILQKFQWI 10 14 YPTQKLNKM 9 1
KIPEDILQK 5
TABLE-US-00136 TABLE XXXI V12-HLA-B2709-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO:25; 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
RALDGEESL 16 2 ALDGEESLL 11
TABLE-US-00137 TABLE XXXII V1-HLA-B4402-9mers-193P1E1B 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 324
VEDRTSLVL 24 220 LEHFGISEY 23 36 FEDYPMRIL 22 61 PELSNCENF 22 31
GEESDFEDY 21 127 LENQEGIDF 21 174 QEAINSDNY 21 322 LEVEDRTSL 21 154
REYFQKYGY 20 183 KEEPVIVTP 19 265 TPSPIIQQL 19 37 EDYPMRILY 18 390
AVPPSKRFL 18 134 DFIKATKVL 17 148 MDIMKIREY 17 344 PSSPTISSY 17 366
KIPEDILQL 17 21 ETARLQRAL 16 55 KDDVNIPEL 16 90 GKSPRSPQL 16 113
PNPPQAVNL 16 250 EAIDTESRL 16 255 ESRLNDNVF 16 278 AEYTNSPLV 16 281
TNSPLVPTF 16 389 KAVPPSKRF 16 28 ALDGEESDF 15 93 PRSPQLSDF 15 114
NPPQAVNLL 15 184 EEPVIVTPP 15 6 SFCGKLRSL 14 32 EESDFEDYP 14 39
YPMRILYDL 14 49 SEVQTLKDD 14 145 KNSMDIMKI 14 172 HEQEAINSD 14 189
VTPPTKQSL 14 193 TKQSLVKVL 14 213 FECVTPKLE 14 215 CVTPKLEHF 14 297
PSTKNSIAL 14 333 NSDTCFENL 14 359 PTPPEVTKI 14 362 PEVTKIPED 14 367
IPEDILQLL 14 380 SNLATPIAI 14 10 KLRSLASTL 13 99 SDFGLERYI 13 103
LERYIVSQV 13 104 ERYIVSQVL 13 125 ARLENQEGI 13 128 ENQEGIDFI 13 130
QEGIDFIKA 13 131 EGIDFIKAT 13 149 DIMKIREYF 13 152 KIREYFQKY 13 200
VLKTPKCAL 13 226 SEYTMCLNE 13 233 NEDYTMGLK 13 249 EEAIDTESR 13 304
ALVSTNYPL 13 341 LTDPSSPTI 13 347 PTISSYENL 13 348 TISSYENLL 13 368
PEDILQLLS 13 370 DILQLLSKY 13 17 TLDCETARL 12 20 CETARLQRA 12 46
DLHSEVQTL 12 95 SPQLSDFGL 12 98 LSDFGLERY 12 119 VNLLDKARL 12 205
KCALKMDDF 12 212 DFECVTPKL 12 224 GISEYTMCL 12 232 LNEDYTMGL 12 248
SEEAIDTES 12 254 TESRLNDNV 12 261 NVFATPSPI 12 302 SIALVSTNY 12 314
KTNSSSNDL 12 330 LVLNSDTCF 12 352 YENLLRTPT 12 363 EVTKIPEDI 12 3
PIRSFCGKL 11 70 QKTDVKDDL 11 143 MEKNSMDIM 11 169 NSVHEQEAI 11 228
YTMCLNEDY 11 273 LEKSDAEYT 11 286 VPTFCTPGL 11 288 TFCTPGLKI 11 295
KIPSTKNSI 11 338 FENLTDPSS 11 374 LLSKYNSNL 11 378 YNSNLATPI 11 383
ATPIAIKAV 11
TABLE-US-00138 TABLE XXXII V5-HLA-B4402-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 EKSPRSPQL 18 8 SEKSPRSPQ 12
TABLE-US-00139 TABLE XXXII V6-HLA-B4402-9mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 EAIDAESRL 16 9 AESRLNDNV 15 4 EEAIDAESR 13 3 SEEAIDAES 12
TABLE-US-00140 TABLE XXXII V10-HLA-B4402-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 EDILQKFQW 17 11 QWIYPTQKL 15 2 IPEDILQKF 14 3
PEDILQKFQ 13 5 DILQKFQWI 10 6 ILQKFQWIY 10
TABLE-US-00141 TABLE XXXII V12-HLA-B4402-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 GEESLLSKY 22 2 ALDGEESLL 16 6 EESLLSKYN 16 1
RALDGEESL 12
TABLE-US-00142 TABLE XXXIII V1-HLA-B5101-9mers-193P1E1B 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 80 DPPVASSCI 25 180 DNYKEEPVI 22 190 TPPTKQSLV 22
277 DAEYTNSPL 22 114 NPPQAVNLL 21 217 TPKLEHFGI 21 163 SPRVKKNSV 20
367 IPEDILQLL 20 39 YPMRILYDL 19 250 EAIDTESRL 19 35 DFEDYPMRI 18
265 TPSPIIQQL 18 95 SPQLSDFGL 17 286 VPTFCTPGL 17 360 TPPEVTKIP 17
2 DPIRSFCGK 16 133 IDFIKATKV 16 303 IALVSTNYP 16 310 YPLSKTNSS 16
359 PTPPEVTKI 16 380 SNLATPIAI 16 382 LATPIAIKA 16 43 ILYDLHSEV 15
101 FGLERYIVS 15 104 ERYIVSQVL 15 112 LPNPPQAVN 15 115 PPQAVNLLD 15
134 DFIKATKVL 15 158 QKYGYSPRV 15 160 YGYSPRVKK 15 191 PPTKQSLVK 15
261 NVFATPSPI 15 288 TFCTPGLKI 15 296 IPSTKNSIA 15 341 LTDPSSPTI 15
343 DPSSPTISS 15 384 TPIAIKAVP 15 386 IAIKAVPPS 15 392 PPSKRFLKH 15
46 DLHSEVQTL 14 52 QTLKDDVNI 14 60 IPELSNCEN 14 125 ARLENQEGI 14
137 KATKVLMEK 14 206 CALKMDDFE 14 212 DFECVTPKL 14 263 FATPSPIIQ 14
358 TPTPPEVTK 14 378 YNSNLATPI 14 14 LASTLDCET 13 27 RALDGEESD 13
30 DGEESDFED 13 99 SDFGLERYI 13 117 QAVNLLDKA 13 128 ENQEGIDFI 13
142 LMEKNSMDI 13 145 KNSMDIMKI 13 181 NYKEEPVIV 13 192 PTKQSLVKV 13
193 TKQSLVKVL 13 203 TPKCALKMD 13 278 AEYTNSPLV 13 291 TPGLKIPST 13
295 KIPSTKNSI 13 391 VPPSKRFLK 13 396 RFLKHGQNI 13 81 PPVASSCIS 12
83 VASSCISGK 12 100 DFGLERYIV 12 103 LERYIVSQV 12 175 EAINSDNYK 12
185 EPVIVTPPT 12 208 LKMDDFECV 12 238 MGLKNARNN 12 244 RNNKSEEAI 12
262 VFATPSPII 12 283 SPLVPTFCT 12 292 PGLKIPSTK 12 324 VEDRTSLVL 12
356 LRTPTPPEV 12 361 PPEVTKIPE 12 363 EVTKIPEDI 12 389 KAVPPSKRF
12
TABLE-US-00143 TABLE XXXIII V5-HLA-B5101-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 11; 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 VASSCISEK 12 7 ISEKSPRSP 6 9 EKSPRSPQL 6
TABLE-US-00144 TABLE XXXIII V6-HLA-B5101-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; 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 EAIDAESRL 18 8 DAESRLNDN 16 9 AESRLNDNV 9
TABLE-US-00145 TABLE XXXIII V10-HLA-B5101-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO:21; 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 DILQKFQWI 18 14 YPTQKLNKM 17 2 IPEDILQKF 16 11
QWIYPTQKL 8
TABLE-US-00146 TABLE XXXIII V12-HLA-B5101-9mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 RALDGEESL 19 4 DGEESLLSK 15
TABLE-US-00147 TABLE XXXIV V1-HLA-A1-10mers-193P1E1B 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 36
FEDYPMRILY 33 30 DGEESDFEDY 28 147 SMDIMKIREY 27 219 KLEHFGISEY 26
153 IREYFQKYGY 25 173 EQEAINSDNY 25 71 KTDVKDDLSD 22 129 NQEGIDFIKA
20 151 MKIREYFQKY 20 225 ISEYTMCLNE 20 341 LTDPSSPTIS 20 233
NEDYTMGLKN 19 301 NSIALVSTNY 19 78 LSDPPVASSC 18 251 AIDTESRLND 18
367 IPEDILQLLS 18 253 DTESRLNDNV 17 323 EVEDRTSLVL 17 369
EDILQLLSKY 17 97 QLSDFGLERY 16 126 RLENQEGIDF 16 227 EYTMCLNEDY 16
333 NSDTCFENLT 16 368 PEDILQLLSK 16
TABLE-US-00148 TABLE XXXIV V5-HLA-A1-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 ISEKSPRSPQ 15
TABLE-US-00149 TABLE XXXIV V6-HLA-A1-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 AIDAESRLND 18 3 KSEEAIDAES 14 4 SEEAIDAESR 12 9 DAESRLNDNV 11
TABLE-US-00150 TABLE XXXIV V10-HLA-A1-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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 DILQKFQWIY 15 1 TKIPEDILQK 10 3 IPEDILQKFQ 10 4 PEDILQKFQW 10 13
WIYPTQKLNK 10
TABLE-US-00151 TABLE XXXIV V12-HLA-A1-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 DGEESLLSKY 27 3 ALDGEESLLS 21
TABLE-US-00152 TABLE XXXV V1-HLA-A0201-10mers-193P1E1B 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 355
LLRTPTPPEV 25 366 KIPEDILQLL 25 102 GLERYIVSQV 24 231 CLNEDYTMGL 24
141 VLMEKNSMDI 22 373 QLLSKYNSNL 22 16 STLDCETARL 21 42 RILYDLHSEV
21 207 ALKMDDFECV 21 340 NLTDPSSPTI 21 382 LATPIAIKAV 21 13
SLASTLDCET 20 45 YDLHSEVQTL 20 54 LKDDVNIPEL 20 132 GIDFIKATKV 20
264 ATPSPIIQQL 20 321 DLEVEDRTSL 20 110 QVLPNPPQAV 19 118
AVNLLDKARL 19 188 IVTPPTKQSL 19 303 IALVSTNYPL 19 365 TKIPEDILQL 19
77 DLSDPPVASS 18 199 KVLKTPKCAL 18 285 LVPTFCTPGL 18 381 NLATPIAIKA
18 398 LKHGQNIRDV 18 5 RSFCGKLRSL 17 112 LPNPPQAVNL 17 189
VTPPTKQSLV 17 290 CTPGLKIPST 17 294 LKIPSTKNSI 17 374 LLSKYNSNLA 17
389 KAVPPSKRFL 17 99 SDFGLERYIV 16 120 NLLDKARLEN 16 121 LLDKARLENQ
16 147 SMDIMKIREY 16 219 KLEHFGISEY 16 257 RLNDNVFATP 16 276
SDAEYTNSPL 16 9 GKLRSLASTL 15 97 QLSDFGLERY 15 106 YIVSQVLPNP 15
127 LENQEGIDFI 15 162 YSPRVKKNSV 15 211 DDFECVTPKL 15 229
TMCLNEDYTM 15 358 TPTPPEVTKI 15 28 ALDGEESDFE 14 38 DYPMRILYDL 14
43 ILYDLHSEVQ 14 113 PNPPQAVNLL 14 124 KARLENQEGI 14 191 PPTKQSLVKV
14 209 KMDDFECVTP 14 216 VTPKLEHFGI 14 261 NVFATPSPII 14 269
IIQQLEKSDA 14 280 YTNSPLVPTF 14 322 LEVEDRTSLV 14 331 VLNSDTCFEN 14
347 PTISSYENLL 14 2 DPIRSFCGKL 13 34 SDFEDYPMRI 13 74 VKDDLSDPPV 13
101 FGLERYIVSQ 13 111 VLPNPPQAVN 13 135 FIKATKVLME 13 142
LMEKNSMDIM 13 192 PTKQSLVKVL 13 223 FGISEYTMCL 13 239 GLKNARNNKS 13
256 SRLNDNVFAT 13 272 QLEKSDAEYT 13 287 PTFCTPGLKI 13 295
KIPSTKNSIA 13 296 IPSTKNSIAL 13 299 TKNSIALVST 13 302 SIALVSTNYP 13
304 ALVSTNYPLS 13 315 TNSSSNDLEV 13 332 LNSDTCFENL 13 354
NLLRTPTPPE 13 371 ILQLLSKYNS 13 14 LASTLDCETA 12 24 RLQRALDGEE 12
49 SEVQTLKDDV 12 59 NIPELSNCEN 12 79 SDPPVASSCI 12 87 CISGKSPRSP 12
89 SGKSPRSPQL 12 133 IDFIKATKVL 12 144 EKNSMDIMKI 12 176 AINSDNYKEE
12 180 DNYKEEPVIV 12 194 KQSLVKVLKT 12 196 SLVKVLKTPK 12 200
VLKTPKCALK 12 243 ARNNKSEEAI 12 253 DTESRLNDNV 12 277 DAEYTNSPLV 12
297 PSTKNSIALV 12 313 SKTNSSSNDL 12 323 EVEDRTSLVL 12 329
SLVLNSDTCF 12 385 PIAIKAVPPS 12
TABLE-US-00153 TABLE XXXV V5-HLA-A0201-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 CISEKSPRSP 12 9 SEKSPRSPQL 12 2 PVASSCISEK 9 3 VASSCISEKS 7 6
SCISEKSPRS 5
TABLE-US-00154 TABLE XXXV V6-HLA-A0201-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 DAESRLNDNV 12 7 AIDAESRLND 11 8 IDAESRLNDN 11 5 EEAIDAESRL 9 2
NKSEEAIDAE 7 3 KSEEAIDAES 6
TABLE-US-00155 TABLE XXXV-V10 HLA-A0201-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 KIPEDILQKF 17 11 FQWIYPTQKL 14 14 IYPTQKLNKM 13
7 ILQKFQWIYP 12 13 WIYPTQKLNK 12
TABLE-US-00156 TABLE XXXV-V12 HLA-A0201-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 SLLSKYNSNL 24 2 RALDGEESLL 17 3 ALDGEESLLS 15 1
QRALDGEESL 14 4 LDGEESLLSK 11
TABLE-US-00157 TABLE XXXVI-V1 HLA-A0203-10mers-193P1E1B 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 SFCGKLRSLA 10 14 LASTLDCETA 10 19 DCETARLQRA 10
75 KDDLSDPPVA 10 109 SQVLPNPPQA 10 116 PQAVNLLDKA 10 129 NQEGIDFIKA
10 167 KKNSVHEQEA 10 198 VKVLKTPKCA 10 234 EDYTMGLKNA 10 242
NARNNKSEEA 10 255 ESRLNDNVFA 10 269 IIQQLEKSDA 10 295 KIPSTKNSIA 10
374 LLSKYNSNLA 10 378 YNSNLATPIA 10 381 NLATPIAIKA 10 7 FCGKLRSLAS
9 15 ASTLDCETAR 9 20 CETARLQRAL 9 76 DDLSDPPVAS 9 110 QVLPNPPQAV 9
117 QAVNLLDKAR 9 130 QEGIDFIKAT 9 168 KNSVHEQEAI 9 199 KVLKTPKCAL 9
235 DYTMGLKNAR 9 243 ARNNKSEEAI 9 256 SRLNDNVFAT 9 270 IQQLEKSDAE 9
296 IPSTKNSIAL 9 375 LSKYNSNLAT 9 379 NSNLATPIAI 9 382 LATPIAIKAV 9
8 CGKLRSLAST 8 16 STLDCETARL 8 21 ETARLQRALD 8 77 DLSDPPVASS 8 111
VLPNPPQAVN 8 118 AVNLLDKARL 8 131 EGIDFIKATK 8 169 NSVHEQEAIN 8 200
VLKTPKCALK 8 236 YTMGLKNARN 8 244 RNNKSEEAID 8 257 RLNDNVFATP 8 271
QQLEKSDAEY 8 297 PSTKNSIALV 8 376 SKYNSNLATP 8 380 SNLATPIAIK 8 383
ATPIAIKAVP 8
TABLE-US-00158 TABLE XXXVI-V6 HLA-A0203-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; 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 NNKSEEAIDA 10 2 NKSEEAIDAE 9 3 KSEEAIDAES 8
TABLE-US-00159 TABLE XXXVI-V5-HLA-A0203-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00160 TABLE XXXVI-V10-HLA-A0203-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00161 TABLE XXXVI-V12-HLA-A0203-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00162 TABLE XXXVII-V1 HLA-A3-10mers-193P1E1B 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 390
AVPPSKRFLK 27 305 LVSTNYPLSK 26 62 ELSNCENFQK 24 82 PVASSCISGK 24
200 VLKTPKCALK 24 158 QKYGYSPRVK 23 126 RLENQEGIDF 22 196
SLVKVLKTPK 22 219 KLEHFGISEY 22 257 RLNDNVFATP 22 357 RTPTPPEVTK 22
387 AIKAVPPSKR 22 43 ILYDLHSEVQ 21 46 DLHSEVQTLK 21 131 EGIDFIKATK
21 97 QLSDFGLERY 19 102 GLERYIVSQV 19 110 QVLPNPPQAV 19 140
KVLMEKNSMD 19 291 TPGLKIPSTK 19 3 PIRSFCGKLR 18 24 RLQRALDGEE 18
111 VLPNPPQAVN 18 159 KYGYSPRVKK 18 323 EVEDRTSLVL 18 380
SNLATPIAIK 18 386 IAIKAVPPSK 18 10 KLRSLASTLD 17 42 RILYDLHSEV 17
118 AVNLLDKARL 17 120 NLLDKARLEN 17 150 IMKIREYFQK 17 188
IVTPPTKQSL 17 190 TPPTKQSLVK 17 329 SLVLNSDTCF 17 373 QLLSKYNSNL 17
77 DLSDPPVASS 16 185 EPVIVTPPTK 16 199 KVLKTPKCAL 16 368 PEDILQLLSK
16 9 GKLRSLASTL 15 28 ALDGEESDFE 15 115 PPQAVNLLDK 15 135
FIKATKVLME 15 152 KIREYFQKYG 15 163 SPRVKKNSVH 15 165 RVKKNSVHEQ 15
170 SVHEQEAINS 15 186 PVIVTPPTKQ 15 251 AIDTESRLND 15 272
QLEKSDAEYT 15 278 AEYTNSPLVP 15 311 PLSKTNSSSN 15 340 NLTDPSSPTI 15
348 TISSYENLLR 15 354 NLLRTPTPPE 15 27 RALDGEESDF 14 50 EVQTLKDDVN
14 57 DVNIPELSNC 14 66 CENFQKTDVK 14 136 IKATKVLMEK 14 174
QEAINSDNYK 14 193 TKQSLVKVLK 14 207 ALKMDDFECV 14 266 PSPIIQQLEK 14
271 QQLEKSDAEY 14 284 PLVPTFCTPG 14 295 KIPSTKNSIA 14 321
DLEVEDRTSL 14 355 LLRTPTPPEV 14 381 NLATPIAIKA 14 401 GQNIRDVSNK 14
17 TLDCETARLQ 13 87 CISGKSPRSP 13 107 IVSQVLPNPP 13 143 MEKNSMDIMK
13 215 CVTPKLEHFG 13 238 MGLKNARNNK 13 268 PIIQQLEKSD 13 269
IIQQLEKSDA 13 298 STKNSIALVS 13 304 ALVSTNYPLS 13 330 LVLNSDTCFE 13
369 EDILQLLSKY 13 371 ILQLLSKYNS 13 376 SKYNSNLATP 13
TABLE-US-00163 TABLE XXXVII-V5 HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 11; 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 PVASSCISEK 24 7 CISEKSPRSP 12
TABLE-US-00164 TABLE XXXVII-V6 HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 13; 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 AIDAESRLND 17 10 AESRLNDNVF 14 4 SEEAIDAESR 12 3 KSEEAIDAES
TABLE-US-00165 TABLE XXXVII-V10 HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 21; 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
13 WIYPTQKLNK 28 1 TKIPEDILQK 22 10 KFQWIYPTQK 18 2 KIPEDILQKF 16 6
DILQKFQWIY 16
TABLE-US-00166 TABLE XXXVII-V12 HLA-A3-10mers-193P1E1B Each peptide
is a portion of SEQ ID NO: 25; 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 ALDGEESLLS 18 4 LDGEESLLSK 16 9 SLLSKYNSNL 16 2 RALDGEESLL 10 5
DGEESLLSKY 10
TABLE-US-00167 TABLE XXXVIII-V1 HLA-A26-10mers-193P1E1B 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 323 EVEDRTSLVL 30 369 EDILQLLSKY 30 363 EVTKIPEDIL
29 214 ECVTPKLEHF 26 343 DPSSPTISSY 25 57 DVNIPELSNC 23 211
DDFECVTPKL 23 2 DPIRSFCGKL 22 264 ATPSPIIQQL 22 280 YTNSPLVPTF 22
188 IVTPPTKQSL 21 227 EYTMCLNEDY 21 347 PTISSYENLL 21 30 DGEESDFEDY
20 38 DYPMRILYDL 20 50 EVQTLKDDVN 20 173 EQEAINSDNY 20 192
PTKQSLVKVL 20 335 DTCFENLTDP 20 365 TKIPEDILQL 20 73 DVKDDLSDPP 19
199 KVLKTPKCAL 19 249 EEAIDTESRL 19 16 STLDCETARL 18 21 ETARLQRALD
18 118 AVNLLDKARL 18 285 LVPTFCTPGL 18 366 KIPEDILQLL 18 5
RSFCGKLRSL 17 35 DFEDYPMRIL 17 253 DTESRLNDNV 17 321 DLEVEDRTSL 17
37 EDYPMRILYD 16 82 PVASSCISGK 16 131 EGIDFIKATK 16 144 EKNSMDIMKI
16 155 EYFQKYGYSP 16 67 ENFQKTDVKD 15 92 SPRSPQLSDF 15 97
QLSDFGLERY 15 147 SMDIMKIREY 15 151 MKIREYFQKY 15 175 EAINSDNYKE 15
185 EPVIVIPPTK 15 186 PVIVTPPTKQ 15 219 KLEHFGISEY 15 221
EHFGISEYTM 15 250 EAIDTESRLN 15 279 EYTNSPLVPT 15 287 PTFCTPGLKI 15
325 EDRTSLVLNS 15 326 DRTSLVLNSD 15 56 DDVNIPELSN 14 77 DLSDPPVASS
14 165 RVKKNSVHEQ 14 170 SVHEQEAINS 14 234 EDYTMGLKNA 14 261
NVFATPSPII 14 301 NSIALVSTNY 14
TABLE-US-00168 TABLE XXXVIII-V5 HLA-A26-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 11; 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 PVASSCISEK 16 9 SEKSPRSPQL 11 10 EKSPRSPQLS 11 7
CISEKSPRSP 7
TABLE-US-00169 TABLE XXXVIII-V6 HLA-A26-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; 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 EEAIDAESRL 19 6 EAIDAESRLN 15 9 DAESRLNDNV 9
TABLE-US-00170 TABLE XXXVIII-V10 HLA-A26-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 DILQKFQWIY 22 2 KIPEDILQKF 19 5 EDILQKFQWI 14 1
TKIPEDILQK 12
TABLE-US-00171 TABLE XXXVIII-V12 HLA-A26-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 DGEESLLSKY 26
TABLE-US-00172 TABLE XXXIX-V1 HLA-B0702-10mers-193P1E1B 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 296 IPSTKNSIAL 24 112 LPNPPQAVNL 23 2 DPIRSFCGKL
20 346 SPTISSYENL 20 191 PPTKQSLVKV 19 358 TPTPPEVTKI 19 92
SPRSPQLSDF 18 60 IPELSNCENF 17 323 EVEDRTSLVL 14 384 TPIAIKAVPP 14
103 LERYIVSQVL 13 115 PPQAVNLLDK 13 118 AVNLLDKARL 13 133
IDFIKATKVL 13 163 SPRVKKNSVH 13 190 TPPTKQSLVK 13 199 KVLKTPKCAL 13
265 TPSPIIQQLE 13 332 LNSDTCFENL 13 365 TKIPEDILQL 13 367
IPEDILQLLS 13 389 KAVPPSKRFL 13 391 VPPSKRFLKH 13 392 PPSKRFLKHG 13
16 STLDCETARL 12 20 CETARLQRAL 12 54 LKDDVNIPEL 12 113 PNPPQAVNLL
12 114 NPPQAVNLLD 12 185 EPVIVTPPTK 12 188 IVTPPTKQSL 12 192
PTKQSLVKVL 12 194 KQSLVKVLKT 12 211 DDFECVTPKL 12 249 EEAIDTESRL 12
255 ESRLNDNVFA 12 264 ATPSPIIQQL 12 276 SDAEYTNSPL 12 285
LVPTFCTPGL 12 303 IALVSTNYPL 12 321 DLEVEDRTSL 12 343 DPSSPTISSY 12
363 EVTKIPEDIL 12 5 RSFCGKLRSL 11 39 YPMRILYDLH 11 45 YDLHSEVQTL 11
81 PPVASSCISG 11 89 SGKSPRSPQL 11 94 RSPQLSDFGL 11 95 SPQLSDFGLE 11
217 TPKLEHFGIS 11 223 FGISEYTMCL 11 231 CLNEDYTMGL 11 283
SPLVPTFCTP 11 291 TPGLKIPSTK 11 349 ISSYENLLRT 11 360 TPPEVTKIPE 11
361 PPEVTKIPED 11 366 KIPEDILQLL 11
TABLE-US-00173 TABLE XXXIX V5-HLA-B0702-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 11; 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 PPVASSCISE 11 9 SEKSPRSPQL 11
TABLE-US-00174 TABLE XXXIX V6-HLA-B0702-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO:13; 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 EEAIDAESRL 12 10 AESRLNDNVF 11 7 AIDAESRLND 7 1
NNKSEEAIDA 6 9 DAESRLNDNV 6
TABLE-US-00175 TABLE XXXIX V10-HLA-B0702-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 IPEDILQKFQ 12 11 FQWIYPTQKL 11 15 YPTQKLNKMR 10
5 EDILQKFQWI 8 2 KIPEDILQKF 7 8 LQKFQWIYPT 7 14 IYPTQKLNKM 7
TABLE-US-00176 TABLE XXXIX V12-HLA-B0702-10mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 QRALDGEESL 11 2 RALDGEESLL 11 9 SLLSKYNSNL 10 3
ALDGEESLLS 7
TABLE-US-00177 TABLE XL-V1-HLA-B08-10mers-193P1E1B Pos 1234567890
score NoResultsFound.
TABLE-US-00178 TABLE XL-V5-HLA-B08-10mers-193P1E1B Pos 1234567890
score NoResultsFound.
TABLE-US-00179 TABLE XL-V6-HLA-B08-10mers-193P1E1B Pos 1234567890
score NoResultsFound.
TABLE-US-00180 TABLE XL-V10-HLA-B08-10mers-193P1E1B Pos 1234567890
score NoResultsFound.
TABLE-US-00181 TABLE XL-V12-HLA-B08-10mers-193P1E1B Pos 1234567890
score NoResultsFound.
TABLE-US-00182 TABLE XLI-V1-HLA-B1510-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00183 TABLE XLI-V5-HLA-B1510-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00184 TABLE XLI-V6-HLA-B1510-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00185 TABLE XLI-V10-HLA-B1510-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00186 TABLE XLI-V12-HLA-B1510-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00187 TABLE XLII-V1-HLA-B2705-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00188 TABLE XLII-V5-HLA-B2705-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00189 TABLE XLII-V6-HLA-B2705-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00190 TABLE XLII-V10-HLA-B2705-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00191 TABLE XLII-V12-HLA-B2705-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00192 TABLE XLIII-V1-HLA-B2709-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00193 TABLE XLIII-V5-HLA-B2709-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00194 TABLE XLIII-V6-HLA-B2709-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00195 TABLE XLIII-V10-HLA-B2709-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00196 TABLE XLIII-V12-HLA-B2709-10mers-193P1E1B Pos
1234567890 score NoResultsFound.
TABLE-US-00197 TABLE XLIV V1-HLA-B4402-10mers- 193P1E1B 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 36 FEDYPMRILY 24 254 TESRLNDNVF 24 20 CETARLQRAL
23 249 EEAIDTESRL 22 103 LERYIVSQVL 21 127 LENQEGIDFI 21 365
TKIPEDILQL 21 362 PEVTKIPEDI 20 264 ATPSPIIQQL 18 369 EDILQLLSKY 18
113 PNPPQAVNLL 17 130 QEGIDFIKAT 17 278 AEYTNSPLVP 17 133
IDFIKATKVL 16 147 SMDIMKIREY 16 151 MKIREYFQKY 16 2 DPIRSFCGKL 15
54 LKDDVNIPEL 15 112 LPNPPQAVNL 15 183 KEEPVIVTPP 15 294 LKIPSTKNSI
15 296 IPSTKNSIAL 15 323 EVEDRTSLVL 15 324 VEDRTSLVLN 15 347
PTISSYENLL 15 389 KAVPPSKRFL 15 5 RSFCGKLRSL 14 9 GKLRSLASTL 14 16
STLDCETARL 14 32 EESDFEDYPM 14 118 AVNLLDKARL 14 144 EKNSMDIMKI 14
148 MDIMKIREYF 14 184 EEPVIVTPPT 14 192 PTKQSLVKVL 14 199
KVLKTPKCAL 14 211 DDFECVTPKL 14 214 ECVTPKLEHF 14 219 KLEHFGISEY 14
223 FGISEYTMCL 14 226 SEYTMCLNED 14 233 NEDYTMGLKN 14 243
ARNNKSEEAI 14 301 NSIALVSTNY 14 343 DPSSPTISSY 14 366 KIPEDILQLL 14
379 NSNLATPIAI 14 45 YDLHSEVQTL 13 79 SDPPVASSCI 13 97 QLSDFGLERY
13 188 IVTPPTKQSL 13 285 LVPTFCTPGL 13 313 SKTNSSSNDL 13 332
LNSDTCFENL 13 352 YENLLRTPTP 13 358 TPTPPEVTKI 13 368 PEDILQLLSK 13
27 RALDGEESDF 12 34 SDFEDYPMRI 12 35 DFEDYPMRIL 12 38 DYPMRILYDL 12
61 PELSNCENFQ 12 66 CENFQKTDVK 12 89 SGKSPRSPQL 12 92 SPRSPQLSDF 12
126 RLENQEGIDF 12 143 MEKNSMDIMK 12 168 KNSVHEQEAI 12 173
EQEAINSDNY 12 227 EYTMCLNEDY 12 248 SEEAIDTESR 12 280 YTNSPLVPTF 12
287 PTFCTPGLKI 12 322 LEVEDRTSLV 12 329 SLVLNSDTCF 12 338
FENLTDPSSP 12 363 EVTKIPEDIL 12 388 IKAVPPSKRF 12 395 KRFLKHGQNI 12
30 DGEESDFEDY 11 49 SEVQTLKDDV 11 60 IPELSNCENF 11 69 FQKTDVKDDL 11
94 RSPQLSDFGL 11 174 QEAINSDNYK 11 213 FECVTPKLEH 11 231 CLNEDYTMGL
11 261 NVFATPSPII 11 271 QQLEKSDAEY 11 273 LEKSDAEYTN 11 276
SDAEYTNSPL 11 303 IALVSTNYPL 11 321 DLEVEDRTSL 11 340 NLTDPSSPTI 11
346 SPTISSYENL 11 373 QLLSKYNSNL 11
TABLE-US-00198 TABLE XLIV V5-HLA-B4402-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 11; 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 SEKSPRSPQL 22
TABLE-US-00199 TABLE XLIV V6-HLA-B4402-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 13; 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 AESRLNDNVF 27 5 EEAIDAESRL 22 4 SEEAIDAESR
12
TABLE-US-00200 TABLE XLIV V10-HLA-B4402-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 21; 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 PEDILQKFQW 22 5 EDILQKFQWI 15 2 KIPEDILQKF 14 11
FQWIYPTQKL 12 1 TKIPEDILQK 11 6 DILQKFQWIY 11
TABLE-US-00201 TABLE XLIV V12-HLA-B4402-10mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 25; 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 EESLLSKYNS 14 2 RALDGEESLL 13 5 DGEESLLSKY 12 6
GEESLLSKYN 12 9 SLLSKYNSNL 12 1 QRALDGEESL 11 3 ALDGEESLLS 7
TABLE-US-00202 TABLE XLV-V1-HLA-B5101-10mers-193P1E1B Pos
123456789012345 score NoResultsFound.
TABLE-US-00203 TABLE XLV-V5-HLA-B5101-10mers-193P1E1B Pos
123456789012345 score NoResultsFound.
TABLE-US-00204 TABLE XLV-V6-HLA-B5101-10mers-193P1E1B Pos
123456789012345 score NoResultsFound.
TABLE-US-00205 TABLE XLV-V10-HLA-B5101-10mers-193P1E1B Pos
123456789012345 score NoResultsFound.
TABLE-US-00206 TABLE XLV-V12-HLA-B5101-10mers-193P1E1B Pos
123456789012345 score NoResultsFound.
TABLE-US-00207 TABLE XLVI V1-HLA-DRB1- 0101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 130 QEGIDFIKATKVLME 33 124 KARLENQEGIDFIKA 31
168 KNSVHEQEAINSDNY 31 38 DYPMRILYDLHSEVQ 29 300 KNSIALVSTNYPLSK 29
105 RYIVSQVLPNPPQAV 28 375 LSKYNSNLATPIAIK 28 291 TPGLKIPSTKNSIAL
27 23 ARLQRALDGEESDFE 26 116 PQAVNLLDKARLENQ 26 153 IREYFQKYGYSPRVK
26 335 DTCFENLTDPSSPTI 26 8 CGKLRSLASTLDCET 25 229 TMCLNEDYTMGLKNA
25 267 SPIIQQLEKSDAEYT 25 307 STNYPLSKTNSSSND 25 361
PPEVTKIPEDILQLL 25 369 EDILQLLSKYNSNLA 25 379 NSNLATPIAIKAVPP 25 5
RSFCGKLRSLASTLD 24 185 EPVIVTPPTKQSLVK 24 195 QSLVKVLKTPKCALK 24
283 SPLVPTFCTPGLKIP 24 350 SSYENLLRTPTPPEV 24 353 ENLLRTPTPPEVTKI
24 372 LQLLSKYNSNLATPI 24 71 KTDVKDDLSDPPVAS 23 82 PVASSCISGKSPRSP
23 85 SSCISGKSPRSPQLS 23 104 ERYIVSQVLPNPPQA 23 210 MDDFECVTPKLEHFG
23 256 SRLNDNVFATPSPII 23 338 FENLTDPSSPTISSY 23 368
PEDILQLLSKYNSNL 23 41 MRILYDLHSEVQTLK 22 101 FGLERYIVSQVLPNP 22 108
VSQVLPNPPQAVNLL 22 139 TKVLMEKNSMDIMKI 22 259 NDNVFATPSPIIQQL 22
321 DLEVEDRTSLVLNSD 22 376 SKYNSNLATPIAIKA 22 382 LATPIAIKAVPPSKR
22 385 PIAIKAVPPSKRFLK 22 214 ECVTPKLEHFGISEY 21 77 DLSDPPVASSCISGK
20 132 GIDFIKATKVLMEKN 20 286 VPTFCTPGLKIPSTK 20 1 MDPIRSFCGKLRSLA
19 33 ESDFEDYPMRILYDL 19 36 FEDYPMRILYDLHSE 19 98 LSDFGLERYIVSQVL
19 159 KYGYSPRVKKNSVHE 19 327 RTSLVLNSDTCFENL 19 349
ISSYENLLRTPTPPE 19 383 ATPIAIKAVPPSKRF 19 4 IRSFCGKLRSLASTL 18 92
SPRSPQLSDFGLERY 18 131 EGIDFIKATKVLMEK 18 136 IKATKVLMEKNSMD I 18
140 KVLMEKNSMDIMKIR 18 145 KNSMDIMKIREYFQK 18 189 VTPPTKQSLVKVLKT
18 194 KQSLVKVLKTPKCAL 18 197 LVKVLKTPKCALKMD 18 199
KVLKTPKCALKMDDF 18 205 KCALKMDDFECVTPK 18 227 EYTMCLNEDYTMGLK 18
235 DYTMGLKNARNNKSE 18 237 TMGLKNARNNKSEEA 18 265 TPSPIIQQLEKSDAE
18 270 IQQLEKSDAEYTNSP 18 15 ASTLDCETARLQRAL 17 20 CETARLQRALDGEES
17 97 QLSDFGLERYIVSQV 17 102 GLERYIVSQVLPNPP 17 110 QVLPNPPQAVNLLDK
17 137 KATKVLMEKNSMDIM 17 178 NSDNYKEEPVIVTPP 17 186
PVIVTPPTKQSLVKV 17 196 SLVKVLKTPKCALKM 17 207 ALKMDDFECVTPKLE 17
255 ESRLNDNVFATPSPI 17 285 LVPTFCTPGLKIPST 17 296 IPSTKNSIALVSTNY
17 302 SIALVSTNYPLSKTN 17 306 VSTNYPLSKTNSSSN 17 319
SNDLEVEDRTSLVLN 17 393 PSKRFLKHGQNIRDV 17 7 FCGKLRSLASTLDCE 16 12
RSLASTLDCETARLQ 16 40 PMRILYDLHSEVQTL 16 48 HSEVQTLKDDVNIPE 16 57
DVNIPELSNCENFQK 16 72 TDVKDDLSDPPVASS 16 75 KDDLSDPPVASSCIS 16 100
DFGLERYIVSQVLPN 16 107 IVSQVLPNPPQAVNL 16 142 LMEKNSMDIMKIREY 16
147 SMDIMKIREYFQKYG 16 183 KEEPVIVTPPTKQSL 16 184 EEPVIVTPPTKQSLV
16 202 KTPKCALKMDDFECV 16 232 LNEDYTMGLKNARNN 16 240
LKNARNNKSEEAIDT 16 252 IDTESRLNDNVFATP 16 258 LNDNVFATPSPIIQQ 16
260 DNVFATPSPIIQQLE 16 290 CTPGLKIPSTKNDIA 16 293 GLKIPSTKNSIALVS
16 309 NYPLSKTNSSSNDLE 16 318 SSNDLEVEDRTSLVL 16 336
TCFENLTDPSSPTIS 16 351 SYENLLRTPTPPEVT 16 364 VTKIPEDILQLLSKY 16
371 ILQLLSKYNSNLATP 16 377 KYNSNLATPIAIKAV 16 380 SNLATPIAIKAVPPS
16
TABLE-US-00208 TABLE XLVI V5-HLA-DRB1- 0101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 11; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 10 SSCISEKSPRSPQLS 23 2 DLSDPPVASSCISEK 20 6
PPVASSCISEKSPRS 15 7 PVASSCISEKSPRSP 15 12 CISEKSPRSPQLSDF 15 3
LSDPPVASSCISEKS 14 9 ASSCISEKSPRSPQL 14 13 ISEKSPRSPQLSDFG 14
TABLE-US-00209 TABLE XLVI V6-HLA-DRB1- 0101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 4 ARNNKSEEAIDAESR 18 1 LKNARNNKSEEAIDA 16 13
IDAESRLNDNVFATP 16 7 NKSEEAIDAESRLND 14 8 KSEEAIDAESRLNDN 11 14
DAESRLNDNVFATPS 11 2 KNARNNKSEEAIDAE 9 6 NNKSEEAIDAESRLN 8 9
SEEAIDAESRLNDNV 8 10 EEAIDAESRLNDNVF 8 12 AIDAESRLNDNVFAT 8
TABLE-US-00210 TABLE XLVI V10-HLA-DRB1- 0101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 21; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 2 PPEVTKIPEDILQKF 25 13 LQKFQWIYPTQKLNK 23 10
EDILQKFQWIYPTQK 18 14 QKFQWIYPTQKLNKM 18 5 VTKIPEDILQKFQWI 17 1
TPPEVTKIPEDILQK 15 8 IPEDILQKFQWIYPT 15
TABLE-US-00211 TABLE XLVI V12-HLA-DRB1-0101-15mers- 193P1E1B Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 3 ARLQRALDGEESLLS 26 13 ESLLSKYNSNLATPI 24 6
QRALDGEESLLSKYN 23 10 DGEESLLSKYNSNLA 17 12 EESLLSKYNSNLATP 16 9
LDGEESLLSKYNSNL 15
TABLE-US-00212 TABLE XLVII V1-HLA-DRB1- 0301-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 364 VTKIPEDILQLLSKY 31 229 TMCLNEDYTMGLKNA 29
197 LVKVLKTPKCALKMD 28 371 ILQLLSKYNSNLATP 28 51 VQTLKDDVNIPELSN 27
186 PVIVTPPTKQSLVKV 27 116 PQAVNLLDKARLENQ 26 361 PPEVTKIPEDILQLL
26 67 ENFQKTDVKDDLSDP 25 247 KSEEAIDTESRLNDN 24 319 SNDLEVEDRTSLVLN
24 40 PMRILYDLHSEVQTL 23 71 KTDVKDDLSDPPVAS 22 205 KCALKMDDFECVTPK
22 174 QEAINSDNYKEEPVI 21 327 RTSLVLNSDTCFENL 21 329
SLVLNSDTCFENLTD 21 138 ATKVLMEKNSMDIMK 20 346 SPTISSYENLLRTPT 20 13
SLASTLDCETARLQR 19 95 SPQLSDFGLERYIVS 19 118 AVNLLDKARLENQEG 19 124
KARLENQEGIDFIKA 19 145 KNSMDIMKIREYFQK 19 213 FECVTPKLEHFGISE 19
217 TPKLEHFGISEYTMC 19 237 TMGLKNARNNKSEEA 19 249 EEAIDTESRLNDNVF
19 321 DLEVEDRTSLVLNSD 19 369 EDILQLLSKYNSNLA 19 388
IKAVPPSKRFLKHGQ 19 24 RLQRALDGEESDFED 18 44 LYDLHSEVQTLKDDV 18 57
DVNIPELSNCENFQK 18 109 SQVLPNPPQAVNLLD 18 150 IMKIREYFQKYGYSP 18
194 KQSLVKVLKTPKCAL 18 266 PSPIIQQLEKSDAEY 18 271 QQLEKSDAEYTNSPL
18 283 SPLVPTFCTPGLKIP 18 293 GLKIPSTKNSIALVS 18 315
TNSSSNDLEVEDRTS 18 345 SSPTISSYENLLRTP 18 394 SKRFLKHGQNIRDVS 18 18
LDCETARLQRALDGE 17 25 LQRALDGEESDFEDY 17 29 LDGEESDFEDYPMRI 17 33
ESDFEDYPMRILYDL 17 60 IPELSNCENFQKTDV 17 132 GIDFIKATKVLMEKN 17 225
ISEYTMCLNEDYTMG 17 267 SPIIQQLEKSDAEYT 17 387 AIKAVPPSKRFLKHG 17
395 KRFLKHGQNIRDVSN 17 4 IRSFCGKLRSLASTL 16 74 VKDDLSDPPVASSCI 16
108 VSQVLPNPPQAVNLL 16 146 NSMDIMKIREYFQKY 16 147 SMDIMKIREYFQKYG
16 206 CALKMDDFECVTPKL 16 301 NSIALVSTNYPLSKT 16 34 SDFEDYPMRILYDLH
15 117 QAVNLLDKARLENQE 15 274 EKSDAEYTNSPLVPT 15
TABLE-US-00213 TableXLVII-V5-HLA-DRB1- 0301-15mers Each peptide is
a portion of SEQ ID NO: 11; each start position is specified, the
length of peptide is 15 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 10 SSCISEKSPRSPQLS 12 5 DPPVASSCISEKSPR 11 12 CISEKSPRSPQLSDF
10 15 EKSPRSPQLSDFGLE 10 14 SEKSPRSPQLSDFGL 8 7 PVASSCISEKSPRSP 7 8
VASSCISEKSPRSPQ 7 11 SCISEKSPRSPQLSD 7
TABLE-US-00214 TableXLVII-V6-HLA-DRB1- 0301-15mers Each peptide is
a portion of SEQ ID NO: 13; each start position is specified, the
length of peptide is 15 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 8 KSEEAIDAESRLNDN 24 10 EEAIDAESRLNDNVF 19 15 AESRLNDNVFATPSP
13
TABLE-US-00215 TableXLVII-V10-HLA-DRB1- 0301-15mers Each peptide is
a portion of SEQ ID NO: 21; each start position is specified, the
length of peptide is 15 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 5 VTKIPEDILQKFQWI 31 2 PPEVTKIPEDILQKF 26 9 PEDILQKFQWIYPTQ
26
TABLE-US-00216 TableXLVII-V12-HLA-DRB1- 0301-15mers Each peptide is
a portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 15 amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 12 EESLLSKYNSNLATP 28 4 RLQRALDGEESLLSK 26 5 LQRALDGEESLLSKY
18 6 QRALDGEESLLSKYN 13
TABLE-US-00217 TableXLVIII-V1-HLA-DR1- 0401-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 349 ISSYENLLRTPTPPE 28 40 PMRILYDLHSEVQTL 26
41 MRILYDLHSEVQTLK 26 44 LYDLHSEVQTLKDDV 26 57 DVNIPELSNCENFQK 26
229 TMCLNEDYTMGLKNA 26 237 TMGLKNARNNKSEEA 26 283 SPLVPTFCTPGLKIP
26 319 SNDLEVEDRTSLVLN 26 368 PEDILQLLSKYNSNL 26 98 LSDFGLERYIVSQVL
22 132 GIDFIKATKVLMEKN 22 157 FQKYGYSPRVKKNSV 22 179
SDNYKEEPVIVTPPT 22 307 STNYPLSKTNSSSND 22 335 DTCFENLTDPSSPTI 22 8
CGKLRSLASTLDCET 20 15 ASTLDCETARLQRAL 20 26 QRALDGEESDFEDYP 20 38
DYPMRILYDLHSEVQ 20 48 HSEVQTLKDDVNIPE 20 51 VQTLKDDVNIPELSN 20 60
IPELSNCENFQKTDV 20 71 KTDVKDDLSDPPVAS 20 109 SQVLPNPPQAVNLLD 20 116
PQAVNLLDKARLENQ 20 119 VNLLDKARLENQEGI 20 130 QEGIDFIKATKVLME 20
138 ATKVLMEKNSMDIMK 20 147 SMDIMKIREYFQKYG 20 185 EPVIVTPPTKQSLVK
20 194 KQSLVKVLKTPKCAL 20 195 QSLVKVLKTPKCALK 20 205
KCALKMDDFECVTPK 20 249 EEAIDTESRLNDNVF 20 259 NDNVFATPSPIIQQL 20
267 SPIIQQLEKSDAEYT 20 291 TPGLKIPSTKNSIAL 20 293 GLKIPSTKNSIALVS
20 300 KNSIALVSTNYPLSK 20 309 NYPLSKTNSSSNDLE 20 329
SLVLNSDTCFENLTD 20 338 FENLTDPSSPTISSY 20 361 PPEVTKIPEDILQLL 20
364 VTKIPEDILQLLSKY 20 369 EDILQLLSKYNSNLA 20 372 LQLLSKYNSNLATPI
20 388 IKAVPPSKRFLKHGQ 20 5 RSFCGKLRSLASTLD 18 77 DLSDPPVASSCISGK
18 78 LSDPPVASSCISGKS 18 97 QLSDFGLERYIVSQV 18 101 FGLERYIVSQVLPNP
18 106 YIVSQVLPNPPQAVN 18 122 LDKARLENQEGIDFI 18 170
SVHEQEAINSDNYKE 18 182 YKEEPVIVTPPTKQS 18 214 ECVTPKLEHFGISEY 18
221 EHFGISEYTMCLNED 18 234 EDYTMGLKNARNNKS 18 264 ATPSPIIQQLEKSDA
18 280 YTNSPLVPTFCTPGL 18 290 CTPGLKIPSTKNSIA 18 320
NDLEVEDRTSLVLNS 18 325 EDRTSLVLNSDTCFE 18 337 CFENLTDPSSPTISS 18
343 DPSSPTISSYENLLR 18 365 TKIPEDILQLLSKYN 18 376 SKYNSNLATPIAIKA
18 392 PPSKRFLKHGQNIRD 18 4 IRSFCGKLRSLASTL 17 33 ESDFEDYPMRILYDL
16 42 RILYDLHSEVQTLKD 16 103 LERYIVSQVLPNPPQ 16 210 MDDFECVTPKLEHFG
16 225 ISEYTMCLNEDYTMG 16 260 DNVFATPSPIIQQLE 16 277
DAEYTNSPLVPTFCT 16 375 LSKYNSNLATPIAIK 16 394 SKRFLKHGQNIRDVS 16
118 AVNLLDKARLENQEG 15 139 TKVLMEKNSMDIMKI 15 321 DLEVEDRTSLVLNSD
15 371 ILQLLSKYNSNLATP 15 1 MDPIRSFCGKLRSLA 14 11 LRSLASTLDCETARL
14 22 TARLQRALDGEESDF 14 75 KDDLSDPPVASSCIS 14 80 DPPVASSCISGKSPR
14 95 SPQLSDFGLERYIVS 14 100 DFGLERYIVSQVLPN 14 105 RYIVSQVLPNPPQAV
14 108 VSQVLPNPPQAVNLL 14 140 KVLMEKNSMDIMKIR 14 150
IMKIREYFQKYGYSP 14 163 SPRVKKNSVHEQEAI 14 168 KNSVHEQEAINSDNY 14
174 QEAINSDNYKEEPVI 14 184 EEPVIVTPPTKQSLV 14 186 PVIVTPPTKQSLVKV
14 197 LVKVLKTPKCALKMD 14 198 VKVLKTPKCALKMDD 14 207
ALKMDDFECVTPKLE 14 217 TPKLEHFGISEYTMC 14 222 HFGISEYTMCLNEDY 14
227 EYTMCLNEDYTMGLK 14 270 IQQLEKSDAEYTNSP 14 302 SIALVSTNYPLSKTN
14 303 IALVSTNYPLSKTNS 14 328 TSLVLNSDTCFENLT 14 346
SPTISSYENLLRTPT 14 352 YENLLRTPTPPEVTK 14 353 ENLLRTPTPPEVTKI 14
379 NSNLATPIAIKAVPP 14 385 PIAIKAVPPSKRFLK 14 395 KRFLKHGQNIRDVSN
14
TABLE-US-00218 TableXLVIII-V5-HLA-DR1- 0401-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 11; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 2 DLSDPPVASSCISEK 18 3 LSDPPVASSCISEKS 18 9
ASSCISEKSPRSPQL 18 5 DPPVASSCISEKSPR 14 6 PPVASSCISEKSPRS 12 11
SCISEKSPRSPQLSD 12 12 CISEKSPRSPQLSDF 12
TABLE-US-00219 TableXLVIII-V6-HLA-DR1- 0401-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 10 EEAIDAESRLNDNVF 20 9 SEEAIDAESRLNDNV 18 1
LKNARNNKSEEAIDA 12 4 ARNNKSEEAIDAESR 12 6 NNKSEEAIDAESRLN 12 8
KSEEAIDAESRLNDN 12 14 DAESRLNDNVFATPS 12 15 AESRLNDNVFATPSP 12
TABLE-US-00220 TableXLVIII-V10-HLA-DR1- 0401-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO:21; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 13 LQKFQWIYPTQKLNK 22 15 KFQWIYPTQKLNKMR 22 2
PPEVTKIPEDILQKF 20 6 TKIPEDILQKFQWIY 18 5 VTKIPEDILQKFQWI 14 10
EDILQKFQWIYPTQK 14 4 EVTKIPEDILQKFQW 12 14 QKFQWIYPTQKLNKM 12
TABLE-US-00221 TableXLVIII-V12-HLA-DR1- 0401-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO:25; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 6 QRALDGEESLLSKYN 26 13 ESLLSKYNSNLATPI 20 9
LDGEESLLSKYNSNL 18 12 EESLLSKYNSNLATP 15 2 TARLQRALDGEESLL 14 3
ARLQRALDGEESLLS 12 4 RLQRALDGEESLLSK 12 7 RALDGEESLLSKYNS 12 10
DGEESLLSKYNSNLA 12 14 SLLSKYNSNLATPIA 12
TABLE-US-00222 TableXLIX-V1-HLA-DRB1- 1101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 159 KYGYSPRVKKNSVHE 26 267 SPIIQQLEKSDAEYT 26
369 EDILQLLSKYNSNLA 26 4 IRSFCGKLRSLASTL 25 349 ISSYENLLRTPTPPE 24
116 PQAVNLLDKARLENQ 22 335 DTCFENLTDPSSPTI 22 194 KQSLVKVLKTPKCAL
20 233 NEDYTMGLKNARNNK 20 306 VSTNYPLSKTNSSSN 20 132
GIDFIKATKVLMEKN 19 157 FQKYGYSPRVKKNSV 19 38 DYPMRILYDLHSEVQ 18 105
RYIVSQVLPNPPQAV 18 300 KNSIALVSTNYPLSK 18 98 LSDFGLERYIVSQVL 17 153
IREYFQKYGYSPRVK 17 191 PPTKQSLVKVLKTPK 17 210 MDDFECVTPKLEHFG 17
286 VPTFCTPGLKIPSTK 17 2 DPIRSFCGKLRSLAS 16 97 QLSDFGLERYIVSQV 16
144 EKNSMDIMKIREYFQ 16 186 PVIVTPPTKQSLVKV 16 287 PTFCTPGLKIPSTKN
16 307 STNYPLSKTNSSSND 16 19 DCETARLQRALDGEE 15 68 NFQKTDVKDDLSDPP
15 319 SNDLEVEDRTSLVLN 15 361 PPEVTKIPEDILQLL 15 381
NLATPIAIKAVPPSK 15 388 IKAVPPSKRFLKHGQ 15 397 FLKHGQNIRDVSNKE 15 40
PMRILYDLHSEVQTL 14 104 ERYIVSQVLPNPPQA 14 118 AVNLLDKARLENQEG 14
137 KATKVLMEKNSMDIM 14 160 YGYSPRVKKNSVHEQ 14 175 EAINSDNYKEEPVIV
14 195 QSLVKVLKTPKCALK 14 197 LVKVLKTPKCALKMD 14 213
FECVTPKLEHFGISE 14 214 ECVTPKLEHFGISEY 14 249 EEAIDTESRLNDNVF 14
255 ESRLNDNVFATPSPI 14 358 TPTPPEVTKIPEDIL 14 392 PPSKRFLKHGQNIRD
14 8 CGKLRSLASTLDCET 13 41 MRILYDLHSEVQTLK 13 48 HSEVQTLKDDVNIPE 13
85 SSCISGKSPRSPQLS 13 102 GLERYIVSQVLPNPP 13 127 LENQEGIDFIKATKV 13
130 QEGIDFIKATKVLME 13 136 IKATKVLMEKNSMDI 13 145 KNSMDIMKIREYFQK
13 237 TMGLKNARNNKSEEA 13 293 GLKIPSTKNSIALVS 13 302
SIALVSTNYPLSKTN 13 318 SSNDLEVEDRTSLVL 13 365 TKIPEDILQLLSKYN 13
372 LQLLSKYNSNLATPI 13 376 SKYNSNLATPIAIKA 13 379 NSNLATPIAIKAVPP
13 385 PIAIKAVPPSKRFLK 13 5 RSFCGKLRSLASTLD 12 12 RSLASTLDCETARLQ
12 23 ARLQRALDGEESDFE 12 33 ESDFEDYPMRILYDL 12 57 DVNIPELSNCENFQK
12 71 KTDVKDDLSDPPVAS 12 75 KDDLSDPPVASSCIS 12 82 PVASSCISGKSPRSP
12 121 LLDKARLENQEGIDF 12 147 SMDIMKIREYFQKYG 12 150
IMKIREYFQKYGYSP 12 165 RVKKNSVHEQEAINS 12 168 KNSVHEQEAINSDNY 12
181 NYKEEPVIVTPPTKQ 12 185 EPVIVTPPTKQSLVK 12 207 ALKMDDFECVTPKLE
12 225 ISEYTMCLNEDYTMG 12 232 LNEDYTMGLKNARNN 12 256
SRLNDNVFATPSPII 12 277 DAEYTNSPLVPTFCT 12 282 NSPLVPTFCTPGLKI 12
291 TPGLKIPSTKNSIAL 12 350 SSYENLLRTPTPPEV 12 368 PEDILQLLSKYNSNL
12 382 LATPIAIKAVPPSKR 12 383 ATPIAIKAVPPSKRF 12
TABLE-US-00223 TableXLIX-V5-HLA-DRB1- 1101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 11; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 10 SSCISEKSPRSPQLS 13 7 PVASSCISEKSPRSP 12 3
LSDPPVASSCISEKS 8 8 VASSCISEKSPRSPQ 8 9 ASSCISEKSPRSPQL 8 11
SCISEKSPRSPQLSD 8 6 PPVASSCISEKSPRS 7 13 ISEKSPRSPQLSDFG 7 2
DLSDPPVASSCISEK 6 5 DPPVASSCISEKSPR 6
TABLE-US-00224 TableXLIX-V6-HLA-DRB1- 1101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 13; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 10 EEAIDAESRLNDNVF 14 13 IDAESRLNDNVFATP 7 1
LKNARNNKSEEAIDA 6 4 ARNNKSEEAIDAESR 6 6 NNKSEEAIDAESRLN 6 7
NKSEEAIDAESRLND 6 14 DAESRLNDNVFATPS 6
TABLE-US-00225 TableXLIX-V10-HLA-DRB1- 1101E1B Each
peptid-15mers-193P1e is a portion of SEQ ID NO: 21; each start
position is specified, the length of peptide is 15 amino acids, and
the end position for each peptide is the start position plus
fourteen. Pos 123456789012345 score 13 LQKFQWIYPTQKLNK 16 2
PPEVTKIPEDILQKF 15 7 KIPEDILQKFQWIYP 14 10 EDILQKFQWIYPTQK 12 15
KFQWIYPTQKLNKMR 11 5 VTKIPEDILQKFQWI 7 9 PEDILQKFQWIYPTQ 7
TABLE-US-00226 TableXLIX-V12-HLA-DRB1- 1101-15mers-193P1E1B Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 15 amino acids, and the end
position for each peptide is the start position plus fourteen. Pos
123456789012345 score 10 DGEESLLSKYNSNLA 20 6 QRALDGEESLLSKYN 13 13
ESLLSKYNSNLATPI 13 3 ARLQRALDGEESLLS 12
TABLE-US-00227 TABLE L Properties of 193P1E1B Bioinformatic Program
Outcome Variants 1, 5, 6 ORF ORF Finder 805-2043 Protein Length n/a
412 amino acids Transmembrane region TM Pred No TM HMMTop No TM
Sosui No TM, soluble TMHMM No TM Signal Peptide Signal P indicates
no signal pI pI/MW tool pI 5.03 Molecular weight pI/MW tool 46.2
kDa Localization PSORT Mitochondrial 48% PSORT II Nuclear 60.9%
iPSORT No signal motif Motifs Pfam No motif Prints Rhodopsin Blocks
No motif Prosite No motif Variant 9 ORF ORF Finder 989-1981 Protein
Length n/a 330 amino acids Transmembrane region TM Pred No TM
HMMTop No TM Sosui No TM, soluble TMHMM No TM Signal Peptide Signal
P indicates no signal pI pI/MW tool pI 5.17 Molecular weight pI/MW
tool 16.5 kDa Localization PSORT Cytoplasmic 45% PSORT II Nuclear
60.9% iPSORT No signal motif Motifs Pfam No motif Prints No motif
Blocks No motif Prosite No motif Variant 10 ORF ORF Finder 805-1971
Protein Length n/a 388 amino acids Transmembrane region TM Pred No
TM HMMTop No TM Sosui No TM, soluble TMHMM No TM Signal Peptide
Signal P indicates no signal pI pI/MW tool pI 4.8 Molecular weight
pI/MW tool 34.5 kDa Localization PSORT Mitochondrial 48% PSORT II
Nuclear 60.9% iPSORT No signal motif Motifs Pfam No motif Prints No
motif Blocks No motif Prosite No motif Variant 12 ORF ORF Finder
805-1026 Protein Length n/a 73 amino acids Transmembrane region TM
Pred No TM HMMTop No TM Sosui No TM, soluble TMHMM No TM Signal
Peptide Signal P indicates no signal pI pI/MW tool pI 9.4 Molecular
weight pI/MW tool 8.1 kDa Localization PSORT Mitochondrial 48%
PSORT II Nuclear 60.9% iPSORT No signal motif Motifs Pfam No motif
Prints No motif Blocks No motif Prosite No motif
TABLE-US-00228 TABLE LI Nucleotide sequence of transcript variant
193P1E1B v.9 (SEQ ID NO: 93) 1 tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 61 caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 121 catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt 181
tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 361 tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 421 aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 481 cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga 541
ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc
601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg
gcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag
agccgaggac gcgtccggcg 721 ccgagattca aactagtggc gggaggctgt
gagctgagcg gtggggtctg cgtacgcctg 781 gagtccttcc ccgctgtgct
cagcatggac cctatccgga gcttctgcgg gaagctgcgg 841 tctctggcca
gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag 901
gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatc aagaaggcat
961 tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggata
ttatgaaaat 1021 aagagagtat ttccagaagt atggatatag tccacgtgtc
aagaaaaatt cagtacacga 1081 gcaagaagcc attaactctg acccagagtt
gtctaattgt gaaaattttc agaagactga 1141 tgtgaaagat gatctgtctg
atcctcctgt tgcaagcagt tgtatttctg ggaagtctcc 1201 acgtagtcca
caactttcag attttggact tgagcggtac atcgtatccc aagttctacc 1261
aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaa ccccacctac
1321 caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa
tggatgattt 1381 tgagtgtgta actcctaaat tagaacactt tggtatctct
gaatatacta tgtgtttaaa 1441 tgaagattac acaatgggac ttaaaaatgc
gaggaataat aaaagtgagg aggccataga 1501 tacagaatcc aggctcaatg
ataatgtttt tgccactccc agccccatca tccagcagtt 1561 ggaaaaaagt
gatgccgaat ataccaactc tcctttggta cctacattct gtactcctgg 1621
tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaatt acccattatc
1681 aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgt
tggttttaaa 1741 ttcagacaca tgctttgaga atttaacaga tccctcttca
cctacgattt cttcttatga 1801 gaatctgctc agaacaccta cacctccaga
agtaactaaa attccagaag atattctcca 1861 gcttttatca aaatacaact
caaacctagc tactccaata gcaattaaag cagtgccacc 1921 cagtaaaagg
ttccttaaac atggacagaa catccgagat gtcagcaaca aagaaaactg 1981
aaattccagt ggatctatcc aacacagaaa ctgaacaaaa tgagatgaaa gccgagctgg
2041 accgatttta acattcacat tgccctgcct ctgtccccct ttaaacgttg
acccatttta 2101 aagacaaaca tgaacattaa catcataata tgctttttat
gaagtttcaa taaggtttaa 2161 ccttagtctt gttgacatgt agcccagtca
ttcactcttt aaggactatt agtgtttcat 2221 tgatactaaa ttacccagct
taatcaacag aatggtttaa gtagtaccag gaagtaggac 2281 aagtaatttc
aaaaatataa aggtgtttgc tactcagatg aggccgcccc tgaccttctg 2341
gccagagaga cattgctgcc agccagctct gccttcccat catctccttt caggaccgtc
2401 ccacaccttt tacttgctca gtgctgtctg aagatgcagt tgctgtttgc
aaacaacagg 2461 aacaccagtt aaactaatta ggaaacagag ggagatttcc
aggcctgggt aactatatac 2521 tgtgaccatt ggcggttgag accggtcttc
aaccagtgga accccgaact ctgctgtcag 2581 ggtgtggact tcggtgctct
tccaagtttt cacctggggg ggggagctaa ccccctatgt 2641 tcacgccttc
tattcccatt ggcgctgaac tcttaaggtc actctggtcg cttgtgaccc 2701
cgtaaccctg atgtacccct ctaaaaggtg aggggc
TABLE-US-00229 TABLE LII Nucleotide sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 94) and 193P1E1B v.9 (SEQ ID NO: 95) Score = 1744
bits (907), Expect = 0.0Identities = 907/907 (100%) Strand =
Plus/Plus ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## Score = 3519 bits (1830), Expect =
0.0Identities = 1830/1830 (100%) Strand = Plus/Plus ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047##
TABLE-US-00230 TABLE LIII Peptide sequences of protein coded by
193P1E1B v.9 (SEQ ID NO: 96) MEKNSMDIMK IREYFQKYGY SPRVKKNSVH
EQEAINSDPE LSNCENFQKT DVKDDLSDPP 60 VASSCISGKS PRSPQLSDFG
LERYIVSQVL PNPPQAVNNY KEEPVIVTPP TKQSLVKVLK 120 TPKCALKMDD
FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAI DTESRLNDNV 180
FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPL SKTNSSSNDL
240 EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDIL
QLLSKYNSNL 300 ATPIAIKAVP PSKRFLKHGQ NIRDVSNKEN 330
TABLE-US-00231 TABLE LIV Amino acid sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 97) and 193P1E1B v.9 (SEQ ID NO: 98) Score = 665
bits (1716), Expect = Identities = 330/330 (100%), Positives =
330/330 (100%) V.1: 83
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP V.9: 1
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 60
V.1: 143
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK V.9:
61 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120
V.1: 203
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV V.9:
121 TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV
180 V.1: 263
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL V.9:
181 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL
240 V.1: 323
EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 382
EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL V.9:
241 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL
300 V.1: 383 ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 412
ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN V.9: 301
ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 330
TABLE-US-00232 TABLE LV Nucleotide sequence of transcript variant
193P1E1B v.10 (SEQ ID NO: 99) 1 tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 61 caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 121 catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt 181
tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 361 tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 421 aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 481 cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga 541
ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc
601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg
gcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag
agccgaggac gcgtccggcg 721 ccgagattca aactagtggc gggaggctgt
gagctgagcg gtggggtctg cgtacgcctg 781 gagtccttcc ccgctgtgct
cagcatggac cctatccgga gcttctgcgg gaagctgcgg 841 tctctggcca
gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag 901
gaaagcgact ttgaagatta tccaatgaga attttatatg accttcattc agaagttcag
961 actctaaagg atgatgttaa tattcttctt gataaagcaa gattggaaaa
tcaagaaggc 1021 attgatttca taaaggcaac aaaagtacta atggaaaaaa
attcaatgga tattatgaaa 1081 ataagagagt atttccagaa gtatggatat
agtccacgtg tcaagaaaaa ttcagtacac 1141 gagcaagaag ccattaactc
tgacccagag ttgtctaatt gtgaaaattt tcagaagact 1201 gatgtgaaag
atgatctgtc tgatcctcct gttgcaagca gttgtatttc tgggaagtct 1261
ccacgtagtc cacaactttc agattttgga cttgagcggt acatcgtatc ccaagttcta
1321 ccaaaccctc cacaggcagt gaacaactat aaggaagagc ccgtaattgt
aaccccacct 1381 accaaacaat cactagtaaa agtactaaaa actccaaaat
gtgcactaaa aatggatgat 1441 tttgagtgtg taactcctaa attagaacac
tttggtatct ctgaatatac tatgtgttta 1501 aatgaagatt acacaatggg
acttaaaaat gcgaggaata ataaaagtga ggaggccata 1561 gatacagaat
ccaggctcaa tgataatgtt tttgccactc ccagccccat catccagcag 1621
ttggaaaaaa gtgatgccga atataccaac tctcctttgg tacctacatt ctgtactcct
1681 ggtttgaaaa ttccatctac aaagaacagc atagctttgg tatccacaaa
ttacccatta 1741 tcaaaaacaa atagttcatc aaatgatttg gaagttgaag
atcgtacttc gttggtttta 1801 aattcagaca catgctttga gaatttaaca
gatccctctt cacctacgat ttcttcttat 1861 gagaatctgc tcagaacacc
tacacctcca gaagtaacta aaattccaga agatattctc 1921 cagaaattcc
agtggatcta tccaacacag aaactgaaca aaatgagatg aaagccgagc 1981
tggaccgatt ttaacattca cattgccctg cctctgtccc cctttaaacg ttgacccatt
2041 ttaaagacaa acatgaacat taacatcata atatgctttt tatgaagttt
caataaggtt 2101 taaccttagt cttgttgaca tgtagcccag tcattcactc
tttaaggact attagtgttt 2161 cattgatact aaattaccca gcttaatcaa
cagaatggtt taagtagtac caggaagtag 2221 gacaagtaat ttcaaaaata
taaaggtgtt tgctactcag atgaggccgc ccctgacctt 2281 ctggccagag
agacattgct gccagccagc tctgccttcc catcatctcc tttcaggacc 2341
gtcccacacc ttttacttgc tcagtgctgt ctgaagatgc agttgctgtt tgcaaacaac
2401 aggaacacca gttaaactaa ttaggaaaca gagggagatt tccaggcctg
ggtaactata 2461 tactgtgacc attggcggtt gagaccggtc ttcaaccagt
ggaaccccga actctgctgt 2521 cagggtgtgg acttcggtgc tcttccaagt
tttcacctgg gggggggagc taacccccta 2581 tgttcacgcc ttctattccc
attggcgctg aactcttaag gtcactctgg tcgcttgtga 2641 ccccgtaacc
ctgatgtacc cctctaaaag gtgaggggc
TABLE-US-00233 TABLE LVI Nucleotide sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 100) and 193P1E1B v.10 (SEQ ID NO: 101) Score =
3698 bits (1923), Expect = 0.0Identities = 1923/1923 (100%) Strand
= Plus/Plus ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080## Score 1456 bits
(757), Expect = 0.0Identities = 757/757 (100%) Strancd = Plus/Plus
##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093##
TABLE-US-00234 TABLE LVII Peptide sequences of protein coded by
193P1E1B v.10 (SEQ ID NO: 102) MDPIRSFCGK LRSLASTLDC ETARLQRALD
GEESDFEDYP MRILYDLHSE VQTLKDDVNI 60 LLDKARLENQ EGIDFIKATK
VLMEKNSMDI MKIREYFQKY GYSPRVKKNS VHEQEAINSD 120 PELSNCENFQ
KTDVKDDLSD PPVASSCISG KSPRSPQLSD FGLERYIVSQ VLPNPPQAVN 180
NYKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL EHFGISEYTM CLNEDYTMGL
240 KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY TNSPLVPTFC
TPGLKIPSTK 300 NSIALVSTNY PLSKTNSSSN DLEVEDRTSL VLNSDTCFEN
LTDPSSPTIS SYENLLRTPT 360 PPEVTKIPED ILQKFQWIYP TQKLNKMR 388
TABLE-US-00235 TABLE LVIII Amino acid sequence alignment of
193P1E1B v.1 (SEQ ID NO: 103) and 193P1E1B v.10 (SEQ ID NO: 104)
Score = 749 bits (1935), Expect = 0.0 Identities = 373/373 (100%),
Positives = 373/373 (100%) V.1: 1
MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI 60
MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI V.10:
1 MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI 60
V.1: 61
LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD 120
LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD V.10:
61 LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD 120
V.1: 121
PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN 180
PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN V.10:
121 PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN
180 V.1: 181
NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL 240
NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL V.10:
181 NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL
240 V.1: 241
KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK 300
KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK V.10:
241 KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK
300 V.1: 301
NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT 360
NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT V.10:
301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT
360 V.1: 361 PPEVTKIPEDILQ 373 PPEVTKIPEDILQ V.10: 361
PPEVTKIPEDILQ 373
TABLE-US-00236 TABLE LIX Nucleotide sequence of transcript variant
193P1E1B v.11 (SEQ ID NO: 105) 1 tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 61 caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 121 catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt 181
tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 361 tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 421 aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 481 cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga 541
ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc
601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg
gcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag
agccgaggac gcgtccggcg 721 ccgagattca aactagtggc gggaggctgt
gagctgagcg gtggggtctg cgtacgcctg 781 gagtccttcc ccgctgtgct
cagcatggac cctatccgga gcttctgcgg gaagctgcgg 841 tctctggcca
gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag 901
gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatc aagaaggcat
961 tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggata
ttatgaaaat 1021 aagagagtat ttccagaagt atggatatag tccacgtgtc
aagaaaaatt cagtacacga 1081 gcaagaagcc attaactctg acccagagtt
gtctaattgt gaaaattttc agaagactga 1141 tgtgaaagat gatctgtctg
atcctcctgt tgcaagcagt tgtatttctg ggaagtctcc 1201 acgtagtcca
caactttcag attttggact tgagcggtac atcgtatccc aagttctacc 1261
aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaa ccccacctac
1321 caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa
tggatgattt 1381 tgagtgtgta actcctaaat tagaacactt tggtatctct
gaatatacta tgtgtttaaa 1441 tgaagattac acaatgggac ttaaaaatgc
gaggaataat aaaagtgagg aggccataga 1501 tacagaatcc aggctcaatg
ataatgtttt tgccactccc agccccatca tccagcagtt 1561 ggaaaaaagt
gatgccgaat ataccaactc tcctttggta cctacattct gtactcctgg 1621
tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaatt acccattatc
1681 aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgt
tggttttaaa 1741 ttcagacaca tgctttgaga atttaacaga tccctcttca
cctacgattt cttcttatga 1801 gaatctgctc agaacaccta cacctccaga
agtaactaaa attccagaag atattctcca 1861 gaaattccag tggatctatc
caacacagaa actgaacaaa atgagatgaa agccgagctg 1921 gaccgatttt
aacattcaca ttgccctgcc tctgtccccc tttaaacgtt gacccatttt 1981
aaagacaaac atgaacatta acatcataat atgcttttta tgaagtttca ataaggttta
2041 accttagtct tgttgacatg tagcccagtc attcactctt taaggactat
tagtgtttca 2101 ttgatactaa attacccagc ttaatcaaca gaatggttta
agtagtacca ggaagtagga 2161 caagtaattt caaaaatata aaggtgtttg
ctactcagat gaggccgccc ctgaccttct 2221 ggccagagag acattgctgc
cagccagctc tgccttccca tcatctcctt tcaggaccgt 2281 cccacacctt
ttacttgctc agtgctgtct gaagatgcag ttgctgtttg caaacaacag 2341
gaacaccagt taaactaatt aggaaacaga gggagatttc caggcctggg taactatata
2401 ctgtgaccat tggcggttga gaccggtctt caaccagtgg aaccccgaac
tctgctgtca 2461 gggtgtggac ttcggtgctc ttccaagttt tcacctgggg
gggggagcta accccctatg 2521 ttcacgcctt ctattcccat tggcgctgaa
ctcttaaggt cactctggtc gcttgtgacc 2581 ccgtaaccct gatgtacccc
tctaaaaggt gaggggc
TABLE-US-00237 TABLE LX Nucleotide sequence slignment of 193P1E1B
v.1 (SEQ ID NO: 106) and 193P1E1B v.11 (SEQ ID NO: 107) Score =
1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand =
Plus/Plus ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108## ##STR00109## Score = 1836 bits (955), Expect =
0.0Identities = 955/955 (100%) Strand = Plus/Plus ##STR00110##
##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115##
##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120##
##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125##
Score = 1456 bits (757), Expect = 0.0Identities = 757/757 (100%)
Strand = Plus/Plus ##STR00126## ##STR00127## ##STR00128##
##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133##
##STR00134## ##STR00135## ##STR00136## ##STR00137##
##STR00138##
TABLE-US-00238 TABLE LXI Peptide sequences of protein coded by
193P1E1B v.11 (SEQ ID NO: 108) MEKNSMDIMK IREYFQKYGY SPRVKKNSVH
EQEAINSDPE LSNCENFQKT DVKDDLSDPP 60 VASSCISGKS PRSPQLSDFG
LERYIVSQVL PNPPQAVNNY KEEPVIVTPP TKQSLVKVLK 120 TPKCALKMDD
FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAI DTESRLNDNV 180
FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPL SKTNSSSNDL
240 EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDIL
QKFQWIYPTQ 300 KLNKMR 306
TABLE-US-00239 TABLE LXII Amino acid sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 109) and 193P1E1B v.11 (SEQ ID NO: 110) Score = 589
bits (1518), Expect = e-167 Identities = 291/291 (100%), Positives
= 291/291 (100%) V.1: 83
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP V.11:
1 MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 60
V.1: 143
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK V.11:
61 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120
V.1: 203
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV V.11:
121 TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV
180 V.1: 263
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL V.11:
181 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL
240 V.1: 323 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ
373 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ V.11: 241
EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ 291
TABLE-US-00240 TABLE LXIII Nucleotide sequence of transcript
variant 193P1E1B v.12 (SEQ ID NO: 111) 1 tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 61 caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact 121
catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 241 cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 361 tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 421 aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 481
cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 721 ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 781 gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg 841
tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag
901 gaaagccttt tatcaaaata caactcaaac ctagctactc caatagcaat
taaagcagtg 961 ccacccagta aaaggttcct taaacatgga cagaacatcc
gagatgtcag caacaaagaa 1021 aactgaaatt ccagtggatc tatccaacac
agaaactgaa caaaatgaga tgaaagccga 1081 gctggaccga ttttaacatt
cacattgccc tgcctctgtc cccctttaaa cgttgaccca 1141 ttttaaagac
aaacatgaac attaacatca taatatgctt tttatgaagt ttcaataagg 1201
tttaacctta gtcttgttga catgtagccc agtcattcac tctttaagga ctattagtgt
1261 ttcattgata ctaaattacc cagcttaatc aacagaatgg tttaagtagt
accaggaagt 1321 aggacaagta atttcaaaaa tataaaggtg tttgctactc
agatgaggcc gcccctgacc 1381 ttctggccag agagacattg ctgccagcca
gctctgcctt cccatcatct cctttcagga 1441 ccgtcccaca ccttttactt
gctcagtgct gtctgaagat gcagttgctg tttgcaaaca 1501 acaggaacac
cagttaaact aattaggaaa cagagggaga tttccaggcc tgggtaacta 1561
tatactgtga ccattggcgg ttgagaccgg tcttcaacca gtggaacccc gaactctgct
1621 gtcagggtgt ggacttcggt gctcttccaa gttttcacct ggggggggga
gctaaccccc 1681 tatgttcacg ccttctattc ccattggcgc tgaactctta
aggtcactct ggtcgcttgt 1741 gaccccgtaa ccctgatgta cccctctaaa
aggtgagggg c
TABLE-US-00241 TABLE LXIV Nucleotide sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 112) and 193P1E1B v.12 (SEQ ID NO: 113) Score =
1742 bits (906), Expect = 0.0Identities = 906/906 (100%) Strand =
Plus/Plus ##STR00139## ##STR00140## ##STR00141## ##STR00142##
##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147##
##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152##
##STR00153## ##STR00154## Score = 1683 bits (875), Expect =
0.0Identities = 875/875 (100%) Strand = Plus/Plus ##STR00155##
##STR00156## ##STR00157## ##STR00158## ##STR00159## ##STR00160##
##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165##
##STR00166## ##STR00167## ##STR00168## ##STR00169##
TABLE-US-00242 TABLE LXV Peptide sequences of protein coded by
193P1E1B v.12 (SEQ ID NO: 114) MDPIRSFCGK LRSLASTLDC ETARLQRALD
GEESLLSKYN SNLATPIAIK AVPPSKRFLK 60 HGQNIRDVSN KEN 73
TABLE-US-00243 TABLE LXVI Amino acid sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 115) and 193P1E1B v.12 (SEQ ID NO: 116) Score =
72.0 bits (175), Expect = e-12 Identities = 35/39 (89%), Positives
= 35/39 (89%) V.1: 1 MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDY 39 MDP
IRSFCGKLRSLASTLDCETARLQRALDGEES Y V.12: 1
MDPIRSFCGKLRSLASTLDCETARLQRALDGEESLLSKY 39 Score = 80.9 bits (198),
Expect = 4e-15 Identities = 39/39 (100%), Positives = 39/39 (100%)
V.1: 374 LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 412
LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN V.12: 35
LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 73
TABLE-US-00244 TABLE LXVII Nucleotide sequence of transcript
variant 193P1E1B v.13 (SEQ ID NO: 117) 1 tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 61 caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact 121
catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 241 cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 361 tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 421 aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 481
cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 721 ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 781 gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg 841
tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag
901 gaaagcggtg cgtgaggcgg gcggccaggg cacgactttg aagattatcc
aatgagaatt 961 ttatatgacc ttcattcaga agttcagact ctaaaggatg
atgttaatat tcttcttgat 1021 aaagcaagat tggaaaatca agaaggcatt
gatttcataa aggcaacaaa agtactaatg 1081 gaaaaaaatt caatggatat
tatgaaaata agagagtatt tccagaagta tggatatagt 1141 ccacgtgtca
agaaaaattc agtacacgag caagaagcca ttaactctga cccagagttg 1201
tctaattgtg aaaattttca gaagactgat gtgaaagatg atctgtctga tcctcctgtt
1261 gcaagcagtt gtatttctgg gaagtctcca cgtagtccac aactttcaga
ttttggactt 1321 gagcggtaca tcgtatccca agttctacca aaccctccac
aggcagtgaa caactataag 1381 gaagagcccg taattgtaac cccacctacc
aaacaatcac tagtaaaagt actaaaaact 1441 ccaaaatgtg cactaaaaat
ggatgatttt gagtgtgtaa ctcctaaatt agaacacttt 1501 ggtatctctg
aatatactat gtgtttaaat gaagattaca caatgggact taaaaatgcg 1561
aggaataata aaagtgagga ggccatagat acagaatcca ggctcaatga taatgttttt
1621 gccactccca gccccatcat ccagcagttg gaaaaaagtg atgccgaata
taccaactct 1681 cctttggtac ctacattctg tactcctggt ttgaaaattc
catctacaaa gaacagcata 1741 gctttggtat ccacaaatta cccattatca
aaaacaaata gttcatcaaa tgatttggaa 1801 gttgaagatc gtacttcgtt
ggttttaaat tcagacacat gctttgagaa tttaacagat 1861 ccctcttcac
ctacgatttc ttcttatgag aatctgctca gaacacctac acctccagaa 1921
gtaactaaaa ttccagaaga tattctccag cttttatcaa aatacaactc aaacctagct
1981 actccaatag caattaaagc agtgccaccc agtaaaaggt tccttaaaca
tggacagaac 2041 atccgagatg tcagcaacaa agaaaactga aattccagtg
gatctatcca acacagaaac 2101 tgaacaaaat gagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc 2161 tgtccccctt taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat 2221 gctttttatg
aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat 2281
tcactcttta aggactatta gtgtttcatt gatactaaat tacccagctt aatcaacaga
2341 atggtttaag tagtaccagg aagtaggaca agtaatttca aaaatataaa
ggtgtttgct 2401 actcagatga ggccgcccct gaccttctgg ccagagagac
attgctgcca gccagctctg 2461 ccttcccatc atctcctttc aggaccgtcc
cacacctttt acttgctcag tgctgtctga 2521 agatgcagtt gctgtttgca
aacaacagga acaccagtta aactaattag gaaacagagg 2581 gagatttcca
ggcctgggta actatatact gtgaccattg gcggttgaga ccggtcttca 2641
accagtggaa ccccgaactc tgctgtcagg gtgtggactt cggtgctctt ccaagttttc
2701 acctgggggg gggagctaac cccctatgtt cacgccttct attcccattg
gcgctgaact 2761 cttaaggtca ctctggtcgc ttgtgacccc gtaaccctga
tgtacccctc taaaaggtga 2821 ggggc
TABLE-US-00245 TABLE LXVIII Nucleotide sequence alignment of
193P1E1B v.1 (SEQ ID NO: 118) and 193P1E1B v.13 (SEQ ID NO: 119)
Score = 1744 bits (907), Expect = 0.0Identities = 907/907 (100%)
Strand = Plus/Plus ##STR00170## ##STR00171## ##STR00172##
##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177##
##STR00178## ##STR00179## ##STR00180## ##STR00181## ##STR00182##
##STR00183## ##STR00184## ##STR00185## Score = 3640 bits (1893),
Expect = 0.0Identities = 1893/1893 (100%) Strand = Plus/Plus
##STR00186## ##STR00187## ##STR00188## ##STR00189## ##STR00190##
##STR00191## ##STR00192## ##STR00193## ##STR00194## ##STR00195##
##STR00196## ##STR00197## ##STR00198## ##STR00199## ##STR00200##
##STR00201## ##STR00202## ##STR00203## ##STR00204## ##STR00205##
##STR00206## ##STR00207## ##STR00208## ##STR00209## ##STR00210##
##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215##
##STR00216## ##STR00217## Score = 1744 bits (907), Expect =
0.0Identities = 907/907 (100%) Strand = Plus/Plus ##STR00218##
##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223##
##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228##
##STR00229## ##STR00230## ##STR00231## ##STR00232##
##STR00233##
TABLE-US-00246 TABLE LXIX Peptide sequences of protein coded by
193P1E1B v.13 (SEQ ID NO: 120) MRILYDLHSE VQTLKDDVNI LLDKARLENQ
EGIDFIKATK VLMEKNSMDI MKIREYFQKY 60 GYSPRVKKNS VHEQEAINSD
PELSNCENFQ KTDVKDDLSD PPVASSCISG KSPRSPQLSD 120 FGLERYIVSQ
VLPNPPQAVN NYKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL 180
EHFGISEYTM CLNEDYTMGL KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY
240 TNSPLVPTFC TPGLKIPSTK NSIALVSTNY PLSKTNSSSN DLEVEDRTSL
VLNSDTCFEN 300 LTDPSSPTIS SYENLLRTPT PPEVTKIPED ILQLLSKYNS
NLATPIAIKA VPPSKRFLKH 360 GQNIRDVSNK EN 372
TABLE-US-00247 TABLE LXX Amino acid sequence alignment of 193P1E1B
v.1 (SEQ ID NO: 121) and 193P1E1B v.13 (SEQ ID NO: 122) Score = 745
bits (1923), Expect = 0.0 Identities = 372/372 (100%), Positives =
372/372 (100%) V.1: 41
MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY 100
MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY V.13:
1 MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY 60
V.1: 101
GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 160
GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD V.13:
61 GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 120
V.1: 161
FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL 220
FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL V.13:
121 FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL
180 V.1: 221
EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY 280
EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY V.13:
181 EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY
240 V.1: 281
TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN 340
TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN V.13:
241 TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN
300 V.1: 341
LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH 400
LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH V.13:
301 LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH
360 V.1: 401 GQNIRDVSNKEN 412 GQNIRDVSNKEN V.13: 361 GQNIRDVSNKEN
372
Sequence CWU 1
1
2591227DNAHomo sapiensmisc_feature177n = A,T,C or G 1gatccactgg
aatttcagtt ttctttgttg ctgacatctc ggatgttctg tccatgttta 60gggaaccttt
tactgggtgg cactgcttta attgctattg gagtagctag gtttgagttg
120tattttgata gaagctggag aatatcttct ggaattttag ttacttctgg
agggggnagg 180ttctgagcag attctcataa gaagaaatcg taggtgaaag agggatc
22722798DNAHomo sapiensCDS(805)...(2043) 2tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg
831 Met Asp Pro Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc
agc acg ctg gac tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala
Ser Thr Leu Asp Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg
gac gga gag gaa agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu
Asp Gly Glu Glu Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta
tat gac ctt cat tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu
Tyr Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat
att ctt ctt gat aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn
Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat
ttc ata aag gca aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp
Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80
85att atg aaa ata aga gag tat ttc cag aag tat gga tat agt cca cgt
1119Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro
Arg90 95 100 105gtc aag aaa aat tca gta cac gag caa gaa gcc att aac
tct gac cca 1167Val Lys Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn
Ser Asp Pro 110 115 120gag ttg tct aat tgt gaa aat ttt cag aag act
gat gtg aaa gat gat 1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr
Asp Val Lys Asp Asp 125 130 135ctg tct gat cct cct gtt gca agc agt
tgt att tct gag aag tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser
Cys Ile Ser Glu Lys Ser Pro 140 145 150cgt agt cca caa ctt tca gat
ttt gga ctt gag cgg tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp
Phe Gly Leu Glu Arg Tyr Ile Val Ser 155 160 165caa gtt cta cca aac
cct cca cag gca gtg aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn
Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta
att gta acc cca cct acc aaa caa tca cta gta aaa gta cta 1407Pro Val
Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val Leu 190 195
200aaa act cca aaa tgt gca cta aaa atg gat gat ttt gag tgt gta act
1455Lys Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr
205 210 215cct aaa tta gaa cac ttt ggt atc tct gaa tat act atg tgt
tta aat 1503Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys
Leu Asn 220 225 230gaa gat tac aca atg gga ctt aaa aat gcg agg aat
aat aaa agt gag 1551Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn
Asn Lys Ser Glu 235 240 245gag gcc ata gat aca gaa tcc agg ctc aat
gat aat gtt ttt gcc act 1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn
Asp Asn Val Phe Ala Thr250 255 260 265ccc agc ccc atc atc cag cag
ttg gaa aaa agt gat gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln
Leu Glu Lys Ser Asp Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta
cct aca ttc tgt act cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val
Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag
aac agc ata gct ttg gta tcc aca aat tac cca tta tca 1743Ser Thr Lys
Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa
aca aat agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys
Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320
325ttg gtt tta aat tca gac aca tgc ttt gag aat tta aca gat ccc tct
1839Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro
Ser330 335 340 345tca cct acg att tct tct tat gag aat ctg ctc aga
aca cct aca cct 1887Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg
Thr Pro Thr Pro 350 355 360cca gaa gta act aaa att cca gaa gat att
ctc cag ctt tta tca aaa 1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile
Leu Gln Leu Leu Ser Lys 365 370 375tac aac tca aac cta gct act cca
ata gca att aaa gca gtg cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro
Ile Ala Ile Lys Ala Val Pro Pro 380 385 390agt aaa agg ttc ctt aaa
cat gga cag aac atc cga gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys
His Gly Gln Asn Ile Arg Asp Val Ser Asn 395 400 405aaa gaa aac tga
aattccagtg gatctatcca acacagaaac tgaacaaaat 2083Lys Glu
Asn410gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt 2143taaacgttga cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg 2203aagtttcaat aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta 2263aggactatta gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag 2323tagtaccagg
aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
2383ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc 2443atctcctttc aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt 2503gctgtttgca aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca 2563ggcctgggta actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa 2623ccccgaactc
tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
2683gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca 2743ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 27983412PRTHomo sapiens 3Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe
Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val
Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg
Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 41042798DNAHomo
sapiensCDS(805)...(2043) 4tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacagggcag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg 831 Met Asp Pro
Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc agc acg ctg gac
tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala Ser Thr Leu Asp
Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg gac gga gag gaa
agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu Asp Gly Glu Glu
Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta tat gac ctt cat
tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu Tyr Asp Leu His
Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat att ctt ctt gat
aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn Ile Leu Leu Asp
Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat ttc ata aag gca
aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80 85att atg aaa ata
aga gag tat ttc cag aag tat gga tat agt cca cgt 1119Ile Met Lys Ile
Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg90 95 100 105gtc aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca 1167Val Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro 110 115
120gag ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat
1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp
125 130 135ctg tct gat cct cct gtt gca agc agt tgt att tct gag aag
tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys
Ser Pro 140 145 150cgt agt cca caa ctt tca gat ttt gga ctt gag cgg
tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val Ser 155 160 165caa gtt cta cca aac cct cca cag gca gtg
aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn Pro Pro Gln Ala Val
Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta att gta acc cca cct
acc aaa caa tca cta gta aaa gta cta 1407Pro Val Ile Val Thr Pro Pro
Thr Lys Gln Ser Leu Val Lys Val Leu 190 195 200aaa act cca aaa tgt
gca cta aaa atg gat gat ttt gag tgt gta act 1455Lys Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr 205 210 215cct aaa tta
gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat 1503Pro Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn 220 225 230gaa
gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa agt gag 1551Glu
Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu 235 240
245gag gcc ata gat aca gaa tcc agg ctc aat gat aat gtt ttt gcc act
1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala
Thr250 255 260 265ccc agc ccc atc atc cag cag ttg gaa aaa agt gat
gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp
Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta cct aca ttc tgt act
cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val Pro Thr Phe Cys Thr
Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag aac agc ata gct ttg
gta tcc aca aat tac cca tta tca 1743Ser Thr Lys Asn Ser Ile Ala Leu
Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa aca aat agt tca tca
aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys Thr Asn Ser Ser Ser
Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320 325ttg gtt tta aat
tca gac aca tgc ttt gag aat tta aca gat ccc tct 1839Leu Val Leu Asn
Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser330 335 340 345tca
cct acg att tct tct tat gag aat ctg ctc aga aca cct aca cct 1887Ser
Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr Pro 350 355
360cca gaa gta act aaa att cca gaa gat att ctc cag ctt tta tca aaa
1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser Lys
365 370 375tac aac tca aac cta gct act cca ata gca att aaa gca gtg
cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val
Pro Pro 380 385 390agt aaa agg ttc ctt aaa cat gga cag aac atc cga
gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile Arg
Asp Val Ser Asn 395 400 405aaa gaa aac tga aattccagtg gatctatcca
acacagaaac tgaacaaaat 2083Lys Glu Asn410gagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc tgtccccctt 2143taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat gctttttatg
2203aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat
tcactcttta 2263aggactatta gtgtttcatt gatactaaat tacccagctt
aatcaacaga atggtttaag 2323tagtaccagg aagtaggaca agtaatttca
aaaatataaa ggtgtttgct actcagatga 2383ggccgcccct gaccttctgg
ccagagagac attgctgcca gccagctctg ccttcccatc 2443atctcctttc
aggaccgtcc cacacctttt acttgctcag tgctgtctga agatgcagtt
2503gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg
gagatttcca 2563ggcctgggta actatatact gtgaccattg gcggttgaga
ccggtcttca accagtggaa 2623ccccgaactc tgctgtcagg gtgtggactt
cggtgctctt ccaagttttc acctgggggg 2683gggagctaac cccctatgtt
cacgccttct attcccattg gcgctgaact cttaaggtca 2743ctctggtcgc
ttgtgacccc gtaaccctga tgtacccctc taaaaggtga ggggc 27985412PRTHomo
sapiens 5Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu
Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn
Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp
Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile
Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser
Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile Val
Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn Tyr
Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln Ser
Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys Met
Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215
220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly
Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile
Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro
Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr
Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu
Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr
Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp
Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330
335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr
340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys
Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr Asn Ser
Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro Pro Ser
Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg Asp Val
Ser Asn Lys Glu Asn 405 41062798DNAHomo sapiensCDS(805)...(2043)
6tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag
60caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc cggctgtgct cagc atg gac cct atc cgg agc
ttc tgc ggg 831 Met Asp Pro Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg
tct ctg gcc agc acg ctg gac tgc gag acg gcc cgg ctg 879Lys Leu Arg
Ser Leu Ala Ser Thr Leu Asp Cys Glu Thr Ala Arg Leu10 15 20 25cag
cga gcg ctg gac gga gag gaa agc gac ttt gaa gat tat cca atg 927Gln
Arg Ala Leu Asp Gly Glu Glu Ser Asp Phe Glu Asp Tyr Pro Met 30 35
40aga att tta tat gac ctt cat tca gaa gtt cag act cta aag gat gat
975Arg Ile Leu Tyr Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp Asp
45 50 55gtt aat att ctt ctt gat aaa gca aga ttg gaa aat caa gaa ggc
att 1023Val Asn Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly
Ile 60 65 70gat ttc ata aag gca aca aaa gta cta atg gaa aaa aat tca
atg gat 1071Asp Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser
Met Asp 75 80 85att atg aaa ata aga gag tat ttc cag aag tat gga tat
agt cca cgt 1119Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr
Ser Pro Arg90 95 100 105gtc aag aaa aat tca gta cac gag caa gaa gcc
att aac tct gac cca 1167Val Lys Lys Asn Ser Val His Glu Gln Glu Ala
Ile Asn Ser Asp Pro 110 115 120gag ttg tct aat tgt gaa aat ttt cag
aag act gat gtg aaa gat gat 1215Glu Leu Ser Asn Cys Glu Asn Phe Gln
Lys Thr Asp Val Lys Asp Asp 125 130 135ctg tct gat cct cct gtt gca
agc agt tgt att tct gag aag tct cca 1263Leu Ser Asp Pro Pro Val Ala
Ser Ser Cys Ile Ser Glu Lys Ser Pro 140 145 150cgt agt cca caa ctt
tca gat ttt gga ctt gag cgg tac atc gta tcc 1311Arg Ser Pro Gln Leu
Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val Ser 155 160 165caa gtt cta
cca aac cct cca cag gca gtg aac aac tat aag gaa gag 1359Gln Val Leu
Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu Glu170 175 180
185ccc gta att gta acc cca cct acc aaa caa tca cta gta aaa gta cta
1407Pro Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val Leu
190 195 200aaa act cca aaa tgt gca cta aaa atg gat gat ttt gag tgt
gta act 1455Lys Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys
Val Thr 205 210 215cct aaa tta gaa cac ttt ggt atc tct gaa tat act
atg tgt tta aat 1503Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr
Met Cys Leu Asn 220 225 230gaa gat tac aca atg gga ctt aaa aat gcg
agg aat aat aaa agt gag 1551Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala
Arg Asn Asn Lys Ser Glu 235 240 245gag gcc ata gat aca gaa tcc agg
ctc aat gat aat gtt ttt gcc act 1599Glu Ala Ile Asp Thr Glu Ser Arg
Leu Asn Asp Asn Val Phe Ala Thr250 255 260 265ccc agc ccc atc atc
cag cag ttg gaa aaa agt gat gcc gaa tat acc 1647Pro Ser Pro Ile Ile
Gln Gln Leu Glu Lys Ser Asp Ala Glu Tyr Thr 270 275 280aac tct cct
ttg gta cct aca ttc tgt act cct ggt ttg aaa att cca 1695Asn Ser Pro
Leu Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile Pro 285 290 295tct
aca aag aac agc ata gct ttg gta tcc aca aat tac cca tta tca 1743Ser
Thr Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu Ser 300 305
310aaa aca aat agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg
1791Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser
315 320 325ttg gtt tta aat tca gac aca tgc ttt gag aat tta aca gat
ccc tct 1839Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp
Pro Ser330 335 340 345tca cct acg att tct tct tat gag aat ctg ctc
aga aca cct aca cct 1887Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu
Arg Thr Pro Thr Pro 350 355 360cca gaa gta act aaa att cca gaa gat
att ctc cag ctt tta tca aaa 1935Pro Glu Val Thr Lys Ile Pro Glu Asp
Ile Leu Gln Leu Leu Ser Lys 365 370 375tac aac tca aac cta gct act
cca ata gca att aaa gca gtg cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr
Pro Ile Ala Ile Lys Ala Val Pro Pro 380 385 390agt aaa agg ttc ctt
aaa cat gga cag aac atc cga gat gtc agc aac 2031Ser Lys Arg Phe Leu
Lys His Gly Gln Asn Ile Arg Asp Val Ser Asn 395 400 405aaa gaa aac
tga aattccagtg gatctatcca acacagaaac tgaacaaaat 2083Lys Glu
Asn410gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt 2143taaacgttga cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg 2203aagtttcaat aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta 2263aggactatta gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag 2323tagtaccagg
aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
2383ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc 2443atctcctttc aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt 2503gctgtttgca aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca 2563ggcctgggta actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa 2623ccccgaactc
tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
2683gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca 2743ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 27987412PRTHomo sapiens 7Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe
Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val
Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg
Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 41082798DNAHomo
sapiensCDS(805)...(2043) 8tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct caga atg gac cct atc cgg agc ttc tgc ggg 831 Met Asp Pro
Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc agc acg ctg gac
tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala Ser Thr Leu Asp
Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg gac gga gag gaa
agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu Asp Gly Glu Glu
Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta tat gac ctt cat
tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu Tyr Asp Leu His
Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat att ctt ctt gat
aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn Ile Leu Leu Asp
Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat ttc ata aag gca
aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80 85att atg aaa ata
aga gag tat ttc cag aag tat gga tat agt cca cgt 1119Ile Met Lys Ile
Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg90 95 100 105gtc aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca 1167Val Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro 110 115
120gag ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat
1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp
125 130 135ctg tct gat cct cct gtt gca agc agt tgt att tct gag aag
tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys
Ser Pro 140 145 150cgt agt cca caa ctt tca gat ttt gga ctt gag cgg
tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val Ser 155 160 165caa gtt cta cca aac cct cca cag gca gtg
aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn Pro Pro Gln Ala Val
Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta att gta acc cca cct
acc aaa caa tca cta gta aaa gta cta 1407Pro Val Ile Val Thr Pro Pro
Thr Lys Gln Ser Leu Val Lys Val Leu 190 195 200aaa act cca aaa tgt
gca cta aaa atg gat gat ttt gag tgt gta act 1455Lys Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr 205 210 215cct aaa tta
gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat 1503Pro Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn 220 225 230gaa
gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa agt gag 1551Glu
Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu 235 240
245gag gcc ata gat aca gaa tcc agg ctc aat gat aat gtt ttt gcc act
1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala
Thr250 255 260 265ccc agc ccc atc atc cag cag ttg gaa aaa agt gat
gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp
Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta cct aca ttc tgt act
cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val Pro Thr Phe Cys Thr
Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag aac agc ata gct ttg
gta tcc aca aat tac cca tta tca 1743Ser Thr Lys Asn Ser Ile Ala Leu
Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa aca aat agt tca tca
aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys Thr Asn Ser Ser Ser
Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320 325ttg gtt tta aat
tca gac aca tgc ttt gag aat tta aca gat ccc tct 1839Leu Val Leu Asn
Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser330 335 340 345tca
cct acg att tct tct tat gag aat ctg ctc aga aca cct aca cct 1887Ser
Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr Pro 350 355
360cca gaa gta act aaa att cca gaa gat att ctc cag ctt tta tca aaa
1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser Lys
365 370 375tac aac tca aac cta gct act cca ata gca att aaa gca gtg
cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val
Pro Pro 380 385 390agt aaa agg ttc ctt aaa cat gga cag aac atc cga
gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile Arg
Asp Val Ser Asn 395 400 405aaa gaa aac tga aattccagtg gatctatcca
acacagaaac tgaacaaaat 2083Lys Glu Asn410gagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc tgtccccctt 2143taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat gctttttatg
2203aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat
tcactcttta 2263aggactatta gtgtttcatt gatactaaat tacccagctt
aatcaacaga atggtttaag 2323tagtaccagg aagtaggaca agtaatttca
aaaatataaa ggtgtttgct actcagatga 2383ggccgcccct gaccttctgg
ccagagagac attgctgcca gccagctctg ccttcccatc 2443atctcctttc
aggaccgtcc cacacctttt acttgctcag tgctgtctga agatgcagtt
2503gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg
gagatttcca 2563ggcctgggta actatatact gtgaccattg gcggttgaga
ccggtcttca accagtggaa 2623ccccgaactc tgctgtcagg gtgtggactt
cggtgctctt ccaagttttc acctgggggg 2683gggagctaac cccctatgtt
cacgccttct attcccattg gcgctgaact cttaaggtca 2743ctctggtcgc
ttgtgacccc gtaaccctga tgtacccctc taaaaggtga ggggc 27989412PRTHomo
sapiens 9Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu
Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn
Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp
Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile
Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150
155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro
Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val
Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr
Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr
Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys
Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg
Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu
Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265
270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr
275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser
Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn
Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser
Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp
Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg
Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile
Leu Gln Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala Thr 370 375 380Pro
Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu Lys His385 390
395 400Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu Asn 405
410102798DNAHomo sapiensCDS(805)...(2043) 10tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg
831 Met Asp Pro Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc
agc acg ctg gac tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala
Ser Thr Leu Asp Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg
gac gga gag gaa agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu
Asp Gly Glu Glu Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta
tat gac ctt cat tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu
Tyr Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat
att ctt ctt gat aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn
Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat
ttc ata aag gca aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp
Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80
85att atg aaa ata aga gag tat ttc cag aag tat gga tat agt cca cgt
1119Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro
Arg90 95 100 105gtc aag aaa aat tca gta cac gag caa gaa gcc att aac
tct gac cca 1167Val Lys Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn
Ser Asp Pro 110 115 120gag ttg tct aat tgt gaa aat ttt cag aag act
gat gtg aaa gat gat 1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr
Asp Val Lys Asp Asp 125 130 135ctg tct gat cct cct gtt gca agc agt
tgt att tct ggg aag tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser
Cys Ile Ser Gly Lys Ser Pro 140 145 150cgt agt cca caa ctt tca gat
ttt gga ctt gag cgg tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp
Phe Gly Leu Glu Arg Tyr Ile Val Ser 155 160 165caa gtt cta cca aac
cct cca cag gca gtg aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn
Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta
att gta acc cca cct acc aaa caa tca cta gta aaa gta cta 1407Pro Val
Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val Leu 190 195
200aaa act cca aaa tgt gca cta aaa atg gat gat ttt gag tgt gta act
1455Lys Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr
205 210 215cct aaa tta gaa cac ttt ggt atc tct gaa tat act atg tgt
tta aat 1503Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys
Leu Asn 220 225 230gaa gat tac aca atg gga ctt aaa aat gcg agg aat
aat aaa agt gag 1551Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn
Asn Lys Ser Glu 235 240 245gag gcc ata gat aca gaa tcc agg ctc aat
gat aat gtt ttt gcc act 1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn
Asp Asn Val Phe Ala Thr250 255 260 265ccc agc ccc atc atc cag cag
ttg gaa aaa agt gat gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln
Leu Glu Lys Ser Asp Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta
cct aca ttc tgt act cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val
Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag
aac agc ata gct ttg gta tcc aca aat tac cca tta tca 1743Ser Thr Lys
Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa
aca aat agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys
Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320
325ttg gtt tta aat tca gac aca tgc ttt gag aat tta aca gat ccc tct
1839Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro
Ser330 335 340 345tca cct acg att tct tct tat gag aat ctg ctc aga
aca cct aca cct 1887Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg
Thr Pro Thr Pro 350 355 360cca gaa gta act aaa att cca gaa gat att
ctc cag ctt tta tca aaa 1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile
Leu Gln Leu Leu Ser Lys 365 370 375tac aac tca aac cta gct act cca
ata gca att aaa gca gtg cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro
Ile Ala Ile Lys Ala Val Pro Pro 380 385 390agt aaa agg ttc ctt aaa
cat gga cag aac atc cga gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys
His Gly Gln Asn Ile Arg Asp Val Ser Asn 395 400 405aaa gaa aac tga
aattccagtg gatctatcca acacagaaac tgaacaaaat 2083Lys Glu
Asn410gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt 2143taaacgttga cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg 2203aagtttcaat aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta 2263aggactatta gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag 2323tagtaccagg
aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
2383ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc 2443atctcctttc aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt 2503gctgtttgca aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca 2563ggcctgggta actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa 2623ccccgaactc
tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
2683gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca 2743ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 279811412PRTHomo sapiens 11Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe
Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val
Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg
Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Gly Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 410122798DNAHomo
sapiensCDS(805)...(2043) 12tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg 831 Met Asp Pro
Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc agc acg ctg gac
tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala Ser Thr Leu Asp
Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg gac gga gag gaa
agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu Asp Gly Glu Glu
Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta tat gac ctt cat
tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu Tyr Asp Leu His
Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat att ctt ctt gat
aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn Ile Leu Leu Asp
Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat ttc ata aag gca
aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80 85att atg aaa ata
aga gag tat ttc cag aag tat gga tat agt cca cgt 1119Ile Met Lys Ile
Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg90 95 100 105gtc aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca 1167Val Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro 110 115
120gag ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat
1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp
125 130 135ctg tct gat cct cct gtt gca agc agt tgt att tct gag aag
tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys
Ser Pro 140 145 150cgt agt cca caa ctt tca gat ttt gga ctt gag cgg
tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val Ser 155 160 165caa gtt cta cca aac cct cca cag gca gtg
aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn Pro Pro Gln Ala Val
Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta att gta acc cca cct
acc aaa caa tca cta gta aaa gta cta 1407Pro Val Ile Val Thr Pro Pro
Thr Lys Gln Ser Leu Val Lys Val Leu 190 195 200aaa act cca aaa tgt
gca cta aaa atg gat gat ttt gag tgt gta act 1455Lys Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr 205 210 215cct aaa tta
gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat 1503Pro Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn 220 225 230gaa
gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa agt gag 1551Glu
Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu 235 240
245gag gcc ata gat gca gaa tcc agg ctc aat gat aat gtt ttt gcc act
1599Glu Ala Ile Asp Ala Glu Ser Arg Leu Asn Asp Asn Val Phe Ala
Thr250 255 260 265ccc agc ccc atc atc cag cag ttg gaa aaa agt gat
gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp
Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta cct aca ttc tgt act
cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val Pro Thr Phe Cys Thr
Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag aac agc ata gct ttg
gta tcc aca aat tac cca tta tca 1743Ser Thr Lys Asn Ser Ile Ala Leu
Val Ser Thr Asn Tyr Pro Leu Ser 300 305
310aaa aca aat agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg
1791Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser
315 320 325ttg gtt tta aat tca gac aca tgc ttt gag aat tta aca gat
ccc tct 1839Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp
Pro Ser330 335 340 345tca cct acg att tct tct tat gag aat ctg ctc
aga aca cct aca cct 1887Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu
Arg Thr Pro Thr Pro 350 355 360cca gaa gta act aaa att cca gaa gat
att ctc cag ctt tta tca aaa 1935Pro Glu Val Thr Lys Ile Pro Glu Asp
Ile Leu Gln Leu Leu Ser Lys 365 370 375tac aac tca aac cta gct act
cca ata gca att aaa gca gtg cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr
Pro Ile Ala Ile Lys Ala Val Pro Pro 380 385 390agt aaa agg ttc ctt
aaa cat gga cag aac atc cga gat gtc agc aac 2031Ser Lys Arg Phe Leu
Lys His Gly Gln Asn Ile Arg Asp Val Ser Asn 395 400 405aaa gaa aac
tga aattccagtg gatctatcca acacagaaac tgaacaaaat 2083Lys Glu
Asn410gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt 2143taaacgttga cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg 2203aagtttcaat aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta 2263aggactatta gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag 2323tagtaccagg
aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
2383ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc 2443atctcctttc aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt 2503gctgtttgca aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca 2563ggcctgggta actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa 2623ccccgaactc
tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
2683gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca 2743ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 279813412PRTHomo sapiens 13Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe
Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val
Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg
Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Ala Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 410142798DNAHomo
sapiensCDS(805)...(2043) 14tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg 831 Met Asp Pro
Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc agc acg ctg gac
tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala Ser Thr Leu Asp
Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg gac gga gag gaa
agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu Asp Gly Glu Glu
Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta tat gac ctt cat
tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu Tyr Asp Leu His
Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat att ctt ctt gat
aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn Ile Leu Leu Asp
Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat ttc ata aag gca
aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80 85att atg aaa ata
aga gag tat ttc cag aag tat gga tat agt cca cgt 1119Ile Met Lys Ile
Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg90 95 100 105gtc aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca 1167Val Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro 110 115
120gag ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat
1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp
125 130 135ctg tct gat cct cct gtt gca agc agt tgt att tct gag aag
tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys
Ser Pro 140 145 150cgt agt cca caa ctt tca gat ttt gga ctt gag cgg
tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val Ser 155 160 165caa gtt cta cca aac cct cca cag gca gtg
aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn Pro Pro Gln Ala Val
Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta att gta acc cca cct
acc aaa caa tca cta gta aaa gta cta 1407Pro Val Ile Val Thr Pro Pro
Thr Lys Gln Ser Leu Val Lys Val Leu 190 195 200aaa act cca aaa tgt
gca cta aaa atg gat gat ttt gag tgt gta act 1455Lys Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr 205 210 215cct aaa tta
gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat 1503Pro Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn 220 225 230gaa
gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa agt gag 1551Glu
Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu 235 240
245gag gcc ata gat aca gaa tcc agg ctc aat gat aat gtt ttt gcc act
1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala
Thr250 255 260 265ccc agc ccc atc atc cag cag ttg gaa aaa agt gat
gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp
Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta cct aca ttc tgt act
cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val Pro Thr Phe Cys Thr
Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag aac agc ata gct ttg
gta tcc aca aat tac cca tta tca 1743Ser Thr Lys Asn Ser Ile Ala Leu
Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa aca aat agt tca tca
aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys Thr Asn Ser Ser Ser
Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320 325ttg gtt tta aat
tca gac aca tgc ttt gag aat tta aca gat ccc tct 1839Leu Val Leu Asn
Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser330 335 340 345tca
cct acg att tct tct tat gag aat ctg ctc aga aca cct aca cct 1887Ser
Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr Pro 350 355
360cca gaa gta act aaa att cca gaa gat att ctc cag ctt tta tca aaa
1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser Lys
365 370 375tac aac tca aac cta gct act cca ata gca att aaa gca gtg
cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val
Pro Pro 380 385 390agt aaa agg ttc ctt aaa cat gga cag aac atc cga
gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile Arg
Asp Val Ser Asn 395 400 405aaa gaa aac tga aattccagtg gatctatcca
acacagaaac tgaacaaaat 2083Lys Glu Asn410gagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc tgtccccctt 2143taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat gctttttatg
2203aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat
tcactcttta 2263aggattatta gtgtttcatt gatactaaat tacccagctt
aatcaacaga atggtttaag 2323tagtaccagg aagtaggaca agtaatttca
aaaatataaa ggtgtttgct actcagatga 2383ggccgcccct gaccttctgg
ccagagagac attgctgcca gccagctctg ccttcccatc 2443atctcctttc
aggaccgtcc cacacctttt acttgctcag tgctgtctga agatgcagtt
2503gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg
gagatttcca 2563ggcctgggta actatatact gtgaccattg gcggttgaga
ccggtcttca accagtggaa 2623ccccgaactc tgctgtcagg gtgtggactt
cggtgctctt ccaagttttc acctgggggg 2683gggagctaac cccctatgtt
cacgccttct attcccattg gcgctgaact cttaaggtca 2743ctctggtcgc
ttgtgacccc gtaaccctga tgtacccctc taaaaggtga ggggc 279815412PRTHomo
sapiens 15Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu
Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn
Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp
Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile
Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150
155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro
Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val
Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr
Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr
Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys
Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg
Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu
Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265
270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr
275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser
Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn
Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser
Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp
Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg
Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile
Leu Gln Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala Thr 370 375 380Pro
Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu Lys His385 390
395 400Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu Asn 405
410162798DNAHomo sapiensCDS(805)...(2043) 16tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg
831 Met Asp Pro Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc
agc acg ctg gac tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala
Ser Thr Leu Asp Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg
gac gga gag gaa agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu
Asp Gly Glu Glu Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta
tat gac ctt cat tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu
Tyr Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat
att ctt ctt gat aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn
Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat
ttc ata aag gca aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp
Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80
85att atg aaa ata aga gag tat ttc cag aag tat gga tat agt cca cgt
1119Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro
Arg90 95 100 105gtc aag aaa aat tca gta cac gag caa gaa gcc att aac
tct gac cca 1167Val Lys Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn
Ser Asp Pro 110 115 120gag ttg tct aat tgt gaa aat ttt cag aag act
gat gtg aaa gat gat 1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr
Asp Val Lys Asp Asp 125 130 135ctg tct gat cct cct gtt gca agc agt
tgt att tct gag aag tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser
Cys Ile Ser Glu Lys Ser Pro 140 145 150cgt agt cca caa ctt tca gat
ttt gga ctt gag cgg tac atc gta tcc 1311Arg Ser Pro Gln Leu Ser Asp
Phe Gly Leu Glu Arg Tyr Ile Val Ser 155 160 165caa gtt cta cca aac
cct cca cag gca gtg aac aac tat aag gaa gag 1359Gln Val Leu Pro Asn
Pro
Pro Gln Ala Val Asn Asn Tyr Lys Glu Glu170 175 180 185ccc gta att
gta acc cca cct acc aaa caa tca cta gta aaa gta cta 1407Pro Val Ile
Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val Leu 190 195 200aaa
act cca aaa tgt gca cta aaa atg gat gat ttt gag tgt gta act 1455Lys
Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr 205 210
215cct aaa tta gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat
1503Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn
220 225 230gaa gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa
agt gag 1551Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys
Ser Glu 235 240 245gag gcc ata gat aca gaa tcc agg ctc aat gat aat
gtt ttt gcc act 1599Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn
Val Phe Ala Thr250 255 260 265ccc agc ccc atc atc cag cag ttg gaa
aaa agt gat gcc gaa tat acc 1647Pro Ser Pro Ile Ile Gln Gln Leu Glu
Lys Ser Asp Ala Glu Tyr Thr 270 275 280aac tct cct ttg gta cct aca
ttc tgt act cct ggt ttg aaa att cca 1695Asn Ser Pro Leu Val Pro Thr
Phe Cys Thr Pro Gly Leu Lys Ile Pro 285 290 295tct aca aag aac agc
ata gct ttg gta tcc aca aat tac cca tta tca 1743Ser Thr Lys Asn Ser
Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu Ser 300 305 310aaa aca aat
agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg 1791Lys Thr Asn
Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser 315 320 325ttg
gtt tta aat tca gac aca tgc ttt gag aat tta aca gat ccc tct 1839Leu
Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser330 335
340 345tca cct acg att tct tct tat gag aat ctg ctc aga aca cct aca
cct 1887Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr
Pro 350 355 360cca gaa gta act aaa att cca gaa gat att ctc cag ctt
tta tca aaa 1935Pro Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu
Leu Ser Lys 365 370 375tac aac tca aac cta gct act cca ata gca att
aaa gca gtg cca ccc 1983Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile
Lys Ala Val Pro Pro 380 385 390agt aaa agg ttc ctt aaa cat gga cag
aac atc cga gat gtc agc aac 2031Ser Lys Arg Phe Leu Lys His Gly Gln
Asn Ile Arg Asp Val Ser Asn 395 400 405aaa gaa aac tga aattccagtg
gatctatcca acacagaaac tgaacaaaat 2083Lys Glu Asn410gagatgaaag
ccgagctgga ccgattttaa cattcacatt gccctgcctc tgtccccctt
2143taaacgttga cccattttaa agacaaacat gaacattaac atcataatat
gctttttatg 2203aagtttcaat aaggtttaac cttagtcttg ttgacatgta
gcccagtcat tcactcttta 2263aggactatta gtgtttcatt gatactaaat
tacccagctt aatcaacaga atggtttaag 2323tagtaccagg aagtaggaca
agtaatttca aaaatataaa ggtgtttgct actcagatga 2383ggctgcccct
gaccttctgg ccagagagac attgctgcca gccagctctg ccttcccatc
2443atctcctttc aggaccgtcc cacacctttt acttgctcag tgctgtctga
agatgcagtt 2503gctgtttgca aacaacagga acaccagtta aactaattag
gaaacagagg gagatttcca 2563ggcctgggta actatatact gtgaccattg
gcggttgaga ccggtcttca accagtggaa 2623ccccgaactc tgctgtcagg
gtgtggactt cggtgctctt ccaagttttc acctgggggg 2683gggagctaac
cccctatgtt cacgccttct attcccattg gcgctgaact cttaaggtca
2743ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc taaaaggtga ggggc
279817412PRTHomo sapiens 17Met Asp Pro Ile Arg Ser Phe Cys Gly Lys
Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu
Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro
Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys
Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln
Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu
Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys
Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu
Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120
125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala
130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu
Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val
Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu
Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys
Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe
Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu
Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235
240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser
245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile
Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro
Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser
Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu
Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu
Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu
Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu
Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360
365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala Thr
370 375 380Pro Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu
Lys His385 390 395 400Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu
Asn 405 410182736DNAHomo sapiensCDS(989)...(1981) 18tatcatctgt
gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac cctatccgga
gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg
gcccggctgc agcgagcgct ggacggagag 900gaaagcggat gatgttaata
ttcttcttga taaagcaaga ttggaaaatc aagaaggcat 960tgatttcata
aaggcaacaa aagtacta atg gaa aaa aat tca atg gat att 1012 Met Glu
Lys Asn Ser Met Asp Ile 1 5atg aaa ata aga gag tat ttc cag aag tat
gga tat agt cca cgt gtc 1060Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr
Gly Tyr Ser Pro Arg Val 10 15 20aag aaa aat tca gta cac gag caa gaa
gcc att aac tct gac cca gag 1108Lys Lys Asn Ser Val His Glu Gln Glu
Ala Ile Asn Ser Asp Pro Glu25 30 35 40ttg tct aat tgt gaa aat ttt
cag aag act gat gtg aaa gat gat ctg 1156Leu Ser Asn Cys Glu Asn Phe
Gln Lys Thr Asp Val Lys Asp Asp Leu 45 50 55tct gat cct cct gtt gca
agc agt tgt att tct gag aag tct cca cgt 1204Ser Asp Pro Pro Val Ala
Ser Ser Cys Ile Ser Glu Lys Ser Pro Arg 60 65 70agt cca caa ctt tca
gat ttt gga ctt gag cgg tac atc gta tcc caa 1252Ser Pro Gln Leu Ser
Asp Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln 75 80 85gtt cta cca aac
cct cca cag gca gtg aac aac tat aag gaa gag ccc 1300Val Leu Pro Asn
Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro 90 95 100gta att
gta acc cca cct acc aaa caa tca cta gta aaa gta cta aaa 1348Val Ile
Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val Leu Lys105 110 115
120act cca aaa tgt gca cta aaa atg gat gat ttt gag tgt gta act cct
1396Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr Pro
125 130 135aaa tta gaa cac ttt ggt atc tct gaa tat act atg tgt tta
aat gaa 1444Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu
Asn Glu 140 145 150gat tac aca atg gga ctt aaa aat gcg agg aat aat
aaa agt gag gag 1492Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn
Lys Ser Glu Glu 155 160 165gcc ata gat aca gaa tcc agg ctc aat gat
aat gtt ttt gcc act ccc 1540Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp
Asn Val Phe Ala Thr Pro 170 175 180agc ccc atc atc cag cag ttg gaa
aaa agt gat gcc gaa tat acc aac 1588Ser Pro Ile Ile Gln Gln Leu Glu
Lys Ser Asp Ala Glu Tyr Thr Asn185 190 195 200tct cct ttg gta cct
aca ttc tgt act cct ggt ttg aaa att cca tct 1636Ser Pro Leu Val Pro
Thr Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser 205 210 215aca aag aac
agc ata gct ttg gta tcc aca aat tac cca tta tca aaa 1684Thr Lys Asn
Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys 220 225 230aca
aat agt tca tca aat gat ttg gaa gtt gaa gat cgt act tcg ttg 1732Thr
Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser Leu 235 240
245gtt tta aat tca gac aca tgc ttt gag aat tta aca gat ccc tct tca
1780Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser
250 255 260cct acg att tct tct tat gag aat ctg ctc aga aca cct aca
cct cca 1828Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr
Pro Pro265 270 275 280gaa gta act aaa att cca gaa gat att ctc cag
ctt tta tca aaa tac 1876Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln
Leu Leu Ser Lys Tyr 285 290 295aac tca aac cta gct act cca ata gca
att aaa gca gtg cca ccc agt 1924Asn Ser Asn Leu Ala Thr Pro Ile Ala
Ile Lys Ala Val Pro Pro Ser 300 305 310aaa agg ttc ctt aaa cat gga
cag aac atc cga gat gtc agc aac aaa 1972Lys Arg Phe Leu Lys His Gly
Gln Asn Ile Arg Asp Val Ser Asn Lys 315 320 325gaa aac tga
aattccagtg gatctatcca acacagaaac tgaacaaaat 2021Glu Asn
330gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt 2081taaacgttga cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg 2141aagtttcaat aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta 2201aggactatta gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag 2261tagtaccagg
aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
2321ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc 2381atctcctttc aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt 2441gctgtttgca aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca 2501ggcctgggta actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa 2561ccccgaactc
tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
2621gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca 2681ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 273619330PRTHomo sapiens 19Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala Ile Asn
Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys Thr Asp
Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55 60Cys Ile
Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65 70 75
80Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln Ala
85 90 95Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro Thr
Lys 100 105 110Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala
Leu Lys Met 115 120 125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu
His Phe Gly Ile Ser 130 135 140Glu Tyr Thr Met Cys Leu Asn Glu Asp
Tyr Thr Met Gly Leu Lys Asn145 150 155 160Ala Arg Asn Asn Lys Ser
Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn Val
Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys Ser
Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200
205Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val
210 215 220Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn
Asp Leu225 230 235 240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn
Ser Asp Thr Cys Phe 245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro
Thr Ile Ser Ser Tyr Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro
Pro Glu Val Thr Lys Ile Pro Glu Asp 275 280 285Ile Leu Gln Leu Leu
Ser Lys Tyr Asn Ser Asn Leu Ala Thr Pro Ile 290 295 300Ala Ile Lys
Ala Val Pro Pro Ser Lys Arg Phe Leu Lys His Gly Gln305 310 315
320Asn Ile Arg Asp Val Ser Asn Lys Glu Asn 325 330202679DNAHomo
sapiensCDS(805)...(1971) 20tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagc atg gac cct atc cgg agc ttc tgc ggg 831 Met Asp Pro
Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg tct ctg gcc agc acg ctg gac
tgc gag acg gcc cgg ctg 879Lys Leu Arg Ser Leu Ala Ser Thr Leu Asp
Cys Glu Thr Ala Arg Leu10 15 20 25cag cga gcg ctg gac gga gag gaa
agc gac ttt gaa gat tat cca atg 927Gln Arg Ala Leu Asp Gly Glu Glu
Ser Asp Phe Glu Asp Tyr Pro Met 30 35 40aga att tta tat gac ctt cat
tca gaa gtt cag act cta aag gat gat 975Arg Ile Leu Tyr Asp Leu His
Ser Glu Val Gln Thr Leu Lys Asp Asp 45 50 55gtt aat att ctt ctt gat
aaa gca aga ttg gaa aat caa gaa ggc att 1023Val Asn Ile Leu Leu Asp
Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile 60 65 70gat ttc ata aag gca
aca aaa gta cta atg gaa aaa aat tca atg gat 1071Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 75 80 85att atg aaa ata
aga gag tat ttc cag aag tat gga tat agt cca cgt 1119Ile Met Lys Ile
Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg90 95 100 105gtc aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca 1167Val Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro 110 115
120gag ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat
1215Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp
125 130 135ctg tct gat cct cct gtt gca agc agt tgt att tct gag aag
tct cca 1263Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys
Ser Pro 140 145 150cgt agt
cca caa ctt tca gat ttt gga ctt gag cgg tac atc gta tcc 1311Arg Ser
Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val Ser 155 160
165caa gtt cta cca aac cct cca cag gca gtg aac aac tat aag gaa gag
1359Gln Val Leu Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu
Glu170 175 180 185ccc gta att gta acc cca cct acc aaa caa tca cta
gta aaa gta cta 1407Pro Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu
Val Lys Val Leu 190 195 200aaa act cca aaa tgt gca cta aaa atg gat
gat ttt gag tgt gta act 1455Lys Thr Pro Lys Cys Ala Leu Lys Met Asp
Asp Phe Glu Cys Val Thr 205 210 215cct aaa tta gaa cac ttt ggt atc
tct gaa tat act atg tgt tta aat 1503Pro Lys Leu Glu His Phe Gly Ile
Ser Glu Tyr Thr Met Cys Leu Asn 220 225 230gaa gat tac aca atg gga
ctt aaa aat gcg agg aat aat aaa agt gag 1551Glu Asp Tyr Thr Met Gly
Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu 235 240 245gag gcc ata gat
aca gaa tcc agg ctc aat gat aat gtt ttt gcc act 1599Glu Ala Ile Asp
Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala Thr250 255 260 265ccc
agc ccc atc atc cag cag ttg gaa aaa agt gat gcc gaa tat acc 1647Pro
Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp Ala Glu Tyr Thr 270 275
280aac tct cct ttg gta cct aca ttc tgt act cct ggt ttg aaa att cca
1695Asn Ser Pro Leu Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile Pro
285 290 295tct aca aag aac agc ata gct ttg gta tcc aca aat tac cca
tta tca 1743Ser Thr Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro
Leu Ser 300 305 310aaa aca aat agt tca tca aat gat ttg gaa gtt gaa
gat cgt act tcg 1791Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu
Asp Arg Thr Ser 315 320 325ttg gtt tta aat tca gac aca tgc ttt gag
aat tta aca gat ccc tct 1839Leu Val Leu Asn Ser Asp Thr Cys Phe Glu
Asn Leu Thr Asp Pro Ser330 335 340 345tca cct acg att tct tct tat
gag aat ctg ctc aga aca cct aca cct 1887Ser Pro Thr Ile Ser Ser Tyr
Glu Asn Leu Leu Arg Thr Pro Thr Pro 350 355 360cca gaa gta act aaa
att cca gaa gat att ctc cag aaa ttc cag tgg 1935Pro Glu Val Thr Lys
Ile Pro Glu Asp Ile Leu Gln Lys Phe Gln Trp 365 370 375atc tat cca
aca cag aaa ctg aac aaa atg aga tga aagccgagct 1981Ile Tyr Pro Thr
Gln Lys Leu Asn Lys Met Arg 380 385ggaccgattt taacattcac attgccctgc
ctctgtcccc ctttaaacgt tgacccattt 2041taaagacaaa catgaacatt
aacatcataa tatgcttttt atgaagtttc aataaggttt 2101aaccttagtc
ttgttgacat gtagcccagt cattcactct ttaaggacta ttagtgtttc
2161attgatacta aattacccag cttaatcaac agaatggttt aagtagtacc
aggaagtagg 2221acaagtaatt tcaaaaatat aaaggtgttt gctactcaga
tgaggccgcc cctgaccttc 2281tggccagaga gacattgctg ccagccagct
ctgccttccc atcatctcct ttcaggaccg 2341tcccacacct tttacttgct
cagtgctgtc tgaagatgca gttgctgttt gcaaacaaca 2401ggaacaccag
ttaaactaat taggaaacag agggagattt ccaggcctgg gtaactatat
2461actgtgacca ttggcggttg agaccggtct tcaaccagtg gaaccccgaa
ctctgctgtc 2521agggtgtgga cttcggtgct cttccaagtt ttcacctggg
ggggggagct aaccccctat 2581gttcacgcct tctattccca ttggcgctga
actcttaagg tcactctggt cgcttgtgac 2641cccgtaaccc tgatgtaccc
ctctaaaagg tgaggggc 267921388PRTHomo sapiens 21Met Asp Pro Ile Arg
Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys
Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp
Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu
Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala
Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Lys Phe Gln Trp Ile
Tyr Pro Thr Gln Lys Leu 370 375 380Asn Lys Met Arg385222617DNAHomo
sapiensCDS(989)...(1909) 22tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagcggat gatgttaata ttcttcttga taaagcaaga
ttggaaaatc aagaaggcat 960tgatttcata aaggcaacaa aagtacta atg gaa aaa
aat tca atg gat att 1012 Met Glu Lys Asn Ser Met Asp Ile 1 5atg aaa
ata aga gag tat ttc cag aag tat gga tat agt cca cgt gtc 1060Met Lys
Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val 10 15 20aag
aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca gag 1108Lys
Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu25 30 35
40ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat gat ctg
1156Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp Leu
45 50 55tct gat cct cct gtt gca agc agt tgt att tct ggg aag tct cca
cgt 1204Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys Ser Pro
Arg 60 65 70agt cca caa ctt tca gat ttt gga ctt gag cgg tac atc gta
tcc caa 1252Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val
Ser Gln 75 80 85gtt cta cca aac cct cca cag gca gtg aac aac tat aag
gaa gag ccc 1300Val Leu Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys
Glu Glu Pro 90 95 100gta att gta acc cca cct acc aaa caa tca cta
gta aaa gta cta aaa 1348Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu
Val Lys Val Leu Lys105 110 115 120act cca aaa tgt gca cta aaa atg
gat gat ttt gag tgt gta act cct 1396Thr Pro Lys Cys Ala Leu Lys Met
Asp Asp Phe Glu Cys Val Thr Pro 125 130 135aaa tta gaa cac ttt ggt
atc tct gaa tat act atg tgt tta aat gaa 1444Lys Leu Glu His Phe Gly
Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu 140 145 150gat tac aca atg
gga ctt aaa aat gcg agg aat aat aaa agt gag gag 1492Asp Tyr Thr Met
Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu 155 160 165gcc ata
gat aca gaa tcc agg ctc aat gat aat gtt ttt gcc act ccc 1540Ala Ile
Asp Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala Thr Pro 170 175
180agc ccc atc atc cag cag ttg gaa aaa agt gat gcc gaa tat acc aac
1588Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp Ala Glu Tyr Thr
Asn185 190 195 200tct cct ttg gta cct aca ttc tgt act cct ggt ttg
aaa att cca tct 1636Ser Pro Leu Val Pro Thr Phe Cys Thr Pro Gly Leu
Lys Ile Pro Ser 205 210 215aca aag aac agc ata gct ttg gta tcc aca
aat tac cca tta tca aaa 1684Thr Lys Asn Ser Ile Ala Leu Val Ser Thr
Asn Tyr Pro Leu Ser Lys 220 225 230aca aat agt tca tca aat gat ttg
gaa gtt gaa gat cgt act tcg ttg 1732Thr Asn Ser Ser Ser Asn Asp Leu
Glu Val Glu Asp Arg Thr Ser Leu 235 240 245gtt tta aat tca gac aca
tgc ttt gag aat tta aca gat ccc tct tca 1780Val Leu Asn Ser Asp Thr
Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser 250 255 260cct acg att tct
tct tat gag aat ctg ctc aga aca cct aca cct cca 1828Pro Thr Ile Ser
Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro265 270 275 280gaa
gta act aaa att cca gaa gat att ctc cag aaa ttc cag tgg atc 1876Glu
Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Lys Phe Gln Trp Ile 285 290
295tat cca aca cag aaa ctg aac aaa atg aga tga aagccgagct
ggaccgattt 1929Tyr Pro Thr Gln Lys Leu Asn Lys Met Arg 300
305taacattcac attgccctgc ctctgtcccc ctttaaacgt tgacccattt
taaagacaaa 1989catgaacatt aacatcataa tatgcttttt atgaagtttc
aataaggttt aaccttagtc 2049ttgttgacat gtagcccagt cattcactct
ttaaggacta ttagtgtttc attgatacta 2109aattacccag cttaatcaac
agaatggttt aagtagtacc aggaagtagg acaagtaatt 2169tcaaaaatat
aaaggtgttt gctactcaga tgaggccgcc cctgaccttc tggccagaga
2229gacattgctg ccagccagct ctgccttccc atcatctcct ttcaggaccg
tcccacacct 2289tttacttgct cagtgctgtc tgaagatgca gttgctgttt
gcaaacaaca ggaacaccag 2349ttaaactaat taggaaacag agggagattt
ccaggcctgg gtaactatat actgtgacca 2409ttggcggttg agaccggtct
tcaaccagtg gaaccccgaa ctctgctgtc agggtgtgga 2469cttcggtgct
cttccaagtt ttcacctggg ggggggagct aaccccctat gttcacgcct
2529tctattccca ttggcgctga actcttaagg tcactctggt cgcttgtgac
cccgtaaccc 2589tgatgtaccc ctctaaaagg tgaggggc 261723306PRTHomo
sapiens 23Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys
Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro
Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro
Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg Tyr Ile Val Ser Gln
Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn Asn Tyr Lys Glu Glu
Pro Val Ile Val Thr Pro Pro Thr Lys 100 105 110Gln Ser Leu Val Lys
Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met 115 120 125Asp Asp Phe
Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly Ile Ser 130 135 140Glu
Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn145 150
155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser Arg
Leu 165 170 175Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln
Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu
Val Pro Thr Phe Cys 195 200 205Thr Pro Gly Leu Lys Ile Pro Ser Thr
Lys Asn Ser Ile Ala Leu Val 210 215 220Ser Thr Asn Tyr Pro Leu Ser
Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230 235 240Glu Val Glu Asp
Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys Phe 245 250 255Glu Asn
Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr Glu Asn 260 265
270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro Glu Asp
275 280 285Ile Leu Gln Lys Phe Gln Trp Ile Tyr Pro Thr Gln Lys Leu
Asn Lys 290 295 300Met Arg305241781DNAHomo sapiensCDS(805)...(1026)
24tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag
60caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagc atg gac cct atc cgg agc
ttc tgc ggg 831 Met Asp Pro Ile Arg Ser Phe Cys Gly 1 5aag ctg cgg
tct ctg gcc agc acg ctg gac tgc gag acg gcc cgg ctg 879Lys Leu Arg
Ser Leu Ala Ser Thr Leu Asp Cys Glu Thr Ala Arg Leu10 15 20 25cag
cga gcg ctg gac gga gag gaa agc ctt tta tca aaa tac aac tca 927Gln
Arg Ala Leu Asp Gly Glu Glu Ser Leu Leu Ser Lys Tyr Asn Ser 30 35
40aac cta gct act cca ata gca att aaa gca gtg cca ccc agt aaa agg
975Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg
45 50 55ttc ctt aaa cat gga cag aac atc cga gat gtc agc aac aaa gaa
aac 1023Phe Leu Lys His Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu
Asn 60 65 70tga aattccagtg gatctatcca acacagaaac tgaacaaaat
gagatgaaag 1076ccgagctgga ccgattttaa cattcacatt gccctgcctc
tgtccccctt taaacgttga 1136cccattttaa agacaaacat gaacattaac
atcataatat gctttttatg aagtttcaat 1196aaggtttaac cttagtcttg
ttgacatgta gcccagtcat tcactcttta aggactatta 1256gtgtttcatt
gatactaaat tacccagctt aatcaacaga atggtttaag tagtaccagg
1316aagtaggaca agtaatttca aaaatataaa ggtgtttgct actcagatga
ggccgcccct 1376gaccttctgg ccagagagac attgctgcca gccagctctg
ccttcccatc atctcctttc 1436aggaccgtcc cacacctttt acttgctcag
tgctgtctga agatgcagtt gctgtttgca 1496aacaacagga acaccagtta
aactaattag gaaacagagg gagatttcca ggcctgggta 1556actatatact
gtgaccattg gcggttgaga ccggtcttca accagtggaa ccccgaactc
1616tgctgtcagg gtgtggactt cggtgctctt ccaagttttc acctgggggg
gggagctaac 1676cccctatgtt cacgccttct attcccattg gcgctgaact
cttaaggtca ctctggtcgc 1736ttgtgacccc gtaaccctga tgtacccctc
taaaaggtga ggggc 17812573PRTHomo sapiens 25Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Leu Leu
Ser Lys Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala 35 40 45Ile Lys Ala
Val Pro Pro Ser Lys Arg Phe Leu Lys His Gly Gln Asn 50 55 60Ile Arg
Asp Val Ser Asn Lys Glu Asn65 70262825DNAHomo
sapiensCDS(952)...(2070) 26tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac cctatccgga
gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg
gcccggctgc agcgagcgct ggacggagag 900gaaagcggtg cgtgaggcgg
gcggccaggg cacgactttg aagattatcc a atg aga 957 Met Arg 1att tta tat
gac ctt cat tca gaa gtt cag act cta aag gat gat gtt 1005Ile Leu Tyr
Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp Asp Val 5 10 15aat att
ctt ctt gat aaa gca aga ttg gaa aat caa gaa ggc att gat 1053Asn Ile
Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp 20 25 30ttc
ata aag gca aca aaa gta cta atg gaa aaa aat tca atg gat att 1101Phe
Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp Ile35 40 45
50atg aaa ata aga gag tat ttc cag aag tat gga tat agt cca cgt gtc
1149Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val
55 60 65aag aaa aat tca gta cac gag caa gaa gcc att aac tct gac cca
gag 1197Lys Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp Pro
Glu 70 75 80ttg tct aat tgt gaa aat ttt cag aag act gat gtg aaa gat
gat ctg 1245Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp
Asp Leu 85 90 95tct gat cct cct gtt gca agc agt tgt att tct ggg aag
tct cca cgt 1293Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys
Ser Pro Arg 100 105 110agt cca caa ctt tca gat ttt gga ctt gag cgg
tac atc gta tcc caa 1341Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val Ser Gln115 120 125 130gtt cta cca aac cct cca cag gca
gtg aac aac tat aag gaa gag ccc 1389Val Leu Pro Asn Pro Pro Gln Ala
Val Asn Asn Tyr Lys Glu Glu Pro 135 140 145gta att gta acc cca cct
acc aaa caa tca cta gta aaa gta cta aaa 1437Val Ile Val Thr Pro Pro
Thr Lys Gln Ser Leu Val Lys Val Leu Lys 150 155 160act cca aaa tgt
gca cta aaa atg gat gat ttt gag tgt gta act cct 1485Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val Thr Pro 165 170 175aaa tta
gaa cac ttt ggt atc tct gaa tat act atg tgt tta aat gaa 1533Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu 180 185
190gat tac aca atg gga ctt aaa aat gcg agg aat aat aaa agt gag gag
1581Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu
Glu195 200 205 210gcc ata gat aca gaa tcc agg ctc aat gat aat gtt
ttt gcc act ccc 1629Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn Val
Phe Ala Thr Pro 215 220 225agc ccc atc atc cag cag ttg gaa aaa agt
gat gcc gaa tat acc aac 1677Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser
Asp Ala Glu Tyr Thr Asn 230 235 240tct cct ttg gta cct aca ttc tgt
act cct ggt ttg aaa att cca tct 1725Ser Pro Leu Val Pro Thr Phe Cys
Thr Pro Gly Leu Lys Ile Pro Ser 245 250 255aca aag aac agc ata gct
ttg gta tcc aca aat tac cca tta tca aaa 1773Thr Lys Asn Ser Ile Ala
Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys 260 265 270aca aat agt tca
tca aat gat ttg gaa gtt gaa gat cgt act tcg ttg 1821Thr Asn Ser Ser
Ser Asn Asp Leu Glu Val Glu Asp Arg Thr Ser Leu275 280 285 290gtt
tta aat tca gac aca tgc ttt gag aat tta aca gat ccc tct tca 1869Val
Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser 295 300
305cct acg att tct tct tat gag aat ctg ctc aga aca cct aca cct cca
1917Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro
310 315 320gaa gta act aaa att cca gaa gat att ctc cag ctt tta tca
aaa tac 1965Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser
Lys Tyr 325 330 335aac tca aac cta gct act cca ata gca att aaa gca
gtg cca ccc agt 2013Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala
Val Pro Pro Ser 340 345 350aaa agg ttc ctt aaa cat gga cag aac atc
cga gat gtc agc aac aaa 2061Lys Arg Phe Leu Lys His Gly Gln Asn Ile
Arg Asp Val Ser Asn Lys355 360 365 370gaa aac tga aattccagtg
gatctatcca acacagaaac tgaacaaaat 2110Glu Asngagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc tgtccccctt 2170taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat gctttttatg
2230aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat
tcactcttta 2290aggactatta gtgtttcatt gatactaaat tacccagctt
aatcaacaga atggtttaag 2350tagtaccagg aagtaggaca agtaatttca
aaaatataaa ggtgtttgct actcagatga 2410ggccgcccct gaccttctgg
ccagagagac attgctgcca gccagctctg ccttcccatc 2470atctcctttc
aggaccgtcc cacacctttt acttgctcag tgctgtctga agatgcagtt
2530gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg
gagatttcca 2590ggcctgggta actatatact gtgaccattg gcggttgaga
ccggtcttca accagtggaa 2650ccccgaactc tgctgtcagg gtgtggactt
cggtgctctt ccaagttttc acctgggggg 2710gggagctaac cccctatgtt
cacgccttct attcccattg gcgctgaact cttaaggtca 2770ctctggtcgc
ttgtgacccc gtaaccctga tgtacccctc taaaaggtga ggggc 282527372PRTHomo
sapiens 27Met Arg Ile Leu Tyr Asp Leu His Ser Glu Val Gln Thr Leu
Lys Asp1 5 10 15Asp Val Asn Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn
Gln Glu Gly 20 25 30Ile Asp Phe Ile Lys Ala Thr Lys Val Leu Met Glu
Lys Asn Ser Met 35 40 45Asp Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys
Tyr Gly Tyr Ser Pro 50 55 60Arg Val Lys Lys Asn Ser Val His Glu Gln
Glu Ala Ile Asn Ser Asp65 70 75 80Pro Glu Leu Ser Asn Cys Glu Asn
Phe Gln Lys Thr Asp Val Lys Asp 85 90 95Asp Leu Ser Asp Pro Pro Val
Ala Ser Ser Cys Ile Ser Gly Lys Ser 100 105 110Pro Arg Ser Pro Gln
Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val 115 120 125Ser Gln Val
Leu Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu 130 135 140Glu
Pro Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val145 150
155 160Leu Lys Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys
Val 165 170 175Thr Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr
Met Cys Leu 180 185 190Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala
Arg Asn Asn Lys Ser 195 200 205Glu Glu Ala Ile Asp Thr Glu Ser Arg
Leu Asn Asp Asn Val Phe Ala 210 215 220Thr Pro Ser Pro Ile Ile Gln
Gln Leu Glu Lys Ser Asp Ala Glu Tyr225 230 235 240Thr Asn Ser Pro
Leu Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile 245 250 255Pro Ser
Thr Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu 260 265
270Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr
275 280 285Ser Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr
Asp Pro 290 295 300Ser Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu
Arg Thr Pro Thr305 310 315 320Pro Pro Glu Val Thr Lys Ile Pro Glu
Asp Ile Leu Gln Leu Leu Ser 325 330 335Lys Tyr Asn Ser Asn Leu Ala
Thr Pro Ile Ala Ile Lys Ala Val Pro 340 345 350Pro Ser Lys Arg Phe
Leu Lys His Gly Gln Asn Ile Arg Asp Val Ser 355 360 365Asn Lys Glu
Asn 37028412PRTHomo sapiens 28Met Asp Pro Ile Arg Ser Phe Cys Gly
Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg
Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr
Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu
Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn
Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met
Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln
Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His 100 105
110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn
115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro
Val Ala 130 135 140Ser Ser Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro
Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser
Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys
Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu
Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys Met Asp
Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215 220Ile
Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230
235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu
Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile
Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser
Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro
Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro
Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val
Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe
Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345
350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro
355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr Asn Ser Asn Leu
Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg
Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg Asp Val Ser Asn
Lys Glu Asn 405 41029412PRTHomo sapiens 29Met Asp Pro Ile Arg Ser
Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu
Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe
Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val
Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg
Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 41030412PRTHomo sapiens 30Met Asp
Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr
Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25
30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His
35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp
Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala
Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys
Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys
Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro
Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys
Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser
Gly Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly
Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170
175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro
180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys
Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu
Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu
Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys
Ser Glu Glu Ala Ile Asp Ala Glu Ser 245 250 255Arg Leu Asn Asp Asn
Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys
Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe
Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295
300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser
Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu
Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser
Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr
Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu
Leu Ser Lys Tyr Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile
Lys Ala Val Pro Pro Ser Lys Arg Phe Leu Lys
His385 390 395 400Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu Asn
405 41031330PRTHomo sapiens 31Met Glu Lys Asn Ser Met Asp Ile Met
Lys Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val
Lys Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro
Glu Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp
Asp Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Glu Lys
Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg
Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro Thr Lys 100 105
110Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met
115 120 125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
Ile Ser 130 135 140Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu Lys Asn145 150 155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200 205Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val 210 215 220Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230
235 240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys
Phe 245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser
Tyr Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr
Lys Ile Pro Glu Asp 275 280 285Ile Leu Gln Leu Leu Ser Lys Tyr Asn
Ser Asn Leu Ala Thr Pro Ile 290 295 300Ala Ile Lys Ala Val Pro Pro
Ser Lys Arg Phe Leu Lys His Gly Gln305 310 315 320Asn Ile Arg Asp
Val Ser Asn Lys Glu Asn 325 33032388PRTHomo sapiens 32Met Asp Pro
Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu
Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu
Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40
45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys
50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr
Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile
Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys
Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu
Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp
Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu
Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu
Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln
Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185
190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu
195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His
Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr
Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu
Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe
Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp
Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr
Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu
Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310
315 320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp
Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile
Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu
Val Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Lys Phe Gln Trp
Ile Tyr Pro Thr Gln Lys Leu 370 375 380Asn Lys Met
Arg38533306PRTHomo sapiens 33Met Glu Lys Asn Ser Met Asp Ile Met
Lys Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val
Lys Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro
Glu Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp
Asp Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys
Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg
Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro Thr Lys 100 105
110Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met
115 120 125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
Ile Ser 130 135 140Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu Lys Asn145 150 155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200 205Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val 210 215 220Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230
235 240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys
Phe 245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser
Tyr Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr
Lys Ile Pro Glu Asp 275 280 285Ile Leu Gln Lys Phe Gln Trp Ile Tyr
Pro Thr Gln Lys Leu Asn Lys 290 295 300Met Arg3053473PRTHomo
sapiens 34Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala
Thr Pro Ile Ala 35 40 45Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu
Lys His Gly Gln Asn 50 55 60Ile Arg Asp Val Ser Asn Lys Glu Asn65
7035372PRTHomo sapiens 35Met Arg Ile Leu Tyr Asp Leu His Ser Glu
Val Gln Thr Leu Lys Asp1 5 10 15Asp Val Asn Ile Leu Leu Asp Lys Ala
Arg Leu Glu Asn Gln Glu Gly 20 25 30Ile Asp Phe Ile Lys Ala Thr Lys
Val Leu Met Glu Lys Asn Ser Met 35 40 45Asp Ile Met Lys Ile Arg Glu
Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro 50 55 60Arg Val Lys Lys Asn Ser
Val His Glu Gln Glu Ala Ile Asn Ser Asp65 70 75 80Pro Glu Leu Ser
Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp 85 90 95Asp Leu Ser
Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys Ser 100 105 110Pro
Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val 115 120
125Ser Gln Val Leu Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu
130 135 140Glu Pro Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val
Lys Val145 150 155 160Leu Lys Thr Pro Lys Cys Ala Leu Lys Met Asp
Asp Phe Glu Cys Val 165 170 175Thr Pro Lys Leu Glu His Phe Gly Ile
Ser Glu Tyr Thr Met Cys Leu 180 185 190Asn Glu Asp Tyr Thr Met Gly
Leu Lys Asn Ala Arg Asn Asn Lys Ser 195 200 205Glu Glu Ala Ile Asp
Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala 210 215 220Thr Pro Ser
Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp Ala Glu Tyr225 230 235
240Thr Asn Ser Pro Leu Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile
245 250 255Pro Ser Thr Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr
Pro Leu 260 265 270Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val
Glu Asp Arg Thr 275 280 285Ser Leu Val Leu Asn Ser Asp Thr Cys Phe
Glu Asn Leu Thr Asp Pro 290 295 300Ser Ser Pro Thr Ile Ser Ser Tyr
Glu Asn Leu Leu Arg Thr Pro Thr305 310 315 320Pro Pro Glu Val Thr
Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser 325 330 335Lys Tyr Asn
Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val Pro 340 345 350Pro
Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile Arg Asp Val Ser 355 360
365Asn Lys Glu Asn 37036373PRTHomo sapiens 36Met Asp Pro Ile Arg
Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys
Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp
Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu
Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala
Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Gly Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln 37037373PRTHomo
sapiens 37Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu
Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn
Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp
Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile
Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150
155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro
Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val
Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr
Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr
Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys
Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg
Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu
Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265
270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr
275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser
Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn
Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser
Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp
Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg
Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile
Leu Gln 37038373PRTHomo sapiens 38Met Asp Pro Ile Arg Ser Phe Cys
Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala
Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp
Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr
Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu
Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu
Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe
Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His 100 105
110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn
115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro
Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro
Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser
Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys
Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu
Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys Met Asp
Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215 220Ile
Ser Glu
Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235
240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser
245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile
Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro
Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser
Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu
Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu
Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu
Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu
Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360
365Glu Asp Ile Leu Gln 37039373PRTHomo sapiens 39Met Asp Pro Ile
Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp
Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser
Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser
Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55
60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65
70 75 80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu
Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser
Val His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser
Asn Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu
Ser Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser
Pro Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg
Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val
Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr
Lys Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln 37040388PRTHomo
sapiens 40Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu
Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn
Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp
Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met
Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser
Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile
Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150
155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro
Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val
Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr
Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr
Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys
Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg
Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu
Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265
270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr
275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser
Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn
Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser
Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp
Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg
Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile
Leu Gln Lys Phe Gln Trp Ile Tyr Pro Thr Gln Lys Leu 370 375 380Asn
Lys Met Arg38541388PRTHomo sapiens 41Met Asp Pro Ile Arg Ser Phe
Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr
Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu
Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln
Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu
Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val
Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90
95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His
100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys
Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp
Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro Arg
Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile
Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn
Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln
Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys
Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215
220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly
Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile
Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro
Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr
Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu
Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr
Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp
Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330
335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr
340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys
Ile Pro 355 360 365Glu Asp Ile Leu Gln Lys Phe Gln Trp Ile Tyr Pro
Thr Gln Lys Leu 370 375 380Asn Lys Met Arg3854239PRTHomo sapiens
42Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1
5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly
Glu 20 25 30Glu Ser Leu Leu Ser Lys Tyr 354339PRTHomo sapiens 43Met
Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10
15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu
20 25 30Glu Ser Asp Phe Glu Asp Tyr 354447PRTHomo sapiens 44Val Asn
Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly Ile1 5 10 15Asp
Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met Asp 20 25
30Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro 35 40
454549PRTHomo sapiens 45Ile Gly Val Leu Ile Asp Arg Ile Arg Ile His
Asn Pro His Ile Leu1 5 10 15Gly Cys Ile Ala Gly Asp Asp Thr Ile Leu
Ile Leu Ser Lys Asn Lys 20 25 30Glu Asp Ala Leu Glu Val Asn Asn Tyr
Phe Gln Gln Tyr Leu Tyr His 35 40 45Pro46129PRTHomo sapiens 46Leu
Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu Glu1 5 10
15Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His Ser
20 25 30Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys
Ala 35 40 45Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr
Lys Val 50 55 60Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg
Glu Tyr Phe65 70 75 80Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys
Asn Ser Val His Glu 85 90 95Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu
Ser Asn Cys Glu Asn Phe 100 105 110Gln Lys Thr Asp Val Lys Asp Asp
Leu Ser Asp Pro Pro Val Ala Ser 115 120 125Ser47127PRTHomo sapiens
47Val Asp Cys Leu Ser Ser His Phe Gln Glu Leu Ser Ile Tyr Gln Asp1
5 10 15Gln Glu Gln Arg Ile Leu Lys Phe Leu Glu Glu Leu Gly Glu Gly
Lys 20 25 30Ala Thr Thr Ala His Asp Leu Ser Gly Lys Leu Gly Thr Pro
Lys Lys 35 40 45Glu Ile Asn Arg Val Leu Tyr Ser Leu Ala Lys Lys Gly
Lys Leu Gln 50 55 60Lys Glu Ala Gly Thr Pro Pro Leu Trp Lys Ile Ala
Val Ser Thr Gln65 70 75 80Ala Trp Asn Gln His Ser Gly Val Val Arg
Pro Asp Gly His Ser Gln 85 90 95Gly Ala Pro Asn Ser Asp Pro Ser Leu
Glu Pro Glu Asp Arg Asn Ser 100 105 110Thr Ser Val Ser Glu Asp Leu
Leu Glu Pro Phe Ile Ala Val Ser 115 120 1254814PRTClostridium
tetaniTetanus toxoid 48Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly
Ile Thr Glu1 5 104921PRTPlasmodium falciparum 49Asp Ile Glu Lys Lys
Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe1 5 10 15Asn Val Val Asn
Ser 205016PRTStreptococcus aureus 50Gly Ala Val Asp Ser Ile Leu Gly
Gly Val Ala Thr Tyr Gly Ala Ala1 5 10 155113PRTArtificial
SequenceVARIANT3Xaa = cyclohexylalanine, phenylalanine, or tyrosine
51Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa1 5
105214DNAArtificial SequencePrimer 52ttttgatcaa gctt
145342DNAArtificial SequencePrimer 53ctaatacgac tcactatagg
gctcgagcgg ccgcccgggc ag 425412DNAArtificial SequencePrimer
54gatcctgccc gg 125540DNAArtificial SequencePrimer 55gtaatacgac
tcactatagg gcagcgtggt cgcggccgag 405610DNAArtificial SequencePrimer
56gatcctcggc 105722DNAArtificial SequencePrimer 57ctaatacgac
tcactatagg gc 225822DNAArtificial SequencePrimer 58tcgagcggcc
gcccgggcag ga 225920DNAArtificial SequencePrimer 59agcgtggtcg
cggccgagga 206025DNAArtificial SequencePrimer 60atatcgccgc
gctcgtcgtc gacaa 256126DNAArtificial SequencePrimer 61agccacacgc
agctcattgt agaagg 266224DNAArtificial SequenceFlag Sequence -
Epitope tag 62gattacaagg atgacgacga taag 24634PRTHomo sapiens 63Asn
Lys Ser Glu1644PRTHomo sapiens 64Asn Ser Ser Ser1654PRTHomo sapiens
65Asn Leu Thr Asp1664PRTHomo sapiens 66Lys Lys Asn Ser1674PRTHomo
sapiens 67Ser Thr Leu Asp1684PRTHomo sapiens 68Ser Asp Phe
Glu1694PRTHomo sapiens 69Thr Leu Lys Asp1704PRTHomo sapiens 70Ser
Val His Glu1714PRTHomo sapiens 71Ser Asp Pro Glu1724PRTHomo sapiens
72Ser Asn Cys Glu1734PRTHomo sapiens 73Ser Asp Ala Glu1744PRTHomo
sapiens 74Ser Ser Asn Asp1754PRTHomo sapiens 75Thr Cys Phe
Glu1764PRTHomo sapiens 76Ser Ser Tyr Glu1774PRTHomo sapiens 77Thr
Pro Pro Glu1784PRTHomo sapiens 78Ser Asn Lys Glu1796PRTHomo sapiens
79Gly Leu Lys Asn Ala Arg1 580412PRTHomo sapiens 80Met Asp Pro Ile
Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp
Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser
Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser
Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Pro Glu Leu Ser 50 55
60Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp65
70 75 80Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys Ser Pro Arg Ser
Pro 85 90 95Gln Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln
Val Leu 100 105 110Pro Asn Pro Pro Gln Ala Val Asn Leu Leu Asp Lys
Ala Arg Leu Glu 115 120 125Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala
Thr Lys Val Leu Met Glu 130 135 140Lys Asn Ser Met Asp Ile Met Lys
Ile Arg Glu Tyr Phe Gln Lys Tyr145 150 155 160Gly Tyr Ser Pro Arg
Val Lys Lys Asn Ser Val His Glu Gln Glu Ala 165 170 175Ile Asn Ser
Asp Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr
Lys Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser
Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg Asp Val
Ser Asn Lys Glu Asn 405 4108117PRTHomo sapiens 81Pro Val Ala Ser
Ser Cys Ile Ser Glu Lys Ser Pro Arg Ser Pro Gln1 5 10
15Leu8219PRTHomo sapiens 82Pro Pro Val Ala Ser Ser Cys Ile Ser Glu
Lys Ser Pro Arg Ser Pro1 5 10 15Gln Leu Ser8329PRTHomo sapiens
83Asp Asp Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Glu Lys1
5 10 15Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu 20
258417PRTHomo sapiens 84Asn Lys Ser Glu Glu Ala Ile Asp Ala Glu Ser
Arg Leu Asn Asp Asn1 5 10 15Val8519PRTHomo sapiens 85Asn Asn Lys
Ser Glu Glu Ala Ile Asp Ala Glu Ser Arg Leu Asn Asp1 5 10 15Asn Val
Phe8629PRTHomo sapiens 86Leu Lys Asn Ala Arg Asn Asn Lys Ser Glu
Glu Ala Ile Asp Ala Glu1 5 10 15Ser Arg Leu Asn Asp Asn Val Phe Ala
Thr Pro Ser Pro 20 258723PRTHomo sapiens 87Lys Ile Pro Glu Asp Ile
Leu Gln Lys Phe Gln Trp Ile Tyr Pro Thr1 5 10 15Gln Lys Leu Asn Lys
Met Arg 208824PRTHomo sapiens 88Thr Lys Ile Pro Glu Asp Ile Leu Gln
Lys Phe Gln Trp Ile Tyr Pro1 5 10 15Thr Gln Lys Leu Asn Lys Met Arg
208929PRTHomo sapiens 89Thr Pro Pro Glu Val Thr Lys Ile Pro Glu Asp
Ile Leu Gln Lys Phe1 5 10 15Gln Trp Ile Tyr Pro Thr Gln Lys Leu Asn
Lys Met Arg 20 259016PRTHomo sapiens 90Arg Ala Leu Asp Gly Glu Glu
Ser Leu Leu Ser Lys Tyr Asn Ser Asn1 5 10 159118PRTHomo sapiens
91Gln Arg Ala Leu Asp Gly Glu Glu Ser Leu Leu Ser Lys Tyr Asn Ser1
5 10 15Asn Leu9228PRTHomo sapiens 92Glu Thr Ala Arg Leu Gln Arg Ala
Leu Asp Gly Glu Glu Ser Leu Leu1 5 10 15Ser Lys Tyr Asn Ser Asn Leu
Ala Thr Pro Ile Ala 20 25932736DNAHomo sapiens 93tatcatctgt
gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac cctatccgga
gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg
gcccggctgc agcgagcgct ggacggagag 900gaaagcggat gatgttaata
ttcttcttga taaagcaaga ttggaaaatc aagaaggcat 960tgatttcata
aaggcaacaa aagtactaat ggaaaaaaat tcaatggata ttatgaaaat
1020aagagagtat ttccagaagt atggatatag tccacgtgtc aagaaaaatt
cagtacacga 1080gcaagaagcc attaactctg acccagagtt gtctaattgt
gaaaattttc agaagactga 1140tgtgaaagat gatctgtctg atcctcctgt
tgcaagcagt tgtatttctg ggaagtctcc 1200acgtagtcca caactttcag
attttggact tgagcggtac atcgtatccc aagttctacc 1260aaaccctcca
caggcagtga acaactataa ggaagagccc gtaattgtaa ccccacctac
1320caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa
tggatgattt 1380tgagtgtgta actcctaaat tagaacactt tggtatctct
gaatatacta tgtgtttaaa 1440tgaagattac acaatgggac ttaaaaatgc
gaggaataat aaaagtgagg aggccataga 1500tacagaatcc aggctcaatg
ataatgtttt tgccactccc agccccatca tccagcagtt 1560ggaaaaaagt
gatgccgaat ataccaactc tcctttggta cctacattct gtactcctgg
1620tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaatt
acccattatc 1680aaaaacaaat agttcatcaa atgatttgga agttgaagat
cgtacttcgt tggttttaaa 1740ttcagacaca tgctttgaga atttaacaga
tccctcttca cctacgattt cttcttatga 1800gaatctgctc agaacaccta
cacctccaga agtaactaaa attccagaag atattctcca 1860gcttttatca
aaatacaact caaacctagc tactccaata gcaattaaag cagtgccacc
1920cagtaaaagg ttccttaaac atggacagaa catccgagat gtcagcaaca
aagaaaactg 1980aaattccagt ggatctatcc aacacagaaa ctgaacaaaa
tgagatgaaa gccgagctgg 2040accgatttta acattcacat tgccctgcct
ctgtccccct ttaaacgttg acccatttta 2100aagacaaaca tgaacattaa
catcataata tgctttttat gaagtttcaa taaggtttaa 2160ccttagtctt
gttgacatgt agcccagtca ttcactcttt aaggactatt agtgtttcat
2220tgatactaaa ttacccagct taatcaacag aatggtttaa gtagtaccag
gaagtaggac 2280aagtaatttc aaaaatataa aggtgtttgc tactcagatg
aggccgcccc tgaccttctg 2340gccagagaga cattgctgcc agccagctct
gccttcccat catctccttt caggaccgtc 2400ccacaccttt tacttgctca
gtgctgtctg aagatgcagt tgctgtttgc aaacaacagg 2460aacaccagtt
aaactaatta ggaaacagag ggagatttcc aggcctgggt aactatatac
2520tgtgaccatt ggcggttgag accggtcttc aaccagtgga accccgaact
ctgctgtcag 2580ggtgtggact tcggtgctct tccaagtttt cacctggggg
ggggagctaa ccccctatgt 2640tcacgccttc tattcccatt ggcgctgaac
tcttaaggtc actctggtcg cttgtgaccc 2700cgtaaccctg atgtacccct
ctaaaaggtg aggggc 2736942737DNAHomo sapiens 94tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg
gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg gcccggctgc
agcgagcgct ggacggagag 900gaaagcggga tgatgttaat attcttcttg
ataaagcaag attggaaaat caagaaggca 960ttgatttcat aaaggcaaca
aaagtactaa tggaaaaaaa ttcaatggat attatgaaaa 1020taagagagta
tttccagaag tatggatata gtccacgtgt caagaaaaat tcagtacacg
1080agcaagaagc cattaactct gacccagagt tgtctaattg tgaaaatttt
cagaagactg 1140atgtgaaaga tgatctgtct gatcctcctg ttgcaagcag
ttgtatttct gggaagtctc 1200cacgtagtcc acaactttca gattttggac
ttgagcggta catcgtatcc caagttctac 1260caaaccctcc acaggcagtg
aacaactata aggaagagcc cgtaattgta accccaccta 1320ccaaacaatc
actagtaaaa gtactaaaaa ctccaaaatg tgcactaaaa atggatgatt
1380ttgagtgtgt aactcctaaa ttagaacact ttggtatctc tgaatatact
atgtgtttaa 1440atgaagatta cacaatggga cttaaaaatg cgaggaataa
taaaagtgag gaggccatag 1500atacagaatc caggctcaat gataatgttt
ttgccactcc cagccccatc atccagcagt 1560tggaaaaaag tgatgccgaa
tataccaact ctcctttggt acctacattc tgtactcctg 1620gtttgaaaat
tccatctaca aagaacagca tagctttggt atccacaaat tacccattat
1680caaaaacaaa tagttcatca aatgatttgg aagttgaaga tcgtacttcg
ttggttttaa 1740attcagacac atgctttgag aatttaacag atccctcttc
acctacgatt tcttcttatg 1800agaatctgct cagaacacct acacctccag
aagtaactaa aattccagaa gatattctcc 1860agcttttatc aaaatacaac
tcaaacctag ctactccaat agcaattaaa gcagtgccac 1920ccagtaaaag
gttccttaaa catggacaga acatccgaga tgtcagcaac aaagaaaact
1980gaaattccag tggatctatc caacacagaa actgaacaaa atgagatgaa
agccgagctg 2040gaccgatttt aacattcaca ttgccctgcc tctgtccccc
tttaaacgtt gacccatttt 2100aaagacaaac atgaacatta acatcataat
atgcttttta tgaagtttca ataaggttta 2160accttagtct tgttgacatg
tagcccagtc attcactctt taaggactat tagtgtttca 2220ttgatactaa
attacccagc ttaatcaaca gaatggttta agtagtacca ggaagtagga
2280caagtaattt caaaaatata aaggtgtttg ctactcagat gaggccgccc
ctgaccttct 2340ggccagagag acattgctgc cagccagctc tgccttccca
tcatctcctt tcaggaccgt 2400cccacacctt ttacttgctc agtgctgtct
gaagatgcag ttgctgtttg caaacaacag 2460gaacaccagt taaactaatt
aggaaacaga gggagatttc caggcctggg taactatata 2520ctgtgaccat
tggcggttga gaccggtctt caaccagtgg aaccccgaac tctgctgtca
2580gggtgtggac ttcggtgctc ttccaagttt tcacctgggg gggggagcta
accccctatg 2640ttcacgcctt ctattcccat tggcgctgaa ctcttaaggt
cactctggtc gcttgtgacc 2700ccgtaaccct gatgtacccc tctaaaaggt gaggggc
2737952737DNAHomo sapiens 95tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagcggga tgatgttaat attcttcttg ataaagcaag
attggaaaat caagaaggca 960ttgatttcat aaaggcaaca aaagtactaa
tggaaaaaaa ttcaatggat attatgaaaa 1020taagagagta tttccagaag
tatggatata gtccacgtgt caagaaaaat tcagtacacg 1080agcaagaagc
cattaactct gacccagagt tgtctaattg tgaaaatttt cagaagactg
1140atgtgaaaga tgatctgtct gatcctcctg ttgcaagcag ttgtatttct
gggaagtctc 1200cacgtagtcc acaactttca gattttggac ttgagcggta
catcgtatcc caagttctac 1260caaaccctcc acaggcagtg aacaactata
aggaagagcc cgtaattgta accccaccta 1320ccaaacaatc actagtaaaa
gtactaaaaa ctccaaaatg tgcactaaaa atggatgatt 1380ttgagtgtgt
aactcctaaa ttagaacact ttggtatctc tgaatatact atgtgtttaa
1440atgaagatta cacaatggga cttaaaaatg cgaggaataa taaaagtgag
gaggccatag 1500atacagaatc caggctcaat gataatgttt ttgccactcc
cagccccatc atccagcagt 1560tggaaaaaag tgatgccgaa tataccaact
ctcctttggt acctacattc tgtactcctg 1620gtttgaaaat tccatctaca
aagaacagca tagctttggt atccacaaat tacccattat 1680caaaaacaaa
tagttcatca aatgatttgg aagttgaaga tcgtacttcg ttggttttaa
1740attcagacac atgctttgag aatttaacag atccctcttc acctacgatt
tcttcttatg 1800agaatctgct cagaacacct acacctccag aagtaactaa
aattccagaa gatattctcc 1860agcttttatc aaaatacaac tcaaacctag
ctactccaat agcaattaaa gcagtgccac 1920ccagtaaaag gttccttaaa
catggacaga acatccgaga tgtcagcaac aaagaaaact 1980gaaattccag
tggatctatc caacacagaa actgaacaaa atgagatgaa agccgagctg
2040gaccgatttt aacattcaca ttgccctgcc tctgtccccc tttaaacgtt
gacccatttt 2100aaagacaaac atgaacatta acatcataat atgcttttta
tgaagtttca ataaggttta 2160accttagtct tgttgacatg tagcccagtc
attcactctt taaggactat tagtgtttca 2220ttgatactaa attacccagc
ttaatcaaca gaatggttta agtagtacca ggaagtagga 2280caagtaattt
caaaaatata aaggtgtttg ctactcagat gaggccgccc ctgaccttct
2340ggccagagag acattgctgc cagccagctc tgccttccca tcatctcctt
tcaggaccgt 2400cccacacctt ttacttgctc agtgctgtct gaagatgcag
ttgctgtttg caaacaacag 2460gaacaccagt taaactaatt aggaaacaga
gggagatttc caggcctggg taactatata 2520ctgtgaccat tggcggttga
gaccggtctt caaccagtgg aaccccgaac tctgctgtca 2580gggtgtggac
ttcggtgctc ttccaagttt tcacctgggg gggggagcta accccctatg
2640ttcacgcctt ctattcccat tggcgctgaa ctcttaaggt cactctggtc
gcttgtgacc 2700ccgtaaccct gatgtacccc tctaaaaggt gaggggc
273796330PRTHomo sapiens 96Met Glu Lys Asn Ser Met Asp Ile Met Lys
Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val Lys
Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro Glu
Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp Asp
Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys Ser
Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn Asn
Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro Thr Lys 100 105 110Gln
Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met 115 120
125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly Ile Ser
130 135 140Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu
Lys Asn145 150 155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp
Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn Val Phe Ala Thr Pro Ser
Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu Tyr Thr
Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200 205Thr Pro Gly Leu Lys
Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val 210 215 220Ser Thr Asn
Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230 235
240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys Phe
245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr
Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys
Ile Pro Glu Asp 275 280 285Ile Leu Gln Leu Leu Ser Lys Tyr Asn Ser
Asn Leu Ala Thr Pro Ile 290 295 300Ala Ile Lys Ala Val Pro Pro Ser
Lys Arg Phe Leu Lys His Gly Gln305 310 315 320Asn Ile Arg Asp Val
Ser Asn Lys Glu Asn 325 33097330PRTHomo sapiens 97Met Glu Lys Asn
Ser Met Asp Ile Met Lys Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly
Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala
Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys
Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55
60Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65
70 75 80Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln
Ala 85 90 95Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro
Thr Lys 100 105 110Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys
Ala Leu Lys Met 115 120 125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu
Glu His Phe Gly Ile Ser 130 135 140Glu Tyr Thr Met Cys Leu Asn Glu
Asp Tyr Thr Met Gly Leu Lys Asn145 150 155 160Ala Arg Asn Asn Lys
Ser Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn
Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys
Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200
205Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val
210 215 220Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn
Asp Leu225 230 235 240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn
Ser Asp Thr Cys Phe 245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro
Thr Ile Ser Ser Tyr Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro
Pro Glu Val Thr Lys Ile Pro Glu Asp 275 280 285Ile Leu Gln Leu Leu
Ser Lys Tyr Asn Ser Asn Leu Ala Thr Pro Ile 290 295 300Ala Ile Lys
Ala Val Pro Pro Ser Lys Arg Phe Leu Lys His Gly Gln305 310 315
320Asn Ile Arg Asp Val Ser Asn Lys Glu Asn 325 33098330PRTHomo
sapiens 98Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys
Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro
Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro
Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg Tyr Ile Val Ser Gln
Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn Asn Tyr Lys Glu Glu
Pro Val Ile Val Thr Pro Pro Thr Lys 100 105 110Gln Ser Leu Val Lys
Val
Leu Lys Thr Pro Lys Cys Ala Leu Lys Met 115 120 125Asp Asp Phe Glu
Cys Val Thr Pro Lys Leu Glu His Phe Gly Ile Ser 130 135 140Glu Tyr
Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn145 150 155
160Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu
165 170 175Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln
Leu Glu 180 185 190Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val
Pro Thr Phe Cys 195 200 205Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys
Asn Ser Ile Ala Leu Val 210 215 220Ser Thr Asn Tyr Pro Leu Ser Lys
Thr Asn Ser Ser Ser Asn Asp Leu225 230 235 240Glu Val Glu Asp Arg
Thr Ser Leu Val Leu Asn Ser Asp Thr Cys Phe 245 250 255Glu Asn Leu
Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr Glu Asn 260 265 270Leu
Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro Glu Asp 275 280
285Ile Leu Gln Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala Thr Pro Ile
290 295 300Ala Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu Lys His
Gly Gln305 310 315 320Asn Ile Arg Asp Val Ser Asn Lys Glu Asn 325
330992679DNAHomo sapiens 99tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagcgact ttgaagatta tccaatgaga attttatatg
accttcattc agaagttcag 960actctaaagg atgatgttaa tattcttctt
gataaagcaa gattggaaaa tcaagaaggc 1020attgatttca taaaggcaac
aaaagtacta atggaaaaaa attcaatgga tattatgaaa 1080ataagagagt
atttccagaa gtatggatat agtccacgtg tcaagaaaaa ttcagtacac
1140gagcaagaag ccattaactc tgacccagag ttgtctaatt gtgaaaattt
tcagaagact 1200gatgtgaaag atgatctgtc tgatcctcct gttgcaagca
gttgtatttc tgggaagtct 1260ccacgtagtc cacaactttc agattttgga
cttgagcggt acatcgtatc ccaagttcta 1320ccaaaccctc cacaggcagt
gaacaactat aaggaagagc ccgtaattgt aaccccacct 1380accaaacaat
cactagtaaa agtactaaaa actccaaaat gtgcactaaa aatggatgat
1440tttgagtgtg taactcctaa attagaacac tttggtatct ctgaatatac
tatgtgttta 1500aatgaagatt acacaatggg acttaaaaat gcgaggaata
ataaaagtga ggaggccata 1560gatacagaat ccaggctcaa tgataatgtt
tttgccactc ccagccccat catccagcag 1620ttggaaaaaa gtgatgccga
atataccaac tctcctttgg tacctacatt ctgtactcct 1680ggtttgaaaa
ttccatctac aaagaacagc atagctttgg tatccacaaa ttacccatta
1740tcaaaaacaa atagttcatc aaatgatttg gaagttgaag atcgtacttc
gttggtttta 1800aattcagaca catgctttga gaatttaaca gatccctctt
cacctacgat ttcttcttat 1860gagaatctgc tcagaacacc tacacctcca
gaagtaacta aaattccaga agatattctc 1920cagaaattcc agtggatcta
tccaacacag aaactgaaca aaatgagatg aaagccgagc 1980tggaccgatt
ttaacattca cattgccctg cctctgtccc cctttaaacg ttgacccatt
2040ttaaagacaa acatgaacat taacatcata atatgctttt tatgaagttt
caataaggtt 2100taaccttagt cttgttgaca tgtagcccag tcattcactc
tttaaggact attagtgttt 2160cattgatact aaattaccca gcttaatcaa
cagaatggtt taagtagtac caggaagtag 2220gacaagtaat ttcaaaaata
taaaggtgtt tgctactcag atgaggccgc ccctgacctt 2280ctggccagag
agacattgct gccagccagc tctgccttcc catcatctcc tttcaggacc
2340gtcccacacc ttttacttgc tcagtgctgt ctgaagatgc agttgctgtt
tgcaaacaac 2400aggaacacca gttaaactaa ttaggaaaca gagggagatt
tccaggcctg ggtaactata 2460tactgtgacc attggcggtt gagaccggtc
ttcaaccagt ggaaccccga actctgctgt 2520cagggtgtgg acttcggtgc
tcttccaagt tttcacctgg gggggggagc taacccccta 2580tgttcacgcc
ttctattccc attggcgctg aactcttaag gtcactctgg tcgcttgtga
2640ccccgtaacc ctgatgtacc cctctaaaag gtgaggggc 26791002680DNAHomo
sapiens 100tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac
cacaggacag 60caattagact tttaagtatt ggggggttta gagctctaga tattcgatat
gcagactact 120catgtttgtt tgttttaata aagactggtc caaaggctca
ttttcacaca agctacagtt 180tttcagttcc aggaccaggt aaagatggtc
agctccgtga tccataaaat ccaagggtga 240cgactcagga ttaggaccat
ttcttggtga cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg
ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc
360tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg
ctcagccagt 420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt
ctgcttggcg cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc
actgacggcc gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg
gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac
gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga
660gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac
gcgtccggcg 720ccgagattca aactagtggc gggaggctgt gagctgagcg
gtggggtctg cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac
cctatccgga gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga
ctgcgagacg gcccggctgc agcgagcgct ggacggagag 900gaaagcgact
ttgaagatta tccaatgaga attttatatg accttcattc agaagttcag
960actctaaagg atgatgttaa tattcttctt gataaagcaa gattggaaaa
tcaagaaggc 1020attgatttca taaaggcaac aaaagtacta atggaaaaaa
attcaatgga tattatgaaa 1080ataagagagt atttccagaa gtatggatat
agtccacgtg tcaagaaaaa ttcagtacac 1140gagcaagaag ccattaactc
tgacccagag ttgtctaatt gtgaaaattt tcagaagact 1200gatgtgaaag
atgatctgtc tgatcctcct gttgcaagca gttgtatttc tgggaagtct
1260ccacgtagtc cacaactttc agattttgga cttgagcggt acatcgtatc
ccaagttcta 1320ccaaaccctc cacaggcagt gaacaactat aaggaagagc
ccgtaattgt aaccccacct 1380accaaacaat cactagtaaa agtactaaaa
actccaaaat gtgcactaaa aatggatgat 1440tttgagtgtg taactcctaa
attagaacac tttggtatct ctgaatatac tatgtgttta 1500aatgaagatt
acacaatggg acttaaaaat gcgaggaata ataaaagtga ggaggccata
1560gatacagaat ccaggctcaa tgataatgtt tttgccactc ccagccccat
catccagcag 1620ttggaaaaaa gtgatgccga atataccaac tctcctttgg
tacctacatt ctgtactcct 1680ggtttgaaaa ttccatctac aaagaacagc
atagctttgg tatccacaaa ttacccatta 1740tcaaaaacaa atagttcatc
aaatgatttg gaagttgaag atcgtacttc gttggtttta 1800aattcagaca
catgctttga gaatttaaca gatccctctt cacctacgat ttcttcttat
1860gagaatctgc tcagaacacc tacacctcca gaagtaacta aaattccaga
agatattctc 1920caggaaattc cagtggatct atccaacaca gaaactgaac
aaaatgagat gaaagccgag 1980ctggaccgat tttaacattc acattgccct
gcctctgtcc ccctttaaac gttgacccat 2040tttaaagaca aacatgaaca
ttaacatcat aatatgcttt ttatgaagtt tcaataaggt 2100ttaaccttag
tcttgttgac atgtagccca gtcattcact ctttaaggac tattagtgtt
2160tcattgatac taaattaccc agcttaatca acagaatggt ttaagtagta
ccaggaagta 2220ggacaagtaa tttcaaaaat ataaaggtgt ttgctactca
gatgaggccg cccctgacct 2280tctggccaga gagacattgc tgccagccag
ctctgccttc ccatcatctc ctttcaggac 2340cgtcccacac cttttacttg
ctcagtgctg tctgaagatg cagttgctgt ttgcaaacaa 2400caggaacacc
agttaaacta attaggaaac agagggagat ttccaggcct gggtaactat
2460atactgtgac cattggcggt tgagaccggt cttcaaccag tggaaccccg
aactctgctg 2520tcagggtgtg gacttcggtg ctcttccaag ttttcacctg
ggggggggag ctaaccccct 2580atgttcacgc cttctattcc cattggcgct
gaactcttaa ggtcactctg gtcgcttgtg 2640accccgtaac cctgatgtac
ccctctaaaa ggtgaggggc 26801012680DNAHomo sapiens 101tatcatctgt
gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac cctatccgga
gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg
gcccggctgc agcgagcgct ggacggagag 900gaaagcgact ttgaagatta
tccaatgaga attttatatg accttcattc agaagttcag 960actctaaagg
atgatgttaa tattcttctt gataaagcaa gattggaaaa tcaagaaggc
1020attgatttca taaaggcaac aaaagtacta atggaaaaaa attcaatgga
tattatgaaa 1080ataagagagt atttccagaa gtatggatat agtccacgtg
tcaagaaaaa ttcagtacac 1140gagcaagaag ccattaactc tgacccagag
ttgtctaatt gtgaaaattt tcagaagact 1200gatgtgaaag atgatctgtc
tgatcctcct gttgcaagca gttgtatttc tgggaagtct 1260ccacgtagtc
cacaactttc agattttgga cttgagcggt acatcgtatc ccaagttcta
1320ccaaaccctc cacaggcagt gaacaactat aaggaagagc ccgtaattgt
aaccccacct 1380accaaacaat cactagtaaa agtactaaaa actccaaaat
gtgcactaaa aatggatgat 1440tttgagtgtg taactcctaa attagaacac
tttggtatct ctgaatatac tatgtgttta 1500aatgaagatt acacaatggg
acttaaaaat gcgaggaata ataaaagtga ggaggccata 1560gatacagaat
ccaggctcaa tgataatgtt tttgccactc ccagccccat catccagcag
1620ttggaaaaaa gtgatgccga atataccaac tctcctttgg tacctacatt
ctgtactcct 1680ggtttgaaaa ttccatctac aaagaacagc atagctttgg
tatccacaaa ttacccatta 1740tcaaaaacaa atagttcatc aaatgatttg
gaagttgaag atcgtacttc gttggtttta 1800aattcagaca catgctttga
gaatttaaca gatccctctt cacctacgat ttcttcttat 1860gagaatctgc
tcagaacacc tacacctcca gaagtaacta aaattccaga agatattctc
1920caggaaattc cagtggatct atccaacaca gaaactgaac aaaatgagat
gaaagccgag 1980ctggaccgat tttaacattc acattgccct gcctctgtcc
ccctttaaac gttgacccat 2040tttaaagaca aacatgaaca ttaacatcat
aatatgcttt ttatgaagtt tcaataaggt 2100ttaaccttag tcttgttgac
atgtagccca gtcattcact ctttaaggac tattagtgtt 2160tcattgatac
taaattaccc agcttaatca acagaatggt ttaagtagta ccaggaagta
2220ggacaagtaa tttcaaaaat ataaaggtgt ttgctactca gatgaggccg
cccctgacct 2280tctggccaga gagacattgc tgccagccag ctctgccttc
ccatcatctc ctttcaggac 2340cgtcccacac cttttacttg ctcagtgctg
tctgaagatg cagttgctgt ttgcaaacaa 2400caggaacacc agttaaacta
attaggaaac agagggagat ttccaggcct gggtaactat 2460atactgtgac
cattggcggt tgagaccggt cttcaaccag tggaaccccg aactctgctg
2520tcagggtgtg gacttcggtg ctcttccaag ttttcacctg ggggggggag
ctaaccccct 2580atgttcacgc cttctattcc cattggcgct gaactcttaa
ggtcactctg gtcgcttgtg 2640accccgtaac cctgatgtac ccctctaaaa
ggtgaggggc 2680102388PRTHomo sapiens 102Met Asp Pro Ile Arg Ser Phe
Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr
Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu
Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln
Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu
Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val
Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90
95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His
100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys
Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp
Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Gly Lys Ser Pro Arg
Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile
Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn
Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln
Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys
Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215
220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly
Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile
Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro
Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr
Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu
Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser Thr
Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315 320Asp
Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330
335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr
340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys
Ile Pro 355 360 365Glu Asp Ile Leu Gln Lys Phe Gln Trp Ile Tyr Pro
Thr Gln Lys Leu 370 375 380Asn Lys Met Arg385103373PRTHomo sapiens
103Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1
5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly
Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp
Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu
Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile
Lys Ala Thr Lys65 70 75 80Val Leu Met Glu Lys Asn Ser Met Asp Ile
Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg
Val Lys Lys Asn Ser Val His 100 105 110Glu Gln Glu Ala Ile Asn Ser
Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp
Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala 130 135 140Ser Ser Cys
Ile Ser Gly Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp145 150 155
160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro Pro
165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr
Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val Leu Lys Thr Pro
Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu Cys Val Thr Pro
Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr Thr Met Cys Leu
Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys Asn Ala Arg Asn
Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn
Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu
Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280
285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala
290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser
Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val
Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser
Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro
Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln
370104373PRTHomo sapiens 104Met Asp Pro Ile Arg Ser Phe Cys Gly Lys
Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu
Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Pro
Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu Val Gln Thr Leu Lys
Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala Arg Leu Glu Asn Gln
Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75 80Val Leu Met Glu
Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr 85 90 95Phe Gln Lys
Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His 100 105 110Glu
Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn 115 120
125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala
130 135 140Ser Ser Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro Gln Leu
Ser
Asp145 150 155 160Phe Gly Leu Glu Arg Tyr Ile Val Ser Gln Val Leu
Pro Asn Pro Pro 165 170 175Gln Ala Val Asn Asn Tyr Lys Glu Glu Pro
Val Ile Val Thr Pro Pro 180 185 190Thr Lys Gln Ser Leu Val Lys Val
Leu Lys Thr Pro Lys Cys Ala Leu 195 200 205Lys Met Asp Asp Phe Glu
Cys Val Thr Pro Lys Leu Glu His Phe Gly 210 215 220Ile Ser Glu Tyr
Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu225 230 235 240Lys
Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser 245 250
255Arg Leu Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln
260 265 270Leu Glu Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val
Pro Thr 275 280 285Phe Cys Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys
Asn Ser Ile Ala 290 295 300Leu Val Ser Thr Asn Tyr Pro Leu Ser Lys
Thr Asn Ser Ser Ser Asn305 310 315 320Asp Leu Glu Val Glu Asp Arg
Thr Ser Leu Val Leu Asn Ser Asp Thr 325 330 335Cys Phe Glu Asn Leu
Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr 340 345 350Glu Asn Leu
Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro 355 360 365Glu
Asp Ile Leu Gln 3701052617DNAHomo sapiens 105tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg
gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg gcccggctgc
agcgagcgct ggacggagag 900gaaagcggat gatgttaata ttcttcttga
taaagcaaga ttggaaaatc aagaaggcat 960tgatttcata aaggcaacaa
aagtactaat ggaaaaaaat tcaatggata ttatgaaaat 1020aagagagtat
ttccagaagt atggatatag tccacgtgtc aagaaaaatt cagtacacga
1080gcaagaagcc attaactctg acccagagtt gtctaattgt gaaaattttc
agaagactga 1140tgtgaaagat gatctgtctg atcctcctgt tgcaagcagt
tgtatttctg ggaagtctcc 1200acgtagtcca caactttcag attttggact
tgagcggtac atcgtatccc aagttctacc 1260aaaccctcca caggcagtga
acaactataa ggaagagccc gtaattgtaa ccccacctac 1320caaacaatca
ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa tggatgattt
1380tgagtgtgta actcctaaat tagaacactt tggtatctct gaatatacta
tgtgtttaaa 1440tgaagattac acaatgggac ttaaaaatgc gaggaataat
aaaagtgagg aggccataga 1500tacagaatcc aggctcaatg ataatgtttt
tgccactccc agccccatca tccagcagtt 1560ggaaaaaagt gatgccgaat
ataccaactc tcctttggta cctacattct gtactcctgg 1620tttgaaaatt
ccatctacaa agaacagcat agctttggta tccacaaatt acccattatc
1680aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgt
tggttttaaa 1740ttcagacaca tgctttgaga atttaacaga tccctcttca
cctacgattt cttcttatga 1800gaatctgctc agaacaccta cacctccaga
agtaactaaa attccagaag atattctcca 1860gaaattccag tggatctatc
caacacagaa actgaacaaa atgagatgaa agccgagctg 1920gaccgatttt
aacattcaca ttgccctgcc tctgtccccc tttaaacgtt gacccatttt
1980aaagacaaac atgaacatta acatcataat atgcttttta tgaagtttca
ataaggttta 2040accttagtct tgttgacatg tagcccagtc attcactctt
taaggactat tagtgtttca 2100ttgatactaa attacccagc ttaatcaaca
gaatggttta agtagtacca ggaagtagga 2160caagtaattt caaaaatata
aaggtgtttg ctactcagat gaggccgccc ctgaccttct 2220ggccagagag
acattgctgc cagccagctc tgccttccca tcatctcctt tcaggaccgt
2280cccacacctt ttacttgctc agtgctgtct gaagatgcag ttgctgtttg
caaacaacag 2340gaacaccagt taaactaatt aggaaacaga gggagatttc
caggcctggg taactatata 2400ctgtgaccat tggcggttga gaccggtctt
caaccagtgg aaccccgaac tctgctgtca 2460gggtgtggac ttcggtgctc
ttccaagttt tcacctgggg gggggagcta accccctatg 2520ttcacgcctt
ctattcccat tggcgctgaa ctcttaaggt cactctggtc gcttgtgacc
2580ccgtaaccct gatgtacccc tctaaaaggt gaggggc 26171062619DNAHomo
sapiens 106tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac
cacaggacag 60caattagact tttaagtatt ggggggttta gagctctaga tattcgatat
gcagactact 120catgtttgtt tgttttaata aagactggtc caaaggctca
ttttcacaca agctacagtt 180tttcagttcc aggaccaggt aaagatggtc
agctccgtga tccataaaat ccaagggtga 240cgactcagga ttaggaccat
ttcttggtga cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg
ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc
360tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg
ctcagccagt 420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt
ctgcttggcg cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc
actgacggcc gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg
gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac
gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga
660gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac
gcgtccggcg 720ccgagattca aactagtggc gggaggctgt gagctgagcg
gtggggtctg cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac
cctatccgga gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga
ctgcgagacg gcccggctgc agcgagcgct ggacggagag 900gaaagcggga
tgatgttaat attcttcttg ataaagcaag attggaaaat caagaaggca
960ttgatttcat aaaggcaaca aaagtactaa tggaaaaaaa ttcaatggat
attatgaaaa 1020taagagagta tttccagaag tatggatata gtccacgtgt
caagaaaaat tcagtacacg 1080agcaagaagc cattaactct gacccagagt
tgtctaattg tgaaaatttt cagaagactg 1140atgtgaaaga tgatctgtct
gatcctcctg ttgcaagcag ttgtatttct gggaagtctc 1200cacgtagtcc
acaactttca gattttggac ttgagcggta catcgtatcc caagttctac
1260caaaccctcc acaggcagtg aacaactata aggaagagcc cgtaattgta
accccaccta 1320ccaaacaatc actagtaaaa gtactaaaaa ctccaaaatg
tgcactaaaa atggatgatt 1380ttgagtgtgt aactcctaaa ttagaacact
ttggtatctc tgaatatact atgtgtttaa 1440atgaagatta cacaatggga
cttaaaaatg cgaggaataa taaaagtgag gaggccatag 1500atacagaatc
caggctcaat gataatgttt ttgccactcc cagccccatc atccagcagt
1560tggaaaaaag tgatgccgaa tataccaact ctcctttggt acctacattc
tgtactcctg 1620gtttgaaaat tccatctaca aagaacagca tagctttggt
atccacaaat tacccattat 1680caaaaacaaa tagttcatca aatgatttgg
aagttgaaga tcgtacttcg ttggttttaa 1740attcagacac atgctttgag
aatttaacag atccctcttc acctacgatt tcttcttatg 1800agaatctgct
cagaacacct acacctccag aagtaactaa aattccagaa gatattctcc
1860aggaaattcc agtggatcta tccaacacag aaactgaaca aaatgagatg
aaagccgagc 1920tggaccgatt ttaacattca cattgccctg cctctgtccc
cctttaaacg ttgacccatt 1980ttaaagacaa acatgaacat taacatcata
atatgctttt tatgaagttt caataaggtt 2040taaccttagt cttgttgaca
tgtagcccag tcattcactc tttaaggact attagtgttt 2100cattgatact
aaattaccca gcttaatcaa cagaatggtt taagtagtac caggaagtag
2160gacaagtaat ttcaaaaata taaaggtgtt tgctactcag atgaggccgc
ccctgacctt 2220ctggccagag agacattgct gccagccagc tctgccttcc
catcatctcc tttcaggacc 2280gtcccacacc ttttacttgc tcagtgctgt
ctgaagatgc agttgctgtt tgcaaacaac 2340aggaacacca gttaaactaa
ttaggaaaca gagggagatt tccaggcctg ggtaactata 2400tactgtgacc
attggcggtt gagaccggtc ttcaaccagt ggaaccccga actctgctgt
2460cagggtgtgg acttcggtgc tcttccaagt tttcacctgg gggggggagc
taacccccta 2520tgttcacgcc ttctattccc attggcgctg aactcttaag
gtcactctgg tcgcttgtga 2580ccccgtaacc ctgatgtacc cctctaaaag
gtgaggggc 26191072619DNAHomo sapiens 107tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg
gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg gcccggctgc
agcgagcgct ggacggagag 900gaaagcggga tgatgttaat attcttcttg
ataaagcaag attggaaaat caagaaggca 960ttgatttcat aaaggcaaca
aaagtactaa tggaaaaaaa ttcaatggat attatgaaaa 1020taagagagta
tttccagaag tatggatata gtccacgtgt caagaaaaat tcagtacacg
1080agcaagaagc cattaactct gacccagagt tgtctaattg tgaaaatttt
cagaagactg 1140atgtgaaaga tgatctgtct gatcctcctg ttgcaagcag
ttgtatttct gggaagtctc 1200cacgtagtcc acaactttca gattttggac
ttgagcggta catcgtatcc caagttctac 1260caaaccctcc acaggcagtg
aacaactata aggaagagcc cgtaattgta accccaccta 1320ccaaacaatc
actagtaaaa gtactaaaaa ctccaaaatg tgcactaaaa atggatgatt
1380ttgagtgtgt aactcctaaa ttagaacact ttggtatctc tgaatatact
atgtgtttaa 1440atgaagatta cacaatggga cttaaaaatg cgaggaataa
taaaagtgag gaggccatag 1500atacagaatc caggctcaat gataatgttt
ttgccactcc cagccccatc atccagcagt 1560tggaaaaaag tgatgccgaa
tataccaact ctcctttggt acctacattc tgtactcctg 1620gtttgaaaat
tccatctaca aagaacagca tagctttggt atccacaaat tacccattat
1680caaaaacaaa tagttcatca aatgatttgg aagttgaaga tcgtacttcg
ttggttttaa 1740attcagacac atgctttgag aatttaacag atccctcttc
acctacgatt tcttcttatg 1800agaatctgct cagaacacct acacctccag
aagtaactaa aattccagaa gatattctcc 1860aggaaattcc agtggatcta
tccaacacag aaactgaaca aaatgagatg aaagccgagc 1920tggaccgatt
ttaacattca cattgccctg cctctgtccc cctttaaacg ttgacccatt
1980ttaaagacaa acatgaacat taacatcata atatgctttt tatgaagttt
caataaggtt 2040taaccttagt cttgttgaca tgtagcccag tcattcactc
tttaaggact attagtgttt 2100cattgatact aaattaccca gcttaatcaa
cagaatggtt taagtagtac caggaagtag 2160gacaagtaat ttcaaaaata
taaaggtgtt tgctactcag atgaggccgc ccctgacctt 2220ctggccagag
agacattgct gccagccagc tctgccttcc catcatctcc tttcaggacc
2280gtcccacacc ttttacttgc tcagtgctgt ctgaagatgc agttgctgtt
tgcaaacaac 2340aggaacacca gttaaactaa ttaggaaaca gagggagatt
tccaggcctg ggtaactata 2400tactgtgacc attggcggtt gagaccggtc
ttcaaccagt ggaaccccga actctgctgt 2460cagggtgtgg acttcggtgc
tcttccaagt tttcacctgg gggggggagc taacccccta 2520tgttcacgcc
ttctattccc attggcgctg aactcttaag gtcactctgg tcgcttgtga
2580ccccgtaacc ctgatgtacc cctctaaaag gtgaggggc 2619108306PRTHomo
sapiens 108Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys
Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro
Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro
Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg Tyr Ile Val Ser Gln
Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn Asn Tyr Lys Glu Glu
Pro Val Ile Val Thr Pro Pro Thr Lys 100 105 110Gln Ser Leu Val Lys
Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met 115 120 125Asp Asp Phe
Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly Ile Ser 130 135 140Glu
Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn145 150
155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser Arg
Leu 165 170 175Asn Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln
Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu
Val Pro Thr Phe Cys 195 200 205Thr Pro Gly Leu Lys Ile Pro Ser Thr
Lys Asn Ser Ile Ala Leu Val 210 215 220Ser Thr Asn Tyr Pro Leu Ser
Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230 235 240Glu Val Glu Asp
Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys Phe 245 250 255Glu Asn
Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr Glu Asn 260 265
270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys Ile Pro Glu Asp
275 280 285Ile Leu Gln Lys Phe Gln Trp Ile Tyr Pro Thr Gln Lys Leu
Asn Lys 290 295 300Met Arg305109291PRTHomo sapiens 109Met Glu Lys
Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr
Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val His Glu Gln 20 25 30Glu
Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn Cys Glu Asn Phe Gln 35 40
45Lys Thr Asp Val Lys Asp Asp Leu Ser Asp Pro Pro Val Ala Ser Ser
50 55 60Cys Ile Ser Gly Lys Ser Pro Arg Ser Pro Gln Leu Ser Asp Phe
Gly65 70 75 80Leu Glu Arg Tyr Ile Val Ser Gln Val Leu Pro Asn Pro
Pro Gln Ala 85 90 95Val Asn Asn Tyr Lys Glu Glu Pro Val Ile Val Thr
Pro Pro Thr Lys 100 105 110Gln Ser Leu Val Lys Val Leu Lys Thr Pro
Lys Cys Ala Leu Lys Met 115 120 125Asp Asp Phe Glu Cys Val Thr Pro
Lys Leu Glu His Phe Gly Ile Ser 130 135 140Glu Tyr Thr Met Cys Leu
Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn145 150 155 160Ala Arg Asn
Asn Lys Ser Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu 165 170 175Asn
Asp Asn Val Phe Ala Thr Pro Ser Pro Ile Ile Gln Gln Leu Glu 180 185
190Lys Ser Asp Ala Glu Tyr Thr Asn Ser Pro Leu Val Pro Thr Phe Cys
195 200 205Thr Pro Gly Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala
Leu Val 210 215 220Ser Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser
Ser Asn Asp Leu225 230 235 240Glu Val Glu Asp Arg Thr Ser Leu Val
Leu Asn Ser Asp Thr Cys Phe 245 250 255Glu Asn Leu Thr Asp Pro Ser
Ser Pro Thr Ile Ser Ser Tyr Glu Asn 260 265 270Leu Leu Arg Thr Pro
Thr Pro Pro Glu Val Thr Lys Ile Pro Glu Asp 275 280 285Ile Leu Gln
290110291PRTHomo sapiens 110Met Glu Lys Asn Ser Met Asp Ile Met Lys
Ile Arg Glu Tyr Phe Gln1 5 10 15Lys Tyr Gly Tyr Ser Pro Arg Val Lys
Lys Asn Ser Val His Glu Gln 20 25 30Glu Ala Ile Asn Ser Asp Pro Glu
Leu Ser Asn Cys Glu Asn Phe Gln 35 40 45Lys Thr Asp Val Lys Asp Asp
Leu Ser Asp Pro Pro Val Ala Ser Ser 50 55 60Cys Ile Ser Gly Lys Ser
Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly65 70 75 80Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro Gln Ala 85 90 95Val Asn Asn
Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro Thr Lys 100 105 110Gln
Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu Lys Met 115 120
125Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly Ile Ser
130 135 140Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met Gly Leu
Lys Asn145 150 155 160Ala Arg Asn Asn Lys Ser Glu Glu Ala Ile Asp
Thr Glu Ser Arg Leu 165 170 175Asn Asp Asn Val Phe Ala Thr Pro Ser
Pro Ile Ile Gln Gln Leu Glu 180 185 190Lys Ser Asp Ala Glu Tyr Thr
Asn Ser Pro Leu Val Pro Thr Phe Cys 195 200 205Thr Pro Gly Leu Lys
Ile Pro Ser Thr Lys Asn Ser Ile Ala Leu Val 210 215 220Ser Thr Asn
Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu225 230 235
240Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr Cys Phe
245 250 255Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser Ser Tyr
Glu Asn 260 265 270Leu Leu Arg Thr Pro Thr Pro Pro Glu Val Thr Lys
Ile Pro Glu Asp 275 280 285Ile Leu Gln 2901111781DNAHomo sapiens
111tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac
cacaggacag 60caattagact tttaagtatt ggggggttta gagctctaga tattcgatat
gcagactact 120catgtttgtt tgttttaata aagactggtc caaaggctca
ttttcacaca agctacagtt 180tttcagttcc aggaccaggt aaagatggtc
agctccgtga tccataaaat ccaagggtga 240cgactcagga ttaggaccat
ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg
gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg gcccggctgc
agcgagcgct ggacggagag 900gaaagccttt tatcaaaata caactcaaac
ctagctactc caatagcaat taaagcagtg 960ccacccagta aaaggttcct
taaacatgga cagaacatcc gagatgtcag caacaaagaa 1020aactgaaatt
ccagtggatc tatccaacac agaaactgaa caaaatgaga tgaaagccga
1080gctggaccga ttttaacatt cacattgccc tgcctctgtc cccctttaaa
cgttgaccca 1140ttttaaagac aaacatgaac attaacatca taatatgctt
tttatgaagt ttcaataagg 1200tttaacctta gtcttgttga catgtagccc
agtcattcac tctttaagga ctattagtgt 1260ttcattgata ctaaattacc
cagcttaatc aacagaatgg tttaagtagt accaggaagt 1320aggacaagta
atttcaaaaa tataaaggtg tttgctactc agatgaggcc gcccctgacc
1380ttctggccag agagacattg ctgccagcca gctctgcctt cccatcatct
cctttcagga 1440ccgtcccaca ccttttactt gctcagtgct gtctgaagat
gcagttgctg tttgcaaaca 1500acaggaacac cagttaaact aattaggaaa
cagagggaga tttccaggcc tgggtaacta 1560tatactgtga ccattggcgg
ttgagaccgg tcttcaacca gtggaacccc gaactctgct 1620gtcagggtgt
ggacttcggt gctcttccaa gttttcacct ggggggggga gctaaccccc
1680tatgttcacg ccttctattc ccattggcgc tgaactctta aggtcactct
ggtcgcttgt 1740gaccccgtaa ccctgatgta cccctctaaa aggtgagggg c
17811121781DNAHomo sapiens 112tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagccttt tatcaaaata caactcaaac ctagctactc
caatagcaat taaagcagtg 960ccacccagta aaaggttcct taaacatgga
cagaacatcc gagatgtcag caacaaagaa 1020aactgaaatt ccagtggatc
tatccaacac agaaactgaa caaaatgaga tgaaagccga 1080gctggaccga
ttttaacatt cacattgccc tgcctctgtc cccctttaaa cgttgaccca
1140ttttaaagac aaacatgaac attaacatca taatatgctt tttatgaagt
ttcaataagg 1200tttaacctta gtcttgttga catgtagccc agtcattcac
tctttaagga ctattagtgt 1260ttcattgata ctaaattacc cagcttaatc
aacagaatgg tttaagtagt accaggaagt 1320aggacaagta atttcaaaaa
tataaaggtg tttgctactc agatgaggcc gcccctgacc 1380ttctggccag
agagacattg ctgccagcca gctctgcctt cccatcatct cctttcagga
1440ccgtcccaca ccttttactt gctcagtgct gtctgaagat gcagttgctg
tttgcaaaca 1500acaggaacac cagttaaact aattaggaaa cagagggaga
tttccaggcc tgggtaacta 1560tatactgtga ccattggcgg ttgagaccgg
tcttcaacca gtggaacccc gaactctgct 1620gtcagggtgt ggacttcggt
gctcttccaa gttttcacct ggggggggga gctaaccccc 1680tatgttcacg
ccttctattc ccattggcgc tgaactctta aggtcactct ggtcgcttgt
1740gaccccgtaa ccctgatgta cccctctaaa aggtgagggg c
17811131781DNAHomo sapiens 113tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagccttt tatcaaaata caactcaaac ctagctactc
caatagcaat taaagcagtg 960ccacccagta aaaggttcct taaacatgga
cagaacatcc gagatgtcag caacaaagaa 1020aactgaaatt ccagtggatc
tatccaacac agaaactgaa caaaatgaga tgaaagccga 1080gctggaccga
ttttaacatt cacattgccc tgcctctgtc cccctttaaa cgttgaccca
1140ttttaaagac aaacatgaac attaacatca taatatgctt tttatgaagt
ttcaataagg 1200tttaacctta gtcttgttga catgtagccc agtcattcac
tctttaagga ctattagtgt 1260ttcattgata ctaaattacc cagcttaatc
aacagaatgg tttaagtagt accaggaagt 1320aggacaagta atttcaaaaa
tataaaggtg tttgctactc agatgaggcc gcccctgacc 1380ttctggccag
agagacattg ctgccagcca gctctgcctt cccatcatct cctttcagga
1440ccgtcccaca ccttttactt gctcagtgct gtctgaagat gcagttgctg
tttgcaaaca 1500acaggaacac cagttaaact aattaggaaa cagagggaga
tttccaggcc tgggtaacta 1560tatactgtga ccattggcgg ttgagaccgg
tcttcaacca gtggaacccc gaactctgct 1620gtcagggtgt ggacttcggt
gctcttccaa gttttcacct ggggggggga gctaaccccc 1680tatgttcacg
ccttctattc ccattggcgc tgaactctta aggtcactct ggtcgcttgt
1740gaccccgtaa ccctgatgta cccctctaaa aggtgagggg c 178111473PRTHomo
sapiens 114Met Asp Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu
Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu
Asp Gly Glu 20 25 30Glu Ser Leu Leu Ser Lys Tyr Asn Ser Asn Leu Ala
Thr Pro Ile Ala 35 40 45Ile Lys Ala Val Pro Pro Ser Lys Arg Phe Leu
Lys His Gly Gln Asn 50 55 60Ile Arg Asp Val Ser Asn Lys Glu Asn65
7011577PRTHomo sapiens 115Met Asp Pro Ile Arg Ser Phe Cys Gly Lys
Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys Glu Thr Ala Arg Leu
Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp Phe Glu Asp Tyr Leu
Leu Ser Lys Tyr Asn Ser Asn Leu 35 40 45Ala Thr Pro Ile Ala Ile Lys
Ala Val Pro Pro Ser Lys Arg Phe Leu 50 55 60Lys His Gly Gln Asn Ile
Arg Asp Val Ser Asn Lys Glu65 70 7511678PRTHomo sapiens 116Met Asp
Pro Ile Arg Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr
Leu Asp Cys Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25
30Glu Ser Leu Leu Ser Lys Tyr Leu Leu Ser Lys Tyr Asn Ser Asn Leu
35 40 45Ala Thr Pro Ile Ala Ile Lys Ala Val Pro Pro Ser Lys Arg Phe
Leu 50 55 60Lys His Gly Gln Asn Ile Arg Asp Val Ser Asn Lys Glu
Asn65 70 751172825DNAHomo sapiens 117tatcatctgt gactgaggaa
atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt
ggggggttta gagctctaga tattcgatat gcagactact 120catgtttgtt
tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt
180tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat
ccaagggtga 240cgactcagga ttaggaccat ttcttggtga cattgagatg
gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg
ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg
cgctcctgct cccgggtcat gtagggcatg ctcagccagt 420aatggttctc
cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca
480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg
ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa
gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc
gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag
agaggactgc gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca
aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg
780gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg
gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg gcccggctgc
agcgagcgct ggacggagag 900gaaagcggtg cgtgaggcgg gcggccaggg
cacgactttg aagattatcc aatgagaatt 960ttatatgacc ttcattcaga
agttcagact ctaaaggatg atgttaatat tcttcttgat 1020aaagcaagat
tggaaaatca agaaggcatt gatttcataa aggcaacaaa agtactaatg
1080gaaaaaaatt caatggatat tatgaaaata agagagtatt tccagaagta
tggatatagt 1140ccacgtgtca agaaaaattc agtacacgag caagaagcca
ttaactctga cccagagttg 1200tctaattgtg aaaattttca gaagactgat
gtgaaagatg atctgtctga tcctcctgtt 1260gcaagcagtt gtatttctgg
gaagtctcca cgtagtccac aactttcaga ttttggactt 1320gagcggtaca
tcgtatccca agttctacca aaccctccac aggcagtgaa caactataag
1380gaagagcccg taattgtaac cccacctacc aaacaatcac tagtaaaagt
actaaaaact 1440ccaaaatgtg cactaaaaat ggatgatttt gagtgtgtaa
ctcctaaatt agaacacttt 1500ggtatctctg aatatactat gtgtttaaat
gaagattaca caatgggact taaaaatgcg 1560aggaataata aaagtgagga
ggccatagat acagaatcca ggctcaatga taatgttttt 1620gccactccca
gccccatcat ccagcagttg gaaaaaagtg atgccgaata taccaactct
1680cctttggtac ctacattctg tactcctggt ttgaaaattc catctacaaa
gaacagcata 1740gctttggtat ccacaaatta cccattatca aaaacaaata
gttcatcaaa tgatttggaa 1800gttgaagatc gtacttcgtt ggttttaaat
tcagacacat gctttgagaa tttaacagat 1860ccctcttcac ctacgatttc
ttcttatgag aatctgctca gaacacctac acctccagaa 1920gtaactaaaa
ttccagaaga tattctccag cttttatcaa aatacaactc aaacctagct
1980actccaatag caattaaagc agtgccaccc agtaaaaggt tccttaaaca
tggacagaac 2040atccgagatg tcagcaacaa agaaaactga aattccagtg
gatctatcca acacagaaac 2100tgaacaaaat gagatgaaag ccgagctgga
ccgattttaa cattcacatt gccctgcctc 2160tgtccccctt taaacgttga
cccattttaa agacaaacat gaacattaac atcataatat 2220gctttttatg
aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat
2280tcactcttta aggactatta gtgtttcatt gatactaaat tacccagctt
aatcaacaga 2340atggtttaag tagtaccagg aagtaggaca agtaatttca
aaaatataaa ggtgtttgct 2400actcagatga ggccgcccct gaccttctgg
ccagagagac attgctgcca gccagctctg 2460ccttcccatc atctcctttc
aggaccgtcc cacacctttt acttgctcag tgctgtctga 2520agatgcagtt
gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg
2580gagatttcca ggcctgggta actatatact gtgaccattg gcggttgaga
ccggtcttca 2640accagtggaa ccccgaactc tgctgtcagg gtgtggactt
cggtgctctt ccaagttttc 2700acctgggggg gggagctaac cccctatgtt
cacgccttct attcccattg gcgctgaact 2760cttaaggtca ctctggtcgc
ttgtgacccc gtaaccctga tgtacccctc taaaaggtga 2820ggggc
28251183707DNAHomo sapiens 118tatcatctgt gactgaggaa atccctatct
tcctatcaga ctaatgaaac cacaggacag 60caattagact tttaagtatt ggggggttta
gagctctaga tattcgatat gcagactact 120catgtttgtt tgttttaata
aagactggtc caaaggctca ttttcacaca agctacagtt 180tttcagttcc
aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga
240cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg
tccgcaatga 300atctatgcgg ggggaacttg gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc 360tgcgcaccgc ggcgtggccg cgctcctgct
cccgggtcat gtagggcatg ctcagccagt 420aatggttctc cgcctcgatc
tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca 480cgaaccgcgg
ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
540ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc 600gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga 660gagacttcgg ctctcgcgag agaggactgc
gcctgcgcag agccgaggac gcgtccggcg 720ccgagattca aactagtggc
gggaggctgt gagctgagcg gtggggtctg cgtacgcctg 780gagtccttcc
ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg
840tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
ggacggagag 900gaaagcgcga ctttgaagat tatccaatga gaattttata
tgaccttcat tcagaagttc 960agactctaaa ggatgatgtt aatattcttc
ttgataaagc aagattggaa aatcaagaag 1020gcattgattt cataaaggca
acaaaagtac taatggaaaa aaattcaatg gatattatga 1080aaataagaga
gtatttccag aagtatggat atagtccacg tgtcaagaaa aattcagtac
1140acgagcaaga agccattaac tctgacccag agttgtctaa ttgtgaaaat
tttcagaaga 1200ctgatgtgaa agatgatctg tctgatcctc ctgttgcaag
cagttgtatt tctgggaagt 1260ctccacgtag tccacaactt tcagattttg
gacttgagcg gtacatcgta tcccaagttc 1320taccaaaccc tccacaggca
gtgaacaact ataaggaaga gcccgtaatt gtaaccccac 1380ctaccaaaca
atcactagta aaagtactaa aaactccaaa atgtgcacta aaaatggatg
1440attttgagtg tgtaactcct aaattagaac actttggtat ctctgaatat
actatgtgtt 1500taaatgaaga ttacacaatg ggacttaaaa atgcgaggaa
taataaaagt gaggaggcca 1560tagatacaga atccaggctc aatgataatg
tttttgccac tcccagcccc atcatccagc 1620agttggaaaa aagtgatgcc
gaatatacca actctccttt ggtacctaca ttctgtactc 1680ctggtttgaa
aattccatct acaaagaaca gcatagcttt ggtatccaca aattacccat
1740tatcaaaaac aaatagttca tcaaatgatt tggaagttga agatcgtact
tcgttggttt 1800taaattcaga cacatgcttt gagaatttaa cagatccctc
ttcacctacg atttcttctt 1860atgagaatct gctcagaaca cctacacctc
cagaagtaac taaaattcca gaagatattc 1920tccagctttt atcaaaatac
aactcaaacc tagctactcc aatagcaatt aaagcagtgc 1980cacccagtaa
aaggttcctt aaacatggac agaacatccg agatgtcagc aacaaagaaa
2040actgaaattc cagtggatct atccaacaca gaaactgaac aaaatgagat
gaaagccgag 2100ctggaccgat tttaacattc acattgccct gcctctgtcc
ccctttaaac gttgacccat 2160tttaaagaca aacatgaaca ttaacatcat
aatatgcttt ttatgaagtt tcaataaggt 2220ttaaccttag tcttgttgac
atgtagccca gtcattcact ctttaaggac tattagtgtt 2280tcattgatac
taaattaccc agcttaatca acagaatggt ttaagtagta ccaggaagta
2340ggacaagtaa tttcaaaaat ataaaggtgt ttgctactca gatgaggccg
cccctgacct 2400tctggccaga gagacattgc tgccagccag ctctgccttc
ccatcatctc ctttcaggac 2460cgtcccacac cttttacttg ctcagtgctg
tctgaagatg cagttgctgt ttgcaaacaa 2520caggaacacc agttaaacta
attaggaaac agagggagat ttccaggcct gggtaactat 2580atactgtgac
cattggcggt tgagaccggt cttcaaccag tggaaccccg aactctgctg
2640tcagggtgtg gacttcggtg ctcttccaag ttttcacctg ggggggggag
ctaaccccct 2700atgttcacgc cttctattcc cattggcgct gaactcttaa
ggtcactctg gtcgcttgtg 2760accccgtaac cctgatgtac ccctctaaaa
ggtgaggggc tatcatctgt gactgaggaa 2820atccctatct tcctatcaga
ctaatgaaac cacaggacag caattagact tttaagtatt 2880ggggggttta
gagctctaga tattcgatat gcagactact catgtttgtt tgttttaata
2940aagactggtc caaaggctca ttttcacaca agctacagtt tttcagttcc
aggaccaggt 3000aaagatggtc agctccgtga tccataaaat ccaagggtga
cgactcagga ttaggaccat 3060ttcttggtga cattgagatg gtcgagctgg
tccgcaatga atctatgcgg ggggaacttg 3120gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc tgcgcaccgc ggcgtggccg 3180cgctcctgct
cccgggtcat gtagggcatg ctcagccagt aatggttctc cgcctcgatc
3240tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca cgaaccgcgg
ccgccggtgc 3300ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
ggagcgcagt caggaacatg 3360gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc gccagcagac gccgtggcgt 3420aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga gagacttcgg ctctcgcgag 3480agaggactgc
gcctgcgcag agccgaggac gcgtccggcg ccgagattca aactagtggc
3540gggaggctgt gagctgagcg gtggggtctg cgtacgcctg gagtccttcc
ccgctgtgct 3600cagcatggac cctatccgga gcttctgcgg gaagctgcgg
tctctggcca gcacgctgga 3660ctgcgagacg gcccggctgc agcgagcgct
ggacggagag gaaagcg 37071193707DNAHomo sapiens 119tatcatctgt
gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag 60caattagact
tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact
120catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca
agctacagtt 180tttcagttcc aggaccaggt aaagatggtc agctccgtga
tccataaaat ccaagggtga 240cgactcagga ttaggaccat ttcttggtga
cattgagatg gtcgagctgg tccgcaatga 300atctatgcgg ggggaacttg
gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc 360tgcgcaccgc
ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt
420aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg
cgcaacgaca 480cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc
gggaatgcgg ccgcgccaga 540ggagcgcagt caggaacatg gtgcctgccg
cgctgctcaa gactctgcgt ctccgcggcc 600gccagcagac gccgtggcgt
aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga 660gagacttcgg
ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg
720ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg
cgtacgcctg 780gagtccttcc ccgctgtgct cagcatggac cctatccgga
gcttctgcgg gaagctgcgg 840tctctggcca gcacgctgga ctgcgagacg
gcccggctgc agcgagcgct ggacggagag 900gaaagcgcga ctttgaagat
tatccaatga gaattttata tgaccttcat tcagaagttc 960agactctaaa
ggatgatgtt aatattcttc ttgataaagc aagattggaa aatcaagaag
1020gcattgattt cataaaggca acaaaagtac taatggaaaa aaattcaatg
gatattatga 1080aaataagaga gtatttccag aagtatggat atagtccacg
tgtcaagaaa aattcagtac 1140acgagcaaga agccattaac tctgacccag
agttgtctaa ttgtgaaaat tttcagaaga 1200ctgatgtgaa agatgatctg
tctgatcctc ctgttgcaag cagttgtatt tctgggaagt 1260ctccacgtag
tccacaactt tcagattttg gacttgagcg gtacatcgta tcccaagttc
1320taccaaaccc tccacaggca gtgaacaact ataaggaaga gcccgtaatt
gtaaccccac 1380ctaccaaaca atcactagta aaagtactaa aaactccaaa
atgtgcacta aaaatggatg 1440attttgagtg tgtaactcct aaattagaac
actttggtat ctctgaatat actatgtgtt 1500taaatgaaga ttacacaatg
ggacttaaaa atgcgaggaa taataaaagt gaggaggcca 1560tagatacaga
atccaggctc aatgataatg tttttgccac tcccagcccc atcatccagc
1620agttggaaaa aagtgatgcc gaatatacca actctccttt ggtacctaca
ttctgtactc 1680ctggtttgaa aattccatct acaaagaaca gcatagcttt
ggtatccaca aattacccat 1740tatcaaaaac aaatagttca tcaaatgatt
tggaagttga agatcgtact tcgttggttt 1800taaattcaga cacatgcttt
gagaatttaa cagatccctc
ttcacctacg atttcttctt 1860atgagaatct gctcagaaca cctacacctc
cagaagtaac taaaattcca gaagatattc 1920tccagctttt atcaaaatac
aactcaaacc tagctactcc aatagcaatt aaagcagtgc 1980cacccagtaa
aaggttcctt aaacatggac agaacatccg agatgtcagc aacaaagaaa
2040actgaaattc cagtggatct atccaacaca gaaactgaac aaaatgagat
gaaagccgag 2100ctggaccgat tttaacattc acattgccct gcctctgtcc
ccctttaaac gttgacccat 2160tttaaagaca aacatgaaca ttaacatcat
aatatgcttt ttatgaagtt tcaataaggt 2220ttaaccttag tcttgttgac
atgtagccca gtcattcact ctttaaggac tattagtgtt 2280tcattgatac
taaattaccc agcttaatca acagaatggt ttaagtagta ccaggaagta
2340ggacaagtaa tttcaaaaat ataaaggtgt ttgctactca gatgaggccg
cccctgacct 2400tctggccaga gagacattgc tgccagccag ctctgccttc
ccatcatctc ctttcaggac 2460cgtcccacac cttttacttg ctcagtgctg
tctgaagatg cagttgctgt ttgcaaacaa 2520caggaacacc agttaaacta
attaggaaac agagggagat ttccaggcct gggtaactat 2580atactgtgac
cattggcggt tgagaccggt cttcaaccag tggaaccccg aactctgctg
2640tcagggtgtg gacttcggtg ctcttccaag ttttcacctg ggggggggag
ctaaccccct 2700atgttcacgc cttctattcc cattggcgct gaactcttaa
ggtcactctg gtcgcttgtg 2760accccgtaac cctgatgtac ccctctaaaa
ggtgaggggc tatcatctgt gactgaggaa 2820atccctatct tcctatcaga
ctaatgaaac cacaggacag caattagact tttaagtatt 2880ggggggttta
gagctctaga tattcgatat gcagactact catgtttgtt tgttttaata
2940aagactggtc caaaggctca ttttcacaca agctacagtt tttcagttcc
aggaccaggt 3000aaagatggtc agctccgtga tccataaaat ccaagggtga
cgactcagga ttaggaccat 3060ttcttggtga cattgagatg gtcgagctgg
tccgcaatga atctatgcgg ggggaacttg 3120gaagtggcgg ccgcctttat
ggcctcgaag gcctccctcc tgcgcaccgc ggcgtggccg 3180cgctcctgct
cccgggtcat gtagggcatg ctcagccagt aatggttctc cgcctcgatc
3240tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca cgaaccgcgg
ccgccggtgc 3300ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga
ggagcgcagt caggaacatg 3360gtgcctgccg cgctgctcaa gactctgcgt
ctccgcggcc gccagcagac gccgtggcgt 3420aagcgcaccc gtctcgcggg
gtctccgggg gcctcggcga gagacttcgg ctctcgcgag 3480agaggactgc
gcctgcgcag agccgaggac gcgtccggcg ccgagattca aactagtggc
3540gggaggctgt gagctgagcg gtggggtctg cgtacgcctg gagtccttcc
ccgctgtgct 3600cagcatggac cctatccgga gcttctgcgg gaagctgcgg
tctctggcca gcacgctgga 3660ctgcgagacg gcccggctgc agcgagcgct
ggacggagag gaaagcg 3707120372PRTHomo sapiens 120Met Arg Ile Leu Tyr
Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp1 5 10 15Asp Val Asn Ile
Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu Gly 20 25 30Ile Asp Phe
Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn Ser Met 35 40 45Asp Ile
Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro 50 55 60Arg
Val Lys Lys Asn Ser Val His Glu Gln Glu Ala Ile Asn Ser Asp65 70 75
80Pro Glu Leu Ser Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp
85 90 95Asp Leu Ser Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys
Ser 100 105 110Pro Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg
Tyr Ile Val 115 120 125Ser Gln Val Leu Pro Asn Pro Pro Gln Ala Val
Asn Asn Tyr Lys Glu 130 135 140Glu Pro Val Ile Val Thr Pro Pro Thr
Lys Gln Ser Leu Val Lys Val145 150 155 160Leu Lys Thr Pro Lys Cys
Ala Leu Lys Met Asp Asp Phe Glu Cys Val 165 170 175Thr Pro Lys Leu
Glu His Phe Gly Ile Ser Glu Tyr Thr Met Cys Leu 180 185 190Asn Glu
Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg Asn Asn Lys Ser 195 200
205Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala
210 215 220Thr Pro Ser Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp Ala
Glu Tyr225 230 235 240Thr Asn Ser Pro Leu Val Pro Thr Phe Cys Thr
Pro Gly Leu Lys Ile 245 250 255Pro Ser Thr Lys Asn Ser Ile Ala Leu
Val Ser Thr Asn Tyr Pro Leu 260 265 270Ser Lys Thr Asn Ser Ser Ser
Asn Asp Leu Glu Val Glu Asp Arg Thr 275 280 285Ser Leu Val Leu Asn
Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro 290 295 300Ser Ser Pro
Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr Pro Thr305 310 315
320Pro Pro Glu Val Thr Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser
325 330 335Lys Tyr Asn Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala
Val Pro 340 345 350Pro Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile
Arg Asp Val Ser 355 360 365Asn Lys Glu Asn 370121372PRTHomo sapiens
121Met Arg Ile Leu Tyr Asp Leu His Ser Glu Val Gln Thr Leu Lys Asp1
5 10 15Asp Val Asn Ile Leu Leu Asp Lys Ala Arg Leu Glu Asn Gln Glu
Gly 20 25 30Ile Asp Phe Ile Lys Ala Thr Lys Val Leu Met Glu Lys Asn
Ser Met 35 40 45Asp Ile Met Lys Ile Arg Glu Tyr Phe Gln Lys Tyr Gly
Tyr Ser Pro 50 55 60Arg Val Lys Lys Asn Ser Val His Glu Gln Glu Ala
Ile Asn Ser Asp65 70 75 80Pro Glu Leu Ser Asn Cys Glu Asn Phe Gln
Lys Thr Asp Val Lys Asp 85 90 95Asp Leu Ser Asp Pro Pro Val Ala Ser
Ser Cys Ile Ser Gly Lys Ser 100 105 110Pro Arg Ser Pro Gln Leu Ser
Asp Phe Gly Leu Glu Arg Tyr Ile Val 115 120 125Ser Gln Val Leu Pro
Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu 130 135 140Glu Pro Val
Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val Lys Val145 150 155
160Leu Lys Thr Pro Lys Cys Ala Leu Lys Met Asp Asp Phe Glu Cys Val
165 170 175Thr Pro Lys Leu Glu His Phe Gly Ile Ser Glu Tyr Thr Met
Cys Leu 180 185 190Asn Glu Asp Tyr Thr Met Gly Leu Lys Asn Ala Arg
Asn Asn Lys Ser 195 200 205Glu Glu Ala Ile Asp Thr Glu Ser Arg Leu
Asn Asp Asn Val Phe Ala 210 215 220Thr Pro Ser Pro Ile Ile Gln Gln
Leu Glu Lys Ser Asp Ala Glu Tyr225 230 235 240Thr Asn Ser Pro Leu
Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile 245 250 255Pro Ser Thr
Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr Pro Leu 260 265 270Ser
Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val Glu Asp Arg Thr 275 280
285Ser Leu Val Leu Asn Ser Asp Thr Cys Phe Glu Asn Leu Thr Asp Pro
290 295 300Ser Ser Pro Thr Ile Ser Ser Tyr Glu Asn Leu Leu Arg Thr
Pro Thr305 310 315 320Pro Pro Glu Val Thr Lys Ile Pro Glu Asp Ile
Leu Gln Leu Leu Ser 325 330 335Lys Tyr Asn Ser Asn Leu Ala Thr Pro
Ile Ala Ile Lys Ala Val Pro 340 345 350Pro Ser Lys Arg Phe Leu Lys
His Gly Gln Asn Ile Arg Asp Val Ser 355 360 365Asn Lys Glu Asn
370122372PRTHomo sapiens 122Met Arg Ile Leu Tyr Asp Leu His Ser Glu
Val Gln Thr Leu Lys Asp1 5 10 15Asp Val Asn Ile Leu Leu Asp Lys Ala
Arg Leu Glu Asn Gln Glu Gly 20 25 30Ile Asp Phe Ile Lys Ala Thr Lys
Val Leu Met Glu Lys Asn Ser Met 35 40 45Asp Ile Met Lys Ile Arg Glu
Tyr Phe Gln Lys Tyr Gly Tyr Ser Pro 50 55 60Arg Val Lys Lys Asn Ser
Val His Glu Gln Glu Ala Ile Asn Ser Asp65 70 75 80Pro Glu Leu Ser
Asn Cys Glu Asn Phe Gln Lys Thr Asp Val Lys Asp 85 90 95Asp Leu Ser
Asp Pro Pro Val Ala Ser Ser Cys Ile Ser Gly Lys Ser 100 105 110Pro
Arg Ser Pro Gln Leu Ser Asp Phe Gly Leu Glu Arg Tyr Ile Val 115 120
125Ser Gln Val Leu Pro Asn Pro Pro Gln Ala Val Asn Asn Tyr Lys Glu
130 135 140Glu Pro Val Ile Val Thr Pro Pro Thr Lys Gln Ser Leu Val
Lys Val145 150 155 160Leu Lys Thr Pro Lys Cys Ala Leu Lys Met Asp
Asp Phe Glu Cys Val 165 170 175Thr Pro Lys Leu Glu His Phe Gly Ile
Ser Glu Tyr Thr Met Cys Leu 180 185 190Asn Glu Asp Tyr Thr Met Gly
Leu Lys Asn Ala Arg Asn Asn Lys Ser 195 200 205Glu Glu Ala Ile Asp
Thr Glu Ser Arg Leu Asn Asp Asn Val Phe Ala 210 215 220Thr Pro Ser
Pro Ile Ile Gln Gln Leu Glu Lys Ser Asp Ala Glu Tyr225 230 235
240Thr Asn Ser Pro Leu Val Pro Thr Phe Cys Thr Pro Gly Leu Lys Ile
245 250 255Pro Ser Thr Lys Asn Ser Ile Ala Leu Val Ser Thr Asn Tyr
Pro Leu 260 265 270Ser Lys Thr Asn Ser Ser Ser Asn Asp Leu Glu Val
Glu Asp Arg Thr 275 280 285Ser Leu Val Leu Asn Ser Asp Thr Cys Phe
Glu Asn Leu Thr Asp Pro 290 295 300Ser Ser Pro Thr Ile Ser Ser Tyr
Glu Asn Leu Leu Arg Thr Pro Thr305 310 315 320Pro Pro Glu Val Thr
Lys Ile Pro Glu Asp Ile Leu Gln Leu Leu Ser 325 330 335Lys Tyr Asn
Ser Asn Leu Ala Thr Pro Ile Ala Ile Lys Ala Val Pro 340 345 350Pro
Ser Lys Arg Phe Leu Lys His Gly Gln Asn Ile Arg Asp Val Ser 355 360
365Asn Lys Glu Asn 370123412PRTHomo sapiens 123Met Asp Pro Ile Arg
Ser Phe Cys Gly Lys Leu Arg Ser Leu Ala Ser1 5 10 15Thr Leu Asp Cys
Glu Thr Ala Arg Leu Gln Arg Ala Leu Asp Gly Glu 20 25 30Glu Ser Asp
Phe Glu Asp Tyr Pro Met Arg Ile Leu Tyr Asp Leu His 35 40 45Ser Glu
Val Gln Thr Leu Lys Asp Asp Val Asn Ile Leu Leu Asp Lys 50 55 60Ala
Arg Leu Glu Asn Gln Glu Gly Ile Asp Phe Ile Lys Ala Thr Lys65 70 75
80Val Leu Met Glu Lys Asn Ser Met Asp Ile Met Lys Ile Arg Glu Tyr
85 90 95Phe Gln Lys Tyr Gly Tyr Ser Pro Arg Val Lys Lys Asn Ser Val
His 100 105 110Glu Gln Glu Ala Ile Asn Ser Asp Pro Glu Leu Ser Asn
Cys Glu Asn 115 120 125Phe Gln Lys Thr Asp Val Lys Asp Asp Leu Ser
Asp Pro Pro Val Ala 130 135 140Ser Ser Cys Ile Ser Glu Lys Ser Pro
Arg Ser Pro Gln Leu Ser Asp145 150 155 160Phe Gly Leu Glu Arg Tyr
Ile Val Ser Gln Val Leu Pro Asn Pro Pro 165 170 175Gln Ala Val Asn
Asn Tyr Lys Glu Glu Pro Val Ile Val Thr Pro Pro 180 185 190Thr Lys
Gln Ser Leu Val Lys Val Leu Lys Thr Pro Lys Cys Ala Leu 195 200
205Lys Met Asp Asp Phe Glu Cys Val Thr Pro Lys Leu Glu His Phe Gly
210 215 220Ile Ser Glu Tyr Thr Met Cys Leu Asn Glu Asp Tyr Thr Met
Gly Leu225 230 235 240Lys Asn Ala Arg Asn Asn Lys Ser Glu Glu Ala
Ile Asp Thr Glu Ser 245 250 255Arg Leu Asn Asp Asn Val Phe Ala Thr
Pro Ser Pro Ile Ile Gln Gln 260 265 270Leu Glu Lys Ser Asp Ala Glu
Tyr Thr Asn Ser Pro Leu Val Pro Thr 275 280 285Phe Cys Thr Pro Gly
Leu Lys Ile Pro Ser Thr Lys Asn Ser Ile Ala 290 295 300Leu Val Ser
Thr Asn Tyr Pro Leu Ser Lys Thr Asn Ser Ser Ser Asn305 310 315
320Asp Leu Glu Val Glu Asp Arg Thr Ser Leu Val Leu Asn Ser Asp Thr
325 330 335Cys Phe Glu Asn Leu Thr Asp Pro Ser Ser Pro Thr Ile Ser
Ser Tyr 340 345 350Glu Asn Leu Leu Arg Thr Pro Thr Pro Pro Glu Val
Thr Lys Ile Pro 355 360 365Glu Asp Ile Leu Gln Leu Leu Ser Lys Tyr
Asn Ser Asn Leu Ala Thr 370 375 380Pro Ile Ala Ile Lys Ala Val Pro
Pro Ser Lys Arg Phe Leu Lys His385 390 395 400Gly Gln Asn Ile Arg
Asp Val Ser Asn Lys Glu Asn 405 4101246PRTArtificial
SequencePeptide motif 124Thr Ile Leu Val Met Ser1
51257PRTArtificial SequencePeptide motif 125Leu Ile Val Met Ala Thr
Gln1 51266PRTArtificial SequencePeptide motif 126Ile Val Met Ala
Thr Leu1 51277PRTArtificial SequencePeptide motif 127Val Ser Met
Ala Thr Leu Ile1 51288PRTArtificial SequencePeptide motif 128Tyr
Phe Trp Ile Val Leu Met Thr1 51296PRTArtificial SequencePeptide
motif 129Phe Ile Tyr Trp Leu Met1 51308PRTArtificial
SequencePeptide motif 130Val Ile Leu Phe Met Trp Tyr Ala1
51318PRTArtificial SequencePeptide motif 131Phe Tyr Leu Trp Met Ile
Val Ala1 51328PRTArtificial SequencePeptide motif 132Phe Trp Tyr
Leu Ile Met Val Ala1 51338PRTArtificial SequencePeptide motif
133Phe Trp Tyr Leu Ile Val Met Ala1 51346PRTArtificial
SequencePeptide motif 134Gln Leu Ile Val Met Pro1
51358PRTArtificial SequencePeptide motif 135Phe Trp Tyr Met Ile Val
Leu Ala1 51367PRTArtificial SequencePeptide motif 136Leu Met Val
Gln Ile Ala Thr1 51376PRTArtificial SequencePeptide motif 137Val
Leu Ile Met Ala Thr1 513811PRTArtificial SequencePeptide motif
138Leu Met Val Ile Ser Ala Thr Phe Cys Gly Asp1 5
101396PRTArtificial SequencePeptide motif 139Lys Tyr Arg His Phe
Ala1 514012PRTArtificial SequencePeptide motif 140Val Thr Met Leu
Ile Ser Ala Gly Asn Cys Asp Phe1 5 101414PRTArtificial
SequencePeptide motif 141Lys Arg Tyr His11424PRTArtificial
SequencePeptide motif 142Tyr Phe Trp Met11434PRTArtificial
SequencePeptide motif 143Phe Leu Ile Trp11447PRTArtificial
SequencePeptide motif 144Met Val Thr Ala Leu Ile Ser1
51458PRTArtificial SequencePeptide motif 145Met Val Ala Leu Phe Ile
Ser Thr1 51467PRTArtificial SequencePeptide motif 146Ala Val Thr
Met Ser Leu Ile1 51478PRTArtificial SequencePeptide motif 147Leu
Met Phe Trp Tyr Ala Ile Val1 51488PRTArtificial SequencePeptide
motif 148Leu Met Phe Trp Tyr Ile Val Ala1 51498PRTArtificial
SequencePeptide motif 149Leu Ile Val Phe Trp Tyr Ala Met1
51508PRTArtificial SequencePeptide motif 150Ile Met Phe Trp Tyr Ala
Leu Val1 51519PRTArtificial SequencePeptide motif 151Ala Thr Ile
Val Leu Met Phe Trp Tyr1 51527PRTArtificial SequencePeptide motif
152Phe Met Tyr Leu Ile Val Trp1 51539PRTArtificial SequencePeptide
motif 153Val Ser Thr Cys Pro Ala Leu Ile Met1 51547PRTArtificial
SequencePeptide motif 154Met Phe Leu Ile Val Trp Tyr1
51554PRTArtificial SequencePeptide motif 155Pro Ala Met
Gln11569PRTArtificial SequencePeptide motif 156Val Met Ala Thr Ser
Pro Leu Ile Cys1 51577PRTArtificial SequencePeptide motif 157Met
Phe Leu Ile Val Trp Tyr1 51589PRTArtificial SequencePeptide motif
158Ile Val Met Ser Ala Cys Thr Pro Leu1 51596PRTArtificial
SequencePeptide motif 159Leu Ile Val Met Phe Tyr1
51607PRTArtificial SequencePeptide motif 160Leu Ile Val Met Phe Ala
Tyr1 51616PRTArtificial SequencePeptide motif 161Asp Asn Gln Glu
Ser Thr1 51627PRTArtificial SequencePeptide motif 162Met Phe Leu
Ile Val Trp Tyr1 51639PRTArtificial SequencePeptide motif 163Val
Met Ser Thr Ala Cys Pro Leu Ile1 51646PRTArtificial SequencePeptide
motif 164Thr Ile Leu Val Met Ser1 51657PRTArtificial
SequencePeptide motif 165Leu Ile Val Met Ala Thr Gln1
51666PRTArtificial SequencePeptide motif 166Leu Ile Val Met Ala
Thr1 51677PRTArtificial SequencePeptide motif 167Val Ser Met Ala
Thr Leu Ile1 51688PRTArtificial SequencePeptide motif 168Tyr Phe
Trp Ile Val Leu Met Thr1 51696PRTArtificial SequencePeptide motif
169Phe Ile Tyr Trp Leu Met1 51704PRTArtificial SequencePeptide
motif 170Leu Ile Val Met11718PRTArtificial SequencePeptide motif
171Val Ile Leu Phe Met Trp Tyr Ala1 51728PRTArtificial
SequencePeptide motif 172Phe Tyr Leu Trp Met Ile Val Ala1
51738PRTArtificial SequencePeptide motif 173Phe Trp Tyr Leu Ile Met
Val Ala1 51748PRTArtificial SequencePeptide
motif 174Phe Trp Tyr Leu Ile Val Met Ala1 51756PRTArtificial
SequencePeptide motif 175Gln Leu Ile Val Met Pro1
51768PRTArtificial SequencePeptide motif 176Phe Trp Tyr Met Ile Val
Leu Ala1 51774PRTArtificial SequencePeptide motif 177Gly Phe Tyr
Trp11784PRTArtificial SequencePeptide motif 178Asp Glu Gln
Asn11798PRTArtificial SequencePeptide motif 179Arg His Lys Leu Ile
Val Met Pro1 51804PRTArtificial SequencePeptide motif 180Gly Arg
His Lys11818PRTArtificial SequencePeptide motif 181Ala Ser Thr Cys
Leu Ile Val Met1 51824PRTArtificial SequencePeptide motif 182Asp
Glu Ala Ser11834PRTArtificial SequencePeptide motif 183Gly Ser Thr
Cys11844PRTArtificial SequencePeptide motif 184Ala Ser Thr
Cys11854PRTArtificial SequencePeptide motif 185Leu Ile Val
Met11869PRTArtificial SequencePeptide motif 186Arg His Lys Asp Glu
Pro Tyr Phe Trp1 51875PRTArtificial SequencePeptide motif 187Asp
Glu Ala Gln Asn1 51885PRTArtificial SequencePeptide motif 188Tyr
Phe Trp Gln Asn1 51895PRTArtificial SequencePeptide motif 189Pro
Ala Ser Thr Cys1 51908PRTArtificial SequencePeptide motif 190Arg
His Lys Gly Leu Ile Val Met1 51916PRTArtificial SequencePeptide
motif 191Arg His Lys Tyr Phe Trp1 51927PRTArtificial
SequencePeptide motif 192Ser Thr Cys Leu Ile Val Met1
51934PRTArtificial SequencePeptide motif 193Asp Glu Ala
Ser11949PRTArtificial SequencePeptide motif 194Arg His Lys Asp Glu
Pro Tyr Phe Trp1 51954PRTArtificial SequencePeptide motif 195Pro
Arg His Lys11967PRTArtificial SequencePeptide motif 196Leu Met Ile
Val Gln Ala Thr1 51976PRTArtificial SequencePeptide motif 197Val
Leu Ile Met Ala Thr1 51985PRTArtificial SequencePeptide motif
198Asp Glu Arg Lys His1 51995PRTArtificial SequencePeptide motif
199Asp Glu Arg Lys His1 52007PRTArtificial SequencePeptide motif
200Leu Met Ile Val Gln Ala Thr1 52014PRTArtificial SequencePeptide
motif 201Leu Val Ile Met12027PRTArtificial SequencePeptide motif
202Phe Tyr Trp Leu Val Ile Met1 52036PRTArtificial SequencePeptide
motif 203Val Leu Ile Met Ala Thr1 52044PRTArtificial
SequencePeptide motif 204Arg Lys His Ala12055PRTArtificial
SequencePeptide motif 205Asp Glu Arg Lys His1 520611PRTArtificial
SequencePeptide motif 206Leu Met Val Ile Ser Ala Thr Phe Cys Gly
Asp1 5 102077PRTArtificial SequencePeptide motif 207Pro Arg His Lys
Tyr Phe Trp1 52086PRTArtificial SequencePeptide motif 208Lys Tyr
Arg His Phe Ala1 520912PRTArtificial SequencePeptide motif 209Val
Thr Leu Met Ile Ser Ala Gly Asn Cys Asp Phe1 5 102104PRTArtificial
SequencePeptide motif 210Lys Arg Tyr His12116PRTArtificial
SequencePeptide motif 211Tyr Phe Trp Arg His Lys1
52124PRTArtificial SequencePeptide motif 212Tyr Phe Trp
Met12134PRTArtificial SequencePeptide motif 213Phe Leu Ile
Trp12145PRTArtificial SequencePeptide motif 214Asp Glu Arg His Lys1
52154PRTArtificial SequencePeptide motif 215Tyr Phe Trp
Met12164PRTArtificial SequencePeptide motif 216Tyr Phe Trp
Pro12174PRTArtificial SequencePeptide motif 217Phe Leu Ile
Trp12187PRTArtificial SequencePeptide motif 218Met Val Thr Ala Leu
Ile Ser1 52198PRTArtificial SequencePeptide motif 219Met Val Ala
Leu Phe Ile Ser Thr1 52204PRTArtificial SequencePeptide motif
220Ala Tyr Phe Trp12216PRTArtificial SequencePeptide motif 221Tyr
Phe Trp Ser Thr Cys1 52227PRTArtificial SequencePeptide motif
222Ala Val Thr Met Ser Leu Ile1 52237PRTArtificial SequencePeptide
motif 223Tyr Phe Trp Leu Ile Val Met1 52246PRTArtificial
SequencePeptide motif 224Arg His Lys Phe Trp Tyr1
52258PRTArtificial SequencePeptide motif 225Leu Met Phe Trp Tyr Ala
Ile Val1 52265PRTArtificial SequencePeptide motif 226Asp Glu Gln
Asn Pro1 52277PRTArtificial SequencePeptide motif 227Phe Trp Tyr
Leu Ile Val Met1 52288PRTArtificial SequencePeptide motif 228Leu
Met Phe Trp Tyr Ile Val Ala1 52297PRTArtificial SequencePeptide
motif 229Leu Ile Val Met Phe Trp Tyr1 52308PRTArtificial
SequencePeptide motif 230Leu Ile Val Phe Trp Tyr Ala Met1
523111PRTArtificial SequencePeptide motif 231Ala Gly Pro Asp Glu
Arg His Lys Ser Thr Cys1 5 102324PRTArtificial SequencePeptide
motif 232Asp Glu Gln Asn12337PRTArtificial SequencePeptide motif
233Leu Ile Val Met Phe Trp Tyr1 52347PRTArtificial SequencePeptide
motif 234Leu Ile Val Met Phe Trp Tyr1 52358PRTArtificial
SequencePeptide motif 235Ile Met Phe Trp Tyr Ala Leu Val1
52365PRTArtificial SequencePeptide motif 236Ala Gly Pro Gln Asn1
52375PRTArtificial SequencePeptide motif 237Arg His Lys Gln Asn1
52387PRTArtificial SequencePeptide motif 238Phe Trp Tyr Leu Ile Val
Met1 52394PRTArtificial SequencePeptide motif 239Leu Ile Val
Met12405PRTArtificial SequencePeptide motif 240Ala Leu Ile Val Met1
52415PRTArtificial SequencePeptide motif 241Phe Trp Tyr Ala Pro1
52429PRTArtificial SequencePeptide motif 242Ala Thr Ile Val Leu Met
Phe Trp Tyr1 52436PRTArtificial SequencePeptide motif 243Gly Pro
Gln Asn Asp Glu1 52446PRTArtificial SequencePeptide motif 244Gly
Asp Glu Ser Thr Cys1 52455PRTArtificial SequencePeptide motif
245Arg His Lys Asp Glu1 52465PRTArtificial SequencePeptide motif
246Gln Asn Asp Gly Glu1 52474PRTArtificial SequencePeptide motif
247Asp Glu Ala Ser12488PRTArtificial SequencePeptide motif 248Ala
Ile Leu Met Val Phe Trp Tyr1 52497PRTArtificial SequencePeptide
motif 249Ala Ile Leu Met Val Ser Thr1 52506PRTArtificial
SequencePeptide motif 250Ala Ile Leu Met Val Thr1
52516PRTArtificial SequencePeptide motif 251Ala Ile Leu Met Val
Thr1 52528PRTArtificial SequencePeptide motif 252Tyr Phe Trp Ile
Val Leu Met Thr1 52536PRTArtificial SequencePeptide motif 253Phe
Ile Tyr Trp Leu Met1 52548PRTArtificial SequencePeptide motif
254Phe Trp Tyr Leu Ile Met Val Ala1 52556PRTArtificial
SequencePeptide motif 255Thr Ile Leu Val Met Ser1
52566PRTArtificial SequencePeptide motif 256Phe Tyr Leu Trp Met
Ile1 52576PRTArtificial SequencePeptide motif 257Gln Leu Ile Val
Met Pro1 52586PRTArtificial SequencePeptide motif 258Phe Trp Tyr
Met Ile Val1 52596PRTArtificial SequencePeptide motif 259Phe Trp
Tyr Leu Ile Val1 5
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