U.S. patent application number 10/236878 was filed with the patent office on 2006-04-06 for nucleic acid and corresponding protein entitled steap-1 useful in treatment and detection of cancer.
Invention is credited to Pia M. Challita-Eid, Mary Faris, Wangmao Ge, Aya Jakobovits, Arthur B. Raitano.
Application Number | 20060073150 10/236878 |
Document ID | / |
Family ID | 26981182 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073150 |
Kind Code |
A1 |
Faris; Mary ; et
al. |
April 6, 2006 |
Nucleic acid and corresponding protein entitled STEAP-1 useful in
treatment and detection of cancer
Abstract
A novel gene 08P1D4 (also designated STEAP-1) and its encoded
protein, and variants thereof, are described wherein STEAP-1
exhibits tissue specific expression in normal adult tissue, and is
aberrantly expressed in the cancers listed in Table I.
Consequently, STEAP-1 provides a diagnostic, prognostic,
prophylactic and/or therapeutic target for cancer. The STEAP-1 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 STEAP-1 can be
used in active or passive immunization.
Inventors: |
Faris; Mary; (Los Angeles,
CA) ; Ge; Wangmao; (Culver City, CA) ;
Raitano; Arthur B.; (Los Angeles, CA) ; Challita-Eid;
Pia M.; (Encino, CA) ; Jakobovits; Aya;
(Beverly Hills, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
26981182 |
Appl. No.: |
10/236878 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60317840 |
Sep 6, 2001 |
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60370387 |
Apr 5, 2002 |
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Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4748 20130101; C07K 14/705 20130101; A61K 51/1045 20130101;
A61K 39/00 20130101; A61K 2039/505 20130101; A61P 35/00 20180101;
C07K 2319/00 20130101; C07K 14/723 20130101; G01N 33/57484
20130101; A61P 43/00 20180101; C07K 16/30 20130101; A61P 37/04
20180101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1.-32. (canceled)
33. A method of inhibiting growth of cancer cells that express a
protein of FIG. 2 (SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, or 35), the method comprising:
administering to the cells an effective amount of a substance that
modulates the status of a protein of FIG. 2 (SEQ ID NO: 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35).
34. The method of claim 52 wherein substance is an antibody or a
fragment thereof that specifically binds to a polypeptide of FIG. 2
(SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, or 35).
35. The method of claim 33 wherein said substance is a
STEAP-1-related protein.
36. The method of claim 33 wherein said substance is a
polynucleotide comprising a coding sequence for a STEAP-1-related
protein or comprising a polynucleotide complementary to a coding
sequence for a STEAP-1-related protein.
37. The method of claim 33 wherein said substance is a ribozyme
that cleaves a polynucleotide that encodes a protein of FIG. 2 (SEQ
ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
or 35).
38. The method of claim 33 wherein said cancer cells express a
protein of FIG. 2 and a particular HLA molecule, and the method
comprises administering human T cells to said cancer cells, wherein
said T cells specifically recognize a peptide subsequence of a
protein of FIG. 2 while the subsequence is n to context of the
particular HLA molecule.
39. A method of claim 33, the method comprising steps of:
administering a vector that delivers a nucleotide that encodes a
single chain monoclonal antibody, whereby the encoded single chain
monoclonal antibody is expressed intracellularly within cancer
cells that express a protein of FIG. 2 (SEQ ID NO: 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35).
40.-51. (canceled)
52. The method of claim 33 wherein said substance comprises an
antibody or an antibody fragment that specifically binds to a
protein of FIG. 2 (SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, or 35).
53. The method of claim 34 wherein said substance is a monoclonal
antibody or a fragment thereof.
54. The method of claim 53 wherein said substance is a human
antibody, a humanized antibody or a chimeric antibody, of a
fragment thereof.
55. The method of claim 53 wherein said cancer is selected from the
group consisting of prostate cancer, gastric cancer colorectal
cancer; multiple myeloma, B-cell lymphoma, leukemia, breast cancer,
kidney cancer, bladder cancer, and lung cancer.
56. The method of claim 55 wherein said cancer is metastatic.
57. The method of claim 56 wherein said cancer is metastatic
prostate cancer.
58. The method of claim 34 wherein the growth of said cancer cells
is inhibited in a human patient.
59. The method of claim 58 wherein said human patient has been
diagnosed with prostate cancer.
60. The method of claim 59 wherein said prostate cancer is advanced
or metastatic prostate cancer.
61. The method of claim 59 wherein said human patient has received
one or more rounds of chemotherapy prior to the administration of
said antibody or antibody fragment.
62. The method of claim 58 wherein said antibody or antibody
fragment is administered in conjunction with radiation,
chemotherapy or hormone ablation.
63. The method of claim 58 wherein said antibody or antibody
fragment is conjugated to a toxin, a radioisotope, or a
chemotherapeutic agent.
64. The method of claim 63 wherein said chemotherapeutic agent is a
maytansinoid.
65. The method of claim 58 wherein said human patient is diagnosed
with breast cancer.
66. The method of claim 65 wherein said antibody or antibody
fragment is co-administered with another cancer therapeutic
agent.
67. The method of claim 66 wherein the other cancer therapeutic
agent is trastuzumab or paclitaxel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application No. 60/317,840, filed Sep. 6, 2001 and U.S.
provisional patent application No. 60/370,387 filed Apr. 5, 2002.
This application relates to U.S. patent application No. 60/087,520,
filed Jun. 1, 1998 and U.S. patent application No. 60/091,183,
filed Jun. 30, 1998 and U.S. patent application Ser. No.
09/323,873, filed Jun. 1, 1999 and U.S. patent application Ser. No.
10/011,095, filed Dec. 6, 2001 and U.S. patent application Ser. No.
10/010,667, filed Dec. 6, 2001 and U.S. patent application Ser. No.
09/455,486, filed Dec. 6, 1999 and U.S. patent application No.
60/296,656, filed Jun. 6, 2001, and U.S. patent application Ser.
No. 10/165,044, filed Jun. 6, 2002. The contents of each
application listed in this paragraph are fully incorporated by
reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The invention described herein relates to a gene and its
encoded protein, termed 08P1D4 or STEAP-1, expressed in certain
cancers, and to diagnostic and therapeutic methods and compositions
useful in the management of cancers that express STEAP-1.
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 sequelae 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 STEAP-1,
that has now been found to be over-expressed in the cancer(s)
listed in Table I. Northern blot expression analysis of STEAP-1
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 STEAP-1 are provided. The
tissue-related profile of STEAP-1 in normal adult tissues, combined
with the over-expression observed in the tissues listed in Table I,
shows that STEAP-1 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 STEAP-1 genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding STEAP-1-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 STEAP-1-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 STEAP-1
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the STEAP-1 genes, mRNAs, or to
STEAP-1-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding STEAP-1. Recombinant DNA
molecules containing STEAP-1 polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of STEAP-1 gene products are also provided. The
invention further provides antibodies that bind to STEAP-1 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 STEAP-1 polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express STEAP-1. A typical embodiment of this invention
provides methods for monitoring STEAP-1 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 STEAP-1 such as cancers of tissues listed in Table I,
including therapies aimed at inhibiting the transcription,
translation, processing or function of STEAP-1 as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
STEAP-1 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 STEAP-1.
Preferably, the carrier is a uniquely human carrier. In another
aspect of the invention, the agent is a moiety that is
immunoreactive with STEAP-1 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 STEAP-1 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 STEAP-1 as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
STEAP-1. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of STEAP-1 (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for STEAP-1 production) or a ribozyme effective to lyse
STEAP-1 mRNA.
[0032] Note that to determine the starting position of any peptide
set forth in Tables V-XVIII and XXII to LI (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 LXI. 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 LXI. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X-1" to each position in Tables V-XVIII and XXII to LI 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 V-XVIII and XXII to LI
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 V-XVIII and at least once in tables
XXII to LI, 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;
[0037] 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;
[0038] 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
[0039] v) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn
profile of FIG. 9.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1. The STEAP-1 SSH sequence of 436 nucleotides.
[0041] FIG. 2. The cDNA and amino acid sequence of STEAP-1 variant
1 (also called "STEAP-1 v.1" or "STEAP-1 variant 1") is shown in
FIG. 2A. The start methionine is underlined. The open reading frame
extends from nucleic acid 66-1085 including the stop codon. The
cDNA and amino acid sequence of STEAP-1 variant 2 (also called
"STEAP-1 v.2") is shown in FIG. 2B. The codon for the start
methionine is underlined. The open reading frame extends from
nucleic acid 96-872 including the stop codon. The cDNA and amino
acid sequence of STEAP-1 variant 3 (also called "STEAP-1 v.3") is
shown in FIG. 2C. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 96-944 including
the stop codon. The cDNA and amino acid sequence of STEAP-1 variant
4 (also called "STEAP-1 v.4") is shown in FIG. 2D. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 96-872 including the stop codon. The cDNA and
amino acid sequence of STEAP-1 variant 5 (also called "STEAP-1
v.5") is shown in FIG. 2E. The codon for the start methionine is
underlined. The open reading frame extends from nucleic acid 96-872
including the stop codon. The cDNA and amino acid sequence of
STEAP-1 variant 6 (also called "STEAP-1 v.6") is shown in FIG. 2F.
The codon for the start methionine is underlined. The open reading
frame extends from nucleic acid 96-872 including the stop codon.
The cDNA and amino acid sequence of STEAP-1 variant 7 (also called
"STEAP-1 v.7") is shown in FIG. 2G. The codon for the start
methionine is underlined. The open reading frame extends from
nucleic acid 96-872 including the stop codon. The cDNA and amino
acid sequence of STEAP-1 variant 8 (also called "STEAP-1 v.8") is
shown in FIG. 2H. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 96-872 including
the stop codon. The cDNA and amino acid sequence of STEAP-1 variant
9 (also called "STEAP-1 v.9") is shown in FIG. 21. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 96-872 including the stop codon. The cDNA and
amino acid sequence of STEAP-1 variant 10 (also called "STEAP-1
v.10") is shown in FIG. 2J. The codon for the start methionine is
underlined. The open reading frame extends from nucleic acid 96-872
including the stop codon. The cDNA and amino acid sequence of
STEAP-1 variant 11 (also called "STEAP-1 v.11") is shown in FIG.
2K. The codon for the start methionine is underlined. The open
reading frame extends from nucleic acid 96-872 including the stop
codon. The cDNA and amino acid sequence of STEAP-1 variant 12 (also
called "STEAP-1 v.12") is shown in FIG. 2L. The codon for the start
methionine is underlined. The open reading frame extends from
nucleic acid 96-872 including the stop codon. The cDNA and amino
acid sequence of STEAP-1 variant 13 (also called "STEAP-1 v.13") is
shown in FIG. 2M. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 96-872 including
the stop codon. The cDNA and amino acid sequence of STEAP-1 variant
14 (also called "STEAP-1 v.14") is shown in FIG. 2N. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 96-872 including the stop codon. The cDNA and
amino acid sequence of STEAP-1 variant 15 (also called "STEAP-1
v.15") is shown in FIG. 20. The codon for the start methionine is
underlined. The open reading frame extends from nucleic acid 96-872
including the stop codon. The cDNA and amino acid sequence of
STEAP-1 variant 16 (also called "STEAP-1 v.16") is shown in FIG.
2P. The codon for the start methionine is underlined. The open
reading frame extends from nucleic acid 96-872 including the stop
codon. The cDNA and amino acid sequence of STEAP-1 variant 17 (also
called "STEAP-1 v.17") is shown in FIG. 2Q. The codon for the start
methionine is underlined. The open reading frame extends from
nucleic acid 96-872 including the stop codon. As used herein, a
reference to STEAP-1 includes all variants thereof, including those
shown in FIGS. 10 and 12.
[0042] FIG. 3. Amino acid sequence of STEAP-1 v.1 is shown in FIG.
3A; it has 339 amino acids. The amino acid sequence of STEAP-1 v.2
is shown in FIG. 3B; it has 258 amino acids. The amino acid
sequence of STEAP-1 v.3 is shown in FIG. 3C; it has 282 amino
acids. The amino acid sequence of STEAP-1 v.4 is shown in FIG. 3D;
it has 258 amino acids. As used herein, a reference to STEAP-1
includes all variants thereof, including those shown in FIG.
11.
[0043] FIG. 4. The amino acid sequence alignment of STEAP-1 v.1
with mouse TNF.alpha.-induced adipose-related protein (gi/16905133)
is shown in FIG. 4A. The amino acid sequence alignment of STEAP-1
v.1 with rat pHyde protein (gi/21717665/) is shown in FIG. 4B. FIG.
4C shows Homology of STEAP-1 to mouse six transmembrane epithelial
antigen of the prostate (gi|20820492|).
[0044] FIG. 5. (5) Hydrophilicity amino acid profile of STEAP-1
variant 1. (5A) Hydrophilicity amino acid profile of STEAP-1
variant 3, 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 URL ch/cgi-bin/protscale.pl)
through the ExPasy molecular biology server.
[0045] FIG. 6. (6) Hydropathicity amino acid profile of STEAP-1
variant 1. (6A) Hydropathicity amino acid profile of STEAP-1
variant 3, 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 URL expasy.ch/cgi-bin/protscale.pl)
through the ExPasy molecular biology server.
[0046] FIG. 7. (7) Percent accessible residues amino acid profile
of STEAP-1 variant 1. (7A) Percent accessible residues amino acid
profile of STEAP-1 variant 3, 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 URL expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0047] FIG. 8. (8) Average flexibility amino acid profile of
STEAP-1 variant 1. (8A) Average flexibility amino acid profile of
STEAP-1 variant 3, 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 URL expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0048] FIG. 9. (9) Beta-turn amino acid profile of STEAP-1 variant
1. (9A) Beta-turn amino acid profile of STEAP-1 variant 3,
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 URL expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0049] FIG. 10. Schematic alignment of SNP variants of 8P1D4.
Variants 8P1D4 v.4 through v.17 are variants with single nucleotide
differences as compared to 8P1D4 v.2. Though these SNP variants are
shown separately, they could also occur in any combinations and in
any transcript variants that contains the base pairs, e.g., 8P1D4
v.1 and v.3. Numbers correspond to those of 8P1D4 v.2. Black box
shows the same sequence as 8P1D4 v.2. SNPs are indicated above the
box.
[0050] FIG. 11. Exon compositions of transcript variants of 8P1D4.
This figure shows the structure of the transcript variants without
poly A tail. Variants 8P1D4 v.1, v.2 and v.3 are transcript
variants that share the same exons 2 and 3. The first exon of 8P1D4
v.1 is 30 bases shorter at 5' end than the first exons of the other
two transcript variants. The fourth exon of 8P1D4 v.2 is the same
as the combined exon 4, intron 4 and exon 5 of 8P1D4 v.1. Compared
with 8P1D4 v.1, variant 8P1D4 v.3 has an additional exon spliced
out from intron 4 of 8P1D4 v.1. Lengths of introns and exons are
not proportional.
[0051] FIG. 12. Schematic alignment of protein variants of 8P1D4.
Protein variants correspond to nucleotide variants. Nucleotide
variants 8P1D4 v.5 through v.17 in FIG. 10 code for the same
protein as 8P1D4 v.2. Proteins translated from transcript variants
8P1D4 v.1 and v.3 as shown in FIG. 11 may contain amino acid F
(Phe) or L (Leu) at position 169. Single amino acid differences
were indicated above the boxes. Black boxes represent the same
sequence as 8P1D4 v.1. Boxes with different patterns of filling
show different sequences. Numbers underneath the box correspond to
8P1D4 v.1
[0052] FIG. 13. FIGS. 13(a)-(c). Secondary structure and
transmembrane domains prediction for 8P1D4 protein variants. The
secondary structure of 8P1D4 protein variants 1 (SEQ ID NO: 93), 2
(SEQ ID NO: 94), and 3 (SEQ ID NO: 95); (FIGS. 13a-13c,
respectively) were predicted using the HNN--Hierarchical Neural
Network method (Guermeur, 1997,
http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page npsa_nn.html),
accessed from the ExPasy molecular biology server located on the
World Wide Web at (.expasy.ch/tools/). This method predicts the
presence and location of alpha helices, extended strands, and
random coils from the primary protein sequence. The percent of the
protein in a given secondary structure is also listed. FIGS. 13(d),
13(f), and 13(h): Schematic representations of the probability of
existence of transmembrane regions and orientation of 8P1D4 variant
1-3, (FIGS. 13(d), 13(f) and 13(h) respectively, based on the
TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K.
Hofmann, W. Stoffel. TMBASE--A database of membrane spanning
protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS.
13(e), 13(g), and 13(i): Schematic representations of the
probability of the existence of transmembrane regions and the
extracellular and intracellular orientation of 8P1D4 variants 1-3,
FIGS. 13(e), 13(g), and 13(i) respectively, based on the TMHMM
algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L.
Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov
model for predicting transmembrane helices in protein sequences. In
Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular
Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R.
Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press,
1998). The TMpred and TMHMM algorithms are accessed from the ExPasy
molecular biology server located on the World Wide Web at
(.expasy.ch/tools/).
[0053] FIG. 14. Expression of STEAP-1 in stomach cancer patient
specimen. RNA was extracted from normal stomach (N) and from 10
different stomach cancer patient specimens (T). Northern blot with
10 .mu.g of total RNA/lane was probed with STEAP-1 sequence.
Results show strong expression of an approximately 1.6 kb STEAP-1
in the stomach tumor tissues. The lower panel represents ethidium
bromide staining of the blot showing quality of the RNA
samples.
[0054] FIG. 15 STEAP-1 expression in rectum cancer patient tissues.
RNA was extracted from normal rectum (N), rectum cancer patient
tumors (T), and rectum cancer metastasis (M). Northern blots with
10 .mu.g of total RNA were probed with the STEAP-1 sequence.
Results show strong expression of STEAP-1 in the rectum cancer
patient tissues. The lower panel represents ethidium bromide
staining of the blot showing quality of the RNA samples.
[0055] FIG. 16. Expression of STEAP-1 in human umbilical vein
endothelial cells (HUVEC). First strand cDNA was prepared from
HUVEC cells, LAPC-4AD and LAPC-9AD prostate cancer xenografts, as
well as from human brain tissues. Normalization was performed by
PCR using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to STEAP-1, was performed at 27 and 30 cycles of
amplification (A). As a control, PCR using primers to actin is
shown in (B). Results show strong expression of STEAP-1 in HUVEC
cells similar to the expression detected in prostate cancer
xenograft tissues. Expression of STEAP-1 in HUVEC cells indicates
that targeting STEAP-1 may also target endothelial cells of the
neovasculature of the tumors.
[0056] FIG. 17. Shows that STEAP-1 increased calcium flux in
response to LPA.
[0057] FIG. 18. Shows that STEAP-1-mediated calcium transport
regulates prostate cancer growth by regulating intracellular levels
of calcium.
[0058] FIG. 19. The figure demonstrates that STEAP-1 mediates the
transfer of the small molecule calcein between adjacent cells, and
thereby regulates cell-cell communication in prostate cancer cells.
The results demonstrated that while PC3 control cells (no
detectable STEAP-1 protein expression) exhibit little calcein
transfer, the expression of STEAP-1 allows the transfer of small
molecules between cells, whereby the initially red recipient cells
take on a brownish color, and co-localize the red and green
molecules.
[0059] FIG. 20. The figure demonstrates that STEAP-1 mediates the
transfer of the small molecule calcein between adjacent cells, and
thereby regulates cell-cell communication in prostate cancer cells.
The figure shows the time-dependent manner of cell-cell
communication mediated by STEAP-1, where little transfer in
PC3-STEAP-1 cells is seen at 6 hours, and much transfer is seen at
24 hours.
[0060] FIG. 21. This figure shows whether expression of STEAP-1 is
sufficient to regulate specific signaling pathways not otherwise
active in resting PC3 cells, the effect of these genes on the
activation of the p38 MAPK cascade was investigated in the prostate
cancer cell line PC3. FIG. 21A shows that while expression of the
control neo gene has no effect on p38 phosphorylation, expression
of STEAP-1 in PC3 cells is sufficient to induce the activation of
the p38 pathway. FIG. 21B shows the results were verified using
western blotting with an anti-p38 Ab, which shows equal protein
loading on the gels.
[0061] FIG. 22. This figure shows the sufficiency of expression of
STEAP-1 in the prostate cancer cell line PC3 to activate the
mitogenic MAPK pathway, namely the ERK cascade. FIG. 22A shows that
while expression of the control neo gene has no effect on ERK
phosphorylation, expression of STEAP-1 in PC3 cells is sufficient
to induce an increase in ERK phosphorylation. FIG. 22B shows these
results were verified using anti-ERK western blotting and confirm
the activation of the ERK pathway by STEAP-1 and STEAP-2.
[0062] FIG. 23. Shows necessary expression of STEAP-1 on Donor and
Recipient Cells.
[0063] FIG. 24. Shows that the introduction of STEAP-1 specific
RNAi reduces the expression of STEAP-1 in recombinant 3T3 and Rat-1
cells. Whole cell immunostaining revealed that the STEAP-1 RNAi
reduced STEAP-1 expression in Rat-1 and 3T3 cells. This reduction
was confirmed by Western blot analysis where STEAP-1 protein was
substantially reduced in STEAP-1 RNAi treated cells relative to
control and untreated cells.
[0064] FIG. 25. Shows that RNAi reduces the endogenous expression
of STEAP-1 in the prostate cancer and LNCaP cell line.
DETAILED DESCRIPTION OF THE INVENTION
[0065] Outline of Sections
[0066] I.) Definitions
[0067] II.) STEAP-1 Polynucleotides
[0068] II.A.) Uses of STEAP-1 Polynucleotides
[0069] II.A.1.) Monitoring of Genetic Abnormalities
[0070] II.A.2.) Antisense Embodiments
[0071] II.A.3.) Primers and Primer Pairs [0072] II.A.4.) Isolation
of STEAP-1-Encoding Nucleic Acid Molecules
[0073] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0074] III.) STEAP-1-related Proteins [0075] III.A.) Motif-bearing
Protein Embodiments [0076] III.B.) Expression of STEAP-1-related
Proteins [0077] III.C.) Modifications of STEAP-1-related Proteins
[0078] III.D.) Uses of STEAP-1-related Proteins
[0079] IV.) STEAP-1 Antibodies
[0080] V.) STEAP-1 Cellular Immune Responses
[0081] VI.) STEAP-1 Transgenic Animals
[0082] VII.) Methods for the Detection of STEAP-1
[0083] VIII.) Methods for Monitoring the Status of STEAP-1-related
Genes and Their Products
[0084] IX.) Identification of Molecules That Interact With
STEAP-1
[0085] X.) Therapeutic Methods and Compositions [0086] X.A.)
Anti-Cancer Vaccines
[0087] X.B.) STEAP-1 as a Target for Antibody-Based Therapy
[0088] X.C.) STEAP-1 as a Target for Cellular Immune Responses
[0089] X.C.1. Minigene Vaccines [0090] X.C.2. Combinations of CTL
Peptides with Helper Peptides [0091] X.C.3. Combinations of CTL
Peptides with T Cell Priming Agents
[0092] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides [0093] X.D.) Adoptive Immunotherapy
[0094] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0095] XI.) Diagnostic and Prognostic Embodiments of STEAP-1.
[0096] XII.) Inhibition of STEAP-1 Protein Function [0097] XII.A.)
Inhibition of STEAP-1 With Intracellular Antibodies [0098] XII.B.)
Inhibition of STEAP-1 with Recombinant Proteins [0099] XII.C.)
Inhibition of STEAP-1 Transcription or Translation
[0100] XII.D.) General Considerations for Therapeutic
Strategies
[0101] XIII.) KITS/Articles of Manufacture
[0102] I.) Definitions:
[0103] 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.
[0104] The terms "advanced prostate cancer", "locally advanced
prostate cancer", "advanced disease" and "locally advanced disease"
mean prostate cancers that have extended through the prostate
capsule, and are meant to include stage C disease under the
American Urological Association (AUA) system, stage C.sub.1-C.sub.2
disease under the Whitmore-Jewett system, and stage T3-T4 and N+
disease under the TNM (tumor, node, metastasis) system. In general,
surgery is not recommended for patients with locally advanced
disease, and these patients have substantially less favorable
outcomes compared to patients having clinically localized
(organ-confined) prostate cancer. Locally advanced disease is
clinically identified by palpable evidence of induration beyond the
lateral border of the prostate, or asymmetry or induration above
the prostate base. Locally advanced prostate cancer is presently
diagnosed pathologically following radical prostatectomy if the
tumor invades or penetrates the prostatic capsule, extends into the
surgical margin, or invades the seminal vesicles.
[0105] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence STEAP-1 (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 STEAP-1. 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.
[0106] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a STEAP-1-related protein). For example, an analog
of a STEAP-1 protein can be specifically bound by an antibody or T
cell that specifically binds to STEAP-1.
[0107] 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-STEAP-1 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.
[0108] 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-STEAP-1 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-STEAP-1 antibody compositions with polyepitopic
specificity.
[0109] 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."
[0110] The term "cytotoxic agent" refers to a substance that
inhibits or prevents the expression activity of cells, function of
cells and/or causes destruction of cells. The term is intended to
include radioactive isotopes chemotherapeutic agents, and toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof. Examples of cytotoxic agents include, but
are not limited to auristatins, auromycins, maytansinoids, yttrium,
bismuth, ricin, ricin A-chain, combrestatin, duocarmycins,
dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065,
ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,
diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A
chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,
retstrictocin, phenomycin, enomycin, curicin, crotin,
calicheamicin, 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.
[0111] 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.
[0112] "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).
[0113] 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.
[0114] 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 STEAP-1 genes or that encode
polypeptides other than STEAP-1 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated STEAP-1 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the STEAP-1 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated STEAP-1 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0115] 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.
[0116] The terms "metastatic prostate cancer" and "metastatic
disease" mean prostate cancers that have spread to regional lymph
nodes or to distant sites, and are meant to include stage D disease
under the AUA system and stage T.times.N.times.M+ under the TNM
system. As is the case with locally advanced prostate cancer,
surgery is generally not indicated for patients with metastatic
disease, and hormonal (androgen ablation) therapy is a preferred
treatment modality. Patients with metastatic prostate cancer
eventually develop an androgen-refractory state within 12 to 18
months of treatment initiation. Approximately half of these
androgen-refractory patients die within 6 months after developing
that status. The most common site for prostate cancer metastasis is
bone. Prostate cancer bone metastases are often osteoblastic rather
than osteolytic (i.e., resulting in net bone formation). Bone
metastases are found most frequently in the spine, followed by the
femur, pelvis, rib cage, skull and humerus. Other common sites for
metastasis include lymph nodes, lung, liver and brain. Metastatic
prostate cancer is typically diagnosed by open or laparoscopic
pelvic lymphadenectomy, whole body radionuclide scans, skeletal
radiography, and/or bone lesion biopsy.
[0117] 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.
[0118] A "{dot over (m)}otif", as in biological motif of a
STEAP-1-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.
[0119] 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.
[0120] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0121] 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).
[0122] 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".
[0123] An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the HLA
molecule. One to three, usually two, primary anchor residues within
a peptide of defined length generally defines a "motif" for an
immunogenic peptide. These residues are understood to fit in close
contact with peptide binding groove of an HLA molecule, with their
side chains buried in specific pockets of the binding groove. In
one embodiment, for example, the primary anchor residues for an HLA
class I molecule are located at position 2 (from the amino terminal
position) and at the carboxyl terminal position of a 8, 9, 10, 11,
or 12 residue peptide epitope in accordance with the invention. In
another embodiment, for example, the primary anchor residues of a
peptide that will bind an HLA class II molecule are spaced relative
to each other, rather than to the termini of a peptide, where the
peptide is generally of at least 9 amino acids in length. The
primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the
primary and/or secondary anchor positions shown in Table IV. Such
analogs are used to modulate the binding affinity and/or population
coverage of a peptide comprising a particular HLA motif or
supermotif.
[0124] "Radioisotopes" include, but are not limited to the
following (non-limiting exemplary uses are also set forth):
TABLE-US-00001 Examples of Medical Isotopes: Isotope Description of
use Actinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227
Parent of Radium-223 (Ra-223) which is an alpha emitter used to
treat metastases in the skeleton (AC-227) resulting from cancer
(i.e., breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212 See Thorium-228 (Th-228) (Bi-212) Bismuth-213 See
Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancer detection (Cd-109)
Cobalt-60 Radiation source for radiotherapy of cancer, for food
irradiators, and for sterilization of medical (Co-60) supplies
Copper-64 A positron emitter used for cancer therapy and SPECT
imaging (Cu-64) Copper-67 Beta/gamma emitter used in cancer
radioimmunotherapy and diagnostic studies (i.e., breast and (Cu-67)
colon cancers, and lymphoma) Dysprosium-166 Cancer
radioimmunotherapy (Dy-166) Erbium-169 Rheumatoid arthritis
treatment, particularly for the small joints associated with
fingers and toes (Er-169) Europium-152 Radiation source for food
irradiation and for sterilization of medical supplies (Eu-152)
Europium-154 Radiation source for food irradiation and for
sterilization of medical supplies (Eu-154) Gadolinium-153
Osteoporosis detection and nuclear medical quality assurance
devices (Gd-153) Gold-198 Implant and intracavity therapy of
ovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiple
myeloma treatment in targeted skeletal therapy, cancer
radioimmunotherapy, bone marrow (Ho-166) ablation, and rheumatoid
arthritis treatment Iodine-125 Osteoporosis detection, diagnostic
imaging, tracer drugs, brain cancer treatment, radiolabeling,
(I-125) tumor imaging, mapping of receptors in the brain,
interstitial radiation therapy, brachytherapy for treatment of
prostate cancer, determination of glomerular filtration rate (GFR),
determination of plasma volume, detection of deep vein thrombosis
of the legs Iodine-131 Thyroid function evaluation, thyroid disease
detection, treatment of thyroid cancer as well as other (I-131)
non-malignant thyroid diseases (i.e., Graves disease, goiters, and
hyperthyroidism), treatment of leukemia, lymphoma, and other forms
of cancer (e.g., breast cancer) using radioimmunotherapy
Iridium-192 Brachytherapy, brain and spinal cord tumor treatment,
treatment of blocked arteries (i.e., (Ir-192) arteriosclerosis and
restenosis), and implants for breast and prostate tumors
Lutetium-177 Cancer radioimmnunotherapy and treatment of blocked
arteries (i.e., arteriosclerosis and restenosis) (Lu-177)
Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used for
imaging the brain, liver, lungs, heart, and (Mo-99) other organs.
Currently, Tc-99m is the most widely used radioisotope used for
diagnostic imaging of various cancers and diseases involving the
brain, heart, liver, lungs; also used in detection of deep vein
thrombosis of the legs Osmium-194 Cancer radioimmunotherapy
(Os-194) Palladium-103 Prostate cancer treatment (Pd-103)
Platinum-195m Studies on biodistribution and metabolism of
cisplatin, a chemotherapeutic drug (Pt-195m) Phosphorus-32
Polycythemia rubra vera (blood cell disease) and leukemia
treatment, bone cancer (P-32) diagnosis/treatment; colon,
pancreatic, and liver cancer treatment; radiolabeling nucleic acids
for in vitro research, diagnosis of superficial tumors, treatment
of blocked arteries (i.e., arteriosclerosis and restenosis), and
intracavity therapy Phosphorus-33 Leukemia treatment, bone disease
diagnosis/treatment, radiolabeling, and treatment of blocked (P-33)
arteries (i.e., arteriosclerosis and restenosis) Radium-223 See
Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,
rheumatoid arthritis treatment, and diagnosis and treatment of
lymphoma (Re-186) and bone, breast, colon, and liver cancers using
radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using
radioimmunotherapy, bone cancer pain relief, treatment of (Re-188)
rheumatoid arthritis, and treatment of prostate cancer Rhodium-105
Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer
treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy and bone
cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapy
and bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used in
brain studies, imaging of adrenal cortex by gamma-scintigraphy,
lateral (Se-75) locations of steroid secreting tumors, pancreatic
scanning, detection of hyperactive parathyroid glands, measure rate
of bile acid loss from the endogenous pool Strontium-85 Bone cancer
detection and brain scans (Sr-85) Strontium-89 Bone cancer pain
relief, multiple myeloma treatment, and osteoblastic therapy
(Sr-89) Technetium-99m See Molybdenum-99 (Mo-99) (Tc-99m)
Thorium-228 Parent of Bismuth-212 (Bi-212) which is an alpha
emitter used in cancer radioimmunotherapy (Th-228) Thorium-229
Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213
(Bi-213) which are alpha (Th-229) emitters used in cancer
radioimmunotherapy Thulium-170 Gamma source for blood irradiators,
energy source for implanted medical devices (Tm-170) Tin-117m
Cancer immunotherapy and bone cancer pain relief (Sn-117m)
Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for
cancer diagnostics/treatment, bone cancer pain (W-188) relief,
rheumatoid arthritis treatment, and treatment of blocked arteries
(i.e., arteriosclerosis and restenosis) Xenon-127 Neuroimaging of
brain disorders, high resolution SPECT studies, pulmonary function
tests, and (Xe-127) cerebral blood flow studies Ytterbium-175
Cancer radioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained
from irradiating Yttrium-89 (Y-89) for liver cancer treatment
(Y-90) Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90)
which is used for cancer radioimmunotherapy (i.e., (Y-91) lymphoma,
breast, colon, kidney, lung, ovarian, prostate, pancreatic, and
inoperable liver cancers)
[0125] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0126] Non-limiting examples of small molecules include compounds
that bind or interact with STEAP-1, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit STEAP-1
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, STEAP-1 protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions.
[0127] "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).
[0128] "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.
[0129] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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 STEAP-1
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.
[0134] The "STEAP-1-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 STEAP-1 proteins or fragments thereof, as well as fusion
proteins of a STEAP-1 protein and a heterologous polypeptide are
also included. Such STEAP-1 proteins are collectively referred to
as the STEAP-1-related proteins, the proteins of the invention, or
STEAP-1. The term "STEAP-1-related protein" refers to a polypeptide
fragment or a STEAP-1 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.
[0135] II.) STEAP-1 Polynucleotides
[0136] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of a STEAP-1 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding a STEAP-1-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to a STEAP-1 gene
or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to a STEAP-1 gene, mRNA, or to a
STEAP-1 encoding polynucleotide (collectively, "STEAP-1
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0137] Embodiments of a STEAP-1 polynucleotide include: a STEAP-1
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of STEAP-1 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 STEAP-1 nucleotides comprise, without
limitation: [0138] (I) a polynucleotide comprising, consisting
essentially of, or consisting of a sequence as shown in FIG. 2A,
wherein T can also be U; [0139] (II) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2A, from nucleotide residue number 66 through nucleotide
residue number 1085, including the stop codon, wherein T can also
be U; [0140] (III) a polynucleotide comprising, consisting
essentially of, or consisting of the sequence as shown in FIG. 2B,
from nucleotide residue number 96 through nucleotide residue number
872, including the stop codon, wherein T can also be U; [0141] (IV)
a polynucleotide comprising, consisting essentially of, or
consisting of the sequence as shown in FIG. 2C, from nucleotide
residue number 96 through nucleotide residue number 944, including
the a stop codon, wherein T can also be U; [0142] (V) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2D, from nucleotide residue number
96 through nucleotide residue number 872, including the stop codon,
wherein T can also be U; [0143] (VI) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2E, from nucleotide residue number 96 through nucleotide
residue number 872, including the stop codon, wherein T can also be
U; [0144] (VII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2F, from
nucleotide residue number 96 through nucleotide residue number 872,
including the stop codon, wherein T can also be U; [0145] (VIII) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2G, from nucleotide residue number
96 through nucleotide residue number 872, including the stop codon,
wherein T can also be U; [0146] (IX) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2H, from nucleotide residue number 96 through nucleotide
residue number 872, including the stop codon, wherein T can also be
U; [0147] (X) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 21, from
nucleotide residue number 96 through nucleotide residue number 872,
including the stop codon, wherein T can also be U; [0148] (XII) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2J, from nucleotide residue number
96 through nucleotide residue number 872, including the stop codon,
wherein T can also be U; [0149] (XIII) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2K, from nucleotide residue number 96 through nucleotide
residue number 872, including the stop codon, wherein T can also be
U; [0150] (XIV) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2L, from
nucleotide residue number 96 through nucleotide residue number 872,
including the stop codon, wherein T can also be U; [0151] (XV) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2M, from nucleotide residue number
96 through nucleotide residue number 872, including the stop codon,
wherein T can also be U; [0152] (XVI) a polynucleotide comprising,
consisting essentially of, or consisting of the sequence as shown
in FIG. 2N, from nucleotide residue number 96 through nucleotide
residue number 872, including the stop codon, wherein T can also be
U; [0153] (XVII) a polynucleotide comprising, consisting
essentially of, or consisting of the sequence as shown in FIG. 20,
from nucleotide residue number 96 through nucleotide residue number
872, including the stop codon, wherein T can also be U; [0154]
(XVIII) a polynucleotide comprising, consisting essentially of, or
consisting of the sequence as shown in FIG. 2P, from nucleotide
residue number 96 through nucleotide residue number 872, including
the stop codon, wherein T can also be U; [0155] (XIX) a
polynucleotide comprising, consisting essentially of, or consisting
of the sequence as shown in FIG. 2Q, from nucleotide residue number
96 through nucleotide residue number 872, including the stop codon,
wherein T can also be U; [0156] (XX) a polynucleotide that encodes
a STEAP-1-related protein that is at least 90% homologous to an
entire amino acid sequence shown in FIG. 2A-Q; [0157] (XXI) a
polynucleotide that encodes a STEAP-1-related protein that is at
least 90% identical to an entire amino acid sequence shown in FIG.
2A-Q; [0158] (XXII) a polynucleotide that encodes at least one
peptide set forth in Tables V-XVIII and XXII-LI; [0159] (XXIII) a
polynucleotide that encodes a peptide region of at least 5 amino
acids of a peptide of FIG. 3A in any whole number increment up to
339 that includes an amino acid position having a value greater
than 0.5 in the Hydrophilicity profile of FIG. 5; [0160] (XXIV) a
polynucleotide that encodes a peptide region of at least 5 amino
acids of a peptide of FIG. 3A in any whole number increment up to
339 that includes an amino acid position having a value less than
0.5 in the Hydropathicity profile of FIG. 6; [0161] (XV) a
polynucleotide that encodes a peptide region of at least 5 amino
acids of a peptide of FIG. 3A in any whole number increment up to
339 that includes an amino acid position having a value greater
than 0.5 in the Percent Accessible Residues profile of FIG. 7;
[0162] (XVI) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 339 that includes an amino acid position having a
value greater than 0.5 in the Average Flexibility profile of FIG.
8; [0163] (XXVII) a polynucleotide that encodes a peptide region of
at least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 339 that includes an amino acid position having a
value greater than 0.5 in the Beta-turn profile of FIG. 9; [0164]
(XXVIII) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XIX). [0165] (XXIX) a peptide
that is encoded by any of (I)-(XIX); and [0166] (XXX) a
polynucleotide of any of (I)-(XIX) or peptide of (XXIII-XXVII)
together with a pharmaceutical excipient and/or in a human unit
dose form.
[0167] As used herein, a range is understood to disclose
specifically all whole unit positions thereof.
[0168] Typical embodiments of the invention disclosed herein
include STEAP-1 polynucleotides that encode specific portions of
STEAP-1 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0169] (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, 330, 339 or more contiguous amino acids of STEAP-1
variant 1; the maximal lengths relevant for other variants are:
variant 2, 258 amino acids; variant 3, 282 amino acids, and variant
4, 258 amino acids.
[0170] 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 STEAP-1 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the STEAP-1
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the STEAP-1 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the STEAP-1 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the STEAP-1 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the STEAP-1
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the STEAP-1 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the STEAP-1 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 STEAP-1 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.
[0171] Polynucleotides encoding relatively long portions of a
STEAP-1 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 STEAP-1 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 STEAP-1
sequence as shown in FIG. 2.
[0172] Additional illustrative embodiments of the invention
disclosed herein include STEAP-1 polynucleotide fragments encoding
one or more of the biological motifs contained within a STEAP-1
protein "or variant" sequence, including one or more of the
motif-bearing subsequences of a STEAP-1 protein "or variant" set
forth in Tables V-XVIII and XXII-LI. In another embodiment, typical
polynucleotide fragments of the invention encode one or more of the
regions of STEAP-1 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 STEAP-1
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.
[0173] Note that to determine the starting position of any peptide
set forth in Tables V-XVIII and Tables XXII to LI (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 LXI. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X minus I" to each position in Tables V-XVIII 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.
[0174] II.A.) Uses of STEAP-1 Polynucleotides
[0175] II.A.1.) Monitoring of Genetic Abnormalities
[0176] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human STEAP-1 gene maps to
the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of STEAP-1." For example, because the STEAP-1
gene maps to this chromosome, polynucleotides that encode different
regions of the STEAP-1 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(34): 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 STEAP-1 proteins provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes STEAP-1 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)).
[0177] Furthermore, as STEAP-1 was shown to be highly expressed in
bladder and other cancers, STEAP-1 polynucleotides are used in
methods assessing the status of STEAP-1 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the STEAP-1 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 STEAP-1 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.
[0178] II.A.2.) Antisense Embodiments
[0179] 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 STEAP-1. 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 STEAP-1 polynucleotides and polynucleotide
sequences disclosed herein.
[0180] 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., STEAP-1. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The STEAP-1 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 STEAP-1 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).
[0181] The STEAP-1 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 STEAP-1 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 STEAP-1 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, STEAP-1 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to STEAP-1 mRNA. Optionally, STEAP-1 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
STEAP-1. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of STEAP-1 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12:
510-515 (1996).
[0182] II.A.3.) Primers and Primer Pairs
[0183] 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 STEAP-1 polynucleotide in a sample and as a means for
detecting a cell expressing a STEAP-1 protein.
[0184] Examples of such probes include polypeptides comprising all
or part of the human STEAP-1 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying STEAP-1
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 STEAP-1 mRNA.
[0185] The STEAP-1 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
STEAP-1 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 STEAP-1
polypeptides; as tools for modulating or inhibiting the expression
of the STEAP-1 gene(s) and/or translation of the STEAP-1
transcript(s); and as therapeutic agents.
[0186] The present invention includes the use of any probe as
described herein to identify and isolate a STEAP-1 or STEAP-1
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.
[0187] II.A.4.) Isolation of STEAP-1-Encoding Nucleic Acid
Molecules
[0188] The STEAP-1 cDNA sequences described herein enable the
isolation of other polynucleotides encoding STEAP-1 gene
product(s), as well as the isolation of polynucleotides encoding
STEAP-1 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a STEAP-1 gene product as
well as polynucleotides that encode analogs of STEAP-1-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding a STEAP-1 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 STEAP-1 gene cDNAs can be identified by
probing with a labeled STEAP-1 cDNA or a fragment thereof. For
example, in one embodiment, a STEAP-1 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 STEAP-1 gene.
A STEAP-1 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with STEAP-1
DNA probes or primers.
[0189] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0190] The invention also provides recombinant DNA or RNA molecules
containing a STEAP-1 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).
[0191] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a STEAP-1
polynucleotide, fragment, analog or homologue thereof within a
suitable prokaryotic or eukaryotic host cell. Examples of suitable
eukaryotic host cells include a yeast cell, a plant cell, or an
animal cell, such as a mammalian cell or an insect cell (e.g., a
baculovirus-infectible cell such as an Sf9 or HighFive cell).
Examples of suitable mammalian cells include various prostate
cancer cell lines such as DU145 and TsuPr1, other transfectable or
transducible prostate cancer cell lines, primary cells (PrEC), as
well as a number of mammalian cells routinely used for the
expression of recombinant proteins (e.g., COS, CHO, 293, 293T
cells). More particularly, a polynucleotide comprising the coding
sequence of STEAP-1 or a fragment, analog or homolog thereof can be
used to generate STEAP-1 proteins or fragments thereof using any
number of host-vector systems routinely used and widely known in
the art.
[0192] A wide range of host-vector systems suitable for the
expression of STEAP-1 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, STEAP-1
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 STEAP-1 protein or fragment thereof. Such
host-vector systems can be employed to study the functional
properties of STEAP-1 and STEAP-1 mutations or analogs.
[0193] Recombinant human STEAP-1 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a STEAP-1-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding STEAP-1 or fragment, analog or homolog thereof, a
STEAP-1-related protein is expressed in the 293T cells, and the
recombinant STEAP-1 protein is isolated using standard purification
methods (e.g., affinity purification using anti-STEAP-1
antibodies). In another embodiment, a STEAP-1 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 STEAP-1 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 STEAP-1 coding sequence can be used for the generation
of a secreted form of recombinant STEAP-1 protein.
[0194] As discussed herein, redundancy in the genetic code permits
variation in STEAP-1 gene sequences. In particular, it is known in
the art that specific host species often have specific codon
preferences, and thus one can adapt the disclosed sequence as
preferred for a desired host. For example, preferred analog codon
sequences typically have rare codons (i.e., codons having a usage
frequency of less than about 20% in known sequences of the desired
host) replaced with higher frequency codons. Codon preferences for
a specific species are calculated, for example, by utilizing codon
usage tables available on the INTERNET such as at URL
dna.affrc.go.jp/.about.nakamura/codon.html.
[0195] 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)).
[0196] III.) STEAP-1-Related Proteins
[0197] Another aspect of the present invention provides
STEAP-1-related proteins. Specific embodiments of STEAP-1 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human STEAP-1 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of STEAP-1 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of STEAP-1 shown in FIG. 2 or FIG. 3.
[0198] Embodiments of a STEAP-1 polypeptide include: a STEAP-1
polypeptide having a sequence shown in FIG. 2, a peptide sequence
of a STEAP-1 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 STEAP-1 peptides comprise, without
limitation: [0199] (I) an protein comprising, consisting
essentially of, or consisting of an amino acid sequence as shown in
FIG. 2A-Q or FIG. 3A-D; [0200] (II) a 101P3A11-related protein that
is at least 90% homologous to an entire amino acid sequence shown
in FIG. 2A-Q; [0201] (III) a 101P3A11-related protein that is at
least 90% identical to an entire amino acid sequence shown in FIG.
2A-Q or 3A-D; [0202] (IV) a protein that comprises at least one
peptide set forth in Tables V-XVIII or Tables XXII to LI,
optionally with a proviso that it is not an entire protein of FIG.
2; [0203] (V) a protein that comprises at least one peptide set
forth in Tables V-XVIII, collectively, which peptide is also set
forth in Tables XXII to LI, collectively, optionally with a proviso
that it is not an entire protein of FIG. 2; [0204] (VI) a protein
that comprises at least two peptides selected from the peptides set
forth in Tables V-XVIII and XXII to LI collectively, optionally
with a proviso that it is not an entire protein of FIG. 2; [0205]
(VII) a protein that comprises at least two peptides selected from
the peptides set forth in Tables V-XVIII and XXII to LI
collectively, with a proviso that the protein is not a contiguous
sequence from an amino acid sequence of FIG. 2; [0206] (VIII) a
protein that comprises at least one peptide selected from the
peptides set forth in Tables V-XVIII; and at least one peptide set
forth in Tables XXII to LI, with a proviso that the protein is not
a contiguous sequence from an amino acid sequence of FIG. 2; [0207]
(IX) a polypeptide comprising at least 5 amino acids of a protein
of FIG. 3A, 3B, 3C, or 3D in any whole number increment up to 339,
258, 282, or 258 respectively that includes an amino acid position
having a value greater than 0.5 in the Hydrophilicity profile of
FIG. 5; [0208] (X) a polypeptide comprising at least 5 amino acids
of a protein of FIG. 3A, 3B, 3C, or 3D in any whole number
increment up to 339, 258, 282, or 258, respectively, that includes
an amino acid position having a value less than 0.5 in the
Hydropathicity profile of FIG. 6; [0209] (XI) a polypeptide
comprising at least 5 amino acids of a protein of FIG. 3A, 3B, 3C,
or 3D in any whole number increment up to 339, 258, 282, or 258,
respectively, that includes an amino acid position having a value
greater than 0.5 in the Percent Accessible Residues profile of FIG.
7; [0210] (XII) a polypeptide comprising at least 5 amino acids of
a protein of FIG. 3A, 3B, 3C, or 3D in any whole number increment
up to 339, 258, 282, or 258, respectively, that includes an amino
acid position having a value greater than 0.5 in the Average
Flexibility profile of FIG. 8; [0211] (XIII) a polypeptide
comprising at least 5 amino acids of a protein of FIG. 3A, 3B, 3C,
or 3D in any whole number increment up to 339, 258, 282, or 258,
respectively, that includes an amino acid position having a value
greater than 0.5 in the Beta-turn profile of FIG. 9; [0212] (XIV) a
peptide that occurs at least twice in Tables V-XVIII and XXII to
LI, collectively; [0213] (XV) a peptide that occurs at least three
times in Tables V-XVIII and XXII to LI, collectively; [0214] (XVI)
a peptide that occurs at least four times in Tables V-XVIII and
XXII to LI, collectively; [0215] (XVII) a peptide that occurs at
least five times in Tables V-XVIII and XXII to LI, collectively;
[0216] (XVIII) a peptide that occurs at least once in Tables
V-XVIII, and at least once in tables XXII to LI; [0217] (XIX) a
peptide which comprises one two, three, four, or five of the
following characteristics, or an oligonucleotide encoding such
peptide: [0218] 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; [0219] ii) a region of at least S
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; [0220] 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; [0221]
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, [0222] 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; [0223] (XX) a peptide of (I)-(XIX) together with a
pharmaceutical excipient and/or in a human unit dose form.
[0224] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0225] Typical embodiments of the invention disclosed herein
include 101P3A11 polynucleotides that encode specific portions of
101P3A11 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0226] (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, 330, 339 or more contiguous amino acids of STEAP-1
variant 1; the maximal lengths relevant for other variants are:
variant 2, 258 amino acids; variant 3, 282 amino acids, and variant
4, 258 amino acids.
[0227] In general, naturally occurring allelic variants of human
STEAP-1 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
STEAP-1 protein contain conservative amino acid substitutions
within the STEAP-1 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of STEAP-1. One class of STEAP-1 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular STEAP-1 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.
[0228] 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).
[0229] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of STEAP-1 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. STEAP-1 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
STEAP-1 variant DNA.
[0230] 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.
[0231] As defined herein, STEAP-1 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a STEAP-1 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
STEAP-1 variant also specifically binds to a STEAP-1 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 STEAP-1 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.
[0232] Other classes of STEAP-1-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 STEAP-1
protein variants or analogs comprises one or more of the STEAP-1
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of STEAP-1
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.
[0233] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a STEAP-1 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 STEAP-1 protein shown in
FIG. 2 or FIG. 3.
[0234] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of a STEAP-1 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a STEAP-1 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of a
STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of a STEAP-1 protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a STEAP-1 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 60 to about
amino acid 70 of a STEAP-1 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 70 to about amino acid
80 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 80 to about amino acid 90 of a
STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 90 to about amino acid 100 of a STEAP-1 protein
shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a
STEAP-1 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 STEAP-1 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.
[0235] STEAP-1-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
STEAP-1-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a STEAP-1 protein
(or variants, homologs or analogs thereof).
[0236] III.A.) Motif-Bearing Protein Embodiments
[0237] Additional illustrative embodiments of the invention
disclosed herein include STEAP-1 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a STEAP-1 polypeptide sequence set forth in FIG. 2 or FIG.
3. Various motifs are known in the art, and a protein can be
evaluated for the presence of such motifs by a number of publicly
available Internet sites (see, e.g., URL addresses:
pfam.wustl.edu/;
http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;
psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/;
ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html;
Epimatrix.TM. and Epimer.TM., Brown University,
brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dcrt.nih.gov/.).
[0238] Motif bearing subsequences of all STEAP-1 variant proteins
are set forth and identified in Tables V-XVIII and XXII-LI.
[0239] Table XIX sets forth several frequently occurring motifs
based on pfam searches (see URL address pfam.wustl.edu/). The
columns of Table XIX 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.
[0240] Polypeptides comprising one or more of the STEAP-1 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the STEAP-1 motifs discussed above are associated with growth
dysregulation and because STEAP-1 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)).
[0241] In another embodiment, proteins of the invention comprise
one or more of the immunoreactive epitopes identified in accordance
with art-accepted methods, such as the peptides set forth in Tables
V-XVIII and XXII-LI. CTL epitopes can be determined using specific
algorithms to identify peptides within a STEAP-1 protein that are
capable of optimally binding to specified HLA alleles (e.g., Table
IV; Epimatrix.TM. and Epimer.TM., Brown University, URL
brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying
peptides that have sufficient binding affinity for HLA molecules
and which are correlated with being immunogenic epitopes, are well
known in the art, and are carried out without undue
experimentation. In addition, processes for identifying peptides
that are immunogenic epitopes, are well known in the art, and are
carried out without undue experimentation either in vitro or in
vivo.
[0242] Also known in the art are principles for creating analogs of
such epitopes in order to modulate immunogenicity. For example, one
begins with an epitope that bears a CTL or HTL motif (see, e.g.,
the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
The epitope is analoged by substituting out an amino acid at one of
the specified positions, and replacing it with another amino acid
specified for that position. For example, one can substitute out a
deleterious residue in favor of any other residue, such as a
preferred residue as defined in Table IV; substitute a
less-preferred residue with a preferred residue as defined in Table
IV; or substitute an originally-occurring preferred residue with
another preferred residue as defined in Table IV. Substitutions can
occur at primary anchor positions or at other positions in a
peptide; see, e.g., Table IV.
[0243] 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.
[0244] Related embodiments of the invention include polypeptides
comprising combinations of the different motifs set forth in Table
XX, and/or, one or more of the predicted CTL epitopes of Tables
V-XVII and XXII-XLVII, and/or, one or more of the predicted HTL
epitopes of Tables XLVIII-LI, 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 ammo acid
residues, preferably 5 to about 50 amino acid residues.
[0245] STEAP-1-related proteins are embodied in many forms,
preferably in isolated form A purified STEAP-1 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of STEAP-1 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a STEAP-1-related proteins include
purified STEAP-1-related proteins and functional, soluble
STEAP-1-related proteins. In one embodiment, a functional, soluble
STEAP-1 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0246] The invention also provides STEAP-1 proteins comprising
biologically active fragments of a STEAP-1 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting STEAP-1 protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting STEAP-1 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.
[0247] STEAP-1-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-STEAP-1 antibodies or T cells or in identifying cellular
factors that bind to STEAP-1. 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.
[0248] CTL epitopes can be determined using specific algorithms to
identify peptides within a STEAP-1 protein that are capable of
optimally binding to specified HLA alleles (e.g., by using the
SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/;
the listings in Table IV(A)-(E); Epimatrix.TM. and Epimer.TM.,
Brown University, URL
(brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and
BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide
epitopes from STEAP-1 that are presented in the context of human
MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35
were predicted (see, e.g., Tables V-XVIII, XXII-LI). Specifically,
the complete amino acid sequence of the STEAP-1 protein and
relevant portions of other variants, i.e., for HLA Class I
predictions 9 flanking residues on either side of a point mutation,
and for H LA Class II predictions 14 flanking residues on either
side of a point mutation, were entered into the HLA Peptide Motif
Search algorithm found in the Bioinformatics and Molecular Analysis
Section (BIMAS) web site listed above; in addition to the site
SYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.
[0249] 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 STEAP-1 predicted binding peptides are shown in Tables
V-XVIII and XXII-LI herein. In Tables V-XVIII 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
XLVIII-LI, 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.
[0250] 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.
[0251] It is to be appreciated that every epitope predicted by the
BIMAS site, Epimer.TM. and Epimatrix.TM. sites, or specified by the
HLA class I or class II motifs available in the art or which become
part of the art such as set forth in Table IV (or determined using
World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS,
bimas.dcrt.nih.gov/) are to be "applied" to a STEAP-1 protein in
accordance with the invention. As used in this context "applied"
means that a STEAP-1 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 STEAP-1 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.
[0252] III.B.) Expression of STEAP-1-Related Proteins
[0253] In an embodiment described in the examples that follow,
STEAP-1 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 STEAP-1 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 STEAP-1 protein in transfected cells. The
secreted HIS-tagged STEAP-1 in the culture media can be purified,
e.g., using a nickel column using standard techniques.
[0254] III.C.) Modifications of STEAP-1-Related Proteins
[0255] Modifications of STEAP-1-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 STEAP-1 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a STEAP-1 protein. Another type of
covalent modification of a STEAP-1 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 STEAP-1 comprises linking a STEAP-1 polypeptide to
one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the
manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0256] The STEAP-1-related proteins of the present invention can
also be modified to form a chimeric molecule comprising STEAP-1
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 STEAP-1 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 STEAP-1. A chimeric
molecule can comprise a fusion of a STEAP-1-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 STEAP-1 protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
STEAP-1-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 STEAP-1 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0257] III.D.) Uses of STEAP-1-Related Proteins
[0258] The proteins of the invention have a number of different
specific uses. As STEAP-1 is highly expressed in prostate and other
cancers, STEAP-1-related proteins are used in methods that assess
the status of STEAP-1 gene products in normal versus cancerous
tissues, thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of a STEAP-1 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 STEAP-1-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within a STEAP-1 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,
STEAP-1-related proteins that contain the amino acid residues of
one or more of the biological motifs in a STEAP-1 protein are used
to screen for factors that interact with that region of
STEAP-1.
[0259] STEAP-1 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of a STEAP-1 protein), for identifying agents or cellular
factors that bind to STEAP-1 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.
[0260] Proteins encoded by the STEAP-1 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 STEAP-1 gene product. Antibodies raised against a STEAP-1
protein or fragment thereof are useful in diagnostic and prognostic
assays, and imaging methodologies in the management of human
cancers characterized by expression of STEAP-1 protein, such as
those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. STEAP-1-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0261] Various immunological assays useful for the detection of
STEAP-1 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
STEAP-1-expressing cells (e.g., in radioscintigraphic imaging
methods). STEAP-1 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
[0262] IV.) STEAP-1 Antibodies
[0263] Another aspect of the invention provides antibodies that
bind to STEAP-1-related proteins. Preferred antibodies specifically
bind to a STEAP-1-related protein and do not bind (or bind weakly)
to peptides or proteins that are not STEAP-1-related proteins. For
example, antibodies that bind STEAP-1 can bind STEAP-1-related
proteins such as the homologs or analogs thereof.
[0264] STEAP-1 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 STEAP-1 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 STEAP-1 is involved, such as
advanced or metastatic prostate cancers.
[0265] The invention also provides various immunological assays
useful for the detection and quantification of STEAP-1 and mutant
STEAP-1-related proteins. Such assays can comprise one or more
STEAP-1 antibodies capable of recognizing and binding a
STEAP-1-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.
[0266] 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.
[0267] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing STEAP-1 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled STEAP-1
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of STEAP-1 expressing cancers such as
prostate cancer.
[0268] STEAP-1 antibodies are also used in methods for purifying a
STEAP-1-related protein and for isolating STEAP-1 homologues and
related molecules. For example, a method of purifying a
STEAP-1-related protein comprises incubating a STEAP-1 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a STEAP-1-related protein under conditions that
permit the STEAP-1 antibody to bind to the STEAP-1-related protein;
washing the solid matrix to eliminate impurities; and eluting the
STEAP-1-related protein from the coupled antibody. Other uses of
STEAP-1 antibodies in accordance with the invention include
generating anti-idiotypic antibodies that mimic a STEAP-1
protein.
[0269] 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 STEAP-1-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 STEAP-1 can also be used, such as a
STEAP-1 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 STEAP-1-related
protein is synthesized and used as an immunogen.
[0270] In addition, naked DNA immunization techniques known in the
art are used (with or without purified STEAP-1-related protein or
STEAP-1 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0271] The amino acid sequence of a STEAP-1 protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
STEAP-1 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a STEAP-1 amino acid
sequence are used to identify hydrophilic regions in the STEAP-1
structure. Regions of a STEAP-1 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-A92. 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 STEAP-1 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, carbodimide reagents are used; in
other instances linking reagents such as those supplied by Pierce
Chemical Co., Rockford, Ill., are effective. Administration of a
STEAP-1 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.
[0272] STEAP-1 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
STEAP-1-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.
[0273] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a STEAP-1 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
STEAP-1 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.
[0274] 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 STEAP-1 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 STEAP-1
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued
19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and,
U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the
in vitro manipulation required with phage display technology and
efficiently produces high affinity authentic human antibodies.
[0275] Reactivity of STEAP-1 antibodies with a STEAP-1-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, STEAP-1-related proteins,
STEAP-1-expressing cells or extracts thereof. A STEAP-1 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 STEAP-1 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).
[0276] V.) STEAP-1 Cellular Immune Responses
[0277] 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.
[0278] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the
study of single amino acid substituted antigen analogs and the
sequencing of endogenously bound, naturally processed peptides,
critical residues that correspond to motifs required for specific
binding to HLA antigen molecules have been identified and are set
forth in Table IV (see also, e.g., Southwood, et al., J. Immunol.
160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al, SYFPEITHI, access via World Wide Web at URL
syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.
Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,
1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;
Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et
al., Cell 74:929-937, 1993; Kondo et al, J. Immunol. 155:4307-4312,
1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et
al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.
Immunogenetics 1999 November; 50(3-4):201-12, Review).
[0279] 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.)
[0280] 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).
[0281] 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.
[0282] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0283] 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.
[0284] 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.
[0285] 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.5, Cr release involving
peptide-sensitized targets, T cell proliferation, or lymphokine
release.
[0286] VI.) STEAP-1 Transgenic Animals
[0287] Nucleic acids that encode a STEAP-1-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 STEAP-1 can be used to clone genomic DNA
that encodes STEAP-1. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode STEAP-1. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. No.
4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued
26 Sep. 1989. Typically, particular cells would be targeted for
STEAP-1 transgene incorporation with tissue-specific enhancers.
[0288] Transgenic animals that include a copy of a transgene
encoding STEAP-1 can be used to examine the effect of increased
expression of DNA that encodes STEAP-1. 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.
[0289] Alternatively, non-human homologues of STEAP-1 can be used
to construct a STEAP-1 "knock out" animal that has a defective or
altered gene encoding STEAP-1 as a result of homologous
recombination between the endogenous gene encoding STEAP-1 and
altered genomic DNA encoding STEAP-1 introduced into an embryonic
cell of the animal. For example, cDNA that encodes STEAP-1 can be
used to clone genomic DNA encoding STEAP-1 in accordance with
established techniques. A portion of the genomic DNA encoding
STEAP-1 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 STEAP-1
polypeptide.
[0290] VII.) Methods for the Detection of STEAP-1
[0291] Another aspect of the present invention relates to methods
for detecting STEAP-1 polynucleotides and STEAP-1-related proteins,
as well as methods for identifying a cell that expresses STEAP-1.
The expression profile of STEAP-1 makes it a diagnostic marker for
metastasized disease. Accordingly, the status of STEAP-1 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 STEAP-1 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.
[0292] More particularly, the invention provides assays for the
detection of STEAP-1 polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable STEAP-1 polynucleotides
include, for example, a STEAP-1 gene or fragment thereof, STEAP-1
mRNA, alternative splice variant STEAP-1 mRNAs, and recombinant DNA
or RNA molecules that contain a STEAP-1 polynucleotide. A number of
methods for amplifying and/or detecting the presence of STEAP-1
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0293] In one embodiment, a method for detecting a STEAP-1 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 STEAP-1 polynucleotides as sense and
antisense primers to amplify STEAP-1 cDNAs therein; and detecting
the presence of the amplified STEAP-1 cDNA. Optionally, the
sequence of the amplified STEAP-1 cDNA can be determined.
[0294] In another embodiment, a method of detecting a STEAP-1 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using STEAP-1
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified STEAP-1 gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
STEAP-1 nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0295] The invention also provides assays for detecting the
presence of a STEAP-1 protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a STEAP-1-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 STEAP-1-related
protein in a biological sample comprises first contacting the
sample with a STEAP-1 antibody, a STEAP-1-reactive fragment
thereof, or a recombinant protein containing an antigen-binding
region of a STEAP-1 antibody; and then detecting the binding of
STEAP-1-related protein in the sample.
[0296] Methods for identifying a cell that expresses STEAP-1 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a STEAP-1 gene comprises
detecting the presence of STEAP-1 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 STEAP-1 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for STEAP-1, 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 STEAP-1 gene comprises detecting the presence of
STEAP-1-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 STEAP-1-related proteins
and cells that express STEAP-1-related proteins.
[0297] STEAP-1 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate STEAP-1 gene
expression. For example, STEAP-1 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 STEAP-1 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 STEAP-1 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0298] VIII.) Methods for Monitoring the Status of STEAP-1-Related
Genes and Their Products
[0299] 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 STEAP-1 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 STEAP-1 in a biological
sample of interest can be compared, for example, to the status of
STEAP-1 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 STEAP-1 in the
biological sample (as compared to the normal sample) provides
evidence of dysregulated cellular growth. In addition to using a
biological sample that is not affected by a pathology as a normal
sample, one can also use a predetermined normative value such as a
predetermined normal level of mRNA expression (see, e.g., Grever et
al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No.
5,837,501) to compare STEAP-1 status in a sample.
[0300] 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 STEAP-1
expressing cells) as well as the level, and biological activity of
expressed gene products (such as STEAP-1 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of STEAP-1
comprises a change in the location of STEAP-1 and/or STEAP-1
expressing cells and/or an increase in STEAP-1 mRNA and/or protein
expression.
[0301] STEAP-1 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 STEAP-1 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 STEAP-1 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 STEAP-1 gene), Northern analysis and/or PCR analysis of STEAP-1
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of STEAP-1 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
STEAP-1 proteins and/or associations of STEAP-1 proteins with
polypeptide binding partners). Detectable STEAP-1 polynucleotides
include, for example, a STEAP-1 gene or fragment thereof, STEAP-1
mRNA, alternative splice variants, STEAP-1 mRNAs, and recombinant
DNA or RNA molecules containing a STEAP-1 polynucleotide.
[0302] The expression profile of STEAP-1 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 STEAP-1 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining STEAP-1 status and diagnosing
cancers that express STEAP-1, such as cancers of the tissues listed
in Table I. For example, because STEAP-1 mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of STEAP-1 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with STEAP-1 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0303] The expression status of STEAP-1 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 STEAP-1 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.
[0304] As described above, the status of STEAP-1 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of STEAP-1 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 STEAP-1
expressing cells (e.g. those that express STEAP-1 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when STEAP-1-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 STEAP-1 in a biological sample are often associated with
dysregulated cellular growth. Specifically, one indicator of
dysregulated cellular growth is the metastases of cancer cells from
an organ of origin (such as the prostate) to a different area of
the body (such as a lymph node). In this context, evidence of
dysregulated cellular growth is important for example because
occult lymph node metastases can be detected in a substantial
proportion of patients with prostate cancer, and such metastases
are associated with known predictors of disease progression (see,
e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al.,
Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J. Urol
1995 August 154(2 Pt 1):474-8).
[0305] In one aspect, the invention provides methods for monitoring
STEAP-1 gene products by determining the status of STEAP-1 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 STEAP-1 gene products in a corresponding normal
sample. The presence of aberrant STEAP-1 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.
[0306] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in STEAP-1 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
STEAP-1 mRNA can, for example, be evaluated in tissues including
but not limited to those listed in Table I. The presence of
significant STEAP-1 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 STEAP-1 mRNA or
express it at lower levels.
[0307] In a related embodiment, STEAP-1 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of STEAP-1 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of STEAP-1 expressed in a corresponding
normal sample. In one embodiment, the presence of STEAP-1 protein
is evaluated, for example, using immunohistochemical methods.
STEAP-1 antibodies or binding partners capable of detecting STEAP-1
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0308] In a further embodiment, one can evaluate the status of
STEAP-1 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
STEAP-1 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in STEAP-1 indicates a potential loss of function or
increase in tumor growth.
[0309] 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 STEAP-1 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No.
5,952,170 issued 17 Jan. 1995).
[0310] Additionally, one can examine the methylation status of a
STEAP-1 gene in a biological sample. Aberrant demethylation and/or
hypermethylation of CpG islands in gene 5' regulatory regions
frequently occurs in immortalized and transformed cells, and can
result in altered expression of various genes. For example,
promoter hypermethylation of the pi-class glutathione S-transferase
(a protein expressed in normal prostate but not expressed in
>90% of prostate carcinomas) appears to permanently silence
transcription of this gene and is the most frequently detected
genomic alteration in prostate carcinomas (De Marzo et al., Am. J.
Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is
present in at least 70% of cases of high-grade prostatic
intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.
Biomarkers Prev., 1998, 7:531-536). In another example, expression
of the LAGE-1 tumor specific gene (which is not expressed in normal
prostate but is expressed in 25-50% of prostate cancers) is induced
by deoxy-azacytidine in lymphoblastoid cells, suggesting that
tumoral expression is due to demethylation (Lethe et al., Int. J.
Cancer 76(6): 903-908 (1998)). A variety of assays for examining
methylation status of a gene are well known in the art. For
example, one can utilize, in Southern hybridization approaches,
methylation-sensitive restriction enzymes that cannot cleave
sequences that contain methylated CpG sites to assess the
methylation status of CpG islands. In addition, MSP (methylation
specific PCR) can rapidly profile the methylation status of all the
CpG sites present in a CpG island of a given gene. This procedure
involves initial modification of DNA by sodium bisulfite (which
will convert all unmethylated cytosines to uracil) followed by
amplification using primers specific for methylated versus
unmethylated DNA. Protocols involving methylation interference can
also be found for example in Current Protocols In Molecular
Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
[0311] Gene amplification is an additional method for assessing the
status of STEAP-1. 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.
[0312] 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 STEAP-1 expression.
The presence of RT-PCR amplifiable STEAP-1 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).
[0313] 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 STEAP-1 mRNA or STEAP-1 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of STEAP-1 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of STEAP-1
in prostate or other tissue is examined, with the presence of
STEAP-1 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 STEAP-1 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 STEAP-1 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0314] 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
STEAP-1 mRNA or STEAP-1 protein expressed by tumor cells, comparing
the level so determined to the level of STEAP-1 mRNA or STEAP-1
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of STEAP-1 mRNA or STEAP-1 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 STEAP-1 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of STEAP-1 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.
[0315] 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 STEAP-1 mRNA or STEAP-1 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of STEAP-1 mRNA or STEAP-1 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of STEAP-1 mRNA or STEAP-1 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 STEAP-1 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
STEAP-1 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.
[0316] 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 STEAP-1 gene and STEAP-1 gene products (or
perturbations in STEAP-1 gene and STEAP-1 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 STEAP-1 gene
and STEAP-1 gene products (or perturbations in STEAP-1 gene and
STEAP-1 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.
[0317] In one embodiment, methods for observing a coincidence
between the expression of STEAP-1 gene and STEAP-1 gene products
(or perturbations in STEAP-1 gene and STEAP-1 gene products) and
another factor associated with malignancy entails detecting the
overexpression of STEAP-1 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
STEAP-1 mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of STEAP-1 and PSA mRNA in prostate tissue is examined, where the
coincidence of STEAP-1 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.
[0318] Methods for detecting and quantifying the expression of
STEAP-1 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 STEAP-1 mRNA include in situ hybridization using
labeled STEAP-1 riboprobes, Northern blot and related techniques
using STEAP-1 polynucleotide probes, RT-PCR analysis using primers
specific for STEAP-1, 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 STEAP-1 mRNA expression. Any number of primers
capable of amplifying STEAP-1 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
STEAP-1 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0319] IX.) Identification of Molecules that Interact with
STEAP-1
[0320] The STEAP-1 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with STEAP-1, as well as
pathways activated by STEAP-1 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999,
U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No.
5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21
Dec. 1999. Algorithms are also available in the art for
genome-based predictions of protein function (see, e.g., Marcotte,
et al., Nature 402: 4 Nov. 1999, 83-86).
[0321] Alternatively one can screen peptide libraries to identify
molecules that interact with STEAP-1 protein sequences. In such
methods, peptides that bind to STEAP-1 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 STEAP-1 protein(s).
[0322] 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 STEAP-1 protein sequences are disclosed for example
in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No.
5,733,731 issued 31 Mar. 1998.
[0323] Alternatively, cell lines that express STEAP-1 are used to
identify protein-protein interactions mediated by STEAP-1. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). STEAP-1 protein can be immunoprecipitated from
STEAP-1-expressing cell lines using anti-STEAP-1 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of STEAP-1 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.
[0324] Small molecules and ligands that interact with STEAP-1 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 STEAP-1'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
STEAP-1-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses STEAP-1 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2.sup.nd Ed., Sinauer Assoc., Sunderland,
Mass., 1992). Moreover, ligands that regulate STEAP-1 function can
be identified based on their ability to bind STEAP-1 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 STEAP-1 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
STEAP-1.
[0325] An embodiment of this invention comprises a method of
screening for a molecule that interacts with a STEAP-1 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a STEAP-1 amino acid
sequence, allowing the population of molecules and the STEAP-1
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the STEAP-1 amino acid sequence, and then separating molecules
that do not interact with the STEAP-1 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 STEAP-1 amino acid sequence. The identified
molecule can be used to modulate a function performed by STEAP-1.
In a preferred embodiment, the STEAP-1 amino acid sequence is
contacted with a library of peptides.
[0326] X.) Therapeutic Methods and Compositions
[0327] The identification of STEAP-1 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, STEAP-1 functions as a transcription factor
involved in activating tumor-promoting genes or repressing genes
that block tumorigenesis.
[0328] Accordingly, therapeutic approaches that inhibit the
activity of a STEAP-1 protein are useful for patients suffering
from a cancer that expresses STEAP-1. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a STEAP-1
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a STEAP-1 gene or translation of STEAP-1 mRNA.
[0329] X.A.) Anti-Cancer Vaccines
[0330] The invention provides cancer vaccines comprising a
STEAP-1-related protein or STEAP-1-related nucleic acid. In view of
the expression of STEAP-1, cancer vaccines prevent and/or treat
STEAP-1-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).
[0331] Such methods can be readily practiced by employing a
STEAP-1-related protein, or a STEAP-1-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the STEAP-1 immunogen (which typically comprises a
number of antibody or T cell epitopes). Skilled artisans understand
that a wide variety of vaccine systems for delivery of
immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., Cancer Immunol
Immunother 2000 June 49(3):123-32) Briefly, such methods of
generating an immune response (e.g. humoral and/or cell-mediated)
in a mammal, comprise the steps of: exposing the mammal's immune
system to an immunoreactive epitope (e.g. an epitope present in a
STEAP-1 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 STEAP-1 immunogen contains
a biological motif, see e.g., Tables V-XVIII and XXII-LI, or a
peptide of a size range from STEAP-1 indicated in FIG. 5, FIG. 6,
FIG. 7, FIG. 8, and FIG. 9.
[0332] The entire STEAP-1 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.
[0333] In patients with STEAP-1-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.
[0334] Cellular Vaccines:
[0335] CTL epitopes can be determined using specific algorithms to
identify peptides within STEAP-1 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University (URL
brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and,
BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL
syfpeithi.bmi-heidelberg.coni/). In a preferred embodiment, a
STEAP-1 immunogen contains one or more amino acid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables V-XVIII and XXII-LI 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.
[0336] Antibody-Based Vaccines
[0337] 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 STEAP-1 protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to STEAP-1 in a host,
by contacting the host with a sufficient amount of at least one
STEAP-1 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval thereafter re-contacting the host
with the STEAP-1 B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a STEAP-1-related protein or a man-made
multiepitopic peptide comprising: administering STEAP-1 immunogen
(e.g. a STEAP-1 protein or a peptide fragment thereof, a STEAP-1
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 STEAP-1 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 STEAP-1
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 STEAP-1, in
order to generate a response to the target antigen.
[0338] Nucleic Acid Vaccines:
[0339] 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 STEAP-1. Constructs comprising DNA encoding a
STEAP-1-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 STEAP-1 protein/immunogen.
Alternatively, a vaccine comprises a STEAP-1-related protein.
Expression of the STEAP-1-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a STEAP-1 protein. Various
prophylactic and therapeutic genetic immunization techniques known
in the art can be used (for review, see information and references
published at Internet address genweb.com). Nucleic acid-based
delivery is described, for instance, in Wolff et. al., Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples
of DNA-based delivery technologies include "naked DNA", facilitated
(bupivicaine, polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0340] 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
STEAP-1-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0341] 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.
[0342] Thus, gene delivery systems are used to deliver a
STEAP-1-related nucleic acid molecule. In one embodiment, the
full-length human STEAP-1 cDNA is employed. In another embodiment,
STEAP-1 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0343] Ex Vivo Vaccines
[0344] 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
STEAP-1 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 STEAP-1 peptides to T cells in the context of MHC class
I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with STEAP-1 peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete STEAP-1 protein. Yet another
embodiment involves engineering the overexpression of a STEAP-1
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 STEAP-1 can also be engineered to express immune
modulators, such as GM-CSF, and used as immunizing agents.
[0345] X.B.) STEAP-1 as a Target for Antibody-Based Therapy
[0346] STEAP-1 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 STEAP-1 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of STEAP-1-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 STEAP-1 are useful
to treat STEAP-1-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0347] STEAP-1 antibodies can be introduced into a patient such
that the antibody binds to STEAP-1 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 STEAP-1, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0348] 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 STEAP-1 sequence shown in FIG. 2 or FIG.
3. In addition, skilled artisans understand that it is routine to
conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al.
Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or
therapeutic agents are delivered directly to cells, such as by
conjugating them to antibodies specific for a molecule expressed by
that cell (e.g. STEAP-1), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0349] 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-STEAP-1
antibody) that binds to a marker (e.g. STEAP-1) 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 STEAP-1, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
STEAP-1 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.
[0350] Cancer immunotherapy using anti-STEAP-1 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.
55:43984403), 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, STEAP-1 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).
[0351] Although STEAP-1 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.
[0352] Although STEAP-1 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.
[0353] Cancer patients can be evaluated for the presence and level
of STEAP-1 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative STEAP-1 imaging, or other
techniques that reliably indicate the presence and degree of
STEAP-1 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.
[0354] Anti-STEAP-1 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-STEAP-1 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-STEAP-1 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express STEAP-1.
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-STEAP-1 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.
[0355] 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 STEAP-1 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0356] Therapeutic methods of the invention contemplate the
administration of single anti-STEAP-1 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-STEAP-1 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-STEAP-1 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0357] Anti-STEAP-1 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-STEAP-1 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.
[0358] 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-STEAP-1 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 STEAP-1 expression in the patient, the
extent of circulating shed STEAP-1 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.
[0359] Optionally, patients should be evaluated for the levels of
STEAP-1 in a given sample (e.g. the levels of circulating STEAP-1
antigen and/or STEAP-1 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).
[0360] Anti-idiotypic anti-STEAP-1 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a STEAP-1-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-STEAP-1 antibodies that mimic an epitope on a STEAP-1-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.
[0361] X.C.) STEAP-1 as a Target for Cellular Immune Responses
[0362] 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.
[0363] Carriers that can be used with vaccines of the invention are
well known in the art, and include, e.g., thyroglobulin, albumins
such as human serum albumin, tetanus toxoid, polyamino acids such
as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B
virus core protein, and the like. The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water,
or saline, preferably phosphate buffered saline. The vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, as
disclosed herein, CTL responses can be primed by conjugating
peptides of the invention to lipids, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS).
Moreover, an adjuvant such as a synthetic
cytosine-phosphorothiolated-guanine-containing (CpG)
oligonucleotides has been found to increase CTL responses 10- to
100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547
(2000)).
[0364] 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 STEAP-1 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0365] 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).
[0366] 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.
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] X.C.1. Minigene Vaccines
[0376] 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.
[0377] 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 STEAP-1, the PADRE.RTM. universal helper T cell
epitope or multiple HTL epitopes from STEAP-1 (see e.g., Tables
V-XVIII and XXII to LI), and an endoplasmic reticulum-translocating
signal sequence can be engineered. A vaccine may also comprise
epitopes that are derived from other TAAs.
[0378] 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.
[0379] 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.
[0380] 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.
[0381] 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.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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.
[0386] 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.
[0387] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0388] 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.
[0389] 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.
[0390] 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.
[0391] 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.
[0392] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0393] 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.
[0394] 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.
[0395] 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: 44), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 45), and Streptococcus 18 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 46).
Other examples include peptides bearing a DR 14-7 supermotif, or
either of the DR3 motifs.
[0396] 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: 47), 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.
[0397] 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.
[0398] X.C3. Combinations of CTL Peptides with T Cell Priming
Agents
[0399] 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.
[0400] 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.
[0401] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0402] 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.
[0403] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to STEAP-1. 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 STEAP-1.
[0404] X.D. Adoptive Immunotherapy
[0405] Antigenic STEAP-1-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.
[0406] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0407] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses STEAP-1. 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.
[0408] 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 STEAP-1. 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.
[0409] For therapeutic use, administration should generally begin
at the first diagnosis of STEAP-1-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 STEAP-1, a vaccine comprising
STEAP-1-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0410] 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.
[0411] 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.
[0412] 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.
[0413] 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.
[0414] 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.
[0415] 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.
[0416] 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.
[0417] 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.
[0418] 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.
[0419] For antibodies, a treatment generally involves repeated
administration of the anti-STEAP-1 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-STEAP-1
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
STEAP-1 expression in the patient, the extent of circulating shed
STEAP-1 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-Ig, 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.
[0420] 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.
[0421] 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' 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.6 cells/m.sup.2.
[0422] 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.
[0423] 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.
[0424] 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%.
[0425] 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.
[0426] XI.) Diagnostic and Prognostic Embodiments of STEAP-1.
[0427] As disclosed herein, STEAP-1 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 STEAP-1 in normal tissues, and patient
specimens").
[0428] STEAP-1 can be analogized to a prostate associated antigen
PSA, the archetypal marker that has been used by medical
practitioners for years to identify and monitor the presence of
prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2):
503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306
(1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):
1635-1640(1999)). A variety of other diagnostic markers are also
used in similar contexts including p53 and K-ras (see, e.g.,
Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto
et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this
disclosure of STEAP-1 polynucleotides and polypeptides (as well as
STEAP-1 polynucleotide probes and anti-STEAP-1 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.
[0429] Typical embodiments of diagnostic methods which utilize the
STEAP-1 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
STEAP-1 polynucleotides described herein can be utilized in the
same way to detect STEAP-1 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 STEAP-1
polypeptides described herein can be utilized to generate
antibodies for use in detecting STEAP-1 overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0430] 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 STEAP-1 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
STEAP-1-expressing cells (lymph node) is found to contain
STEAP-1-expressing cells such as the STEAP-1 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0431] Alternatively STEAP-1 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 STEAP-1 or
express STEAP-1 at a different level are found to express STEAP-1
or have an increased expression of STEAP-1 (see, e.g., the STEAP-1
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 STEAP-1) such as PSA, PSCA
etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237
(1996)).
[0432] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, STEAP-1 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 STEAP-1 in normal tissues, and patient
specimens," where a STEAP-1 polynucleotide fragment is used as a
probe to show the expression of STEAP-1 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 STEAP-1 polynucleotide shown in
FIG. 2 or variant thereof) under conditions of high stringency.
[0433] 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. STEAP-1
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
STEAP-1 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 STEAP-1
polypeptide shown in FIG. 3).
[0434] As shown herein, the STEAP-1 polynucleotides and
polypeptides (as well as the STEAP-1 polynucleotide probes and
anti-STEAP-1 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 1. Diagnostic
assays that measure the presence of STEAP-1 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 STEAP-1 polynucleotides and polypeptides (as well as the
STEAP-1 polynucleotide probes and anti-STEAP-1 antibodies used to
identify the presence of these molecules) need to be employed to
confirm a metastases of prostatic origin.
[0435] Finally, in addition to their use in diagnostic assays, the
STEAP-1 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 STEAP-1 gene maps (see the Example entitled "Chromosomal
Mapping of STEAP-1" below). Moreover, in addition to their use in
diagnostic assays, the STEAP-1-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).
[0436] Additionally, STEAP-1-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of STEAP-1. 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 STEAP-1 antigen. Antibodies or other molecules that react with
STEAP-1 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0437] XII.) Inhibition of STEAP-1 Protein Function
[0438] The invention includes various methods and compositions for
inhibiting the binding of STEAP-1 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
STEAP-1 function.
[0439] XII.A.) Inhibition of STEAP-1 with Intracellular
Antibodies
[0440] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to STEAP-1 are introduced
into STEAP-1 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-STEAP-1 antibody is
expressed intracellularly, binds to STEAP-1 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).
[0441] 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.
[0442] In one embodiment, intrabodies are used to capture STEAP-1
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such STEAP-1
intrabodies in order to achieve the desired targeting. Such STEAP-1
intrabodies are designed to bind specifically to a particular
STEAP-1 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a STEAP-1 protein are used to prevent STEAP-1
from gaining access to the nucleus, thereby preventing it from
exerting any biological activity within the nucleus (e.g.,
preventing STEAP-1 from forming transcription complexes with other
factors).
[0443] 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).
[0444] XII.B.) Inhibition of STEAP-1 with Recombinant Proteins
[0445] In another approach, recombinant molecules bind to STEAP-1
and thereby inhibit STEAP-1 function. For example, these
recombinant molecules prevent or inhibit STEAP-1 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 STEAP-1 specific antibody
molecule. In a particular embodiment, the STEAP-1 binding domain of
a STEAP-1 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two STEAP-1 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 STEAP-1, whereby the dimeric fusion protein
specifically binds to STEAP-1 and blocks STEAP-1 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0446] XII.C.) Inhibition of STEAP-1 Transcription or
Translation
[0447] The present invention also comprises various methods and
compositions for inhibiting the transcription of the STEAP-1 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of STEAP-1 mRNA into protein.
[0448] In one approach, a method of inhibiting the transcription of
the STEAP-1 gene comprises contacting the STEAP-1 gene with a
STEAP-1 antisense polynucleotide. In another approach, a method of
inhibiting STEAP-1 mRNA translation comprises contacting a STEAP-1
mRNA with an antisense polynucleotide. In another approach, a
STEAP-1 specific ribozyme is used to cleave a STEAP-1 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
STEAP-1 gene, such as STEAP-1 promoter and/or enhancer elements.
Similarly, proteins capable of inhibiting a STEAP-1 gene
transcription factor are used to inhibit STEAP-1 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.
[0449] Other factors that inhibit the transcription of STEAP-1 by
interfering with STEAP-1 transcriptional activation are also useful
to treat cancers expressing STEAP-1. Similarly, factors that
interfere with STEAP-1 processing are useful to treat cancers that
express STEAP-1. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0450] XII.D.) General Considerations for Therapeutic
Strategies
[0451] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing STEAP-1 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other STEAP-1 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding STEAP-1 antisense polynucleotides, ribozymes,
factors capable of interfering with STEAP-1 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0452] 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.
[0453] 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 STEAP-1 to a binding partner, etc.
[0454] In vivo, the effect of a STEAP-1 therapeutic composition can
be evaluated in a suitable animal model. For example, xenogenic
prostate cancer models can be used, wherein human prostate cancer
explants or passaged xenograft tissues are introduced into immune
compromised animals, such as nude or SCID mice (Klein et al., 1997,
Nature Medicine 3: 402-408). For example, PCT Patent Application
WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft
models of human prostate cancer capable of recapitulating the
development of primary tumors, micrometastasis, and the formation
of osteoblastic metastases characteristic of late stage disease.
Efficacy can be predicted using assays that measure inhibition of
tumor formation, tumor regression or metastasis, and the like.
[0455] 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.
[0456] 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).
[0457] 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.
[0458] 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.
[0459] XIII.) Kits/Articles of Manufacture
[0460] 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.
[0461] 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.
[0462] 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.
[0463] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0464] 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.
[0465] 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 STEAP-1 and modulating the function of
STEAP-1.
[0466] 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/or
dextrose solution. It can further include other materials desirable
from a commercial and user standpoint, including other buffers,
diluents, filters, stirrers, needles, syringes, and/or package
inserts with indications and/or instructions for use.
EXAMPLES
[0467] 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 STEAP Gene
Materials and Methods
LAPC Xenografts:
[0468] LAPC xenografts were obtained from Dr. Charles Sawyers
(UCLA) and generated as described (Klein et al, 1997, Nature Med.
3: 402-408; Craft et al., 1999, Cancer Res. 59: 5030-5036).
Androgen dependent and independent LAPC-4 xenografts (LAPC-4 AD and
AI, respectively) and LAPC-9 xenografts (LAPC-9 AD and A1,
respectively) were grown in intact male SCID mice or in castrated
males, respectively, and were passaged as small tissue chunks in
recipient males. LAPC-4 AI xenografts were derived from LAPC-4 AD
tumors and LAPC-9 AI xenografts were derived from LAPC-9 AD tumors.
To generate the AI xenografts, male mice bearing LAPC AD tumors
were castrated and maintained for 2-3 months. After the LAPC tumors
re-grew, the tumors were harvested and passaged in castrated males
or in female SCID mice.
[0469] LAPC-4 AD xenografts were grown intratibially as follows.
LAPC-4 AD xenograft tumor tissue grown subcutaneously was minced
into 1-2 mm.sup.3 sections while the tissue was bathed in 1.times.
Iscoves medium, minced tissue was then centrifuged at 1.3K rpm for
4 minutes, the supernatant was resuspended in 10 ml ice cold
1.times. Iscoves medium and centrifuged at 1.3K rpm for 4 minutes.
The pellet was then resuspended in 1.times. Iscoves with 1% pronase
E and incubated for 20 minutes at room temperature with mild
rocking agitation followed by incubation on ice for 24 minutes.
Filtrate was centrifuged at 1.3K rpm for 4 minutes, and the pronase
was removed from the aspirated pellet by resuspending in 10 ml
Iscoves and re-centrifuging. Clumps of cells were then plated in
PrEGM medium and grown overnight. The cells were then harvested,
filtered, washed 2.times. RPMI, and counted. Approximately 50,000
cells were mixed with and equal volume of ice-cold Matrigel on ice,
and surgically injected into the proximal tibial metaphyses of SCID
mice via a 27 gauge needle. After 10-12 weeks, LAPC-4 tumors
growing in bone marrow were recovered.
Cell Lines and Tissues:
[0470] Human cell lines (e.g., HeLa) were obtained from the ATCC
and were maintained in DMEM with 5% fetal calf serum. Human tissues
for RNA and protein analyses were obtained from the Human Tissue
Resource Center (HTRC) at the UCLA (Los Angeles, Calif.) and from
QualTek, Inc. (Santa Barbara, Calif.).
RNA Isolation:
[0471] Tumor tissue and cell lines 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.
Oligonucleotides:
[0472] The following HPLC purified oligonucleotides were used.
TABLE-US-00002 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 48)
5'TTTTGATCAAGCTT.sub.303' Adaptor 1: (SEQ ID NO: 49)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 50)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO: 51)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 52)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO: 53)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO: 54)
5'TCGAGCGGCCGCCCGGGCAGGA3' Nested primer (NP)2: (SEQ ID NO: 55)
5'AGCGTGGTCGCGGCCGAGGA3'
Suppression Subtractive Hybridization:
[0473] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes, which may be up-regulated in
androgen dependent prostate cancer compared to benign prostatic
hyperplasia (BPH).
[0474] Double stranded cDNAs corresponding to the LAPC-4 AD
xenograft (tester) and the BPH tissue (driver) were synthesized
from 2 .mu.g of poly(A)+ RNA isolated from xenograft and BPH
tissue, as described above, using CLONTECH's PCR-Select cDNA
Subtraction Kit and 1 ng of oligonucleotide RSACDN 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 Rsa I
for 3 hrs. at 37.degree. C. Digested cDNA was extracted with
phenol/chloroform (1:1) and ethanol precipitated.
[0475] Driver cDNA (BPH) was generated by combining in a 4 to 1
ratio Rsa I digested BPH cDNA with digested cDNA from mouse liver,
in order to ensure that murine genes were subtracted from the
tester cDNA (LAPC-4 AD).
[0476] Tester cDNA (LAPC-4 AD) was generated by diluting 1 .mu.l of
Rsa I digested LAPC-4 AD cDNA (400 ng) in 5 .mu.l of water. The
diluted cDNA (2 .mu.l, 160 ng) was then ligated to 2 .mu.l of
adaptor 1 and adaptor 2 (10 .mu.M), in separate ligation reactions,
in a total volume of 10 .mu.l at 16.degree. C. overnight, using 400
u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 .mu.l
of 0.2 M EDTA and heating at 72.degree. C. for 5 min.
[0477] The first hybridization was performed by adding 1.5 .mu.l
(600 ng) of driver cDNA to each of two tubes containing 1.5 .mu.l
(20 ng) adaptor 1- and adaptor 2-ligated tester cDNA. In a final
volume of 4 .mu.l, the samples were overlayed 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.
PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0478] To amplify gene fragments resulting from SSH reactions, two
PCR amplifications were performed. In the primary PCR reaction 1
.mu.l of the diluted final hybridization mix was added to 1 .mu.l
of PCR primer 1 (10 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5
.mu.l 10.times. reaction buffer (CLONTECH) and 0.5 .mu.l 50.times.
Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25
.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,
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0479] 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 NP 1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0480] 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.
RT-PCR Expression Analysis:
[0481] First strand cDNAs were 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 and
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 was increased to 200
.mu.l with water prior to normalization. First strand cDNAs from 16
different normal human tissues were obtained from Clontech.
[0482] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 56) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 57) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) was 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 was 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 was 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 were required to
achieve equal band intensities in all tissues after 22 cycles of
PCR.
[0483] To determine expression levels of the 8P1D4 gene, 5 .mu.l of
normalized first strand cDNA was analyzed by PCR using 25, 30, and
35 cycles of amplification using the following primer pairs:
TABLE-US-00003 (SEQ ID NO: 58) 5' ACT TTG TTG ATG ACG AGG ATT GGA
3' (SEQ ID NO: 59) 5' CAG AAC TTC AGC ACA GAG AGG AAC 3'
Semi quantitative expression analysis was achieved by comparing the
PCR products at cycle numbers that give light band intensities.
Results
[0484] Several SSH experiments were conduced as described in the
Materials and Methods, supra, and led to the isolation of numerous
candidate gene fragment clones. All candidate clones were sequenced
and subjected to homology analysis against all sequences in the
major public gene and EST databases in order to provide information
on the identity of the corresponding gene and to help guide the
decision to analyze a particular gene for differential expression.
In general, gene fragments which had no homology to any known
sequence in any of the searched databases, and thus considered to
represent novel genes, as well as gene fragments showing homology
to previously sequenced expressed sequence tags (ESTs), were
subjected to differential expression analysis by RT-PCR and/or
Northern analysis.
[0485] One of the cDNA clones, designated 8P1D4, was 436 bp in
length and showed homology to an EST sequence in the NCI-CGAP tumor
gene database. The full length cDNA encoding the 8P1D4 gene was
subsequently isolated using this cDNA and re-named STEAP-1. The
8P1D4 cDNA nucleotide sequence corresponds to nucleotide residues
150 through 585 in the STEAP-1 cDNA sequence as shown in FIG. 1A-B.
Another clone, designated 28P3EI, 561 bp in length showed homology
to a number of EST sequences in the NCI-CGAP tumor gene database or
in other databases. Part of the 28P3 .mu.l sequence (356 bp) is
identical to an EST derived from human fetal tissue. After the
full-length STEAP-1 cDNA was obtained and sequenced, it became
apparent that this clone also corresponds to STEAP-1 (more
specifically, to residues 622 through the 3' end of the STEAP-1
nucleotide sequence as shown in FIG. 1).
[0486] Differential expression analysis by RT-PCR using primers
derived from the 8P1D4 cDNA clone showed that the 8P1D4 (STEAP-1)
gene is expressed at approximately equal levels in normal prostate
and the LAPC-4 and LAPC-9 xenografts. Further RT-PCR expression
analysis of first strand cDNAs from 16 normal tissues showed
greatest levels of 8P1D4 expression in prostate. Substantially
lower level expression in several other normal tissues (i.e.,
colon, ovary, small intestine, spleen and testis) was detectable
only at 30 cycles of amplification in brain, pancreas, colon and
small intestine.
Example 2
Isolation of Full Length STEAP-1 Encoding cDNA
[0487] The 436 bp 8P1D4 gene fragment (Example 1) was used to
isolate additional cDNAs encoding the 8P1D4/STEAP-1 gene. Briefly,
a normal human prostate cDNA library (Clontech) was screened with a
labeled probe generated from the 436 bp 8P1D4 cDNA. One of the
positive clones, clone 10, is 1195 bp in length and encodes a 339
amino acid protein having nucleotide and encoded amino acid
sequences bearing no significant homology to any known human genes
or proteins (homology to a rat Kidney Injury Protein recently
described in International Application WO98/53071). The encoded
protein contains at least 6 predicted transmembrane motifs implying
a cell surface orientation (see FIG. 1A-B, predicted transmembrane
motifs underlined). These structural features led to the
designation "STEAP", for "Six Transmembrane Epithelial Antigen of
the Prostate".
[0488] Subsequent identification of additional "STEAP" proteins led
to the re-designation of the 8P1D4 gene product as "STEAP-1". The
STEAP-1 cDNA and encoded amino acid sequences are shown in FIG.
2A-Q. STEAP-1 cDNA clone 10 was deposited with the American Type
Culture Collection ("ATCC") (10801 University Blvd., Manassas, Va.
20110-2209 USA) as plasmid 8P1D4 clone 10.1 on Aug. 26, 1998 as
ATCC Accession Number 98849. The STEAP-1 cDNA clone can be excised
therefrom using EcoRI/XbaI double digest (EcoRI at the 5'end, XbaI
at the 3'end).
Example 3
Chromosomal Mapping of STEAP-1
[0489] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are available
including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics
7:22; Research Genetics, Huntsville, Ala.), human-rodent somatic
cell hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0490] STEAP-1 maps to chromosome 7q2.sup.1 using STEAP-1 sequence
and the NCBI BLAST tool: (located on the World Wide Web at
(.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs)).
Example 4
Expression Analysis of STEAP-1
[0491] Expression of STEAP-1 in stomach cancer patient specimens is
shown in FIG. 14. RNA was extracted from normal stomach (N) and
from 10 different stomach cancer patient specimens (T). Northern
blot with 10 .mu.g of total RNA/lane was probed with STEAP-1
sequence. Results show strong expression of an approximately 1.6 kb
STEAP-1 in the stomach tumor tissues. The lower panel represents
ethidium bromide staining of the blot showing quality of the RNA
samples.
[0492] FIG. 15 shows that STEAP-1 was expressed in rectum cancer
patient tissues. RNA was extracted from normal rectum (N), rectum
cancer patient tumors (T), and rectum cancer metastasis (M).
Northern blots with 10 .mu.g of total RNA were probed with the
STEAP-1 sequence. Results show strong expression of STEAP-1 in the
rectum cancer patient tissues. The lower panel represents ethidium
bromide staining of the blot showing quality of the RNA
samples.
[0493] Expression of STEAP-1 by RT-PCR demonstrated that STEAP-1 is
strongly expressed in human umbilical vein endothelial cells
(HUVEC) (FIG. 16). First strand cDNA was prepared from HUVEC cells,
LAPC-4AD and LAPC-9AD prostate cancer xenografts, as well as from
human brain tissues. Normalization was performed by PCR using
primers to actin and GAPDH. Semi-quantitative PCR, using primers to
STEAP-1, was performed at 27 and 30 cycles of amplification (FIG.
16A). As a control, PCR using primers to actin is shown in FIG.
16B. Results show strong expression of STEAP-1 in HUVEC cells
similar to the expression detected in prostate cancer xenograft
tissues. Expression of STEAP-1 in HUVEC cells indicates that
targeting STEAP-1 may also target endothelial cells of the
neovasculature of the tumors.
Example 5
Transcript Variants of 8P1D4
[0494] 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.
[0495] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified by full-length cloning experiment,
or by use of full-length transcript and EST sequences. First, all
human ESTs were grouped into clusters which show direct or indirect
identity with each other. Second, ESTs in the same cluster were
further grouped into sub-clusters and assembled into a consensus
sequence. The original gene sequence is compared to the consensus
sequence(s) or other full-length sequences. Each consensus sequence
is a potential splice variant for that gene (see, e.g., Kan, Z., et
al., Gene structure prediction and alternative splicing analysis
using genomically aligned ESTs, Genome Research, 2001 May,
11(5):889-900.) 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.
[0496] Moreover, computer programs are available in the art that
identify transcript variants based on genomic sequences.
Genomic-based transcript variant identification programs include
FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in
Drosophila genomic DNA," Genome Research. 2000 April;
10(4):516-22); Grail (URL
compbio.orn1.gov/Grail-bin/EmptyGrailForm) and GenScan (URL
genes.mit.edu/GENSCAN.html). For a general discussion of splice
variant identification protocols see, e.g., Southan, C., A genomic
perspective on human proteases, FEBS Lett. 2001 Jun. 8;
498(2-3):214-8; de Souza, S. J., et al., Identification of human
chromosome 22 transcribed sequences with ORF expressed sequence
tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.
[0497] 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).
[0498] 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 8P1D4 has a particular expression profile related to cancer.
Alternative transcripts and splice variants of 8P1D4 may also be
involved in cancers in the same or different tissues, thus serving
as tumor-associated markers/antigens.
[0499] The exon composition of the original transcript, designated
as 8P1D4 v.1, is shown in Table LXI. Using the full-length gene and
EST sequences, two transcript variants were identified, designated
as 8P1D4 v.2 and v.3. Compared with 8P1D4 v.1, transcript variant
8P1D4 v.2 did not splice out intron 4 of 8P1D4 v.1 and variant
8P1D4 v.3 spliced out one additional exon from intron 4 of 8P1D4
v.1, as shown in FIG. 11. Theoretically, each different combination
of exons in spatial order, e.g. exons 2 and 3, is a potential
splice variant. FIG. 11 shows the schematic alignment of exons of
the transcript variants.
[0500] Tables LIII through LX are set forth on a variant by variant
bases. Tables LIII and LVII show the nucleotide sequence of the
transcript variants. Table LIV shows the alignment of the
transcript variant with the nucleic acid sequence of 8P1D4 v. I.
Table LVIII shows the alignment of the transcript variant with the
nucleic acid sequence of 8P1D4 v.2. Tables LV and LIX lays out
amino acid translation of the transcript variant for the identified
reading frame orientation. Table LVI displays the alignment of the
amino acid sequence encoded by the splice variant with that of
8P1D4 v.1. Table LX displays the alignment of the amino acid
sequence encoded by the splice variant with that of 8P1D4 v.2.
Example 6
Single Nucleotide Polymorphisms of 8P1D4
[0501] 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).
[0502] 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).
[0503] Using the methods described above, fourteen SNPs were
identified in the transcript from clone GTH9, designated as 8P1D4
v.2, at positions 602 (C/G), 386 (C/T), 1087 (T/G), 1447 (T/C),
1621 (A/T), 1625 (G/T), 1716 (C/A), 2358 (C/T), 2646 (T/G), 2859
(T/G), 2908 (A/T), 3006 (G/C), 3107 (C/T), and 3180 (A/T). The
transcripts or proteins with alternative alleles were designated as
variants 8P1D4 v.4, v.5, v.6, v.7, v.8, v.9, v.10, v.11, v.12,
v.13, v.14, v.15, v.16 and v.17, respectively. FIG. 10 shows the
schematic alignment of the SNP variants. FIG. 12 shows the
schematic alignment of protein variants, corresponding to
nucleotide variants. 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 8P 1D4 v.1 and
v.3) that contains the sequence context of the SNPs. E.g., the
first two SNP were also on 8P1D4 v.3 at the same positions, but at
572 and 356, respectively, on 8P1D4 v.1.
Example 7
Production of Recombinant 8P1D4 in Prokaryotic Systems
[0504] To express recombinant 8P1D4 and 8P1D4 variants in
prokaryotic cells, the full or partial length 8P1D4 and 8P1D4
variant cDNA sequences are cloned into any one of a variety of
expression vectors known in the art. The full length cDNA, 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 8P1D4,
variants, or analogs thereof are used.
[0505] A. In Vitro Transcription and Translation Constructs:
[0506] pCRII: To generate 8P1D4 sense and anti-sense RNA probes for
RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad
Calif.) are generated encoding either all or fragments of the 8P1D4
cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert
to drive the transcription of 8P1D4 RNA for use as probes in RNA in
situ hybridization experiments. These probes are used to analyze
the cell and tissue expression of 8P1D4 at the RNA level.
Transcribed 8P1D4 RNA representing the cDNA amino acid coding
region of the 8P1D4 gene is used in in vitro translation systems
such as the TnT.TM. Coupled Reticulolysate System (Promega, Corp.,
Madison, Wis.) to synthesize 8P1D4 protein.
[0507] B. Bacterial Constructs:
[0508] pGEX Constructs: To generate recombinant 8P1D4 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the 8P1D4 cDNA or variants are cloned into
the GST-fusion vector of the pGEX family (Amersham Pharmacia
Biotech, Piscataway, N.J.). These constructs allow controlled
expression of recombinant 8P1D4 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 8P1D4-related protein. The ampicillin resistance gene and
pBR322 origin permits selection and maintenance of the pGEX
plasmids in E. coli.
[0509] pMAL Constructs: To generate, in bacteria, recombinant 8P1D4
proteins that are fused to maltose-binding protein (MBP), all or
parts of the 8P1D4 cDNA protein coding sequence are fused to the
MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New
England Biolabs, Beverly, Mass.). These constructs allow controlled
expression of recombinant 8P1D4 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 8P1D4. The pMAL-c2X and pMAL-p2X vectors are
optimized to express the recombinant protein in the cytoplasm or
periplasm respectively. Periplasm expression enhances folding of
proteins with disulfide bonds.
[0510] pET Constructs: To express 8P1D4 in bacterial cells, all or
parts of the 8P1D4 cDNA protein coding sequence are cloned into the
pET family of vectors (Novagen, Madison, Wis.). These vectors allow
tightly controlled expression of recombinant 8P1D4 protein in
bacteria with and without fusion to proteins that enhance
solubility, such as NusA and thioredoxin (Trx), and epitope tags,
such as 6.times.His and S-Tag.TM. that aid purification and
detection of the recombinant protein. For example, constructs are
made utilizing pET NusA fusion system 43.1 such that regions of the
8P1D4 protein are expressed as amino-terminal fusions to NusA.
[0511] C. Yeast Constructs:
[0512] pESC Constructs: To express 8P1D4 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 8P1D4 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 8P1D4. In addition, expression in
yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations, that are found when expressed
in eukaryotic cells.
[0513] pESP Constructs: To express 8P1D4 in the yeast species
Saccharomyces pombe, all or parts of the 8P1D4 cDNA protein coding
sequence are cloned into the pESP family of vectors. These vectors
allow controlled high level of expression of a 8P 1D4 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 STEAP-1 in Eukaryotic Systems
[0514] A. Mammalian Constructs:
[0515] To express recombinant STEAP-1 in eukaryotic cells, the full
or partial length STEAP-1 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 STEAP-1 are expressed in these constructs,
amino acids 1 to 339 of STEAP-1 v.1, v.4, amino acids 1 to 258 of
v.2, amino acids 1 to 282 of v.3, or any 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50 or more contiguous amino acids from STEAP-1, variants, or
analogs thereof. In certain embodiments a region of a specific
variant of STEAP-1 is expressed that encodes an amino acid at a
specific position which differs from the amino acid of any other
variant found at that position. In other embodiments, a region of a
variant of STEAP-1 is expressed that lies partly or entirely within
a sequence that is unique to that variant.
[0516] 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-STEAP-1 polyclonal serum,
described herein.
[0517] pcDNA4/HisMax Constructs: To express STEAP-1 in mammalian
cells, a STEAP-1 ORF, or portions thereof, of STEAP-1 are cloned
into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.).
Protein expression is driven from the cytomegalovirus (CMV)
promoter and the SP 16 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.
[0518] pcDNA3.1/MycHis Constructs: To express STEAP-1 in mammalian
cells, a STEAP-1 ORF, or portions thereof, of STEAP-1 with a
consensus Kozak translation initiation site was cloned into
pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein
expression is driven from the cytomegalovirus (CMV) promoter. The
recombinant proteins have the myc epitope and 6.times.His epitope
fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also
contains the bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene was 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.
[0519] pcDNA3.1/CT-GFP-TOPO Construct: To express STEAP-1 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, a STEAP-1 ORF, or portions thereof, with a
consensus Kozak translation initiation site are cloned into
pcDNA3.1/CT-GFP-TOPO (Invitrogen, Calif.). 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. I/NT-GFP-TOPO spanning the entire length of a STEAP-1
protein.
[0520] PAPtag: A STEAP-1 ORF, or portions thereof, is cloned into
pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct
generates an alkaline phosphatase fusion at the carboxyl-terminus
of a STEAP-1 protein while fusing the IgGK signal sequence to the
amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgGK signal sequence is fused to
the amino-terminus of a STEAP-1 protein. The resulting recombinant
STEAP-1 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 STEAP-1 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.
[0521] ptag5: A STEAP-1 ORF, or portions thereof, was cloned into
pTag-5. This vector is similar to pAPtag but without the alkaline
phosphatase fusion. This construct generated STEAP-1 protein with
an amino-terminal IgGK signal sequence and myc and 6.times.His
epitope tags at the carboxyl-terminus that facilitate detection and
affinity purification. The resulting recombinant STEAP-1 protein
was 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 STEAP-1 proteins.
Protein expression was driven from the CMV promoter. The Zeocin
resistance gene present in the vector allowed for selection of
mammalian cells expressing the protein, and the ampicillin
resistance gene permits selection of the plasmid in E. coli.
[0522] PsecFc: A STEAP-1 ORF, or portions thereof, was also cloned
into psecFc. The psecFc vector was assembled by cloning the human
immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2
(Invitrogen, California). This construct generated an IgG1 Fc
fusion at the carboxyl-terminus of the STEAP-1 proteins, while
fusing the IgGK signal sequence to N-terminus. STEAP-1 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant STEAP-1 proteins were optimized for secretion into the
media of transfected mammalian cells, and can were used as
immunogens or to identify proteins such as ligands or receptors
that interact with STEAP-1 protein. Protein expression is driven
from the CMV promoter. The hygromycin resistance gene present in
the vector allowed for selection of mammalian cells that express
the recombinant protein, and the ampicillin resistance gene permits
selection of the plasmid in E. coli.
[0523] pSR.alpha. Constructs: To generate mammalian cell lines that
express STEAP-1 constitutively, STEAP-1 ORF, or portions thereof,
of STEAP-1 were cloned into pSR.alpha. constructs. Amphotropic and
ecotropic retroviruses were generated by transfection of pSR.alpha.
constructs into the 293T-10A1 packaging line or co-transfection of
pSR.alpha. and a helper plasmid (containing deleted packaging
sequences) into the 293 cells, respectively. The retrovirus was
used to infect a variety of mammalian cell lines, resulting in the
integration of the cloned gene, STEAP-1, into the host cell-lines.
Protein expression was driven from a long terminal repeat (LTR).
The Neomycin resistance gene present in the vector allowed 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 were
thereafter be used for infection and generation of various cell
lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1
cells.
[0524] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
STEAP-1 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: 60) is added to cloning primer at the 3' end of
the ORF. Additional pSR.alpha. constructs were made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length STEAP-1 proteins.
[0525] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of STEAP-1. High virus titer
leading to high level expression of STEAP-1 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. A STEAP-1 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, STEAP-1 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.
[0526] Regulated Expression Systems: To control expression of
STEAP-1 in mammalian cells, coding sequences of STEAP-1, 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 STEAP-1. These
vectors are thereafter used to control expression of STEAP-1 in
various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0527] B. Baculovirus Expression Systems
[0528] To generate recombinant STEAP-1 proteins in a baculovirus
expression system, STEAP-1 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-STEAP-1 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. Recombinant STEAP-1 protein is then
generated by infection of HighFive insect cells (Invitrogen) with
purified baculovirus. Recombinant STEAP-1 protein can be detected
using anti-STEAP-1 or anti-His-tag antibody. STEAP-1 protein can be
purified and used in various cell-based assays or as immunogen to
generate polyclonal and monoclonal antibodies specific for
STEAP-1.
Example 9
Antigenicity Profiles and Secondary Structure
[0529] FIGS. 5-9 and FIGS. 5a-9a depict graphically five amino acid
profiles of the 8P1D4 variants 1 and 3 respectively, each
assessment available by accessing the ProtScale website (URL
www.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology
server.
[0530] 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 the 8P1D4 protein. Each of the above amino acid profiles
of 8P1D4 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.
[0531] Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and
Percentage Accessible Residues (FIG. 7) profiles were used to
determine stretches of hydrophilic amino acids (i.e., values
greater than 0.5 on the Hydrophilicity and Percentage Accessible
Residues profile, and values less than 0.5 on the Hydropathicity
profile). Such regions are likely to be exposed to the aqueous
environment, be present on the surface of the protein, and thus
available for immune recognition, such as by antibodies.
[0532] 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.
[0533] Antigenic sequences of the 8P1D4 protein and of the variant
proteins indicated, e.g., by the profiles set forth in FIG. 5, FIG.
6, FIG. 7, FIG. 8, and/or FIG. 9 are used to prepare immunogens,
either peptides or nucleic acids that encode them, to generate
therapeutic and diagnostic anti-8P1D4 antibodies. The immunogen can
be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous
amino acids, or the corresponding nucleic acids that encode them,
from the 8P1D4 protein variants listed in FIGS. 2 and 3. In
particular, peptide immunogens of the invention can comprise, a
peptide region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value greater than 0.5 in the Hydrophilicity profile of FIG. 5; a
peptide region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value less than 0.5 in the Hydropathicity profile of FIG. 6; a
peptide region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value greater than 0.5 in the Percent Accessible Residues profile
of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2
and 3 in any whole number increment that includes an amino acid
position having a value greater than 0.5 in the Average Flexibility
profile on FIG. 8; and, a peptide region of at least 5 amino acids
of FIGS. 2 and 3 in any whole number increment that includes an
amino acid position having a value greater than 0.5 in the
Beta-turn profile of FIG. 9. Peptide immunogens of the invention
can also comprise nucleic acids that encode any of the
forgoing.
[0534] 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.
[0535] The secondary structures of 8P1D4 variant 1 and variant 3,
namely the predicted presence and location of alpha helices,
extended strands, and random coils, are predicted from the
respective primary amino acid sequences using the HNN--Hierarchical
Neural Network method (Guermeur, 1997,
http://pbil.ibcp.fr/cgi-bin/npsa_automat.p1?page npsa_nn.html),
accessed from the ExPasy molecular biology server
(http://www.expasy.ch/tools/). The analysis indicates that 8P1D4
variant 1 is composed of 64.60% alpha helix, 4.72% extended strand,
and 30.68% random coil (FIG. 13a). 8P1D4 variant 2 is composed of
62.79% alpha helix, 3.10% extended strand, and 34.11% random coil
(FIG. 13b). 8P1D4 variant 3 is composed of 58.87% alpha helix,
5.32% extended strand, and 35.82% random coil (FIG. 13c).
[0536] Analysis for the potential presence of transmembrane domains
in 8P1D4 variants were carried out using a variety of transmembrane
prediction algorithms accessed from the ExPasy molecular biology
server (http://www.expasy.ch/tools/). Shown graphically are the
results of analysis of variant 1 depicting the presence and
location of 6 transmembrane domains using the TMpred program (FIG.
13d) and TMHMM program (FIG. 13e). Also shown are the results of
analysis of variant 2 depicting the presence and location of 4
transmembrane domains using TMpred (FIG. 13f) and 3 transmembrane
domains using TMHMM (FIG. 13g). Analysis of variant 3 predicts the
presence of 4 transmembrane domains using the TMpred (Figure h) and
3 transmembrane domains with TMHMM (FIG. 13i). The results of each
program, namely the amino acids encoding the transmembrane domains
are summarized in Table XX.
Example 10
Generation of 8P1D4 Polyclonal Antibodies
[0537] 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 8P1D4 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 the Example entitled
"Antigenicity Profiles"). 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 8P1D4 and variants).
[0538] For example, 8P1D4 recombinant bacterial fusion proteins or
peptides containing hydrophilic, flexible, beta-turn regions of
8P1D4 variant proteins 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 1-40, amino acids
143-165, amino acids 180-220, of 8P1D4 variants 1, 2, and 3, amino
acids 312-339 of 8P1D4 variant 1, and amino acids 250-282 of 8P1D4
variant 3. 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 250-282 of 8P1D4 variant 3 is
conjugated to KLH and used to immunize a rabbit. Alternatively the
immunizing agent may include all or portions of the 8P1D4 variant
proteins, analogs or fusion proteins thereof. For example, the
8P1D4 variant 1 amino acid sequence can be fused using recombinant
DNA techniques to any one of a variety of fusion protein partners
that are well known in the art, such as glutathione-S-transferase
(GST) and HIS tagged fusion proteins. Such fusion proteins are
purified from induced bacteria using the appropriate affinity
matrix.
[0539] In one embodiment, a GST-fusion protein encoding amino acids
250-282, encompassing the second predicted extracellular loop, is
produced, purified, and used as immunogen. Other recombinant
bacterial fusion proteins that may be employed include maltose
binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin
constant region (see the section entitled "Production of 8P1D4 in
Prokaryotic Systems" and Current Protocols In Molecular Biology,
Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley,
P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and
Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).
[0540] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the Example entitled "Production of
Recombinant 8P1D4 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, the predicted extracellular loop
of variant 1, amino acids 185-218, 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 8P1D4 protein is then used as immunogen.
[0541] 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).
[0542] 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.
[0543] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with a KLH-conjugated
peptide encoding amino acids 250-282 of variant 3, the full-length
8P1D4 variant 1 cDNA is cloned into pcDNA 3.1 myc-his expression
vector (Invitrogen, see the Example entitled "Production of
Recombinant 8P1D4 in Eukaryotic Systems"). After transfection of
the constructs into 293T cells, cell lysates are probed with the
anti-8P1D4 serum and with anti-His antibody (Santa Cruz
Biotechnologies, Santa Cruz, Calif.) to determine specific
reactivity to denatured 8P1D4 protein using the Western blot
technique. The immune serum is then tested by the Western blot
technique against 293T-8P1D4 cells. In addition, the immune serum
is tested by fluorescence microscopy, flow cytometry and
immunoprecipitation against 293T and other recombinant
8P1D4-expressing cells to determine specific recognition of native
protein. Western blot, immunoprecipitation, fluorescent microscopy,
and flow cytometric techniques using cells that endogenously
express 8P1D4 are also carried out to test reactivity and
specificity.
[0544] Anti-serum from rabbits immunized with 8P 1D4 variant fusion
proteins, such as GST and MBP fusion proteins, are purified by
depletion of antibodies reactive to the fusion partner sequence by
passage over an affinity column containing the fusion partner
either alone or in the context of an irrelevant fusion protein. For
example, antiserum derived from a GST-8P1D4 fusion protein encoding
amino acids 250-282 of variant 3 is first purified by passage over
a column of GST protein covalently coupled to AffiGel matrix
(BioRad, Hercules, Calif.). The antiserum is then affinity purified
by passage over a column composed of a MBP-fusion protein also
encoding amino acids 250-282 covalently coupled to AffiGel matrix.
The serum is then further purified by protein G affinity
chromatography to isolate the IgG fraction. Sera from other
His-tagged antigens and peptide immunized rabbits as well as fusion
partner depleted sera are affinity purified by passage over a
column matrix composed of the original protein immunogen or free
peptide.
Example 11
Generation of 8P1D4 Monoclonal Antibodies (mAbs)
[0545] In one embodiment, therapeutic mAbs to 8P1D4 variants
comprise those that react with epitopes specific for each variant
protein or specific to sequences in common between the variants
that would disrupt or modulate the biological function of the 8P1D4
variants, for example those that would disrupt the interaction with
ligands and substrates or disrupt its biological activity.
Immunogens for generation of such mAbs include those designed to
encode or contain the entire 8P1D4 protein variant sequence,
regions of the 8P1D4 protein variants predicted to be antigenic
from computer analysis of the amino acid sequence (see, e.g., FIG.
5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled
"Antigenicity Profiles"). Immunogens include peptides, recombinant
bacterial proteins, and mammalian expressed Tag 5 proteins and
human and murine IgG FC fusion proteins. In addition, cells
engineered to express high levels of a respective 8P1D4 variant,
such as 293T-8P1D4 variant 1 or 300.19-8P1D4 variant 1 murine Pre-B
cells, are used to immunize mice.
[0546] To generate mAbs to an 8P1D4 variant, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen or 10.sup.7 8P1D4-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 an 8P1D4 variant sequence is
used to immunize mice by direct injection of the plasmid DNA. For
example amino acids 185-218, is cloned into the Tag5 mammalian
secretion vector and the recombinant vector is used as immunogen.
In another example the same amino acids are cloned into an
Fc-fusion secretion vector in which the 8P1D4 variant 1 sequence is
fused at the amino-terminus to an IgK leader sequence and at the
carboxyl-terminus to the coding sequence of the human or murine IgG
Fc region. This recombinant vector is then used as immunogen. The
plasmid immunization protocols are used in combination with
purified proteins expressed from the same vector and with cells
expressing the respective 8P1D4 variant.
[0547] 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).
[0548] In one embodiment for generating 8P1D4 variant 3 specific
monoclonal antibodies, a GST-8P1D4 variant 3 antigen encoding amino
acids 250-282, is expressed and purified from bacteria. Balb C mice
are initially immunized intraperitoneally with 25 .mu.g of the
GST-8P1D4 variant 3 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 an MBP-fusion protein encoding
amino acids 250-282 determines the titer of serum from immunized
mice. Reactivity and specificity of serum to full length 8P1D4
variant 3 protein is monitored by Western blotting,
immunoprecipitation and flow cytometry using 293T cells transfected
with an expression vector encoding the 8P 1D4 variant 3 cDNA
compared to cells transfected with variant 1 and 2 cDNA (see e.g.,
the Example entitled "Production of Recombinant 8P1D4 in Eukaryotic
Systems"). Other recombinant 8P1D4 variant 3-expressing cells or
cells endogenously expressing 8P1D4 variant 3 are also used. Mice
showing the strongest specific reactivity to 8P1D4 variant 3 are
rested and given a final injection of GST-antigen in PBS and then
sacrificed four days later. The spleens of the sacrificed nice are
harvested and fused to SPO/2 myeloma cells using standard
procedures (Harlow and Lane, 1988). Supernatants from HAT selected
growth wells are screened by ELISA, Western blot,
immunoprecipitation, fluorescent microscopy, and flow cytometry to
identify 8P1D4 specific antibody-producing clones. The binding
affinity of a 8P1D4 monoclonal antibody is determined using
standard technologies. Affinity measurements quantify the strength
of antibody to epitope binding and are used to help define which
8P1D4 monoclonal antibodies preferred for diagnostic or therapeutic
use, as appreciated by one of skill in the art. The BIAcore system
(Uppsala, Sweden) is a preferred method for determining binding
affinity. The BIAcore system uses surface plasmon resonance (SPR,
Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,
Methods in Enzymology 295: 268) to monitor biomolecular
interactions in real time. BIAcore analysis conveniently generates
association rate constants, dissociation rate constants,
equilibrium dissociation constants, and affinity constants.
Example 12
HLA Class I and Class II Binding Assays
[0549] 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.
[0550] 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.
[0551] 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
[0552] 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.
[0553] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0554] The searches performed to identify the motif-bearing peptide
sequences in the Example entitled "Antigenicity Profiles" and
Tables V-XVIII and XXII-LI employ the protein sequence data from
the gene product of STEAP-1 set forth in FIGS. 2 and 3, the
specific peptides used to generate the tables are listed in Table
LXI.
[0555] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
STEAP-1 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.
[0556] Identified A2-, A3-, and DR-supermotif sequences are scored
using polynomial algorithms to predict their capacity to bind to
specific HLA-Class I or Class II molecules. These polynomial
algorithms account for the impact of different amino acids at
different positions, and are essentially based on the premise that
the overall affinity (or .DELTA.G) of peptide-HLA molecule
interactions can be approximated as a linear polynomial function of
the type: ".DELTA.G"=a.sub.1i.times.a.sub.2i.times.a.sub.3i . . .
.times.a.sub.ni
[0557] where a.sub.ji is a coefficient which represents the effect
of the presence of a given amino acid (i) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial
assumption of this method is that the effects at each position are
essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at position i in the
peptide, it is assumed to contribute a constant amount j to the
free energy of binding of the peptide irrespective of the sequence
of the rest of the peptide.
[0558] 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.
[0559] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0560] Protein sequences from STEAP-1 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).
[0561] 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.
[0562] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0563] The STEAP-1 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.
[0564] Selection of HLA-B7 Supermotif Bearing Epitopes
[0565] The STEAP-1 protein(s) scanned above is also analyzed for
the presence of 8-, 9-10-, or 11-mer peptides with the
HLA-B7-supermotif. Corresponding peptides are synthesized and
tested for binding to HLA-B*0702, the molecule encoded by the most
common B7-supertype allele (i.e., the prototype B7 supertype
allele). Peptides binding B*0702 with IC.sub.50 of .ltoreq.500 nM
are identified using standard methods. These peptides are then
tested for binding to other common B7-supertype molecules (e.g.,
B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to
three or more of the five B7-supertype alleles tested are thereby
identified.
[0566] Selection of A1 and A24 Motif-Bearing Epitopes
[0567] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the STEAP-1 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0568] 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
[0569] 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:
[0570] Target Cell Lines for Cellular Screening:
[0571] 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.
[0572] Primary CTL Induction Cultures:
[0573] 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.
[0574] 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.
[0575] 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.
[0576] 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 101 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.
[0577] Measurement of CTL Lytic Activity by .sup.51Cr Release.
[0578] 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.
[0579] 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/mm (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.
[0580] 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.
[0581] In situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-Specific and Endogenous Recognition
[0582] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH8.2) overnight
at 4.degree. C. The plates are washed with Ca.sup.2+,
Mg.sup.2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for
two hours, after which the CTLs (100 .mu.l/well) and targets (1001
.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.
[0583] 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.
[0584] CTL Expansion.
[0585] 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.
[0586] Cultures are expanded in the absence of anti-CD3+ 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.
[0587] Immunogenicity of A2 Supermotif-Bearing Peptides
[0588] 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.
[0589] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses STEAP-1. 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.
[0590] Evaluation of A*03/A11 Immunogenicity
[0591] 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.
[0592] Evaluation of B7 Immunogenicity
[0593] 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.
[0594] 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
[0595] 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.
[0596] Analoging at Primary Anchor Residues
[0597] 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.
[0598] 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.
[0599] 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.
[0600] 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).
[0601] 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.
[0602] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0603] 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.
[0604] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate .ltoreq.500 nM binding capacity are then confirmed as
having A3-supertype cross-reactivity.
[0605] 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).
[0606] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0607] 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.
[0608] Analoging at Secondary Anchor Residues
[0609] 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.
[0610] 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 STEAP-1-expressing tumors.
[0611] Other Analoging Strategies
[0612] 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).
[0613] 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 STEAP-1-Derived Sequences with
HLA-DR Binding Motifs
[0614] 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.
[0615] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0616] To identify STEAP-1-derived, HLA class II HTL epitopes, a
STEAP-1 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).
[0617] 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.
[0618] The STEAP-1-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. STEAP-1-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0619] Selection of DR3 Motif Peptides
[0620] 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.
[0621] To efficiently identify peptides that bind DR3, target
STEAP-1 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.
[0622] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0623] 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 STEAP-1-Derived HTL Epitopes
[0624] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0625] 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 STEAP-1-expressing
tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0626] 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.
[0627] 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].
[0628] 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).
[0629] Population coverage achieved by combining the A2-, A3- and
B7-supertypes is approximately 86% in five major ethnic groups.
Coverage may be extended by including peptides bearing the A1 and
A24 motifs. On average, A1 is present in 12% and A24 in 29% of the
population across five different major ethnic groups (Caucasian,
North American Black, Chinese, Japanese, and Hispanic). Together,
these alleles are represented with an average frequency of 39% in
these same ethnic populations. The total coverage across the major
ethnicities when A1 and A24 are combined with the coverage of the
A2-, A3- and B7-supertype alleles is >95%. An analogous approach
can be used to estimate population coverage achieved with
combinations of class II motif-bearing epitopes.
[0630] 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.
[0631] 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
[0632] 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.
[0633] 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 STEAP-1
expression vectors.
[0634] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
STEAP-1 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-A
1 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
[0635] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a STEAP-1-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a STEAP-1-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.
[0636] 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.
[0637] 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)
[0638] 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.
[0639] 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.
[0640] 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
STEAP-1-Specific Vaccine
[0641] 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.
[0642] 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.
[0643] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with STEAP-1 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear STEAP-1. For example, if it has been observed
that patients who spontaneously clear STEAP-1-expressing cells
generate an immune response to at least three (3) epitopes from
STEAP-1 antigen, then at least three epitopes should be included
for HLA class I. A similar rationale is used to determine HLA class
II epitopes.
[0644] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 nM or less for an HLA class I molecule, or for
class II, an IC.sub.50 of 1000 nM or less; or HLA Class I peptides
with high binding scores from the BIMAS web site, at URL
bimas.dcrt.nih.gov/.
[0645] 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.
[0646] 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 STEAP-1, 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.
[0647] 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 STEAP-1.
Example 22
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0648] 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.
[0649] 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 STEAP-1, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from STEAP-1 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.
[0650] 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.
[0651] 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.
[0652] 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.
[0653] 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.
[0654] For example, a minigene is prepared as follows. For a first
PCR reaction, 5 .mu.g of each of two oligonucleotides are annealed
and extended: In an example using eight oligonucleotides, i.e.,
four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are
combined in 100 .mu.l reactions containing Pfu polymerase buffer
(1x=10 mM KCL, 10 mM (NH4).sub.2SO.sub.4, 20 mM Tris-chloride, pH
8.75, 2 mM MgSO.sub.4, 0.1% Triton X-100, 100 .mu.g/ml BSA), 0.25
mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer
products are gel-purified, and two reactions containing the product
of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed,
and extended for 10 cycles. Half of the two reactions are then
mixed, and 5 cycles of annealing and extension carried out before
flanking primers are added to amplify the full length product. The
full-length product is gel-purified and cloned into pCR-blunt
(Invitrogen) and individual clones are screened by sequencing.
Example 23
The Plasmid Construct and the Decree to which it Induces
Immunogenicity
[0655] 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).
[0656] 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.
[0657] 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.
[0658] 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.
[0659] 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.
[0660] 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.
[0661] 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).
[0662] 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.
[0663] 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
[0664] Vaccine compositions of the present invention can be used to
prevent STEAP-1 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
STEAP-1-associated tumor.
[0665] 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 STEAP-1-associated disease.
[0666] 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 STEAP-1
Sequences
[0667] A native STEAP-1 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.
[0668] The vaccine composition will include, for example, multiple
CTL epitopes from STEAP-1 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.
[0669] 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 STEAP-1, 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.
[0670] 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
[0671] The STEAP-1 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
STEAP-1 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from STEAP-1 as well as tumor-associated antigens that are
often expressed with a target cancer associated with STEAP-1
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
[0672] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to STEAP-1. 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.
[0673] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, STEAP-1 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising a STEAP-1
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.
[0674] 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 STEAP-1 epitope, and thus the
status of exposure to STEAP-1, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
[0675] 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 STEAP-1-associated disease or who have been
vaccinated with a STEAP-1 vaccine.
[0676] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
STEAP-1 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.
[0677] 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.
[0678] 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).
[0679] 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).
[0680] Cytotoxicity assays are performed in the following manner.
Target cells consist of either allogeneic HLA-matched or autologous
EBV-transformed B lymphoblastoid cell line that are incubated
overnight with the synthetic peptide epitope of the invention at 10
.mu.M, and labeled with 100 .mu.Ci of .sup.51Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0681] 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.
[0682] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to STEAP-1 or a STEAP-1 vaccine.
[0683] 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 STEAP-1
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
[0684] 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:
[0685] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0686] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0687] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0688] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0689] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0690] 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.
[0691] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0692] 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.
[0693] The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing STEAP-1
[0694] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses STEAP-1. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express STEAP-1, 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:
[0695] 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.
[0696] 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 STEAP-1.
[0697] 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 STEAP-1-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
[0698] 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.
[0699] 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 34 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.
[0700] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against STEAP-1 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0701] 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
STEAP-1 protein from which the epitopes in the vaccine are
derived.
[0702] 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/IL4. After
pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
[0703] 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.
[0704] 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.
[0705] Ex Vivo Activation of CTL/HTL Responses
[0706] Alternatively, ex vivo CTL or HTL responses to STEAP-1
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
[0707] 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. STEAP-1.
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.
[0708] 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
STEAP-1 to isolate peptides corresponding to STEAP-1 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.
[0709] 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
[0710] Sequences complementary to the STEAP-1-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring STEAP-1. 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 STEAP-1. 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 STEAP-1-encoding
transcript.
Example 35
Purification of Naturally-Occurring or Recombinant STEAP-1 Using
STEAP-1-Specific Antibodies
[0711] Naturally occurring or recombinant STEAP-1 is substantially
purified by immunoaffinity chromatography using antibodies specific
for STEAP-1. An immunoaffinity column is constructed by covalently
coupling anti-STEAP-1 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.
[0712] Media containing STEAP-1 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of STEAP-1 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/STEAP-1 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 STEAP-1
[0713] STEAP-1, or biologically active fragments thereof, are
labeled with 121 l Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
STEAP-1, washed, and any wells with labeled STEAP-1 complex are
assayed. Data obtained using different concentrations of STEAP-1
are used to calculate values for the number, affinity, and
association of STEAP-1 with the candidate molecules.
Example 37
In Vivo Assay for STEAP-1 Tumor Growth Promotion
[0714] The effect of the STEAP-1 protein on tumor cell growth is
evaluated in vivo by evaluating tumor development and growth of
cells expressing or lacking STEAP-1. For example, SCID mice are
injected subcutaneously on each flank with 1.times.10.sup.6 of
either 3T3, or prostate cancer cell lines (e.g. PC3 cells)
containing tkNeo empty vector or STEAP-1. At least two strategies
may be used: (1) Constitutive STEAP-1 expression under regulation
of a promoter such as a constitutive promoter obtained from the
genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus
2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, provided such promoters are compatible
with the host cell systems, and (2) Regulated expression under
control of an inducible vector system, such as ecdysone,
tetracycline, etc., provided such promoters are compatible with the
host cell systems. Tumor volume is then monitored by caliper
measurement at the appearance of palpable tumors and followed over
time to determine if STEAP-1-expressing cells grow at a faster rate
and whether tumors produced by STEAP-1-expressing cells demonstrate
characteristics of altered aggressiveness (e.g. enhanced
metastasis, vascularization, reduced responsiveness to
chemotherapeutic drugs).
[0715] Additionally, mice can be implanted with 1.times.10.sup.5 of
the same cells orthotopically to determine if STEAP-1 has an effect
on local growth in the prostate, and whether STEAP-1 affects the
ability of the cells to metastasize, specifically to lymph nodes,
and bone (Miki T et al, Oncol Res. 2001; 12:209; Fu X et al, Int J
Cancer. 1991, 49:938). The effect of STEAP on bone tumor formation
and growth may be assessed by injecting prostate tumor cells
intratibially.
[0716] The assay is also useful to determine the STEAP-1 inhibitory
effect of candidate therapeutic compositions, such as for example,
STEAP-1 intrabodies, STEAP-1 antisense molecules and ribozymes.
Example 38
STEAP-1 Monoclonal Antibody-Mediated Inhibition of Prostate Tumors
In Vivo
[0717] The significant expression of STEAP-1 in cancer tissues and
surface localization, together with its restrictive expression in
normal tissues makes STEAP-1 a good target for antibody therapy.
Similarly, STEAP-1 is a target for T cell-based immunotherapy.
Thus, the therapeutic efficacy of anti-STEAP-1 mAbs in human
prostate cancer xenograft mouse models is evaluated by using
recombinant cell lines such as PC3-STEAP-1, and 3T3-STEAP-1 (see,
e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): 16-23), as
well as human prostate xenograft models such as LAPC 9AD (Saffran
et al PNAS 1999, 10:1073-1078).
[0718] Antibody efficacy on tumor growth and metastasis formation
is studied, e.g., in a mouse orthotopic prostate cancer xenograft
models. The antibodies can be unconjugated, as discussed in this
Example, or can be conjugated to a therapeutic modality, as
appreciated in the art. Anti-STEAP-1 mAbs inhibit formation of both
lung and prostate xenografts. Anti-STEAP-1 mAbs also retard the
growth of established orthotopic tumors and prolonged survival of
tumor-bearing mice. These results indicate the utility of
anti-STEAP-1 mAbs in the treatment of local and advanced stages
prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078
or world wide web URL pnas.org/cgi/doi/10.1073/pnas.051624698).
[0719] Administration of the anti-STEAP-1 mAbs led to retardation
of established orthotopic tumor growth and inhibition of metastasis
to distant sites, resulting in a significant prolongation in the
survival of tumor-bearing mice. These studies indicate that STEAP-1
as an attractive target for immunotherapy and demonstrate the
therapeutic potential of anti-STEAP-1 mAbs for the treatment of
local and metastatic prostate cancer. This example demonstrates
that unconjugated STEAP-1 monoclonal antibodies are effective to
inhibit the growth of human prostate tumor xenografts grown in SCID
mice; accordingly a combination of such efficacious monoclonal
antibodies is also effective.
[0720] Tumor Inhibition Using Multiple Unconjugated STEAP-1
mAbs
[0721] Materials and Methods
[0722] STEAP-1 Monoclonal Antibodies:
[0723] Monoclonal antibodies are raised against STEAP-1 as
described in the Example entitled "Generation of STEAP-1 Monoclonal
Antibodies (mAbs)." The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation for their capacity to
bind STEAP-1. Epitope mapping data for the anti-STEAP-1 mAbs, as
determined by ELISA and Western analysis, recognize epitopes on the
STEAP-1 protein. Immunohistochemical analysis of prostate cancer
tissues and cells with these antibodies is performed.
[0724] 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 UM-UC3 and CaLu1 tumor xenografts.
[0725] Cell Lines and Xenografts
[0726] The prostate cancer cell lines, PC3 and LNCaP cell line as
well as the fibroblast line NIH 3T3 (American Type Culture
Collection) are maintained in RPMI and DMEM respectively,
supplemented with L-glutamine and 10% FBS.
[0727] PC3-STEAP-1 and 3T3-STEAP-1 cell populations are generated
by retroviral gene transfer as described in Hubert, R. S., et al.,
Proc Natl Acad Sci USA, 1999. 96(25): 14523.
[0728] The LAPC-9 xenograft, which expresses a wild-type androgen
receptor and produces prostate-specific antigen (PSA), is passaged
in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID)
mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., Nat
Med. 1999, 5:280). Single-cell suspensions of LAPC-9 tumor cells
are prepared as described in Craft, et al.
[0729] Xenograft Mouse Models.
[0730] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10.sup.6 cancer cells mixed at a 1:1 dilution with Matrigel
(Collaborative Research) in the right flank of male SCID mice. To
test antibody efficacy on tumor formation, i.e. antibody injections
are started on the same day as tumor-cell injections. As a control,
mice are injected with either purified mouse IgG (ICN) or PBS; or a
purified monoclonal antibody that recognizes an irrelevant antigen
not expressed in human cells. In preliminary studies, no difference
is found between mouse IgG or PBS on tumor growth. Tumor sizes are
determined by caliper measurements, and the tumor volume is
calculated as length.times.width.times.height. Mice with
Subcutaneous tumors greater than 1.5 cm in diameter are
sacrificed.
[0731] Orthotopic injections are performed under anesthesia by
using ketamine/xylazine. For prostate orthotopic studies, an
incision is made through the abdomen to expose the prostate and
LAPC or PC3 tumor cells (5.times.10.sup.5) mixed with Matrigel are
injected into the prostate capsule in a 10-.mu.l volume. To monitor
tumor growth, mice are palpated and blood is collected on a weekly
basis to measure PSA levels. The mice are segregated into groups
for the appropriate treatments, with anti-STEAP-1 or control mAbs
being injected i.p.
[0732] Anti-STEAP-1 mAbs Inhibit Growth of STEAP-1-Expressing
Xenograft-Cancer Tumors
[0733] The effect of anti-STEAP-1 mAbs on tumor formation is tested
by using LNCaP and LAPC9 orthotopic models. As compared with the
s.c. tumor model, the orthotopic model, which requires injection of
tumor cells directly in the mouse prostate, 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). The features make the orthotopic model more
representative of human disease progression and allowed us to
follow the therapeutic effect of mAbs on clinically relevant end
points.
[0734] Accordingly, tumor cells are injected into the mouse
prostate, and 2 days later, the mice are segregated into two groups
and treated with either: a) 200-500 .mu.g, of anti-STEAP-1 Ab, or
b) PBS three times per week for two to five weeks.
[0735] A major advantage of the orthotopic cancer models is the
ability to study the development of metastases. Formation of
metastasis in mice bearing established orthotopic tumors is studies
by IHC analysis on lung sections using an antibody against a
tumor-specific cell-surface protein such as anti-CK20 for prostate
cancer (Lin S et al, Cancer Detect Prev. 2001; 25:202).
[0736] Another advantage of xenograft cancer models is the ability
to study neovascularization and angiogenesis. Tumor growth is
partly dependent on new blood vessel development. Although the
capillary system and developing blood network is of host origin,
the initiation and architecture of the neovasculature is regulated
by the xenograft tumor (Davidoff A M et al, Clin Cancer Res. 2001;
7:2870; Solesvik O et al, Eur J Cancer Clin Oncol. 1984, 20:1295).
The effect of antibody and small molecule on neovascularization is
studied in accordance with procedures known in the art, such as by
IHC analysis of tumor tissues and their surrounding
microenvironment.
[0737] Mice bearing established orthotopic tumors are administered
1000 .mu.g injections of either anti-STEAP-1 mAb or PBS over a
4-week period. Mice in both groups are allowed to establish a high
tumor burden, to ensure a high frequency of metastasis formation in
mouse lungs. Mice then are killed and their bladders, livers, bone
and lungs are analyzed for the presence of tumor cells by IHC
analysis. These studies demonstrate a broad anti-tumor efficacy of
anti-STEAP-1 antibodies on initiation and progression of prostate
cancer in xenograft mouse models. Anti-STEAP-1 antibodies inhibit
tumor formation of tumors as well as retarding the growth of
already established tumors and prolong the survival of treated
mice. Moreover, anti-STEAP-1 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. Thus, anti-STEAP-1 mAbs
are efficacious on major clinically relevant end points (tumor
growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-STEAP-1 Antibodies in
Humans
[0738] Anti-STEAP-1 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-STEAP-1 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of STEAP-1 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-STEAP-1 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0739] As determined by flow cytometry, anti-STEAP-1 mAb
specifically binds to carcinoma cells. Thus, anti-STEAP-1
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 STEAP-1. Shedding or release of an extracellular
domain of STEAP-1 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of STEAP-1 by
anti-STEAP-1 antibodies in serum and/or urine samples from suspect
patients.
[0740] Anti-STEAP-1 antibodies that specifically bind STEAP-1 are
used in therapeutic applications for the treatment of cancers that
express STEAP-1. Anti-STEAP-1 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-STEAP-1 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 "STEAP-1 Monoclonal Antibody-mediated Inhibition
of Bladder and Lung Tumors In Vivo"). Either conjugated and
unconjugated anti-STEAP-1 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-STEAP-1 Antibodies In Vivo
[0741] Antibodies are used in accordance with the present invention
which recognize an epitope on STEAP-1, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including STEAP-1 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
[0742] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-STEAP-1 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-STEAP-1
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-STEAP-1 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).
[0743] II.) Monotherapy: In connection with the use of the
anti-STEAP-1 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.
[0744] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-STEAP-1 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 STEAP-1. In connection with the use of the anti-STEAP-1
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)-STEAP-1 antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses STEAP-1 (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
[0745] Dose and Route of Administration
[0746] 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-STEAP-1
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-STEAP-1 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-STEAP-1 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-STEAP-1 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.
[0747] Three distinct delivery approaches are useful for delivery
of anti-STEAP-1 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.
[0748] Clinical Development Plan (CDP)
[0749] Overview: The CDP follows and develops treatments of
anti-STEAP-1 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-STEAP-1 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
STEAP-1 expression levels in their tumors as determined by
biopsy.
[0750] 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 STEAP-1. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-STEAP-1 antibodies are
found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-STEAP-1
Antibody and Chemotherapeutic Agent
[0751] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-STEAP-1 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-STEAP-1 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-STEAP-1 antibody
with dosage of antibody escalating from approximately about 25
mg/m.sup.2 to about 275 mg/m.sup.2 over the course of the treatment
in accordance with the following schedule: TABLE-US-00004 Day 0 Day
7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 mg/m.sup.2 75 mg/m.sup.2
125 mg/m.sup.2 175 mg/m.sup.2 225 mg/m.sup.2 275 mg/m.sup.2
Chemotherapy + + + + + + (standard dose)
[0752] 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 STEAP-1. 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.
[0753] The anti-STEAP-1 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-STEAP-1
Antibody
[0754] Anti-STEAP-1 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-STEAP-1
antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-STEAP-1
Antibody
[0755] 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-STEAP-1 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 STEAP-1 to Known Sequences
[0756] The human STEAP protein does not exhibit a high degree of
homology to any known human proteins. The prototype member of the
STEAP family, STEAP-1v.1, is a type IIIa membrane protein
consisting of 339 amino acids. STEAP-1 is a six transmembrane
domain protein, with intracellular N- and C-termini.
[0757] The STEAP-1 gene is somewhat homologous to a previously
cloned gene, namely mouse TNFa-induced adipose-related protein
(gi|16905133|). STEAP-1v.1 shares 40% identities and 63% homology
with TIARP. In addition, STEAP-1 shows homology to rat pHyde
protein, with 49% identity and 71% homology to that protein. The
TIARP protein is a 6 transmembrane protein of unknown function
which localizes to the cell surface (Moldes M et al, J. Biol. Chem
2001, 276:33938). Rat pHyde is a protein expressed in Dunning rats
prostate cancer lines. Overexpression of pHyde in DU145 cells
reduces their tumor growth properties (Steiner M et al, Cancer Res
2000, 60:4419). In addition, STEAP1 shows significant homology to
mouse six transmembrane epithelial antigen of the prostate (gi
20820492).
[0758] Motif analysis revealed the presence of several protein
functional motifs (Table XXI). Prints prediction identified a
Transforming protein P21 ras signature, and Fibronectin type III
repeat signature, while Blocks prediction identified a Half-A-TPR
repeat, and an arsenical pump membrane protein signature. The
fibronectin type III repeat region is a 100 amino acid domain,
which mediated binding to DNA, heparin, basement membrane and the
cell surface. The predominant role of these repeats is binding to
the cell surface, allowing cell adhesion and mediating signaling
event. Half-A-TPR repeat is a motif found in several RNA processing
proteins. The arsenical pump functions in the efflux of ion and
small molecules (Walmsley A R et al. J Biol Chem 2001,
276:6378-91). The motifs can participate in tumor growth and
progression by enhancing the initial stages of tumorigenesis, such
as tumor take or establishment of a tumor, by allowing adhesion to
basement membranes and surrounding cells, by mediating cell
communication and transport of ions.
[0759] Accordingly, when STEAP-1 functions as a regulator of tumor
establishment, tumor formation, tumor growth, cell signaling or as
a modulator of transcription involved in activating genes
associated with survival, invasion, tumorigenesis or proliferation,
STEAP-1 is used for therapeutic, diagnostic, prognostic and/or
preventative purposes. In addition, when a molecule, such as a
variant or SNP of STEAP-1 is expressed in cancerous tissues, such
as those listed in Table I, they are used for therapeutic,
diagnostic, prognostic and/or preventative purposes.
Example 45
Regulation of Transcription
[0760] The localization of STEAP-1 coupled to the presence of RNA
processing motifs within its sequence indicate that STEAP-1
modulates the transcriptional regulation of eukaryotic genes.
Regulation of gene expression is confirmed, e.g., by studying gene
expression in cells expressing or lacking STEAP-1. For this
purpose, two types of experiments are performed.
[0761] In the first set of experiments, RNA from parental and
STEAP-1-expressing cells are extracted and hybridized to
commercially available gene arrays (Clontech) (Smid-Koopman E et
al. Br J Cancer. 2000. 83:246). Resting cells as well as cells
treated with FBS, androgen or growth factors are compared.
Differentially expressed genes are identified in accordance with
procedures known in the art. The differentially expressed genes are
then mapped to biological pathways (Chen K et al. Thyroid. 2001.
11:41.).
[0762] 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.
[0763] Thus, STEAP-1 plays a role in gene regulation, and it is
used as a target for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 46
Identification and Confirmation of Potential Signal Transduction
Pathways
[0764] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J. Neurochem. 2001; 76:217-223). Fibronectin in
particular has been associated with the MAPK signaling cascade that
control cell mitogenesis (Jiang F, Jia Y, Cohen I. Blood. 2002,
99:3579). In addition, the STEAP-1 protein contains several
phosphorylation sites (see Table XXI) indicating an association
with specific signaling cascades. Using immunoprecipitation and
Western blotting techniques, proteins are identified that associate
with STEAP-1 and mediate signaling events. Several pathways known
to play a role in cancer biology can be regulated by STEAP-1,
including phospholipid pathways such as PI3K, AKT, etc, adhesion
and migration pathways, including FAK, Rho, Rac-1, .beta.-catenin,
etc, as well as mitogenic/survival cascades such as ERK, p38, etc
(Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999, 274:801;
Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).). In order
to determine whether expression of STEAP-1 is sufficient to
regulate specific signaling pathways not otherwise active in
resting PC3 cells, the effect of these genes on the activation of
the p38 MAPK cascade was investigated in the prostate cancer cell
line PC3 (FIG. 21A-B). Activation of the p38 kinase is dependent on
its phosphorylation on tyrosine and serine residues. Phosphorylated
p38 can be distinguished from the non-phosphorylated state by a
Phospho-p38 mAb. This phospho-specific Ab was used to study the
phosphorylation state of p38 in engineered PC3 cell lines.
[0765] PC3 cells stably expressing STEAP-1 neo were grown overnight
in either 1% or 10% FBS. Whole cell lysates were analyzed by
western blotting. PC3 cells treated with the known p38 activators,
NaSaI or TNF, were used as a positive control. The results show
that while expression of the control neo gene has no effect on p38
phosphorylation, expression of STEAP-1 in PC3 cells is sufficient
to induce the activation of the p38 pathway (FIG. 21A). The results
were verified using western blotting with an anti-p38 Ab, which
shows equal protein loading on the gels (FIG. 21B).
[0766] In another set of experiments, the sufficiency of expression
of STEAP-1 in the prostate cancer cell line PC3 to activate the
mitogenic MAPK pathway, namely the ERK cascade, was examined (FIG.
22A-B). Activation of ERK is dependent on its phosphorylation on
tyrosine and serine residues. Phosphorylated ERK can be
distinguished from the non-phosphorylated state by a Phospho-ERK
mAb. This phospho-specific Ab was used to study the phosphorylation
state of ERK in engineered PC3 cell lines. PC3 cells, expressing an
activated form of Ras, were used as a positive control.
[0767] The results show that while expression of the control neo
gene has no effect on ERK phosphorylation, expression of STEAP-1 in
PC3 cells is sufficient to induce an increase in ERK
phosphorylation (FIG. 22A). These results were verified using
anti-ERK western blotting (FIG. 22B) and confirm the activation of
the ERK pathway by STEAP-1 and STEAP-2.
[0768] Since FBS contains several components that may contribute to
receptor-mediated ERK activation, we examined the effect of STEAP-1
in low and optimal levels of FBS. PC3 cells expressing neo or
STEAP-1 were grown in either 0.1% or 10% FBS overnight. The cells
were analyzed by anti-Phospho-ERK western blotting. This experiment
shows that STEAP-1 induces the phosphorylation of ERK in 0.1% FBS,
and confirms that expression of STEAP-1 is sufficient to induce
activation of the ERK signaling cascade in the absence of
additional stimuli.
[0769] To confirm that STEAP-1 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. [0770] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress [0771] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation [0772] 3. AP-1-luc, FOS/JIJN; MAPK/SAPK/PKC;
growth/apoptosis/stress [0773] 4. ARE-luc, androgen receptor;
steroids/MAPK; growth/differentiation/apoptosis [0774] 5. p53-luc,
p53; SAPK; growth/differentiation/apoptosis [0775] 6. CRE-luc,
CREB/ATF2; PKA/p38; growth/apoptosis/stress [0776] 7. TCF-luc,
TCF/Lef; .beta.-catenin, Adhesion/invasion
[0777] 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.
[0778] Signaling pathways activated by STEAP-1 are mapped and used
for the identification and validation of therapeutic targets. When
STEAP-1 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 47
Involvement in Tumor Progression
[0779] Based on the documented role of transforming protein in
transformation, and progression, the STEAP-1 gene can contribute to
the growth and transformation of cancer cells. The role of STEAP-1
in tumor growth is confirmed in a variety of primary and
transfected cell lines including prostate cell lines, as well as
NIH 3T3 cells engineered to stably express STEAP-1. Parental cells
lacking STEAP-1 and cells expressing STEAP-1 are evaluated for cell
growth using a well-documented proliferation assay (Fraser S P,
Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E,
Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).
[0780] To confirm the role of STEAP-1 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH-3T3 cells lacking STEAP-1 are compared to NIH-3T3
cells expressing STEAP-1, using a soft agar assay under stringent
and more permissive conditions (Song Z. et al. Cancer Res. 2000;
60:6730).
[0781] To confirm the role of STEAP-1 in invasion and metastasis of
cancer cells, a well-established assay is used, e.g., a Transwell
Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010).
Control cells, including prostate, breast and kidney cell lines
lacking STEAP-1 are compared to cells expressing STEAP-1. Cells are
loaded with the fluorescent dye, calcein, and plated in the top
well of the Transwell insert coated with a basement membrane
analog. Invasion is determined by fluorescence of cells in the
lower chamber relative to the fluorescence of the entire cell
population.
[0782] STEAP-1 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing STEAP-1 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 STEAP-1, including normal and tumor prostate
cells. Engineered and parental cells are treated with various
chemotherapeutic agents, such as etoposide, taxol, etc, and protein
synthesis inhibitors, such as cycloheximide. Cells are stained with
annexin V-FITC and cell death is measured by FACS analysis. The
modulation of cell death by STEAP-1 can play a critical role in
regulating tumor progression and tumor load.
[0783] When STEAP-1 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
[0784] 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,
STEAP-1 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 STEAP-1 in angiogenesis,
enhancement or inhibition, is confirmed.
[0785] For example, endothelial cells engineered to express STEAP-1
are evaluated using tube formation and proliferation assays. The
effect of STEAP-1 is also confirmed in animal models in vivo. For
example, cells either expressing or lacking STEAP-1 are implanted
subcutaneously in immunocompromised mice. Endothelial cell
migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. STEAP-1 affects angiogenesis, and
it is used as a target for diagnostic, prognostic, preventative
and/or therapeutic purposes
Example 49
Involvement in Protein-Protein Interactions
[0786] Fibronectin motifs have been shown to mediate interaction
with other proteins, including cell surface protein. Using
immunoprecipitation techniques as well as two yeast hybrid systems,
proteins are identified that associate with STEAP-1.
Immunoprecipitates from cells expressing STEAP-1 and cells lacking
STEAP-1 are compared for specific protein-protein associations.
[0787] Studies are performed to confirm the extent of association
of STEAP-1 with effector molecules, such as nuclear proteins,
transcription factors, kinases, phosphates etc. Studies comparing
STEAP-1 positive and STEAP-1 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.
[0788] 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 STEAP-1-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
calorimetric reporter activity. Specific association with effector
molecules and transcription factors directs one of skill to the
mode of action of STEAP-1, 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 STEAP-1.
[0789] Thus it is found that STEAP-1 associates with proteins and
small molecules. Accordingly, STEAP-1 and these proteins and small
molecules are used for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 50
Involvement of STEAP-1 in Small Molecule Transport and Cell-Cell
Communication
[0790] Cell-cell communication is essential in maintaining organ
integrity and homeostasis, both of which become deregulated during
tumor formation and progression. Intercellular communications can
be measured using two types of assays (J. Biol. Chem. 2000,
275:25207). In the first assay, cells loaded with a fluorescent dye
are incubated in the presence of unlabeled recipient cells and the
cell populations are examined under fluorescent microscopy. This
qualitative assay measures the exchange of dye between adjacent
cells. In the second assay system, donor and recipient cell
populations are treated as above and quantitative measurements of
the recipient cell population are performed by FACS analysis. Using
these two assay systems, cells expressing STEAP-1 are compared to
controls that do not express STEAP-1, and it is found that STEAP-1
enhances cell communications. FIG. 19 and FIG. 20 demonstrate that
STEAP-1 mediates the transfer of the small molecule calcein between
adjacent cells, and thereby regulates cell-cell communication in
prostate cancer cells. In this experiment, recipient PC3 cells were
labeled with dextran-Texas Red and donor PC3 cells were labeled
with calcein AM (green). The donor (green) and recipient (red)
cells were co-cultured at 37.degree. C. and analyzed by microscopy
for the co-localization of Texas red and calcein. The results
demonstrated that while PC3 control cells (no detectable STEAP-1
protein expression) exhibit little calcein transfer, the expression
of STEAP-1 allows the transfer of small molecules between cells
(FIG. 19), whereby the initially red recipient cells take on a
brownish color, and co-localize the red and green molecules. Small
molecules and/or antibodies that modulate cell-cell communication
mediated by STEAP-1 are used as therapeutics for cancers that
express STEAP-1. FIG. 20 shows the time-dependent manner of
cell-cell communication mediated by STEAP-1, where little transfer
in PC3-STEAP-1 cells is seen at 6 hours, and much transfer is seen
at 24 hours. FIG. 23 demonstrates that expression of STEAP-1 is
necessary on both donor and recipient populations for the transfer
of small molecules to take place. In this experiment, donor cells
labeled with dextran red were incubated with recipient cells
labeled with calcein (green). However, the donor and recipient
populations were alternated, such that a PC3 donor population was
incubated with PC3 recipient, or with PC3-STEAP-1 recipient
populations. Similarly, a PC3-STEAP-1 donor population was
co-cultured with a PC3 or a PC3-STEAP-1 recipient population. The
results show that co-culturing of control PC3 and PC3 cells fail to
mediate calcein transfer. Similarly, co-incubation of control PC3
and PC3-STEAP-1 does not allow the transfer of calcein. However,
co-culturing PC3-STEAP-1 donor and PC3-STEAP-1 recipient cells
mediates small molecule transfer as depicted by co-localization of
green and red pigments in the same cells. Taken together, the data
shown in FIGS. 19, 20 and 23 demonstrate that STEAP-1 mediates
small molecule transfer and regulates cell-cell communication by
forming inter-cellular communication channels that are similar in
function to gap junctions.
[0791] Thus, because STEAP-1 functions in cell-cell communication
and small molecule transport, it is used as a target or marker for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 51
Involvement of STEAP-1 in Ion Transport
[0792] The localization and topology of STEAP-1 supports its
function as a transporter. To confirm that STEAP-1 functions as an
ion channel, FACS analysis and fluorescent microscopy techniques
were used (Gergely L, et al., Clin Diagn Lab Immunol. 1997; 4:70;
Skryma R, et al., J. Physiol. 2000, 527: 71). Using FACS analysis
and commercially available indicators (Molecular Probes), parental
cells and cells expressing STEAP-1 were compared for their ability
to transport calcium; prostate cancer PC3 cell lines were used in
these studies. PC3 and PC3-STEAP-1 cells were loaded with calcium
responsive indicators Fluo4 and Fura red, incubated in the presence
or absence of calcium and lipophosphatidic acid (LPA), and analyzed
by flow cytometry. FIG. 17 shows that STEAP-1 increased calcium
flux in response to LPA. This data validates an important mechanism
by which cancer cells are regulated. This is particularly true in
the case of calcium, as calcium channel inhibitors have been
reported to induce the death of certain cancer cells, including
prostate cancer cell lines (Batra S, Popper L D, Hartley-Asp B.
Prostate. 1991, 19: 299).
[0793] Furthermore, FIG. 18 shows that STEAP-1-mediated calcium
transport regulates prostate cancer growth by regulating
intracellular levels of calcium. In this study, control PC3 and
PC3-STEAP-1 cells were grown in the presence or absence of ion
channel inhibitors, namely amiloride, NDGA and TEA, which inhibit
the transport of sodium, calcium and potassium, respectively.
STEAP-1 expression rendered PC3 cells sensitive to treatment with
the Ca++ channel inhibitor NDGA. While PC3 control cells
demonstrated 100% growth in the presence of the inhibitor, NDGA, at
50 .mu.M inhibited the proliferation of PC3-STEAP-1 cells by 42%
and 10 example 51 .mu.M NDGA inhibited proliferation 20%. These
findings confirm the importance of STEAP-1 as regulator of calcium
transport in the proliferation of cancer cells that express this
protein. Thus, STEAP-1 is used in therapeutic modalities that
control the proliferation of prostate cancer cells. Because STEAP-1
functions in ion transport, it is used as a target or marker for
diagnostic, prognostic, preventative and therapeutic purposes.
Example 52
RNA Interference (RNAi)
[0794] RNA interference (RNAi) makes use of sequence specific
double stranded RNA to prevent gene expression. Small interfering
RNA (siRNA) are transfected into mammalian cells and thereby
mediate sequence specific mRNA degradation. (Elbashir, et al,
Nature, 2001; vol. 411: 494-498). The following siRNA
oligonucleotide sequences were used: TABLE-US-00005 (SEQ ID NO: 96)
STEAP-1 (1) target: GACAUCACAAACCAAGAAG (corresponding to
nucleotides 16-34 of STEAP-1 ORF) (SEQ ID NO: 97) STEAP-1 (2)
target: GUGGAUAGAUAUAAAACAA (corresponding to nucleotides 893-911
of STEAP-1 ORF)
[0795] The sense strand of STEAP-1 (1) is labeled at 3' with
fluorescein, 6-FAM (ABS 494 nm, EMM 525 nm, green). The siRNA is
dissolved in RNA-free sterile buffer (100 mM KOAc, 30 mM HEPES KOH,
2 mM MOAc, at pH 7.4) to make 20 .mu.M stock (200.times.). The
siRNa is transfected into LNCaP, 3T3-STEAP-1, and Rat-1-STEAP-1
cells seeded on 6-well plates with oligofectamine reagent
(Invitrogen). The final concentration of siRNA was 100.times.nM.
The following oligonucleotide was used as a control to rule out any
non-specific effect of STEAP-1 siRNA oligonucleotides:
TABLE-US-00006 (SEQ ID NO: 98) Control: GCCUUCCCAUUGUGCUCCC
(corresponding to nucleotides 91-109 of ORF)
[0796] Protein expression was detected 24 hours after transfection
by immunostaining followed by flow cytometry. In addition,
confirmation of altered gene expression was performed by Western
blotting. The results (FIG. 24) show that the introduction of
STEAP-1 specific RNAi reduced the expression of STEAP-1 in
recombinant 3T3 and Rat-1 cells. 3T3 and Rat-1 cells were
permeabilized and stained with anti-STEAP-1 polyclonal antibodies.
Whole cell immunostaining revealed that the STEAP-1 RNAi reduced
STEAP-1 expression in Rat-1 and 3T3 cells. This reduction was
confirmed by Western blot analysis where STEAP-1 protein was
substantially reduced in STEAP-1 RNAi treated cells relative to
control and untreated cells. Additionally, RNAi reduces the
endogenous expression of STEAP-1 in the prostate cancer and LNCaP
cell line as shown in FIG. 25.
[0797] Accordingly, the RNA oligonucleotide sequences are used in
therapeutic and prophylactic applications. Moreover, the RNA
oligonucleotide sequences are used to assess how modulating the
expression of a STEAP-1 gene affects function of cancer cells
and/or tissues.
Example 53
Modulation of STEAP-1 Function
[0798] Ion transport plays an important role regulating cell growth
intracellular permeability, molecular trafficking and signal
transduction (Minke B. Cell Mol Neurobiol. 2001, 21:629; Golovina
et al, Am J Physiol Heart Circ Physiol. 2001, 280:H746) these are
functions that are especially relevant to the neoplastic condition.
Cell-cell communication regulates homeostasis, cell proliferation
and cell death (Evans W H, Martin P E. Mol Membr Biol. 2002 19:121;
Carruba G, et al, Ann N Y Acad. Sci. 2002, 963:156) these functions
too are especially relevant to the neoplastic condition.
[0799] Using control cell lines and cell lines expressing STEAP-1,
inhibitors of STEAP-1 function are identified. For example, PC3 and
PC3-STEAP-1 cells can be incubated in the presence and absence of
mAb or small molecule inhibitors. The effect of these mAb or small
molecule inhibitors are investigated using the ion flux, cell
communication, proliferation and signaling assays described
above.
[0800] Signal transduction and biological output mediated by
transporters can be modulated through various mechanisms, including
inhibition of receptor and ligand binding, ion antagonists, protein
interactions, regulation of ion and small molecule transport, etc
(Tang W et al, Front Biosci 2002, 7:1583). Using control cell lines
and cell lines expressing STEAP-1, modulators (inhibitors or
enhancers) of STEAP-1 function are identified. For example, PC3 and
PC3-STEAP-1 cells are incubated in the presence and absence of mAb
or small molecule modulators. In view of the functions of STEAP-1
disclosed herein, modulators that are ion channel blockers used in
the context of the present invention include such compounds as
amlodipine, azulene, dihydropyridines, thianines, nifedine,
verapamil and their derivatives (Tanaka Y, Shigenobu K. Cardiovasc
Drug Rev. 2001, 19:297; Djuric D, Mitrovic V, Jakovljevic V.
Arzneimittelforschung. 2002, 52:365; Kourie J I, Wood H B. Prog
Biophys Mol. Biol. 2000; 73:91); and, modulators that are
inhibitors of cell communication used in the context of the present
invention include such compounds as beta-glycyrrhetinic acid,
retinoids, TPA (Krutovskikh V A et al, Oncogene. 2002, 21:1989;
Rudkin et al, J Surg Res. 2002, 103:183; Ruch J et al, J Cell
Biochem. 2001, 83:163). Accordingly, the effect(s) of mAb or small
molecule inhibitors are investigated using the ion flux, cell
communication, proliferation and signaling assays described
Examples above.
[0801] When mAb and small molecules modulate, e.g., inhibit, the
transport and tumorigenic function of STEAP-1, they are used for
preventative, prognostic, diagnostic and/or therapeutic
purposes.
[0802] 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.
[0803] 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.
[0804] Tables: TABLE-US-00007 TABLE I Tissues that Express STEAP-1
When Malignant Stomach Rectum
[0805] TABLE-US-00008 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
[0806] TABLE-US-00009 TABLE III Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution
matrix (block substitution matrix). The higher the value, the more
likely a substitution is found in related, natural proteins. (See
world wide web URL ikp.unibe.ch/manual/blosum62.html) A C D E F G H
I K L M N P Q R S T V W Y . 4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1
-1 1 0 0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1
-2 -2 C 6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D 5 -3 -2
0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3
-2 -2 -1 1 3 F 6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G 8 -3 -1
-3 -2 1 -2 0 0 -1 -2 -3 -2 2 H 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I
5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L 5
-2 -2 0 -1 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N 7 -1 -2 -1 -1 -2
-4 -3 P 5 1 0 -1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 5 0
-2 -2 T 4 -3 -1 V 11 2 W 7 Y
[0807] TABLE-US-00010 TABLE IV HLA Class I/II Motifs/Supermotifs
(A): HLA Class I Supermotifs/Motifs SUPERMOTIFS POSITION POSITION
POSITION 2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary
Anchor) A1 TILVMS FWY A2 LIVMATQ IVMATL A3 VSMATLI RK A24 YFWIVLMT
FIYWLM B7 P VILFMWYA B27 RHK FYLWMIVA B44 ED FWYLIMVA B58 ATS
FWYLIVMA B62 QLIVMP FWYMIVLA MOTIFS A1 TSM Y A1 DEAS Y A2.1 LMVQIAT
VLIMAT A3 LMVISATFCGD KYRHFA A11 VTMLISAGNCDF KRYH A24 YFWM FLIW
A*3101 MVTALIS RK A*3301 MVALFIST RK A*6801 AVTMSLI RK B*0702 P
LMFWYAIV B*3501 P LMFWYIVA B51 P LIVFWYAM B*5301 P IMFWYALV B*5401
P ATIVLMFWY Bolded residues are preferred, italicized residues are
less preferred: A peptide is considered motif-bearing if it has
primary anchors at each primary anchor position for a motif or
supermotif as specified in the above table. (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 (C): HLA Class II Motifs MOTIFS 1.degree. anchor 1
2 3 4 5 1.degree. anchor 6 7 8 9 DR4 preferred FMYLIVW M T I
VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ
VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY
M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1.degree.
anchor 1 2 3 1.degree. anchor 4 5 1.degree. anchor 6 Motif a
preferred LIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR
Supermotif MFLIVWY VMSTACPLI Italicized residues indicate less
preferred or "tolerated" residues (D): HLA Class I Supermotifs
SUPER- POSITION: MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1.degree.
Anchor 1.degree. Anchor TILVMS FWY A2 1.degree. Anchor 1.degree.
Anchor {overscore (LIVMATQ)} LIVMAT A3 Preferred 1.degree. Anchor
YFW YFW YFW P 1.degree. Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK
deleterious DE (3/5); DE P (5/5) (4/5) A24 1.degree. Anchor
1.degree. Anchor {overscore (YFWIVLMT)} FIYWLM B7 Preferred FWY
(5/5) 1.degree. Anchor FWY FWY 1.degree. Anchor LIVM (3/5) P (4/5)
(3/5) {overscore (VILFMWYA)} deleterious DE (3/5); DE G QN DE
P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27
1.degree. Anchor 1.degree. Anchor RHK {overscore (FYLWMIVA)} B44
1.degree. Anchor 1.degree. Anchor ED {overscore (FWYLIMVA)} B58
1.degree. Anchor 1.degree. Anchor ATS {overscore (FWYLIVMA)} B62
1.degree. Anchor 1.degree. Anchor QLIVMP {overscore (FWYMIVLA)}
Italicized residues indicate less preferred or "tolerated" residues
(E): HLA Class I Motifs POSITION 9 or C- C- 1 2 3 4 5 6 7 8
terminus terminus A1 preferred GFYW 1.degree. Anchor DEA YFW P DEQN
YFW 1.degree. Anchor 9-mer STM Y deleterious DE RHKLIVMP A G A A1
preferred GRHK ASTCLIVM 1.degree. Anchor GSTC ASTC LIVM DE
1.degree. Anchor 9-mer DEAS Y deleterious A RHKDEPYFW DE PQN RHK PG
GP A1 preferred YFW 1.degree. Anchor DEAQN A YFWQN PASTC GDE P
1.degree. Anchor 10-mer STM Y deleterious GP RHKGLIVM DE RHK QNA
RHKYFW RHK A A1 preferred YFW STCLIVM 1.degree. Anchor A YFW PG G
YFW 1.degree. Anchor 10-mer DEAS Y deleterious RHK RHKDEPYFW P G
PRHK QN A2.1 preferred YFW 1.degree. Anchor YFW STC YFW A P
1.degree. Anchor 9-mer LMIVQAT VLIMAT deleterious DEP DERKH RKH
DERKH POSITION: C- 1 2 3 4 5 6 7 8 9 terminus A2.1 pre- AYFW
1.degree. Anchor LVIM G G FYWLVIM 1.degree. Anchor 10-mer ferred
LMIVQAT VLIMAT dele- DEP DE RKHA P RKH DERKH RKH te- rious A3 pre-
RHK 1.degree. Anchor YFW PRHKYFW A YFW P 1.degree. Anchor ferred
{overscore (LMVISATFCGD)} KYRHFA dele- DEP DE te- rious A11 pre- A
1.degree. Anchor YFW YFW A YFW YFW P 1.degree. Anchor ferred
{overscore (VTLMISAGNCD)} KRYH dele- DEP F A G te- rious A24 pre-
YFWRHK 1.degree. Anchor STC YFW YFW 1.degree. Anchor 9-mer ferred
YFWM FLIW dele- DEG DE G QNP DERHK G AQN te- rious A24 Pre-
1.degree. Anchor P YFWP P 1.degree. Anchor 10-mer ferred YFWM FLIW
Dele- GDE QN RHK DE A QN DEA te- rious A3101 Pre- RHK 1.degree.
Anchor YFW P YFW YFW AP 1.degree. Anchor ferred MVTALIS RK Dele-
DEP DE ADE DE DE DE te- rious A3301 Pre- 1.degree. Anchor YFW AYFW
1.degree. Anchor ferred {overscore (MVALFIST)} RK Dele- GP DE te-
rious A6801 Pre- YFWSTC 1.degree. Anchor YFWLIV YFW P 1.degree.
Anchor ferred AVTMSLI M RK dele- GP DEG RHK A te- rious B0702 Pre-
RHKFWY 1.degree. Anchor RHK RHK RHK RHK PA 1.degree. Anchor ferred
P {overscore (LMFWYAIV)} dele- DEQNP DEP DE DE GDE QN DE te- rious
B3501 Pre- FWYLIVM 1.degree. Anchor FWY FWY 1.degree. Anchor ferred
P {overscore (LMFWYIVA)} B51 Pre- LIVMFWY 1.degree. Anchor FWY STC
FWY G FWY 1.degree. Anchor ferred P {overscore (LIVFWYAM)} dele-
AGPDER DE G DEQN GDE te- HKSTC rious B5301 pre- LIVMFWY 1.degree.
Anchor FWY STC FWY LIVMFWY FWY 1.degree. Anchor ferred P {overscore
(IMFWYALV)} dele- AGPQN G RHKQN DE te- rious B5401 pre- FWY
1.degree. Anchor FWYLIVM LIVM ALIVM FWYAP 1.degree. Anchor ferred P
{overscore (ATIVLMFWY)} dele- GPQNDE GDESTC RHKDE DE QNDGE DE te-
rious Italicized residues indicate less preferred or "tolerated"
residues. The information in this Table is specific for 9-mers
unless otherwise specified.
[0808] TABLE-US-00011 TABLE V -V1-HLA-A1-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 3; each startposition 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 32 TGETSMLKR 11.250 180 LSYPMRRSY 7.500 22
LEEDDYLHK 4.500 9 NQEELWKMK 2.700 80 IIASLTFLY 2.500 122 SITLLALVY
2.500 7 ITNQEELWK 2.500 31 DTGETSMLK 2.500 277 WIDIKQFVW 2.500 324
WEDVIKINK 2.500 99 ATSHQQYFY 2.500 244 SLTWREFHY 2.500 153
WLDKWMLTR 2.500 21 NLEEDDYLH 1.800 100 TSHQQYFYK 1.500 286
YTPPTFMIA 1.250 84 LTFLYTLLR 1.250 108 KIPILVINK 1.000 307
ILFLPCLRK 1.000 208 WIEHDVWRM 0.900 178 YSLSYPMRR 0.750 78
AAIIASLTF 0.500 51 HADEFDCPS 0.500 301 VLIFKSILF 0.500 148
KKFPHWLDK 0.500 296 FLPIVVLIF 0.500 215 RMEIYVSLG 0.450 297
LPIVVLIFK 0.250 247 WREFHYIQS 0.225 203 NKEDAWIEH 0.225 138
IVQLHNGTK 0.200 140 QLHNGTKYK 0.200 268 HALIFAWNK 0.200 236
TSIPSVSDS 0.150 64 TQELFPQWH 0.135 265 GTIHALIFA 0.125 174
LHAIYSLSY 0.125 264 LGTIHALIF 0.125 210 EHDVWRMEI 0.125 94
VIHPLATSH 0.100 60 ELQHTQELF 0.100 306 SILFLPCLR 0.100 240
SVSDSLTWR 0.100 68 FPQWHLPIK 0.100 116 KVLPMVSIT 0.100 98 LATSHQQYF
0.100 308 LFLPCLRKK 0.100 91 LREVIHPLA 0.090 139 VQLHNGTKY 0.075
241 VSDSLTWRE 0.075 256 KLGIVSLLL 0.050 125 LLALVYLPG 0.050 227
LAILALLAV 0.050 88 YTLLREVIH 0.050 293 IAVFLPIVV 0.050 223
GIVGLAILA 0.050 123 ITLLALVYL 0.050 224 IVGLAILAL 0.050 97
PLATSHQQY 0.050 170 FFAVLHAIY 0.050 261 SLLLGTIHA 0.050 63
HTQELFPQW 0.050 112 LVINKVLPM 0.050 258 GIVSLLLGT 0.050 171
FAVLHAIYS 0.050 81 LASLTFLYT 0.050 49 TAHADEFDC 0.050 135 IAAIVQLHN
0.050 266 TIHALIFAW 0.050 232 LLAVTSIPS 0.050 221 SLGIVGLAI 0.050
165 GLLSFFFAV 0.050 117 VLPMVSITL 0.050 120 MVSITLLAL 0.050 226
GLAILALLA 0.050 323 GWEDVTKIN 0.045 35 TSMLKRPVL 0.030 121
VSITLLALV 0.030 167 LSFFFAVLH 0.030 82 ASLTFLYTL 0.030 305
KSILFLPCL 0.030 34 ETSMLKRPV 0.025 19 RRNLEEDDY 0.025 129 VYLPGVIAA
0.025 24 EDDYLHKDT 0.025 182 YPMRRSYRY 0.025 71 WHLPIKIAA 0.025 285
WYTPPTFMI 0.025 291 FMIAVFLPI 0.025 163 QFGLLSFFF 0.025 29
HKDTGETSM 0.025 54 EFDCPSELQ 0.025 55 FDCPSELQH 0.025 211 HDVWRMEIY
0.025 161 RKQFGLLSF 0.025 142 HNGTKYKKF 0.025 10 QEELWKMKP 0.022
172 AVLHAIYSL 0.020 321 RHGWEDVTK 0.020 133 GVIAAIVQL 0.020
[0809] TABLE-US-00012 TABLE V -V2-HLA-A1-9mers: STEAP-1 Each
peptide is a portionof SEQ ID NO: 5; each startposition 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 WREFHYIQV 0.225 3 EFHYIQVNN 0.001 2 REFHYIQVN
0.001 4 FHYIQVNNI 0.001
[0810] TABLE-US-00013 TABLE V -V3-HLA-A1-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 20 LWDPCLTRF 2.500 13 KSDVPESLW 0.750 16
VPESLWDPC 0.225 1 WREFHYIQI 0.225 19 SLWDPCLTR 0.050 4 FHYIQIIHK
0.050 18 ESLWDPCLT 0.015 22 DPCLTREKG 0.013 25 LTRFKGLNL 0.013 8
QIIHKKSDV 0.010 6 YIQIIHKKS 0.010 5 HYIQIIHKK 0.010 21 WDPCLTRFK
0.010 14 SDVPESLWD 0.003 3 EFHYIQIIH 0.003 28 FKGLNLIQS 0.003 15
DVPESLWDP 0.001 24 CLTRFKGLN 0.001 23 PCLTRFKGL 0.001 2 REFHYIQII
0.001 12 KKSDVPESL 0.001 27 RFKGLNLIQ 0.000 7 IQIIHKKSD 0.000 17
PESLWDPCL 0.000 9 IIHKKSDVP 0.000 11 HKKSDVPES 0.000 26 TRFKGLNLI
0.000 10 LHKKSDVPE 0.000
[0811] TABLE-US-00014 TABLE V -V4-HLA-A1-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAV 0.050 8 SLFFAVLHA 0.050 7 LSLFFAVLH
0.030 3 QFGLLSLFF 0.025 6 LLSLFFAVL 0.020 2 KQFGLLSLF 0.015 4
FGLLSLFFA 0.013 1 RKQFGLLSL 0.003 9 LFFAVLHAI 0.001
[0812] TABLE-US-00015 TABLE VI -V1-HLA-A1-10mers: STEAP-1 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 21 NLEEDDYLHK 90.000 277 WIDIKQFVWY 50.000 241
VSDSLTWREF 7.500 323 GWEDVTKINK 4.500 243 DSLTWREFHY 3.750 121
VSITLLALVY 3.750 99 ATSHQQYFYK 2.500 173 VLHAIYSLSY 2.500 79
AIIASLTFLY 2.500 215 RMEIYVSLGI 2.250 180 LSYPMRRSYR 1.500 210
EHDVWRMEIY 1.250 286 YTPPTFMIAV 1.250 31 DTGETSMLKR 1.250 296
FLPIVVLIFK 1.000 153 WLDKWMLTRK 1.000 98 LATSHQQYFY 1.000 306
SILFLPCLRK 1.000 6 DITNQEELWK 1.000 247 WREFHYIQSK 0.900 77
IAAIIASLTF 0.500 138 IVQLHNGTKY 0.500 300 VVLIFKSILF 0.500 263
LLGTIHALIF 0.500 107 YKIPILVINK 0.500 116 KVLPMVSITL 0.500 83
SLTFLYTLLR 0.500 179 SLSYPMRRSY 0.500 66 ELFPQWHLPI 0.500 10
QEELWKMKPR 0.450 140 QLHNGTKYKK 0.400 310 LPCLRKKILK 0.250 295
VFLPIVVLIF 0.250 91 LREVIHPLAT 0.225 195 WAYQQVQQNK 0.200 307
ILFLPCLRKK 0.200 176 AIYSLSYPMR 0.200 137 ALVQLHNGTK 0.200 305
KSILFLPCLR 0.150 236 TSIPSVSDSL 0.150 64 TQELFPQWHL 0.135 58
PSELQHTQEL 0.135 48 QTAHADEFDC 0.125 96 HPLATSHQQY 0.125 54
EFDCPSELQH 0.125 265 GTIHALIFAW 0.125 94 VIHPLATSHQ 0.100 51
HADEFDCPSE 0.100 208 WIEHDVWRME 0.090 167 LSFFFAVLHA 0.075 162
KQFGLLSFFF 0.075 260 VSLLLGTIHA 0.075 220 VSLGIVGLAI 0.075 9
NQEELWKMKP 0.068 80 IIASLTFLYT 0.050 226 GLAILALLAV 0.050 292
MIAVFLPIVV 0.050 223 GIVGLAILAL 0.050 169 FFFAVLHAIY 0.050 34
ETSMLKRPVL 0.050 30 KDTGETSMLK 0.050 217 EIYVSLGIVG 0.050 124
TLLALVYLPG 0.050 8 TNQEELWKMK 0.050 252 YIQSKLGIVS 0.050 111
ILVLNKVLPM 0.050 256 KLGIVSLLLG 0.050 301 VLIFKSILFL 0.050 231
ALLAVTSLPS 0.050 134 VIAAIVQLHN 0.050 128 LVYLPGVIAA 0.050 287
TPPTFMIAVF 0.050 181 SYPMRRSYRY 0.050 52 ADEFDCPSEL 0.045 139
VQLHNGTKYK 0.030 100 TSHQQYFYKI 0.030 35 TSMLKRPVLL 0.030 7
ITNQEELWKM 0.025 4 RKDITNQEEL 0.025 29 HKDTGETSML 0.025 204
KEDAWIEHDV 0.025 235 VTSIPSVSDS 0.025 23 EEDDYLHKDT 0.025 285
WYTPPTFMIA 0.025 152 HWLDKWMLTR 0.025 157 WMLTRKQFGL 0.025 24
EDDYLHKDTG 0.025 324 WEDVTKINKT 0.025 177 IYSLSYPMRR 0.025 63
HTQELFPQWH 0.025 32 TGETSMLKRP 0.022 232 LLAVTSIPSV 0.020 43
LLHLHQTAHA 0.020 227 LAILALLAVT 0.020 130 YLPGVIAAIV 0.020 262
LLLGTIHALI 0.020 294 AVFLPIVVLI 0.020 224 IVGLAILALL 0.020 166
LLSFFFAVLH 0.020 127 ALVYLPGVIA 0.020
[0813] TABLE-US-00016 TABLE VI -V2-HLA-A1-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 2 WREFHYIQVN 0.045 3 REFHYIQVNN 0.001 4
EFHYIQVNNI 0.001 1 TWREFHYIQV 0.000
[0814] TABLE-US-00017 TABLE VI -V3-HLA-A1-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 21 LWDPCLTRFK 5.000 19 ESLWDPCLTR 0.750 17
VPESLWDPCL 0.450 14 KSDVPESLWD 0.375 4 EFHYIQIIHK 0.050 2
WREFHYIQII 0.045 16 DVPESLWDPC 0.010 5 FHYIQIIHKK 0.010 20
SLWDPCLTRE 0.010 25 CLTRFKGLNL 0.005 3 REFHYIQILH 0.003 22
WDPCLTRFKG 0.003 26 LTRFKGLNLI 0.003 23 DPCLTRFKGL 0.003 8
IQIIHKKSDV 0.002 24 PCLTREKGLN 0.001 9 QIIHKKSDVP 0.001 7
YIQIIHKKSD 0.001 15 SDVPESLWDP 0.001 6 HYIQIIHKKS 0.001 13
KKSDVPESLW 0.001 1 TWREFHYIQI 0.000 28 RFKGLNLIQS 0.000 27
TRFKGLNLIQ 0.000 10 IIHKKSDVPE 0.000 12 HKKSDVPESL 0.000 18
PESLWDPCLT 0.000 11 IHKKSDVPES 0.000
[0815] TABLE-US-00018 TABLE VI -V4-HLA-A1-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 8 LSLFFAVLHA 0.075 3 KQFGLLSLFF 0.075 10
LFFAVLHAIY 0.050 6 GLLSLFFAVL 0.020 7 LLSLFFAVLH 0.020 5 FGLLSLFFAV
0.013 9 SLFFAVLHAI 0.010 2 RKQFGLLSLF 0.005 4 QFGLLSLFFA 0.003 1
TRKQFGLLSL 0.000
[0816] TABLE-US-00019 TABLE VII -V1-HLA-A2-9mers: STEAP-1 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 165 GLLSFFFAV 10776.470 86 FLYTLLREV 470.951 262
LLLGTIHAL 309.050 302 LIFKSILFL 233.719 158 MLTRKQFGL 210.633 192
LLNWAYQQV 177.358 130 YLPGVIAAI 110.379 166 LLSFFFAVL 107.503 256
KLGIVSLLL 74.768 291 FMIAVFLPI 65.854 292 MIAVFLPIV 58.258 36
SMLKRPVLL 57.085 270 LIFAWNKWI 49.627 309 FLPCLRKKI 47.991 113
VINKVLPMV 37.393 79 AIIASLTFL 37.157 117 VLPMVSITL 36.316 164
FGLLSFFFA 33.454 83 SLTFLYTLL 32.044 42 VLLHLHQTA 31.249 90
LLREVLHPL 26.027 76 KIAAIIASL 23.646 123 ITLLALVYL 23.608 245
LTWREFHYI 21.365 212 DVWRMEIYV 20.836 219 YVSLGIVGL 19.776 261
SLLLGTIHA 18.382 300 VVLIFKSIL 18.318 263 LLGTIHALI 17.736 172
AVLHAIYSL 15.907 126 LALVYLPGV 13.975 65 QELFPQWHL 11.527 221
SLGIVGLAI 10.433 272 FAWNKWLDI 9.405 116 KVLPMVSIT 7.963 120
MVSITLLAL 7.309 224 IVGLAILAL 7.309 294 AVFLPIVVL 6.522 201
QQNKEDAWI 5.818 20 RNLEEDDYL 5.701 252 YIQSKLGIV 5.393 157
WMLTRKQFG 5.038 226 GLAILALLA 4.968 82 ASLTFLYTL 4.510 27 YLHKDTGET
4.456 133 GVIAAIVQL 4.299 305 KSILFLPCL 4.266 287 TPPTFMIAV 3.968
233 LAVTSIPSV 3.777 227 LAILALLAV 3.777 228 AILALLAVT 3.575 208
WIEHDVWRM 3.469 103 QQYFYKIPI 3.385 299 LVVLIFKSI 2.949 258
GIVSLLLGT 2.357 8 TNQEELWKM 2.294 176 AIYSLSYPM 2.186 319 KIRHGWEDV
2.119 230 LALLAVTSI 2.096 118 LPMVSITLL 2.017 225 VGLAILALL 2.017
81 IASLTFLYT 1.839 312 CLRKKILKI 1.824 109 IPILVINKV 1.775 121
VSITLLALV 1.775 112 LVINKVLPM 1.700 59 SELQHTQEL 1.537 237
SIPSVSDSL 1.439 162 KQFGLLSFF 1.238 137 AIVQLHNGT 1.025 279
DIKQFVWYT 0.970 128 LVYLPGVIA 0.970 265 GTIHALIFA 0.873 150
FPHWLDKWM 0.820 72 HLPIKIAAI 0.816 276 KWIDLKQFV 0.762 105
YFYKIPILV 0.711 223 GIVGLAILA 0.683 293 IAVFLPIVV 0.673 127
ALVYLPGVI 0.556 53 DEFDCPSEL 0.473 259 IVSLLLGTI 0.464 244
SLTWREFHY 0.432 49 TAHADEFDC 0.400 216 MEIYVSLGI 0.345 296
FLPIVVLIF 0.323 131 LPGVIAAIV 0.316 214 WRMEIYVSL 0.311 169
FFFAVLHAI 0.284 217 EIYVSLGIV 0.281 255 SKLGIVSLL 0.252 283
FVWYTPPTF 0.249 286 YTPPTFMIA 0.247 222 LGIVGLAIL 0.224 199
QVQQNKEDA 0.213 35 TSMLKRPVL 0.212 69 PQWHLPIKI 0.193 191 KLLNWAYQQ
0.193 173 VLHAIYSLS 0.190 125 LLALVYLPG 0.190
[0817] TABLE-US-00020 TABLE VII -V2-HLA-A2-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 4 FHYIQVNNI 0.245 2 REFHYIQVN 0.005 1 WREFHYIQV
0.004 3 EFHYIQVNN 0.000
[0818] TABLE-US-00021 TABLE VII -V3-HLA-A2-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 QIIHKKSDV 9.563 2 REFHYIQLI 0.708 19 SLWDPCLTR
0.507 12 KKSDVPESL 0.209 6 YIQIIHKKS 0.047 25 LTRFKGLNL 0.038 24
CLTRFKGLN 0.037 23 PCLTRFKGL 0.022 18 ESLWDPCLT 0.013 26 TRFKGLNLI
0.012 17 PESLWDPCL 0.009 15 DVPESLWDP 0.007 16 VPESLWDPC 0.003 7
IQIIHKKSD 0.003 28 FKGLNLIQS 0.001 9 IIHKKSDVP 0.001 13 KSDVPESLW
0.001 1 WREEHYIQI 0.001 4 FHYIQIIHK 0.000 22 DPCLTRFKG 0.000 14
SDVPESLWD 0.000 21 WDPCLTRFK 0.000 20 LWDPCLTRF 0.000 3 EFHYIQIIH
0.000 10 IHKKSDVPE 0.000 11 HKKSDVPES 0.000 5 HYIQIIHKK 0.000 27
RFKGLNLIQ 0.000
[0819] TABLE-US-00022 TABLE VII -V4-HLA-A2-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAV 2835.913 6 LLSLFFAVL 107.503 8
SLFFAVLHA 42.278 4 FGLLSLFFA 40.497 2 KQFGLLSLF 0.225 9 LFFAVLHAI
0.105 1 RKQFGLLSL 0.068 7 LSLFFAVLH 0.012 3 QFGLLSLFF 0.000
[0820] TABLE-US-00023 TABLE VII -V1-HLA-A2-10mers: STEAP-1 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 157 WMLTRKQFGL 1523.030 191 KLLNWAYQQV 1351.048
291 FMIAVFLPIV 828.006 301 VLIFKSILFL 459.398 164 FGLLSFFFAV
323.593 125 LLALVYLPGV 271.948 232 LLAVTSIPSV 271.948 165
GLLSFFFAVL 233.977 261 SLLLGTIHAL 181.794 89 TLLREVIHPL 181.794 226
GLAILALLAV 159.970 130 YLPGVIAAIV 131.175 244 SLTWREFHYI 109.023
283 FVWYTPPTFM 91.283 117 VLPMVSITLL 83.527 108 KIPILVFNKV 76.985
262 LLLGTIHALI 65.622 269 ALIFAWNKWI 57.380 229 ILALLAVTSI 40.792
309 FLPCLRKKIL 40.289 286 YTPPTFMIAV 38.837 122 SIILLALVYL 37.157
86 FLYTLLREVI 30.852 116 KVLPMVSITL 24.206 120 MVSITLLALV 23.795
112 LVINKVLPMV 23.795 221 SLGIVGLAIL 20.145 111 ILVINKVLPM 19.425
980 IIASLTFLYT 18.203 200 VQQNKEDAWI 9.890 281 KQFVWYTPPT 9.784 158
MLTRKQFGLL 8.970 253 IQSKLGIVSL 8.469 43 LLHLHQTAHA 8.446 294
AVFLPIVVLI 7.769 224 IVGLAILALL 7.309 223 GIVGLAILAL 6.756 292
MIAVFLPIVV 6.666 103 QQYFYKIPIL 6.536 278 IDIKQFVWYT 6.343 128
LVYLPGVIAA 5.439 299 IVVLIFKSIL 4.951 150 FPHWLDKWML 4.811 275
NKWIDIKQFV 4.410 81 IASLTFLYTL 4.408 78 AAIIASLTFL 3.753 76
KIAAIIASLT 3.586 171 FAVLHAIYSL 3.139 162 KQFGLLSFFF 3.121 37
MLKRPVLLHL 2.923 66 ELFPQWHLPI 2.895 90 LLREVIHPLA 2.632 127
ALVYLPGVIA 2.037 7 ITNQEELWKM 2.023 145 TKYKKFPHWL 1.984 204
KEDAWIEHDV 1.893 248 REFHYIQSKL 1.537 209 IEHDVWRMEI 1.414 304
FKSILFLPCL 1.193 64 TQELFPQWHL 1.040 237 SIPSVSDSLT 1.025 296
FLPIVVLIFK 0.867 119 PMVSITLLAL 0.781 82 ASLTFLYTLL 0.774 264
LGTIHALIFA 0.697 324 WEDVTKINKT 0.613 256 KLGIVSLLLG 0.600 219
YVSLGIVGLA 0.598 35 TSMLKRPVLL 0.516 215 RMEIYVSLGI 0.482 166
LLSFFFAVLH 0.481 293 IAVFLPIVVL 0.476 167 LSFFFAVLHA 0.469 42
VLLHLHQTAH 0.469 36 SMLKRPVLLH 0.458 258 GIVSLLLGTI 0.429 220
VSLGIVGLAI 0.428 124 TLLALVYLPG 0.414 48 QTAHADEFDC 0.400 68
FPQWHLPIKI 0.313 71 WHLPIKIAAI 0.284 216 MEIYVSLGIV 0.282 231
ALLAVTSIPS 0.276 33 GETSMLKRPV 0.270 198 QQVQQNKEDA 0.247 163
QFGLLSFFFA 0.209 260 VSLLLGTIHA 0.204 225 VGLAILALLA 0.204 118
LPMVSITLLA 0.204 277 WIDIKQFVWY 0.197 257 LGIVSLLLGT 0.190 302
LIFKSILFLP 0.172 27 YLHKDTGETS 0.168 227 LAILALLAVT 0.166 72
HLPIKIAAII 0.145 318 LKIRHGWEDV 0.137 100 TSHQQYFYKI 0.129 173
VLHAIYSLSY 0.127 75 IKIAAIIASL 0.116 40 RPVLLHLHQT 0.112
[0821] TABLE-US-00024 TABLE VIII -V2-HLA-A2-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 3 REFHYIQVNN 0.005 1 TWREFHYIQV 0.005 4
EFHYIQVNNI 0.001 2 WREFHYLQVN 0.000
[0822] TABLE-US-00025 TABLE VIII -V3-HLA-A2-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 25 CLTRFKGLNL 21.362 8 IQIIHKKSDV 11.988 20
SLWDPCLTRF 5.833 16 DVPESLWDPC 0.111 26 LTRFKGLNLI 0.043 23
DPCLTRFKGL 0.020 17 VPESLWDPCL 0.015 7 YIQIIHKKSD 0.009 10
IIHKKSDVPE 0.003 18 PESLWDPCLT 0.003 22 WDPCLTRFKG 0.003 3
REFHYIQILH 0.002 13 ICKSDVPESLW 0.001 14 KSDVPESLWD 0.001 1
TWREFHYIQI 0.001 9 QIIHKKSDVP 0.000 2 WREFHYIQII 0.000 15
SDVPESLWDP 0.000 5 FHYIQIIHKK 0.000 19 ESLWDPCLTR 0.000 24
PCLTRFKGLN 0.000 12 HKKSDVPESL 0.000 11 IHKKSDVPES 0.000 28
RFKGLNLIQS 0.000 27 TRFKGLNLIQ 0.000 4 EFHYIQIIHK 0.000 21
LWDPCLTRFK 0.000 6 HYIQIIHKKS 0.000
[0823] TABLE-US-00026 TABLE VIII -V4-HLA-A2-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAVL 233.977 9 SLFFAVLHAI 88.783 5
FGLLSLFFAV 85.156 3 KQFGLLSLFF 0.538 7 LLSLFFAVLH 0.481 8
LSLFFAVLHA 0.469 4 QFGLLSLFFA 0.253 1 TRKQFGLLSL 0.000 2 RKQFGLLSLF
0.000 10 LFFAVLHAIY 0.000
[0824] TABLE-US-00027 TABLE IX -V1-A3-9mers: STEAP-1 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
307 ILFLPCLRK 300.000 153 WLDKWMLTR 36.000 108 KIPILVINK 36.000 165
GLLSFFFAV 24.300 296 FLPIVVLIF 18.000 244 SLTWREFHY 12.000 140
QLHNGTKYK 10.000 291 FMIAVFLPI 8.100 301 VLIFKSILF 6.000 84
LTFLYTLLR 6.000 12 ELWKMKPRR 6.000 256 KLGIVSLLL 5.400 166
LLSFFFAVL 5.400 268 HALIFAWNK 2.700 90 LLREVIHPL 2.025 162
KQFGLLSFF 2.025 262 LLLGTLHAL 2.025 130 YLPGVIAAI 2.025 7 ITNQEELWK
2.000 37 MLKRPVLLH 1.800 221 SLGIVGLAI 1.800 158 MLTRKQFGL 1.800
117 VLPMVSITL 1.800 312 CLRKKILKI 1.800 248 REFHYIQSK 1.350 297
LPIVVLIFK 1.350 226 GLAILALLA 1.200 80 IIASLTFLY 1.200 283
FVWYTPPTF 1.000 148 KKFPHWLDK 0.900 31 DTGETSMLK 0.900 72 HLPIKIAAI
0.900 21 NLEEDDYLH 0.900 263 LLGTIHALI 0.900 302 LIFKSILFL 0.900 36
SMLKRPVLL 0.900 191 KLLNWAYQQ 0.810 122 SITLLALVY 0.090 294
AVFLPIVVL 0.675 60 ELQHTQELF 0.600 306 SILFLPCLR 0.600 261
SLLLGTIHA 0.600 138 IVQLHNGTK 0.600 99 ATSHQQYFY 0.600 240
SVSDSLTWR 0.600 83 SLTFLYTLL 0.600 103 QQYFYKIPI 0.540 9 NQEELWKMK
0.450 270 LIFAWNKWI 0.450 245 LTWREFHYI 0.450 76 KIAAIIASL 0.405
172 AVLHAIYSL 0.405 133 GVIAAIVQL 0.405 97 PLATSHQQY 0.400 176
AIYSLSYPM 0.300 68 FPQWHLPIK 0.300 100 TSHQQYFYK 0.300 309
FLPCLRKKI 0.300 269 ALIFAWNKW 0.300 42 VLLHLHQTA 0.300 127
ALVYLPGVI 0.270 300 VVLIFKSIL 0.270 124 TLLALVYLP 0.270 223
GIVGLAILA 0.270 299 IVVLIFKSI 0.203 116 KVLPMVSIT 0.203 43
LLHLHQTAH 0.200 183 PMRRSYRYK 0.200 192 LLNWAYQQV 0.200 178
YSLSYPMRR 0.180 139 VQLHNGTKY 0.180 224 IVGLAILAL 0.180 120
MVSITLLAL 0.180 182 YPMRRSYRY 0.180 208 WIEHDVWRM 0.180 112
LVINKVLPM 0.180 89 TLLREVIHP 0.180 215 RMEIYVSLG 0.180 128
LVYLPGVIA 0.150 86 FLYTLLREV 0.150 258 GIVSLLLGT 0.135 265
GTIHALIFA 0.135 316 KILKIRHGW 0.135 79 AIIASLTFL 0.135 324
WEDVTK1NK 0.120 22 LEEDDYLHK 0.120 273 AWNKWIDIK 0.090 141
LHNGTKYKK 0.090 237 SIPSVSDSL 0.090 292 MIAVFLPIV 0.090 286
YTPPTFMIA 0.090 119 PMVSITLLA 0.090 219 YVSLGIVGL 0.090 134
VIAAIVQLH 0.090 15 KMKPRRNLE 0.090 144 GTKYKKFPH 0.090 279
DIKQFVWYT 0.081 232 LLAVTSIPS 0.080 123 ITLLALVYL 0.068
[0825] TABLE-US-00028 TABLE IX -V2-HLA-A3-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 4 FHYIQVNNI 0.009 2 REFHYIQVN 0.001 1 WREFHYIQV
0.000 3 EFHYIQVNN 0.000
[0826] TABLE-US-00029 TABLE IX -V3-HLA-A3-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 SLWDPCLTR 60.000 4 FHYIQIIHK 0.300 5 HYIQIIHKK
0.068 25 LTRFKGLNL 0.060 2 REFHYIQII 0.041 8 QIIHKKSDV 0.030 24
CLTRFKGLN 0.018 20 LWDPCLTRF 0.005 13 KSDVPESLW 0.005 26 TRFKGLNLI
0.005 15 DVPESLWDP 0.004 6 YIQIIHKKS 0.003 12 KKSDVPESL 0.003 21
WDPCLTRFK 0.002 16 VPESLWDPC 0.002 9 IIHKKSDVP 0.002 23 PCLTRFKGL
0.001 1 WREFHYIQI 0.001 17 PESLWDPCL 0.001 7 IQIIHKKSD 0.000 18
ESLWDPCLT 0.000 22 DPCLTRFKG 0.000 3 EFHYIQIIH 0.000 28 FKGLNLIQS
0.000 11 HKKSDVPES 0.000 14 SDVPESLWD 0.000 27 RFKGLNLIQ 0.000 10
IHKKSDVPE 0.000
[0827] TABLE-US-00030 TABLE IX -V4-HLA-A3-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAV 8.100 8 SLFFAVLHA 6.000 6 LLSLFFAVL
5.400 2 KQFGLLSLF 2.025 9 LFFAVLHAI 0.007 7 LSLFFAVLH 0.005 3
QFGLLSLFF 0.004 1 RKQFGLLSL 0.002 4 FGLLSLFFA 0.001
[0828] TABLE-US-00031 TABLE X -V1-HLA-A3-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 aminoacids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 140 QLHNGTKYKK 60.000 21 NLEEDDYLHK 60.000 153
WLDKWMLTRK 30.000 165 GLLSFFFAVL 24.300 83 SLTFLYTLLR 24.000 173
VLHAIYSLSY 24.000 307 ILFLPCLRKK 15.000 306 SILFLPCLRK 9.000 66
ELFPQWHLPI 8.100 312 CLRKKILKIR 4.500 272 FAWNKWIDIK 4.500 162
KQFGLLSFFF 4.050 263 LLGTIHALIF 4.000 99 ATSHQQYFYK 3.000 86
FLYTLLREVI 3.000 157 WMLTRKQFGL 2.700 37 MLKRPVLLHL 2.700 36
SMLKRPVLLH 2.700 301 VLIFKSILFL 2.700 262 LLLGTIHALI 2.025 89
TLLREVIHPL 2.025 176 AIYSLSYPMR 2.000 229 ILALLAVTSI 1.800 79
AIIASLTFLY 1.800 226 GLAILALLAV 1.800 244 SLTWREFHYI 1.800 111
ILVINKVLPM 1.800 215 RMEIYVSLGI 1.800 195 VVAYQQVQQN 1.500 247
WREFHYIQSK 0.090 261 SLLLGTIHAL 1.350 305 KSILFLPCLR 0.090 269
ALLFAWNKWI 1.350 291 FMIAVFLPIV 1.350 116 KVLPMVSITL 1.215 223
GIVGLAILAL 1.215 6 DITNQEELWK 1.200 277 WIDIKQFVWY 1.200 117
VLPMVSITLL 0.900 137 AIVQLHNGTK 0.900 72 HLPIKIAAII 0.900 221
SLGIVGLAIL 0.900 125 LLALVYLPGV 0.900 191 KLLNWAYQQV 0.900 128
LVYLPGVIAA 0.600 300 VVLIFKSILF 0.600 166 LLSFFFAVLH 0.600 183
PMRRSYRYKL 0.540 158 MLTRKQFGLL 0.540 130 YLPGVIAAIV 0.450 139
VQLHNGTKYK 0.450 90 LLREVIHPLA 0.450 127 ALVYLPGVIA 0.450 133
GVIAAIVQLH 0.405 138 IVQLHNGTKY 0.400 179 SLSYPMRRSY 0.400 310
LPCLRKKILK 0.400 42 VLLHLHQTAH 0.300 309 FLPCLRKKIL 0.300 232
LLAVTSLPSV 0.300 31 DTGETSMLKR 0.270 107 YKIPILVINK 0.270 119
PMVSITLLAL 0.270 299 IVVLIFKSIL 0.270 265 GTIHALLFAW 0.203 97
PLATSHQQYF 0.200 43 LLHLHQTAHA 0.200 267 IHALIFAWNK 0.180 30
KDTGETSMLK 0.180 256 KLGIVSLLLG 0.180 15 KMKPRRNLEE 0.180 283
FVWYTPPTFM 0.150 180 LSYPMRRSYR 0.150 224 IVGLAILALL 0.135 103
QQYFYKIPIL 0.135 281 KQFVWYTPPT 0.135 286 YTPPTFMIAV 0.135 108
KIPILVINKV 0.135 258 GIVSLLLGTI 0.121 323 GWEDVTKINK 0.120 231
ALLAVTSIPS 0.120 98 LATSHQQYFY 0.120 102 HQQYFYKIPI 0.108 319
KIRHGWEDVT 0.090 124 TLLALVYLPG 0.090 122 SLILLALVYL 0.090 144
GTKYKKFPHW 0.090 64 TQELFPQWHL 0.081 81 LASLTFLYTL 0.081 100
TSHQQYFYKI 0.081 112 LVINKVLPMV 0.068 302 LIFKSILFLP 0.068 96
HPLATSHQQY 0.060 192 LLNVVAYQQV 0.060 206 DAWIEHDVWR 0.060 80
IIASLTFLYT 0.060 317 ILKIRHGWED 0.060 188 YRYKLLNWAY 0.060
[0829] TABLE-US-00032 TABLE X -V2-HLA-A3-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 1 TWREFHYIQV 0.001 4 EFHYIQVNNI 0.001 3
REFHYIQVNN 0.000 2 WREFHYIQVN 0.000
[0830] TABLE-US-00033 TABLE X -V3-HLA-A3-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 20 SLWDPCLTRF 15.000 25 CLTRFKGLNL 1.200 5
FHYIQILHKK 0.225 26 LTRFKGLNLI 0.045 19 ESLWDPCLTR 0.027 17
TESLVVDPCL 0.018 4 EFHYIQIIHK 0.018 8 IQIIHKKSDV 0.009 3 REFHYIQIIH
0.009 16 DVPESLWDPC 0.009 21 LWDPCLTRFK 0.003 9 QIIHKKSDVP 0.003 2
WREFHYIQII 0.003 23 DPCLTRFKGL 0.003 10 IIHKIKSDVPE 0.002 1
TWREFHYIQI 0.002 7 YIQIIHKKSD 0.001 13 KKSDVPESLW 0.001 12
HKKSDVPESL 0.001 14 KSDVPESLWD 0.001 15 SDVPESLWDP 0.000 27
TRFKGLNLIQ 0.000 28 REKGLNLIQS 0.000 11 IHKIKSDVPES 0.000 22
WDPCLTRFKG 0.000 6 HYIQIIHKKS 0.000 18 PESLWDPCLT 0.000 24
PCLTRFKGLN 0.000
[0831] TABLE-US-00034 TABLE X -V4-HLA-A3-10mers: STEAP-1 Each
peptide is a portion of NO:9; each start position is specified, the
length of peptide is amino acids, and the endposition for each
peptide is the start plus nine. Start Subsequence Score 6
GLLSLFFAVL 24.300 9 SLFFAVLHAI 6.750 3 KQFGLLSLFF 2.700 7
LLSLFFAVLH 0.600 10 LFFAVLHAIY 0.020 8 LSLFFAVLHA 0.009 2
RKQFGLLSLF 0.003 5 FGLLSLFFAV 0.003 1 TRKQFGLLSL 0.002 4 QFGLLSLFFA
0.001
[0832] TABLE-US-00035 TABLE X -V4-HLA-A3-10mers: STEAP-1 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 108 KIPILVINK 2.400 138 IVQLHNGTK 2.000 7
ITNQEELWK 2.000 307 ILFLPCLRK 1.600 84 LTFLYTLLR 0.800 268
HALIFAWNK 0.600 297 LPIVVLIFK 0.600 31 DTGETSMLK 0.600 196
AYQQVQQNK 0.400 240 SVSDSLTWR 0.400 248 REFHYIQSK 0.360 9 NQEELWKMK
0.300 148 KKFPHWLDK 0.240 140 QLHNGTKYK 0.200 68 FPQWHLPIK 0.200
153 WLDKWMLTR 0.160 22 LEEDDYLHK 0.120 306 SILFLPCLR 0.120 324
WEDVTKINK 0.120 144 GTKYKKEPH 0.090 133 GVIAAIVQL 0.090 265
GTIHALIFA 0.090 177 IYSLSYPMR 0.080 181 SYPMRRSYR 0.080 321
RHGWEDVTK 0.060 311 PCLRKKILK 0.060 100 TSHQQYFYK 0.060 172
AVLHAIYSL 0.060 112 LVThKVLPM 0.060 165 GLLSFFFAV 0.054 12
ELWKMKPRR 0.048 294 AVFLPIVVL 0.040 141 LHNGTKYKK 0.040 128
LVYLPGVIA 0.040 283 FVWYTPPTF 0.040 120 MVSITLLAL 0.040 224
IVGLAILAL 0.040 162 KQFGLLSFF 0.036 223 GIVGLAILA 0.036 308
LFLPCLRKK 0.030 300 VVLIFKSIL 0.030 189 RYKLLNWAY 0.024 212
DVWRMEIYV 0.024 103 QQYFYKIPI 0.024 256 KLGIVSLLL 0.024 226
GLAILALLA 0.024 219 YVSKGUVGL 0.020 154 LDKWMLTRK 0.020 273
AWNKWIDLK 0.020 259 IVSLLLGTI 0.020 99 ATSHQQYFY 0.020 286
YTPPTFMIA 0.020 199 QVQQNKEDA 0.020 245 LTWREFHYI 0.020 316
KILKIRHGW 0.018 302 LIFKSILFL 0.016 176 AIYSLSYPM 0.016 299
IVVLIFKSI 0.015 123 ITLLALVYL 0.015 88 YTLLREVIH 0.015 178
YSLSYPMRR 0.012 244 SLTWREFHY 0.012 291 FMIAVFLPI 0.012 80
IIASLTFLY 0.012 277 WIDIKQFVW 0.012 319 KIRHGWEDV 0.012 146
KYKKFPHWL 0.012 285 WYTPPTFMI 0.012 266 TIHALIFAW 0.012 261
SLLLGTIHA 0.012 301 VLIFKSILF 0.012 76 KIAAIIASL 0.012 129
VYLPGVIAA 0.012 158 MLTRKQFGL 0.012 251 HYIQSKLGI 0.012 327
VTKINKTEI 0.010 63 HTQELFPQW 0.010 159 LTRKQFGLL 0.010 116
KVLPMVSIT 0.009 139 VQLHNGTKY 0.009 187 SYRYKLLNW 0.008 122
SITLLALVY 0.008 106 FYKIPILVI 0.008 32 TGETSMLKR 0.008 312
CLRKKILKI 0.008 105 YFYKIPILV 0.008 208 WIEHDVWRM 0.008 37
MLKRPVLLH 0.008 182 YPMRRSYRY 0.008 117 VLPMVSITL 0.008 168
SFFFAVLHA 0.008 104 QYFYKIPIL 0.008 296 FLPIVVLIF 0.008 21
NLEEDDYLH 0.008 272 FAWNKWIDI 0.008 221 SLGIVGLAI 0.008 207
AWIEHDVWR 0.006 262 LLLGTIHAL 0.006 36 SMLKRPVLL 0.006 163
QFGLLSFFF 0.006
[0833] TABLE-US-00036 TABLE XI -V2-HLA-A11-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position for plus eight.
Start Subsequence Score 4 FHYIQVNNI 0.000 1 WREFHYIQV 0.000 2
REFHYIQVN 0.000 3 EFHYIQVNN 0.000
[0834] TABLE-US-00037 TABLE XI -V3-HLA-A11-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 HYIQIIHKK 0.600 19 SLWDPCLTR 0.320 4 FHYIQIIHK
0.080 25 LTRFKGLNL 0.020 8 QIIHKKSDV 0.006 2 REFHYIQII 0.004 21
WDPCLTRFK 0.002 15 DVPESLWDP 0.001 27 RFKGLNLLQ 0.001 3 EFHYIQIIH
0.001 12 KKSDVPESL 0.001 13 KSDVPESLW 0.001 7 IQIIHKKSD 0.000 26
TRFKGLNLI 0.000 24 CLTRFKGLN 0.000 9 IIHKKSDVP 0.000 1 WREFHYIQI
0.000 6 YIQIIHKKS 0.000 20 LWDPCLTRF 0.000 16 VPESLWDPC 0.000 22
DPCLTRFKG 0.000 23 PCLTRFKGL 0.000 14 SDVPESLWD 0.000 17 PESLWDPCL
0.000 28 FKGLNLIQS 0.000 11 HKKSDVPES 0.000 10 IHKKSDVPE 0.000 18
ESLWDPCLT 0.000
[0835] TABLE-US-00038 TABLE XI -V4-HLA-A11-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start plus eight. Start
Subsequence Score 5 GLLSLFFAV 0.054 2 KQFGLLSLF 0.036 8 SLFFAVLHA
0.016 6 LLSLFFAVL 0.004 9 LFFAVLHAI 0.004 3 QFGLLSLFF 0.004 1
RKQFGLLSL 0.001 4 FGLLSLFFA 0.001 7 LSLFFAVLH 0.000 TABLE XII
-V1-HLA-A11-10mers: STEAP-1 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 99 ATSHQQYFYK 3.000 306
SILFLPCLRK 1.200 296 FLPIVVLIFK 0.800 21 NLEEDDYLHK 0.800 140
QLHNGTKYKK 0.800 137 AIVQLHNGTK 0.600 139 VQLHNGTKYK 0.450 195
WAYQQVQQNK 0.400 153 WLDKWMLTRK 0.400 272 FAWNKWIDIK 0.400 310
LPCLRKKILK 0.400 6 DITNQEELWK 0.240 67 LFPQWHLPLK 0.200 116
KVLPMVSITL 0.180 177 IYSLSYPMRR 0.160 83 SLTFLYTLLR 0.160 176
AIYSLSYPMR 0.160 265 GTIHALIFAW 0.135 30 KDTGETSMLK 0.120 323
GWEDVTKINK 0.120 31 DTGETSMLKR 0.120 162 KQFGLLSFFF 0.108 133
GVTAAIVQLH 0.090 307 ILFLPCLRKK 0.080 128 LVYLPGVIAA 0.080 107
YKIPILVINK 0.060 300 VVLIFKSILF 0.060 147 YKKFPHWLDK 0.040 294
AVFLPIVVLI 0.040 182 YPMRRSYRYK 0.040 267 IHALIFAWNK 0.040 312
CLRKKILKIR 0.040 283 FVWYTPPTFM 0.040 223 GIVGLAILAL 0.036 144
GTKYKKFPHW 0.030 299 IVVLIFKSIL 0.030 112 LVINKVLPMV 0.030
[0836] TABLE-US-00039 TABLE XII -V1-HLA-A11-10mers: STEAP-1 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 206 DAWIEHDVWR 0.024 226 GLAILALLAV 0.024 215
RMEIYVSLGI 0.024 120 MVSITLLALV 0.020 8 TNQEELWKMK 0.020 320
IRHGWEDVTK 0.020 7 ITNQEELWKM 0.020 286 YTPPTFMIAV 0.020 138
IVQLHNGTKY 0.020 199 QVQQNKEDAW 0.020 219 YVSLGIVGLA 0.020 247
WREFHYIQSK 0.020 259 IVSLLLGTIH 0.020 224 IVGLAILALL 0.020 305
KSILFLPCLR 0.018 157 WMLTRKQFGL 0.018 79 AIIASLTFLY 0.018 191
KLLNWAYQQV 0.018 258 GIVSLLLGTI 0.018 165 GLLSFFFAVL 0.018 104
QYFYKIPILV 0.016 152 HWLDKWMLTR 0.012 102 HQQYFYKIPI 0.012 36
SMLKRPVLLH 0.012 108 KIPILVINKV 0.012 64 TQELFPQWHL 0.012 103
QQYFYKIPIL 0.012 301 VLIFKSILFL 0.012 111 ILVINKVLPM 0.012 63
HTQELFPQWH 0.010 198 QQVQQNKEDA 0.009 93 EVIHPLATSH 0.009 285
WYTPPTFMIA 0.008 181 SYPMRRSYRY 0.008 86 FLYTLLREVI 0.008 37
MLKRPVLLHL 0.008 292 MIAVFLPIVV 0.008 180 LSYPMRRSYR 0.008 173
VLHAIYSLSY 0.008 118 LPMVSITLLA 0.008 105 YFYKIPILVI 0.008 263
LLGTIHALIF 0.008 290 TFMIAVFLPI 0.008 200 VQQNKEDAWI 0.006 171
FAVLHAIYSL 0.006 73 LPIKIAAIIA 0.006 262 LLLGTIHALI 0.006 129
VYLPGVIAAI 0.006 291 FMIAVFLPIV 0.006 253 IQSKLGIVSL 0.006 42
VLLHLHQTAH 0.006 163 QFGLLSFFFA 0.006 326 DVTKINKTEI 0.006 89
TLLREVIHPL 0.006 251 HYIQSKLGIV 0.006 295 VFLPIVVLIF 0.006 10
QEELWKMKPR 0.006 127 ALVYLPGVIA 0.006 261 SLLLGTIHAL 0.006 218
IYVSLGIVGL 0.006 175 HAIYSLSYPM 0.906 11 EELWKMKPRR 0.005 66
ELFPQWHLPI 0.005
[0837] TABLE-US-00040 TABLE XII -V2-HLA-A11-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 4 EFHYIQVNNI 0.001 1 TWREFHYIQV 0.000 3
REFHYIQVNN 0.000 2 WREFHYIQVN 0.000
[0838] TABLE-US-00041 TABLE XII -V3-HLA-A3-l0mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start plus nine. Start Subsequence
Score 4 EFHYIQIIHK 0.120 5 FHYIQIIHKK 0.040 26 LTRFKGLNLI 0.010 8
IQIIHKKSDV 0.009 25 CLTRFKGLNL 0.008 20 SLWDPCLTRF 0.008 3
REFHYIQIIH 0.007 19 ESLWDPCLTR 0.004 21 LWDPCLTRFK 0.002 17
VPESLWDPCL 0.002 28 RFKGLNLIQS 0.001 9 QIHIKKSDVP 0.001 13
KKSDVPESLW 0.001 16 DVPESLWDPC 0.001 10 IIHKKSDVPE 0.000 1
TWREFHYIQI 0.000 6 HYIQIIHKKS 0.000 23 DPCLTRFKGL 0.000 2
WREFHYIQII 0.000 12 HKKSDVPESL 0.000 7 YIQIIHKKSD 0.000 14
KSDVPESLWD 0.000 27 TRFKGLNLIQ 0.000 22 WDPCLTRFKG 0.000 15
SDVPESLWDP 0.000 24 PCLTRFKGLN 0.000 11 IHKKSDVPES 0.000 18
PESLWPDCTL 0.000
[0839] TABLE-US-00042 TABLE XII -V4-HLA-A3-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 GLLSLFFAVL 24.300 9 SLFFAVLHAI 6.750 3
KQFGLLSLFF 2.700 7 LLSLFFAVLH 0.600 10 LFFAVLHAIY 0.020 8
LSLFFAVLHA 0.009 2 RKQFGLLSLF 0.003 5 FGLLSLFFAV 0.003 1 TRKQFGLLSL
0.002 4 QFGLLSLFFA 0.001
[0840] TABLE-US-00043 TABLE XIII -V1-HLA-A24-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 acids, and the end position
for each peptide is the start position plus eight. Start
Subsequence Score 146 KYKKFPHWL 400.000 104 QYFYKIPIL 200.000 251
HYIQSKLGI 75.000 87 LYTLLREVI 60.000 285 WYTPPTFMI 60.000 106
FYKIPILVI 50.000 249 EFHYIQSKL 30.800 20 RNLEEDDYL 14.400 295
VFLPIVVLI 12.600 189 RYKLLNWAY 12.000 163 QFGLLSFFF 12.000 305
KSILFLPCL 12.000 256 KLGIVSLLL 11.200 76 KIAAIIASL 11.200 129
VYLPGVIAA 9.000 225 VGLAILALL 8.400 237 SIPSVSDSL 8.400 117
VLPMVSITL 8.400 67 LFPQWHLPI 7.500 169 FFFAVLHAI 7.000 90 LLREVIHPL
6.720 82 ASLTFLYTL 6.000 222 LGIVGLAIL 6.000 118 LPMVSITLL 6.000 35
TSMLKRPVL 6.000 262 LLLGTIHAL 6.000 79 AIIASLTFL 6.000 156
KWMLTRKQF 6.000 36 SMLKRPVLL 6.000 133 GVIAAIVQL 6.000 172
AVLHAIYSL 6.000 123 ITLLALVYL 6.000 187 SYRYKLLNW 5.000 159
LTRKQFGLL 4.800 166 LLSFFFAVL 4.800 162 KQFGLLSFF 4.800 83
SLTFLYTLL 4.800 296 FLPIVVLIF 4.200 302 LIFKSILFL 4.000 310
LPCLRKKIL 4.000 158 MLTRKQFGL 4.000 224 IVGLAILAL 4.000 294
AVFLPIVVL 4.000 120 MVSITLLAL 4.000 219 YVSLGIVGL 4.000 254
QSKLGIVSL 4.000 60 ELQHTQELF 3.000 301 VLIFKSILF 3.000 78 AAIIASLTF
3.000 299 IVVLIFKSI 2.520 291 FMIAVFLPI 2.520 98 LATSHQQYF 2.400 47
HQTAHADEF 2.200 142 HNGTKYKKF 2.200 73 LPIKIAAII 2.100 130
YLPGVIAAI 2.100 283 FVWYTPPTF 2.000 264 LGTIHALIF 2.000 309
FLPCLRKKI 1.980 127 ALVYLPGVI 1.800 149 KFPHWLDKW 1.650 230
LALLAVTSI 1.500 201 QQNKEDAWI 1.500 72 HLPIKIAAI 1.500 263
LLGTIHALI 1.400 221 SLGIVGLAI 1.400 322 HGWEDVTKI 1.320 5 KDITNQEEL
1.320 196 AYQQVQQNK 1.260 245 LTWREFHYI 1.200 259 IVSLLLGTI 1.200
327 VTKINKTEI 1.100 312 CLRKKILKI 1.100 214 WRMEIYVSL 1.008 270
LIFAWNKWI 1.000 272 FAWNKWIDI 1.000 103 QQYFYKIPI 1.000 8 TNQEELWKM
0.990 218 IYVSLGIVG 0.900 255 SKIGIVSLL 0.840 110 PILVINKVL 0.840
185 RRSYRYKLL 0.800 30 KDTGETSML 0.800 59 SELQHTQEL 0.792 208
WIEHDVWRM 0.750 282 QFVWYTPPT 0.750 181 SYPMRRSYR 0.750 112
LVINKVLPM 0.750 26 DYLHKDTGE 0.750 105 YFYKIPILV 0.700 38 LKRPVLLHL
0.672 331 NKTEICSQL 0.672 276 KWIDIKQFV 0.605 170 FFAVLHAIY 0.600
176 AIYSLSYPM 0.600 150 FPHWLDKWM 0.600 161 RKQFGLLSF 0.600 14
WKMKPRRNL 0.600 65 QELFPQWHL 0.600
[0841] TABLE-US-00044 TABLE XIII -V2-HLA-A24-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 3 EFHYIQVNN 0.700 4 FHYIQVNNI 0.120 2 REFHYIQVN
0.024 1 WREFHYIQV 0.015
[0842] TABLE-US-00045 TABLE XIII -VI-HLA-A24-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 25 LTRFKGLNL 4.000 20 LWDPCLTRF 2.400 12
KKSDVPESL 1.344 5 HYIQIIHKK 1.155 23 PCLTRFKGL 0.720 2 REFHYIQII
0.240 6 YIQIIHKKS 0.231 13 KSDVPESLW 0.200 16 VPESLWDPC 0.180 8
QIIHKKSDV 0.150 1 WREFHYIQI 0.150 18 ESLWDPCLT 0.150 27 RFKGLNLIQ
0.120 26 TRFKGLNLI 0.120 24 CLTRFKGLN 0.100 3 EFHYIQIIH 0.070 17
PESLWDPCL 0.040 15 DVPESLWDP 0.018 7 IQIIHKKSD 0.015 19 SLWDPCLTR
0.012 11 HKKSDVPES 0.011 22 DPCLTRFKG 0.011 9 ILHKKSDVP 0.010 28
FKGLNLIQS 0.010 21 WDPCLTRFK 0.002 14 SDVPESLWD 0.002 10 IHKKSDVPE
0.001 4 FHYIQIIHK 0.001
[0843] TABLE-US-00046 TABLE XIII -V4-HLA-A24-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 QFGLLSLFF 12.000 9 LFFAVLHAI 7.000 2 KQFGLLSLF
4.800 6 LLSLFFAVL 4.800 1 RKQFGLLSL 1.200 5 GLLSLFFAV 0.216 4
FGLLSLFFA 0.150 8 SLFFAVLHA 0.100 7 LSLFFAVLH 0.015
[0844] TABLE-US-00047 TABLE XIV- V1-HLA-A24-10mers: STEAP-1 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 218 IYVSLGIVGL 300.000 129 VYLPGVIAAI 126.000 295
VFLPIVVLIF 25.200 116 KVLPMVSITL 20.160 282 QFVWYTPPTF 15.000 290
TFMIAVFLPI 12.600 308 LFLPCLRKKI 11.880 236 TSIPSVSDSL 10.080 149
KFPHWLDKWM 9.000 89 TLLREVIHPL 8.400 109 IPILVINKVL 8.400 26
DYLHKDTGET 8.250 181 SYPMRRSYRY 7.500 251 HYIQSKLGIV 7.500 187
SYRYKLLNWA 7.200 285 WYTPPTFMIA 7.200 82 ASLTFLYTLL 7.200 165
GLLSFFFAVL 7.200 104 QYFYKIPILV 7.000 106 FYKIPILVIN 7.000 168
SFFFAVLHAI 7.000 261 SLLLGTIHAL 6.000 293 IAVFLPIVVL 6.000 299
IVVLIFKSIL 6.000 157 WMLTRKQFGL 6.000 223 GIVGLAILAL 6.000 171
FAVLHAIYSL 6.000 309 FLPCLRKKIL 6.000 35 TSMLKRPVLL 6.000 117
VLPMVSITLL 6.000 78 AAIIASLTFL 6.000 301 VLIFKSILFL 6.000 64
TQELFPQWHL 6.000 213 VWRMEIYVSL 5.600 254 QSKLGIVSLL 5.600 37
MLKRPVLLHL 5.600 330 INKTEICSQL 5.600 224 IVGLAILALL 5.600 271
IFAWNKWIDI 5.000 105 YFYKIPILVI 5.000 158 MLTRKQFGLL 4.800 162
KQFGLLSFFF 4.800 215 RMEIYVSLGI 4.200 103 QQYFYKIPIL 4.000 253
IQSKLGIVSL 4.000 122 SITLLALVYL 4.000 81 IASLTFLYTL 4.000 34
ETSMLKRPVL 4.000 13 LWKMKPRRNL 4.000 221 SLGIVGLAIL 4.000 150
FPHWLDKWML 4.000 287 TPPTFMIAVF 3.000 300 VVLIFKSILF 3.000 241
VSDSLTWREF 2.200 262 LLLGTIHALI 2.100 72 HLPIKLAAII 2.100 220
VSLGIVGLAI 2.100 274 WNKWIDIKQF 2.000 263 LLGTIHALIF 2.000 77
IAAIIASLTF 2.000 68 FPQWHLPIKI 1.980 258 GIVSLLLGTI 1.800 126
LALVYLPGVI 1.800 102 HQQYFYKIPI 1.500 200 VQQNKEDAWI 1.500 269
ALIFAWNKWI 1.500 294 AVFLPIVVLI 1.400 100 TSHQQYFYKI 1.320 248
REFHYIQSKL 1.232 86 FLYTLLREVI 1.200 244 SLTWREFHYI 1.200 66
ELFPQWHLPI 1.200 19 RRNLEEDDYL 1.200 326 DVTKINKTEI 1.100 146
KYKKFPHWLD 1.000 189 RYKLLNWAYQ 1.000 284 VWYTPPTFMI 1.000 114
INKVLPMVSI 1.000 229 ILALLAVTSI 1.000 196 AYQQVQQNKE 0.990 85
TFLYTLLREV 0.990 207 AWIEHDVWRM 0.900 175 HAIYSLSYPM 0.900 4
RKDITNQEEL 0.880 255 SKLGIVSLLL 0.840 75 IKIAAIIASL 0.840 7
ITNQEELWKM 0.825 58 PSELQHTQEL 0.792 111 ILVINKVLPM 0.750 161
RKQFGLLSFF 0.720 303 IFKSILFLPC 0.720 52 ADEFDCPSEL 0.660 119
PMVSITLLAL 0.600 163 QFGLLSFFFA 0.600 169 FFFAVLHAIY 0.600 132
PGVIAAIVQL 0.600 288 PPTFMIAVFL 0.560 108 KIPILVINKV 0.554 170
FFAVLHAIYS 0.500 87 LYTLLREVIH 0.500
[0845] TABLE-US-00048 TABLE XIV -V2-HLA-A24-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for eachpeptide is the start position plus nine. Start
Subsequence Score 4 EFHYIQVNNI 6.000 1 TWREFHYIQV 0.120 3
REFHYLQVNN 0.028 2 WREFHYIQVN 0.018
[0846] TABLE-US-00049 TABLE XIV -V3-HLA-A24-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 HYIQIIHKKS 11.550 17 VPESLWDPCL 6.000 23
DPCLTRFKGL 4.800 25 CLTRFKGLNL 4.000 20 SLWDPCLTRF 2.400 1
TWREFHYIQI 1.200 26 LTRFKGLNLI 1.200 28 RFKGLNLIQS 1.000 12
HKKSDVPESL 0.560 16 DVPESLWDPC 0.216 2 WREFHYIQII 0.180 8
IQIIHKKSDV 0.150 4 EFHYIQILHK 0.050 13 KKSDVPESLW 0.024 14
KSDVPESLWD 0.020 19 ESLWDPCLTR 0.015 24 PCLTRFKGLN 0.015 9
QIIHKKSDVP 0.015 7 YIQIIIIKKSD 0.015 21 LWDPCLTRFK 0.014 11
IHKKSDVPES 0.011 10 IIHKKSDVPE 0.010 3 REFHYIQIIH 0.003 15
SDVPESLWDP 0.002 22 WDPCLTRFKG 0.002 5 FHYIQIIHKK 0.002 27
TRFKGLNLIQ 0.001 18 PESLWDPCLT 0.001
[0847] TABLE-US-00050 TABLE XIV -V4-HLA-A24-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAVL 7.200 3 KQFGLLSLFF 4.800 9
SLFFAVLHAI 1.400 2 RKQFGLLSLF 0.720 10 LFFAVLHAIY 0.600 4
QFGLLSLFFA 0.500 1 TRKQFGLLSL 0.400 5 FGLLSLFFAV 0.216 8 LSLFFAVLHA
0.150 7 LLSLFFAVLH 0.010
[0848] TABLE-US-00051 TABLE XV -V1-HLA-B7-9mers: STEAP-1 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 118 LPMVSITLL 240.000 294 AVFLPIVVL 90.000 310
LPCLRKKIL 80.000 172 AVLHAIYSL 60.000 159 LTRKQFGLL 40.000 90
LLREVIHPL 40.000 120 MVSITLLAL 20.000 219 YVSLGIVGL 20.000 224
IVGLAALAL 20.000 150 FPHWLDKWM 20.000 133 GVIAAIVQL 20.000 300
VVLIFKSIL 20.000 35 TSMLKRPVL 18.000 79 AIIASLTFL 12.000 82
ASLTFLYTL 12.000 73 LPIKIAAII 8.000 184 MRRSYRYKL 6.000 112
LVJNKVLPM 5.000 38 LKRPVLLHL 4.000 312 CLRKKILKI 4.000 305
KSILFLPCL 4.000 225 VGLAILALL 4.000 158 MLTRKQFGL 4.000 254
QSKLGIVSL 4.000 262 LLLGTIHAL 4.000 123 ITLLALVYL 4.000 287
TPPTFMIAV 4.000 131 LPGVIAAIV 4.000 166 LLSFFFAVL 4.000 302
LIFKSILFL 4.000 222 LGIVGLAIL 4.000 117 VLPMVSITL 4.000 256
KLGIVSLLL 4.000 237 SIPSVSDSL 4.000 76 KIAAIIASL 4.000 83 SLTFLYTLL
4.000 36 SMLKRPVLL 4.000 20 RNLEEDDYL 4.000 109 IPILVINKV 4.000 176
AIYSLSYPM 3.000 14 WKMXPRRNL 2.700 319 KIRHGWEDV 2.000 259
IVSLLLGTI 2.000 17 KPRRNLEED 2.000 299 IVVLIFKSI 2.000 238
IPSVSDSLT 2.000 182 YPMRRSYRY 1.200 272 FAWNKWIDI 1.200 214
WRMEIYVSL 1.200 230 LALLAVTSI 1.200 127 ALVYLPOVI 1.200 8 TNQEELWKM
1.000 212 DVWRMEIYV 1.000 128 LVYLPGVIA 0.750 227 LAILALLAV 0.600
293 IAVFLPIVV 0.600 126 LALVYLPGV 0.600 309 FLPCLRKKI 0.600 65
QELFPQWHL 0.600 233 LAVTSIPSV 0.600 199 QVQQNKEDA 0.500 116
KVLPMVSIT 0.500 331 NKTEICSQL 0.400 291 FMIAVFLPI 0.400 146
KYKKFPHWL 0.400 322 HGWEDVTKI 0.400 263 LLGTIHALI 0.400 245
LTWREFHYI 0.400 221 SLGIVGLAI 0.400 59 SELQHTQEL 0.400 289
PTFMIAVFL 0.400 30 KDTGETSML 0.400 53 DEFDCPSEL 0.400 201 QQNKEDAWI
0.400 270 LIFAWNKWI 0.400 249 EFHYIQSKL 0.400 110 PILVINKVL 0.400
104 QYFYKIPIL 0.400 103 QQYFYKIPI 0.400 5 KDITNQEEL 0.400 185
RRSYRYKLL 0.400 72 HLPIKJAAI 0.400 130 YLPGVIAAI 0.400 327
VTK1NKTEI 0.400 255 SKLGIVSLL 0.400 234 AVTSIPSVS 0.300 49
TAHADEFDC 0.300 77 IAAIIASLT 0.300 228 AILALLAVT 0.300 81 IASLTFLYT
0.300 137 AIVQLHNGT 0.300 208 WIEHDVWRM 0.300 252 YIQSKLGIV 0.200
217 EIYVSLGIV 0.200 68 FPQWHLPIK 0.200 40 RPVLLHLHQ 0.200 121
VSITLLALV 0.200 292 MIAVFLPIV 0.200 57 CPSELQHTQ 0.200 192
LLNWAYQQV 0.200
[0849] TABLE-US-00052 TABLE XV -V2-HLA-B7-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 4 FHYIQVNNI 0.040 1 WREFHYIQV 0.006 2 REFHYIQVN
0.002 3 EFHYIQVNN 0.002
[0850] TABLE-US-00053 TABLE XV -V1-HLA-B7-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Start
Subsequence Score 6 LLSLFFAVL 4.000 1 RKQFGLLSL 0.400 5 GLLSLFFAV
0.200 4 FGLLSLFFA 0.100 8 SLFFAVLHA 0.100 9 LFFAVLHAI 0.040 2
KQFGLLSLF 0.020 7 LSLFFAVLH 0.010 3 QFGLLSLFF 0.002
[0851] TABLE-US-00054 TABLE XVI -V1-HLA-B7-10mers: STEAP-1 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 109 IPILVINKVL 80.000 150 FPHWLDKWML 80.000 78
AAIIASLTFL 36.000 116 KVLPMVSITL 20.000 299 IVVLIFKSIL 20.000 224
IVGLAILALL 20.000 293 IAVFLPIVVL 18.000 82 ASLTFLYTLL 12.000 81
IASLTFLYTL 12.000 171 FAVLHAIYSL 12.000 35 TSMLKRPVLL 12.000 68
FPQWHLPIKI 8.000 288 PPTFMIAVFL 8.000 283 FVWYTPPTFM 7.500 183
PMRRSYRYKL 6.000 294 AVFLPIVVLI 6.000 118 LPMVSITLLA 6.000 34
ETSMLKRPVL 6.000 261 SLLLGTIHAL 4.000 165 GLLSFFFAVL 4.000 117
VLPMVSITLL 4.000 213 VWRMEIYVSL 4.000 184 MRRSYRYKLL 4.000 254
QSKLGIVSLL 4.000 330 INKTEICSQL 4.000 221 SLGIVGLAIL 4.000 309
FLPCLRKKIL 4.000 236 TSIPSVSDSL 4.000 37 MLKRPVLLHL 4.000 301
VLIFKSILFL 4.000 122 SITLLALVYL 4.000 253 IQSKLGIVSL 4.000 103
QQYFYKIPIL 4.000 223 GIVGLAILAL 4.000 157 WMLTRKQFGL 4.000 89
TLLREVIHPL 4.000 158 MLTRKQFGLL 4.000 175 HAIYSLSYPM 3.000 326
DVTKINKTEI 2.000 17 KPRRNLEEDD 2.000 73 LPIKIAAIIA 2.000 40
RPVLLHLHQT 2.000 64 TQELFPQWHL 1.800 269 ALIFAWNKWI 1.200 126
LALVYLPGVI 1.200 90 LLREVIHPLA 1.000 111 ILVINKVLPM 1.000 7
ITNQEELWKM 1.000 319 KIRHGWEDVT 1.000 112 LVINKVLPMV 1.000 120
MVSITLLALV 1.000 13 LWKMKPRRNL 0.900 136 AAIVQLHNGT 0.900 114
INKVLPMVSI 0.600 182 YPMRRSYRYK 0.600 86 FLYTLLREVI 0.600 219
YVSLGIVGLA 0.500 128 LVYLPGVIAA 0.500 127 ALVYLPGVIA 0.450 220
VSLGIVGLAI 0.400 66 ELFPQWHLPI 0.400 145 TKYKKFPHWL 0.400 255
SKLGIVSLLL 0.400 304 FKSILFLPCL 0.400 200 VQQNKEDAWI 0.400 218
IYVSLGIVGL 0.400 287 TPPTFMLAVF 0.400 297 LPWVVLIFKS 0.400 258
GIVSLLLGTI 0.400 262 LLLGTIHALI 0.400 244 SLTWREFHNI 0.400 238
IPSVSDSLTW 0.400 229 ILALLAVTSI 0.400 75 IKIAAIIASL 0.400 100
TSHQQYFYKI 0.400 96 HPLATSHQQY 0.400 119 PMVSITLLAL 0.400 102
HQQYFYKIPI 0.400 248 REFHYIQSKL 0.400 19 RRNLEEDDYL 0.400 132
PGVIAAIVQL 0.400 72 HLPIKIAAII 0.400 52 ADEFDGPSEL 0.360 207
AWIEHDVWRM 0.300 172 AVLHAIYSLS 0.300 227 LAILALLAVT 0.300 234
AVTSIPSVSD 0.225 226 GLAILALLAV 0.200 130 YLPGVIAAIV 0.200 108
KIPILVINKV 0.200 291 FMIAVFLPIV 0.200 164 FGLLSFFFAV 0.200 159
LTRKQFGLLS 0.200 57 CPSELQHTQE 0.200 125 LLALVYLPGV 0.200 292
MIAVFLPIVV 0.200 286 YTPPTFMIAV 0.200 310 LPCLRKKILK 0.200 191
KLLNWAYQQV 0.200
[0852] TABLE-US-00055 TABLE XVI -V2-HLA-B7-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Start
Subsequence Score 1 TWREFHYIQV 0.200 4 EFHYIQVNNI 0.040 3
REFHYIQVNN 0.002 2 WREFHYIQVN 0.001
[0853] TABLE-US-00056 TABLE XVI -V3-HLA-B7-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of amino acids, and the end position for
eachpeptide is the start plus nine. Start Subsequence Score 23
DPCLTRFKGL 80.000 17 VPESLWDPCL 24.000 25 CLTRFKGLNL 4.000 26
LTRFKGLNLI 4.000 16 DVPESLWDPC 0.500 1 TWREFHYIQI 0.400 12
HKKSDVPESL 0.400 8 LQIIHKKSDV 0.200 20 SLWDPCLTRF 0.020 19
ESLWDPCLTR 0.015 2 WREFHYIQII 0.012 10 IIHKKSDVPE 0.010 9
QIIHKKSDVP 0.010 7 YIQIIHKKSD 0.010 13 KKSDVPESLW 0.003 14
KSDVPESLWD 0.003 11 IHKKSDVPES 0.002 24 PCLTRFKGLN 0.002 6
HYIQIIHKKS 0.002 28 RFKGLNLIQS 0.002 22 WDPCLTRFKG 0.002 18
PESLWDPCLT 0.001 3 REFHYIQIIH 0.001 5 FHYIQIIHKK 0.001 15
SDVPESLWDP 0.001 27 TRFKGLNLIQ 0.001 4 EFHYIQIIHK 0.001 21
LWDPCLTRFK 0.000
[0854] TABLE-US-00057 TABLE XVI -V4-HLA-B7-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAVL 4.000 9 SLFFAVLHAI 0.400 1
TRKQFGLLSL 0.400 5 FGLLSLFFAV 0.200 8 LSLFFAVLHA 0.100 3 KQFGLLSLFF
0.020 7 LLSLFFAVLH 0.010 4 QFGLLSLFFA 0.010 2 RKQFGLLSLF 0.002 10
LFFAVLHAIY 0.002
[0855] TABLE-US-00058 TABLE XVII -V1-HLA-B35-9mers: STEAP-1 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 150 FPHWLDKWM 40.000 182 YPMRRSYRY 40.000 310
LPCLRKKIL 20.000 118 LPMVSITLL 20.000 254 QSKLGIVSL 15.000 305
KSILFLPCL 10.000 180 LSYPMRRSY 10.000 73 LPIKTAAII 8.000 90
LLREVIHPL 6.000 20 RNLEEDDYL 6.000 8 TNQEELWKM 6.000 82 ASLTFLYTL
5.000 35 TSMLKRPVL 5.000 131 LPGVIAAIV 4.000 109 IPILVINKV 4.000
287 TPPTFMIAV 4.000 78 AAIIASLTF 3.000 159 LTRKQFGLL 3.000 98
LATSHQQYF 3.000 206 DAWIEHDVW 2.250 122 SITLLALVY 2.000 176
AIYSLSYPM 2.000 162 KQFGLLSFF 2.000 112 LVINKVLPM 2.000 76
KIAAIIASL 2.000 139 VQLHNGTKY 2.000 80 IIASLTFLY 2.000 244
SLTWREFHY 2.000 238 IPSVSDSLT 2.000 256 KLGIVSLLL 2.000 99
ATSHQQYFY 2.000 288 PPTFMIAVF 2.000 319 KIRHGWEDV 1.200 189
RYKLLNWAY 1.200 17 KPRRNLEED 1.200 272 FAWNKWIDI 1.200 327
VTKINKTEI 1.200 312 CLRKKILKI 1.200 230 LALLAVTSI 1.200 322
HGWEDVTKI 1.200 262 LLLGTHIAL 1.000 172 AVLHAIYSL 1.000 166
LLSFFFAVL 1.000 219 YVSLGIVGL 1.000 224 IVGLAILAL 1.000 133
GVLAAIVQL 1.000 47 HQTAHADEF 1.000 222 LGIVGLAIL 1.000 63 HTQELFPQW
1.000 294 AVFLPIVVL 1.000 186 RSYRYKLLN 1.000 60 ELQHTQELF 1.000
264 LGTIHALIF 1.000 316 KJLKIRHGW 1.000 283 FVWYTPPTF 1.000 225
VGLAILALL 1.000 158 MLTRKQFGL 1.000 121 VSITLLALV 1.000 79
AIIASLTFL 1.000 36 SMLKRPVLL 1.000 300 VVLIFKSIL 1.000 296
FLPIVVLIF 1.000 117 VLPMVSITL 1.000 301 VLIFKSILF 1.000 237
SIPSVSDSL 1.000 123 ITLLALVYL 1.000 142 HNGTKYKKF 1.000 120
MVSITLLAL 1.000 302 LIFKSILFL 1.000 83 SLTFLYTLL 1.000 208
WIEHDVWRM 0.900 201 QQNKEDAWI 0.600 146 KYKKFPHWL 0.600 233
LAVTSIPSV 0.600 293 IAVFLPIVV 0.600 19 RRNLEEDDY 0.600 245
LTWREFHYI 0.600 126 LALVYLPGV 0.600 227 LAILALLAV 0.600 200
VQQNKEDAW 0.500 220 VSLGIVGLA 0.500 269 ALIFAWNKW 0.500 236
TSIPSVSDS 0.500 6 DITNQEELW 0.500 266 TIHALIFAW 0.500 49 TAHADEFDC
0.450 221 SLGIVGLAI 0.400 259 IVSLLLGTI 0.400 72 HLPIKIAAI 0.400
263 LLGTIHALI 0.400 130 YLPGVIAAI 0.400 299 IVVLIFKSI 0.400 103
QQYFYKIPI 0.400 309 FLPCLRKKI 0.400 40 RPVLLHLHQ 0.400 270
LIFAWNKWI 0.400 127 ALVYLPGVI 0.400 291 FMIAVFLPI 0.400 57
CPSELQHTQ 0.400 239 PSVSDSLTW 0.375
[0856] TABLE-US-00059 TABLE XVII -V2-HLA-B35-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is for
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 FHYIQVNNI 0.040 2 REFHYIQVN 0.020 3 EFHYIQVNN
0.010 1 WREFHYIQV 0.006
[0857] TABLE-US-00060 TABLE XVII -V3-HLA-B35-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 25 LTRFKGLNL 3.000 13 KSDVPESLW 1.500 18
ESLWDPCLT 0.750 16 VPESLWDPC 0.600 12 KKSDVPESL 0.400 8 QIIHKKSDV
0.200 22 DPCLTRFKG 0.200 6 YIQLIHKKS 0.100 24 CLTRFKGLN 0.100 23
PCLTRFKGL 0.100 2 REFHYIQII 0.080 11 HKKSDVPES 0.045 26 TRFKGLNLI
0.040 20 LWDPCLTRF 0.030 19 SLWDPCLTR 0.020 15 DVPESLWDP 0.020 1
WREFHYIQI 0.012 9 IIHKKSDVP 0.010 28 FKGLNLIQS 0.010 17 PESLWDPCL
0.010 7 IQIIHKKSD 0.010 27 RFKGLNLIQ 0.006 10 IHKKSDVPE 0.003 14
SDVPESLWD 0.002 3 EFHYIQIIH 0.001 5 HYIQIIHKK 0.001 4 FHYIQIIHK
0.001 21 WDPCLTRFK 0.001
[0858] TABLE-US-00061 TABLE XVII -V4-HLA-B35-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 KQFGLLSLF 2.000 6 LLSLFFAVL 1.000 1 RKQFGLLSL
0.200 5 GLLSLFFAV 0.200 8 SLFFAVLHA 0.100 4 FGLLSLFFA 0.100 3
QFGLLSLFF 0.100 7 LSLFFAVLH 0.050 9 LFFAVLHAI 0.040
[0859] TABLE-US-00062 TABLE XVIII -V1-HLA-B35-10mers: STEAP-1 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 96 HPLATSHQQY 40.000 150 FPHWLDKWML 30.000 109
IPILVINKVL 20.000 287 TPPTFMIAVF 20.000 238 IPSVSDSLTW 15.000 254
QSKLGIVSLL 15.000 243 DSLTWREFHY 10.000 121 VSITLLALVY 10.000 68
FPQWHLPIKI 8.000 175 HAIYSLSYPM 6.000 98 LATSHQQYFY 6.000 236
TSIPSVSDSL 5.000 35 TSMLKRPVLL 5.000 186 RSYRYKLLNW 5.000 82
ASLTFLYTLL 5.000 274 WNKWIDLKQF 4.500 40 RPVLLHLHQT 4.000 171
FAVLHAIYSL 3.000 330 INKTEICSQL 3.000 81 IASLTFLYTL 3.000 78
AAIIASLTFL 3.000 37 MLKRPVLLHL 3.000 77 LAAIIASLTF 3.000 7
ITNQEELWKM 3.000 293 IAVFLPIVVL 3.000 179 SLSYPMRRSY 2.000 297
LPIVVLIFKS 2.000 73 LPIKIAAIIA 2.000 283 FVWYTPPTFM 2.000 162
KQFGLLSFFF 2.000 116 KVLPMVSITL 2.000 220 VSLGIVGLAI 2.000 138
IVQLHNGTKY 2.000 118 LPMVSITLLA 2.000 288 PPTFMIAVPL 2.000 100
TSHQQYFYKI 2.000 111 ILVINKVLPM 2.000 79 AIIASLTFLY 2.000 173
VLHAIYSLSY 2.000 241 VSDSLTWREF 1.500 268 HALIFAWNKW 1.500 144
GTKYKKFPHW 1.500 114 INKVLPMVSI 1.200 17 KIPRNLEEDD 1.200 28
LHKDTGETSM 1.200 126 LALVYLPGVI 1.200 301 VLIFKSILFL 1.000 253
IQSKLGIVSL 1.000 103 QQYFYKIPIL 1.000 224 IVGLAILALL 1.000 122
SITLLALVYL 1.000 263 LLGTIHALIF 1.000 221 SLGIVGLAIL 1.000 223
GIVGLAILAL 1.000 158 MLTRKQFGLL 1.000 89 TLLREVIHPL 1.000 157
WMLTRKQFGL 1.000 299 IVVLIFKSIL 1.000 165 GLLSFFFAVL 1.000 261
SLLLGTIHAL 1.000 309 FLPCLRKKIL 1.000 300 VVLIFKSILF 1.000 34
ETSMLKRPVL 1.000 117 VLPMVSITLL 1.000 200 VQQNKEDAWI 0.600 277
WIDIKQFVWY 0.600 90 LLREVIHPLA 0.600 207 AWIEHDVWRM 0.600 244
SLTWREFHYI 0.600 319 KIRHGWEDVT 0.600 178 YSLSYPMRRS 0.500 167
LSFFFAVLHA 0.500 265 GTIHALIFAW 0.500 260 VSLLLGTIHA 0.500 199
QVQQNKEDAW 0.500 269 ALIFAWNKWI 0.400 191 KLLNWAYQQV 0.400 149
KFPHWLDKWM 0.400 294 AVFLPIVVLI 0.400 86 FLYTLLREVI 0.400 66
ELFPQWHLPI 0.400 258 GIVSLLLGTI 0.400 262 LLLGTIHALI 0.400 326
DVTKLNKTEI 0.400 57 CPSELQHTQE 0.400 102 HQQYFYKIPI 0.400 72
HLPIKIAAII 0.400 108 KIPILVINKV 0.400 229 ILALLAVTSI 0.400 327
VTKINKTEIC 0.300 213 VWRMEIYVSL 0.300 227 LAILALLAVT 0.300 184
MRRSYRYKLL 0.300 159 LTRKQFGLLS 0.300 233 LAVTSIPSVS 0.300 136
AAIVQLHNGT 0.300 64 TQELFPQWHL 0.300 160 TRKQFGLLSF 0.300 13
LWKMKPRRNL 0.300 183 PMRRSYRYKL 0.300
[0860] TABLE-US-00063 TABLE XVIII -V2-HLA-B35-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of amino acids, and the end position for
eachpeptide is the start plus nine. Start Subsequence Score 1
TWREFHYIQV 0.120 4 EFHYIQVNNI 0.040 3 REFHYIQVNN 0.020 2 WREFHYIQVN
0.003
[0861] TABLE-US-00064 TABLE XVIII -V3-HLA-B35-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 23 DPCLTRFKGL 20.000 17 VPESLWDPCL 6.000 20
SLWDPCLTRF 2.000 26 LTRFKGLNLI 1.200 25 CLTRFKGLNL 1.000 12
HKKSDVPESL 0.300 1 TWREFHYIQI 0.240 8 IQIIHKKSDV 0.200 16
DVPESLWDPC 0.200 13 KKSDVPESLW 0.200 28 RFKGLNLIQS 0.060 19
ESLWDPCLTR 0.050 11 IHKKSDVPES 0.045 14 KSDVPESLWD 0.045 2
WREFHYIQII 0.012 6 HYIQIIHKKS 0.010 9 QIIHKKSDVP 0.010 24
PCLTRFKGLN 0.010 10 IIHKKSDVPE 0.010 7 YIQIIHKKSD 0.010 3
REFHYIQIIH 0.002 18 PESLWDPCLT 0.002 22 WDPCLTRFKG 0.001 5
FHYIQIIHKK 0.001 27 TRFKGLNLIQ 0.001 4 EFHYIQIIHK 0.001 15
SDVPESLWDP 0.001 21 LWDPCLTRFK 0.000
[0862] TABLE-US-00065 TABLE XVIII -V4-HLA-B35-10mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 KQFGLLSLFF 2.000 6 GLLSLFFAVL 1.000 8
LSLFFAVLHA 0.500 9 SLFFAVLHAI 0.400 1 TRKQFGLLSL 0.300 10
LFFAVLHAIY 0.200 2 RKQFGLLSLF 0.200 5 FGLLSLFFAV 0.200 4 QFGLLSLFFA
0.010 7 LLSLFFAVLH 0.010
[0863] TABLE-US-00066 TABLE XIX 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 terminal)/b6/petB
superoxide 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
Ubiquinone/plastoquinone translocation 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 (20 copies) formation of connective tissue.
The sequence consists of the G-X-Y and the polypeptide chains forms
a triple helix. fn3 20% Fibronectin type III 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, (rhodopsin family) with the
N-terminus located extracellularly while the C-terminus is
cytoplasmic. Signal through G proteins
[0864] TABLE-US-00067 TABLE XX Motifs and Post-translational
Modifications of STEAP-1 N-glycosylation site 143-146 NGTK (SEQ ID
NO: 61) 331-334 NKTE (SEQ ID NO: 62) Protein kinase C
phosphorylation site 3-5 SrK 160-162 TrK 187-189 SyR 246-248 TwR
Casein kinase II phosphorylation site 3-6 SrkD (SEQ ID NO: 63) 8-11
TnqE (SEQ ID NO: 64) 240-243 SvsD (SEQ ID NO: 65) 246-249 TwrE (SEQ
ID NO: 66) Tyrosine kinase phosphorylation site 19-27 RRNLEEDDY
(SEQ ID NO: 67) N-myristoylation site 133-138 GVLAAI (SEQ ID NO:
68) 265-270 GTIHAL (SEQ ID NO: 69) Bipartite nuclear targeting
sequence 4-20 RKDITNQEELWKMKPRR (SEQ ID NO: 70)
[0865] TABLE-US-00068 TABLE XXI Properties of STEAP-1 Bioinformatic
URL (Located on the World Wide Web Program at) Outcome ORF ORF
finder 1193 bp Protein length 339 aa Transmembrane region TM Pred
(.ch.embnet.org/) 6 TM at aa 73-91, 120-141, 163-181, 218-236,
253-274, 286-304 HMMTop (.enzim.hu/hmmtop/) 6 TM at aa 73-90,
117-139, 164-182, 220-238, 257-274, 291-309 Sosui
(.genome.ad.jp/SOSui/) 6 TM at aa 70-92, 114-136, 163-184, 219-241,
255-273, 292-313 TMHMM (.cbs.dtu.dk/services/TMHMM) 6 TM at aa
73-95, 117-139, 164-182, 218-240, 252-274, 289-311 Signal Peptide
Signal P (.cbs.dtu.dk/services/SignalP/) potential cleavage between
aa 136 and 137 pI pI/MW tool (.expasy.ch/tools/) 9.2 pI Molecular
weight pI/MW tool (.expasy.ch/tools/) 39.8 kD Localization PSORT
http://psort.nibb.ac.jp/ 60% plama membrane, 40% golgi, 30%
endoplasmic reticulum PSORT II http://psort.nibb.ac.jp/ 66%
endoplasmic reticulum, 11% mitochondria, 11% plasma membrane Motifs
Pfam (.sanger.ac.uk/Pfam/) none Prints (.biochem.ucl.ac.uk/)
Transforming protein P21 ras signature, Fibronectin type III repeat
signature Blocks (.blocks.fhcrc.org/) Half-A-TPR repeat, Arsenical
pump membrane protein signature, M protein repeat
[0866] TABLE-US-00069 TABLE XXII -V1-HLA-A1- 9mers:STEAP-1 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 99 ATSHQQYFY 24 122 SITLLALVY 24 180 LSYPMRRSY 24
174 LHAIYSLSY 22 278 IDIKQFVWY 20 139 VQLHNGTKY 19 32T GETSMLKR 18
244 SLTWREFHY 18 80 IIASLTFLY 17 153 WLDKWMLTR 17 182 YPMRRSYRY 17
22 LEEDDYLHK 16 58 PSELQHTQE 16 97 PLATSHQQY 16 170 FFAVLHAIY 16
189 RYKLLNWAY 16 19 RRNLEEDDY 15 84 LTFLYTLLR 15 211 HDVWRMEIY 15
241 VSDSLTWRE 15 286 YTPPTFMIA 15 203 NKEDAWIEH 14 215 RMEIYVSLG 14
247 WREFHYIQS 14 324 WEDVTKINK 14 21 NLEEDDYLH 13 23 EEDDYLHKD 13
54 EFDCPSELQ 13 159 LTRKQFGLL 13 186 RSYRYKLLN 13 7 ITNQEELWK 12 10
QEELWKMKP 12 29 HKDTGETSM 12 38 LKRPVLLHL 12 91 LREVIHPLA 12 129
VYLPGVIAA 12 221 SLGIVGLAI 12 291 FMIAVFLPI 12 323 GWEDVTKIN 12 4
RKDITNQEE 11 9 NQEELWKMK 11 37 MLKRPVLLH 11 52 ADEFDCPSE 11 55
FDCPSELQH 11 208 WIEHDVWRM 11 210 EHDVWRMEI 11 256 KLGIVSLLL 11 277
WIDIKQFVW 11 296 FLPIVVLIF 11
[0867] TABLE-US-00070 TABLE XXII -V1-HLA-A1- Each peptide is a
portion of SEQ ID NO: 5; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide start position plus eight. Pos 123456789 score 2 WREFHYIQV
14
[0868] TABLE-US-00071 TABLE XXII -V3-HLA-A1- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 13 KSDVPESLW 17 1 WREEHYIQI 14 20 LWDPCLTRF 14 25
LTRFKGLNL 13 14 SDVPESLWD 12 16 VPESLWDPC 11 19 SLWDPCLTR 10 27
RFKGLNLIQ 8
[0869] TABLE-US-00072 TABLE XXII -V4-HLA-A1-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 SLFFAVLHA 10 1 RKQFGLLSL 8 3 QFGLLSLFF 6 7
LSLFFAVLH 6 6 LLSLFFAVL 5 5 GLLSLFFAV 4
[0870] TABLE-US-00073 TABLE XXIII -V1-HLA-A0201- 9mers:STEAP-1 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 262 LLLGTIHAL 32 76 KIAAIIASL 29 86 FLYTLLREV 29 90
LLREVIHPL 29 130 YLPGVIAAI 29 72 HLPIKIAAI 26 79 AIIASLTFL 26 292
MIAVFLPIV 26 302 LIFKSILFL 26 312 CLRKKILKI 26 113 VINKVLPMV 25 126
LALVVLPGV 25 165 GLLSFFFAV 25 172 AVLHAIYSL 25 219 YVSLGIVGL 25 252
YIQSKLGIV 25 36 SMLKRPVLL 24 123 ITLLALVYL 24 166 LLSFFFAVL 24 227
LAILALLAV 24 294 AVFLPIVVL 24 117 VLPMVSITL 23 133 GVIAAIVQL 23 192
LLNWAYQQV 23 228 AILALLAVT 23 233 LAVTSIPSV 23 82 ASLTFLYTL 22 221
SLGIVGLAI 22 222 LGIVGLAIL 22 224 IVGLAILAL 22 230 LALLAVTSI 22 237
SIPSVSDSL 22 256 KLGIVSLLL 22 261 SLLLGTIHA 22 263 LLGTIHALI 22 83
SLTFLYTLL 21 109 IPILVINKV 21 158 MLTRKQFGL 21 259 IVSLLLGTI 21 309
FLPCLRKKI 21 319 KIRHGWEDV 21 27 YLHKDTGET 20 42 VLLHLHQTA 20 120
MVSITLLAL 20 121 VSITLLALV 20 127 ALVYLPGVI 20 217 EIYVSLGIV 20 255
SKLGIVSLL 20 258 GIVSLLLGT 20 291 FMIAVFLPI 20 38 LKRPVLLHL 19 225
VGLAILALL 19 226 GLAILALLA 19 229 ILALLAVTS 19 254 QSKLGIVSL 19 295
VFLPIVVLI 19 305 KSILFLPCL 19 59 SELQHTQEL 18 112 LVINKVLPM 18 124
TLLALVYLP 18 270 LIFAWNKWI 18 300 VVLIFKSIL 18 116 KVLPMVSIT 17 118
LPMVSITLL 17 137 AIVQLHNGT 17 176 AIYSLSYPM 17 214 WRMEIYVSL 17 322
HGWEDVTKI 17 89 TLLREVIHP 16 104 QYFYKIPIL 16 110 PILVINKVL 16 129
VYLPGVIAA 16 159 LTRKQFGLL 16 169 FFFAVLHAI 16 223 GIVGLAILA 16 231
ALLAVTSIP 16 245 LTWREFHYI 16 269 ALIFAWNKW 16 293 IAVFLPIVV 16 296
ELPIVVLIF 16 5 KDITNQEEL 15 37 MLKRPVLLH 15 80 IIASLTFLY 15 125
LLALVYLPG 15 134 VIAAIVQLH 15 208 WIEHDVWRM 15 265 GTIHALIFA 15 272
FAWNKWIDI 15 279 DIKQFVVYT 15 299 IVVLIFKSI 15 307 ILFLPCLRK 15
[0871] TABLE-US-00074 TABLE XXIII -V2-HLA-A0201- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 5 FHYIQVNNI 15 2 WREFHYIQV 8
[0872] TABLE-US-00075 TABLE XXII -V3-HLA-A0201- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 QIIHKKSDV 21 19 SLWDPCLTR 18 25 LTRFKGLNL 17 26
TRFKGLNLI 16 2 REFHYIQII 14 12 KKSDVPESL 14 6 YIQIIHKKS 11 9
IIHKKSDVP 11 24 CLTRFKGLN 10
[0873] TABLE-US-00076 TABLE XXIII -V4-HLA-0201- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 GLLSLFFAV 25 6 LLSLFFAVL 25 8 SLFFAVLHA 21 1
RKQFGLLSL 18 9 LFFAVLHAI 17
[0874] TABLE-US-00077 TABLE XXIV -V2-HLA-A202- 9mers:STEAP-1 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 NoResultsfound.
[0875] TABLE-US-00078 TABLE XXIV -V2-HLA-A0202- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score NoResultsfound.
[0876] TABLE-US-00079 TABLE XXIV -V3-HLA-A0202- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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. Each
peptide is a portion of SEQ ID NO: 7; 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 NoResultsfound.
[0877] TABLE-US-00080 TABLE XXIV -V4-HLA-A0202- 9mers:STEAP-1
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 NoResultsfound.
[0878] TABLE-US-00081 TABLE XXV -V1-A0203- 9mers:STEAP-1 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 Noresultsfound.
[0879] TABLE-US-00082 TABLE XXV -V2-HLA-A0203- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score NoResultsfound.
[0880] TABLE-US-00083 TABLE XXV -V3-HLA-A0203- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 NoResultsfound.
[0881] TABLE-US-00084 TABLE XXV -V4-HLA-A0203- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 NoResultsfound.
[0882] TABLE-US-00085 TABLE XXVI -V1-HLA-A3- 9mers:STEAP-1 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 posifion plus eight. Pos
123456789 score 138 IVQLHNGTK 28 122 SITLLALVY 26 307 ILFLPCLRK 26
140 QLHNGTKYK 25 229 ILALLAVTS 25 108 KIPILVINK 22 128 LVYLPGVIA 22
133 GVIAAIVQL 22 191 KLLNWAYQQ 22 228 AILALLAVT 22 294 AVFLPIVVL 22
321 RHGWEDVTK 22 37 MLKRPVLLH 21 94 VIHPLATSH 21 116 KVLPMVSIT 21
153 WLDKWMLTR 21 231 ALLAVTSIP 21 234 AVTSIPSVS 21 93 EVIHPLATS 20
97 PLATSHQQY 20 221 SLGIVGLAI 20 76 KIAAIIASL 19 127 ALVYLPGVI 19
148 KKFPHWLDK 19 244 SLTWREFHY 19 283 FVWYTPPTF 19 296 FLPIVVLIF 19
78 AAIIASLTF 18 112 LVINKVLPM 18 226 GLAILALLA 18 240 SVSDSLTWR 18
256 KLGIVSLLL 18 259 IVSLLLGTI 18 301 VLIFKSILF 18 317 ILKIRHGWE 18
43 LLHLHQTAH 17 79 AIIASLTFL 17 80 IIASLTFLY 17 111 ILVINKVLP 17
130 YLPGVIAAI 17 166 LLSFFFAVL 17 172 AVLHAIYSL 17 219 YVSLGIVGL 17
224 IVGLAILAL 17 312 CLRKKILKI 17 7 ITNQEELWK 16 41 PVLLHLHQT 16 42
VLLHLHQTA 16 72 HLPIKIAAI 16 125 LLALVYLPG 16 180 LSYPMRRSY 16 183
PMRRSYRYK 16 207 AWIEHDVWR 16 248 REFHYIQSK 16 261 SLLLGTIHA 16 268
HALIFAWNK 16 269 ALIFAWNKW 16 278 IDIKQFVWY 16 300 VVLIFKSIL 16 306
SILFLPCLR 16 311 PCLRKKILK 16 319 KIRHGWEDV 16 21 NLEEDDYLH 15 89
TLLREVIHP 15 90 LLREVIHPL 15 117 VLPMVSITL 15 120 MVSITLLAL 15 134
VIAAIVQLH 15 161 RKQFGLLSF 15 173 VLHAIYSLS 15 176 AIYSLSYPM 15 262
LLLGTIHAL 15 299 IVVLIFKSI 15 308 LFLPCLRKK 15 12 ELWKMKPRR 14 22
LEEDDYLHK 14 45 HLHQTAHAD 14 60 ELQHTQELF 14 86 FLYTLLREV 14 110
PILVINKVL 14 124 TLLALVYLP 14 158 MLTRKQFGL 14 186 RSYRYKLLN 14 196
AYQQVQQNK 14 277 WIDIKQFVW 14 297 LPIVVLIFK 14 316 KILKIRHGW 14 326
DVTKINKTE 14 55 FDCPSELQH 13 66 ELFPQWHLP 13 74 PIKIAAIIA 13 88
YTLLREVIH 13 165 GLLSFFFAV 13 174 LHAIYSLSY 13 189 RYKLLNWAY 13 192
LLNWAYQQV 13 212 DVWRMEIYV 13 217 EIYVSLGIV 13 223 GIVGLAILA 13 263
LLGTIHALI 13 273 AWNKWIDIK 13
[0883] TABLE-US-00086 TABLE XXVI -V2-HLA-A3- 9mers:STEAP-1 start
position plus eight. Pos 123456789 score 3 REFHYIQVN 8 5 FHYIQVNNI
5 1 TWREFHYIQ 4 4 EFHYIQVNN 4 2 WREFHYIQV 3
[0884] TABLE-US-00087 TABLE XXVI -V3-HLA-A3-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 19 SLWDPCLTR 23 8 QIIHKKSDV 17 4 FHYIQIIHK 15 9
IIHKKSDVP 15 24 CLTRFKGLN 14 5 HYIQIIHKK 13 15 DVPESLWDP 11
[0885] TABLE-US-00088 TABLE XXVI -V4-HLA-A3- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVL 18 8 SLFFAVLHA 14 5 GLLSLFFAV 13 7
LSLFFAVLH 13 2 KQFGLLSLF 12 1 RKQFGLLSL 11 3 QFGLLSLFF 10
[0886] TABLE-US-00089 TABLE XXVII V1-HLA-A26- 9mers:STEAP-1 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 133 GVIAAIVQL 26 302 LIFKSILFL 26 76 KIAAIIASL 25
79 AIIASLTFL 25 112 LVINKVLPM 25 294 AVFLPIVVL 25 60 ELQHTQELF 24
159 LTRKQFGLL 24 219 YVSLGIVGL 24 80 IIASLTFLY 23 93 EVIHPLATS 23
120 MVSITLLAL 23 123 ITLLALVYL 23 172 AVLHAIYSL 23 208 WIEHDVWRM 23
224 IVGLAILAL 23 262 LLLGTIHAL 23 296 FLPIVVLIF 23 90 LLREVIHPL 22
97 PLATSHQQY 22 249 EFHYIQSKL 22 279 DIKQFVWYT 22 83 SLTFLYTLL 21
99 ATSHQQYFY 21 122 SITLLALVY 21 166 LLSFFFAVL 21 217 EIYVSLGIV 21
237 SIPSVSDSL 21 283 FVWYTPPTF 21 289 PTFMIAVFL 21 301 VLIFKSILF 21
162 KQFGLLSFF 20 176 AIYSLSYPM 20 31 DTGETSMLK 19 53 DEFDCPSEL 19
63 HTQELFPQW 19 66 ELFPQWHLP 19 170 FFAVLHAIY 19 244 SLTWREFHY 19
278 IDIKQFVWY 19 300 VVLIFKSIL 19 110 PILVINKVL 18 158 MLTRKQFGL 18
286 YTPPTFMIA 18 6 DITNQEELW 17 12 ELWKMKPRR 17 34 ETSMLKRPV 17 108
KIPILVINK 17 113 VINKVLPMV 17 116 KVLPMVSIT 17 117 VLPMVSITL 17 130
YLPGVIAAI 17 163 QFGLLSFFF 17 258 GIVSLLLGT 17 326 DVTKINKTE 17 72
HLPIKIAAI 16 104 QYFYKIPIL 16 134 VIAAIVQLH 16 142 HNGTKYKKF 16 212
DVWRMEIYV 16 214 WRMEIYVSL 16 228 AILALLAVT 16 240 SVSDSLTWR 16 252
YIQSKLGIV 16 256 KLGIVSLLL 16 265 GTIHALIFA 16 266 TIHALIFAW 16 292
MIAVFLPIV 16 298 PIVVLIEKS 16 8 TNQEELWKM 15 38 LKRPVLLHL 15 41
PVLLHLHQT 15 161 RKQFGLLSF 15 222 LGIVGLAIL 15 254 QSKLGIVSL 15 255
SKLGIVSLL 15 259 IVSLLLGTI 15 275 NKWIDIKQF 15 288 PPTFMIAVF 15 299
IVVLIFKSI 15 305 KSILFLPCL 15 312 CLRKKILKI 15 56 DCPSELQHT 14 86
FLYTLLREV 14 94 VIHPLATSH 14 153 WLDKWMLTR 14 165 GLLSFFFAV 14 192
LLNWAYQQV 14 211 HDVWRMEIY 14 245 LTWREFHYI 14 23 EEDDYLHKD 13 37
MLKRPVLLH 13 54 EFDCPSELQ 13 89 TLLREVIHP 13 118 LPMVSITLL 13 124
TLLALVYLP 13 125 LLALVYLPG 13 137 AIVQLHNGT 13 168 SFFFAVLHA 13 169
FFFAVLHAI 13 173 VLHAIYSLS 13 179 SLSYPMRRS 13 180 LSYPMRRSY 13 189
RYKLLNWAY 13 223 GIVGLAILA 13 225 VGLAILALL 13 295 VFLPIVVLI 13 308
LFLPCLRKK 13 2 ESRKDITNQ 12 30 KDTGETSML 12 78 AAIIASLTF 12 82
ASLTFLYTL 12 84 LTFLYTLLR 12 98 LATSHQQYF 12 144 GTKYKKFPH 12 149
KFPHWLDKW 12 174 LHAIYSLSY 12 185 RRSYRYKLL 12 234 AVTSIPSVS 12
235 VTSIPSVSD 12 270 LIFAWNKWI 12 277 WIDIKQFVW 12 303 IFKSILFLP 12
316 KILKIRHGW 12 325 EDVTKINKT 12 327 VTKINKTEI 12 331 NKTEICSQL
12
[0887] TABLE-US-00090 TABLE XXVII -V2-HLA-A26- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 4 EFHYIQVNN 14 3 REFHYIQVN 7 5 FHYIQVNNI 6
[0888] TABLE-US-00091 TABLE XXVII -V4-HLA-A26- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each pepUde is the start position plus eight. Pos
123456789 score 15 DVPESLWDP 24 25 LTRFKGLNL 20 20 LWDPCLTRF 18 8
QIIHKKSDV 14 23 PCLTRFKGL 13 3 EFHYIQIIH 12 9 IIHKKSDVP 12 6
YIQIIHKKS 11
[0889] TABLE-US-00092 TABLE XXVII -V4-HLA-26- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVL 21 2 KQFGLLSLF 20 3 QFGLLSLFF 17 5
GLLSLFFAV 15 8 SLFFAVLHA 15 1 RKQFGLLSL 13 9 LFFAVLHAI 13
[0890] TABLE-US-00093 TABLE XXVII -V1-HLA-B0702- 9mers:STEAP-1 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 118 LPMVSITLL 22 310 LPCLRKKIL 20 238 IPSVSDSLT 19
73 LPIKIAAII 18 131 LPGVIAAIV 18 288 PPTFMIAVF 18 294 AVFLPIVVL 18
109 IPILVINKV 17 287 TPPTFMIAV 17 14 WKMKPRRNL 16 150 FPHWLDKWM 16
38 LKRPVLLHL 15 79 AIIASLTFL 15 120 MVSITLLAL 15 166 LLSFFFAVL 15
224 IVGLAILAL 15 30 KDTGETSML 14 35 TSMLKRPVL 14 76 KIAAIIASL 14
159 LTRKQFGLL 14 219 YVSLGIVGL 14 256 KLGIVSLLL 14 302 LIFKSILFL 14
17 KPRRNLEED 13 36 SMLKRPVLL 13 40 RPVLLHLHQ 13 65 QELFPQWHL 13 90
LLREVIHPL 13 123 ITLLALVYL 13 133 GVIAAIVQL 13 184 MRRSYRYKL 13 185
RRSYRYKLL 13 214 WRMEIYVSL 13 255 SKLGIVSLL 13 262 LLLGTIHAL 13 289
PTFMIAVFL 13 57 CPSELQHTQ 12 81 IASLTFLYT 12 82 ASLTFLYTL 12 106
FYKIPILVI 12 128 LVYLPGVIA 12 146 KYKKFPHWL 12 172 AVLHAIYSL 12 225
VGLAILALL 12 254 QSKLGIVSL 12 305 KSILFLPCL 12 5 KDITNQEEL 11 20
RNLEEDDYL 11 59 SELQHTQEL 11 68 FPQWHLPIK 11 83 SLTFLYTLL 11 96
HPLATSHQQ 11 104 QYFYKIPIL 11 110 PILVINKVL 11 115 NKVLPMVSI 11 182
YPMRRSYRY 11 221 SLGIVGLAI 11 222 LGIVGLAIL 11 228 AILALLAVT 11 249
EFHYIQSKL 11 284 VWYTPPTFM 11 285 WYTPPTFMI 11 291 FMIAVFLPI 11 297
LPIVVLIFK 11 312 CLRKKILKI 11 34 ETSMLKRPV 10 53 DEFDCPSEL 10 78
AAIIASLTF 10 92 REVIHPLAT 10 112 LVINKVLPM 10 117 VLPMVSITL 10 130
YLPGVIAAI 10 151 PHWLDKWML 10 158 MLTRKQFGL 10 162 KQFGLLSFF 10 237
SIPSVSDSL 10 295 VELPIVVLI 10 300 VVLIFKSIL 10 320 IRHGWEDVT 10 331
NKTEICSQL 10
[0891] TABLE-US-00094 TABLE XXVIII -V2-HLA-B0702- 9mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 5 FHYIQVNNI 7 2 WREFHYIQV 6 4 EFHYIQVNN 3
[0892] TABLE-US-00095 TABLE XXVIII -V3-HLA-B0702- mers:STEAP-1 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 25 LTRFKGLNL 15 12 KKSDVPESL 13 17 PESLWDPCL 13 16
VPESLWDPC 11 22 DPCLTRFKG 11 23 PCLTRFKGL 11 20 LWDPGLTRF 9 2
REFHYIQII 7 18 ESLWDPCLT 7 26 TRFKGLNLI 7
[0893] TABLE-US-00096 TABLE XXVIII -V4-HLA-B0702- 9mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVL 15 1 RKQFGLLSL 13 3 QFGLLSLFF 10 2
KQFGLLSLF 9 8 SLFFAVLHA 9 5 GLLSLFFAV 8 9 LFFAVLHAI 8 4 FGLLSLFFA
7
[0894] TABLE-US-00097 TABLE XXIX -V1-HLA-B08- 9mers:STEAP-1 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 312 CLRKKILKI 29 158 MLTRKQFGL 26 310 LPCLRKKIL 26
72 HLPIKIAAI 25 90 LLREVIHPL 25 317 ILKIRHGWE 25 254 QSKLGIVSL 23
301 VLIFKSILF 23 104 QYFYKIPIL 22 144 GTKYKKFPH 22 35T SAALKRPVL 21
36 SMLKRPVLL 21 272 FAWNKWIDI 21 13 LWKMKPRRN 20 146 KYKKFPHWL 20
185 RRSYRYKLL 20 187 SYRYKLLNW 19 15 KMKPRRNLE 18 83 SLTFLYTLL 18
106 FYKIPILVI 18 118 LPMVSITLL 18 142 HNGTKYKKF 18 262 LLLGTIHAL 18
327 VTKINKTEI 18 37 MLKRPVLLH 17 110 PILVINKVL 17 117 VLPMVSITL 17
38 LKRPVLLHL 16 159 LTRKQFGLL 16 166 LLSFFFAVL 16 184 MRRSYRYKL 16
237 SIPSVSDSL 16 244 SLTWREFHY 16 256 KLGIVSLLL 16 76 KIAAIIASL 15
221 SLGIVGLAI 15 279 DIKQFVWYT 15 302 LIFKSILFL 15 60 ELQHTQELF 14
74 PIKIAAIIA 14 79 AIIASLTFL 14 130 YLPGVIAAI 14 182 YPMRRSYRY 14
224 IVGLAILAL 14 252 YIQSKLGIV 14 277 WIDIKQFVW 14 296 FLPIVVLIF 14
309 FLPCLRKKI 14 11 EELWKMKPR 13 17 KPRRNLEED 13 59 SELQHTQEL 13
123 ITLLALVYL 13 127 ALVYLPGVI 13 133 GVIAAIVQL 13 189 RYKLLNWAY 13
214 WRMEIYVSL 13 219 YVSLGIVGL 13 249 EFHYIQSKL 13 255 SKLGIVSLL 13
288 PPTFMIAVF 13 294 AVFLPIVVL 13 325 EDVTKINKT 13
[0895] TABLE-US-00098 TABLE XXIX -V2-HLA-B08- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 1 TWREFHYIQ 8 5 FHYIQVNNI 8 4 EFHYIQVNN 4
[0896] TABLE-US-00099 TABLE XXIX -V1-HLA-B08- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 25 LTRFKGLNL 26 23 PCLTRFKGL 19 9 IIHKKSDVP 15 8
QIIHKKSDV 14 11 HKKSDVPES 13 10 IHKKSDVPE 12
[0897] TABLE-US-00100 TABLE XXIX -V4-B08- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVL 17 1 RKQFGLLSL 10 8 SLFFAVLHA 9 5
GLLSLFFAV 8
[0898] TABLE-US-00101 TABLE XXIX -V1-HLA-B1510- 9mers:STEAP-1 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 151 PHWLDKWML 21 219 YVSLGIVGL 16 294 AVFLPIVVL 16
14 WKMKPRRNL 15 35 TSMLKRPVL 15 36 SMLKRPVLL 14 71 WHLPIKIAA 14 123
ITLLALVYL 14 133 GVIAAIVQL 14 210 EHDVWRMEI 14 224 IVGLAILAL 14 254
QSKLGIVSL 14 53 DEFDCPSEL 13 110 PILVINKVL 13 166 LLSFFFAVL 13 214
WRMEIYVSL 13 262 LLLGTIHAL 13 267 IHALIFAWN 13 321 RHGWEDVTK 13 5
KDITNQEEL 12 28 LHKDTGETS 12 38 LKRPVLLHL 12 50 AHADEEDOP 12 59
SELQHTQEL 12 76 KIAAIIASL 12 90 LLREVIHPL 12 95 IHPLATSHQ 12 104
QYFYKIPIL 12 118 LPMVSITLL 12 120 MVSITLLAL 12 141 LHNGTKYKK 12 146
KYKKFPHWL 12 222 LGIVGLAIL 12 255 SKLGIVSLL 12 289 PTFMIAVFL 12 302
LIFKSILFL 12 20 RNLEEDDYL 11 30 KDTGETSML 11 62 QHTQELFPQ 11 65
QELFPQWHL 11 82 ASLTFLYTL 11 101 SHQQYFYKI 11 117 VLPMVSITL 11 159
LTRKQFGLL 11 172 AVLHAIYSL 11 174 LHAIYSLSY 11 184 MRRSYRYKL 11 185
RRSYRYKLL 11 249 EFHYIQSKL 11 300 VVLIFKSIL 11 305 KSILFLPCL 11 310
LPCLRKKIL 11 331 NKTEICSQL 11 44 LHLHQTAHA 10 46 LHQTAHADE 10 79
AIIASLTFL 10 83 SLTFLYTLL 10 158 MLTRKQFGL 10 208 WIEHDVWRM 10 225
VGLAILALL 10 237 SIPSVSDSL 10 242 SDSLTWREF 10 250 FHYIQSKLG 10 256
KLGIVSLLL 10 283 FVWYTPPTF 9 288 PPTFMIAVF 9
[0899] TABLE-US-00102 TABLE XXX +HC,1-V2-HLA-B1510- 9mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each pepfide is the start position plus eight. Pos
123456789 score 5 FHYIQVNNI 11
[0900] TABLE-US-00103 TABLE XXX -V4-HLA-B1510- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVL 13 1 RKQFGLLSL 11 2 KQFGLLSLF 7 3
QFGLLSLFF 6
[0901] TABLE-US-00104 TABLE XXXI -V1-HLA-B2705- 9mers:STEAP-1 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 19 RRNLEEDDY 24 185 RRSYRYKLL 24 214 WRMEIYVSL 24
313 LRKKILKIR 24 184 MRRSYRYKL 23 39 KRPVLLHLH 22 248 REFHYIQSK 21
294 AVFLPIVVL 21 133 GVIAAIVQL 20 162 KQFGLLSFF 20 302 LIFKSILFL 20
20 RNLEEDDYL 19 161 RKQFGLLSF 19 307 ILFLPCLRK 19 76 KIAAIIASL 18
104 QYFYKIPIL 18 172 AVLHAIYSL 18 196 AYQQVQQNK 18 254 QSKLGIVSL 18
321 RHGWEDVTK 18 5 KDITNQEEL 17 79 AIIASLTFL 17 108 KIPILVINK 17
148 KKFPHWLDK 17 188 YRYKLLNWA 17 219 YVSLGIVGL 17 255 SKLGIVSLL 17
262 LLLGTIHAL 17 305 KSILFLPCL 17 314 RKKILKIRH 17 12 ELWKMKPRR 16
30 KDTGETSML 16 65 QELFPQWHL 16 78 AAIIASLTF 16 82 ASLTFLYTL 16 118
LPMVSITLL 16 123 ITLLALVYL 16 141 LHNGTKYKK 16 189 RYKLLNWAY 16 207
AWIEHDVWR 16 222 LGIVGLAIL 16 224 IVGLAILAL 16 249 EFHYIQSKL 16 268
HALIFAWNK 16 273 AWNKWIDIK 16 289 PTFMIAVFL 16 308 LFLPCLRKK 16 7
ITNQEELWK 15 14 WKMKPRRNL 15 36 SMLKRPVLL 15 53 DEFDCPSEL 15 59
SELQHTQEL 15 84 LTFLYTLLR 15 90 LLREVIHPL 15 110 PILVINKVL 15 154
LDKWMLTRK 15 178 YSLSYPMRR 15 225 VGLAILALL 15 256 KLGIVSLLL 15 288
PPTFMIAVF 15 297 LPIVVLIFK 15 301 VLIFKSILF 15 311 PCLRKKILK 15 312
CLRKKILKI 15 8 TNQEELWKM 14 32 TGETSMLKR 14 35 TSMLKRPVL 14 38
LKRPVLLHL 14 94 VIHPLATSH 14 117 VLPMVSITL 14 138 IVQLHNGTK 14 139
VQLHNGTKY 14 146 KYKKFPHWL 14 153 WLDKWMLTR 14 156 KWMLTRKQF 14 176
AIYSLSYPM 14 177 IYSLSYPMR 14 240 SVSDSLTWR 14 260 VSLLLGTIH 14 275
NKWIDIKQF 14 278 IDIKQFVWY 14 300 VVLIFKSIL 14 320 IRHGWEDVT 14 324
WEDVTKINK 14 331 NKTEICSQL 14 3 SRKDITNQE 13 11 EELWKMKPR 13 29
HKDTGETSM 13 31 DTGETSMLK 13 43 LLHLHQTAH 13 73 LPIKIAAII 13 83
SLTFLYTLL 13 112 LVINKVLPM 13 122 SITLLALVY 13 130 YLPGVIAAI 13 142
HNGTKYKKF 13 144 GTKYKKFPH 13 151 PHWLDKWML 13 158 MLTRKQFGL 13 160
TRKQFGLLS 13 163 QFGLLSFFF 13 167 LSFFFAVLH 13 230 LALLAVTSI 13 237
SIPSVSDSL 13 251 HYIQSKLGI 13 284 VWYTPPTFM 13 296 FLPIVVLIF 13 306
SILFLPCLR 13 9 NQEELWKMK 12 47 HQTAHADEF 12 55 FDCPSELQH 12 60
ELQHTQELF 12 68 FPQWHLPIK 12 88 YTLLREVIH 12 91 LREVIHPLA 12 98
LATSHQQYF 12 99 ATSHQQYFY 12 100 TSHQQYFYK 12 106 FYKIPILVI 12
120 MVSITLLAL 12 140 QLHNGTKYK 12 159 LTRKQFGLL 12 166 LLSFFFAVL 12
180 LSYPMRRSY 12 182 YPMRRSYRY 12 208 WIEHDVWRM 12 242 SDSLTWREF 12
247 WREFHYIQS 12 264 LGTIHALIF 12 283 FVWYTPPTF 12 295 VFLPIVVLI 12
310 LPCLRKKIL 12 322 HGWEDVTKI 12 18 PRRNLEEDD 11 21 NLEEDDYLH 11
22 LEEDDYLHK 11 37 MLKRPVLLH 11 72 HLPIKIAAI 11 101 SHQQYFYKI 11
109 IPILVINKV 11 115 NKVLPMVSI 11 169 FFFAVLHAI 11 174 LHAIYSLSY 11
181 SYPMRRSYR 11 183 PMRRSYRYK 11 201 QQNKEDAWI 11 216 MEIYVSLGI 11
259 IVSLLLGTI 11 299 IVVLIFKSI 11
[0902] TABLE-US-00105 TABLE XXXI -V2-HLA-B2705- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 2 WREFHYIQV 12 5 FHYIQVNNI 12 3 REFHYIQVN 11 4
EFHYIQVNN 6
[0903] TABLE-US-00106 TABLE XXXI -V3-HLA-B2705- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 26 TRFKGLNLI 25 5 HYIQIIHKK 20 1 WREEHYIQI 18 2
REFHYIQII 16 4 FHYIQIIHK 16 20 LWDPCLTRF 16 12 KKSDVPESL 14 25
LTRFKGLNL 14 17 PESLWDPCL 13 19 SLWDPCLTR 13 3 EFHYIQIIH 12 23
PCLTRFKGL 12
[0904] TABLE-US-00107 TABLE XXXI -V4-HLA-B2705- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 KQFGLLSLF 20 1 RKQFGLLSL 19 3 QFGLLSLFF 14 6
LLSLFFAVL 13 7 LSLFFAVLH 12 9 LFFAVLHAI 11
[0905] TABLE-US-00108 TABLE XXXII -V1-HLA-B2709- 9mers:STEAP-1 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 185 RRSYRYKLL 24 184 MRRSYRYKL 22 214 WRMEIYVSL 22
20 RNLEEDDYL 16 133 GVIAAIVQL 16 289 PTFMIAVFL 15 19 RRNLEEDDY 14
82 ASLTFLYTL 14 123 ITLLALVYL 14 165 GLLSFFFAV 14 172 AVLHAIYSL 14
255 SKLGIVSLL 14 294 AVFLPIVVL 14 295 VFLPIVVLI 14 305 KSILFLPDL 14
5 KDITNQEEL 13 30 KDTGETSML 13 36 SMLKRPVLL 13 76 KIAAIIASL 13 104
QYFYKIPIL 13 110 PILVINKVL 13 161 RKQFGLLSF 13 176 AIYSLSYPM 13 225
VGLAILALL 13 256 KLGIVSLLL 13 302 LIFKSILFL 13 331 NKTEICSQL 13 3
SRKDITNQE 12 38 LKRPVLLHL 12 39 KRPVLLHLH 12 53 DEFDCPSEL 12 59
SELQHTQEL 12 65 QELFPQWHL 12 79 AIIASLTFL 12 91 LREVIHPLA 12 146
KYKKFPHWL 12 162 KQFGLLSFF 12 188 YRYKLLNWA 12 224 IVGLAILAL 12 262
LLLGTIHAL 12 276 KWIDIKQFV 12 300 WLIFKSILI 2 14 WKMKPRRNL 11 35
TSMLKRPVL 11 73 LPIKIAAII 11 78 AAIIASLTF 11 83 SLTFLYTLL 11 105
YFYKIPILV 11 112 LVINKVLPM 11 120 MVSITLLAL 11 126 LALVYLPGV 11 151
PHWLDKWML 11 156 KWMLTRKQF 11 158 MLTRKQFGL 11 160 TRKQFGLLS 11 208
WIEHDVWRM 11 219 YVSLGIVGL 11 222 LGIVGLAIL 11 230 LALLAVTSI 11 237
SIPSVSDSL 11 247 WREFHYIQS 11 249 EFHYIQSKL 11 254 QSKLGIVSL 11 264
LGTIHALIF 11 299 IVVLIFKSI 11 313 LRKKILKIR 11 320 IRHGWEDVT 11
[0906] TABLE-US-00109 TABLE XXXII -V2-HLA-B2709- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 2 WREFHYIQV 19 5 FHYIQVNNI 12
[0907] TABLE-US-00110 TABLE XXXII -V3-B2709-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 26 TRFKGLNLI 20 1 WREFHYIQI 19 2 REFHYIQII 14 12
KKSDVPESL 12 23 PCLTRFKGL 12 25 LTRFKGLNL 12 17 PESLWDPCL 11 20
LWDPCLTRF 10 8 QIIHKKSDV 9
[0908] TABLE-US-00111 TABLE XXXII -V4-HLA-B2709- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 RKQFGLLSL 15 5 GLLSLFFAV 14 2 KQFGLLSLF 12 6
LLSLFFAVL 10 9 LFFAVLHAI 10 3 QFGLLSLFF 9
[0909] TABLE-US-00112 TABLE XXXII -V1-HLA-B4402- 9mers:STEAP-1 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 53 DEFDCPSEL 24 59 SELQHTQEL 23 216 MEIYVSLGI 22 65
QELFPQWHL 21 294 AVFLPIVVL 21 78 AAIIASLTF 18 133 GVIAAIVQL 18 262
LLLGTIHAL 18 269 ALIFAWNKW 18 23 EEDDYLHKD 17 79 AIIASLTFL 17 82
ASLTFLYTL 17 156 KWMLTRKQF 17 172 AVLHAIYSL 17 224 IVGLAILAL 17 275
NKWIDIKQF 17 5 KDITNQEEL 16 36 SMLKRPVLL 16 219 YVSLGIVGL 16 255
SKLGIVSLL 16 295 VFLPIVVLI 16 302 LIFKSILFL 16 305 KSILFLPCL 16 11
EELWKMKPR 15 38 LKRPVLLHL 15 76 KIAAIIASL 15 104 QYFYKIPIL 15 110
PILVINKVL 15 118 LPMVSITLL 15 120 MVSITLLAL 15 122 SITLLALVY 15 130
YLPGVIAAI 15 162 KQFGLLSFF 15 180 LSYPMRRSY 15 185 RRSYRYKLL 15 204
KEDAWIEHD 15 248 REFHYIQSK 15 254 QSKLGIVSL 15 266 TIHALIFAW 15 278
IDIKQFVWY 15 289 PTFMIAVFL 15 301 VLIFKSILF 15 316 KILKIRHGW 15 1
MESRKDITN 14 35 TSMLKRPVL 14 72 HLPIKIAAI 14 90 LLREVIHPL 14 92
REVIHPLAT 14 99 ATSHQQYFY 14 123 ITLLALVYL 14 142 HNGTKYKKF 14 149
KFPHWLDKW 14 169 FFFAVLHAI 14 222 LGIVGLAIL 14 225 VGLAILALL 14 242
SDSLTWREF 14 249 EFHYIQSKL 14 288 PPTFMIAVF 14 310 LPCLRKKIL 14 14
WKMKPRRNL 13 30 KDTGETSML 13 33 GETSMLKRP 13 60 ELQHTQELF 13 73
LPIKIAAII 13 106 FYKIPILVI 13 117 VLPMVSITL 13 127 ALVYLPGVI 13 139
VQLHNGTKY 13 146 KYKKFPHWL 13 166 LLSFFFAVL 13 182 YPMRRSYRY 13 187
SYRYKLLNW 13 189 RYKLLNWAY 13 200 VQQNKEDAW 13 214 WRMEIYVSL 13 256
KLGIVSLLL 13 291 FMIAVFLPI 13 296 FLPIVVLIF 13 299 IVVLIFKSI 13 309
FLPCLRKKI 13 312 CLRKKILKI 13 331 NKTEICSQL 13 6 DITNQEELW 12 10
QEELWKMKP 12 22 LEEDDYLHK 12 63 HTQELFPQW 12 80 IIASLTFLY 12 83
SLTFLYTLL 12 97 PLATSHQQY 12 159 LTRKQFGLL 12 161 RKQFGLLSF 12 184
MRRSYRYKL 12 209 IEHDVWRME 12 210 EHDVWRMEI 12 221 SLGIVGLAI 12 237
SIPSVSDSL 12 239 PSVSDSLTW 12 244 SLTWREFHY 12 251 HYIQSKLGI 12 259
IVSLLLGTI 12 264 LGTIHALIF 12 270 LIFAWNKWI 12 277 WIDIKQFVW 12 283
FVWYTPPTF 12 285 WYTPPTFMI 12 324 WEDVTKINK 12 19 RRNLEEDDY 11 20
RNLEEDDYL 11 47 HQTAHADEF 11 69 PQWHLPIKI 11 87 LYTLLREVI 11 101
SHQQYFYKI 11 115 NKVLPMVSI 11 145 TKYKKFPHW 11 151 PHWLDKWML 11 158
MLTRKQFGL 11 163 QFGLLSFFF 11 174 LHAIYSLSY 11 206 DAWIEHDVW 11
211 HDVWRMEIY 11 230 LALLAVTSI 11 245 LTWREFHYI 11 263 LLGTIHALI 11
300 VVLIFKSIL 11
[0910] TABLE-US-00113 TABLE XXXIII -V2-HLA-B4402- 9mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. Pos
123456789 score 3 REFHYIQVN 16 5 FHYIQVNNI 10
[0911] TABLE-US-00114 TABLE XXXIII -V3-B4402- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 REEHYIQII 23 17 PESLWDPCL 22 23 PCLTRFKGL 16 20
LWDPCLTRF 15 12 KKSDVPESL 14 13 KSDVPESLW 13 26 TRFKGLNLI 13 25
LTRFKGLNL 11
[0912] TABLE-US-00115 TABLE XXXIII -V4-HLA-B4402- 9mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 9; 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 KQFGLLSLF 16 6 LLSLFFAVL 14 9 LFFAVLHAI 14 1
RKQFGLLSL 12 3 QFGLLSLFF 12
[0913] TABLE-US-00116 TABLE XXXIV -V1-B5101- 9mers:STEAP-1 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 230 LALLAVTSI 27 109 IPILVINKV 26 293 IAVFLPIVV 25
73 LPIKIAAII 24 126 LALVYLPGV 24 272 FAWNKWIDI 24 322 HGWEDVTKI 24
233 LAVTSIPSV 23 227 LAILALLAV 22 287 TPPTFMIAV 22 118 LPMVSITLL 21
131 LPGVIAAIV 21 310 LPCLRKKIL 19 206 DAWIEHDVW 18 105 YFYKIPILV 17
130 YLPGVIAAI 17 225 VGLAILALL 17 86 FLYTLLREV 16 103 QQYFYKIPI 16
106 FYKIPILVI 16 135 IAAIVQLHN 16 169 FFFAVLHAI 16 195 WAYQQVQQN 16
270 LIFAWNKWI 16 291 FMIAVFLPI 16 295 VFLPIVVLI 16 297 LPIVVLIFK 16
312 CLRKKILKI 16 69 PQWHLPIKI 15 81 IASLTFLYT 15 87 LYTLLREVI 15
212 DVWRMEIYV 15 222 LGIVGLAIL 15 259 IVSLLLGTI 15 288 PPTFMIAVF 15
309 FLPCLRKKI 15 57 CPSELQHTQ 14 67 LFPQWHLPI 14 96 HPLATSHQQ 14
101 SHQQYFYKI 14 127 ALVYLPGVI 14 216 MEIYVSLGI 14 217 EIYVSLGIV 14
245 LTWREFHYI 14 299 IVVLIFKSI 14 38 LKRPVLLHL 13 53 DEFDCPSEL 13
72 HLPIKIAAI 13 77 IAAIIASLT 13 78 AAIIASLTF 13 90 LLREVIHPL 13 110
PILVINKVL 13 113 VINKVLPMV 13 117 VLPMVSITL 13 171 FAVLHAIYS 13 182
YPMRRSYRY 13 221 SLGIVGLAI 13 238 IPSVSDSLT 13 262 LLLGTIHAL 13 263
LLGTIHALI 13 268 HALIFAWNK 13 302 LIFKSILFL 13
[0914] TABLE-US-00117 TABLE XXXIV -V2-HLA-B5101- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start posifion plus eight. Pos
123456789 score 5 FHYIQVNNI 18 2 WREFHYIQV 9
[0915] TABLE-US-00118 TABLE XXXIV -V3-HLA-B5101- 9mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 22 DPCLTRFKG 17 26 TRFKGLNLI 15 2 REFHYIQII 13 1
WREFHYIQI 11 16 VPESLWDPC 11 23 PCLTRFKGL 11 12 KKSDVPESL 10 25
LTRFKGLNL 10 8 QIIHKKSDV 9 15 DVPESLWDP 9 4 FHYIQIIHK 8
[0916] TABLE-US-00119 TABLE XXXIV -V4-B5101-9mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LFFAVLHAI 17 6 LLSLFFAVL 12 4 FGLLSLFFA 11 5
GLLSLFFAV 11 1 RKQFGLLSL 8
[0917] TABLE-US-00120 TABLE XXXV -V1-HLA-A1- 10mers:STEAP-1 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 277 WIDIKQFVWY 28 121 VSITLLALVY 27 210 EHDVWRMEIY
25 173 VLHAIYSLSY 23 179 SLSYPMRRSY 19 243 DSLTWREFHY 19 79
AIIASLTFLY 18 138 IVQLHNGTKY 18 188 YRYKLLNWAY 18 21 NLEEDDYLHK 17
181 SYPMRRSYRY 17 54 EFDCPSELQH 16 91 LREVIHPLAT 16 169 FFFAVLHAIY
16 215 RMEIYVSLGI 16 286 YTPPTFMIAV 16 18 PRRNLEEDDY 15 96
HPLATSHQQY 15 98 LATSHQQYFY 15 159 LTRKQFGLLS 15 241 VSDSLTWREF 15
31 DTGETSMLKR 14 58 PSELQHTQEL 14 323 GWEDVTKINK 14 88 YTLLREVIHP
13 220 VSLGIVGLAI 13
[0918] TABLE-US-00121 TABLE XXXV -V2-HLA-A1- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 2 WREFHYIQVN 11
[0919] TABLE-US-00122 TABLE XXXV -V3-HLA-A1- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 14 KSDVPESLWD 20 21 LWDPCLTRFK 14 17 VPESLWDPCL 11
2 WREFHYIQII 10 19 ESLWDPGLTR 10
[0920] TABLE-US-00123 TABLE XXXV -V4-HLA-A1- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LFFAVLHAIY 15 8 LSLFFAVLHA 11 3 KQFGLLSLFF
7
[0921] TABLE-US-00124 TABLE XXXVI -V1-A0201- 10mers:STEAP-1 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 232 LLAVTSIPSV 31 125 LLALVYLPGV 30 261 SLLLGTIHAL
30 226 GLAILALLAV 29 221 SLGIVGLAIL 28 229 ILALLAVTSI 28 89
TLLREVIHPL 27 223 GIVGLAILAL 27 301 VLIFKSILFL 27 108 KIPILVINKV 26
130 YLPGVIAAIV 26 262 LLLGTIHALI 26 291 FMIAVFLPIV 26 117
VLPMVSITLL 25 122 SITLLALVYL 25 165 GLLSFFFAVL 25 37 MLKRPVLLHL 24
81 IASLTFLYTL 23 112 LVINKVLPMV 23 191 KLLNWAYQQV 23 258 GIVSLLLGTI
23 75 IKIAAIIASL 22 78 AAIIASLTFL 22 171 FAVLHAIYSL 22 224
IVGLAILALL 22 111 ILVINKVLPM 21 116 KVLPMVSITL 21 119 PMVSITLLAL 21
157 WMLTRKQFGL 21 293 IAVFLPIVVL 21 294 AVFLPIVVLI 21 72 HLPIKIAAII
20 86 FLYTLLREVI 20 120 MVSITLLALV 20 129 VYLPGVIAAI 20 244
SLTWREFHYI 20 253 IQSKLGIVSL 20 269 ALIFAWNKWI 20 292 MIAVFLPIVV 20
309 FLPCLRKKIL 20 66 ELFPQWHLPI 19 71 WHLPIKIAAI 19 90 LLREVIHPLA
19 158 MLTRKQFGLL 19 218 IYVSLGIVGL 19 286 YTPPTFMIAV 19 307
ILFLPCLRKK 19 43 LLHLHQTAHA 18 183 PMRRSYRYKL 18 227 LAILALLAVT 18
7 ITNQEELWKM 17 36 SMLKRPVLLH 17 80 IIASLTFLYT 17 85 TFLYTLLREV 17
127 ALVYLPGVIA 17 215 RMEIYVSLGI 17 255 SKLGIVSLLL 17 105
YFYKIPILVI 16 126 LALVYLPGVI 16 128 LVYLPGVIAA 16 168 SFFFAVLHAI 16
228 AILALLAVTS 16 236 TSIPSVSDSL 16 251 HYIQSKLGIV 16 256
KLGIVSLLLG 16 299 IVVLIFKSIL 16 304 FKSILFLPCL 16 329 KINKTEICSQ 16
76 KIAAIIASLT 15 103 QQYFYKIPIL 15 213 VWRMEIYVSL 15 216 MEIYVSLGIV
15 220 VSLGIVGLAI 15 296 FLPIVVLIFK 15 302 LIFKSILFLP 15 308
LFLPCLRKKI 15
[0922] TABLE-US-00125 TABLE XXXVI -V2-HLA-A0201- 10mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 1 TWREFHYIQV 12 4 EFHYIQVNNI 9
[0923] TABLE-US-00126 TABLE XXVI -V3-HLA-A0201- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the +UZ,5/28 start position plus nine.
Pos 1234567890 score 25 CLTRFKGLNL 20 20 SLWDPCLTRF 18 26
LTRFKGLNLI 18 8 IQIIHKKSDV 14 10 IIHKKSDVPE 12 7 YIQIIHKKSD 11 12
HKKSDVPESL 11 17 VPESLWDPCL 11 1 TWREFHYIQI 10 2 WREFHYIQII 10 9
QIIHKKSDVP 10 5 FHYIQIIHKK 9 23 DPCLTRFKGL 9
[0924] TABLE-US-00127 TABLE XXXVI -V4-HLA-A0201- 10mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 9, 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 SLFFAVLHAI 26 6 GLLSLFFAVL 25 1 TRKQFGLLSL 16 5
FGLLSLFFAV 14 7 LLSLFFAVLH 14 8 LSLFFAVLHA 12
[0925] TABLE-US-00128 TABLE XXXVII -V1-HLA-A0202- 10mers:STEAP-1
Pos 1234567890 score NoResultsfound.
[0926] TABLE-US-00129 TABLE XXXVII -V2-HLA-A0202- 10mers:STEAP-1
Pos 1234567890 score NoResultsfound.
[0927] TABLE-US-00130 TABLE XXXVII -V3-HLA-A0202- 10mers:STEAP-1
Pos 1234567890 score NoResultsfound.
[0928] TABLE-US-00131 TABLE XXXVII -V4-HLA-A0202- 10mers:STEAP-1
Pos 1234567890 score NoResultsfound.
[0929] TABLE-US-00132 TABLE XXXVIII -V1-HLA-A0203- 10mers:STEAP-1
Each peptide isa portion of SEQ ID NO: 9, 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 70 QWHLPIKIAA 19 128 LVYLPGVIAA 19 43 LLHLHQTAHA
18 71 WHLPIKIAAI 17 129 VYLPGVIAAI 17 41 PVLLHLHQTA 10 69
PQWHLPIKIA 10 73 LPIKIAAIIA 10 90 LLREVIHPLA 10 118 LPMVSITLLA 10
127 ALVYLPGVIA 10 163 QFGLLSFFFA 10 167 LSFFFAVLHA 10 187
SYRYKLLNWA 10 198 QQVQQNKEDA 10 219 YVSLGIVGLA 10 222 LGIVGLAILA 10
225 VGLAILALLA 10 260 VSLLLGTIHA 10 264 LGTIHALIFA 10 285
WYTPPTFMIA 10 42 VLLHLHQTAH 9 44 LHLHQTAHAD 9 74 PIKIAAIIAS 9 91
LREVIHPLAT 9 119 PMVSITLLAL 9 164 FGLLSFFFAV 9 168 SFFFAVLHAI 9 188
YRYKLLNWAY 9 199 QVQQNKEDAW 9 220 VSLGIVGLAI 9 223 GIVGLAILAL 9 226
GLAILALLAV 9 261 SLLLGTIHAL 9 265 GTIHALIFAW 9 286 YTPPTFMIAV 9
[0930] TABLE-US-00133 TABLE XXXVIII -V2-HLA-A0203- 10mers: STEAP-1
Pos 123456789 score NoResultsfound.
[0931] TABLE-US-00134 TABLE XXXVIII -V3-HLA-A0203- 10mers: STEAP-1
Pos 123456789 score NoResultsfound.
[0932] TABLE-US-00135 TABLE XXXVIII -V4-HLA-A0203- 10mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 9; 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 QFGLLSLFFA 10 8 LSLFFAVLHA 10 5 FGLLSLFFAV 9 9
SLFFAVLHAI 9 6 GLLSLFFAVL 8 10 LFFAVLHAIY 8
[0933] TABLE-US-00136 TABLE XXXVIII -V1-HLA-A0203- 10mers:STEAP-1
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 137 AIVQLHNGTK 26 228 AILALLAVTS 26 21 NLEEDDYLHK
24 306 SILFLPCLRK 23 6 DITNQEELWK 23 93 EVIHPLATSH 23 138
IVQLHNGTKY 23 140 QLHNGTKYKK 23 173 VLHAIYSLSY 23 307 ILFLPCLRKK 23
79 AIIASLTFLY 22 116 KVLPMVSITL 22 320 IRHGWEDVTK 22 153 WLDKWMLTRK
21 166 LLSFFFAVLH 21 259 IVSLLLGTIH 21 296 FLPIVVLIFK 21 76
KIAAIIASLT 20 127 ALVYLPGVIA 20 176 AIYSLSYPMR 20 179 SLSYPMRRSY 20
263 LLGTIHALIF 20 277 WIDIKQFVWY 20 86 FLYTLLREVI 19 121 VSITLLALVY
19 133 GVIAAIVQLH 19 165 GLLSFFFAVL 19 191 KLLNWAYQQV 19 217
EIYVSLGIVG 19 234 AVTSIPSVSD 19 42 VLLHLHQTAH 18 83 SLTFLYTLLR 18
130 YLPGVIAAIV 18 172 AVLHAIYSLS 18 294 AVFLPIVVLI 18 319
KIRHGWEDVT 18 27 YLHKDTGETS 17 37 MLKRPVLLHL 17 41 PVLLHLHQTA 17 72
HLPIKIAAII 17 124 TLLALVYLPG 17 212 DVWRMEIYVS 17 229 ILALLAVTSI 17
231 ALLAVTSIPS 17 256 KLGIVSLLLG 17 262 LLLGTIHALI 17 267
IHALIFAWNK 17 269 ALIFAWNKWI 17 300 VVLIFKSILF 17 312 CLRKKILKIR 17
317 ILKIRHGWED 17 30 KDTGETSMLK 16 66 ELFPQWHLPI 16 107 YKIPILVINK
16 111 ILVINKVLPM 16 128 LVYLPGVIAA 16 224 IVGLAILALL 16 226
GLAILALLAV 16 299 IVVLIFKSIL 16 301 VLIFKSILFL 16 77 IAAIIASLTF 15
89 TLLREVIHPL 15 97 PLATSHQQYF 15 112 LVINKVLPMV 15 180 LSYPMRRSYR
15 221 SLGIVGLAIL 15 252 YIQSKLGIVS 15 261 SLLLGTIHAL 15 316
KILKIRHGWE 15 43 LLHLHQTAHA 14 80 IIASLTFLYT 14 90 LLREVIHPLA 14 94
VIHPLATSHQ 14 110 PILVINKVLP 14 113 VINKVLPMVS 14 120 MVSITLLALV 14
122 SITLLALVYL 14 134 VIAAIVQLHN 14 147 YKKFPHWLDK 14 152
HWLDKWMLTR 14 158 MLTRKQFGLL 14 186 RSYRYKLLNW 14 192 LLNWAYQQVQ 14
195 WAYQQVQQNK 14 223 GIVGLAILAL 14 266 TIHALIFAWN 14 292
MIAVFLPIVV 14 45 HLHQTAHADE 13 60 ELQHTQELFP 13 96 HPLATSHQQY 13 99
ATSHQQYFYK 13 182 YPMRRSYRYK 13 199 QVQQNKEDAW 13 232 LLAVTSIPSV 13
258 GIVSLLLGTI 13 279 DIKQFVWYTP 13 329 KINKTEICSQ 13 8 TNQEELWKMK
12 75 IKIAAIIASL 12 92 REVIHPLATS 12 108 KIPILVINKV 12 125
LLALVYLPGV 12 139 VQLHNGTKYK 12 219 YVSLGIVGLA 12 237 SIPSVSDSLT 12
240 SVSDSLTWRE 12 247 WREFHYIQSK 12 283 FVWYTPPTFM 12 295
VFLPIVVLIF 12 305 KSILFLPCLR 12
[0934] TABLE-US-00137 TABLE XXXIX -V2-HLA-A3- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 3 REFHYIQVNN 8 1 TWREFHYIQV 7 2 WREFHYIQVN 4
[0935] TABLE-US-00138 TABLE XXXIX -V3-HLA-A3- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 25 CLTRFKGLNL 19 9 QIIHKKSDVP 17 20 SLWDPCLTRF 17
5 FHYIQIIHKK 15 10 IIHKKSDVPE 14 19 ESLWDPCLTR 13 21 LWDPCLTRFK 13
7 YIQIIHKKSD 11 16 DVPESLWDPC 11 4 EFHYIQIIHK 10 28 RFKGLNLIQS 10 8
IQIIHKKSDV 9
[0936] TABLE-US-00139 TABLE XXXIX -V4-A3- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 LLSLFFAVLH 22 6 GLLSLFFAVL 19 3 KQFGLLSLFF 12 9
SLFFAVLHAI 12 2 RKQFGLLSLF 11 10 LFFAVLHAIY 11
[0937] TABLE-US-00140 TABLE XXXIX -V1-HLA-A26- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 3; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 7 ITNQEELWKM 24 31 DTGETSMLKR 23 122 SITLLALVYL 23
224 IVGLAILALL 23 37 MLKRPVLLHL 22 79 AIIASLTFLY 22 165 GLLSFFFAVL
22 295 VFLPIVVLIF 22 301 VLIFKSILFL 22 117 VLPMVSITLL 21 138
IVQLHNGTKY 21 210 EHDVWRMEIY 21 221 SLGIVGLAIL 21 261 SLLLGTIHAL 21
89 TLLREVIHPL 20 97 PLATSHQQYF 20 111 ILVINKVLPM 20 116 KVLPMVSITL
20 158 MLTRKQFGLL 20 212 DVWRMEIYVS 20 300 VVLIFKSILF 20 93
EVIHPLATSH 19 169 FFFAVLHAIY 19 173 VLHAIYSLSY 19 179 SLSYPMRRSY 19
299 IVVLIFKSIL 19 66 ELFPQWHLPI 18 112 LVINKVLPMV 18 263 LLGTIHALIF
18 279 DIKQFVWYTP 18 283 FVWYTPPTFM 18 286 YTPPTFMIAV 18 326
DVTKINKTEI 18 84 LTFLYTLLRE 17 133 GVIAAIVQLH 17 141 LHNGTKYKKF 17
144 GTKYKKFPHW 17 155 DKWMLTRKQF 17 160 TRKQFGLLSF 17 176
AIYSLSYPMR 17 207 AWIEHDVWRM 17 243 DSLTWREFHY 17 265 GTIHALIFAW 17
282 QFVWYTPPTF 17 294 AVFLPIVVLI 17 302 LIFKSILFLP 17 309
FLPCLRKKIL 17 6 DITNQEELWK 16 12 ELWKMKPRRN 16 74 PIKIAAIIAS 16 75
IKIAAIIASL 16 80 IIASLTFLYT 16 108 KIPILVINKV 16 217 EIYVSLGIVG 16
274 WNKWIDIKQF 16 287 TPPTFMIAVF 16 289 PTFMIAVFLP 16 329
KINKTEICSQ 16 21 NLEEDDYLHK 15 88 YTLLREVIHP 15 120 MVSITLLALV 15
125 LLALVYLPGV 15 128 LVYLPGVIAA 15 149 KFPHWLDKWM 15 153
WLDKWMLTRK 15 161 RKQFGLLSFF 15 172 AVLHAIYSLS 15 218 IYVSLGIVGL 15
232 LLAVTSIPSV 15 235 VTSIPSVSDS 15 253 IQSKLGIVSL 15 258
GIVSLLLGTI 15 266 TIHALIFAWN 15 298 PIVVLIFKSI 15 307 ILFLPCLRKK 15
54 EFDCPSELQH 14 60 ELQHTQELFP 14 78 AAIIASLTFL 14 94 VIHPLATSHQ 14
99 ATSHQQYFYK 14 119 PMVSITLLAL 14 123 ITLLALVYLP 14 132 PGVIAAIVQL
14 162 KQFGLLSFFF 14 191 KLLNWAYQQV 14 213 VWRMEIYVSL 14 219
YVSLGIVGLA 14 226 GLAILALLAV 14 229 ILALLAVTSI 14 254 QSKLGIVSLL 14
63 HTQELFPQWH 13 67 LFPQWHLPIK 13 103 QQYFYKIPIL 13 168 SFFFAVLHAI
13 171 FAVLHAIYSL 13 184 MRRSYRYKLL 13 234 AVTSIPSVSD 13 236
TSIPSVSDSL 13 237 SIPSVSDSLT 13 241 VSDSLTWREF 13 293 IAVFLPIVVL 13
296 FLPIVVLIFK 13 304 FKSILFLPCL 13 312 CLRKKILKIR 13 76 KIAAIIASLT
12 81 IASLTFLYTL 12 82 ASLTFLYTLL 12 96 HPLATSHQQY 12 113
VINKVLPMVS 12 121 VSITLLALVY 12 124 TLLALVYLPG 12 130 YLPGVIAAIV 12
134 VIAAIVQLHN 12 181 SYPMRRSYRY 12 188 YRYKLLNWAY 12 199
QVQQNKEDAW 12 240 SVSDSLTWRE 12 244 SLTWREFHYI 12 248 REFHYIQSKL
12
249 EFHYIQSKLG 12 256 KLGIVSLLLG 12 270 LIFAWNKWID 12 303
IFKSILFLPC 12
[0938] TABLE-US-00141 TABLE XL -V2-HLA-A26- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 4 EFHYIQVNNI 17 1 TWREFHYIQV 7
[0939] TABLE-US-00142 TABLE XL -V3-HLA-A26- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 20 SLWDPCLTRF 24 16 DVPESLWDPC 19 23 DPCLTRFKGL 19
4 EFHYIQIIHK 17 25 CLTRFKGLNL 17 26 LTRFKGLNLI 14 28 RFKGLNLIQS 14
9 QIIHKKSDVP 12 10 IIHKKSDVPE 12 7 YIQIIHKKSD 11 12 HKKSDVPESL
11
[0940] TABLE-US-00143 TABLE XL -V4-A26- 10mers:STEAP-1 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 start position plus nine. Pos 1234567890 score 6 GLLSLFFAVL
22 10 LFFAVLHAIY 19 1 TRKQFGLLSL 15 2 RKQFGLLSLF 15 9 SLFFAVLHAI 15
3 KQFGLLSLFF 14 4 QFGLLSLFFA 10
[0941] TABLE-US-00144 TABLE XLI -V1-HLA-B0702- 10mers:STEAP-1 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 288 PPTFMIAVFL 23 109 IPILVINKVL 22 118 LPMVSITLLA
20 150 FPHWLDKWML 20 287 TPPTFMIAVF 18 40 RPVLLHLHQT 17 73
LPIKIAAIIA 17 34 ETSMLKRPVL 16 68 FPQWHLPIKI 16 253 IQSKLGIVSL 16
223 GIVGLAILAL 15 238 IPSVSDSLTW 15 78 AAIIASLTFL 14 213 VWRMEIYVSL
14 255 SKLGIVSLLL 14 293 IAVFLPIVVL 14 17 KPRRNLEEDD 13 35
TSMLKRPVLL 13 57 CPSELQHTQE 13 75 IKIAAIIASL 13 81 IASLTFLYTL 13 82
ASLTFLYTLL 13 119 PMVSITLLAL 13 165 GLLSFFFAVL 13 183 PMRRSYRYKL 13
218 IYVSLGIVGL 13 224 IVGLAILALL 13 301 VLIFKSILFL 13 304
FKSILFLPCL 13 4 RKDITNQEEL 12 29 HKDTGETSML 12 37 MLKRPVLLHL 12 52
ADEFDCPSEL 12 116 KVLPMVSITL 12 122 SITLLALVYL 12 131 LPGVIAAIVQ 12
132 PGVIAAIVQL 12 184 MRRSYRYKLL 12 221 SLGIVGLAIL 12 13 LWKMKPRRNL
11 19 RRNLEEDDYL 11 58 PSELQHTQEL 11 66 ELFPQWHLPI 11 80 IIASLTFLYT
11 89 TLLREVIHPL 11 96 HPLATSHQQY 11 103 QQYFYKIPIL 11 111
ILVINKVLPM 11 117 VLPMVSITLL 11 145 TKYKKFPHWL 11 158 MLTRKQFGLL 11
182 YPMRRSYRYK 11 220 VSLGIVGLAI 11 236 TSIPSVSDSL 11 248
REFHYIQSKL 11 254 QSKLGIVSLL 11 261 SLLLGTIHAL 11 290 TFMIAVFLPI 11
294 AVELPIVVLI 11 299 IVVLIFKSIL 11 330 INKTEICSQL 11
[0942] TABLE-US-00145 TABLE XLI -V2-HLA-B0702- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 1 TWREFHYIQV 8 4 EFHYIQVNNI 8 3 REFHYIQVNN 3
[0943] TABLE-US-00146 TABLE XLI -V3-HLA-B0702- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each pepfide is the start position plus nine. Pos
1234567890 score 17 VPESLWDPCL 21 23 DPCLTRFKGL 21 25 CLTRFKGLNL 12
12 HKKSDVPESL 10
[0944] TABLE-US-00147 TABLE XLI -V4-HLA-B0702- 10mers:STEAP-1 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 GLLSLFFAVL 13 1 TRKQFGLLSL 12 3 KQFGLLSLFF 12 8
LSLFFAVLHA 9 4 QFGLLSLFFA 8 5 FGLLSLFFAV 8 2 RKQFGLLSLF 7 9
SLFFAVLHAI 7
[0945] TABLE-US-00148 TABLE XLII -V1-HLA-B08- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0946] TABLE-US-00149 TABLE XLII -V2-HLA-B08-10mers: STEAP-1 Pos
123456789 score NoResuftsfound.
[0947] TABLE-US-00150 TABLE XLII -V3-HLA-B08- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0948] TABLE-US-00151 TABLE XLII -V4-HLA-B08- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0949] TABLE-US-00152 TABLE XLIII -V1-HLA-B1510- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0950] TABLE-US-00153 TABLE XLIII -V2-HLA-B1510- 10mers:STEAP-1 Pos
123456789 score NoResultsfound.
[0951] TABLE-US-00154 TABLE XLIII -V3-HLA-B1510- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0952] TABLE-US-00155 TABLE XLIII -V4-HLA-B1510- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0953] TABLE-US-00156 TABLE XLIV -V1-HLA-B2705- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0954] TABLE-US-00157 TABLE XLIV -V2-HLA-B2705- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0955] TABLE-US-00158 TABLE XLIV -V3-HLA-B2705 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0956] TABLE-US-00159 TABLE XLIV -V4-HLA-B2705- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0957] TABLE-US-00160 TABLE XLV -V1-HLA-B2709- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0958] TABLE-US-00161 TABLE XLV -V2-HLA-B2709- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0959] TABLE-US-00162 TABLE XLV -V3-HLA-B2709- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0960] TABLE-US-00163 TABLE XLV -V4-HLA-B2709- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0961] TABLE-US-00164 TABLE XLVI -V1-HLA- B4402:STEAP-1 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 59 SELQHTQELF 23 248 REFHYIQSKL 23 209 IEHDVWRMEI
20 261 SLLLGTIHAL 19 79 AIIASLTFLY 18 148 KKFPHWLDKW 18 265
GTIHALIFAW 18 71 WHLPIKIAAI 17 78 AAIIASLTFL 17 109 IPILVINKVL 17
116 KVLPMVSITL 17 121 VSITLLALVY 17 129 VYLPGVIAAI 17 223
GIVGLAILAL 17 294 AVFLPIVVLI 17 301 VLIFKSILFL 17 315 KKILKIRHGW 17
5 KDITNQEELW 16 23 EEDDYLHKDT 16 66 ELFPQWHLPI 16 75 IKIAAIIASL 16
82 ASLTFLYTLL 16 89 TLLREVIHPL 16 236 TSIPSVSDSL 16 269 ALIFAWNKWI
16 274 WNKWIDIKQF 16 276 KWIDIKQFVW 16 287 TPPTFMIAVF 16 295
VFLPIVVLIF 16 308 LFLPCLRKKI 16 324 WEDVTKINKT 16 1 MESRKDITNQ 15
34 ETSMLKRPVL 15 35 TSMLKRPVLL 15 117 VLPMVSITLL 15 132 PGVIAAIVQL
15 168 SFFFAVLHAI 15 253 IQSKLGIVSL 15 255 SKLGIVSLLL 15 293
IAVFLPIVVL 15 311 PCLRKKILKI 15 22 LEEDDYLHKD 14 52 ADEFDCPSEL 14
53 DEFDCPSELQ 14 81 IASLTFLYTL 14 119 PMVSITLLAL 14 122 SITLLALVYL
14 141 LHNGTKYKKF 14 155 DKWMLTRKQF 14 162 KQFGLLSFFF 14 171
FAVLHAIYSL 14 179 SLSYPMRRSY 14 216 MEIYVSLGIV 14 218 IYVSLGIVGL 14
10 QEELWKMKPR 13 11 EELWKMKPRR 13 29 HKDTGETSML 13 37 MLKRPVLLHL 13
65 QELFPQWHLP 13 103 QQYFYKIPIL 13 105 YFYKIPILVI 13 158 MLTRKQFGLL
13 165 GLLSFFFAVL 13 183 PMRRSYRYKL 13 184 MRRSYRYKLL 13 186
RSYRYKLLNW 13 204 KEDAWIEHDV 13 205 EDAWIEHDVW 13 210 EHDVWRMEIY 13
220 VSLGIVGLAI 13 224 IVGLAILALL 13 238 PSVSDSLTVV 13 254
QSKLGIVSLL 13 263 LLGTIHALIF 13 268 HALIFAWNKW 13 277 WIDIKQFVWY 13
300 VVLIFKSILF 13 309 FLPCLRKKIL 13 13 LWKMKPRRNL 12 46 LHQTAHADEF
12 58 PSELQHTQEL 12 62 QHTQELFPQW 12 77 IAAIIASLTF 12 92 REVIHPLATS
12 96 HPLATSHQQY 12 138 IVQLHNGTKY 12 145 TKYKKFPHWL 12 157
WMLTRKQFGL 12 160 TRKQFGLLSF 12 169 FFFAVLHAIY 12 181 SYPMRRSYRY 12
188 YRYKLLNWAY 12 199 QVQQNKEDAW 12 221 SLGIVGLAIL 12 241
VSDSLTWREF 12 243 DSLTWREFHY 12 282 QFVWYTPPTF 12 288 PPTFMIAVFL 12
304 FKSILFLPCL 12 330 INKTEICSQL 12 4 RKDITNQEEL 11 18 PRRNLEEDDY
11 19 RRNLEEDDYL 11 33 GETSMLKRPV 11 64 TQELFPQWHL 11 68 FPQWHLPIKI
11 86 FLYTLLREVI 11 97 PLATSHQQYF 11 126 LALVYLPGVI 11 144
GTKYKKFPHW 11 161 RKQFGLLSFF 11 173 VLHAIYSLSY 11 213 VWRMEIYVSL 11
244 SLTWREFHYI 11 262 LLLGTIHALI 11 290 TFMIAVFLPI 11 298
PIVVLIFKSI 11 299 IVVLIFKSIL 11
[0962] TABLE-US-00165 TABLE XLVI -V2-HLA-B4402- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. Pos
1234567890 score 3 REFHYIQVNN 13 4 EFHYIQVNNI 12
[0963] TABLE-US-00166 TABLE XLVI -V3-HLA-B4402- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 KKSDVPESLW 14 18 PESLWDPCLT 14 23 DPCLTRFKGL 14
3 REFHYIQIIH 13 20 SLWDPCLTRF 13 25 CLTRFKGLNL 12 2 WREEHYIQII 11
12 HKKSDVPESL 11 26 LTRFKGLNLI 11 1 TWREFHYIQI 10 17 VPESLWDPCL 10
4 EFHYIQIIHK 6 6 HYIQIIHKKS 6 21 LWDPCLTRFK 6
[0964] TABLE-US-00167 TABLE XLVI -V4-B4402- 10mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 KQFGLLSLFF 15 9 SLFFAVLHAI 15 6 GLLSLFFAVL 13 1
TRKQFGLLSL 12 2 RKQFGLLSLF 12 10 LFFAVLHAIY 12 5 FGLLSLFFAV 5 7
LLSLFFAVLH 5
[0965] TABLE-US-00168 TABLE XLVI -V1-HLA-B5101- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0966] TABLE-US-00169 TABLE XLVII -V2-HLA-B5101- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0967] TABLE-US-00170 TABLE XLVII -V3-HLA-B5101- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0968] TABLE-US-00171 TABLE XLVII -V4-HLA-B5101- 10mers:STEAP-1 Pos
1234567890 score NoResultsfound.
[0969] TABLE-US-00172 TABLE XLVIII -V1-HLA-DRB10101- 15mers:STEAP-1
Each peptide is a portion of NO: 3; each start position specified,
the length of peptide amino acids, and the end position for each
peptide is the start position plus fourteen. Pos 123456789012345
score 168 SFFFAVLHAIYSLSY 34 125 LLALVYLPGVIAAIV 33 226
GLAILALLAVTSIPS 32 111 ILVINKVLPMVSITL 31 128 LVYLPGVIAAIVQLH 30
216 MEIYVSLGIVGLAIL 30 261 SLLLGTIHALIFAWN 30 106 FYKiPILVINKVLPM
28 114 INKVLPMVSITLLAL 28 229 ILALLAVTSIPSVSD 28 70 QWHLPIKIAAIIASL
27 161 RKQFGLLSFFFAVLH 27 213 VWRMEIYVSLGIVGL 27 224
IVGLAILALLAVTSI 27 25 DDYLHKDTGETSMLK 26 103 QQYFYKIPILVINKV 26 120
MVSITLLALVYLPGV 26 157 WMLTRKQFGLLSFFF 26 219 YVSLGIVGLAILALL 26
227 LAILALLAVTSIPSV 26 257 LGIVSLLLGTIHALI 26 296 FLPIVVLIFKSILFL
26 324 WEDVTKINKTEICSQ 26 123 ITLLALVYLPGVIAA 25 221
SLGIVGLAILALLAV 25 256 KLGIVSLLLGTIHAL 25 280 IKQFVVYTPPTFMIA 25
298 PIVVLIFKSILFLPC 25 307 ILFLPCLRKKILKIR 25 71 WHLPIKIAAIIASLT 24
81 IASLTFLYTLLREVI 24 84 LTFLYTLLREVIHPL 24 88 YTLLREVIHPLATSH 24
102 HQQYFYKIPILVINK 24 115 NKVLPMVSITLLALV 24 250 FHYIQSKLGIVSLLL
24 258 GIVSLLLGTIHALIF 24 282 QFVWYTPPTFMIAVF 24 286
YTPPTFMIAVFLPIV 24 293 IAVFLPIVVLIFKSI 24 297 LPIVVLIFKSILFLP 24 10
QEELWKMKPRRNLEE 23 41 PVLLHLHQTAHADEF 23 75 IKIXAIIASLTFLYT 23 117
VLPMVSITLLALVYL 23 210 EHDVWRMEIYVSLGI 23 247 WREFHYIQSKLGIVS 23
281 KQFVWYTPPTFMIAV 23 64 TQELFPQWHLPIKIA 22 74 PIKIAAIIASLTFLY 22
92 REVIHPLATSHQQYF 22 108 KIPILVINKVLPMVS 22 160 TRKQFGLLSFFFAVL 22
218 IYVSLGIVGLAILAL 22 223 GIVGLAILALLAVTS 22 289 PTFMIAVFLPIVVLI
22 290 TFMIAVFLPIVVLIF 22 11 EELWKMKPRRNLEED 21 56 DCPSELQHTQELFPQ
21 211 HDVWRMEIYVSLGIV 21 232 LLAVTSIPSVSDSLT 21 288
PPTFMIAVFLPIVVL 21 72 HLPIKIAAIIASLTF 20 85 TFLYTLLREVIHPLA 20 109
IPILVINKVLPMVSI 20 171 FAVLHAIYSLSYPMR 20 185 RRSYRYKLLNWAYQQ 20
187 SYRYKLLNWAYQQVQ 20 244 SLTWREFHYIQSKLG 20 253 IQSKLGIVSLLLGTI
20 306 SILFLPCLRKKILKI 20 315 KKILKIRHGWEDVTK 20 68 FPQWHLPIKIAAIIA
19 83 SLTFLYTLLREVIHP 19 118 LPMVSITLLALVYLP 19 136 AAIVQLHNGTKYKKF
19 147 YKKFPHWLDKWMLTR 19 166 LLSFFFAVLHAIYSL 19 181
SYPMRRSYRYKLLNW 19 248 REFHYIQSKLGIVSL 19 251 HYIQSKLGIVSLLLG 19
283 FVWYTPPTFMIAVFL 19 302 LIFKSILFLPCLRKK 19 126 LALVYLPGVIAAIVQ
18 127 ALVYLPGVIAAIVQL 18 153 WLDKWMLTRKQFGLL 18 155
DKWMLTRKQFGLLSF 18 205 EDAWIEHDVWRMEIY 18 242 SDSLTWREFHYIQSK 18
249 EFHYIQSKLGIVSLL 18 267 IHALIFAWNKWIDIK 18 269 ALIFAWNKWIDIKQF
18 291 FAAIAVFLPIWLIFK 18 300 VVLIFKSILFLPCLR 18 7 ITNQEELWKMKPRRN
17 32 TGETSMLKRPVLLHL 17 35 TSMLKRPVLLHLHQT 17 39 KRPVLLHLHQTAHAD
17 40 RPVLLHLHQTAHADE 17 67 LFPQWHLPIKIAAII 17 91 LREVIHPLATSHQQY
17 112 LVINKVLPMVSITLL 17 135 IAAIVQLHNGTKYKK 17 156
KWMLTRKQFGLLSFF 17 164 FGLLSFFFAVLHAIY 17 175 HAIYSLSYPMRRSYR 17
179 SLSYPMRRSYRYKLL 17 194 NWAYQQVQQNKEDAW 17 217 EIYVSLGIVGLAILA
17 222 LGIVGLAILALLAVT 17 239 PSVSDSLTWREFHYI 17 254
QSKLGIVSLLLGTIH 17 259 IVSLLLGTIHALIFA 17 277 WIDIKQFVWYTPPTF 17
292 MIAVFLPIVVLIFKS 17 294 AVFLPIVVLIFKSIL 17 299 IVVLIFKSILFLPCL
17 301 VLIFKSILFLPCLRK 17 304 FKSILFLPCLRKKIL 17
4 RKDITNQEELWKMKP 16 24 EDDYLHKDTGETSML 16 31 DTGETSMLKRPVLLH 16 44
LHLHQTAHADEFDCP 16 58 PSELQHTQELFPQWH 16 78 AAIIASLTFLYTLLR 16 87
LYTLLREVIHPLATS 16 95 IHPLATSHQQYFYKI 16 107 YKIPILVINKVLPMV 16 110
PILVINKVLPMVSIT 16 119 PMVSITLLALVVLPG 16 122 SITLLALVYLPGVIA 16
143 NGTKYKKFPHWLDKW 16 148 KKFPHWLDKWMLTRK 16 163 QFGLLSFFFAVLHAI
16 167 LSFFFAVLHAIYSLS 16 170 FFAVLHAIYSLSYPM 16 174
LHAIYSLSYPMRRSY 16 189 RYKLLNWAYQQVQQN 16 192 LLNWAYQQVQQNKED 16
215 RMEIYVSLGIVGLAI 16 220 VSLGIVGLAILALLA 16 230 LALLAVTSIPSVSDS
16 260 VSLLLGTIHALIFAW 16 264 LGTIHALIFAWNKWI 16 272
FAWNKWIDIKQFVWY 16 274 WNKWIDIKQFVWYTP 16 278 IDIKQFVWYTPPTFM 16
314 RKKILKIRHGWEDVT 16 321 RHGWEDVTKINKTEI 16
[0970] TABLE-US-00173 TABLE XLVIII -V2-HLA-DRB10101- 15mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; each start position is
specified, the length of peptide is 15 amino acids, and the end
posifion for each peptide is the start position plus fourteen. Pos
123456789012345 score 4 SLTWREFHYIQVNNI 20 2 SDSLTWREFHYIQVN 18
[0971] TABLE-US-00174 TABLEXLVIII -V3-HLA-DRB10101- 15mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 7; 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 9 EFHYIQIIHKKSDVP 26 28 DPCLTRFKGLNLIQS 25 12
YIQIIHKKSDVPESL 22 4 SLTWREFHYIQIIHK 20 2 SDSLTWREFHYIQII 18 10
FHYIQIIHKKSDVPE 18 23 PESLWDPCLTRFKGL 17 24 ESLWDPCLTRFKGLN 17 7
WREFHYIQIIHKKSD 16 20 SDVPESLWDPCLTRF 16 15 IIHKKSDVPESLWDP 15 26
LWDPCLTRFKGLNLI 12
[0972] TABLE-US-00175 TABLE XLVIII -V4-HLA-DRB10101- 15mers-STEAP-1
Each peptide is a portion of SEQ ID NO: 9; 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 14 SLFFAVLHAIYSLSY 34 7 RKQFGLLSLFFAVLH 27 3
WMLTRKQFGLLSLFF 26 9 QFGLLSLFFAVLHAI 24 6 TRKQFGLLSLFFAVL 22 1
DKWMLTRKQFGLLSL 18 2 KWMLTRKQFGLLSLF 17 10 FGLLSLFFAVLHAIY 17 12
LLSLFFAVLHAIYSL 17 13 LSLFFAVLHAIYSLS 16
[0973] TABLE-US-00176 TABLE XLIX -V1-HLA-DRB10301- 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 155 DKWMLTRKQFGLLSF 27 181 SYPMRRSYRYKLLNW 26 297
LPIWLIFKSILFLP 26 95 IHPLATSHQQYFYKI 25 177 IYSLSYPMRRSYRYK 24 307
ILFLPCLRKKILKIR 24 19 RRNLEEDDYLHKDTG 22 117 VLPMVSITLLALVYL 22 163
QFGLLSFFFAVLHAI 22 259 IVSLLLGTIHALIFA 22 298 PIVVLIFKSILFLPC 22 77
IAAIIASLTFLYTLL 21 114 INKVLPMVSITLLAL 21 115 NKVLPMVSITLLALV 21
120 MVSITLLALVYLPGV 21 221 SLGIVGLAILALLAV 21 222 LGIVGLAILALLAVT
21 299 IVVLIFKSILFLPCL 21 25 DDYLHKDTGETSMLK 20 35 TSMLKRPVLLHLHQT
20 87 LYTLLREVIHPLATS 20 108 KIPILVINKVLPMVS 20 136 AAIVQLHNGTKYKKF
20 156 KWMLTRKQFGLLSFF 20 206 DAWIEHDVWRMEIYV 20 219
YVSLGIVGLAILALL 20 293 IAVFLPIVVLIFKSI 20 4 RKDITNQEELWKMKP 19 13
LWKMKPRRNLEEDDY 19 40 RPVLLHLHQTAHADE 19 171 FAVLHAIYSLSYPMR 19 261
SLLLGTIHALIFAWN 19 275 NKWIDIKQFVWYTPP 19 306 SILFLPCLRKKILKI 19 11
EELWKMKPRRNLEED 18 50 AHADEFDCPSELQHT 18 167 LSFFFAVLHAIYSLS 18 197
YQQVQQNKEDAWIEH 18 57 CPSELQHTQELFPQW 17 58 PSELQHTQELFPQWH 17 84
LTFLYTLLREVIHPL 17 132 PGVIAAIVQLHNGTK 17 135 IAAIVQLHNGTKYKK 17
138 IVQLHNGTKYKKFPH 17 190 YKLLNWAYQQVQQNK 17 267 IHALIFAWNKWIDIK
17 273 AWNKWIDIKQFVWYT 17 280 IKQFVWYTPPTFMIA 17 317
ILKIRHGWEDVTKIN 17 324 WEDVTKINKTEICSQ 17 16 MKPRRNLEEDDYLHK 16 32
TGETSMLKRPVLLHL 16 52 ADEFDCPSELQHTQE 16 62 QHTQELFPQWHLPIK 16 96
HPLATSHQQYFYKIP 16 107 YKIPILVINKVLPMV 16 186 RSYRYKLLNWAYQQV 16
241 VSDSLTWREFHYIQS 16 268 HALIFAWNKWIDIKQ 16 34 ETSMLKRPVLLHLHQ 15
39 KRPVLLHLHQTAHAD 15 153 WLDKWMLTRKQFGLL 15 246 TWREFHYIQSKLGIV 15
2 ESRKDITNQEELWKM 14 148 KKFPHWLDKWMLTRK 14 226 GLAILALLAVTSIPS 14
232 LLAVTSIPSVSDSLT 14 260 VSLLLGTIHALIFAW 14 304 FKSILFLPCLRKKIL
14 81 IASLTFLYTLLREVI 13 88 YTLLREVIHPLATSH 13 109 IPILVINKVLPMVSI
13 118 LPMVSITLLALVYLP 13 122 SITLLALVYLPGVIA 13 125
LLALVYLPGVIAAIV 13 126 LALVYLPGVIAAIVQ 13 131 LPGVIAAIVQLHNGT 13
149 KFPHWLDKWMLTRKQ 13 170 FFAVLHAIYSLSYPM 13 189 RYKLLNWAYQQVQQN
13 235 VTSIPSVSDSLTWRE 13 237 SIPSVSDSLTWREFH 13 254
QSKLGIVSLLLGTIH 13 256 KLGIVSLLLGTIHAL 13 292 MIAVFLPIVVLIFKS
13
[0974] TABLE-US-00177 TABLE XLIX -V2-DRB1-0301- 15mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; 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 1 VSDSLTWREFHYIQV 16 2 SDSLTWREFHYIQVN 12
[0975] TABLE-US-00178 TABLE XLIX -V3- DRB1030115mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 28 DPCLTRFKGLNLIQS 22 15 IIHKKSDVPESLWDP 20
23 PESLWDPCLTRFKGL 18 1 VSDSLTWREFHYIQI 16 10 FHYIQIIHKKSDVPE 16 22
VPESLWDPCLTRFKG 14 2 SDSLTWREFHYIQII 12 12 YIQIIHKKSDVPESL 12 13
IQIIHKKSDVPESLW 12 19 KSDVPESLWDPCLTR 12
[0976] TABLE-US-00179 TABLE XLIX -V4-HLA-0301- 15mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 1 DKWMLTRKQFGLLSL 27 9 QFGLLSLFFAVLHAI 22 2
KWMLTRKQFGLLSLF 20 13 LSLFFAVLHAIYSLS 18 10 FGLLSLFFAVLHAIY 13
[0977] TABLE-US-00180 TABLE L -V1-HLA-DR1-0401- 15mers:STEAP-1 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 84 LTFLYTLLREVIHPL 26 88 YTLLREVIHPLATSH 26
92 REVIHPLATSHQQYF 26 132 PGVIAAIVQLHNGTK 26 135 IAAIVQLHNGTKYKK 26
190 YKLLNWAYQQVQQNK 26 229 ILALLAVTSIPSVSD 26 267 IHALIFAWNKWIDIK
26 103 QQYFYKIPILVINKV 22 144 GTKYKKFPHWLDKWM 22 161
RKQFGLLSFFFAVLH 22 166 LLSFFFAVLHAIYSL 22 167 LSFFFAVLHAIYSLS 22
168 SFFFAVLHAIYSLSY 22 187 SYRYKLLNWAYQQVQ 22 194 NWAYQQVQQNKEDAW
22 211 HDVWRMEIYVSLGIV 22 216 MEIYVSLGIVGLAIL 22 244
SLTWREFHYIQSKLG 22 247 WREFHYIQSKLGIVS 22 274 WNKWIDIKQFVWYTP 22
282 QFVWYTPPTFMIAVF 22 288 PPTFMIAVFLPIVVL 22 306 SILFLPCLRKKILKI
22 321 RHGWEDVTKINKTEI 22 19 RRNLEEDDYLHKDTG 20 25 DDYLHKDTGETSMLK
20 39 KRPVLLHLHQTAHAD 20 40 RPVLLHLHQTAHADE 20 41 PVLLHLHQTAHADEF
20 64 TQELFPQWHLPIKIA 20 72 HLPIKIAAIIASLTF 20 74 PIKIAAIIASLTFLY
20 78 AAIIASLTFLYTLLR 20 81 IASLTFLYTLLREVI 20 87 LYTLLREVIHPLATS
20 106 FYKIPILVINKVLPM 20 111 ILVINKVLPMVSITL 20 114
INKVLPMVSITLLAL 20 115 NKVLPMVSITLLALV 20 117 VLPMVSITLLALVYL 20
120 MVSITLLALVYLPGV 20 123 ITLLALVYLPGVIAA 20 125 LLALVYLPGVIAAIV
20 128 LVYLPGVIAAIVQLH 20 163 QFGLLSFFFAVLHAI 20 170
FFAVLHAIYSLSYPM 20 171 FAVLHAIYSLSYPMR 20 181 SYPMRRSYRYKLLNW 20
213 VWRMEIYVSLGIVGL 20 219 YVSLGIVGLAILALL 20 221 SLGIVGLAILALLAV
20 222 LGIVGLAILALLAVT 20 224 IVGLAILALLAVTSI 20 226
GLAILALLAVTSIPS 20 227 LAILALLAVTSIPSV 20 232 LLAVTSIPSVSDSLT 20
235 VTSIPSVSDSLTWRE 20 256 KLGIVSLLLGTIHAL 20 260 VSLLLGTIHALIFAW
20 264 LGTIHALIFAWNKWI 20 289 PTFMIAVFLPIVVLI 20 290
TFMIAVFLPIVVLIF 20 294 AVFLPIVVLIFKSIL 20 296 FLPIVVLIFKSILFL 20
297 LPIVVLIFKSILFLP 20 298 PIVVLIFKSILFLPC 20 304 FKSILFLPCLRKKIL
20 307 ILFLPCLRKKILKIR 20 314 RKKILKIRHGWEDVT 20 324
WEDVTKINKTEICSQ 20 28 LHKDTGETSMLKRPV 18 32 TGETSMLKRPVLLHL 18 37
MLKRPVLLHLHQTAH 18 54 EFDCPSELQHTQELF 18 62 QHTQELFPQWHLPIK 18 93
EVIHPLATSHQQYFY 18 107 YKIPILVINKVLPMV 18 207 AWIEHDVWRMEIYVS 18 3
DSLTWREFHYIQSKL 18 253 IQSKLGIVSLLLGTI 18 258 GIVSLLLGTIHALIF 18
320 IRHGWEDVTKINKTE 18 11 EELWKMKPRRNLEED 17 24 EDDYLHKDTGETSML 17
65 QELFPQWHLPIKIAA 16 68 FPQWHLPIKIAAIIA 16 83 SLTFLYTLLREVIHP 16
85 TFLYTLLREVIHPLA 16 104 QYFYKIPILVINKVL 16 127 ALVYLPGVIAAIVQL 16
147 YKKFPHWLDKWMLTR 16 192 LLNWAYQQVQQNKED 16 205 EDAWIEHDVWRMEIY
16 249 EFHYIQSKLGIVSLL 16 269 ALIFAWNKWIDIKQF 16 271
IFAWNKWIDIKQFVW 16 283 FVWYTPPTFMIAVFL 16 293 IAVFLPIVVLIFKSI 16
301 VLIFKSILFLPCLRK 16 155 DKWMLTRKQFGLLSF 15 4 RKDITNQEELWKMKP 14
10 QEELWKMKPRRNLEE 14 35 TSMLKRPVLLHLHQT 14 43 LLHLHQTAHADEFDC 14
77 IAAIIASLTFLYTLL 14 95 IHPLATSHQQYFYKI 14 108 KIPILVINKVLPMVS 14
109 IPILVINKVLPMVSI 14 118 LPMVSITLLALVYLP 14 122 SITLLALVYLPGVIA
14 126 LALVYLPGVIAAIVQ 14 131 LPGVIAAIVQLHNGT 14 138
IVQLHNGTKYKKFPH 14 151 PHWLDKWMLTRKQFG 14 164 FGLLSFFFAVLHAIY 14
174 LHAIYSLSYPMRRSY 14 177 IYSLSYPMRRSYRYK 14 189 RYKLLNWAYQQVQQN
14
206 DAWIEHDVWRMEIYV 14 210 EHDVWRMEIYVSLGI 14 217 EIYVSLGIVGLAILA
14 230 LALLAVTSIPSVSDS 14 238 IPSVSDSLTWREFHY 14 254
QSKLGIVSLLLGTIH 14 257 LGIVSLLLGTIHALI 14 259 IVSLLLGTIHALIFA 14
261 SLLLGTIHALIFAWN 14 277 WIDIKQFVWYTPPTF 14 281 KQFVWYTPPTFMIAV
14 292 MIAVFLPIVVLIFKS 14 305 KSILFLPCLRKKILK 14 317
ILKIRHGWEDVTKIN 14 1 MESRKDITNQEELWK 12 2 ESRKDITNQEELWKM 12 5
KDITNQEELWKMKPR 12 6 DITNQEELWKMKPRR 12 17 KPRRNLEEDDYLHKD 12 21
NLEEDDYLHKDTGET 12 27 YLHKDTGETSMLKRP 12 38 LKRPVLLHLHQTAHA 12 48
QTAHADEFDCPSELQ 12 50 AHADEFDCPSELQHT 12 51 HADEFDCPSELQHTQ 12 55
FDCPSELQHTQELFP 12 56 DCPSELQHTQELFPQ 12 67 LFPQWHLPIKIAAII 12 69
PQWHLPIKIAAIIAS 12 71 WHLPIKIAAIIASLT 12 73 LPIKIAAIIASLTFL 12 75
IKIAAIIASLTFLYT 12 76 KIAAIIASLTFLYTL 12 80 IIASLTFLYILLREV 12 89
TLLREVIHPLATSHQ 12 100 TSHQQYFYKIPILVI 12 105 YFYKIPILVINKVLP 12
112 LVINKVLPMVSITLL 12 116 KVLPMVSITLLALVY 12 129 VYLPGVIAAIVQLHN
12 130 YLPGVIMIVQLHNG 12 134 VIAAIVQLHNGTKYK 12 137 AIVQLHNGTKYKKFP
12 148 KKFPHWLDKWMLTRK 12 149 KFPHWLDKWMLTRKQ 12 152
HWLDKWMLTRKQFGL 12 153 WLDKWMLTRKQFGLL 12 160 TRKQFGLLSFFFAVL 12
165 GLLSFFFAVLHAIYS 12 173 VLHAIYSLSYPMRRS 12 186 RSYRYKLLNWAYQQV
12 191 KLLNWAYQQVQQNKE 12 193 LNWAYQQVQQNKEDA 12 203
NKEDAWIEHDVWRME 12 214 WRMEIYVSLGIVGLA 12 218 IYVSLGIVGLAILAL 12
223 GIVGLAILALLAVTS 12 225 VGLAILALLAVTSIP 12 228 AILALLAVTSIPSVS
12 234 AVTSIPSVSDSLTWR 12 246 TWREFHYIQSKLGIV 12 251
HYIQSKLGIVSLLLG 12 265 GTIHALIFAWNKWID 12 273 AWNKWIDIKQFVWYT 12
279 DIKQFVWYTPPTFMI 12 291 FMIAVFLPIVVLIFK 12 295 VFLPIVVLIFKSILF
12 302 LIFKSILFLPCLRKK 12 319 KIRHGWEDVTKINKT 12
[0978] TABLE-US-00181 TABLE L -V2-HLA-DR10401- 15mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 5; 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 SLTWREFHYIQVNNI 22 3 DSLTWREFHYIQVNN 18
[0979] TABLE-US-00182 TABLE L -V3-HLA-DR1-0401- 15mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 9 EFHYIQIIHKKSDVP 28 4 SLTWREFHYIQIIHK 22 7
WREFHYIQIIHKKSD 22 24 ESLWDPCLTRFKGLN 22 28 DPCLTRFKGLNLIQS 20 3
DSLTWREFHYIQIIH 18 10 FHYIQIIHKKSDVPE 14
[0980] TABLE-US-00183 TABLE L -V4-DR10401-15mers: STEAP-1 Each
peptide is a portion of SEQ ID NO: 9; 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 7 RKQFGLLSLFFAVLH 22 13 LSLFFAVLHAIYSLS 22 14
SLFFAVLHAIYSLSY 22 9 QFGLLSLFFAVLHAI 20 12 LLSLFFAVLHAIYSL 20 1
DKWMLTRKQFGLLSL 15 10 FGLLSLFFAVLHAIY 14 4 MLTRKQFGLLSLFFA 12 6
TRKQFGLLSLFFAVL 12 11 GLLSLFFAVLHAIYS 12
[0981] TABLE-US-00184 TABLE LI -V1-DRB1101- 15mers:STEAP-1 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 108 KIPILVINKVLPMVS 28 88 YTLLREVIHPLATSH 27
167 LSFFFAVLHAIYSLS 26 68 FPQWHLPIKIAAIIA 24 154 LDKWMLTRKQFGLLS 24
321 RHGWEDVTKINKTEI 24 168 SFFFAVLHAIYSLSY 23 247 WREFHYIQSKLGIVS
23 161 RKQFGLLSFFFAVLH 22 324 WEDVTKINKTEICSQ 22 7 ITNQEELWKMKPRRN
21 84 LTFLYTLLREVIHPL 21 297 LPIVVLIFKSILFLP 21 307 ILFLPCLRKKILKIR
21 22 LEEDDYLHKDTGETS 20 39 KRPVLLHLHQTAHAD 20 122 SITLLALVYLPGVIA
20 314 RKKILKIRHGWEDVT 20 11 EELWKMKPRRNLEED 19 85 TFLYTLLREVIHPLA
19 111 ILVINKVLPMVSITL 19 125 LLALVYLPGVIAAIV 19 144
GTKYKKFPHWLDKWM 19 210 EHDVWRMEIYVSLGI 19 254 QSKLGIVSLLLGTIH 19
283 FVWYTPPTFMIAVFL 19 293 IAVFLPIVVLIFKSI 19 306 SILFLPCLRKKILKI
19 226 GLAILALLAVTSIPS 18 229 ILALLAVTSIPSVSD 18 31 DTGETSMLKRPVLLH
17 103 QQYFYKIPILVINKV 17 194 NWAYQQVQQNKEDAW 17 274
WNKWIDIKQFVWYTP 17 185 RRSYRYKLLNWAYQQ 16 187 SYRYKLLNWAYQQVQ 16
216 MEIYVSLGIVGLAIL 16 244 SLTWREFHYIQSKLG 16 268 HALIFAWNKWIDIKQ
16 301 VLIFKSILFLPCLRK 16 311 PCLRKKILKIRHGWE 16 10 QEELWKMKPRRNLEE
15 32 TGETSMLKRPVLLHL 15 37 MLKRPVLLHLHQTAH 15 64 TQELFPQWHLPIKIA
15 81 IASLTFLYTLLREVI 15 106 FYKIPILVINKVLPM 15 138 IVQLHNGTKYKKFPH
15 177 IYSLSYPMRRSYRYK 15 181 SYPMRRSYRYKLLNW 15 203
NKEDAWIEHDVWRME 15 260 VSLLLGTIHALIFAW 15 43 LLHLHQTAHADEFDC 14 55
FDCPSELQHTQELFP 14 114 INKVLPMVSITLLAL 14 120 MVSITLLALVYLPGV 14
132 PGVIAAIVQLHNGTK 14 140 QLHNGTKYKKFPHWL 14 148 KKFPHWLDKWMLTRK
14 153 WLDKWMLTRKQFGLL 14 178 YSLSYPMRRSYRYKL 14 196
AYQQVQQNKEDAWIE 14 207 AWIEHDVWRMEIYVS 14 215 RMEIYVSLGIVGLAI 14
232 LLAVTSIPSVSDSLT 14 277 WIDIKQFVWYTPPTF 14 287 TPPTFMIAVFLPIVV
14 294 AVFLPIVVLIFKSIL 14 295 VFLPIVVLIFKSILF 14 300
VVLIFKSILFLPCLR 14 304 FKSILFLPCLRKKIL 14 67 LFPQWHLPIKIAAII 13 71
WHLPIKIAAIIASLT 13 74 PIKIAAIIASLTFLY 13 115 NKVLPMVSITLLALV 13 128
LVYLPGVIAAIVQLH 13 166 LLSFFFAVLHAIYSL 13 170 FFAVLHAIYSLSYPM 13
212 DVWRMEIYVSLGIVG 13 217 EIYVSLGIVGLAILA 13 219 YVSLGIVGLAILALL
13 223 GIVGLAILALLAVTS 13 235 VTSIPSVSDSLTWRE 13 250
FHYIQSKLGIVSLLL 13 257 LGIVSLLLGTIHALI 13 261 SLLLGTIHALIFAWN 13
289 PTFMIAVFLPIVVLI 13 296 FLPIVVLIFKSILFL 13 312 CLRKKILKIRHGWED
13
[0982] TABLE-US-00185 TABLE LI -V2-HLA-DRB1-1101- 15mers:STEAP-1
Each peptide is a portion of SEQ ID NO: 5; 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 SLTWREFHYIQVNNI 10 3 DSLTWREFHYIQVNN 9 2
SDSLTWREFHYIQVN 8
[0983] TABLE-US-00186 TABLE LI -V3-DRB11101- 15mers:STEAP-1 Each
peptide is a portion of SEQ ID NO: 7; 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 9 EFHYIQIIHKKSDVP 31 25 SLWDPCLTRFKGLNL 21 10
FHYIQIIHKKSDVPE 20 7 WREFHYIQIIHKKSD 16
[0984] TABLE-US-00187 TABLE LI -V4-DRB1101- 15mers:SIEAP-1 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 13 LSLFFAVLHAIYSLS 26 14 SLFFAVLHAIYSLSY 23 7
RKQFGLLSLFFAVLH 22 9 QFGLLSLFFAVLHAI 13
[0985] TABLE-US-00188 TABLE LII Exon compositions of 8P1D4 v.1 Exon
number Start End 1 1 34 2 35 149 3 150 662 4 663 827 5 828 1176
[0986] TABLE-US-00189 TABLE LIII Nucleotide sequence of transcript
variant 8P1D4 v.2 (SEQ ID NO: 81) ggggcccgca cctctgggca gcagcggcag
ccgagactca cggtcaaqct aaggcgaaga 60 gtgggtggct gaagccatac
tattttatag aattaatgga aagcagaaaa qacatcacaa 120 accaagaaga
actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc 180
ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa
240 cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag
gaactctttc 300 cacagtggca cttgccaatt aaaatagctg ctattatagc
atctctyact tttctttaca 360 ctcttctgag ggaagtaatt caccccttag
caacttccca tcaacaatat ttttataaaa 420 ttccaatcct ggtcatcaac
aaagtcttgc caatggtttc catcactctc ttggcattgg 480 tttacctgcc
aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga 540
agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt
600 tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg
cgatcctaca 660 gatacaagtt gctaaactgg gcatatcaac aggtccaaca
aaataaagaa gatgcctgga 720 ttgagcatga tgtttggaga atggagattt
atgtgtctct gggaattgtg ggattggcaa 780 tactggctct gttggctgtg
acatctattc catctgtgag tgactctttg acatggagag 840 aatttcacta
tattcaggta aataatatat aaaataaccc taagaggtaa atcttctttt 900
tgtgtttatg atatagaata tgttgacttt accccataaa aaataacaaa tgtttttcaa
960 cagcaaagat cttatacttg ttccaattaa taatgtgctc tcctgttgtt
ttccctattg 1020 cttctaatta ggacaagtgt ttcctagaca taaataaaag
gcattaaaat attctttgtt 1080 tttttttttt tgtttgtttg ttttttgttt
qtttgtttgt ttttttgaga tyaagtctcg 1140 ctctgttgcc catgctggag
tacagtggca cgatctcggc tcact9caac ctgcgcctcc 1200 tgggttcagq
cgattctctt gcctcagcct cctgagtagc tgggattaca ggcacccatc 1260
accatgtcca gctaattttt gtatttttag tagagacagg gttttcccat gttqgccagg
1320 ctggtctcga tctcctgacc tcaaatqatc cgcccacctc ggcctcccaa
agtgctggga 1380 tgacagttgt gagccaccac actcagcctg ctctttctaa
tatttgaaac ttgttagaca 1440 atttgctacc catctaatgt gatattttag
gaatccaata tgcatggttt attatttctt 1500 aaaaaaaata ttcttttacc
tgtcacctga atttagtaat gccttttatg ttacacaact 1560 tagcactttc
cagaaacaaa aactctctcc ttgaaataat agagttttta tctaccaaag 1620
atatgctagt gtctcatttc aaaggctgct ttttccagct tacattttat atacttactc
1680 acttgaagtt tctaaatatt cttgtaattt taaaactatc tcagatttac
tgaggtttat 1740 cttctqgtgg tagattatcc ataagaagag tgatgtqcca
gaatcactct gggatccttg 1800 tctgacaaga ttcaaaggac taaatttaat
tcagtcatga acactgccaa ttaccgttta 1860 tqggtagaca tctttggaaa
tttccacaag gtcagacatt cgcaactatc ccttctacat 1920 gtccacacgt
atactccaac actttattag gcatctgatt agtttggaaa gtatgcctcc 1980
atctgaatta gtccagtgtg gcttagagtt ggtacaacat tctcacagaa tttcctaatt
2040 ttgtagyttc agcctgataa ccactggagt tctttggtcc tcattaaata
gctttcttca 2100 cacattgctc tgcctgttac acatatgatg aacactgctt
tttaqacttc attaggaatt 2160 taggactgca tcttgacaac tgagcctatt
ctactatatg tacaatacct agcccataat 2220 aqgtatacaa tacacattty
gtaaaactaa ttttcaacca atgacatgta tttttcaact 2280 agtaacctag
aaatgtttca cttaaaatct gagaactggt tacactacaa gttaccttqg 2340
agattcatat atgaaaacgc aaacttagct atttgattgt attcactggg acttaagaat
2400 qcgcctgaat aattgtgagt tcgatttgtt ctggcaggct aatgaccatt
tccagtaaag 2460 tgaatagagg tcagaagtcg tataaaagag gtgttgtcag
aacaccgttg agattacata 2520 ggtgaacaac tatttttaag caactttatt
tgtgtagtga caaagcatcc caatgcaggc 2580 tgaaatgttt catcacatct
ctggatctct ctattttgtg cagacattga aaaaattgtt 2640 catattattt
ccatgttatc agaatatttg attttttaaa aacataggcc aagttcattc 2700
acttcattat tcatttatca aaatcagagt gaatcacatt agtcgccttc acaactgata
2760 aagatcactg aagtcaaatt gatttttgct ataatcttca atctacctat
atttaattga 2820 gaatctaaaa tgtacaaatc attgtgttga ttctgcaytg
atcctgctat aagtaagact 2880 cagtccctga ttttaygtat cctgtgaaaa
gcagaattaa gacaaataca caagagacaa 2940 agcacaaaaa ataaatatca
taaggggatg aacaaaatgg tggagaaaga gtagacaaag 3000 tttttgatca
cctgccttca aagaaaggct gtgaattttg ttcacttaga cagcttggag 3060
acaagaaatt acccaaaagt aaggtgagga ggataggcaa aaagagcaga aagatgtyaa
3120 tggacattgt tgagaaatgt gataggaaaa caatcataga taaaggattt
ccaagcaaca 3180 gagcatatcc agatgaggta gyatyggata aactcttatt
gaaccaatct tcaccaattt 3240 tgtttttctt ttgcagagca agctaggaat
tgtttccctt ctactgggca caatacacgc 3300 attgattttt gcctggaata
agtggataga tataaaacaa tttgtatggt atacacctcc 3360 aacttttatg
atagctgttt tccttccaat tgttgtcctg atatttaaaa gcatactatt 3420
cctgccatgc ttgaggaaga agatactgaa gattagacat ggttgggaag acgtcaccaa
3480 aattaacaaa actgagatat gttcccagtt gtagaattac tgtttacaca
catttttgtt 3540 caatattgat atattttatc accaacattt caagtttgta
tttgttaata aaatgattat 3600 tcaaggaaaa aaaaaaaaaa aaaaaaa 3627
[0987] TABLE-US-00190 TABLE LIV Nucleotide sequence alignment of
8P1D4v.1 (SEQ ID NO: 82) and 8P1D4 v.2 (SEQ ID NO: 83) Score = 1584
bits (824), Expect = 0.0Identities 826/827 (99%) Strand Plus/Plus
8P1D4v.1: 1
ccgagactcacggtcaagctaaggcgaagagtgggtggctgaagccatactattttatag 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 31
ccgagactcacggtcaagctaaggcgaagagtgggtggctgaagccatactattttatag 90
8P1D4v.1: 61
aattaatggaaagcagaaaagacatcacaaaccaagaagaactttggaaaatgaagccta 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 91
aattaatggaaagcagaaaagacatcacaaaccaagaagaactttggaaaatgaagccta 150
8P1D4v.1: 121
ggagaaatttagaagaagacgattatttgcataaggacacgggagagaccagcatgctaa 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 151
ggagaaatttagaagaagacgattatttgcataaggacacgggagagaccagcatgctaa 210
8P1D4v.1: 181
aaagacctgtgcttttgcatttgcaccaaacagcccatgctgatgaatttgactgccctt 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 211
aaagacctgtgcttttgcatttgcaccaaacagcccatgctgatgaatttgactgccctt 270
8P1D4v.1: 241
cagaacttcagcacacacaggaactctttccacagtggcacttgccaattaaaatagctg 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 271
cagaacttcagcacacacaggaactctttccacagtggcacttgccaattaaaatagctg 330
8P1D4v.1: 301
ctattatagcatctctgacttttctttacactcttctgagggaagtaattcaccctttag 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||
8P1D4v.2: 331
ctattatagcatctctgacttttctttacactcttctgagggaagtaattcaccccttag 390
8P1D4v.1: 361
caacttcccatcaacaatatttttataaaattccaatcctggtcatcaacaaagtcttgc 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 391
caacttcccatcaacaatatttttataaaattccaatcctggtcatcaacaaagtcttgc 450
8P1D4V.1: 421
caatggtttccatcactctcttggcattggtttacctgccaggtgtgatagcagcaattg 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 451
caatggtttccatcactctcttggcattggtttacctgccaggtgtgatagcagcaattg 510
8P1D4v.1: 481
tccaacttcataatggaaccaagtataagaagtttccacattggttggataagtggatgt 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 511
tccaacttcataatggaaccaagtataagaagtttccacattggttggataagtggatgt 570
8P1D4v.1: 541
taacaagaaagcagtttgggcttctcagtttcttttttgctgtactgcatgcaatttata 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 571
taacaagaaagcagtttgggcttctcagtttcttttttgctgtactgcatgcaatttata 630
8P1D4v.1: 601
gtctgtcttacccaatgaggcgatcctacagatacaagttgctaaactgggcatatcaac 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 631
gtctgtcttacccaatgaggcgatcctacagatacaagttgctaaactgggcatatcaac 690
8P1D4v.1: 661
aggtccaacaaaataaagaagatgcctggattgagcatgatgtttggagaatggagattt 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 691
aggtccaacaaaataaagaagatgcctggattgagcatgatgtttggagaatggagattt 750
8P1D4v.1: 721
atgtgtctctgggaattgtgggattggcaatactggctctgttggctgtgacatctattc 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.2: 751
atgtgtctctgggaattgtgggattggcaatactggctctgttggctgtgacatctattc 810
8P1D4v.1: 781 catctgtgagtgactctttgacatggagagaatttcactatattcag 827
||||||||||||||||||||||||||||||||||||||||||||||| 8P1D4v.2: 811
catctgtgagtgactctttgacatggagagaatttcactatattcag 857 Score = 714
bits (371), Expect = 0.0Identities = 371/371 (100%) Strand =
Plus/Plus Query: 825
cagagcaagctaggaattgtttcccttctactgggcacaatacacgcattgatttttgcc 884
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3254 cagagcaagctaggaattgtttcccttctactgggcacaatacacgcattgatttttgcc
3313 Query: 885
tggaataagtggatagatataaaacaatttgtatggtatacacctccaacttttatgata 944
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3314 tggaataagtggatagatataaaacaatttgtatggtatacacctccaacttttatgata
3373 Query: 945
gctgttttccttccaattgttgtcctgatatttaaaagcatactattcctgccatgcttg 1004
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3374 gctgttttccttccaattgttgtcctgatatttaaaagcatactattcctgccatgcttg
3433 Query: 1005
aggaagaagatactgaagattagacatggttgggaagacgtcacaaaattaacaaaact 1064
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3434 aggaagaagatactgaagattagacatggttgggaagacgtcaccaaaattaacaaaact
3493 Query: 1065
gagatatgttcccagttgtagaattactgtttacacacatttttgttcaatattgatata 1124
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3494 gagatatgttcccagttgtagaattactgtttacacacatttttgttcaatattgatata
3553 Query: 1125
ttttatcaccaacatttcaagtttgtatttgttaataaaatgattattcaaggaaaaaaa 1184
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:
3554 ttttatcaccaacatttcaagtttgtatttgttaataaaatgattattcaaggaaaaaaa
3613 Query: 1185 aaaaaaaaaaa 1195 ||||||||||| Sbjct: 3614
aaaaaaaaaaa 3624
[0988] TABLE-US-00191 TABLE LV Peptide sequences of protein coded
by 8P1D4 v.2 (SEQ ID NO: 84) MESRKDITNQ EELWKMKPRR NLEEDDYLHK
DTGETSMLKR PVLLHLHQTA HADEFDCPSE 60 LQHTQELFPQ WHLPIKIAAI
IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM 120 VSITLLALVY
LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF FAVLHAIYSL 180
SYPMRRSYRY KLLNWAYQQV QQNKEDAWIE HDVWRMEIYV SLGIVGLAIL ALLAVTSIPS
240 VSDSLTWREF HYIQVNNI 258
[0989] TABLE-US-00192 TABLE LVI Amino acid sequence alignment of
8P1D4v.1 (SEQ ID NO: 85) and 8P1D4v.2 (SEQ ID NO: 86) Score = 521
bits (1342) , Expect = e-147Identities = 254/254 (100%), Positives
= 254/254 (100%) 8P1D4v.1: 1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE 60
MESRKDITNQEELWKNKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
8P1D4v.2: 1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE 60
8P1D4v.1: 61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM 120
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
8P1D4v.2: 61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM 120
8P1D4v.1: 121
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL 180
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
8P1D4v.2: 121
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL 180
8P1D4v.1: 181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS 240
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
8P1D4v.2: 181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS 240
8P1D4v.1: 241 VSDSLTWREFHYIQ 254 VSDSLTWREFHYIQ 8P1D4v.2: 241
VSDSLTWREFHYTQ 254
[0990] TABLE-US-00193 TABLE LVII Nucleotide sequence of transcript
variant 8P1D4v.3 (SEQ ID NO: 87) ggggcccgca cctctgggca gcagcggcag
ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac
tattttatag aattaatgga aagcagaaaa gacatcacaa 120 accaagaaga
actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc 180
ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa
240 cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag
gaactctttc 300 cacagtggca cttgccaatt aaaatagctg ctattatagc
atctctgact tttctttaca 360 ctcttctgag ggaagtaatt caccctttag
caacttccca tcaacaatat ttttataaaa 420 ttccaatcct ggtcatcaac
aaagtcttgc caatggtttc catcactctc ttggcattgg 480 tttacctgcc
aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga 540
agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcaytt
600 tcttttttgC tgtactgcat gcaatttata gtctgtctta cccaatgagg
cgatcctaca 660 gatacaagtt gctaaactgg gcatatcaac aggtccaaca
aaataaagaa gatgcctgga 720 ttgagcatga tgtttggaga atggagattt
atgtgtctct gggaattgtg ggattggcaa 780 tactggctct gttggctgtg
acatctattc catctgtgag tgactctttg acatggagag 840 aatttcacta
tattcagatt atccataaga agagtgatgt gccagaatca ctctgggatc 900
cttgtctgac aagattcaaa ggactaaatt taattcagtc atgaacactg ccaattaccg
960 tttatgggta gacatctttg gaaatttcca caagagcaag ctaggaattg
tttcccttct 1020 actgggcaca atacacgcat tgatttttgc ctggaataag
tggatagata taaaacaatt 1080 tgtatggtat acacctccaa cttttatgat
agctgttttc cttccaattg ttgtcctgat 1140 atttaaaagc atactattcc
tgccatgctt gaggaagaag atactgaaga ttagacatgg 1200 ttgggaagac
gtcaccaaaa ttaacaaaac tgagatatgt tcccagttgt agaattactg 1260
tttacacaca tttttgttca atattgatat attttatcac caacatttca agtttgtatt
1320 tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 1365
[0991] TABLE-US-00194 TABLE LVIII Nucleotide sequence alignment of
8P1D4v.2 (SEQ ID NO: 88) and 8P1D4 v.3 (SEQ ID NO: 89) Score = 1642
bits (854), Expect 0.0Identities = 856/857 (99%) Strand Plus/Plus
8P1D4v.2: 1
ggggcccgcacctctgggcagcagcggcagccgagactcagggtcaagctaaggcgaaga 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1
ggggcccgcacctctgggcagcagcggcagccgagactcacggtcaagctgaggcgaaga 60
8P1D4v.2: 61
gtgggtggctgaagccatactattttatagaattaatggaaagcagaaaagacatcacaa 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 61
gtgggtggctgaagccatactattttatagaattaatggaaagcagaaaagacatcacaa 120
8P1D4V.2: 121
accaagaagaactttggaaaatgaagcctaggagaaatttagaagaagacgattatttgc 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 121
accaagaagaactttggaaaatgaagcctaggagaaatttagaagaagacgattatttgc 180
8P1D4v.2: 181
ataaggacacgggagagaccagcatgctaaaaagacctgtgcttttgcatttgcaccaaa 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 181
ataaggacacgggagagaccagcatgctaaaaagacctgtgcttttgcatttgcaccaaa 240
8P1D4v.2: 241
cagcccatgctgatgaatttgactgcccttcagaacttcagcacacacaggaactctttc 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 241
cagcccatgctgatgaatttgactgcccttcagaacttcagcacacacaggaactctttc 300
8P1D4v.2: 301
cacagtggcacttgccaattaaaatagctgctattatagcatctctgacttttctttaca 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 301
cacagtggcacttgccaattaaaatagctgctattatagcatctctgacttttctttaca 360
8P1D4v.2: 361
ctcttctgagggaagtaattcaccccttagcaacttcccatcaacaatatttttataaaa 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 361
ctcttctgagggaagtaattcaccccttagcaacttcccatcaacaatatttttataaaa 420
8P1D4v.2: 421
ttccaatcctggtcatcaacaaagtcttgccaatggtttccatcactctcttggcattgg 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4V.3: 421
ttccaatcctggtcatcaacaaagtcttgccaatggtttccatcactctcttggcattgg 480
8P1D4v.2: 481
tttacctgccaggtgtgatagcagcaattgtccaacttcataatggaaccaagtataaga 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 481
tttacctgccaggtgtgatagcagcaattgtccaacttcataatggaaccaagtataaga 540
8P1D4v.2: 541
agtttccacattggttggataagtggatgttaacaagaaagcagtttgggcttctcagtt 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 541
agtttccacattggttggataagtggatgttaacaagaaagcagtttgggcttctcagtt 600
8P1D4v.2: 601
tcttttttgctgtactgcatgcaatttatagtctgtcttacccaatgaggcgatcctaca 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 601
tcttttttgctgtactgcatgcaatttatagtctgtcttacccaatgaggcgatcctaca 660
8P104v.2: 661
gatacaagttgctaaactgggcatatcaacaggtccaacaaaataaagaagatgcctgga 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 661
gatacaagttgctaaactgggcatatcaacaggtccaacaaaataaagaagatgcctgga 720
8P1D4v.2: 721
ttgagcatgatgtttggagaatggagatttatgtgtctctgggaattgtgggattggcaa 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 721
ttgagcatgatgtttggagaatggagatttatgtgtctctgggaattgtgggattggcaa 780
8P1D4v.2: 781
tactggctctgttggctgtgacatctattccatctgtgagtgactctttgacatggagag 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 781
tactggctctgttggctgtgacatctattccatctgtgagtgactctttgacatggagag 840
8P1D4v.2: 841 aatttcactatattcag 857 ||||||||||||||||| 8P1D4v.3: 841
aatttcactatattcag 857 Score 267 bits (139), Expect =
2e-68Identities = 139/139 (100%) Strand = Plus/Plus 8P1D4v.2: 1752
agattatccataagaagagtgatgtgccagaatcactctgggatccttgtctgacaagat 1811
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 856
agattatccataagaagagtgatgtgccagaatcactctgggatccttgtctgacaagat 915
8P1D4v.2: 1812
tcaaaggactaaatttaattcagtcatgaacactgccaattaccgtttatgggtagacat 1871
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 916
tcaaaggactaaatttaattcagtcatgaacactgccaattaccgtttatgggtagacat 975
8P1D4v.2: 1872 ctttggaaatttccacaag 1890 |||||||||||||||||||
8P1D4v.3: 976 ctttggaaatttccacaag 994 Score = 717 bits (373),
Expect = 0.0Identities = 373/373 (100%) Strand = Plus/Plus
8P1D4v.2: 3255
agagcaagctaggaattgtttcccttctactgggcacaatacacgcattgatttttgcct 3314
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 993
agagcaagctaggaattgtttcccttctactgggcacaatacacgcattgatttttgcct 1052
8P1D4v.2: 3315
ggaataagtggatagatataaaacaatttgtatggtatacacctccaacttttatgatag 3374
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1053
ggaataagtggatagatataaaacaatttgtatggtatacacctccaacttttatgatag 1112
8P1D4V.2: 3375
ctgttttccttccaattgttgtcctgatatttaaaagcatactattcctgccatgcttga 3434
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1113
ctgttttccttccaattgttgtcctgatatttaaaagcatactattcctgccatgcttga 1172
8P1D4v.2: 3435
ggaagaagatactgaagattagacatggttgggaagacgtcaccaaaattaacaaaactg 3494
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1173
ggaagaagatactgaagattagacatggttgggaagacgtcaccaaaattaacaaaactg 1232
8P1D4v.2: 3495
agatatgttcccagttgtagaattactgtttacacacatttttgttcaatattgatatat 3554
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1233
agatatgttcccagttgtagaattactgtttacacacatttttgttcaatattgatatat 1292
8P1D4v.2: 3555
tttatcaccaacatttcaagtttgtatttgttaataaaatgattattcaaggaaaaaaaa 3614
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
8P1D4v.3: 1293
tttatcaccaacatttcaagtttgtatttgttaataaaatgattattcaaggaaaaaaaa 1352
8P1D4v.2: 3615 aaaaaaaaaaaaa 3627 ||||||||||||| 8P1D4v.3: 1353
aaaaaaaaaaaaa 1365
[0992] TABLE-US-00195 TABLE LIX Peptide sequences of protein coded
by 8P1D4 v.3 (SEQ ID NO: 90) MESRKDITNQ EELWKMKPRR NLEEDDYLhK
DTGETSMLKR PVLLHLHQTA HADEFDCPSE 60 LQHTQELFPQ WhLPIKIAAI
IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM 120 VSITLLALVY
LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF FAVKHAIYSL 180
SYPMRRSYRY KLLNWAYQQV QQNKEDAWIE HDVWRMEIYV SLGIVGLAIL ALLAVTSIPS
240 VSDSLTWREF HYIQIIHKKS DVPESLWDPC LTRFKGLNLI QS 282
[0993] TABLE-US-00196 TABLE LX Amino acid sequence alignment of
8P1D4 v.2 (SEQ ID NO: 91) and 8P1D4 v.3 (SEQ ID NO: 92) Score = 522
bits (1345), Expect = e-147Identities 254/255 (99%), Positives =
255/255 (99%) 8P1D4v.2: 1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE 60
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
8P1D4v.3: 1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE 60
8P1D4v.2: 61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM 120
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
8P1D4v.3: 61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM 120
8P1D4v.2: 121
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL 180
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
8P1D4v.3: 121
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL 180
8P1D4v.2: 181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS 240
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
8P1D4v.3 181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS 240
8P104v.2: 241 VSDSLTWREFHYIQV 255 VSDSLTWREFHYIQ+ 8P1D4v.3: 241
VSDSLTWREFHYIQI 255
[0994] TABLE-US-00197 TABLE LXI SEARCH Peptides STEAP 1 Variant 1
nonamers, decamers and 15-mers: aa 1-339 (SEQ ID NO: 71) MESRKDITNQ
EELWKMKPRR NLEEDDYLHK DTGETSMLKR PVLLHLHQTA HADEFDCPSE 60
LQHTQELFPQ WHLPIKIAAI IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM
120 VSITLLALVY LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF
FAVLHAIYSL 180 SYPMRRSYRY KLLNWAYQQV QONKEDAWIE HDVWRMEIYV
SLGIVGLAIL ALLAVTSIPS 240 VSDSLTWREF HYJQSKLGIV SLLLGTIHAL
IFAWNKWIDI KQFVWYTPPT FMJAVFLPIV 300 VLIFKSILFL PCLRKKILKI
RHGWEDVTKI NKTEICSQL 339 Variant 2: 9-mers aa 247-258 (SEQ ID NO:
72) WREFHYIQVNNI 10-mers aa 246-258 (SEQ ID NO: 73) TWREFHYIQVNNJ
15-mers aa 241-258 (SEQ ID NO: 74) VSDSLTWREFHYIQVNNI Variant 3:
9-mers aa 247- (SEQ ID NO: 75) WREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS
10-mers aa 246- (SEQ ID NO: 76)
TWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS 15-mers aa 241- (SEQ ID NO:
77) VSDSLTWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS Variant 4: 9-mers aa
160-176 (SEQ ID NO: 78) RKQFCLLSLFFAVLHAI 10-mers aa 159-177 (SEQ
ID NO: 79) TRKQFGLLSLFFAVLHAIY 15-mers aa 154-182 (SEQ ID NO: 80)
DKWMLTRKQFGLLSLFFAVLHAIYSLSYP
[0995]
Sequence CWU 1
1
100 1 436 DNA Homo sapiens 1 gtacagcaaa aaagaaactg agaagcccaa
actgctttct tgttaacatc cacttatcca 60 accaatgtgg aaacttctta
tacttggttc cattatgaag ttggacaatt gctgctatca 120 cacctggcag
gtaaaccaat gccaagagag tgatggaaac cattggcaag actttgttga 180
tgaccaggat tggaatttta taaaaatatt gttgatggga agttgctaaa gggtgaatta
240 cttccctcag aagagtgtaa agaaaagtca gagatgctat aatagcagct
attttaattg 300 gcaagtgcca ctgtggaaag agttcctgtg tgtgctgaag
ttctgaaggg cagtcaaatt 360 catcagcatg ggctatttgg tgcaaatgca
aaagcacagg tctttttagc atgctggtct 420 ctcccgtgtc cttatg 436 2 1193
DNA Homo sapiens CDS (66)...(1085) 2 ccgagactca cggtcaagct
aaggcgaaga gtgggtggct gaagccatac tattttatag 60 aatta atg gaa agc
aga aaa gac atc aca aac caa gaa gaa ctt tgg aaa 110 Met Glu Ser Arg
Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys 1 5 10 15 atg aag cct
agg aga aat tta gaa gaa gac gat tat ttg cat aag gac 158 Met Lys Pro
Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp 20 25 30 acg
gga gag acc agc atg cta aaa aga cct gtg ctt ttg cat ttg cac 206 Thr
Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His 35 40
45 caa aca gcc cat gct gat gaa ttt gac tgc cct tca gaa ctt cag cac
254 Gln Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His
50 55 60 aca cag gaa ctc ttt cca cag tgg cac ttg cca att aaa ata
gct gct 302 Thr Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile
Ala Ala 65 70 75 att ata gca tct ctg act ttt ctt tac act ctt ctg
agg gaa gta att 350 Ile Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu
Arg Glu Val Ile 80 85 90 95 cac cct tta gca act tcc cat caa caa tat
ttt tat aaa att cca atc 398 His Pro Leu Ala Thr Ser His Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile 100 105 110 ctg gtc atc aac aaa gtc ttg cca
atg gtt tcc atc act ctc ttg gca 446 Leu Val Ile Asn Lys Val Leu Pro
Met Val Ser Ile Thr Leu Leu Ala 115 120 125 ttg gtt tac ctg cca ggt
gtg ata gca gca att gtc caa ctt cat aat 494 Leu Val Tyr Leu Pro Gly
Val Ile Ala Ala Ile Val Gln Leu His Asn 130 135 140 gga acc aag tat
aag aag ttt cca cat tgg ttg gat aag tgg atg tta 542 Gly Thr Lys Tyr
Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu 145 150 155 aca aga
aag cag ttt ggg ctt ctc agt ttc ttt ttt gct gta ctg cat 590 Thr Arg
Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His 160 165 170
175 gca att tat agt ctg tct tac cca atg agg cga tcc tac aga tac aag
638 Ala Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys
180 185 190 ttg cta aac tgg gca tat caa cag gtc caa caa aat aaa gaa
gat gcc 686 Leu Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu
Asp Ala 195 200 205 tgg att gag cat gat gtt tgg aga atg gag att tat
gtg tct ctg gga 734 Trp Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr
Val Ser Leu Gly 210 215 220 att gtg gga ttg gca ata ctg gct ctg ttg
gct gtg aca tct att cca 782 Ile Val Gly Leu Ala Ile Leu Ala Leu Leu
Ala Val Thr Ser Ile Pro 225 230 235 tct gtg agt gac tct ttg aca tgg
aga gaa ttt cac tat att cag agc 830 Ser Val Ser Asp Ser Leu Thr Trp
Arg Glu Phe His Tyr Ile Gln Ser 240 245 250 255 aag cta gga att gtt
tcc ctt cta ctg ggc aca ata cac gca ttg att 878 Lys Leu Gly Ile Val
Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile 260 265 270 ttt gcc tgg
aat aag tgg ata gat ata aaa caa ttt gta tgg tat aca 926 Phe Ala Trp
Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr 275 280 285 cct
cca act ttt atg ata gct gtt ttc ctt cca att gtt gtc ctg ata 974 Pro
Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile 290 295
300 ttt aaa agc ata cta ttc ctg cca tgc ttg agg aag aag ata ctg aag
1022 Phe Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu
Lys 305 310 315 att aga cat ggt tgg gaa gac gtc acc aaa att aac aaa
act gag ata 1070 Ile Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn
Lys Thr Glu Ile 320 325 330 335 tgt tcc cag ttg tag aattactgtt
tacacacatt tttgttcaat attgatatat 1125 Cys Ser Gln Leu * tttatcacca
acatttcaag tttgtatttg ttaataaaat gattacaagg aaaaaaaaaa 1185
aaaaaaaa 1193 3 339 PRT Homo sapiens 3 Met Glu Ser Arg Lys Asp Ile
Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn
Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr
Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr
Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55
60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile
65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val
Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys
Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser
Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala
Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe
Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln
Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile
Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185
190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp
195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu
Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr
Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu
Phe His Tyr Ile Gln Ser Lys 245 250 255 Leu Gly Ile Val Ser Leu Leu
Leu Gly Thr Ile His Ala Leu Ile Phe 260 265 270 Ala Trp Asn Lys Trp
Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr Phe
Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys
Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile 305 310
315 320 Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile
Cys 325 330 335 Ser Gln Leu 4 3627 DNA Homo sapiens CDS
(96)...(872) 4 ggggcccgca cctctgggca gcagcggcag ccgagactca
cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac tattttatag aatta atg
gaa agc aga aaa gac 113 Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa
gaa gaa ctt tgg aaa atg aag cct agg aga aat tta 161 Ile Thr Asn Gln
Glu Glu Leu Trp Lys Met Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa
gac gat tat ttg cat aag gac acg gga gag acc agc atg cta 209 Glu Glu
Asp Asp Tyr Leu His Lys Asp Thr Gly Glu Thr Ser Met Leu 25 30 35
aaa aga cct gtg ctt ttg cat ttg cac caa aca gcc cat gct gat gaa 257
Lys Arg Pro Val Leu Leu His Leu His Gln Thr Ala His Ala Asp Glu 40
45 50 ttt gac tgc cct tca gaa ctt cag cac aca cag gaa ctc ttt cca
cag 305 Phe Asp Cys Pro Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro
Gln 55 60 65 70 tgg cac ttg cca att aaa ata gct gct att ata gca tct
ctg act ttt 353 Trp His Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser
Leu Thr Phe 75 80 85 ctt tac act ctt ctg agg gaa gta att cac ccc
tta gca act tcc cat 401 Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro
Leu Ala Thr Ser His 90 95 100 caa caa tat ttt tat aaa att cca atc
ctg gtc atc aac aaa gtc ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile
Leu Val Ile Asn Lys Val Leu 105 110 115 cca atg gtt tcc atc act ctc
ttg gca ttg gtt tac ctg cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu
Leu Ala Leu Val Tyr Leu Pro Gly Val 120 125 130 ata gca gca att gtc
caa ctt cat aat gga acc aag tat aag aag ttt 545 Ile Ala Ala Ile Val
Gln Leu His Asn Gly Thr Lys Tyr Lys Lys Phe 135 140 145 150 cca cat
tgg ttg gat aag tgg atg tta aca aga aag cag ttt ggg ctt 593 Pro His
Trp Leu Asp Lys Trp Met Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165
ctc agt ttc ttt ttt gct gta ctg cat gca att tat agt ctg tct tac 641
Leu Ser Phe Phe Phe Ala Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170
175 180 cca atg agg cga tcc tac aga tac aag ttg cta aac tgg gca tat
caa 689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr
Gln 185 190 195 cag gtc caa caa aat aaa gaa gat gcc tgg att gag cat
gat gtt tgg 737 Gln Val Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His
Asp Val Trp 200 205 210 aga atg gag att tat gtg tct ctg gga att gtg
gga ttg gca ata ctg 785 Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val
Gly Leu Ala Ile Leu 215 220 225 230 gct ctg ttg gct gtg aca tct att
cca tct gtg agt gac tct ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile
Pro Ser Val Ser Asp Ser Leu Thr 235 240 245 tgg aga gaa ttt cac tat
att cag gta aat aat ata taa aataacccta 882 Trp Arg Glu Phe His Tyr
Ile Gln Val Asn Asn Ile * 250 255 agaggtaaat cttctttttg tgtttatgat
atagaatatg ttgactttac cccataaaaa 942 ataacaaatg tttttcaaca
gcaaagatct tatacttgtt ccaattaata atgtgctctc 1002 ctgttgtttt
ccctattgct tctaattagg acaagtgttt cctagacata aataaaaggc 1062
attaaaatat tctttgtttt tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt
1122 ttttgagatg aagtctcgct ctgttgccca tgctggagta cagtggcacg
atctcggctc 1182 actgcaacct gcgcctcctg ggttcaggcg attctcttgc
ctcagcctcc tgagtagctg 1242 ggattacagg cacccatcac catgtccagc
taatttttgt atttttagta gagacagggt 1302 tttcccatgt tggccaggct
ggtctcgatc tcctgacctc aaatgatccg cccacctcgg 1362 cctcccaaag
tgctgggatg acagttgtga gccaccacac tcagcctgct ctttctaata 1422
tttgaaactt gttagacaat ttgctaccca tctaatgtga tattttagga atccaatatg
1482 catggtttat tatttcttaa aaaaaatatt cttttacctg tcacctgaat
ttagtaatgc 1542 cttttatgtt acacaactta gcactttcca gaaacaaaaa
ctctctcctt gaaataatag 1602 agtttttatc taccaaagat atgctagtgt
ctcatttcaa aggctgcttt ttccagctta 1662 cattttatat acttactcac
ttgaagtttc taaatattct tgtaatttta aaactatctc 1722 agatttactg
aggtttatct tctggtggta gattatccat aagaagagtg atgtgccaga 1782
atcactctgg gatccttgtc tgacaagatt caaaggacta aatttaattc agtcatgaac
1842 actgccaatt accgtttatg ggtagacatc tttggaaatt tccacaaggt
cagacattcg 1902 caactatccc ttctacatgt ccacacgtat actccaacac
tttattaggc atctgattag 1962 tttggaaagt atgcctccat ctgaattagt
ccagtgtggc ttagagttgg tacaacattc 2022 tcacagaatt tcctaatttt
gtaggttcag cctgataacc actggagttc tttggtcctc 2082 attaaatagc
tttcttcaca cattgctctg cctgttacac atatgatgaa cactgctttt 2142
tagacttcat taggaattta ggactgcatc ttgacaactg agcctattct actatatgta
2202 caatacctag cccataatag gtatacaata cacatttggt aaaactaatt
ttcaaccaat 2262 gacatgtatt tttcaactag taacctagaa atgtttcact
taaaatctga gaactggtta 2322 cactacaagt taccttggag attcatatat
gaaaacgcaa acttagctat ttgattgtat 2382 tcactgggac ttaagaatgc
gcctgaataa ttgtgagttc gatttgttct ggcaggctaa 2442 tgaccatttc
cagtaaagtg aatagaggtc agaagtcgta taaaagaggt gttgtcagaa 2502
caccgttgag attacatagg tgaacaacta tttttaagca actttatttg tgtagtgaca
2562 aagcatccca atgcaggctg aaatgtttca tcacatctct ggatctctct
attttgtgca 2622 gacattgaaa aaattgttca tattatttcc atgttatcag
aatatttgat tttttaaaaa 2682 cataggccaa gttcattcac ttcattattc
atttatcaaa atcagagtga atcacattag 2742 tcgccttcac aactgataaa
gatcactgaa gtcaaattga tttttgctat aatcttcaat 2802 ctacctatat
ttaattgaga atctaaaatg tacaaatcat tgtgttgatt ctgcagtgat 2862
cctgctataa gtaagactca gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga
2922 caaatacaca agagacaaag cacaaaaaat aaatatcata aggggatgaa
caaaatggtg 2982 gagaaagagt agacaaagtt tttgatcacc tgccttcaaa
gaaaggctgt gaattttgtt 3042 cacttagaca gcttggagac aagaaattac
ccaaaagtaa ggtgaggagg ataggcaaaa 3102 agagcagaaa gatgtgaatg
gacattgttg agaaatgtga taggaaaaca atcatagata 3162 aaggatttcc
aagcaacaga gcatatccag atgaggtagg atgggataaa ctcttattga 3222
accaatcttc accaattttg tttttctttt gcagagcaag ctaggaattg tttcccttct
3282 actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata
taaaacaatt 3342 tgtatggtat acacctccaa cttttatgat agctgttttc
cttccaattg ttgtcctgat 3402 atttaaaagc atactattcc tgccatgctt
gaggaagaag atactgaaga ttagacatgg 3462 ttgggaagac gtcaccaaaa
ttaacaaaac tgagatatgt tcccagttgt agaattactg 3522 tttacacaca
tttttgttca atattgatat attttatcac caacatttca agtttgtatt 3582
tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 3627 5 258 PRT
Homo sapiens 5 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu
Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr
Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro
Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe
Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro
Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser
Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro
Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105
110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu
115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His
Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys
Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe
Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro
Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr
Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His
Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val
Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230
235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val
Asn 245 250 255 Asn Ile 6 1365 DNA Homo sapiens CDS (96)...(944) 6
ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga
60 gtgggtggct gaagccatac tattttatag aatta atg gaa agc aga aaa gac
113 Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa
atg aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys
Met Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat
aag gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His
Lys Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt
ttg cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu
Leu His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc
cct tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys
Pro Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg
cac ttg cca att aaa ata gct gct att ata gca tct ctg act ttt 353 Trp
His Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80
85 ctt tac act ctt ctg agg gaa gta att cac cct tta gca act tcc cat
401 Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His
90 95 100 caa caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa
gtc ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys
Val Leu 105 110 115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac
ctg cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr
Leu Pro Gly Val 120 125 130 ata gca gca att gtc caa ctt cat aat gga
acc aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly
Thr Lys Tyr Lys Lys Phe 135 140 145
150 cca cat tgg ttg gat aag tgg atg tta aca aga aag cag ttt ggg ctt
593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg Lys Gln Phe Gly Leu
155 160 165 ctc agt ttc ttt ttt gct gta ctg cat gca att tat agt ctg
tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His Ala Ile Tyr Ser Leu
Ser Tyr 170 175 180 cca atg agg cga tcc tac aga tac aag ttg cta aac
tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn
Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat aaa gaa gat gcc tgg
att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn Lys Glu Asp Ala Trp
Ile Glu His Asp Val Trp 200 205 210 aga atg gag att tat gtg tct ctg
gga att gtg gga ttg gca ata ctg 785 Arg Met Glu Ile Tyr Val Ser Leu
Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230 gct ctg ttg gct gtg
aca tct att cca tct gtg agt gac tct ttg aca 833 Ala Leu Leu Ala Val
Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235 240 245 tgg aga gaa
ttt cac tat att cag att atc cat aag aag agt gat gtg 881 Trp Arg Glu
Phe His Tyr Ile Gln Ile Ile His Lys Lys Ser Asp Val 250 255 260 cca
gaa tca ctc tgg gat cct tgt ctg aca aga ttc aaa gga cta aat 929 Pro
Glu Ser Leu Trp Asp Pro Cys Leu Thr Arg Phe Lys Gly Leu Asn 265 270
275 tta att cag tca tga acactgccaa ttaccgttta tgggtagaca tctttggaaa
984 Leu Ile Gln Ser * 280 tttccacaag agcaagctag gaattgtttc
ccttctactg ggcacaatac acgcattgat 1044 ttttgcctgg aataagtgga
tagatataaa acaatttgta tggtatacac ctccaacttt 1104 tatgatagct
gttttccttc caattgttgt cctgatattt aaaagcatac tattcctgcc 1164
atgcttgagg aagaagatac tgaagattag acatggttgg gaagacgtca ccaaaattaa
1224 caaaactgag atatgttccc agttgtagaa ttactgttta cacacatttt
tgttcaatat 1284 tgatatattt tatcaccaac atttcaagtt tgtatttgtt
aataaaatga ttattcaagg 1344 aaaaaaaaaa aaaaaaaaaa a 1365 7 282 PRT
Homo sapiens 7 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu
Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr
Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro
Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe
Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro
Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser
Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro
Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105
110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu
115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His
Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys
Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe
Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro
Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr
Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His
Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val
Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230
235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ile
Ile 245 250 255 His Lys Lys Ser Asp Val Pro Glu Ser Leu Trp Asp Pro
Cys Leu Thr 260 265 270 Arg Phe Lys Gly Leu Asn Leu Ile Gln Ser 275
280 8 3627 DNA Homo sapiens CDS (96)...(872) 8 ggggcccgca
cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60
gtgggtggct gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113
Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg
aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag
gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg
cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu
His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct
tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro
Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac
ttg cca att aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His
Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85
ctt tac act ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401
Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90
95 100 caa caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc
ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val
Leu 105 110 115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg
cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu
Pro Gly Val 120 125 130 ata gca gca att gtc caa ctt cat aat gga acc
aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr
Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg
tta aca aga aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met
Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc agt ttg ttt ttt gct
gta ctg cat gca att tat agt ctg tct tac 641 Leu Ser Leu Phe Phe Ala
Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga
tcc tac aga tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc
caa caa aat aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210
aga atg gag att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215
220 225 230 gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct
ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser
Leu Thr 235 240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata
taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile
* 250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 9 258 PRT Homo sapiens 9 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Leu Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
10 3627 DNA Homo sapiens CDS (96)...(872) 10 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac cct tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa
caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110
115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg
497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val
120 125 130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag
aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys
Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga
aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg
Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat
gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His
Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga
tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat
aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag
att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230
gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235
240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata taa
aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile *
250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag
attacatagg tgaacaacta tttttaagca actttatttg tgtagtgaca 2562
aagcatccca atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca
2622 gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat
tttttaaaaa 2682 cataggccaa gttcattcac ttcattattc atttatcaaa
atcagagtga atcacattag 2742 tcgccttcac aactgataaa gatcactgaa
gtcaaattga tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga
atctaaaatg tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa
gtaagactca gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922
caaatacaca agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg
2982 gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt
gaattttgtt 3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa
ggtgaggagg ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg
agaaatgtga taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga
gcatatccag atgaggtagg atgggataaa ctcttattga 3222 accaatcttc
accaattttg tttttctttt gcagagcaag ctaggaattg tttcccttct 3282
actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt
3342 tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg
ttgtcctgat 3402 atttaaaagc atactattcc tgccatgctt gaggaagaag
atactgaaga ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac
tgagatatgt tcccagttgt agaattactg 3522 tttacacaca tttttgttca
atattgatat attttatcac caacatttca agtttgtatt 3582 tgttaataaa
atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 3627 11 258 PRT Homo sapiens
11 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu
His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro
Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His
Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe
Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr
Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile
Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125
Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130
135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu
Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala
Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala
Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val
Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250
255 Asn Ile 12 3627 DNA Homo sapiens CDS (96)...(872) 12 ggggcccgca
cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60
gtgggtggct gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113
Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg
aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag
gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg
cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu
His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct
tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro
Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac
ttg cca att aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His
Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85
ctt tac act ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401
Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90
95 100 caa caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc
ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val
Leu 105 110 115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg
cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu
Pro Gly Val 120 125 130 ata gca gca att gtc caa ctt cat aat gga acc
aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr
Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg
tta aca aga aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met
Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct
gta ctg cat gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala
Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga
tcc tac aga tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc
caa caa aat aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210
aga atg gag att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215
220 225 230 gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct
ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser
Leu Thr 235 240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata
taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile
* 250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
ttttgttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 13 258 PRT Homo sapiens 13 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
14 3627 DNA Homo sapiens CDS (96)...(872) 14 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa
caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110
115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg
497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val
120 125 130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag
aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys
Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga
aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg
Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat
gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His
Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga
tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat
aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag
att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230
gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235
240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata taa
aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile *
250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgccaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 15 258 PRT Homo sapiens 15 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
16 3627 DNA Homo sapiens CDS (96)...(872) 16 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa
caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110
115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg
497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val
120 125 130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag
aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys
Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga
aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg
Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat
gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His
Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga
tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat
aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag
att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230
gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235
240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata taa
aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile *
250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagtt atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 17 258 PRT Homo sapiens 17 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
18 3627 DNA Homo sapiens CDS (96)...(872) 18 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa
caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110
115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg
497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val
120 125 130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag
aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys
Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga
aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg
Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat
gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His
Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga
tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat
aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag
att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230
gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235
240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata taa
aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile *
250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat attctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 19 258 PRT Homo sapiens 19 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro
Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser
Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro
Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105
110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu
115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His
Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys
Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe
Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro
Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr
Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His
Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val
Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230
235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val
Asn 245 250 255 Asn Ile 20 3627 DNA Homo sapiens CDS (96)...(872)
20 ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct
aaggcgaaga 60 gtgggtggct gaagccatac tattttatag aatta atg gaa agc
aga aaa gac 113 Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa
ctt tgg aaa atg aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu
Leu Trp Lys Met Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat
tat ttg cat aag gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp
Tyr Leu His Lys Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga
cct gtg ctt ttg cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg
Pro Val Leu Leu His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50
ttt gac tgc cct tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305
Phe Asp Cys Pro Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55
60 65 70 tgg cac ttg cca att aaa ata gct gct att ata gca tct ctg
act ttt 353 Trp His Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu
Thr Phe 75 80 85 ctt tac act ctt ctg agg gaa gta att cac ccc tta
gca act tcc cat 401 Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu
Ala Thr Ser His 90 95 100 caa caa tat ttt tat aaa att cca atc ctg
gtc atc aac aaa gtc ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu
Val Ile Asn Lys Val Leu 105 110 115 cca atg gtt tcc atc act ctc ttg
gca ttg gtt tac ctg cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu
Ala Leu Val Tyr Leu Pro Gly Val 120 125 130 ata gca gca att gtc caa
ctt cat aat gga acc aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln
Leu His Asn Gly Thr Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg
ttg gat aag tgg atg tta aca aga aag cag ttt ggg ctt 593 Pro His Trp
Leu Asp Lys Trp Met Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc
agt ttc ttt ttt gct gta ctg cat gca att tat agt ctg tct tac 641 Leu
Ser Phe Phe Phe Ala Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175
180 cca atg agg cga tcc tac aga tac aag ttg cta aac tgg gca tat caa
689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln
185 190 195 cag gtc caa caa aat aaa gaa gat gcc tgg att gag cat gat
gtt tgg 737 Gln Val Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp
Val Trp 200 205 210 aga atg gag att tat gtg tct ctg gga att gtg gga
ttg gca ata ctg 785 Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly
Leu Ala Ile Leu 215 220 225 230 gct ctg ttg gct gtg aca tct att cca
tct gtg agt gac tct ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro
Ser Val Ser Asp Ser Leu Thr 235 240 245 tgg aga gaa ttt cac tat att
cag gta aat aat ata taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile
Gln Val Asn Asn Ile * 250 255 agaggtaaat cttctttttg tgtttatgat
atagaatatg ttgactttac cccataaaaa 942 ataacaaatg tttttcaaca
gcaaagatct tatacttgtt ccaattaata atgtgctctc 1002 ctgttgtttt
ccctattgct tctaattagg acaagtgttt cctagacata aataaaaggc 1062
attaaaatat tctttgtttt tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt
1122 ttttgagatg aagtctcgct ctgttgccca tgctggagta cagtggcacg
atctcggctc 1182 actgcaacct gcgcctcctg ggttcaggcg attctcttgc
ctcagcctcc tgagtagctg 1242 ggattacagg cacccatcac catgtccagc
taatttttgt atttttagta gagacagggt 1302 tttcccatgt tggccaggct
ggtctcgatc tcctgacctc aaatgatccg cccacctcgg 1362 cctcccaaag
tgctgggatg acagttgtga gccaccacac tcagcctgct ctttctaata 1422
tttgaaactt gttagacaat ttgctaccca tctaatgtga tattttagga atccaatatg
1482 catggtttat tatttcttaa aaaaaatatt cttttacctg tcacctgaat
ttagtaatgc 1542 cttttatgtt acacaactta gcactttcca gaaacaaaaa
ctctctcctt gaaataatag 1602 agtttttatc taccaaagat atgctagtgt
ctcatttcaa aggctgcttt ttccagctta 1662 cattttatat acttactcac
ttgaagtttc taaatattct tgtaatttta aaaatatctc 1722 agatttactg
aggtttatct tctggtggta gattatccat aagaagagtg atgtgccaga 1782
atcactctgg gatccttgtc tgacaagatt caaaggacta aatttaattc agtcatgaac
1842 actgccaatt accgtttatg ggtagacatc tttggaaatt tccacaaggt
cagacattcg 1902 caactatccc ttctacatgt ccacacgtat actccaacac
tttattaggc atctgattag 1962 tttggaaagt atgcctccat ctgaattagt
ccagtgtggc ttagagttgg tacaacattc 2022 tcacagaatt tcctaatttt
gtaggttcag cctgataacc actggagttc tttggtcctc 2082 attaaatagc
tttcttcaca cattgctctg cctgttacac atatgatgaa cactgctttt 2142
tagacttcat taggaattta ggactgcatc ttgacaactg agcctattct actatatgta
2202 caatacctag cccataatag gtatacaata cacatttggt aaaactaatt
ttcaaccaat 2262 gacatgtatt tttcaactag taacctagaa atgtttcact
taaaatctga gaactggtta 2322 cactacaagt taccttggag attcatatat
gaaaacgcaa acttagctat ttgattgtat 2382 tcactgggac ttaagaatgc
gcctgaataa ttgtgagttc gatttgttct ggcaggctaa 2442 tgaccatttc
cagtaaagtg aatagaggtc agaagtcgta taaaagaggt gttgtcagaa 2502
caccgttgag attacatagg tgaacaacta tttttaagca actttatttg tgtagtgaca
2562 aagcatccca atgcaggctg aaatgtttca tcacatctct ggatctctct
attttgtgca 2622 gacattgaaa aaattgttca tattatttcc atgttatcag
aatatttgat tttttaaaaa 2682 cataggccaa gttcattcac ttcattattc
atttatcaaa atcagagtga atcacattag 2742 tcgccttcac aactgataaa
gatcactgaa gtcaaattga tttttgctat aatcttcaat 2802 ctacctatat
ttaattgaga atctaaaatg tacaaatcat tgtgttgatt ctgcagtgat 2862
cctgctataa gtaagactca gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga
2922 caaatacaca agagacaaag cacaaaaaat aaatatcata aggggatgaa
caaaatggtg 2982 gagaaagagt agacaaagtt tttgatcacc tgccttcaaa
gaaaggctgt gaattttgtt 3042 cacttagaca gcttggagac aagaaattac
ccaaaagtaa ggtgaggagg ataggcaaaa 3102 agagcagaaa gatgtgaatg
gacattgttg agaaatgtga taggaaaaca atcatagata 3162 aaggatttcc
aagcaacaga gcatatccag atgaggtagg atgggataaa ctcttattga 3222
accaatcttc accaattttg tttttctttt gcagagcaag ctaggaattg tttcccttct
3282 actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata
taaaacaatt 3342 tgtatggtat acacctccaa cttttatgat agctgttttc
cttccaattg ttgtcctgat 3402 atttaaaagc atactattcc tgccatgctt
gaggaagaag atactgaaga ttagacatgg 3462 ttgggaagac gtcaccaaaa
ttaacaaaac tgagatatgt tcccagttgt agaattactg 3522 tttacacaca
tttttgttca atattgatat attttatcac caacatttca agtttgtatt 3582
tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 3627 21 258 PRT
Homo sapiens 21 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu
Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr
Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro
Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe
Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro
Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser
Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro
Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105
110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu
115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His
Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys
Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe
Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro
Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr
Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His
Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val
Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230
235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val
Asn 245 250 255 Asn Ile 22 3627 DNA Homo sapiens CDS (96)...(872)
22 ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct
aaggcgaaga 60 gtgggtggct gaagccatac tattttatag aatta atg gaa agc
aga aaa gac 113 Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa
ctt tgg aaa atg aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu
Leu Trp Lys Met Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat
tat ttg cat aag gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp
Tyr Leu His Lys Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga
cct gtg ctt ttg cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg
Pro Val Leu Leu His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50
ttt gac tgc cct tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305
Phe Asp Cys Pro Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55
60 65 70 tgg cac ttg cca att aaa ata gct gct att ata gca tct ctg
act ttt 353 Trp His Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu
Thr Phe 75 80 85 ctt tac act ctt ctg agg gaa gta att cac ccc tta
gca act tcc cat 401 Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu
Ala Thr Ser His 90 95 100 caa caa tat ttt tat aaa att cca atc ctg
gtc atc aac aaa gtc ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu
Val Ile Asn Lys Val Leu 105 110 115 cca atg gtt tcc atc act ctc ttg
gca ttg gtt tac ctg cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu
Ala Leu Val Tyr Leu Pro Gly Val 120 125 130 ata gca gca att gtc caa
ctt cat aat gga acc aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln
Leu His Asn Gly Thr Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg
ttg gat aag tgg atg tta aca aga aag cag ttt ggg ctt 593 Pro His Trp
Leu Asp Lys Trp Met Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc
agt ttc ttt ttt gct gta ctg cat gca att tat agt ctg tct tac 641 Leu
Ser Phe Phe Phe Ala Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175
180 cca atg agg cga tcc tac aga tac aag ttg cta aac tgg gca tat caa
689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln
185 190 195 cag gtc caa caa aat aaa gaa gat gcc tgg att gag cat gat
gtt tgg 737 Gln Val Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp
Val Trp 200 205 210 aga atg gag att tat gtg tct ctg gga att gtg gga
ttg gca ata ctg 785 Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly
Leu Ala Ile Leu 215 220 225 230 gct ctg ttg gct gtg aca tct att cca
tct gtg agt gac tct ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro
Ser Val Ser Asp Ser Leu Thr 235 240 245 tgg aga gaa ttt cac tat att
cag gta aat aat ata taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile
Gln Val Asn Asn Ile * 250 255 agaggtaaat cttctttttg tgtttatgat
atagaatatg ttgactttac cccataaaaa 942 ataacaaatg tttttcaaca
gcaaagatct tatacttgtt ccaattaata atgtgctctc 1002 ctgttgtttt
ccctattgct tctaattagg acaagtgttt cctagacata aataaaaggc 1062
attaaaatat tctttgtttt tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt
1122 ttttgagatg aagtctcgct ctgttgccca tgctggagta cagtggcacg
atctcggctc 1182 actgcaacct gcgcctcctg ggttcaggcg attctcttgc
ctcagcctcc tgagtagctg 1242 ggattacagg cacccatcac catgtccagc
taatttttgt atttttagta gagacagggt 1302 tttcccatgt tggccaggct
ggtctcgatc tcctgacctc aaatgatccg cccacctcgg 1362 cctcccaaag
tgctgggatg acagttgtga gccaccacac tcagcctgct ctttctaata 1422
tttgaaactt gttagacaat ttgctaccca tctaatgtga tattttagga atccaatatg
1482 catggtttat tatttcttaa aaaaaatatt cttttacctg tcacctgaat
ttagtaatgc 1542 cttttatgtt acacaactta gcactttcca gaaacaaaaa
ctctctcctt gaaataatag 1602 agtttttatc taccaaagat atgctagtgt
ctcatttcaa aggctgcttt ttccagctta 1662 cattttatat acttactcac
ttgaagtttc taaatattct tgtaatttta aaactatctc 1722 agatttactg
aggtttatct tctggtggta gattatccat aagaagagtg atgtgccaga 1782
atcactctgg gatccttgtc tgacaagatt caaaggacta aatttaattc agtcatgaac
1842 actgccaatt accgtttatg ggtagacatc tttggaaatt tccacaaggt
cagacattcg 1902 caactatccc ttctacatgt ccacacgtat actccaacac
tttattaggc atctgattag 1962 tttggaaagt atgcctccat ctgaattagt
ccagtgtggc ttagagttgg tacaacattc 2022 tcacagaatt tcctaatttt
gtaggttcag cctgataacc actggagttc tttggtcctc 2082 attaaatagc
tttcttcaca cattgctctg cctgttacac atatgatgaa cactgctttt 2142
tagacttcat taggaattta ggactgcatc ttgacaactg agcctattct actatatgta
2202 caatacctag cccataatag gtatacaata cacatttggt aaaactaatt
ttcaaccaat 2262 gacatgtatt tttcaactag taacctagaa atgtttcact
taaaatctga gaactggtta 2322 cactacaagt taccttggag attcatatat
gaaaatgcaa acttagctat ttgattgtat 2382 tcactgggac ttaagaatgc
gcctgaataa ttgtgagttc gatttgttct ggcaggctaa 2442 tgaccatttc
cagtaaagtg aatagaggtc agaagtcgta taaaagaggt gttgtcagaa 2502
caccgttgag attacatagg tgaacaacta tttttaagca actttatttg tgtagtgaca
2562 aagcatccca atgcaggctg aaatgtttca tcacatctct ggatctctct
attttgtgca 2622 gacattgaaa aaattgttca tattatttcc atgttatcag
aatatttgat tttttaaaaa 2682 cataggccaa gttcattcac ttcattattc
atttatcaaa atcagagtga atcacattag 2742 tcgccttcac aactgataaa
gatcactgaa gtcaaattga tttttgctat aatcttcaat 2802 ctacctatat
ttaattgaga atctaaaatg tacaaatcat tgtgttgatt ctgcagtgat 2862
cctgctataa gtaagactca gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga
2922 caaatacaca agagacaaag cacaaaaaat aaatatcata aggggatgaa
caaaatggtg 2982 gagaaagagt agacaaagtt tttgatcacc tgccttcaaa
gaaaggctgt gaattttgtt 3042 cacttagaca gcttggagac aagaaattac
ccaaaagtaa ggtgaggagg ataggcaaaa 3102 agagcagaaa gatgtgaatg
gacattgttg agaaatgtga taggaaaaca atcatagata 3162 aaggatttcc
aagcaacaga gcatatccag atgaggtagg atgggataaa ctcttattga 3222
accaatcttc accaattttg tttttctttt gcagagcaag ctaggaattg tttcccttct
3282 actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata
taaaacaatt 3342 tgtatggtat acacctccaa cttttatgat agctgttttc
cttccaattg ttgtcctgat 3402 atttaaaagc atactattcc tgccatgctt
gaggaagaag atactgaaga ttagacatgg 3462 ttgggaagac gtcaccaaaa
ttaacaaaac tgagatatgt tcccagttgt agaattactg 3522 tttacacaca
tttttgttca atattgatat attttatcac caacatttca agtttgtatt 3582
tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 3627 23 258 PRT
Homo sapiens 23 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu
Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr
Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro
Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe
Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro
Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser
Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro
Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105
110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu
115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His
Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys
Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe
Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro
Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190
Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195
200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly
Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser
Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe
His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 24 3627 DNA Homo
sapiens CDS (96)...(872) 24 ggggcccgca cctctgggca gcagcggcag
ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac
tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser Arg Lys
Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg aga aat
tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg Arg Asn
Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga gag acc
agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly Glu Thr
Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac caa aca
gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His Gln Thr
Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt cag cac
aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu Gln His
Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att aaa ata
gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile Lys Ile
Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act ctt ctg
agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr Leu Leu
Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa caa tat
ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110 115 cca
atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg 497 Pro
Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val 120 125
130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag aag ttt
545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys Lys Phe
135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga aag cag
ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg Lys Gln
Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat gca att
tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His Ala Ile
Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga tac aag
ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys
Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat aaa gaa
gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn Lys Glu
Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag att tat
gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu Ile Tyr
Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230 gct ctg
ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833 Ala Leu
Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235 240 245
tgg aga gaa ttt cac tat att cag gta aat aat ata taa aataacccta 882
Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile * 250 255
agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac cccataaaaa
942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt ccaattaata
atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg acaagtgttt
cctagacata aataaaaggc 1062 attaaaatat tctttgtttt tttttttttg
tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg aagtctcgct
ctgttgccca tgctggagta cagtggcacg atctcggctc 1182 actgcaacct
gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg 1242
ggattacagg cacccatcac catgtccagc taatttttgt atttttagta gagacagggt
1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc aaatgatccg
cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga gccaccacac
tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat ttgctaccca
tctaatgtga tattttagga atccaatatg 1482 catggtttat tatttcttaa
aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542 cttttatgtt
acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag 1602
agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt ttccagctta
1662 cattttatat acttactcac ttgaagtttc taaatattct tgtaatttta
aaactatctc 1722 agatttactg aggtttatct tctggtggta gattatccat
aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc tgacaagatt
caaaggacta aatttaattc agtcatgaac 1842 actgccaatt accgtttatg
ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902 caactatccc
ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag 1962
tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg tacaacattc
2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc actggagttc
tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg cctgttacac
atatgatgaa cactgctttt 2142 tagacttcat taggaattta ggactgcatc
ttgacaactg agcctattct actatatgta 2202 caatacctag cccataatag
gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262 gacatgtatt
tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta 2322
cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat ttgattgtat
2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc gatttgttct
ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc agaagtcgta
taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg tgaacaacta
tttttaagca actttatttg tgtagtgaca 2562 aagcatccca atgcaggctg
aaatgtttca tcacatctct ggatctctct attttgtgca 2622 gacattgaaa
aaattgttca tatgatttcc atgttatcag aatatttgat tttttaaaaa 2682
cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga atcacattag
2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga tttttgctat
aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg tacaaatcat
tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca gtccctgatt
ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca agagacaaag
cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982 gagaaagagt
agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt 3042
cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg ataggcaaaa
3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga taggaaaaca
atcatagata 3162 aaggatttcc aagcaacaga gcatatccag atgaggtagg
atgggataaa ctcttattga 3222 accaatcttc accaattttg tttttctttt
gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca atacacgcat
tgatttttgc ctggaataag tggatagata taaaacaatt 3342 tgtatggtat
acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat 3402
atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga ttagacatgg
3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt tcccagttgt
agaattactg 3522 tttacacaca tttttgttca atattgatat attttatcac
caacatttca agtttgtatt 3582 tgttaataaa atgattattc aaggaaaaaa
aaaaaaaaaa aaaaa 3627 25 258 PRT Homo sapiens 25 Met Glu Ser Arg
Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro
Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30
Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35
40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His
Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile
Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu
Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro
Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly
Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr
Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160
Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165
170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys
Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu
Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr
Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu
Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr
Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 26 3627
DNA Homo sapiens CDS (96)...(872) 26 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa
caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110
115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg
497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val
120 125 130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag
aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys
Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga
aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg
Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat
gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His
Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga
tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat
aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag
att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230
gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235
240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata taa
aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile *
250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagggat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 27 258 PRT Homo sapiens 27 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
28 3627 DNA Homo sapiens CDS (96)...(872) 28 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu
10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga gag acc agc
atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly Glu Thr Ser
Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac caa aca gcc
cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His Gln Thr Ala
His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt cag cac aca
cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu Gln His Thr
Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att aaa ata gct
gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile Lys Ile Ala
Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act ctt ctg agg
gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr Leu Leu Arg
Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa caa tat ttt
tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln Gln Tyr Phe
Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110 115 cca atg
gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg 497 Pro Met
Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val 120 125 130
ata gca gca att gtc caa ctt cat aat gga acc aag tat aag aag ttt 545
Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys Lys Phe 135
140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga aag cag ttt
ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg Lys Gln Phe
Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat gca att tat
agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His Ala Ile Tyr
Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga tac aag ttg
cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu
Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat aaa gaa gat
gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn Lys Glu Asp
Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag att tat gtg
tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu Ile Tyr Val
Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230 gct ctg ttg
gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833 Ala Leu Leu
Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235 240 245 tgg
aga gaa ttt cac tat att cag gta aat aat ata taa aataacccta 882 Trp
Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile * 250 255 agaggtaaat
cttctttttg tgtttatgat atagaatatg ttgactttac cccataaaaa 942
ataacaaatg tttttcaaca gcaaagatct tatacttgtt ccaattaata atgtgctctc
1002 ctgttgtttt ccctattgct tctaattagg acaagtgttt cctagacata
aataaaaggc 1062 attaaaatat tctttgtttt tttttttttg tttgtttgtt
ttttgtttgt ttgtttgttt 1122 ttttgagatg aagtctcgct ctgttgccca
tgctggagta cagtggcacg atctcggctc 1182 actgcaacct gcgcctcctg
ggttcaggcg attctcttgc ctcagcctcc tgagtagctg 1242 ggattacagg
cacccatcac catgtccagc taatttttgt atttttagta gagacagggt 1302
tttcccatgt tggccaggct ggtctcgatc tcctgacctc aaatgatccg cccacctcgg
1362 cctcccaaag tgctgggatg acagttgtga gccaccacac tcagcctgct
ctttctaata 1422 tttgaaactt gttagacaat ttgctaccca tctaatgtga
tattttagga atccaatatg 1482 catggtttat tatttcttaa aaaaaatatt
cttttacctg tcacctgaat ttagtaatgc 1542 cttttatgtt acacaactta
gcactttcca gaaacaaaaa ctctctcctt gaaataatag 1602 agtttttatc
taccaaagat atgctagtgt ctcatttcaa aggctgcttt ttccagctta 1662
cattttatat acttactcac ttgaagtttc taaatattct tgtaatttta aaactatctc
1722 agatttactg aggtttatct tctggtggta gattatccat aagaagagtg
atgtgccaga 1782 atcactctgg gatccttgtc tgacaagatt caaaggacta
aatttaattc agtcatgaac 1842 actgccaatt accgtttatg ggtagacatc
tttggaaatt tccacaaggt cagacattcg 1902 caactatccc ttctacatgt
ccacacgtat actccaacac tttattaggc atctgattag 1962 tttggaaagt
atgcctccat ctgaattagt ccagtgtggc ttagagttgg tacaacattc 2022
tcacagaatt tcctaatttt gtaggttcag cctgataacc actggagttc tttggtcctc
2082 attaaatagc tttcttcaca cattgctctg cctgttacac atatgatgaa
cactgctttt 2142 tagacttcat taggaattta ggactgcatc ttgacaactg
agcctattct actatatgta 2202 caatacctag cccataatag gtatacaata
cacatttggt aaaactaatt ttcaaccaat 2262 gacatgtatt tttcaactag
taacctagaa atgtttcact taaaatctga gaactggtta 2322 cactacaagt
taccttggag attcatatat gaaaacgcaa acttagctat ttgattgtat 2382
tcactgggac ttaagaatgc gcctgaataa ttgtgagttc gatttgttct ggcaggctaa
2442 tgaccatttc cagtaaagtg aatagaggtc agaagtcgta taaaagaggt
gttgtcagaa 2502 caccgttgag attacatagg tgaacaacta tttttaagca
actttatttg tgtagtgaca 2562 aagcatccca atgcaggctg aaatgtttca
tcacatctct ggatctctct attttgtgca 2622 gacattgaaa aaattgttca
tattatttcc atgttatcag aatatttgat tttttaaaaa 2682 cataggccaa
gttcattcac ttcattattc atttatcaaa atcagagtga atcacattag 2742
tcgccttcac aactgataaa gatcactgaa gtcaaattga tttttgctat aatcttcaat
2802 ctacctatat ttaattgaga atctaaaatg tacaaatcat tgtgttgatt
ctgcagtgat 2862 cctgctataa gtaagactca gtccctgatt ttaggtatcc
tgtgataagc agaattaaga 2922 caaatacaca agagacaaag cacaaaaaat
aaatatcata aggggatgaa caaaatggtg 2982 gagaaagagt agacaaagtt
tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt 3042 cacttagaca
gcttggagac aagaaattac ccaaaagtaa ggtgaggagg ataggcaaaa 3102
agagcagaaa gatgtgaatg gacattgttg agaaatgtga taggaaaaca atcatagata
3162 aaggatttcc aagcaacaga gcatatccag atgaggtagg atgggataaa
ctcttattga 3222 accaatcttc accaattttg tttttctttt gcagagcaag
ctaggaattg tttcccttct 3282 actgggcaca atacacgcat tgatttttgc
ctggaataag tggatagata taaaacaatt 3342 tgtatggtat acacctccaa
cttttatgat agctgttttc cttccaattg ttgtcctgat 3402 atttaaaagc
atactattcc tgccatgctt gaggaagaag atactgaaga ttagacatgg 3462
ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt tcccagttgt agaattactg
3522 tttacacaca tttttgttca atattgatat attttatcac caacatttca
agtttgtatt 3582 tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa
3627 29 258 PRT Homo sapiens 29 Met Glu Ser Arg Lys Asp Ile Thr Asn
Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu
Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met
Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His
Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln
Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70
75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile
His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile
Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile
Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala
Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro
His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe
Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr
Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190
Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195
200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly
Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser
Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe
His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 30 3627 DNA Homo
sapiens CDS (96)...(872) 30 ggggcccgca cctctgggca gcagcggcag
ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac
tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser Arg Lys
Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg aga aat
tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg Arg Asn
Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga gag acc
agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly Glu Thr
Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac caa aca
gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His Gln Thr
Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt cag cac
aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu Gln His
Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att aaa ata
gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile Lys Ile
Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act ctt ctg
agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr Leu Leu
Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90 95 100 caa caa tat
ttt tat aaa att cca atc ctg gtc atc aac aaa gtc ttg 449 Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val Leu 105 110 115 cca
atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg cca ggt gtg 497 Pro
Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu Pro Gly Val 120 125
130 ata gca gca att gtc caa ctt cat aat gga acc aag tat aag aag ttt
545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr Lys Tyr Lys Lys Phe
135 140 145 150 cca cat tgg ttg gat aag tgg atg tta aca aga aag cag
ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met Leu Thr Arg Lys Gln
Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct gta ctg cat gca att
tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala Val Leu His Ala Ile
Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga tcc tac aga tac aag
ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg Ser Tyr Arg Tyr Lys
Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc caa caa aat aaa gaa
gat gcc tgg att gag cat gat gtt tgg 737 Gln Val Gln Gln Asn Lys Glu
Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210 aga atg gag att tat
gtg tct ctg gga att gtg gga ttg gca ata ctg 785 Arg Met Glu Ile Tyr
Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215 220 225 230 gct ctg
ttg gct gtg aca tct att cca tct gtg agt gac tct ttg aca 833 Ala Leu
Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser Leu Thr 235 240 245
tgg aga gaa ttt cac tat att cag gta aat aat ata taa aataacccta 882
Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile * 250 255
agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac cccataaaaa
942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt ccaattaata
atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg acaagtgttt
cctagacata aataaaaggc 1062 attaaaatat tctttgtttt tttttttttg
tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg aagtctcgct
ctgttgccca tgctggagta cagtggcacg atctcggctc 1182 actgcaacct
gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg 1242
ggattacagg cacccatcac catgtccagc taatttttgt atttttagta gagacagggt
1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc aaatgatccg
cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga gccaccacac
tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat ttgctaccca
tctaatgtga tattttagga atccaatatg 1482 catggtttat tatttcttaa
aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542 cttttatgtt
acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag 1602
agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt ttccagctta
1662 cattttatat acttactcac ttgaagtttc taaatattct tgtaatttta
aaactatctc 1722 agatttactg aggtttatct tctggtggta gattatccat
aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc tgacaagatt
caaaggacta aatttaattc agtcatgaac 1842 actgccaatt accgtttatg
ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902 caactatccc
ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag 1962
tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg tacaacattc
2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc actggagttc
tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg cctgttacac
atatgatgaa cactgctttt 2142 tagacttcat taggaattta ggactgcatc
ttgacaactg agcctattct actatatgta 2202 caatacctag cccataatag
gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262 gacatgtatt
tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta 2322
cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat ttgattgtat
2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc gatttgttct
ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc agaagtcgta
taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg tgaacaacta
tttttaagca actttatttg tgtagtgaca 2562 aagcatccca atgcaggctg
aaatgtttca tcacatctct ggatctctct attttgtgca 2622 gacattgaaa
aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa 2682
cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga atcacattag
2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga tttttgctat
aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg tacaaatcat
tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca gtccctgatt
ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca agagacaaag
cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982 gagaaagagt
agacaaagtt tttcatcacc tgccttcaaa gaaaggctgt gaattttgtt 3042
cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg ataggcaaaa
3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga taggaaaaca
atcatagata 3162 aaggatttcc aagcaacaga gcatatccag atgaggtagg
atgggataaa ctcttattga 3222 accaatcttc accaattttg tttttctttt
gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca atacacgcat
tgatttttgc ctggaataag tggatagata taaaacaatt 3342 tgtatggtat
acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat 3402
atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga ttagacatgg
3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt tcccagttgt
agaattactg 3522 tttacacaca tttttgttca atattgatat attttatcac
caacatttca agtttgtatt 3582 tgttaataaa atgattattc aaggaaaaaa
aaaaaaaaaa aaaaa 3627 31 258 PRT Homo sapiens 31 Met Glu Ser Arg
Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro
Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30
Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35
40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His
Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile
Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu
Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro
Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly
Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr
Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160
Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165
170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys
Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu
Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr
Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu
Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr
Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 32 3627
DNA Homo sapiens CDS (96)...(872) 32 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113 Met Glu Ser
Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg aag cct agg
aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg
Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag gac acg gga
gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys Asp Thr Gly
Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg cat ttg cac
caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu His Leu His
Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct tca gaa ctt
cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro Ser Glu Leu
Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac ttg cca att
aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His Leu Pro Ile
Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85 ctt tac act
ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401 Leu Tyr Thr
Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser
His 90 95 100 caa caa tat ttt tat aaa att cca atc ctg gtc atc aac
aaa gtc ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn
Lys Val Leu 105 110 115 cca atg gtt tcc atc act ctc ttg gca ttg gtt
tac ctg cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val
Tyr Leu Pro Gly Val 120 125 130 ata gca gca att gtc caa ctt cat aat
gga acc aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn
Gly Thr Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg ttg gat aag
tgg atg tta aca aga aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys
Trp Met Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt
ttt gct gta ctg cat gca att tat agt ctg tct tac 641 Leu Ser Phe Phe
Phe Ala Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg
agg cga tcc tac aga tac aag ttg cta aac tgg gca tat caa 689 Pro Met
Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195
cag gtc caa caa aat aaa gaa gat gcc tgg att gag cat gat gtt tgg 737
Gln Val Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200
205 210 aga atg gag att tat gtg tct ctg gga att gtg gga ttg gca ata
ctg 785 Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile
Leu 215 220 225 230 gct ctg ttg gct gtg aca tct att cca tct gtg agt
gac tct ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser
Asp Ser Leu Thr 235 240 245 tgg aga gaa ttt cac tat att cag gta aat
aat ata taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn
Asn Ile * 250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg
ttgactttac cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct
tatacttgtt ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct
tctaattagg acaagtgttt cctagacata aataaaaggc 1062 attaaaatat
tctttgtttt tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122
ttttgagatg aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc
1182 actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc
tgagtagctg 1242 ggattacagg cacccatcac catgtccagc taatttttgt
atttttagta gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc
tcctgacctc aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg
acagttgtga gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt
gttagacaat ttgctaccca tctaatgtga tattttagga atccaatatg 1482
catggtttat tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc
1542 cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt
gaaataatag 1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa
aggctgcttt ttccagctta 1662 cattttatat acttactcac ttgaagtttc
taaatattct tgtaatttta aaactatctc 1722 agatttactg aggtttatct
tctggtggta gattatccat aagaagagtg atgtgccaga 1782 atcactctgg
gatccttgtc tgacaagatt caaaggacta aatttaattc agtcatgaac 1842
actgccaatt accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg
1902 caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc
atctgattag 1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc
ttagagttgg tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag
cctgataacc actggagttc tttggtcctc 2082 attaaatagc tttcttcaca
cattgctctg cctgttacac atatgatgaa cactgctttt 2142 tagacttcat
taggaattta ggactgcatc ttgacaactg agcctattct actatatgta 2202
caatacctag cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat
2262 gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga
gaactggtta 2322 cactacaagt taccttggag attcatatat gaaaacgcaa
acttagctat ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa
ttgtgagttc gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg
aatagaggtc agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag
attacatagg tgaacaacta tttttaagca actttatttg tgtagtgaca 2562
aagcatccca atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca
2622 gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat
tttttaaaaa 2682 cataggccaa gttcattcac ttcattattc atttatcaaa
atcagagtga atcacattag 2742 tcgccttcac aactgataaa gatcactgaa
gtcaaattga tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga
atctaaaatg tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa
gtaagactca gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922
caaatacaca agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg
2982 gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt
gaattttgtt 3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa
ggtgaggagg ataggcaaaa 3102 agagtagaaa gatgtgaatg gacattgttg
agaaatgtga taggaaaaca atcatagata 3162 aaggatttcc aagcaacaga
gcatatccag atgaggtagg atgggataaa ctcttattga 3222 accaatcttc
accaattttg tttttctttt gcagagcaag ctaggaattg tttcccttct 3282
actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt
3342 tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg
ttgtcctgat 3402 atttaaaagc atactattcc tgccatgctt gaggaagaag
atactgaaga ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac
tgagatatgt tcccagttgt agaattactg 3522 tttacacaca tttttgttca
atattgatat attttatcac caacatttca agtttgtatt 3582 tgttaataaa
atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 3627 33 258 PRT Homo sapiens
33 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu
His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro
Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His
Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe
Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr
Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile
Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125
Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130
135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu
Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala
Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala
Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val
Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250
255 Asn Ile 34 3627 DNA Homo sapiens CDS (96)...(872) 34 ggggcccgca
cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60
gtgggtggct gaagccatac tattttatag aatta atg gaa agc aga aaa gac 113
Met Glu Ser Arg Lys Asp 1 5 atc aca aac caa gaa gaa ctt tgg aaa atg
aag cct agg aga aat tta 161 Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
Lys Pro Arg Arg Asn Leu 10 15 20 gaa gaa gac gat tat ttg cat aag
gac acg gga gag acc agc atg cta 209 Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr Gly Glu Thr Ser Met Leu 25 30 35 aaa aga cct gtg ctt ttg
cat ttg cac caa aca gcc cat gct gat gaa 257 Lys Arg Pro Val Leu Leu
His Leu His Gln Thr Ala His Ala Asp Glu 40 45 50 ttt gac tgc cct
tca gaa ctt cag cac aca cag gaa ctc ttt cca cag 305 Phe Asp Cys Pro
Ser Glu Leu Gln His Thr Gln Glu Leu Phe Pro Gln 55 60 65 70 tgg cac
ttg cca att aaa ata gct gct att ata gca tct ctg act ttt 353 Trp His
Leu Pro Ile Lys Ile Ala Ala Ile Ile Ala Ser Leu Thr Phe 75 80 85
ctt tac act ctt ctg agg gaa gta att cac ccc tta gca act tcc cat 401
Leu Tyr Thr Leu Leu Arg Glu Val Ile His Pro Leu Ala Thr Ser His 90
95 100 caa caa tat ttt tat aaa att cca atc ctg gtc atc aac aaa gtc
ttg 449 Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys Val
Leu 105 110 115 cca atg gtt tcc atc act ctc ttg gca ttg gtt tac ctg
cca ggt gtg 497 Pro Met Val Ser Ile Thr Leu Leu Ala Leu Val Tyr Leu
Pro Gly Val 120 125 130 ata gca gca att gtc caa ctt cat aat gga acc
aag tat aag aag ttt 545 Ile Ala Ala Ile Val Gln Leu His Asn Gly Thr
Lys Tyr Lys Lys Phe 135 140 145 150 cca cat tgg ttg gat aag tgg atg
tta aca aga aag cag ttt ggg ctt 593 Pro His Trp Leu Asp Lys Trp Met
Leu Thr Arg Lys Gln Phe Gly Leu 155 160 165 ctc agt ttc ttt ttt gct
gta ctg cat gca att tat agt ctg tct tac 641 Leu Ser Phe Phe Phe Ala
Val Leu His Ala Ile Tyr Ser Leu Ser Tyr 170 175 180 cca atg agg cga
tcc tac aga tac aag ttg cta aac tgg gca tat caa 689 Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala Tyr Gln 185 190 195 cag gtc
caa caa aat aaa gaa gat gcc tgg att gag cat gat gtt tgg 737 Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His Asp Val Trp 200 205 210
aga atg gag att tat gtg tct ctg gga att gtg gga ttg gca ata ctg 785
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala Ile Leu 215
220 225 230 gct ctg ttg gct gtg aca tct att cca tct gtg agt gac tct
ttg aca 833 Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser
Leu Thr 235 240 245 tgg aga gaa ttt cac tat att cag gta aat aat ata
taa aataacccta 882 Trp Arg Glu Phe His Tyr Ile Gln Val Asn Asn Ile
* 250 255 agaggtaaat cttctttttg tgtttatgat atagaatatg ttgactttac
cccataaaaa 942 ataacaaatg tttttcaaca gcaaagatct tatacttgtt
ccaattaata atgtgctctc 1002 ctgttgtttt ccctattgct tctaattagg
acaagtgttt cctagacata aataaaaggc 1062 attaaaatat tctttgtttt
tttttttttg tttgtttgtt ttttgtttgt ttgtttgttt 1122 ttttgagatg
aagtctcgct ctgttgccca tgctggagta cagtggcacg atctcggctc 1182
actgcaacct gcgcctcctg ggttcaggcg attctcttgc ctcagcctcc tgagtagctg
1242 ggattacagg cacccatcac catgtccagc taatttttgt atttttagta
gagacagggt 1302 tttcccatgt tggccaggct ggtctcgatc tcctgacctc
aaatgatccg cccacctcgg 1362 cctcccaaag tgctgggatg acagttgtga
gccaccacac tcagcctgct ctttctaata 1422 tttgaaactt gttagacaat
ttgctaccca tctaatgtga tattttagga atccaatatg 1482 catggtttat
tatttcttaa aaaaaatatt cttttacctg tcacctgaat ttagtaatgc 1542
cttttatgtt acacaactta gcactttcca gaaacaaaaa ctctctcctt gaaataatag
1602 agtttttatc taccaaagat atgctagtgt ctcatttcaa aggctgcttt
ttccagctta 1662 cattttatat acttactcac ttgaagtttc taaatattct
tgtaatttta aaactatctc 1722 agatttactg aggtttatct tctggtggta
gattatccat aagaagagtg atgtgccaga 1782 atcactctgg gatccttgtc
tgacaagatt caaaggacta aatttaattc agtcatgaac 1842 actgccaatt
accgtttatg ggtagacatc tttggaaatt tccacaaggt cagacattcg 1902
caactatccc ttctacatgt ccacacgtat actccaacac tttattaggc atctgattag
1962 tttggaaagt atgcctccat ctgaattagt ccagtgtggc ttagagttgg
tacaacattc 2022 tcacagaatt tcctaatttt gtaggttcag cctgataacc
actggagttc tttggtcctc 2082 attaaatagc tttcttcaca cattgctctg
cctgttacac atatgatgaa cactgctttt 2142 tagacttcat taggaattta
ggactgcatc ttgacaactg agcctattct actatatgta 2202 caatacctag
cccataatag gtatacaata cacatttggt aaaactaatt ttcaaccaat 2262
gacatgtatt tttcaactag taacctagaa atgtttcact taaaatctga gaactggtta
2322 cactacaagt taccttggag attcatatat gaaaacgcaa acttagctat
ttgattgtat 2382 tcactgggac ttaagaatgc gcctgaataa ttgtgagttc
gatttgttct ggcaggctaa 2442 tgaccatttc cagtaaagtg aatagaggtc
agaagtcgta taaaagaggt gttgtcagaa 2502 caccgttgag attacatagg
tgaacaacta tttttaagca actttatttg tgtagtgaca 2562 aagcatccca
atgcaggctg aaatgtttca tcacatctct ggatctctct attttgtgca 2622
gacattgaaa aaattgttca tattatttcc atgttatcag aatatttgat tttttaaaaa
2682 cataggccaa gttcattcac ttcattattc atttatcaaa atcagagtga
atcacattag 2742 tcgccttcac aactgataaa gatcactgaa gtcaaattga
tttttgctat aatcttcaat 2802 ctacctatat ttaattgaga atctaaaatg
tacaaatcat tgtgttgatt ctgcagtgat 2862 cctgctataa gtaagactca
gtccctgatt ttaggtatcc tgtgaaaagc agaattaaga 2922 caaatacaca
agagacaaag cacaaaaaat aaatatcata aggggatgaa caaaatggtg 2982
gagaaagagt agacaaagtt tttgatcacc tgccttcaaa gaaaggctgt gaattttgtt
3042 cacttagaca gcttggagac aagaaattac ccaaaagtaa ggtgaggagg
ataggcaaaa 3102 agagcagaaa gatgtgaatg gacattgttg agaaatgtga
taggaaaaca atcatagata 3162 aaggatttcc aagcaactga gcatatccag
atgaggtagg atgggataaa ctcttattga 3222 accaatcttc accaattttg
tttttctttt gcagagcaag ctaggaattg tttcccttct 3282 actgggcaca
atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt 3342
tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
3402 atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga
ttagacatgg 3462 ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt
tcccagttgt agaattactg 3522 tttacacaca tttttgttca atattgatat
attttatcac caacatttca agtttgtatt 3582 tgttaataaa atgattattc
aaggaaaaaa aaaaaaaaaa aaaaa 3627 35 258 PRT Homo sapiens 35 Met Glu
Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15
Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20
25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His
Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile
Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr
Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln
Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val
Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu
Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr
Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150
155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His
Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg
Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn
Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu
Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala
Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser
Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile
36 339 PRT Homo sapiens 36 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln
Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu
Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu
Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala
Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu
Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80
Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85
90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile
Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu
Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val
Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp
Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu
Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu
Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn
Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205
Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210
215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro
Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr
Ile Gln Ser Lys 245 250 255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr
Ile His Ala Leu Ile Phe 260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile
Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala
Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu
Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile
305 310 315 320 Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr
Glu Ile Cys 325 330 335 Ser Gln Leu 37 258 PRT Homo sapiens 37 Met
Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10
15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr
20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu
His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu
Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro
Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr
Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His
Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys
Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr
Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140
Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145
150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu
His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr
Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln
Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met
Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp
Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn
Ile 38 282 PRT Homo sapiens 38 Met Glu Ser Arg Lys Asp Ile Thr Asn
Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu
Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met
Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His
Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln
Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70
75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile
His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile
Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile
Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala
Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro
His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe
Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr
Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190
Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195
200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly
Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser
Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe
His Tyr Ile Gln Ile Ile 245 250 255 His Lys Lys Ser Asp Val Pro Glu
Ser Leu Trp Asp Pro Cys Leu Thr 260 265 270 Arg Phe Lys Gly Leu Asn
Leu Ile Gln Ser 275 280 39 258 PRT Homo sapiens 39 Met Glu Ser Arg
Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro
Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30
Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35
40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His
Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile
Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu
Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro
Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly
Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr
Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160
Arg Lys Gln Phe Gly Leu Leu Ser Leu Phe Phe Ala Val Leu His Ala 165
170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys
Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu
Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr
Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu
Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr
Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 40 339
PRT Homo sapiens 40 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu
Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp
Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg
Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu
Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe
Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala
Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95
Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100
105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala
Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu
His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp
Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser
Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr
Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala
Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu
His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220
Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225
230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln
Ser Lys 245 250 255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His
Ala Leu Ile Phe 260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln
Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala Val Phe
Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu
Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile 305 310 315 320 Arg His Gly
Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser
Gln Leu 41 270 PRT Mus musculus 41 Gln Leu Phe Pro Met Trp Arg Phe
Pro Phe Tyr Leu Ser Ser Val Leu 1 5 10 15 Cys Ile Phe Phe Phe Val
Tyr Cys Ala Ile Arg Glu Val Ile Tyr Pro 20 25 30 Tyr Val Asn Gly
Lys Thr Asp Ala Thr Tyr Arg Leu Ala Ile Ser Ile 35 40 45 Pro Asn
Arg Val Phe Pro Ile Thr Ala Leu Ile Leu Leu Ala Leu Val 50 55 60
Tyr Leu Pro Gly Ile Leu Ala Ala Ile Leu Gln Leu Tyr Arg Gly Thr 65
70 75 80 Lys Tyr Arg Arg Phe Pro Asn Trp Leu Asp His Trp Met Leu
Cys Arg 85 90 95 Lys Gln Leu Gly Leu Val Ala Leu Gly Phe Ala Phe
Leu His Val Ile 100 105 110 Tyr Thr Leu Val Ile Pro Ile Arg Tyr Tyr
Val Arg Trp Arg Leu Arg 115 120 125 Asn Ala Thr Ile Thr Gln Ala Leu
Thr Asn Lys Asp Ser Pro Phe Ile 130 135 140 Thr Ser Tyr Ala Trp Ile
Asn Asp Ser Tyr Leu Ala Leu Gly Ile Leu 145 150 155 160 Gly Phe Phe
Leu Phe Leu Leu Leu Gly Ile Thr Ser Leu Pro Ser Val 165 170 175 Ser
Asn Met Val Asn Trp Arg Glu Phe Arg Phe Val Gln Ser Lys Leu 180 185
190 Gly Tyr Leu Thr Leu Val Leu Cys Thr Ala His Thr Leu Val Tyr Gly
195 200 205 Gly Lys Arg Phe Leu Ser Pro Ser Ile Leu Arg Trp Ser Leu
Pro Ser 210 215 220 Ala Tyr Ile Leu Ala Leu Ile Ile Pro Cys Ala Val
Leu Val Leu Lys 225 230 235 240 Cys Ile Leu Ile Met Pro Cys Ile Asp
Lys Thr Leu Thr Arg Ile Arg 245 250 255 Gln Gly Trp Glu Arg Asn Ser
Lys Tyr Thr Gln Ser Ala Leu 260 265 270 42 339 PRT Homo sapiens 42
Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5
10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp
Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His
Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser
Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu
Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu
Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser
His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn
Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val
Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135
140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr
145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val
Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser
Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln
Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg
Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile
Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser
Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250 255
Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe 260
265 270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr
Pro 275 280 285 Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val
Leu Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys
Lys Ile Leu Lys Ile 305 310 315 320 Arg His Gly Trp Glu Asp Val Thr
Lys Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser Gln Leu 43 259 PRT
Rattus norvegicus 43 Leu Leu Pro Ser Trp Lys Val Pro Thr Leu Leu
Ala Leu Gly Leu Ser 1 5 10 15 Thr Gln Ser Tyr Ala Tyr Asn Phe Ile
Arg Asp Val Leu Gln Pro Tyr 20 25 30 Ile Arg Lys Asp Glu Asn Lys
Phe Tyr Lys Met Pro Leu Ser Val Val 35 40 45 Asn Thr Thr Ile Pro
Cys Val Ala Tyr Val Leu Leu Ser Leu Val Tyr 50 55 60 Leu Pro Gly
Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly Thr Lys 65 70 75 80 Tyr
Gln Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln His Arg Lys 85 90
95 Gln Ile Gly Leu Leu Ser Phe Phe Phe Ala Met Leu His Ala Leu Tyr
100 105 110 Ser Phe Cys Leu Pro Leu Arg Arg Ser His Arg Tyr Asp Leu
Val Asn 115 120 125 Leu Ala Val Lys Gln Val Leu Ala Asn Lys Ser Arg
Leu Trp Val Glu 130 135 140 Glu Glu Val Trp Arg Met Glu Ile Tyr Leu
Ser Leu Gly Val Leu Ala 145 150 155 160 Leu Gly Met Leu Ser Leu Leu
Ala Val Thr Ser Ile Pro Ser Ile Ala 165 170 175 Asn Ser Leu Asn Trp
Lys Glu Phe Ser Phe Val Gln Ser Thr Leu Gly 180 185 190 Phe Val Ala
Leu Met Leu Ser Thr Met His Thr Leu Thr Tyr Gly Trp 195 200 205 Thr
Arg Ala Phe Glu Glu Asn His Tyr Lys Phe Tyr Leu Pro Pro Thr 210 215
220 Phe Thr Leu Thr Leu Leu Leu Pro Cys Val Ile Ile Leu Ala Lys Gly
225 230 235 240 Leu Phe Leu Leu Pro Cys Leu Ser His Arg Leu Thr Lys
Ile Arg Arg 245 250 255 Gly Trp Glu 44 14 PRT Tetanus toxoid 44 Gln
Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu 1 5 10 45 21
PRT Plasmodium falciparum 45 Asp Ile Glu Lys Lys Ile Ala Lys Met
Glu Lys Ala Ser Ser Val Phe 1 5 10 15 Asn Val Val Asn Ser 20 46 16
PRT Streptococcus 46 Gly Ala Val Asp Ser Ile Leu Gly Gly Val Ala
Thr Tyr Gly Ala Ala 1 5 10 15 47 13 PRT Artificial Sequence Pan-DR
binding Epitope 47 Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala
Ala 1 5 10 48 14 DNA Artificial Sequence cDNA synthesis primer 48
ttttgatcaa gctt 14 49 42 DNA Artificial Sequence primer 49
ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag 42 50 12 DNA
Artificial Sequence primer 50 gatcctgccc gg 12 51 40 DNA Artificial
Sequence primer 51 gtaatacgac tcactatagg gcagcgtggt cgcggccgag 40
52 10 DNA Artificial Sequence primer 52 gatcctcggc 10 53 22 DNA
Artificial Sequence PCR-primer 53 ctaatacgac tcactatagg gc 22 54 22
DNA Artificial Sequence Nested primer 54 tcgagcggcc gcccgggcag ga
22 55 20 DNA Artificial Sequence Nested primer 55 agcgtggtcg
cggccgagga 20 56 25 DNA Artificial Sequence primer 56 atatcgccgc
gctcgtcgtc gacaa 25 57 26 DNA Artificial Sequence primer 57
agccacacgc agctcattgt agaagg 26 58 24 DNA Artificial Sequence
primer 58 actttgttga tgaccaggat tgga 24 59 24 DNA Artificial
Sequence primer 59 cagaacttca gcacacacag gaac 24 60 24 DNA
Artificial Sequence Epitope Tag 60 gattacaagg atgacgacga taag 24 61
4 PRT Homo sapiens 61 Asn Gly Thr Lys 1 62 4 PRT Homo sapiens 62
Asn Lys Thr Glu 1 63 4 PRT Homo sapiens 63 Ser Arg Lys Asp 1 64 4
PRT Homo sapiens 64 Thr Asn Gln Glu 1 65 4 PRT Homo sapiens 65 Ser
Val Ser Asp 1 66 4 PRT Homo sapiens 66 Thr Trp Arg Glu 1 67 9 PRT
Homo sapiens 67 Arg Arg Asn Leu Glu Glu Asp Asp Tyr 1 5 68 6 PRT
Homo sapiens 68 Gly Val Ile Ala Ala Ile 1 5 69 6 PRT Homo sapiens
69 Gly Thr Ile His Ala Leu 1 5 70 17 PRT Homo sapiens 70 Arg Lys
Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met Lys Pro Arg 1 5 10 15
Arg 71 339 PRT Homo sapiens 71 Met Glu Ser Arg Lys Asp Ile Thr Asn
Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu
Glu Asp Asp
Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg
Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu
Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe
Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala
Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95
Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100
105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala
Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu
His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp
Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser
Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr
Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala
Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu
His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220
Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225
230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln
Ser Lys 245 250 255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His
Ala Leu Ile Phe 260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln
Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala Val Phe
Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu
Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile 305 310 315 320 Arg His Gly
Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser
Gln Leu 72 12 PRT Homo sapiens 72 Trp Arg Glu Phe His Tyr Ile Gln
Val Asn Asn Ile 1 5 10 73 13 PRT Homo sapiens 73 Thr Trp Arg Glu
Phe His Tyr Ile Gln Val Asn Asn Ile 1 5 10 74 18 PRT Homo sapiens
74 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn
1 5 10 15 Asn Ile 75 36 PRT Homo sapiens 75 Trp Arg Glu Phe His Tyr
Ile Gln Ile Ile His Lys Lys Ser Asp Val 1 5 10 15 Pro Glu Ser Leu
Trp Asp Pro Cys Leu Thr Arg Phe Lys Gly Leu Asn 20 25 30 Leu Ile
Gln Ser 35 76 37 PRT Homo sapiens 76 Thr Trp Arg Glu Phe His Tyr
Ile Gln Ile Ile His Lys Lys Ser Asp 1 5 10 15 Val Pro Glu Ser Leu
Trp Asp Pro Cys Leu Thr Arg Phe Lys Gly Leu 20 25 30 Asn Leu Ile
Gln Ser 35 77 42 PRT Homo sapiens 77 Val Ser Asp Ser Leu Thr Trp
Arg Glu Phe His Tyr Ile Gln Ile Ile 1 5 10 15 His Lys Lys Ser Asp
Val Pro Glu Ser Leu Trp Asp Pro Cys Leu Thr 20 25 30 Arg Phe Lys
Gly Leu Asn Leu Ile Gln Ser 35 40 78 17 PRT Homo sapiens 78 Arg Lys
Gln Phe Gly Leu Leu Ser Leu Phe Phe Ala Val Leu His Ala 1 5 10 15
Ile 79 19 PRT Homo sapiens 79 Thr Arg Lys Gln Phe Gly Leu Leu Ser
Leu Phe Phe Ala Val Leu His 1 5 10 15 Ala Ile Tyr 80 29 PRT Homo
sapiens 80 Asp Lys Trp Met Leu Thr Arg Lys Gln Phe Gly Leu Leu Ser
Leu Phe 1 5 10 15 Phe Ala Val Leu His Ala Ile Tyr Ser Leu Ser Tyr
Pro 20 25 81 3627 DNA Homo sapiens 81 ggggcccgca cctctgggca
gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60 gtgggtggct
gaagccatac tattttatag aattaatgga aagcagaaaa gacatcacaa 120
accaagaaga actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc
180 ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat
ttgcaccaaa 240 cagcccatgc tgatgaattt gactgccctt cagaacttca
gcacacacag gaactctttc 300 cacagtggca cttgccaatt aaaatagctg
ctattatagc atctctgact tttctttaca 360 ctcttctgag ggaagtaatt
caccccttag caacttccca tcaacaatat ttttataaaa 420 ttccaatcct
ggtcatcaac aaagtcttgc caatggtttc catcactctc ttggcattgg 480
tttacctgcc aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga
540 agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg
cttctcagtt 600 tcttttttgc tgtactgcat gcaatttata gtctgtctta
cccaatgagg cgatcctaca 660 gatacaagtt gctaaactgg gcatatcaac
aggtccaaca aaataaagaa gatgcctgga 720 ttgagcatga tgtttggaga
atggagattt atgtgtctct gggaattgtg ggattggcaa 780 tactggctct
gttggctgtg acatctattc catctgtgag tgactctttg acatggagag 840
aatttcacta tattcaggta aataatatat aaaataaccc taagaggtaa atcttctttt
900 tgtgtttatg atatagaata tgttgacttt accccataaa aaataacaaa
tgtttttcaa 960 cagcaaagat cttatacttg ttccaattaa taatgtgctc
tcctgttgtt ttccctattg 1020 cttctaatta ggacaagtgt ttcctagaca
taaataaaag gcattaaaat attctttgtt 1080 tttttttttt tgtttgtttg
ttttttgttt gtttgtttgt ttttttgaga tgaagtctcg 1140 ctctgttgcc
catgctggag tacagtggca cgatctcggc tcactgcaac ctgcgcctcc 1200
tgggttcagg cgattctctt gcctcagcct cctgagtagc tgggattaca ggcacccatc
1260 accatgtcca gctaattttt gtatttttag tagagacagg gttttcccat
gttggccagg 1320 ctggtctcga tctcctgacc tcaaatgatc cgcccacctc
ggcctcccaa agtgctggga 1380 tgacagttgt gagccaccac actcagcctg
ctctttctaa tatttgaaac ttgttagaca 1440 atttgctacc catctaatgt
gatattttag gaatccaata tgcatggttt attatttctt 1500 aaaaaaaata
ttcttttacc tgtcacctga atttagtaat gccttttatg ttacacaact 1560
tagcactttc cagaaacaaa aactctctcc ttgaaataat agagttttta tctaccaaag
1620 atatgctagt gtctcatttc aaaggctgct ttttccagct tacattttat
atacttactc 1680 acttgaagtt tctaaatatt cttgtaattt taaaactatc
tcagatttac tgaggtttat 1740 cttctggtgg tagattatcc ataagaagag
tgatgtgcca gaatcactct gggatccttg 1800 tctgacaaga ttcaaaggac
taaatttaat tcagtcatga acactgccaa ttaccgttta 1860 tgggtagaca
tctttggaaa tttccacaag gtcagacatt cgcaactatc ccttctacat 1920
gtccacacgt atactccaac actttattag gcatctgatt agtttggaaa gtatgcctcc
1980 atctgaatta gtccagtgtg gcttagagtt ggtacaacat tctcacagaa
tttcctaatt 2040 ttgtaggttc agcctgataa ccactggagt tctttggtcc
tcattaaata gctttcttca 2100 cacattgctc tgcctgttac acatatgatg
aacactgctt tttagacttc attaggaatt 2160 taggactgca tcttgacaac
tgagcctatt ctactatatg tacaatacct agcccataat 2220 aggtatacaa
tacacatttg gtaaaactaa ttttcaacca atgacatgta tttttcaact 2280
agtaacctag aaatgtttca cttaaaatct gagaactggt tacactacaa gttaccttgg
2340 agattcatat atgaaaacgc aaacttagct atttgattgt attcactggg
acttaagaat 2400 gcgcctgaat aattgtgagt tcgatttgtt ctggcaggct
aatgaccatt tccagtaaag 2460 tgaatagagg tcagaagtcg tataaaagag
gtgttgtcag aacaccgttg agattacata 2520 ggtgaacaac tatttttaag
caactttatt tgtgtagtga caaagcatcc caatgcaggc 2580 tgaaatgttt
catcacatct ctggatctct ctattttgtg cagacattga aaaaattgtt 2640
catattattt ccatgttatc agaatatttg attttttaaa aacataggcc aagttcattc
2700 acttcattat tcatttatca aaatcagagt gaatcacatt agtcgccttc
acaactgata 2760 aagatcactg aagtcaaatt gatttttgct ataatcttca
atctacctat atttaattga 2820 gaatctaaaa tgtacaaatc attgtgttga
ttctgcagtg atcctgctat aagtaagact 2880 cagtccctga ttttaggtat
cctgtgaaaa gcagaattaa gacaaataca caagagacaa 2940 agcacaaaaa
ataaatatca taaggggatg aacaaaatgg tggagaaaga gtagacaaag 3000
tttttgatca cctgccttca aagaaaggct gtgaattttg ttcacttaga cagcttggag
3060 acaagaaatt acccaaaagt aaggtgagga ggataggcaa aaagagcaga
aagatgtgaa 3120 tggacattgt tgagaaatgt gataggaaaa caatcataga
taaaggattt ccaagcaaca 3180 gagcatatcc agatgaggta ggatgggata
aactcttatt gaaccaatct tcaccaattt 3240 tgtttttctt ttgcagagca
agctaggaat tgtttccctt ctactgggca caatacacgc 3300 attgattttt
gcctggaata agtggataga tataaaacaa tttgtatggt atacacctcc 3360
aacttttatg atagctgttt tccttccaat tgttgtcctg atatttaaaa gcatactatt
3420 cctgccatgc ttgaggaaga agatactgaa gattagacat ggttgggaag
acgtcaccaa 3480 aattaacaaa actgagatat gttcccagtt gtagaattac
tgtttacaca catttttgtt 3540 caatattgat atattttatc accaacattt
caagtttgta tttgttaata aaatgattat 3600 tcaaggaaaa aaaaaaaaaa aaaaaaa
3627 82 1193 DNA Homo sapiens 82 ccgagactca cggtcaagct aaggcgaaga
gtgggtggct gaagccatac tattttatag 60 aattaatgga aagcagaaaa
gacatcacaa accaagaaga actttggaaa atgaagccta 120 ggagaaattt
agaagaagac gattatttgc ataaggacac gggagagacc agcatgctaa 180
aaagacctgt gcttttgcat ttgcaccaaa cagcccatgc tgatgaattt gactgccctt
240 cagaacttca gcacacacag gaactctttc cacagtggca cttgccaatt
aaaatagctg 300 ctattatagc atctctgact tttctttaca ctcttctgag
ggaagtaatt caccctttag 360 caacttccca tcaacaatat ttttataaaa
ttccaatcct ggtcatcaac aaagtcttgc 420 caatggtttc catcactctc
ttggcattgg tttacctgcc aggtgtgata gcagcaattg 480 tccaacttca
taatggaacc aagtataaga agtttccaca ttggttggat aagtggatgt 540
taacaagaaa gcagtttggg cttctcagtt tcttttttgc tgtactgcat gcaatttata
600 gtctgtctta cccaatgagg cgatcctaca gatacaagtt gctaaactgg
gcatatcaac 660 aggtccaaca aaataaagaa gatgcctgga ttgagcatga
tgtttggaga atggagattt 720 atgtgtctct gggaattgtg ggattggcaa
tactggctct gttggctgtg acatctattc 780 catctgtgag tgactctttg
acatggagag aatttcacta tattcagagc aagctaggaa 840 ttgtttccct
tctactgggc acaatacacg cattgatttt tgcctggaat aagtggatag 900
atataaaaca atttgtatgg tatacacctc caacttttat gatagctgtt ttccttccaa
960 ttgttgtcct gatatttaaa agcatactat tcctgccatg cttgaggaag
aagatactga 1020 agattagaca tggttgggaa gacgtcacca aaattaacaa
aactgagata tgttcccagt 1080 tgtagaatta ctgtttacac acatttttgt
tcaatattga tatattttat caccaacatt 1140 tcaagtttgt atttgttaat
aaaatgatta caaggaaaaa aaaaaaaaaa aaa 1193 83 3627 DNA Homo sapiens
83 ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct
aaggcgaaga 60 gtgggtggct gaagccatac tattttatag aattaatgga
aagcagaaaa gacatcacaa 120 accaagaaga actttggaaa atgaagccta
ggagaaattt agaagaagac gattatttgc 180 ataaggacac gggagagacc
agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa 240 cagcccatgc
tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc 300
cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact tttctttaca
360 ctcttctgag ggaagtaatt caccccttag caacttccca tcaacaatat
ttttataaaa 420 ttccaatcct ggtcatcaac aaagtcttgc caatggtttc
catcactctc ttggcattgg 480 tttacctgcc aggtgtgata gcagcaattg
tccaacttca taatggaacc aagtataaga 540 agtttccaca ttggttggat
aagtggatgt taacaagaaa gcagtttggg cttctcagtt 600 tcttttttgc
tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca 660
gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa gatgcctgga
720 ttgagcatga tgtttggaga atggagattt atgtgtctct gggaattgtg
ggattggcaa 780 tactggctct gttggctgtg acatctattc catctgtgag
tgactctttg acatggagag 840 aatttcacta tattcaggta aataatatat
aaaataaccc taagaggtaa atcttctttt 900 tgtgtttatg atatagaata
tgttgacttt accccataaa aaataacaaa tgtttttcaa 960 cagcaaagat
cttatacttg ttccaattaa taatgtgctc tcctgttgtt ttccctattg 1020
cttctaatta ggacaagtgt ttcctagaca taaataaaag gcattaaaat attctttgtt
1080 tttttttttt tgtttgtttg ttttttgttt gtttgtttgt ttttttgaga
tgaagtctcg 1140 ctctgttgcc catgctggag tacagtggca cgatctcggc
tcactgcaac ctgcgcctcc 1200 tgggttcagg cgattctctt gcctcagcct
cctgagtagc tgggattaca ggcacccatc 1260 accatgtcca gctaattttt
gtatttttag tagagacagg gttttcccat gttggccagg 1320 ctggtctcga
tctcctgacc tcaaatgatc cgcccacctc ggcctcccaa agtgctggga 1380
tgacagttgt gagccaccac actcagcctg ctctttctaa tatttgaaac ttgttagaca
1440 atttgctacc catctaatgt gatattttag gaatccaata tgcatggttt
attatttctt 1500 aaaaaaaata ttcttttacc tgtcacctga atttagtaat
gccttttatg ttacacaact 1560 tagcactttc cagaaacaaa aactctctcc
ttgaaataat agagttttta tctaccaaag 1620 atatgctagt gtctcatttc
aaaggctgct ttttccagct tacattttat atacttactc 1680 acttgaagtt
tctaaatatt cttgtaattt taaaactatc tcagatttac tgaggtttat 1740
cttctggtgg tagattatcc ataagaagag tgatgtgcca gaatcactct gggatccttg
1800 tctgacaaga ttcaaaggac taaatttaat tcagtcatga acactgccaa
ttaccgttta 1860 tgggtagaca tctttggaaa tttccacaag gtcagacatt
cgcaactatc ccttctacat 1920 gtccacacgt atactccaac actttattag
gcatctgatt agtttggaaa gtatgcctcc 1980 atctgaatta gtccagtgtg
gcttagagtt ggtacaacat tctcacagaa tttcctaatt 2040 ttgtaggttc
agcctgataa ccactggagt tctttggtcc tcattaaata gctttcttca 2100
cacattgctc tgcctgttac acatatgatg aacactgctt tttagacttc attaggaatt
2160 taggactgca tcttgacaac tgagcctatt ctactatatg tacaatacct
agcccataat 2220 aggtatacaa tacacatttg gtaaaactaa ttttcaacca
atgacatgta tttttcaact 2280 agtaacctag aaatgtttca cttaaaatct
gagaactggt tacactacaa gttaccttgg 2340 agattcatat atgaaaacgc
aaacttagct atttgattgt attcactggg acttaagaat 2400 gcgcctgaat
aattgtgagt tcgatttgtt ctggcaggct aatgaccatt tccagtaaag 2460
tgaatagagg tcagaagtcg tataaaagag gtgttgtcag aacaccgttg agattacata
2520 ggtgaacaac tatttttaag caactttatt tgtgtagtga caaagcatcc
caatgcaggc 2580 tgaaatgttt catcacatct ctggatctct ctattttgtg
cagacattga aaaaattgtt 2640 catattattt ccatgttatc agaatatttg
attttttaaa aacataggcc aagttcattc 2700 acttcattat tcatttatca
aaatcagagt gaatcacatt agtcgccttc acaactgata 2760 aagatcactg
aagtcaaatt gatttttgct ataatcttca atctacctat atttaattga 2820
gaatctaaaa tgtacaaatc attgtgttga ttctgcagtg atcctgctat aagtaagact
2880 cagtccctga ttttaggtat cctgtgaaaa gcagaattaa gacaaataca
caagagacaa 2940 agcacaaaaa ataaatatca taaggggatg aacaaaatgg
tggagaaaga gtagacaaag 3000 tttttgatca cctgccttca aagaaaggct
gtgaattttg ttcacttaga cagcttggag 3060 acaagaaatt acccaaaagt
aaggtgagga ggataggcaa aaagagcaga aagatgtgaa 3120 tggacattgt
tgagaaatgt gataggaaaa caatcataga taaaggattt ccaagcaaca 3180
gagcatatcc agatgaggta ggatgggata aactcttatt gaaccaatct tcaccaattt
3240 tgtttttctt ttgcagagca agctaggaat tgtttccctt ctactgggca
caatacacgc 3300 attgattttt gcctggaata agtggataga tataaaacaa
tttgtatggt atacacctcc 3360 aacttttatg atagctgttt tccttccaat
tgttgtcctg atatttaaaa gcatactatt 3420 cctgccatgc ttgaggaaga
agatactgaa gattagacat ggttgggaag acgtcaccaa 3480 aattaacaaa
actgagatat gttcccagtt gtagaattac tgtttacaca catttttgtt 3540
caatattgat atattttatc accaacattt caagtttgta tttgttaata aaatgattat
3600 tcaaggaaaa aaaaaaaaaa aaaaaaa 3627 84 258 PRT Homo sapiens 84
Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5
10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp
Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His
Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser
Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu
Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu
Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser
His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn
Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val
Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135
140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr
145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val
Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser
Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln
Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg
Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile
Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser
Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255
Asn Ile 85 339 PRT Homo sapiens 85 Met Glu Ser Arg Lys Asp Ile Thr
Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu
Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser
Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala
His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60
Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65
70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val
Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys
Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser
Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala
Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe
Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln
Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile
Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185
190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp
195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu
Gly Ile 210
215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro
Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr
Ile Gln Ser Lys 245 250 255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr
Ile His Ala Leu Ile Phe 260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile
Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala
Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu
Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile 305 310 315 320 Arg
His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330
335 Ser Gln Leu 86 258 PRT Homo sapiens 86 Met Glu Ser Arg Lys Asp
Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg
Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu
Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45
Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50
55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala
Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu
Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr
Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val
Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile
Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys
Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys
Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175
Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180
185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala
Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser
Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val
Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg
Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn Ile 87 1365 DNA
Homo sapiens 87 ggggcccgca cctctgggca gcagcggcag ccgagactca
cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac tattttatag
aattaatgga aagcagaaaa gacatcacaa 120 accaagaaga actttggaaa
atgaagccta ggagaaattt agaagaagac gattatttgc 180 ataaggacac
gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa 240
cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc
300 cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact
tttctttaca 360 ctcttctgag ggaagtaatt caccctttag caacttccca
tcaacaatat ttttataaaa 420 ttccaatcct ggtcatcaac aaagtcttgc
caatggtttc catcactctc ttggcattgg 480 tttacctgcc aggtgtgata
gcagcaattg tccaacttca taatggaacc aagtataaga 540 agtttccaca
ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt 600
tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca
660 gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa
gatgcctgga 720 ttgagcatga tgtttggaga atggagattt atgtgtctct
gggaattgtg ggattggcaa 780 tactggctct gttggctgtg acatctattc
catctgtgag tgactctttg acatggagag 840 aatttcacta tattcagatt
atccataaga agagtgatgt gccagaatca ctctgggatc 900 cttgtctgac
aagattcaaa ggactaaatt taattcagtc atgaacactg ccaattaccg 960
tttatgggta gacatctttg gaaatttcca caagagcaag ctaggaattg tttcccttct
1020 actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata
taaaacaatt 1080 tgtatggtat acacctccaa cttttatgat agctgttttc
cttccaattg ttgtcctgat 1140 atttaaaagc atactattcc tgccatgctt
gaggaagaag atactgaaga ttagacatgg 1200 ttgggaagac gtcaccaaaa
ttaacaaaac tgagatatgt tcccagttgt agaattactg 1260 tttacacaca
tttttgttca atattgatat attttatcac caacatttca agtttgtatt 1320
tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 1365 88 3627 DNA
Homo sapiens 88 ggggcccgca cctctgggca gcagcggcag ccgagactca
cggtcaagct aaggcgaaga 60 gtgggtggct gaagccatac tattttatag
aattaatgga aagcagaaaa gacatcacaa 120 accaagaaga actttggaaa
atgaagccta ggagaaattt agaagaagac gattatttgc 180 ataaggacac
gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa 240
cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc
300 cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact
tttctttaca 360 ctcttctgag ggaagtaatt caccccttag caacttccca
tcaacaatat ttttataaaa 420 ttccaatcct ggtcatcaac aaagtcttgc
caatggtttc catcactctc ttggcattgg 480 tttacctgcc aggtgtgata
gcagcaattg tccaacttca taatggaacc aagtataaga 540 agtttccaca
ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt 600
tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca
660 gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa
gatgcctgga 720 ttgagcatga tgtttggaga atggagattt atgtgtctct
gggaattgtg ggattggcaa 780 tactggctct gttggctgtg acatctattc
catctgtgag tgactctttg acatggagag 840 aatttcacta tattcaggta
aataatatat aaaataaccc taagaggtaa atcttctttt 900 tgtgtttatg
atatagaata tgttgacttt accccataaa aaataacaaa tgtttttcaa 960
cagcaaagat cttatacttg ttccaattaa taatgtgctc tcctgttgtt ttccctattg
1020 cttctaatta ggacaagtgt ttcctagaca taaataaaag gcattaaaat
attctttgtt 1080 tttttttttt tgtttgtttg ttttttgttt gtttgtttgt
ttttttgaga tgaagtctcg 1140 ctctgttgcc catgctggag tacagtggca
cgatctcggc tcactgcaac ctgcgcctcc 1200 tgggttcagg cgattctctt
gcctcagcct cctgagtagc tgggattaca ggcacccatc 1260 accatgtcca
gctaattttt gtatttttag tagagacagg gttttcccat gttggccagg 1320
ctggtctcga tctcctgacc tcaaatgatc cgcccacctc ggcctcccaa agtgctggga
1380 tgacagttgt gagccaccac actcagcctg ctctttctaa tatttgaaac
ttgttagaca 1440 atttgctacc catctaatgt gatattttag gaatccaata
tgcatggttt attatttctt 1500 aaaaaaaata ttcttttacc tgtcacctga
atttagtaat gccttttatg ttacacaact 1560 tagcactttc cagaaacaaa
aactctctcc ttgaaataat agagttttta tctaccaaag 1620 atatgctagt
gtctcatttc aaaggctgct ttttccagct tacattttat atacttactc 1680
acttgaagtt tctaaatatt cttgtaattt taaaactatc tcagatttac tgaggtttat
1740 cttctggtgg tagattatcc ataagaagag tgatgtgcca gaatcactct
gggatccttg 1800 tctgacaaga ttcaaaggac taaatttaat tcagtcatga
acactgccaa ttaccgttta 1860 tgggtagaca tctttggaaa tttccacaag
gtcagacatt cgcaactatc ccttctacat 1920 gtccacacgt atactccaac
actttattag gcatctgatt agtttggaaa gtatgcctcc 1980 atctgaatta
gtccagtgtg gcttagagtt ggtacaacat tctcacagaa tttcctaatt 2040
ttgtaggttc agcctgataa ccactggagt tctttggtcc tcattaaata gctttcttca
2100 cacattgctc tgcctgttac acatatgatg aacactgctt tttagacttc
attaggaatt 2160 taggactgca tcttgacaac tgagcctatt ctactatatg
tacaatacct agcccataat 2220 aggtatacaa tacacatttg gtaaaactaa
ttttcaacca atgacatgta tttttcaact 2280 agtaacctag aaatgtttca
cttaaaatct gagaactggt tacactacaa gttaccttgg 2340 agattcatat
atgaaaacgc aaacttagct atttgattgt attcactggg acttaagaat 2400
gcgcctgaat aattgtgagt tcgatttgtt ctggcaggct aatgaccatt tccagtaaag
2460 tgaatagagg tcagaagtcg tataaaagag gtgttgtcag aacaccgttg
agattacata 2520 ggtgaacaac tatttttaag caactttatt tgtgtagtga
caaagcatcc caatgcaggc 2580 tgaaatgttt catcacatct ctggatctct
ctattttgtg cagacattga aaaaattgtt 2640 catattattt ccatgttatc
agaatatttg attttttaaa aacataggcc aagttcattc 2700 acttcattat
tcatttatca aaatcagagt gaatcacatt agtcgccttc acaactgata 2760
aagatcactg aagtcaaatt gatttttgct ataatcttca atctacctat atttaattga
2820 gaatctaaaa tgtacaaatc attgtgttga ttctgcagtg atcctgctat
aagtaagact 2880 cagtccctga ttttaggtat cctgtgaaaa gcagaattaa
gacaaataca caagagacaa 2940 agcacaaaaa ataaatatca taaggggatg
aacaaaatgg tggagaaaga gtagacaaag 3000 tttttgatca cctgccttca
aagaaaggct gtgaattttg ttcacttaga cagcttggag 3060 acaagaaatt
acccaaaagt aaggtgagga ggataggcaa aaagagcaga aagatgtgaa 3120
tggacattgt tgagaaatgt gataggaaaa caatcataga taaaggattt ccaagcaaca
3180 gagcatatcc agatgaggta ggatgggata aactcttatt gaaccaatct
tcaccaattt 3240 tgtttttctt ttgcagagca agctaggaat tgtttccctt
ctactgggca caatacacgc 3300 attgattttt gcctggaata agtggataga
tataaaacaa tttgtatggt atacacctcc 3360 aacttttatg atagctgttt
tccttccaat tgttgtcctg atatttaaaa gcatactatt 3420 cctgccatgc
ttgaggaaga agatactgaa gattagacat ggttgggaag acgtcaccaa 3480
aattaacaaa actgagatat gttcccagtt gtagaattac tgtttacaca catttttgtt
3540 caatattgat atattttatc accaacattt caagtttgta tttgttaata
aaatgattat 3600 tcaaggaaaa aaaaaaaaaa aaaaaaa 3627 89 1365 DNA Homo
sapiens 89 ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct
aaggcgaaga 60 gtgggtggct gaagccatac tattttatag aattaatgga
aagcagaaaa gacatcacaa 120 accaagaaga actttggaaa atgaagccta
ggagaaattt agaagaagac gattatttgc 180 ataaggacac gggagagacc
agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa 240 cagcccatgc
tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc 300
cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact tttctttaca
360 ctcttctgag ggaagtaatt caccctttag caacttccca tcaacaatat
ttttataaaa 420 ttccaatcct ggtcatcaac aaagtcttgc caatggtttc
catcactctc ttggcattgg 480 tttacctgcc aggtgtgata gcagcaattg
tccaacttca taatggaacc aagtataaga 540 agtttccaca ttggttggat
aagtggatgt taacaagaaa gcagtttggg cttctcagtt 600 tcttttttgc
tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca 660
gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa gatgcctgga
720 ttgagcatga tgtttggaga atggagattt atgtgtctct gggaattgtg
ggattggcaa 780 tactggctct gttggctgtg acatctattc catctgtgag
tgactctttg acatggagag 840 aatttcacta tattcagatt atccataaga
agagtgatgt gccagaatca ctctgggatc 900 cttgtctgac aagattcaaa
ggactaaatt taattcagtc atgaacactg ccaattaccg 960 tttatgggta
gacatctttg gaaatttcca caagagcaag ctaggaattg tttcccttct 1020
actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt
1080 tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg
ttgtcctgat 1140 atttaaaagc atactattcc tgccatgctt gaggaagaag
atactgaaga ttagacatgg 1200 ttgggaagac gtcaccaaaa ttaacaaaac
tgagatatgt tcccagttgt agaattactg 1260 tttacacaca tttttgttca
atattgatat attttatcac caacatttca agtttgtatt 1320 tgttaataaa
atgattattc aaggaaaaaa aaaaaaaaaa aaaaa 1365 90 282 PRT Homo sapiens
90 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu
His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro
Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His
Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe
Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr
Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile
Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125
Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130
135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu
Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala
Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala
Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val
Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ile Ile 245 250
255 His Lys Lys Ser Asp Val Pro Glu Ser Leu Trp Asp Pro Cys Leu Thr
260 265 270 Arg Phe Lys Gly Leu Asn Leu Ile Gln Ser 275 280 91 258
PRT Homo sapiens 91 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu
Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp
Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg
Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu
Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe
Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala
Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95
Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100
105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala
Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu
His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp
Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser
Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr
Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala
Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu
His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220
Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225
230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln
Val Asn 245 250 255 Asn Ile 92 282 PRT Homo sapiens 92 Met Glu Ser
Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys
Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25
30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln
35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln
His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys
Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu
Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln
Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu
Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro
Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys
Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155
160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala
165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr
Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys
Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile
Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu
Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu
Thr Trp Arg Glu Phe His Tyr Ile Gln Ile Ile 245 250 255 His Lys Lys
Ser Asp Val Pro Glu Ser Leu Trp Asp Pro Cys Leu Thr 260 265 270 Arg
Phe Lys Gly Leu Asn Leu Ile Gln Ser 275 280 93 339 PRT Homo sapiens
93 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met
1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys
Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu
His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro
Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His
Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe
Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr
Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile
Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125
Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130
135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu
Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala
Val Leu His Ala 165 170
175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu
180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp
Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val
Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala
Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp
Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250 255 Leu Gly Ile Val Ser
Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe 260 265 270 Ala Trp Asn
Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285 Pro
Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295
300 Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile
305 310 315 320 Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr
Glu Ile Cys 325 330 335 Ser Gln Leu 94 258 PRT Homo sapiens 94 Met
Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10
15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr
20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu
His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu
Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro
Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr
Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His
Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys
Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr
Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140
Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145
150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu
His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr
Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln
Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met
Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp
Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Val Asn 245 250 255 Asn
Ile 95 282 PRT Homo sapiens 95 Met Glu Ser Arg Lys Asp Ile Thr Asn
Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu
Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met
Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His
Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln
Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70
75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile
His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile
Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile
Thr Leu Leu Ala Leu 115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala
Ile Val Gln Leu His Asn Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro
His Trp Leu Asp Lys Trp Met Leu Thr 145 150 155 160 Arg Lys Gln Phe
Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175 Ile Tyr
Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190
Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195
200 205 Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly
Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser
Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe
His Tyr Ile Gln Ile Ile 245 250 255 His Lys Lys Ser Asp Val Pro Glu
Ser Leu Trp Asp Pro Cys Leu Thr 260 265 270 Arg Phe Lys Gly Leu Asn
Leu Ile Gln Ser 275 280 96 19 DNA Homo sapiens 96 gacaucacaa
accaagaag 19 97 19 DNA Homo sapiens 97 guggauagau auaaaacaa 19 98
19 DNA Homo sapiens 98 gccuucccau ugugcuccc 19 99 339 PRT Homo
sapiens 99 Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp
Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu
His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val
Leu Leu His Leu His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp
Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln
Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu
Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu
Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110
Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115
120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn
Gly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp
Met Leu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe
Phe Ala Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met
Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln
Gln Val Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp
Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly
Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235
240 Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys
245 250 255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu
Ile Phe 260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val
Trp Tyr Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala Val Phe Leu Pro
Ile Val Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu Pro Cys
Leu Arg Lys Lys Ile Leu Lys Ile 305 310 315 320 Arg His Gly Trp Glu
Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser Gln Leu
100 303 PRT Mus musculus 100 Met Leu Lys Arg Pro Gly Leu Ser His
Leu Gln His Ala Val His Val 1 5 10 15 Asp Ala Phe Asp Cys Pro Ser
Glu Leu Gln His Thr Gln Glu Phe Phe 20 25 30 Pro Asn Trp Arg Leu
Pro Val Lys Val Ala Ala Ile Ile Ser Ser Leu 35 40 45 Thr Phe Leu
Tyr Thr Leu Leu Arg Glu Ile Ile Tyr Pro Leu Val Thr 50 55 60 Ser
Arg Glu Gln Tyr Phe Tyr Lys Ile Pro Ile Leu Val Ile Asn Lys 65 70
75 80 Val Leu Pro Met Val Ala Ile Thr Leu Leu Ala Leu Val Tyr Leu
Pro 85 90 95 Gly Glu Leu Ala Ala Val Val Gln Leu Arg Asn Gly Thr
Lys Tyr Lys 100 105 110 Lys Phe Pro Pro Trp Leu Asp Arg Trp Met Leu
Ala Arg Lys Gln Phe 115 120 125 Gly Leu Leu Ser Phe Phe Phe Ala Val
Leu His Ala Val Tyr Ser Leu 130 135 140 Ser Tyr Pro Met Arg Arg Ser
Tyr Arg Tyr Lys Leu Leu Asn Trp Ala 145 150 155 160 Tyr Lys Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp Val Glu His Asp 165 170 175 Val Trp
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile Val Gly Leu Ala 180 185 190
Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asp Ser 195
200 205 Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys Leu Gly Ile
Val 210 215 220 Ser Leu Leu Leu Gly Thr Val His Ala Leu Val Phe Ala
Trp Asn Lys 225 230 235 240 Trp Val Asp Val Ser Gln Phe Val Trp Tyr
Met Pro Pro Thr Phe Met 245 250 255 Ile Ala Val Phe Leu Pro Thr Leu
Val Leu Ile Cys Lys Ile Ala Leu 260 265 270 Cys Leu Pro Cys Leu Arg
Lys Lys Ile Leu Lys Ile Arg Cys Gly Trp 275 280 285 Glu Asp Val Ser
Lys Ile Asn Arg Thr Glu Met Ala Ser Arg Leu 290 295 300
* * * * *
References