U.S. patent application number 10/418972 was filed with the patent office on 2004-04-29 for nucleic acids and corresponding proteins entitled 251p5g2 useful in treatment and detection of cancer.
Invention is credited to Challita-Eid, Pia M., Faris, Mary, Ge, Wangmao, Jakobovits, Aya, Raitano, Arthur B..
Application Number | 20040081653 10/418972 |
Document ID | / |
Family ID | 31891426 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040081653 |
Kind Code |
A1 |
Raitano, Arthur B. ; et
al. |
April 29, 2004 |
Nucleic acids and corresponding proteins entitled 251P5G2 useful in
treatment and detection of cancer
Abstract
A novel gene 251P5G2 and its encoded protein, and variants
thereof, are described wherein 251P5G2 exhibits tissue specific
expression in normal adult tissue, and is aberrantly expressed in
the cancers listed in Table I. Consequently, 251P5G2 provides a
diagnostic, prognostic, prophylactic and/or therapeutic target for
cancer. The 251P5G2 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 251P5G2 can be used in active or passive
immunization.
Inventors: |
Raitano, Arthur B.; (Los
Angeles, CA) ; Challita-Eid, Pia M.; (Encino, CA)
; Jakobovits, Aya; (Beverly Hills, CA) ; Faris,
Mary; (Los Angeles, CA) ; Ge, Wangmao; (Culver
City, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
31891426 |
Appl. No.: |
10/418972 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60404306 |
Aug 16, 2002 |
|
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60423290 |
Nov 1, 2002 |
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Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 5/0693 20130101; A61K 47/6851 20170801; G01N 33/57484
20130101; A01K 2217/075 20130101; Y02A 50/30 20180101; Y02A 50/466
20180101; C07K 14/435 20130101; C07K 14/47 20130101; A61K 38/00
20130101; A01K 2217/05 20130101; C07K 2319/00 20130101; A61P 37/04
20180101; A61K 2039/505 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 039/395 |
Claims
1. A composition that comprises, consists essentially of, or
consists of: a) a peptide of eight, nine, ten, or eleven contiguous
amino acids of a protein of FIG. 2; b) a peptide of Tables
VIII-XXI; c) a peptide of Tables XXII to XLV; or, d) a peptide of
Tables XLVI to XLIX.
2. A composition of claim 1 that comprises a protein related to a
protein of FIG. 2.
3. A protein of claim 2 that is at least 90, 91, 92, 93, 94, 95,
96, 97, 98, or 99% homologous to an entire amino acid sequence
shown in FIG. 2.
4. A composition of claim 1 wherein the substance comprises a CTL
polypeptide or an analog thereof, from the amino acid sequence of a
protein of FIG. 2.
5. A composition of claim 4 further limited by a proviso that the
epitope is not an entire amino acid sequence of FIG. 2.
6. A composition of claim 1 further limited by a proviso that the
polypeptide is not an entire amino acid sequence of a protein of
FIG. 2.
7. A composition of claim 1 that comprises an antibody polypeptide
epitope from an amino acid sequence of FIG. 2.
8. A composition of claim 7 further limited by a proviso that the
epitope is not an entire amino acid sequence of FIG. 2.
9. A composition of claim 7 wherein the antibody epitope comprises
a peptide region of at least 5 amino acids of FIG. 2 in any whole
number increment up to the end of said peptide, wherein the epitope
comprises an amino acid position selected from: a) an amino acid
position having a value greater than 0.5 in the Hydrophilicity
profile of FIG. 5, b) an amino acid position having a value less
than 0.5 in the Hydropathicity profile of FIG. 6; c) an amino acid
position having a value greater than 0.5 in the Percent Accessible
Residues profile of FIG. 7; d) an amino acid position having a
value greater than 0.5 in the Average Flexibility profile of FIG.
8; e) an amino acid position having a value greater than 0.5 in the
Beta-turn profile of FIG. 9; f) a combination of at least two of a)
through e); g) a combination of at least three of a) through e); h)
a combination of at least four of a) through e); or i) a
combination of five of a) through e).
10. A polynucleotide that encodes a protein of claim 1.
11. A polynucleotide of claim 10 that comprises a nucleic acid
molecule set forth in FIG. 2.
12. A polynucleotide of claim 10 further limited by a proviso that
the encoded protein is not an entire amino acid sequence of FIG.
2.
13. A composition of claim 11 wherein the substance comprises a
polynucleotide that comprises a coding sequence of a nucleic acid
sequence of FIG. 2.
14. A polynucleotide of claim 11 that further comprises an
additional nucleotide sequence that encodes an additional peptide
of claim 1.
15. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 10.
16. A method of generating a mammalian immune response directed to
a protein of FIG. 2, the method comprising: exposing cells of the
mammal's immune system to a portion of a) a 251P5G2-related protein
and/or b) a nucleotide sequence that encodes said protein, whereby
an immune response is generated to said protein.
17. A method of generating an immune response of claim 16, said
method comprising: providing a 251P5G2-related protein that
comprises at least one T cell or at least one B cell epitope; and,
contacting the epitope with a mammalian immune system T cell or B
cell respectively, whereby the T cell or B cell is activated.
18. A method of claim 17 wherein the immune system cell is a B
cell, whereby the induced B cell generates antibodies that
specifically bind to the 251P5G2-related protein.
19. A method of claim 17 wherein the immune system cell is a T cell
that is a cytotoxic T cell (CTL), whereby the activated CTL kills
an autologous cell that expresses the 251P5G2-related protein.
20. A method of claim 17 wherein the immune system cell is a T cell
that is a helper T cell (HTL), whereby the activated HTL secretes
cytokines that facilitate the cytotoxic activity of a cytotoxic T
cell (CTL) or the antibody-producing activity of a B cell.
21. A method for detecting, in a sample, the presence of a
251P5G2-related protein or a 251P5G2-related polynucleotide,
comprising steps of: contacting the sample with a substance that
specifically binds to the 251P5G2-related protein or to the
251P5G2-related polynucleotide, respectively; and, determining that
there is a complex of the substance with the 251P5G2-related
protein or the substance with the 251P5G2-related polynucleotide,
respectively.
22. A method of claim 21 for detecting the presence of a
251P5G2-related protein in a sample comprising steps of: contacting
the sample with an antibody or fragment thereof either of which
specifically bind to the 251P5G2-related protein; and, determining
that there is a complex of the antibody or fragment thereof and the
251P5G2-related protein.
23. A method of claim 21 further comprising a step of: taking the
sample from a patient who has or who is suspected of having
cancer.
24. A method of claim 21 for detecting the presence of a protein of
FIG. 2 mRNA in a sample comprising: producing cDNA from the sample
by reverse transcription using at least one primer; amplifying the
cDNA so produced using 251P5G2 polynucleotides as sense and
antisense pimers, wherein the 251P5G2 polynucleotides used as the
sense and antisense primers serve to amplify a 251P5G2 cDNA; and,
detecting the presence of the amplified 251P5G2 cDNA.
25. A method of claim 21 for monitoring one or more 251P5G2 gene
products in a biological sample from a patient who has or who is
suspected of having cancer, the method comprising: determining the
status of one or more 251P5G2 gene products expressed by cells in a
tissue sample from an individual; comparing the status so
determined to the status of one or more 251P5G2 gene products in a
corresponding normal sample; and, identifying the presence of one
or more aberrant gene products of 251P5G2 in the sample relative to
the normal sample.
26. The method of claim 25 further comprising a step of determining
if there are one or more elevated gene products of a 251P5G2 mRNA
or a 251P5G2 protein, whereby the presence of one or more elevated
gene products in the test sample relative to the normal tissue
sample indicates the presence or status of a cancer.
27. A method of claim 26 wherein the cancer occurs in a tissue set
forth in Table I.
28. A composition comprising: a substance that a) modulates the
status of a protein of FIG. 2, or b) a molecule that is modulated
by a protein of FIG. 2, whereby the status of a cell that expresses
a protein of FIG. 2 is modulated.
29. A composition of claim 28, further comprising a physiologically
acceptable carrier.
30. A pharmaceutical composition that comprises the composition of
claim 28 in a human unit dose form.
31. A composition of claim 28 wherein the substance comprises an
antibody or fragment thereof that specifically binds to a protein
of FIG. 2.
32. An antibody or fragment thereof of claim 31, which is
monoclonal.
33. An antibody of claim 31, which is a human antibody, a humanized
antibody or a chimeric antibody.
34. A non-human transgenic animal that produces an antibody of
claim 31.
35. A hybridoma that produces an antibody of claim 32.
36. A method of delivering a cytotoxic agent or a diagnostic agent
to a cell that expresses a protein of FIG. 2, said method
comprising: providing the cytotoxic agent or the diagnostic agent
conjugated to an antibody or fragment thereof of claim 4; and,
exposing the cell to the antibody-agent or fragment-agent
conjugate.
37. A composition of claim 28 wherein the substance comprises a
polynucleotide that encodes an antibody or fragment thereof, either
of which immunospecifically bind to a protein of FIG. 2.
38. A composition of claim 28 wherein the substance comprises a) a
ribozyme that cleaves a polynucleotide having a 251P5G2 coding
sequence, or b) a nucleic acid molecule that encodes the ribozyme;
and, a physiologically acceptable carrier.
39. A composition of claim 28 wherein the substance comprises human
T cells, wherein said T cells specifically recognize a 251P5G2
peptide subsequence in the context of a particular HLA
molecule.
40. A method of inhibiting growth of cancer cells that express a
protein of FIG. 2, the method comprising: administering to the
cells the composition of claim 28.
41. A method of claim 40 of inhibiting growth of cancer cells that
express a protein of FIG. 2, the method comprising steps of:
administering to said cells an antibody or fragment thereof, either
of which specifically bind to a 251P5G2-related protein.
42. A method of claim 40 of inhibiting growth of cancer cells that
express a protein of FIG. 2, the method comprising steps of:
administering to said cells a 251P5G2-related protein.
43. A method of claim 40 of inhibiting growth of cancer cells that
express a protein of FIG. 2, the method comprising steps of:
administering to said cells a polynucleotide comprising a coding
sequence for a 251P5G2-related protein or comprising a
polynucleotide complementary to a coding sequence for a
251P5G2-related protein.
44. A method of claim 40 of inhibiting growth of cancer cells that
express a protein of FIG. 2, the method comprising steps of:
administering to said cells a ribozyme that cleaves a
polynucleotide that encodes a protein of FIG. 2.
45. A method of claim 40 of inhibiting growth of cancer cells that
express a protein of FIG. 2 and a particular HLA molecule, the
method comprising steps of: 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 in the
context of the particular HLA molecule.
46. A method of claim 40, the method comprising steps of:
administering a vector that delivers a nucleotide that encodes a
single chain monoclonal antibody, whereby the encoded single chain
antibody is expressed intracellularly within cancer cells that
express a protein of FIG. 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional utility patent
application that claims priority from U.S. provisional patent
application USSN 60/404,306, filed Aug. 16, 2002 and this
application claims priority from U.S. provisional patent
application USSN 60/423,290, fled Nov. 01, 2002. The contents of
the applications listed in this paragraph are fully incorporated by
reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The invention described herein relates to genes and their
encoded proteins, termed 251P5G2, expressed in certain cancers, and
to diagnostic and therapeutic methods and compositions useful in
the management of cancers that express 251P5G2.
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 Sep. 2, 1996
(9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. Dec.
7, 1999; 96(25): 14523-8) and prostate stem cell antigen (PSCA)
(Reiter et al., 1998, Proc. Nati. 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 significanuy 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 251P5G2,
that has now been found to be over-expressed in the cancer(s)
listed in Table I. Northern blot expression analysis of 251P5G2
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 251P5G2 are provided. The
tissue-related profile of 251P5G2 in normal adult tissues, combined
with the over-expression observed in the tissues listed in Table I,
shows that 251P5G2 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 251P5G2 genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding 251P5G2-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 251P5G2-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 251P5G2
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the 251P5G2 genes, mRNAs, or to
251P5G2-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding 251P5G2. Recombinant DNA
molecules containing 251P5G2 polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of 251P5G2 gene products are also provided. The
invention further provides antibodies that bind to 251P5G2 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 251P5G2 polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express 251P5G2. A typical embodiment of this invention
provides methods for monitoring 251P5G2 gene products in a tissue
or hematology sample having or suspected of having some form of
growth dysregulabon such as cancer.
[0030] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 251P5G2 such as cancers of tissues listed in Table I,
including. therapies aimed at inhibiting the transcription,
translation, processing or function of 251P5G2 as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
251P5G2 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 251P5G2.
Preferably, the carrier is a uniquely human carrier. In another
aspect of the invention, the agent is a moiety that is
immunoroactive with 251P5G2 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 251P5G2 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 251P5G2 as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
251P5G2. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of 251P5G2 (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for 251P5G2 production) or a ribozyme effective to lyse
251P5G2 mRNA.
[0032] Note that to determine the starting position of any peptide
set forth in Tables VIIl-XXI and XXII to XLIX (collectively HLA
Peptide Tables) respective to its parental protein, e.g., variant
1, variant 2, etc., reference is made to three factors: the
particular variant, the length of the peptide in an HLA Peptide
Table, and the Search Peptides in Table VII. Generally, a unique
Search Peptide is used to obtain HLA peptides of a particular for a
particular variant. The position of each Search Peptide relative to
its respective parent molecule is listed in Table VII. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X-1" to each position in Tables VIII-XXI and XXII to XLIX to
obtain the actual position of the HLA peptides in their parental
molecule. For example, if a particular Search Peptide begins at
position 150 of its parental molecule, one must add 150-1, i.e.,
149 to each HLA peptide amino acid position to calculate the
position of that amino acid in the parent molecule.
[0033] One embodiment of the invention comprises an HLA peptide,
that occurs at least twice in Tables VIII-XXI and XXII to XLIX
collectively, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention comprises an HLA peptide that
occurs at least once in Tables VIII-XXI and at least once in tables
XXI! to XLIX, or an oligonucleotide that encodes the HLA
peptide.
[0034] Another embodiment of the invention is antibody epitopes,
which comprise a peptide regions, or an oligonucleotide encoding
the peptide region, that has one two, three, four, or five of the
following characteristics:
[0035] i) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or greater than 0.5, 0.6,
0.7, 0.8, 0.9, or having a value equal to 1.0, in the
Hydrophilicity profile of FIG. 5;
[0036] ii) a peptide region of at least 5 amino acids of a
particular peptide of FIG. 3, in any whole number increment up to
the full length of that protein in FIG. 3, that includes an amino
acid position having a value equal to or less than 0.5, 0.4, 0.3,
0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity
profile of FIG. 6;
[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 251P5G2 SSH sequence of 162 nucleotides.
[0041] FIG. 2. A) The cDNA and amino acid sequence of 251P5G2
variant 1 (also called "251P5G2v.1" or "251P5G2 variant 1") is
shown in FIG. 2A. The start methionine is underlined. The open
reading frame extends from nucleic acid 722-1489 including the stop
codon.
[0042] B) The cDNA and amino acid sequence of 251P5G2 variant 2
(also called "251P5G2 v.2") is shown in FIG. 2B. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0043] C) The cDNA and amino acid sequence of 251P5G2 variant 3
(also called "251P5G2 v.3") is shown in FIG. 2C. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0044] D) The cDNA and amino acid sequence of 251P5G2 variant 4
(also called "251P5G2 v.4") is shown in FIG. 2D. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0045] E) The cDNA and amino acid sequence of 251P5G2 variant 5
(also called "251P5G2 v.5") is shown in FIG. 2E. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0046] F) The cDNA and amino acid sequence of 251P5G2 variant 6
(also called "251P5G2 v.6") is shown in FIG. 2F. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0047] G) The cDNA and amino acid sequence of 251P5G2 variant 7
(also called "251P5G2 v.7") is shown in FIG. 2G. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0048] H) The cDNA and amino acid sequence of 251P5G2 variant 8
(also called "251P5G2 v.8") is shown in FIG. 2H. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0049] I) The cDNA and amino acid sequence of 251P5G2 variant 9
(also called "251P5G2 v.9") is shown in FIG. 2I. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0050] J) The cDNA and amino acid sequence of 251P5G2 variant 10
(also called "251P5G2 v.10") is shown in FIG. 2J. The codon for the
start methionine is underlined: The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0051] K) The cDNA and amino acid sequence of 251P5G2 variant 11
(also called "251P5G2 v.11") is shown in FIG. 2K. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-1489 including the stop codon.
[0052] L) The cDNA and amino acid sequence of 251P5G2 variant 12
(also called "251P5G2 v.12") is shown in FIG. 2L. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 722-4522 including the stop codon.
[0053] M) The cDNA and amino acid sequence of 251P5G2 variant 13
(also called 251P5G2 v.13) is shown in FIG. 2M. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 1-3801 including the stop codon.
[0054] FIG. 3.
[0055] A) The amino acid sequence of 251P5G2 v.1 is shown in FIG.
3A; it has 255 amino acids.
[0056] B) The amino acid sequence of 251P5G2 v.2 is shown in FIG.
3B; it has 255 amino acids.
[0057] C) The amino acid sequence of 251P5G2 v.3 is shown in FIG.
3C; it has 255 amino acids.
[0058] D) The amino acid sequence of 251P5G2 v.4 is shown in FIG.
3D; it has 255 amino acids.
[0059] E) The amino acid sequence of 251P5G2 v.12 is shown in FIG.
3E; it has 1266 amino acids.
[0060] As used herein, a reference to 251P5G2 includes all variants
thereof, including those shown in FIGS. 2, 3, 10, and 11, unless
the context clearly indicates otherwise.
[0061] FIG. 4. FIG. 4A. Alignment of 251P5G2 v.1 with the mouse
vomeronasal 1 receptor C3. FIG. 4B. Amino acid alignment of 251P5G2
v.12 with the protein XM.sub.--063686 predicted from
GenomeScan.
[0062] FIG. 5. Hydrophilicity amino acid profile of 251P5G2 v.1 and
v.12 determined by computer algorithm sequence analysis using the
method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc.
Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale
website located on the World Wide Web at
(expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0063] FIG. 6. Hydropathicity amino acid profile of 251P5G2 v.1 and
v.12 determined by computer algorithm sequence analysis using the
method of Kyte and Doolittle (Kyte J., Dooliltle R. F., 1982. J.
Mol. Biol. 157:105-132) accessed on the ProtScale website located
on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through
the ExPasy molecular biology server.
[0064] FIG. 7. Percentaccessible residues amino acid profile of
251P5G2 v.1 and v.12 determined by computer algorithm sequence
analysis using the method of Janin (Janin J., 1979 Nature
277:491-492) accessed on the ProtScale website located on the World
Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0065] FIG. 8. Average flexibility amino acid profile of 251P5G2
v.1 and v.12 determined by computer algorithm sequence analysis
using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and
Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255)
accessed on the ProtScale website located on the World Wide Web at
(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0066] FIG. 9. Beta-turn amino acid profile of 251P5G2 v.1 and v.12
determined by computer algorithm sequence analysis using the method
of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering
1:289-294) accessed on the ProtScale website located on the World
Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0067] FIG. 10. Schematic alignment of SNP variants of 251P5G2.
Variants 251P5G2 v.2 through v.11 are variants with single
nucleotide differences. Though these SNP variants are shown
separately, they could also occur in any combinations and in any
transcript variants that contains the base pairs. Numbers
correspond to those of 251P5G2 v.1. Black box shows the same
sequence as 251P5G2 v.1. SNPs are indicated above the box.
[0068] FIG. 11. Schematic alignment of protein variants of 251P5G2.
Protein variants correspond to nucleotide variants. Nucleotide
variants 251P5G2 v.5 through v.11 in FIG. 10 code for the same
protein as 251P5G2 v.1. Nucleotide variants 251P5G2 v.12 and v.13
as shown in FIG. 12 code the same protein. Single amino acid
differences were indicated above the boxes. Black boxes represent
the same sequence as 251P5G2 v.1. Numbers underneath the box
correspond to 251P5G2 v.1.
[0069] FIG. 12. Exon compositions of transcript variants of
251P5G2. Variant 251P5G2 v.12 and v.13 are transcript variants each
with 19 exons. The first two exons of variants 251P5G2 v.12 and
v.13 matches part of variant 251P5G2 v.1. Compared with 251P5G2
v.12, 251P5G2 v.13 has a shorter first exon (starting at base 722)
but the other 18 exons are the same. Numbers in "( )" underneath
the boxes correspond to those of 251P5G2 v.1. Lengths of introns
and exons are not proportional.
[0070] FIG. 13. FIGS. 13(A) (SEQ ID NO: 82) and 13(B) (SEQ ID NO:
83) Secondary structure and transmembrane domains prediction for
251P5G2 protein variants.
[0071] The secondary structure of 251P5G2 protein variants 1 and 12
(FIGS. 31A and 13B, respectively) were predicted using the
HNN--Hierarchical Neural Network method (Guermeur, 1997,
hftp://pbil.ibcp.fr/cgi-bin/npsa_a- utomat.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.
[0072] FIGS. 13(C) and 13(E): Schematic representations of the
probability of existence of transmembrane regions and orientation
of 251P5G2 variants 1 and 12, 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(D) and
(F): Schematic representations of the probability of the existence
of transmembrane regions and the extracellular and intracellular
orientation of 251P5G2 variants 1 and 12, respectively, based on
the TMHMM-algorith.TM. 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
[0073] FIG. 14. Expression of 251P5G2 by RT-PCR. First strand cDNA
was prepared from vital pool 1 (liver, lung and kidney), vital pool
2 (pancreas, colon and stomach), prostate cancer metastasis to
lymph node, prostate cancer pool, bladder cancer pool, and cancer
metastasis pool. Normalization was performed by PCR using primers
to actin and GAPDH. Semi-quantitative PCR, using primers to
251P5G2, was performed at 26 and 30 cycles of amplification.
Results show strong expression of 251P5G2 in prostate cancer
metastasis, prostate cancer pool, and cancer metastasis pool.
Expression of 251P5G2 was also detected in bladder cancer pool, but
not in vital pool 1 and vital pool 2.
[0074] FIG. 15. Expression of 251P5G2 in normal tissues. Two
multiple tissue northern blots (Clontech) both with 2 ug of
mRNA/lane were probed with the 251P5G2 sequence. Size standards in
kilobases (kb) are indicated on the side. Results show weak
expression of 251P5G2 in prostate and testis, but not in any other
normal tissue tested.
[0075] FIG. 16. Expression of 251P5G2 in Patient Cancer Specimens
and Normal Tissues. RNA was extracted from two prostate cancer
metastasis to lymph node isolated from two different patients (Met1
and Met2), as well as from normal bladder (NB), normal kidney (NK),
normal lung (NL) and normal breast (NBr), normal ovary (NO), and
normal pancreas (NPa). Northern blot with 10 Gig of total RNA/lane
was probed with 251P5G2 SSH sequence. Size standards in kilobases
(kb) are indicated on the side. The 251P5G2 transcript was detected
in the prostate cancer metastasis specimens, but not in the normal
tissues tested.
[0076] FIG. 17. Expression of 251P5G2 in Prostate Cancer Patient
Specimens. RNA was extracted from prostate cancer xenografts
(LAPCAAD, LAPC4AI, LAPC-9AD, LAPC-9AI), prostate cancer cell lines
(LNCaP and PC3), normal prostate (N), and prostate cancer patient
tumors (T). Northern blots with 10 ug of total RNA were probed with
the 251P5G2 SSH fragment. Size standards in kilobases are on the
side. Results show expression of 251P5G2 in the LAPC-9AD xenograft
and in prostate tumor tissues. Lower level expression was detected
in the other xenograft tissues and LNCaP cell line but not in PC3.
The lower panel represents ethidium-bromide staining of the gel
confirming the quality of the RNA.
[0077] FIG. 18. Expression of 251P5G2 in Human Normal and Cancer
Tissues. First strand cDNA was prepared from a panel of 13 normal
tissues, prostate cancer pool, bladder cancer pool, kidney cancer
pool, colon cancer pool, lung cancer pool, ovary cancer pool,
breast cancer pool, pancreas cancer pool, 2 different prostate
cancer metastasis specimens to lymph node, and a pool of prostate
cancer LAPC xenografts (LAPC4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI).
Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was
performed at 26 and 30 cycles of amplification. A standard curve
was generated using plasmid DNA containing 251P5G2 of known copy
number. The experiment was performed in duplicate. Results show
strong expression of 251P5G2 in prostate cancer metastasis,
prostate cancer pool, and cancer metastasis pool. Expression of
251P5G2 was also detected in bladder cancer pool. Amongst normal
tissues, very weak expression was detected in hear, prostate,
skeletal muscle and testis but not in any other normal tissue
tested.
[0078] FIG. 19. Expression of 251P5G2 in Prostate Cancer Patient
Specimens. First strand cDNA was prepared from normal prostate,
prostate cancer cell lines (PC3, DU145, LNCaP, 293T), and a panel
of prostate cancer patient specimens. Normalization was performed
by PCR using primers to actin and GAPDH. Semi-quantitative PCR,
using primers to 251P5G2, was performed at 26 and 30 cycles of
amplification. Results show expression of 251P5G2 in 10 out of 19
patient specimens. Very strong expression was detected in 5 out of
the 10 expressing tumors. Expression was also detected in LNCaP but
not in the other cell lines tested nor in normal prostate.
[0079] FIG. 20. Expression of 251P5G2 in Bladder Cancer Patient
Specimens. First strand cDNA was prepared from normal bladder,
bladder cancer cell lines (UM-UC-3, TCCSUP, J82), and a panel of
bladder cancer patient specimens. Normalization was performed by
PCR using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 251P5G2, was performed at 26 and 30 cycles of
amplification. Results show expression of 251P5G2 in 5 out of 9
patient specimens, but not in the cell lines tested nor in normal
bladder.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
[0080] I.) Definitions
[0081] II.) 251P5G2 Polynucleotides
[0082] II.A.) Uses of 251P5G2 Polynucleotides
[0083] II.A.1.) Monitoring of Genetic Abnormalities
[0084] II.A.2.) Antisense Embodiments
[0085] II.A.3.) Primers and Primer Pairs
[0086] II.A.4.) Isolation of 251P5G2-Encoding Nucleic Acid
Molecules
[0087] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0088] III.) 251P5G2-related Proteins
[0089] III.A.) Motif-bearing Protein Embodiments
[0090] III.B.) Expression of 251P5G2-related Proteins
[0091] III.C.) Modifications of 251P5G2-related Proteins
[0092] III.D.) Uses of 251P5G2-related Proteins
[0093] IV.) 251P5G2 Antibodies
[0094] V.) 251P5G2 Cellular Immune Responses
[0095] VI.) 251P5G2 Transgenic Animals
[0096] VII.) Methods for the Detection of 251P5G2
[0097] VIII.) Methods for Monitoring the Status of 251P5G2-related
Genes and Their Products
[0098] IX.) Identification of Molecules That Interact With
251P5G2
[0099] X.) Therapeutic Methods and Compositions
[0100] X.A.) Anti-Cancer Vaccines
[0101] X.B.) 251P5G2 as a Target for Antibody-Based Therapy
[0102] X.C.) 251P5G2 as a Target for Cellular Immune Responses
[0103] X.C.1. Minigene Vaccines
[0104] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0105] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0106] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0107] X.D.) Adoptive Immunotherapy
[0108] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0109] XI.) Diagnostic and Prognostic Embodiments of 251P5G2.
[0110] XII.) Inhibition of 251P5G2 Protein Function
[0111] XII.A.) Inhibition of 251P5G2 With Intracellular
Antibodies
[0112] XII.B.) Inhibition of 251P5G2 with Recombinant Proteins
[0113] XII.C.) Inhibition of 251P5G2 Transcription or
Translation
[0114] XII.D.) General Considerations for Therapeutic
Strategies
[0115] XIII.) Identification, Characterization and Use of
Modulators of 251P5G2
[0116] XIV.) KITS/Articles of Manufacture
[0117] I.) Definitions:
[0118] 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.
[0119] The terms "advanced prostate cancer", "locally advanced
prostate cancer", "advanced disease" and "locally advanced disease"
mean prostate cancers that have extended through the prostate
capsule, and are meant to include stage C disease under the
American Urological Association (AUA) system, stage C1-C2 disease
under the Whitmore-Jewett system, and stage T3-T4 and N+ disease
under the TNM (tumor, node, metastasis) system. In general, surgery
is not recommended for patients with locally advanced disease, and
these patients have substantially less favorable outcomes compared
to patients having clinically localized (organ-confined) prostate
cancer. Locally advanced disease is clinically identified by
palpable evidence of induration beyond the lateral border of the
prostate, or asymmetry or induration above the prostate base.
Locally advanced prostate cancer is presently diagnosed
pathologically following radical prostatectomy if the tumor invades
or penetrates the prostatic capsule, extends into the surgical
margin, or invades the seminal vesicles.
[0120] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 251P5G2 (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 251P5G2. 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.
[0121] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 251P5G2-related protein). For example, an analog
of a 251P5G2 protein can be specifically bound by an antibody or T
cell that specifically binds to 251P5G2.
[0122] 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-251P5G2 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.
[0123] 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-251P5G2 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-251P5G2 antibody compositions with polyepitopic
specificity.
[0124] 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."
[0125] A "combinatorial library" is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide (e.g., mutein) library, is
formed by combining a set of chemical building blocks called amino
acids in every possible way for a given compound length (i.e., the
number of amino acids in a polypeptide compound). Numerous chemical
compounds are synthesized through such combinatorial mixing of
chemical building blocks (Gallop et al., J. Med. Chem. 37(9):
1233-1251 (1994)).
[0126] Preparation and screening of combinatorial libraries is well
known to those of skill in the art. Such combinatorial chemical
libraries include, but are not limited to, peptide libraries (see,
e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493
(1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT
Publication No WO 91/19735), encoded peptides (PCT Publication WO
93/20242), random bio-oligomers (PCT Publication WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et
al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic
syntheses of small compound libraries (Chen et al., J. Amer. Chem.
Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science
261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J.
Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med.
Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,
Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S.
Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,
Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al., Science
274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small
organic molecule libraries (see, e.g., benzodiazepines, Baum,
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506, 337; benzodiazepines,
U.S. Pat. No. 5,288,514; and the like).
[0127] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced
Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A,
Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore,
Bedford, Mass.). A number of well-known robotic systems have also
been developed for solution phase chemistries. These systems
include automated workstations such as the automated synthesis
apparatus developed by Takeda Chemical Industries, LTD. (Osaka,
Japan) and many robotic systems utilizing robotic arms (Zymate H,
Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo
Alto, Calif.), which mimic the manual synthetic operations
performed by a chemist. Any of the above devices are suitable for
use with the present invention. The nature and implementation of
modifications to these devices (if any) so that they can operate as
discussed herein will be apparent to persons skilled in the
relevant art. In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Asinex, Moscow, RU; Tdpos, Inc., St. Louis, Mo.; ChemStar,
Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md.; etc.).
[0128] 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, ccl065,
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.
[0129] The "gene product" is sometimes referred to herein as a
protein or mRNA. For example, a "gene product of the invention" is
sometimes referred to herein as a "cancer amino acid sequence",
"cancer protein", "protein of a cancer listed in Table I", a
"cancer mRNA", "mRNA of a cancer listed in Table I", etc. In one
embodiment, the cancer protein is encoded by a nucleic acid of FIG.
2. The cancer protein can be a fragment, or alternatively, be the
full-length protein to the fragment encoded by the nucleic acids of
FIG. 2. In one embodiment, a cancer amino acid sequence is used to
determine sequence identity or similarity. In another embodiment,
the sequences are naturally occurring allelic variants of a protein
encoded by a nucleic acid of FIG. 2. In another embodiment, the
sequences are sequence variants as further described herein.
[0130] "High throughput screening" assays for the presence,
absence, quantification, or other properties of particular nucleic
acids or protein products are well known to those of skill in the
art. Similarly, binding assays and reporter gene assays are
similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses
high throughput screening methods for proteins; U.S. Pat. No.
5,585,639 discloses high throughput screening methods for nucleic
acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and
5,541,061 disclose high throughput methods of screening for
ligand/antibody binding.
[0131] In addition, high throughput screening systems are
commercially available (see, e.g., Amersham Biosdences, Piscataway,
N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries,
Mentor, OH; Beckman Instruments, Inc. Fullerton, Calif.; Precision
Systems, Inc., Natick, Mass.; etc.). These systems typically
automate entire procedures, including all sample and reagent
pipetling, liquid dispensing, timed incubations, and final readings
of the microplate in detector(s) appropriate for the assay. These
configurable systems provide high throughput and rapid start up as
well as a high degree of flexibility and customization. The
manufacturers of such systems provide detailed protocols for
various high throughput systems. Thus, e.g., Zymark Corp. provides
technical bulletins describing screening systems for detecting the
modulation of gene transcription, ligand binding, and the like.
[0132] 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.
[0133] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see, e.g.,
Stites, et at., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing, Los
Altos, Calif. (1994).
[0134] 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.
[0135] 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 251P5G2 genes or that encode
polypeptides other than 251P5G2 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 251P5G2 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 251P5G2 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 251P5G2 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0136] 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.
[0137] 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.
[0138] The term "modulator" or "test compound" or "drug candidate"
or grammatical equivalents as used herein describe any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., to be tested for the capacity
to directly or indirectly alter the cancer phenotype or the
expression of a cancer sequence, e.g., a nucleic acid or protein
sequences, or effects of cancer sequences (e.g., signaling, gene
expression, protein interaction, etc.) In one aspect, a modulator
will neutralize the effect of a cancer protein of the invention. By
"neutralize" is meant that an activity of a protein is inhibited or
blocked, along with the consequent effect on the cell. In another
aspect, a modulator will neutralize the effect of a gene, and its
corresponding protein, of the invention by normalizing levels of
said protein. In preferred embodiments, modulators alter expression
profiles, or expression profile nucleic acids or proteins provided
herein, or downstream effector pathways. In one embodiment, the
modulator suppresses a cancer phenotype, e.g. to a normal tissue
fingerprint. In another embodiment, a modulator induced a cancer
phenotype. Generally, a plurality of assay mixtures is run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection.
[0139] Modulators, drug candidates or test compounds encompass
numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular
weight of more than 100 and less than about 2,500 Daltons.
Preferred small molecules are less than 2000, or less than 1500 or
less than 1000 or less than 500 D. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Modulators also comprise biomolecules
such as peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof. Particularly preferred are peptides. One class of
modulators are peptides, for example of from about five to about 35
amino acids, with from about five to about 20 amino acids being
preferred, and from about 7 to about 15 being particularly
preferred. Preferably, the cancer modulatory protein is soluble,
includes a non-transmembrane region, and/or, has an N-terminal Cys
to aid in solubility. In one embodiment, the C-terminus of the
fragment is kept as a free acid and the N-terminus is a free amine
to aid in coupling, i.e., to cysteine. In one embodiment, a cancer
protein of the invention is conjugated to an immunogenic agent as
discussed herein. In one embodiment, the cancer protein is
conjugated to BSA. The peptides of the invention, e.g., of
preferred lengths, can be linked to each other or to other amino
acids to create a longer peptide/protein. The modulatory peptides
can be digests of naturally occurring proteins as is outlined
above, random peptides, or "biased" random peptides. In a preferred
embodiment, peptide/protein-based modulators are antibodies, and
fragments thereof, as defined herein.
[0140] Modulators of cancer can also be nucleic acids. Nucleic acid
modulating agents can be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of prokaryotic or eukaryotic genomes can be used in an
approach analogous to that outlined above for proteins.
[0141] 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.
[0142] A "motif", as in biological motif of a 251P5G2-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.
[0143] 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.
[0144] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0145] 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 "oligonuleotide". 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).
[0146] 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".
[0147] An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the HLA
molecule. One to three, usually two, primary anchor residues within
a peptide of defined length generally defines a "motif" for an
immunogenic peptide. These residues are understood to fit in close
contact with peptide binding groove of an HLA molecule, with their
side chains buried in specific pockets of the binding groove. In
one embodiment, for example, the primary anchor residues for an HLA
class I molecule are located at position 2 (from the amino terminal
position) and at the carboxyl terminal position of a 8, 9, 10, 11,
or 12 residue peptide epitope in accordance with the invention.
Alternatively, in another embodiment, the primary anchor residues
of a peptide binds an HLA class II molecule are spaced relative to
each other, rather than to the termini of a peptide, where the
peptide is generally of at least 9 amino acids in length. The
primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the
primary and/or secondary anchor positions shown in Table IV. Such
analogs are used to modulate the binding affinity and/or population
coverage of a peptide comprising a particular HLA motif or
supermotif.
[0148] "Radioisotopes" include, but are not limited to the
following (non-limiting exemplary uses are also set forth):
[0149] Examples of Medical Isotopes:
1 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 (AC-227) skeleton
resulting from cancer (i.e., breast and prostate cancers), and
cancer radioimmunotherapy Bismuth-212 See Thorium-228 (Th-228)
(Bi-212) Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109
Cancer detection (Cd-109) Cobalt-60 Radiation source for
radiotherapy of cancer, for food irradiators, and for sterilization
of (Co-60) medical supplies Copper-64 A positron emitter used for
cancer therapy and SPECT imaging (Cu-64) Copper-67 Beta/gamma
emitter used in cancer radioimmunotherapy and diagnostic studies
(i.e., breast (Cu-67) and colon cancers, and lymphoma)
Dysprosium-166 Cancer radioimmunotherapy (Dy-166) Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints
associated with fingers and (Er-169) toes Europium-152 Radiation
source for food irradiation and for sterilization of medical
supplies (Eu-152) Europium-154 Radiation source for food
irradiation and for sterilization of medical supplies (Eu-154)
Gadolinium-153 Osteoporosis detection and nuclear medical quality
assurance devices (Gd-153) Gold-198 Implant and intracavity therapy
of ovarian, prostate, and brain cancers (Au-198) Holmium-166
Multiple myeloma treatment in targeted skeletal therapy, cancer
radioimmunotherapy, bone (Ho-166) marrow ablation, and rheumatoid
arthritis treatment Iodine-125 Osteoporosis detection, diagnostic
imaging, tracer drugs, brain cancer treatment, (I-125)
radiolabeling, tumor imaging, mapping of receptors in the brain,
interstitial radiation therapy, brachytherapy for treatment of
prostate cancer, determination of glomerular filtration rate (GFR),
determination of plasma volume, detection of deep vein thrombosis
of the legs Iodine-131 Thyroid function evaluation, thyroid disease
detection, treatment of thyroid cancer as well as (I-131) other
non-malignant thyroid diseases (i.e., Graves disease, goiters, and
hyperthyroidism), treatment of leukemia, lymphoma, and other forms
of cancer (e.g., breast cancer) using radioimmunotherapy
Iridium-192 Brachytherapy, brain and spinal cord tumor treatment,
treatment of blocked arteries (i.e., (Ir-192) arteriosclerosis and
restenosis), and implants for breast and prostate tumors
Lutetium-177 Cancer radioimmunotherapy and treatment of blocked
arteries (i.e., arteriosclerosis and (Lu-177) restenosis)
Molybdenum-99 Parent of Technetium-99 m (Tc-99 m) which is used for
imaging the brain, liver, lungs, heart, (Mo-99) and other organs.
Currently, Tc-99 m 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-195 m Studies on biodistribution and metabolism of
cisplatin, a chemotherapeutic drug (Pt-195 m) 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 (P-33) blocked
arteries (i.e., arteriosclerosis and restenosis) Radium-223 See
Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,
rheumatoid arthritis treatment, and diagnosis and treatment of
(Re-186) lymphoma and bone, breast, colon, and liver cancers using
radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using
radioimmunotherapy, bone cancer pain relief, (Re-188) treatment of
rheumatoid arthritis, and treatment of prostate cancer Rhodium-105
Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer
treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy and bone
cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapy
and bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used in
brain studies, imaging of adrenal cortex by gamma-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-99 m See Molybdenum-99 (Mo-99) (Tc-99 m)
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-117 m
Cancer immunotherapy and bone cancer pain relief (Sn-117 m)
Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for
cancer diagnostics/treatment, bone (W-188) cancer pain relief,
rheumatoid arthritis treatment, and treatment of blocked arteries
(i.e., arteriosclerosis and restenosis) Xenon-127 Neuroimaging of
brain disorders, high resolution SPECT studies, pulmonary function
tests, (Xe-127) and 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 (Y-91) (i.e., lymphoma,
breast, colon, kidney, lung, ovarian, prostate, pancreatic, and
inoperable liver cancers)
[0150] By "randomized" or grammatical equivalents as herein applied
to nucleic acids and proteins is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. These random peptides (or nucleic acids, discussed
herein) can incorporate any nucleotide or amino acid at any
position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the
sequence, thus forming a library of randomized candidate bioactive
proteinaceous agents.
[0151] In one embodiment, a library is "fully randomized," with no
sequence preferences or constants at any position. In another
embodiment, the library is a "biased random" library. That is, some
positions within the sequence either are held constant, or are
selected from a limited number of possibilities. For example, the
nucleotides or amino acid residues are randomized within a defined
class, e.g., of hydrophobic amino acids, hydrophilic residues,
sterically biased (either small or large) residues, towards the
creation of nucleic acid binding domains, the creation of
cysteines, for cross-linking, prolines for SH-3 domains, serines,
threonines, tyrosines or histidines for phosphorylation sites,
etc., or to purines, etc.
[0152] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0153] Non-limiting examples of small molecules include compounds
that bind or interact with 251P5G2, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit 251P5G2
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, 251P5G2 protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions
[0154] "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 lnterscience Publishers, (1995).
[0155] "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.
[0156] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles. Overall
phenotypic frequencies of HLA-supertypes in different ethnic
populations are set forth in Table IV (F). The non-limiting
constituents of various supetypes are as follows:
[0157] A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802,
A*6901, A*0207
[0158] A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101
[0159] B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502,
B*5601, B*6701, B*7801, B*0702, B*5101, B*5602
[0160] B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
[0161] A1: A*0102, A*2604, A*3601, A*4301, A*8001
[0162] A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
[0163] B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02,
B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08
[0164] B58: B*1516, B*1517, B*5701, B*5702, B58
[0165] B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513
(B77)
[0166] Calculated population coverage afforded by different
HLA-supertype combinations are set forth in Table IV (G).
[0167] 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.
[0168] 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.
[0169] 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 anywhole unitintegerfrom 1-150 ormore,
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.
[0170] 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 251P5G2
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.
[0171] The "251P5G2-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 251P5G2 proteins or fragments thereof, as well as fusion
proteins of a 251P5G2 protein and a heterologous polypeptide are
also included. Such 251P5G2 proteins are collectively referred to
as the 251P5G2-related proteins, the proteins of the invention, or
251P5G2. The term "251P5G2-related protein" refers to a polypeptide
fragment or a 251P5G2 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more
than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,
70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,
575, or 576 or more amino acids.
[0172] II.) 251P5G2 Polynucleotides
[0173] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of a 251P5G2 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding a 251P5G2-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to a 251P5G2 gene
or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to a 251P5G2 gene, mRNA, or to a
251P5G2 encoding polynucleotide (collectively, "251P5G2
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0174] Embodiments of a 251P5G2 polynucleotide include: a 251P5G2
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 251P5G2 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 251P5G2 nucleotides comprise, without
limitation:
[0175] (I) a polynucleotide comprising, consisting essentially of,
or consisting of a sequence as shown in FIG. 2, wherein T can also
be U;
[0176] (II) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2A, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0177] (III) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2B, from
nucleotide residue number 722 through nucleotide residue number
1489, including the stop codon, wherein T can also be U;
[0178] (IV) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2C, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the a stop codon, wherein T can also be U;
[0179] (V) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2D, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0180] (VI) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2E, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0181] (VII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2F, from
nucleotide residue number 722 through nucleotide residue number
1489, including the stop codon, wherein T can also be U;
[0182] (VIII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2G, from
nucleotide residue number 722 through nucleotide residue number
1489, including the stop codon, wherein T can also be U;
[0183] (IX) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2H, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0184] (X) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2I, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0185] (XI) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2J, from nucleotide
residue number 722 through nucleotide residue number 1489,
including the stop codon, wherein T can also be U;
[0186] (XII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2K, from
nucleotide residue number 722 through nucleotide residue number
1489, including the stop codon, wherein T can also be U;
[0187] (XIII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2L, from
nucleotide residue number 722 through nucleotide residue number
4522, including the stop codon, wherein T can also be U;
[0188] (XIV) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2M, from
nucleotide residue number 1 through nucleotide residue number 3801,
including the stop codon, wherein T can also be U;
[0189] (XV) a polynucleotide that encodes a 251P5G2-related protein
that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%
homologous to an entire amino acid sequence shown in FIGS.
2A-M;
[0190] (XVI) a polynucleotide that encodes a 251P5G2-related
protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identical to an entire amino acid sequence shown in FIGS.
2A-M;
[0191] (XVII) a polynucleotide that encodes at least one peptide
set forth in Tables VIII-XXI and XXII-XLIX;
[0192] (XVIII) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3A-D in any whole number increment up to
255 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35 amino acid position(s) having a value
greater than 0.5 in the Hydrophilicity profile of FIG. 5;
[0193] (XIX) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3A-D in any whole number increment up to
255 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value less than 0.5
in the Hydropathicity profile of FIG. 6;
[0194] (XX) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3A-D in any whole number increment up to
255 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Percent Accessible Residues profile of FIG. 7;
[0195] (XXI) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3A-D in any whole number increment up to
255 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Average Flexibility profile of FIG. 8;
[0196] (XXII) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIGS. 3A-D in any whole number increment up
to 255 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Beta-turn profile of FIG. 9;
[0197] (XXIII) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3E in any whole number increment up to
1266 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Hydrophilicity profile of FIG. 5;
[0198] (XXIV) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3E in any whole number increment up to
1266 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value less than 0.5
in the Hydropathicity profile of FIG. 6;
[0199] (XXV) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3E in any whole number increment up to
1266 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Percent Accessible Residues profile of FIG. 7;
[0200] (XXVI) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3E in any whole number increment up to
1266 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Average Flexibility profile of FIG. 8;
[0201] (XXVII) a polynucleotide that encodes a peptide region of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino
acids of a peptide of FIG. 3E in any whole number increment up to
1266 that indudes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than
0.5 in the Beta-turn profile of FIG. 9
[0202] (XXVIII) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XXVII).
[0203] (XXIX) a peptide that is encoded by any of (I) to (XXVIII);
and
[0204] (XXX) a composition comprising a polynucleotide of any of
(I)-(XXVIII) or peptide of (XXIX) together with a pharmaceutical
excipient and/or in a human unit dose form.
[0205] (XXXI) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a
method to modulate a cell expressing 251P5G2,
[0206] (XXXII) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 251P5G2
[0207] (XXXIII) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 251P5G2, said cell from a cancer of a
tissue listed in Table I;
[0208] (XXIV) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a
method to diagnose, prophylax, prognose, or treat a a cancer;
[0209] (XXXV) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a
method to diagnose, prophylax, prognose, or treat a a cancer of a
tissue listed in Table I; and,
[0210] (XXXVI) a method of using a polynucleotide of any
(I)-(XXVIII) or peptide of (XXIX) or a composiion of (XXX) in a
method to identify or characterize a modulator of a cell expressing
251P5G2.
[0211] As used herein, a range is understood to disclose
specifically all whole unit positions thereof.
[0212] Typical embodiments of the invention disclosed herein
include 251P5G2 polynucleotides that encode specific portions of
251P5G2 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0213] (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, or
255 or more contiguous amino acids of 251P5G2 variant 1; the
maximal lengths relevant for other variants are: variant 2, 255
amino acids; variant 3, 255 amino acids, variant 4, 255 amino
acids, and variant 12, 1266 amino acids.
[0214] 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 251P5G2 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 251P5G2
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 251P5G2 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 251P5G2 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 251P5G2 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 251P5G2
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 251P5G2 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 251P5G2 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 251P5G2 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.
[0215] Polynucleotides encoding relatively long portions of a
251P5G2 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 251P5G2 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 251P5G2
sequence as shown in FIG. 2.
[0216] Additional illustrative embodiments of the invention
disclosed herein include 251P5G2 polynucleotide fragments encoding
one or more of the biological motifs contained within a "251P5G2
protein" or variant sequence, including one or more of the
motif-bearing subsequences of a 251P5G2 protein or varianr set
forth in Tables VIII-XXI and XXII-XLIX. In another embodiment,
typical polynucleotide fragments of the invention encode one or
more of the regions of 251P5G2 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 251P5G2 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.
[0217] Note that to determine the starting position of any peptide
set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively
HLA Peptide Tables) respective to its parental protein, e.g.,
variant 1, variant 2, etc., reference is made to three factors: the
particular variant, the length of the peptide in an HLA Peptide
Table, and the Search Peptides listed in Table VII. Generally, a
unique Search Peptide is used to obtain HLA peptides for a
particular variant. The position of each Search Peptide relative to
its respective parent molecule is listed in Table VII. Accordingly,
if a Search Peptide begins at position "X", one must add the value
"X minus 1" to each position in Tables VIII-XXI and Tables XXII-IL
to obtain the actual position of the HLA peptides in their parental
molecule. For example if a particular Search Peptide begins at
position 150 of its parental molecule, one must add 150-1, i.e.,
149 to each HLA peptide amino acid position to calculate the
position of that amino acid in the parent molecule.
[0218] II.A.) Uses of 251P5G2 Polynucleotides
[0219] II.A.1.) Monitoring of Genetic Abnormalities
[0220] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 251P5G2 gene maps to
the chromosomal location set forth in the Example entitled
"Chromosomal Mapping of 251P5G2." For example, because the 251P5G2
gene maps to this chromosome, polynucleotides that encode different
regions of the 251P5G2 proteins are used to characterize
cytogenefic abnormalities of this chromosomal locale, such as
abnormalities that are identified as being associated with various
cancers. In certain genes, a variety of chromosomal abnormalities
including rearrangements have been identified as frequent
cytogenetic abnormalities in a number of different cancers (see
e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);
Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,
P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding
specific regions of the 251P5G2 proteins provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 251P5G2 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)).
[0221] Furthermore, as 251P5G2 was shown to be highly expressed in
prostate and other cancers, 251P5G2 polynucleotides are used in
methods assessing the status of 251P5G2 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 251P5G2 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 251P5G2 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.
[0222] II.A.2.) Antisense Embodiments
[0223] Other specifically contemplated nucleic acid related
embodiments of the invention disclosed herein are genomic DNA,
cDNAs, dibozymes, 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 indude molecules capable of inhibiting the RNA or
protein expression of 251P5G2. 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 251P5G2 polynucleotides and polynucleotide
sequences disclosed herein.
[0224] 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., 251P5G2. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 251P5G2 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 phosphor6thioates) 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 a., J. Am. Chem. Soc. 112:1253-1254
(1990). Additional 251P5G2 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).
[0225] The 251P5G2 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 251P5G2 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 251P5G2 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 251P5G2 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 251P5G2 mRNA. Optionally, 251P5G2 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
251P5G2. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 251P5G2 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12:
510-515 (1996).
[0226] II.A.3.) Primers and Primer Pairs
[0227] 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 251P5G2 polynucleotide in a sample and as a means for
detecting a cell expressing a 251P5G2 protein.
[0228] Examples of such probes indude polypeptides comprising all
or part of the human 251P5G2 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 251P5G2
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 251P5G2 mRNA.
[0229] The 251P5G2 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
251P5G2 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 251P5G2
polypeptides; as tools for modulating or inhibiting the expression
of the 251P5G2 gene(s) and/or translation of the 251P5G2
transcript(s); and as therapeutic agents.
[0230] The present invention includes the use of any probe as
described herein to identify and isolate a 251P5G2 or 251P5G2
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.
[0231] II.A.4.) Isolation of 251P5G2-Encoding Nucleic Acid
Molecules
[0232] The 251P5G2 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 251P5G2 gene
product(s), as well as the isolation of polynucleotides encoding
251P5G2 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 251P5G2 gene product as
well as polynucleotides that encode analogs of 251P5G2-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding a 251P5G2 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 doning
methodologies can be conveniently employed, using commercially
available cloning systems (e.g., Lambda ZAP Express, Stratagene).
Phage clones containing 251P5G2 gene cDNAs can be identified by
probing with a labeled 251P5G2 cDNA or a fragment thereof. For
example, in one embodiment, a 251P5G2 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 251P5G2 gene.
A 251P5G2 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 251P5G2
DNA probes or primers.
[0233] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0234] The invention also provides recombinant DNA or RNA molecules
containing a 251P5G2 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).
[0235] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a 251P5G2
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 251P5G2 or a fragment, analog or homolog thereof can be
used to generate 251P5G2 proteins or fragments thereof using any
number of host-vector systems routinely used and widely known in
the art.
[0236] A wide range of host-vector systems suitable for the
expression of 251P5G2 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 pSRatkneo (Muller et al.,
1991, MCB 11:1785). Using these expression vectors, 251P5G2 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 251P5G2 protein or fragment thereof. Such host-vector systems
can be employed to study the functional properties of 251P5G2 and
251P5G2 mutations or analogs.
[0237] Recombinant human 251P5G2 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 251P5G2-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 251P5G2 or fragment, analog or homolog thereof, a
251P5G2-related protein is expressed in the 293T cells, and the
recombinant 251P5G2 protein is isolated using standard purification
methods (e.g., affinity purification using anti-251P5G2
antibodies). In another embodiment, a 251P5G2 coding sequence is
subcloned into the retroviral vector pSR.alpha.MSVtkneo and used to
infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293
and rat-1 in order to establish 251P5G2 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 251P5G2 coding sequence can be used for the generation
of a secreted form of recombinant 251P5G2 protein.
[0238] As discussed herein, redundancy in the genetic code permits
variation in 251PSG2 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/-nakamura/codon.html.
[0239] Additional sequence modifications are known to enhance
protein expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exonlintron
splice site signals, transposon-like repeats, and/or other such
well-haracterized 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)).
[0240] III.) 251P5G2-Related Proteins
[0241] Another aspect of the present invention provides
251P5G2-related proteins. Specific embodiments of 251P5G2 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 251P5G2 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 251P5G2 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 251P5G2 shown in FIG. 2 or FIG. 3.
[0242] Embodiments of a 251P5G2 polypeptide include: a 251P5G2
polypeptide having a sequence shown in FIG. 2, a peptide sequence
of a 251P5G2 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 251P5G2 peptides comprise, without
limitation:
[0243] (I) a protein comprising, consisting essentially of, or
consisting of an amino acid sequence as shown in FIGS. 2A-M or
FIGS. 3A-E;
[0244] (II) a 251P5G2-related protein that is at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino
acid sequence shown in FIGS. 2A-M or 3A-E;
[0245] (III) a 251P5G2-related protein that is at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino
acid sequence shown in FIGS. 2A-M or 3A-E;
[0246] (IV) a protein that comprises at least one peptide set forth
in Tables VIII to XLIX, optionally with a proviso that it is not an
entire protein of FIG. 2;
[0247] (V) a protein that comprises at least one peptide set forth
in Tables VIII-XXI, collectively, which peptide is also set forth
in Tables XXII to XLIX, collectively, optionally with a proviso
that it is not an entire protein of FIG. 2;
[0248] (VI) a protein that comprises at least two peptides selected
from the peptides set forth in Tables VIII-XLIX, optionally with a
proviso that it is not an entire protein of FIG. 2;
[0249] (VII) a protein that comprises at least two peptides
selected from the peptides set forth in Tables VII to XLIX
collectively, with a proviso that the protein is not a contiguous
sequence from an amino acid sequence of FIG. 2;
[0250] (VIII) a protein that comprises at least one peptide
selected from the peptides set forth in Tables VII-XXI; and at
least one peptide selected from the peptides set forth in Tables
XXII to XLIX, with a proviso that the protein is not a contiguous
sequence from an amino acid sequence of FIG. 2;
[0251] (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.
3A-D or 3E, in any whole number increment up to 255 or 1266
respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a
value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
[0252] (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A-D
or 3E, in any whole number increment up to 255 or 1266 respectively
that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value
less than 0.5 in the Hydropathicity profile of FIG. 6;
[0253] (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.
3A-D, or 3E, in any whole number increment up to 255 or 1266
respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)
having a value greater than 0.5 in the Percent Accessible Residues
profile of FIG. 7;
[0254] (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.
3A-D or 3E, in any whole number increment up to 255 or 1266
respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)
having a value greater than 0.5 in the Average Flexibility profile
of FIG. 8;
[0255] (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIGS. 3A-D
or 3E in any whole number increment up to 255 or 1266 respectively
that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value
greater than 0.5 in the Beta-turn profile of FIG. 9;
[0256] (XIV) a peptide that occurs at least twice in Tables
VIII-XXI and XXII to XLIX, collectively;
[0257] (XV) a peptide that occurs at least three times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0258] (XVI) a peptide that occurs at least four times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0259] (XVII) a peptide that occurs at least five times in Tables
VIII-XXI and XXII to XLIX, collectively;
[0260] (XVIII) a peptide that occurs at least once in Tables
VIII-XXI, and at least once in, tables XXII to XLIX;
[0261] (XIX) a peptide that occurs at least once in Tables
VIII-XXI, and at least twice in tables XXII to XLIX;
[0262] (XX) a peptide that occurs at least twice in Tables
VIII-XXI, and at least once in tables XXII to XLIX;
[0263] (XXI) a peptide that occurs at least twice in Tables
VIII-XXI, and at least twice in tables XXII to XLIX;
[0264] (XXII) a peptide which comprises one two, three, four, or
five of the following characteristics, or an oligonucleotide
encoding such peptide:
[0265] 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;
[0266] ii) a region of at least 5 amino acids of a particular
peptide of FIG. 3, in any whole number increment up to the full
length of that protein in FIG. 3, that includes an amino acid
position having a value equal to or less than 0.5, 0.4, 0.3, 0.2,
0.1, or having a value equal to 0.0, in the Hydropathicity profile
of FIG. 6;
[0267] 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 indudes 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;
[0268] 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,
[0269] 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;
[0270] (XXIII) a composition comprising a peptide of (I)-(XXII) or
an antibody or binding region thereof together with a
pharmaceutical excipient and/or in a human unit dose form.
[0271] (XXIV) a method of using a peptide of (I)-(XXII), or an
antibody or binding region thereof or a composition of (XXIII) in a
method to modulate a cell expressing 251P5G2,
[0272] (XXV) a method of using a peptide of (I)-(XXII) or an
antibody or binding region thereof or a composition of (XXIII) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 251P5G2
[0273] (XXVI) a method of using a peptide of (I)-(XXII) or an
antibody or binding region thereof or a composition (XXIII) in a
method to diagnose, prophylax, prognose, or treat an individual who
bears a cell expressing 251P5G2, said cell from a cancer of a
tissue listed in Table I;
[0274] (XXVII) a method of using a peptide of (I)-(XXII) or an
antibody or binding region thereof or a composition of (XXIII) in a
method to diagnose, prophylax, prognose, or treat a a cancer;
[0275] (XXVIII) a method of using a peptide of (I)-(XXII) or an
antibody or binding region thereof or a composition of (XXIII) in a
method to diagnose, prophylax, prognose, or treat a a cancer of a
tissue listed in Table I; and,
[0276] (XXIX) a method of using a a peptide of (I)-(XXII) or an
antibody or binding region thereof or a composition (XXIII) in a
method to identify or characterize a modulator of a cell expressing
251P5G2.
[0277] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0278] Typical embodiments of the invention disclosed herein
include 251P5G2 polynucleotides that encode specific portions of
251P5G2 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0279] (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, or
255 or more contiguous amino acids of 251P5G2 variant 1; the
maximal lengths relevant for other variants are: variant 2, 255
amino acids; variant 3, 255 amino acids, variant 4, 255 amino
acids, and variant 12, 1266 amino acids.
[0280] In general, naturally occurring allelic variants of human
251P5G2 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
251P5G2 protein contain conservative amino acid substitutions
within the 251P5G2 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 251P5G2. One dass of 251P5G2 allelic variants are
proteins that share a high degreeof homology with at least a small
region of a particular 251P5G2 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.
[0281] 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 May 19, 1995;
270(20):11882-6).
[0282] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 251P5G2 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 251P5G2 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 251P5G2 variant DNA.
[0283] 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.
[0284] As defined herein, 251P5G2 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 251P5G2 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
251P5G2 variant also specifically binds to a 251P5G2 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 251P5G2 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.
[0285] Other classes of 251P5G2-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 251P5G2
protein variants or analogs comprises one or more of the 251P5G2
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of 251P5G2
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.
[0286] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a 251P5G2 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 251P5G2 protein shown in
FIG. 2 or FIG. 3.
[0287] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of a 251P5G2 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a 251P5G2 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypepbdes
consisting of about amino acid 30 to about amino acid 40 of a
251P5G2 protein shown in FIG. 2 or FIG. 3, polypepbdes consisting
of about amino acid 40 to about amino acid 50 of a 251P5G2 protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 251P5G2 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 60 to about
amino acid 70 of a 251P5G2 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 70 to about amino acid
80 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 80 to about amino acid 90 of a
251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 90 to about amino acid 100 of a 251P5G2 protein
shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a
251P5G2 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 251P5G2 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.
[0288] 251P5G2-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
251P5G2-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a 251P5G2 protein
(or variants, homologs or analogs thereof).
[0289] III.A.) Motif-bearing Protein Embodiments
[0290] Additional illustrative embodiments of the invention
disclosed herein include 251P5G2 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a 251P5G2 polypeptide sequence set forth in FIG. 2 or FIG.
3. Various motifs are known in the art, and a protein can be
evaluated for the presence of such motifs by a number of publicly
available Internet sites (see, e.g., URL addresses:
pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-p-
redict.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/ResearchrTB-HIV- _Lab/epimatrix/epimatrix.html; and
BIMAS, bimas.dcrt.nih.gov/.).
[0291] Motif bearing subsequences of all 251P5G2 variant proteins
are set forth and identified in Tables VIII-XXI and XXII-XLIX.
[0292] Table V sets forth several frequently occurring motifs based
on pfam searches (see URL address pfam.wustl.edu/). The columns of
Table V list (1) motif name abbreviation, (2) percent identity
found amongst the different member of the motif family, (3) motif
name or description and (4) most common function; location
information is included if the motif is relevant for location.
[0293] Polypeptides comprising one or more of the 251P5G2 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 251P5G2 motifs discussed above are associated with growth
dysregulation and because 251P5G2 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 a., 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 my ristoylafion 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)).
[0294] In another embodiment, proteins of the invention comprise
one or more of the immunoreactive epitopes identified in accordance
with art-accepted methods, such as the peptides set forth in Tables
VIII-XXI and XXII-XLIX. CTL epitopes can be determined using
specific algorithms to identify peptdes within a 251P5G2 protein
that are capable of optimally binding to specified HLA alleles
(e.g., Table IV; Epimatrix.TM. and Epimer.TM., Brown University,
URL brown.edu/Research/TB-HIV_Lab/epima- trix/epimatrix.html; and
BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for
identifying peptides that have sufficient binding affinity for HLA
molecules and which are correlated with being immunogenic epitopes,
are well known in the art, and are carried out without undue
experimentation. In addition, processes for identifying peptides
that are immunogenic epitopes, are well known in the art, and are
carried out without undue experimentation either in vitro or in
vivo.
[0295] Also known in the art are principles for creating analogs of
such epitopes in order to modulate immunogenicity. For example, one
begins with an epitope that bears a CTL or HTL motif (see, e.g.,
the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
The epitope is analoged by substituting out an amino acid at one of
the specified positions, and replacing it with another amino acid
specified for that position. For example, on the basis of residues
defined in Table IV, one can substitute out a deleterious residue
in favor of any other residue, such as a preferred residue;
substitute a less-preferred residue with a preferred residue; or
substitute an originally-occurring preferred residue with another
preferred residue. Substitutions can occur at primary anchor
positions or at other positions in a peptide; see, e.g., Table
IV.
[0296] 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.; Selte, Immunogenetics 1999 50(34): 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 a!, 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 etaL, 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.
[0297] Related embodiments of the invention include polypeptdes
comprising combinations of the different motifs set forth in Table
VI, and/or, one or more of the predicted CTL epitopes of Tables
VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL
epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell
binding motifs known in the art. Preferred embodiments contain no
insertions, deletions or substitutions either within the motifs or
within the intervening sequences of the polypeptides. In addition,
embodiments which include a number of either N-terminal and/or
C-terminal amino acid residues on either side of these motifs may
be desirable (to, for example, include a greater portion of the
polypeptide architecture in which the motif is located). Typically,
the number of N-terminal and/or C-terminal amino acid residues on
either side of a motif is between about 1 to about 100 amino acid
residues, preferably 5 to about 50 amino acid residues.
[0298] 251P5G2-related proteins are embodied in many forms,
preferably in isolated form. A purified 251P5G2 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 251P5G2 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 251P5G2-related proteins include
purified 251P5G2-related proteins and functional, soluble
251P5G2-related proteins. In one embodiment, a functional, soluble
251P5G2 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0299] The invention also provides 251P5G2 proteins comprising
biologically active fragments of a 251P5G2 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting 251P5G2 protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting 251P5G2 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.
[0300] 251P5G2-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, Gamier-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-251P5G2
antibodies or T cells or in identifying cellular factors that bind
to 251P5G2. 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 idenfified, 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.
[0301] CTL epitopes can be determined using specific algorithms to
identify peptides within a 251P5G2 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 251P5G2 that are presented in the context of human
MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35
were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX).
Specifically, the complete amino acid sequence of the 251P5G2
protein and relevant portions of other variants, i.e., for HLA
Class I predictions 9 flanking residues on either side of a point
mutation or exon juction, and for HLA Class II predictions 14
flanking residues on either side of a point mutation or exon
junction corresponding to that variant, were entered into the HLA
Peptide Motif Search algorithm found in the Bioinformatics and
Molecular Analysis Section (BIMAS) web site listed above; in
addition to the site SYFPEITHI, at URL
syfpeithi.bmi-heidelberg.com/.
[0302] 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 251P5G2 predicted binding peptides are shown in Tables
VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII,
selected candidates, 9-mers and 10-mers, for each family member are
shown along with their location, the amino acid sequence of each
specific peptide, and an estimated binding score. In Tables
XLVI-XLIX, selected candidates, 15-mers, for each family member are
shown along with their location, the amino acid sequence of each
specific peptide, and an estimated binding score. The binding score
corresponds to the estimated half time of dissociation of complexes
containing the peptide at 37.degree. C. at pH 6.5. Peptides with
the highest binding score are predicted to be the most tightly
bound to HLA Class I on the cell surface for the greatest period of
time and thus represent the best immunogenic targets for T-cell
recognition.
[0303] 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.
[0304] It is to be appreciated that every epitope predicted by the
BIMAS site, Epimer.TM. and Epimatdx.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.dcrtnih.gov/) are to be "applied" to a 251P5G2 protein in
accordance with the invention. As used in this context "applied"
means that a 251P5G2 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 251P5G2 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.
[0305] III.B.) Expression of 251P5G2-related Proteins
[0306] In an embodiment described in the examples that follow,
251P5G2 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 251P5G2 with a C-terminal
6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter
Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK
secretion signal that can be used to facilitate the production of a
secreted 251P5G2 protein in transfected cells. The secreted
HIS-tagged 251P5G2 in the culture media can be purified, e.g.,
using a nickel column using standard techniques.
[0307] III.C.) Modifications of 251P5G2-related Proteins
[0308] Modifications of 251P5G2-related proteins such as covalent
modifications are included within the scope of this invention. One
type of covalent modification indudes reacting targeted amino acid
residues of a 251P5G2 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C- terminal residues of a 251P5G2 protein. Another type of
covalent modification of a 251P5G2 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 251P5G2 comprises linking a 251P5G2 polypeptide to
one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the
manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0309] The 251P5G2-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 251P5G2
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 251P5G2 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 251P5G2. A chimeric
molecule can comprise a fusion of a 251P5G2-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 251P5G2 protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
251P5G2-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 251P5G2 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0310] III.D.) Uses of 251P5G2-related Proteins
[0311] The proteins of the invention have a number of different
specific uses. As 251P5G2 is highly expressed in prostate and other
cancers, 251P5G2-related proteins are used in methods that assess
the status of 251P5G2 gene products in normal versus cancerous
tissues, thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of a 251P5G2 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 251P5G2-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within a 251P5G2 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,
251P5G2-related proteins that contain the amino acid residues of
one or more of the biological motifs in a 251P5G2 protein are used
to screen for factors that interact with that region of
251P5G2.
[0312] 251P5G2 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of a 251P5G2 protein), for identifying agents or cellular
factors that bind to 251P5G2 or a particular structural domain
thereof, and in various therapeutic and diagnostic contexts,
induding but not limited to diagnostic assays, cancer vaccines and
methods of preparing such vaccines.
[0313] Proteins encoded by the 251P5G2 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 251P5G2 gene product Antibodies raised against a 251P5G2
protein or fragment thereof are useful in diagnostic and prognostic
assays, and imaging methodologies in the management of human
cancers characterized by expression of 251P5G2 protein, such as
those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 251P5G2-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0314] Various immunological assays useful for the detection of
251P5G2 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. Antbodies can be labeled
and used as immunological imaging reagents capable of detecting
251P5G2-expressing cells (e.g., in radioscintigraphic imaging
methods). 251P5G2 proteins are also paricularly useful in
generating cancer vaccines, as further described herein.
[0315] IV.) 251P5G2Antibodies
[0316] Another aspect of the invention provides antibodies that
bind to 251P5G2-related proteins. Preferred antibodies specifically
bind to a 251P5G2-related protein and do not bind (or bind weakly)
to peptdes or proteins that are not 251P5G2-related proteins under
physiological conditions. In this context, examples of
physiological conditions include: 1) phosphate buffered saline; 2)
Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or
normal saline (0.9% NaCl); 4) animal serum such as human serum; or,
5) a combination of any of 1) through 4); these reactions
preferably taking place at pH 7.5, alternatively in a range of pH
7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also,
these reactions taking place at a temperature between 4.degree. C.
to 37.degree. C. For example, antibodies that bind 251P5G2 can bind
251P5G2-related proteins such as the homologs or analogs
thereof.
[0317] 251P5G2 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 251P5G2 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 251P5G2 is involved, such as
advanced or metastatic prostate cancers.
[0318] The invention also provides various immunological assays
useful for the detection and quantification of 251P5G2 and mutant
251P5G2-related proteins. Such assays can comprise one or more
251P5G2 antibodies capable of recognizing and binding a
251P5G2-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,
enzymelinked immunosorbent assays (ELISA), enzyme-linked
immunofluorescent assays (ELIFA), and the like.
[0319] Immunological non-antibody assays of the invention also
comprise T cell immunogenicity assays (inhibitory or stimulatory)
as well as major histocompatbility complex (MHC) binding
assays.
[0320] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing 251P5G2 are
also provided by the invention, including but not limited to
radioscinigraphic imaging methods using labeled 251P5G2 antibodies.
Such assays are clinically useful in the detection, monitoring, and
prognosis of 251P5G2 expressing cancers such as prostate
cancer.
[0321] 251P5G2 antibodies are also used in methods for purifying a
251P5G2-related protein and for isolating 251P5G2 homologues and
related molecules. For example, a method of purifying a
251P5G2-related protein comprises incubating a 251P5G2 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 251P5G2-related protein under conditions that
permit the 251P5G2 antibody to bind to the 251P5G2-related protein;
washing the solid matrix to eliminate impurities; and eluting the
251P5G2-related protein from the coupled antibody. Other uses of
251P5G2 antibodies in accordance with the invention include
generating anti-idiotypic antibodies that mimic a 251P5G2
protein.
[0322] 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 251P5G2-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 251P5G2 can also be used, such as a
251P5G2 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 251P5G2-related
protein is synthesized and used as an immunogen.
[0323] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 251P5G2-related protein or
251P5G2 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0324] The amino acid sequence of a 251P5G2 protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
251P5G2 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 251P5G2 amino acid
sequence are used to identify hydrophilic regions in the 251P5G2
structure. Regions of a 251P5G2 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, Gamier-Robson, Kyte-Doolittie, Eisenberg,
Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles
can be generated using the method of Hopp, T. P. and Woods, K. R.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity
profiles can be generated using the method of Kyte, J. and
Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%)
Accessible Residues profiles can be generated using the method of
Janin J., 1979, Nature 277:491-492. Average Flexibility profiles
can be generated using the method of Bhaskaran R., Ponnuswamy P.
K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles
can be generated using the method of Deleage, G., Roux B., 1987,
Protein Engineering 1:289-294. Thus, each region identified by any
of these programs or methods is within the scope of the present
invention. Methods for the generation of 251P5G2 antibodies are
further illustrated by way of the examples provided herein. Methods
for preparing a protein or polypeptide for use as an immunogen are
well known in the art. Also well known in the art are methods for
preparing immunogenic conjugates of a protein with a carrier, such
as BSA, KLH or other carrier protein. In some circumstances, direct
conjugation using, for example, carbodiimide reagents are used; in
other instances linking reagents such as those supplied by Pierce
Chemical Co., Rockford, Ill., are effective. Administration of a
251P5G2 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.
[0325] 251P5G2 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
251P5G2-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.
[0326] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a 251P5G2 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
251P5G2 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.
[0327] 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 251P5G2 monoclonal antibodies can be generated using doning
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 Applicatons 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 251P5G2
monodonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO98/24893, Kucherlapat and Jakobovits et
al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin.
Invest. Drugs 7(4): 607414; U.S. Pat. Nos. 6,162,963 issued Dec.
19, 2000; 6,150,584 issued Nov. 12, 2000; and, 6,114598 issued Sep.
5, 2000). This method avoids the in vitro manipulation required
with phage display technology and efficienty produces high affinity
authentic human antibodies.
[0328] Reactivity of 251P5G2 antibodies with a 251P5G2-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 251P5G2-related proteins,
251P5G2-expressing cells or extracts thereof. A 251P5G2 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 251P5G2 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).
[0329] V.) 251P5G2 Cellular Immune Responses
[0330] 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.
[0331] 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 a., SYFPEITHI, access via World Wide Web at URL
(134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and
Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H.,
Curr. Opin. Immunol. 6:13, 1994; Selle, 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).
[0332] 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.)
[0333] 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).
[0334] 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.
[0335] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0336] 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.
[0337] 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 vftro 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.
[0338] 3) Demonstration of recall T cell responses from immune
individuals who have been either effectively vaccinated and/or from
chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp.
Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997;
Bertoni, R. et al., J. Clin. Invest. 100:503,1997; Threlkeld, S. C.
et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J.
Virol. 71:6011, 1997). Accordingly, recall responses are detected
by culturing PBL from subjects that have been exposed to the
antigen due to disease and thus have generated an immune response
"naturally", or from patients who were vaccinated against the
antigen. PBL from subjects are cultured in vitro for 1-2 weeks in
the presence of test peptide plus antigen presenting cells (APC) to
allow activation of "memory" T cells, as compared to "naive" T
cells. At the end of the culture period, T cell activity is
detected using assays including .sup.51Cr release involving
peptide-sensitized targets, T cell proliferation, or lymphokine
release.
[0339] VI.) 251P5G2 Transgenic Animals
[0340] Nucleic acids that encode a 251P5G2-related protein can also
be used to generate either transgenic animals or "knock out"
animals that, in tum, are useful in the development and screening
of therapeutically useful reagents. In accordance with established
techniques, cDNA encoding 251P5G2 can be used to clone genomic DNA
that encodes 251P5G2. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 251P5G2. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. Nos.
4,736,866 issued Apr. 12, 1988, and 4,870,009 issued Sep. 26, 1989.
Typically, particular cells would be targeted for 251P5G2 transgene
incorporation with tissue-specific enhancers.
[0341] Transgenic animals that include a copy of a transgene
encoding 251P5G2 can be used to examine the effect of increased
expression of DNA that encodes 251P5G2. 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.
[0342] Alternatively, non-human homologues of 251P5G2 can be used
to construct a "251P5G2 " knock outs animal that has a defective or
altered gene encoding 251P5G2 as a result of homologous
recombination between the endogenous gene encoding 251P5G2 and
altered genomic DNA encoding 251P5G2 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 251P5G2 can be
used to clone genomic DNA encoding 251P5G2 in accordance with
established techniques. A portion of the genomic DNA encoding
251P5G2 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
251P5G2 polypeptide.
[0343] VII.) Methods for the Detection of 251P5G2
[0344] Another aspect of the present invention relates to methods
for detecting 251P5G2 polynucleotides and 251P5G2-related proteins,
as well as methods for identfying a cell that expresses 251P5G2.
The expression profile of 251P5G2 makes it a diagnostic marker for
metastasized disease. Accordingly, the status of 251P5G2 gene
products provides information useful for predicting a variety of
factors including suscepubility to advanced stage disease, rate of
progression, and/or tumor aggressiveness. As discussed in detail
herein, the status of 251P5G2 gene products in patient samples can
be analyzed by a variety protocols that are well known in the art
induding immunohistochemical analysis, the variety of Northern
blotting techniques including in situ hybridizabon, RT-PCR analysis
(for example on laser capture micro-dissected samples), Western
blot analysis and tissue array analysis.
[0345] More particularly, the invention provides assays for the
detection of 251P5G2 polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 251P5G2 polynucleotides
include, for example, a 251P5G2 gene or fragment thereof, 251P5G2
mRNA, alternative splice variant 251P5G2 mRNAs, and recombinant DNA
or RNA molecules that contain a 251P5G2 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 251P5G2
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0346] In one embodiment, a method for detecting a 251P5G2 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 251P5G2 polynucleotides as sense and
antisense primers to amplify 251P5G2 cDNAs therein; and detecting
the presence of the amplified 251P5G2 cDNA. Optionally, the
sequence of the amplified 251P5G2 cDNA can be determined.
[0347] In another embodiment, a method of detecting a 251P5G2 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 251P5G2
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 251P5G2 gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
251P5G2 nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0348] The invention also provides assays for detecting the
presence of a 251P5G2 protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a 251P5G2-related
protein are also well known and indude, for example,
immunoprecipitaton, immunohistochemical analysis, Western blot
analysis, molecular binding assays, ELISA, ELIFA and the like. For
example, a method of detecting the presence of a 251P5G2-related
protein in a biological sample comprises first contacting the
sample with a 251P5G2 antibody, a 251P5G2-reactive fragment
thereof, or a recombinant protein containing an antigen-binding
region of a 251P5G2 antibody; and then detecting the binding of
251P5G2-related protein in the sample.
[0349] Methods for identifying a cell that expresses 251P5G2 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 251P5G2 gene comprises
detecting the presence of 251P5G2 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 sdu hybridization using labeled 251P5G2 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 251P5G2, 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 251P5G2 gene comprises detecting the presence of
251P5G2-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 251P5G2-related proteins
and cells that express 251P5G2-related proteins.
[0350] 251P5G2 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 251P5G2 gene
expression. For example, 251P5G2 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 251P5G2 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 251P5G2 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0351] VIII.) Methods for Monitoring the Status of 251P5G2-related
Genes and Their Products
[0352] 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 251P5G2 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 251P5G2 in a biological
sample of interest can be compared, for example, to the status of
251P5G2 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 251P5G2 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. Dec. 9, 1996; 376(2): 306-14 and U.S. Pat.
No. 5,837,501) to compare 251P5G2 status in a sample.
[0353] 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 251P5G2
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 251P5G2 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 251P5G2
comprises a change in the location of 251P5G2 and/or 251P5G2
expressing cells and/or an increase in 251P5G2 mRNA and/or protein
expression.
[0354] 251P5G2 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 251P5G2 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 251P5G2 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 251P5G2 gene), Northern analysis and/or PCR analysis of 251P5G2
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 251P5G2 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
251P5G2 proteins and/or associations of 251P5G2 proteins with
polypeptide binding partners). Detectable 251P5G2 polynucleotides
include, for example, a 251P5G2 gene or fragment thereof, 251P5G2
mRNA, alternative splice variants, 251P5G2 mRNAs, and recombinant
DNA or RNA molecules containing a 251P5G2 polynucleotide.
[0355] The expression profile of 251P5G2 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 251P5G2 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 251P5G2 status and diagnosing
cancers that express 251P5G2, such as cancers of the tissues listed
in Table I. For example, because 251PSG2 mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of 251P5G2 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 251P5G2 dysregulaton, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0356] The expression status of 251P5G2 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 251P5G2 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.
[0357] As described above, the status of 251P5G2 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 251P5G2 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 251P5G2
expressing cells (e.g. those that express 251P5G2 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 251P5G2-expressing cells
arefound in a biological sample that does not normally contain such
cells (such as a lymph node), because such alterations in the
status of 251P5G2 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).
[0358] In one aspect, the invention provides methods for monitoring
251P5G2 gene products by determining the status of 251P5G2 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 251P5G2 gene products in a corresponding normal
sample. The presence of aberrant 251P5G2 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.
[0359] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 251P5G2 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
251P5G2 mRNA can, for example, be evaluated in tissues including
but not limited to those listed in Table I. The presence of
significant 251P5G2 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 251P5G2 mRNA
or express it at lower levels.
[0360] In a related embodiment, 251P5G2 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 251P5G2 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 251P5G2 expressed in a corresponding
normal sample. In one embodiment, the presence of 251P5G2 protein
is evaluated, for example, using immunohistochemical methods.
251P5G2 antibodies or binding partners capable of detecting 251P5G2
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0361] In a further embodiment, one can evaluate the status of
251P5G2 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules. These perturbations can indude 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
251P5G2 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 251P5G2 indicates a potential loss of function or
increase in tumor growth.
[0362] 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 251P5G2 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. Nos. 5,382,510 issued Sep. 7, 1999, and 5,952,170
issued Jan. 17, 1995).
[0363] Additionally, one can examine the methylaton status of a
251P5G2 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 hypermethylaton of the pi-class glutathione S-transferase
(a protein expressed in normal prostate but not expressed in
>90% of prostate carcinomas) appears to permanently silence
transcription of this gene and is the most frequently detected
genomic alteration in prostate carcinomas (De Marzo et al., Am. J.
Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is
present in at least 70% of cases of high-grade prostatic
intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.
Biomarkers Prev., 1998, 7:531-536). In another example, expression
of the LAGE-I tumor specific gene (which is not expressed in normal
prostate but is expressed in 25-50% of prostate cancers) is induced
by deoxy-azacytdine 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
methylaton 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 deave
sequences that contain methylated CpG sites to assess the
methylation status of CpG islands. In addition, MSP (methylation
specific PCR) can rapidly profile the methylaton 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.
[0364] Gene amplification is an additional method for assessing the
status of 251P5G2. 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.
[0365] 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 251P5G2 expression.
The presence of RT-PCR amplifiable 251P5G2 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 indude 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).
[0366] 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 251P5G2 mRNA or 251P5G2 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 251P5G2 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 251P5G2
in prostate or other tissue is examined, with the presence of
251P5G2 in the sample providing an indication of prostate cancer
susceptibility (or the emergence or existence of a prostate tumor).
Similarly, onecan evaluate the integrity 251P5G2 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 251P5G2 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0367] 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
251P5G2 mRNA or 251P5G2 protein expressed by tumor cells, comparing
the level so determined to the level of 251P5G2 mRNA or 251P5G2
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 251P5G2 mRNA or 251P5G2 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 251P5G2 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 251P5G2 nucleotde 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.
[0368] 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 251P5G2 mRNA or 251P5G2 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 251P5G2 mRNA or 251P5G2 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 251P5G2 mRNA or 251P5G2 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 251P5G2 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
251P5G2 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.
[0369] 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 251P5G2 gene and 251P5G2 gene products (or
perturbations in 251P5G2 gene and 251P5G2 gene products) and a
factor that is assodated 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 251P5G2 gene and 251P5G2 gene
products (or perturbations in 251P5G2 gene and 251P5G2 gene
products) and another factor that is associated with malignancy are
useful, for example, because the presence of a set of specic
factors that coincide with disease provides information crucial for
diagnosing and prognosticating the status of a tissue sample.
[0370] In one embodiment, methods for observing a coincidence
between the expression of 251P5G2 gene and 251P5G2 gene products
(or perturbations in 251P5G2 gene and 251P5G2 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 251P5G2 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
251P5G2 mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of 251P5G2 and PSA mRNA in prostate issue is examined, where the
coincidence of 251P5G2 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.
[0371] Methods for detecting and quantifying the expression of
251P5G2 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
quantificaton of 251P5G2 mRNA include in situ hybridization using
labeled 251P5G2 riboprobes, Northern blot and related techniques
using 251P5G2 polynucdeotde probes, RT-PCR analysis using primers
specific for 251P5G2, 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 251P5G2 mRNA expression. Any number of primers
capable of amplifying 251P5G2 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
251P5G2 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0372] IX.) Identification of Molecules That Interact With
251P5G2
[0373] The 251P5G2 protein and nucleic add sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 251P5G2, as well as
pathways activated by 251P5G2 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued Sep. 21,
1999, 5,925,523 issued Jul. 20, 1999, 5,846,722 issued Dec. 8, 1998
and 6,004,746 issued Dec. 21, 1999. Algorithms are also available
in the art for genome-based predictions of protein function (see,
e.g., Marcotte, et al., Nature 402: Nov. 4, 1999, 83-86).
[0374] Alternatively one can screen peptide libraries to identify
molecules that interact with 251P5G2 protein sequences. In such
methods, peptides that bind to 251P5G2 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 251P5G2 protein(s).
[0375] 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 251P5G2 protein sequences are disclosed for example
in U.S. Pat. Nos. 5,723,286 issued Mar. 3, 1998 and 5,733,731
issued Mar. 31, 1998.
[0376] Alternatively, cell lines that express 251P5G2 are used to
identify protein-protein interactions mediated by 251P5G2. Such
interactions can be examined using immunopredipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 251P5G2 protein can be immunoprecipitated from
251P5G2-expressing cell lines using anti-251P5G2 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 251P5G2 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.
[0377] Small molecules and ligands that interact with 251P5G2 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 251P5G2'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
251P5G2-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses 251P5G2 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2.sup.nd Ed., Sinauer Assoc., Sunderland,
Mass., 1992). Moreover, ligands that regulate 251P5G2 function can
be identified based on their ability to bind 251P5G2 and activate a
reporter construct. Typical methods are discussed for example in
U.S. Pat. No. 5,928,868 issued Jul. 27, 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 251P5G2 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
251P5G2.
[0378] An embodiment of this invention comprises a method of
screening for a molecule that interacts with a 251P5G2 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a 251P5G2 amino acid
sequence, allowing the population of molecules and the 251P5G2
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 251P5G2 amino acid sequence, and then separating molecules
that do not interact with the 251P5G2 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 251P5G2 amino acid sequence. The identified
molecule can be used to modulate a function performed by 251P5G2.
In a preferred embodiment, the 251P5G2 amino acid sequence is
contacted with a library of peptides.
[0379] X.) Therapeutic Methods and Compositions
[0380] The identification of 251P5G2 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in cancers such as those listed in Table I, opens a
number of therapeutic approaches to the treatment of such
cancers.
[0381] Of note, targeted antitumor therapies have been useful even
when the targeted protein is expressed on normal tissues, even
vital normal organ tissues. A vital organ is one that is necessary
to sustain life, such as the heart or colon. A non-vital organ is
one that can be removed whereupon the individual is still able to
survive. Examples of non-vital organs are ovary, breast, and
prostate.
[0382] For example, Herceptin.RTM. is an FDA approved
pharmaceutical that has as its active ingredient an antibody which
is immunoreactive with the protein variously known as HER2,
HER2/neu, and erb-b-2. It is marketed by Genentech and has been a
commercially successful antitumor agent. Herceptin sales reached
almost $400 million in 2002. Herceptin is a treatment for HER2
positive metastatic breast cancer. However, the expression of HER2
is not limited to such tumors. The same protein is expressed in a
number of normal tissues. In particular, it is known that HER2/neu
is present in normal kidney and heart, thus these tissues are
present in all human recipients of Herceptin. The presence of
HER2/neu in normal kidney is also confirmed by Latif, Z., et al.,
B.J.U. International (2002) 89:5-9. As shown in this artide (which
evaluated whether renal cell carcinoma should be a preferred
indication for anti-HER2 antibodies such as Herceptin) both protein
and mRNA are produced in benign renal tissues. Notably, HER2/neu
protein was strongly overexpressed in benign renal tissue. Despite
the fact that HER2/neu is expressed in such vital tissues as heart
and kidney, Herceptin is a very useful, FDA approved, and
commercially successful drug. The effect of Herceptin on cardiac
tissue, i.e., "cardiotoxicity," has merely been a side effect to
treatment. When patients were treated with Herceptin alone,
significant cardiotoxicity occurred in a very low percentage of
patients.
[0383] Of particular note, although kidney tissue is indicated to
exhibit normal expression, possibly even higher expression than
cardiac tissue, kidney has no appreciable Herceptin side effect
whatsoever. Moreover, of the diverse array of normal tissues in
which HER2 is expressed, there is very little occurrence of any
side effect. Only cardiac tissue has manifested any appreciable
side effect at all. A tissue such as kidney, where HER2/neu
expression is especially notable, has not been the basis for any
side effect.
[0384] Furthermore, favorable therapeutic effects have been found
for antitumor therapies that target epidermal growth factor
receptor (EGFR). EGFR is also expressed in numerous normal tissues.
There have been very limited side effects in normal tissues
following use of anti-EGFR therapeutics.
[0385] Thus, expression of a target protein in normal tissue, even
vital normal tissue, does not defeat the utility of a targeting
agent for the protein as a therapeutic for certain tumors in which
the protein is also overexpressed.
[0386] Accordingly, therapeutic approaches that inhibit the
activity of a 251P5G2 protein are useful for patients suffering
from a cancer that expresses 251P5G2. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a 251P5G2
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a 251P5G2 gene or translation of 251P5G2 mRNA.
[0387] X.A.) Anti-Cancer Vaccines
[0388] The invention provides cancer vaccines comprising a
251P5G2-related protein or 251P5G2-related nucleic acid. In view of
the expression of 251P5G2, cancer vaccines prevent and/or treat
251P5G2-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 cellrnediated 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).
[0389] Such methods can be readily practiced by employing a
251P5G2-related protein, or a 251P5G2-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 251P5G2 immunogen (which typically comprises a
number of antibody or T cell epitopes). Skilled artisans understand
that a wide variety of vaccine systems for delivery of
immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med 1999 February 31(1):66-78; Maruyama et a., Cancer
Immunol Immunother 2000 Jun 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
251P5G2 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 251P5G2 immunogen contains
a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a
peptide of a size range from 251P5G2 indicated in FIG. 5, FIG. 6,
FIG. 7, FIG. 8, and FIG. 9.
[0390] The entire 251P5G2 protein, immunogenic regions or epitopes
thereof can be combined and delivered by various means. Such
vaccine compositions can indude, 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
at., 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 peptdes, 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 orsyntheticorigin (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.
[0391] In patients with 251P5G2-assodated 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.
[0392] Cellular Vaccines:
[0393] CTL epitopes can be determined using specific algorithms to
identify peptides within 251P5G2 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University (URL
brown.edulResearch/TB-HIV_Lab/epimatrix/epimatrix.html); and,
BIMAS, (URL bimas.dcrtnih.gov/; SYFPEITHI at URL
syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a
251P5G2 immunogen contains one or more amino adid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8,
9, 10 or 11 amino acids specified by an HLA Class I
motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV
(E)) and/or a peptide of at least 9 amino acids that comprises an
HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)).
As is appreciated in the art, the HLA Class I binding groove is
essentially closed ended so that peptides of only a particular size
range can fit into the groove and be bound, generally HLA Class I
epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA
Class II binding groove is essentially open ended; therefore a
peptide of about 9 or more amino acids can be bound by an HLA Class
II molecule. Due to the binding groove differences between HLA
Class I and II, HLA Class I motifs are length specific, i.e.,
position two of a Class I motif is the second amino acid in an
amino to carboxyl direction of the peptide. The amino acid
positions in a Class II motif are relative only to each other, not
the overall peptide, i.e., additional amino acids can be allached
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.
[0394] Antibody-based Vaccines
[0395] 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 251P5G2 protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to 251P5G2 in a host,
by contacting the host with a sufficient amount of at least one
251P5G2 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval thereafter re-contacting the host
with the 251P5G2 B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a 251P5G2-related protein or a man-made
multiepitopic peptide comprising: administering 251P5G2 immunogen
(e.g. a 251P5G2 protein or a peptide fragment thereof, a 251P5G2
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 251P5G2 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 251P5G2
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 251P5G2, in
order to generate a response to the target antigen.
[0396] Nucleic Acid Vaccines:
[0397] 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 251P5G2. Constructs comprising DNA encoding a
251P5G2-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 251P5G2 proteinlimmunogen.
Alternatively, a vaccine comprises a 251P5G2-related protein.
Expression of the 251P5G2-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a 251P5G2 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 particlemediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0398] 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 indude, but are not limited to,
vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus,
adeno-associated virus, lenfivirus, 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
251P5G2-related protein into the patient (e.g., intramuscularly or
intradenmally) to induce an anfi-tumor response.
[0399] 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 Calmelle 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.
[0400] Thus, gene delivery systems are used to deliver a
251P5G2-related nucleic acid molecule. In one embodiment, the
full-length human 251P5G2 cDNA is employed. In another embodiment,
251P5G2 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0401] Ex Vivo Vaccines
[0402] 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 dendritc cells (DC) to present
251P5G2 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 251P5G2 peptides to T cells in the context of MHC class I
or II molecules. In one embodiment, autologous dendritic cells are
pulsed with 251P5G2 peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete 251P5G2 protein. Yet another
embodiment involves engineering the overexpression of a 251P5G2
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 at., 1997, J. Exp. Med. 186:1177-1182).
Cells that express 251P5G2 can also be engineered to express immune
modulators, such as GM-CSF, and used as immunizing agents.
[0403] X.B.) 251P5G2 as a Target for Antibody-based Therapy
[0404] 251P5G2 is an allractive 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 251P5G2 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 251P5G2-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 251P5G2 are useful
to treat 251P5G2-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0405] 251P5G2 antibodies can be introduced into a patient such
that the antibody binds to 251P5G2 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 251P5G2, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0406] 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 251P5G2 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. 251P5G2), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0407] 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-251P5G2
antibody) that binds to a marker (e.g. 251P5G2) 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 251P5G2, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
251P5G2 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.
[0408] Cancer immunotherapy using anti-251P5G2 antibodies can be
done in accordance with various approaches that have been
successfully employed in the treatment of other types of cancer,
including but not limited to colon cancer (Arlen et al., 1998,
Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al.,
1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood
90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.
52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.
Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et
al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,
1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.
55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.
Immunol. 11:117-127). Some therapeutic approaches involve
conjugation of naked antibody to a toxin or radioisotope, such as
the conjugation of Y.sup.91 or I.sup.131 to anti-CD20 antibodies
(e.g., Zevalin.TM., IDEC Pharmaceuticals Corp. or Bexxar.TM.,
Coulter Pharmaceuticals), while others involve co-administration of
antibodies and other therapeutic agents, such as Herceptin.TM.
(trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can
be conjugated to a therapeutic agent. To treat prostate cancer, for
example, 251P5G2 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., Mylotarglm,
Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 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).
[0409] Although 251P5G2 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.
[0410] Although 251P5G2 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.
[0411] Cancer patients can be evaluated for the presence and level
of 251P5G2 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 251P5G2 imaging, or other
techniques that reliably indicate the presence and degree of
251P5G2 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.
[0412] Anti-251P5G2 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-251P5G2 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-251P5G2 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 251P5G2.
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-251P5G2 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.
[0413] 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 251P5G2 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0414] Therapeutic methods of the invention contemplate the
administration of single anti-251P5G2 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-251P5G2 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-251P5G2 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0415] Anti-251P5G2 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-251P5G2 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.
[0416] Based on clinical experience with the Herceptn.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-251P5G2 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 251P5G2 expression in the patient, the
extent of circulating shed 251P5G2 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.
[0417] Optionally, patients should be evaluated for the levels of
251P5G2 in a given sample (e.g. the levels of circulating 251P5G2
antigen and/or 251P5G2 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 andlor ImmunoCyt levels in bladder cancer therapy, or by
analogy, serum PSA levels in prostate cancer therapy).
[0418] Anti-idiotypic anti-251P5G2 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 251P5G2-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-251P5G2 antibodies that mimic an epitope on a 251P5G2-related
protein (see, for example, Wagner et al., 1997, Hybridoma 16: 3340;
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.
[0419] X.C.) 251P5G2 as a Target for Cellular Immune Responses
[0420] 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., recombinanuy or by chemical
synthesis.
[0421] 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-containi- ng (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))
[0422] 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 251P5G2 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0423] 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).
[0424] 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.
[0425] 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.
[0426] 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 frequenuy-expressed TAAs.
[0427] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenidity: 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.
[0428] 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.
[0429] 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.
[0430] 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.
[0431] 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.
[0432] 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.
[0433] X.C.1. Minigene Vaccines
[0434] 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.
[0435] 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 251P5G2, the PADRE.RTM. universal helper T cell
epitope or multiple HTL epitopes from 251P5G2 (see e.g., Tables
VIII-XXI and XXII to XLIX), and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0436] 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.
[0437] 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.
[0438] 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.
[0439] 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.
[0440] 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.
[0441] 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.
[0442] 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.
[0443] 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.
[0444] 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.
[0445] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS) This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO
91/06309; and Feigner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0446] 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 cellslare
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.
[0447] 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.
[0448] 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.
[0449] 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.
[0450] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0451] 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.
[0452] 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 peptde
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.
[0453] 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: 37), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 38), and Streptococcus 18kD
protein at positions 116-131 (GAVDSILGGVATYGM; SEQ ID NO: 39).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0454] 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: XKXVAAWTLKMX
(SEQ ID NO: 40), 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.
[0455] 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.
[0456] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0457] 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 allached 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.
[0458] 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 P3CSS, 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.
[0459] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
andlor HTL Peptides
[0460] 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.
[0461] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 251P5G2. 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 251P5G2.
[0462] X.D. Adoptive Immunotherapy
[0463] Antigenic 251P5G2-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 patients, 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.
[0464] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0465] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 251P5G2. 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.
[0466] 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 251P5G2. 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.
[0467] For therapeutic use, administration should generally begin
at the first diagnosis of 251P5G2-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 251P5G2, a vaccine comprising
251P5G2-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0468] It is generally important to provide an amount of the
peptide epitope delivered by a mode of administration sufficient to
stimulate effectively a cytptoxic T cell response; compositions
which stimulate helper T cell responses can also be given in
accordance with this embodiment of the invention.
[0469] 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.
[0470] 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.
[0471] 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.
[0472] 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.
[0473] 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.
[0474] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH-adjusting and buffering agents, tonicity
adjusting agents, welting agents, preservatives, and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
[0475] 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.
[0476] 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 add
(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.
[0477] For antibodies, a treatment generally involves repeated
administration of the anti-251P5G2 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-251P5G2
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
251P5G2 expression in the patient, the extent of circulating shed
251P5G2 antigen, the desired steady-state concentration level,
frequency of treatment, and the influence of chemotherapeutic or
other agents used in combination with the treatment method of the
invention, as well as the health status of a particular patient.
Non-limiting preferred human unit doses are, for example, 500
.mu.g-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg,
400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800
mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the
dose is in a range of 2-5 mg/kg body weight, e.g., with follow on
weekly doses of 1-3 mg/kg; 0.5mg, 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.
[0478] 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.
[0479] In one embodiment, human unit dose forms of T-cells comprise
a suitable dosage range or effective amount that provides any
therapeutic effect. As appreciated by one of ordinary skill in the
art, a therapeutic effect depends on a number of factors. Dosages
are generally selected by the physician or other health care
professional in accordance with a variety of parameters known in
the art, such as severity of symptoms, history of the patient and
the like. A dose may be about 10.sup.4 cells to about 10.sup.6
cells, about 10.sup.6 cells to about 10.sup.8 cells, about 10.sup.8
to about 10.sup.11 cells, or about 10.sup.8 to about
5.times.10.sup.10 cells. A dose may also about 10.sup.6
cells/m.sup.2 to about 10.sup.10 cells/M.sup.2, or about 10.sup.6
cells/m.sup.2 to about 10.sup.8 cells/m.sup.2.
[0480] 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.
[0481] 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.
[0482] 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%.
[0483] 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.
[0484] XI.) Diagnostic and Prognostic Embodiments of 251P5G2.
[0485] As disclosed herein, 251P5G2 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 251P5G2 in normal tissues, and patient
specimens").
[0486] 251P5G2 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. Aug; 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 251P5G2 polynucleotides and polypeptides (as well as
251P5G2 polynucleotide probes and anti-251P5G2 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.
[0487] Typical embodiments of diagnostic methods which utilize the
251P5G2 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
251P5G2 polynucleotides described herein can be utilized in the
same way to detect 251P5G2 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 251P5G2
polypeptides described herein can be utilized to generate
antibodies for use in detecting 251P5G2 overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0488] 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 251P5G2 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
251P5G2-expressing cells (lymph node) is found to contain
251P5G2-expressing cells such as the 251P5G2 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0489] Alternatively 251P5G2 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 251P5G2 or
express 251P5G2 at a different level are found to express 251P5G2
or have an increased expression of 251P5G2 (see, e.g., the 251P5G2
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 251P5G2) such as PSA, PSCA
etc. (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237
(1996)).
[0490] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, 251P5G2 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 251P5G2 in normal tissues, and patient
specimens," where a 251P5G2 polynucleotide fragment is used as a
probe to show the expression of 251P5G2 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 251P5G2 polynucleotide shown in FIG. 2 or variant
thereof) under conditions of high stringency.
[0491] 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. 251P5G2
polypeptide fragments and polypeptide analogs or variants can also
be used in an analogous manner. This practice of using polypeptide
fragments or polypeptde 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
251P5G2 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 251P5G2
polypeptide shown in FIG. 3).
[0492] As shown herein, the 251P5G2 polynucleotides and
polypeptides (as well as the 251P5G2 polynucleotide probes and
anti-251P5G2 antibodies or T cells used to identify the presence of
these molecules) exhibit specific properties that make them useful
in diagnosing cancers such as those listed in Table I. Diagnostic
assays that measure the presence of 251P5G2 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 prostatc 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 251P5G2 polynucleotides and polypeptides (as well as the
251P5G2 polynucleotide probes and anti-251P5G2 antibodies used to
identify the presence of these molecules) need to be employed to
confirm a metastases of prostatic origin.
[0493] Finally, in addition to their use in diagnostic assays, the
251P5G2 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 251P5G2 gene maps (see the Example entitled "Chromosomal
Mapping of 251P5G2" below). Moreover, in addition to their use in
diagnostic assays, the 251P5G2-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 Jun. 28, 1996; 80(1-2): 63-9).
[0494] Additionally, 251P5G2-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 251P5G2. 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 251P5G2 antigen. Antibodies or other molecules that react with
251P5G2 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0495] XII.) Inhibition of 251P5G2 Protein Function
[0496] The invention includes various methods and compositions for
inhibiting the binding of 251P5G2 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
251P5G2 function.
[0497] XII.A.) Inhibition of 251P5G2 With Intracellular
Antibodies
[0498] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 251P5G2 are introduced
into 251P5G2 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-251P5G2 antibody is
expressed intracellularly, binds to 251P5G2 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 etaaL, 1995,
Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beedi et al., 1994, J.
Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1:
332-337).
[0499] 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.
[0500] In one embodiment, intrabodies are used to capture 251P5G2
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 251P5G2
intrabodies in order to achieve the desired targeting. Such 251P5G2
intrabodies are designed to bind specifically to a particular
251P5G2 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a 251P5G2 protein are used to prevent 251P5G2
from gaining access to the nucleus, thereby preventing it from
exerting any biological activity within the nucleus (e.g.,
preventing 251P5G2 from forming transcription complexes with other
factors).
[0501] 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 promoterlenhancer can be utilized (See, for
example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).
[0502] XII.B.) Inhibition of 251P5G2 with Recombinant Proteins
[0503] In another approach, recombinant molecules bind to 251P5G2
and thereby inhibit 251P5G2 function. For example, these
recombinant molecules prevent or inhibit 251P5G2 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 251P5G2 specific antibody
molecule. In a particular embodiment, the 251P5G2 binding domain of
a 251P5G2 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 251P5G2 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.H.sup.3 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 assodated with the
expression of 251P5G2, whereby the dimeric fusion protein
specifically binds to 251P5G2 and blocks 251P5G2 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0504] XII.C.) Inhibition of 251P5G2 Transcription or
Translation
[0505] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 251P5G2 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 251P5G2 mRNA into protein.
[0506] In one approach, a method of inhibiting the transcription of
the 251P5G2 gene comprises contacting the 251P5G2 gene with a
251P5G2 antisense polynucleotide. In another approach, a method of
inhibiting 251P5G2 mRNA translation comprises contacting a 251P5G2
mRNA with an antisense polynucleotide. In another approach, a
251P5G2 specific ribozyme is used to cleave a 251P5G2 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
251P5G2 gene, such as 251P5G2 promoter and/or enhancer elements.
Similarly, proteins capable of inhibiting a 251P5G2 gene
transcription factor are used to inhibit 251P5G2 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.
[0507] Other factors that inhibit the transcription of 251P5G2 by
interfering with 251P5G2 transcriptional activation are also useful
to treat cancers expressing 251P5G2. Similarly, factors that
interfere with 251P5G2 processing are useful to treat cancers that
express 251P5G2. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0508] XIII.D.) General Considerations for Therapeutic
Strategies
[0509] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 251P5G2 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 251P5G2 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 251P5G2 antisense polynucleotides, fibozymes,
factors capable of interfering with 251P5G2 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0510] 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.
[0511] 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 251P5G2 to a binding partner, etc.
[0512] In vivo, the effect of a 251P5G2 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.
[0513] 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.
[0514] 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 antitumor 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).
[0515] 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.
[0516] 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.
[0517] XIII.) Identification, Characterization and Use of
Modulators of 251P5G2
[0518] Methods to Identify and Use Modulators
[0519] In one embodiment, screening is performed to identify
modulators that induce or suppress a particular expression profile,
suppress or induce specific pathways, preferably generating the
associated phenotype thereby. In another embodiment, having
identified differentially expressed genes important in a particular
state; screens are performed to identify modulators that alter
expression of individual genes, either increase or decrease. In
another embodiment, screening is performed to identify modulators
that alter a biological function of the expression product of a
differentially expressed gene. Again, having identified the
importance of a gene in a particular state, screens are performed
to identify agents that bind and/or modulate the biological
activity of the gene product.
[0520] In addition, screens are done for genes that are induced in
response to a candidate agent. After identifying a modulator (one
that suppresses a cancer expression pattern leading to a normal
expression pattern, or a modulator of a cancer gene that leads to
expression of the gene as in normal tissue) a screen is performed
to identify genes that are specifically modulated in response to
the agent. Comparing expression profiles between normal tissue and
agent-treated cancer tissue reveals genes that are not expressed in
normal tissue or cancer tissue, but are expressed in agent treated
tissue, and vice versa. These agent-specific sequences are
identified and used by methods described herein for cancer genes or
proteins. In particular these sequences and the proteins they
encode are used in marking or identifying agent-treated cells. In
addition, antibodies are raised against the agent-induced proteins
and used to target novel therapeutics to the treated cancer tissue
sample.
[0521] Modulator-related Identification and Screening Assays:
[0522] Gene Expression-related Assays
[0523] Proteins, nucleic acids, and antibodies of the invention are
used in screening assays. The cancer-associated proteins,
antibodies, nucleic acids, modified proteins and cells containing
these sequences are used in screening assays, such as evaluating
the effect of drug candidates on a "gene expression profile,"
expression profile of polypeptides or alteration of biological
function. In one embodiment, the expression profiles are used,
preferably in conjunction with high throughput screening techniques
to allow monitoring for expression profile genes after treatment
with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69
(2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome
Res 6:986-94, 1996).
[0524] The cancer proteins, antibodies, nucleic acids, modified
proteins and cells containing the native or modified cancer
proteins or genes are used in screening assays. That is, the
present invention comprises methods for screening for compositions
which modulate the cancer phenotype or a physiological function of
a cancer protein of the invention. This is done on a gene itself or
by evaluating the effect of drug candidates on a "gene expression
profile" or biological function. In one embodiment, expression
profiles are used, preferably in conjunction with high throughput
screening techniques to allow monitoring after treatment with a
candidate agent, see Zlokamik, supra.
[0525] A variety of assays are executed directed to the genes and
proteins of the invention. Assays are run on an individual nucleic
acid or protein level. That is, having identified a particular gene
as up regulated in cancer, test compounds are screened for the
ability to modulate gene expression or for binding to the cancer
protein of the invention. "Modulation" in this context includes an
increase or a decrease in gene expression. The preferred amount of
modulation will depend on the original change of the gene
expression in normal versus tissue undergoing cancer, with changes
of at least 10%, preferably 50%, more preferably 100-300%, and in
some embodiments 300-1000% or greater. Thus, if a gene exhibits a
4-fold increase in cancer tissue compared to normal tissue, a
decrease of about four-fold is often desired; similarly, a 10-fold
decrease in cancer tissue compared to normal tissue a target value
of a 10-fold increase in expression by the test compound is often
desired. Modulators that exacerbate the type of gene expression
seen in cancer are also useful, e.g., as an upregulated target in
further analyses.
[0526] The amount of gene expression is monitored using nucleic
acid probes and the quantification of gene expression levels, or,
alternatively, a gene product itself is monitored, e.g., through
the use of antibodies to the cancer protein and standard
immunoassays. Proteomics and separation techniques also allow for
quantification of expression.
[0527] Expression Monitoring to Identify Compounds that Modify Gene
Expression
[0528] In one embodiment, gene expression monitoring, i.e., an
expression profile, is monitored simultaneously for a number of
entities. Such profiles will typically involve one or more of the
genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid
probes are attached to biochips to detect and quantify cancer
sequences in a particular cell. Alternatively, PCR can be used.
Thus, a series, e.g., wells of a microtiter plate, can be used with
dispensed primers in desired wells. A PCR reaction can then be
performed and analyzed for each well.
[0529] Expression monitoring is performed to identify compounds
that modify the expression of one or more cancer-associated
sequences, e.g., a polynucleotide sequence set out in FIG. 2.
Generally, a test modulator is added to the cells prior to
analysis. Moreover, screens are also provided to identify agents
that modulate cancer, modulate cancer proteins of the invention,
bind to a cancer protein of the invention, or interfere with the
binding of a cancer protein of the invention and an antibody or
other binding partner.
[0530] In one embodiment, high throughput screening methods involve
providing a library containing a large number of potential
therapeutic compounds (candidate compounds). Such "combinatorial
chemical libraries" are then screened in one or more assays to
identify those library members (particular chemical species or
subclasses) that display a desired characteristic activity. The
compounds thus identified can serve as conventional "lead
compounds," as compounds for screening, or as therapeutics.
[0531] In certain embodiments, combinatorial libraries of potential
modulators are screened for an abilityito bind to a cancer
polypeptide or to modulate activity. Conventionally, new chemical
entities with useful properties are generated by identifying a
chemical compound (called a "lead compound") with some desirable
property or activity, e.g., inhibiting activity, creating variants
of the lead compound, and evaluating the property and activity of
those variant compounds. Often, high throughput screening (HTS)
methods are employed for such an analysis.
[0532] As noted above, gene expression monitoring is conveniently
used to test candidate modulators (e.g., protein, nucleic acid or
small molecule). After the candidate agent has been added and the
cells allowed to incubate for a period, the sample containing a
target sequence to be analyzed is, e.g., added to a biochip.
[0533] If required, the target sequence is prepared using known
techniques. For example, a sample is treated to lyse the cells,
using known lysis buffers, electroporation, etc., with purification
and/or amplification such as PCR performed as appropriate. For
example, an in vitro transcription with labels covalently attached
to the nucleotides is performed. Generally, the nucleic acids are
labeled with biotin-FITC or PE, or with cy3 or cy5.
[0534] The target sequence can be labeled with, e.g., a
fluorescent, a chemiluminescent, a chemical, or a radioactive
signal, to provide a means of detecting the target sequence's
specific binding to a probe. The label also can be an enzyme, such
as alkaline phosphatase or horseradish peroxidase, which when
provided with an appropriate substrate produces a product that is
detected. Alternatively, the label is a labeled compound or small
molecule, such as an enzyme inhibitor, that binds but is not
catalyzed or altered by the enzyme. The label also can be a moiety
or compound, such as, an epitope tag or biotin which specifically
binds to streptavidin. For the example of biotin, the streptavidin
is labeled as described above, thereby, providing a detectable
signal for the bound target sequence. Unbound labeled streptavidin
is typically removed prior to analysis.
[0535] As will be appreciated by those in the art, these assays can
be direct hybridization assays or can comprise "sandwich assays",
which include the use of multiple probes, as is generally outlined
in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117;
5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802;
5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this
embodiment, in general, the target nucleic acid is prepared as
outlined above, and then added to the biochip comprising a
plurality of nucleic acid probes, under conditions that allow the
formation of a hybridization complex.
[0536] A variety of hybridization conditions are used in the
present invention, including high, moderate and low stringency
conditions as outlined above. The assays are generally run under
stringency conditions which allow formation of the label probe
hybridization complex only in the presence of target. Stringency
can be controlled by altering a step parameter that is a
thermodynamic variable, including, but not limited to, temperature,
formamide concentration, salt concentration, chaotropic salt
concentration pH, organic solvent concentration, etc. These
parameters may also be used to control non-specific binding, as is
generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be
desirable to perform certain steps at higher stringency conditions
to reduce non-specific binding.
[0537] The reactions outlined herein can be accomplished in a
variety of ways. Components of the reaction can be added
simultaneously, or sequentially, in different orders, with
preferred embodiments outlined below. In addition, the reaction may
include a variety of other reagents. These include salts, buffers,
neutral proteins, e.g. albumin, detergents, etc. which can be used
to facilitate optimal hybridization and detection, and/or reduce
nonspecific or background interactions. Reagents that otherwise
improve the efficiency of the assay, such as protease inhibitors,
nuclease inhibitors, anti-microbial agents, etc., may also be used
as appropriate, depending on the sample preparation methods and
purity of the target. The assay data are analyzed to determine the
expression levels of individual genes, and changes in expression
levels as between states, forming a gene expression profile.
[0538] Biological Activity-related Assays
[0539] The invention provides methods identify or screen for a
compound that modulates the activity of a cancer-related gene or
protein of the invention. The methods comprise adding a test
compound, as defined above, to a cell comprising a cancer protein
of the invention. The cells contain a recombinant nucleic acid that
encodes a cancer protein of the invention. In another embodiment, a
library of candidate agents is tested on a plurality of cells.
[0540] In one aspect, the assays are evaluated in the presence or
absence or previous or subsequent exposure of physiological
signals, e.g. hormones, antibodies, peptides, antigens, cytokines,
growth factors, action potentials, pharmacological agents including
chemotherapeutics, radiation, carcinogenics, or other cells (i.e.,
cell-cell contacts). In another example, the determinations are
made at different stages of the cell cycle process. In this way,
compounds that modulate genes or proteins of the invention are
identified. Compounds with pharmacological activity are able to
enhance or interfere with the activity of the cancer protein of the
invention. Once identified, similar structures are evaluated to
identify critical structural features of the compound.
[0541] In one embodiment, a method of modulating (e.g., inhibiting)
cancer cell division is provided; the method comprises
administration of a cancer modulator. In another embodiment, a
method of modulating ( e.g., inhibiting) cancer is provided; the
method comprises administration of a cancer modulator. In a further
embodiment, methods of treating cells or individuals with cancer
are provided; the method comprises administration of a cancer
modulator.
[0542] In one embodiment, a method for modulating the status of a
cell that expresses a gene of the invention is provided. As used
herein status comprises such art-accepted parameters such as
growth, proliferation, survival, function, apoptosis, senescence,
location, enzymatic activity, signal transduction, etc. of a cell.
In one embodiment, a cancer inhibitor is an antibody as discussed
above. In another embodiment, the cancer inhibitor is an antisense
molecule. A variety of cell growth, proliferation, and metastasis
assays are known to those of skill in the art, as described
herein.
[0543] High Throughput Screening to Identify Modulators
[0544] The assays to identify suitable modulators are amenable to
high throughput screening. Preferred assays thus detect enhancement
or inhibition of cancer gene transcription, inhibition or
enhancement of polypeptide expression, and inhibition or
enhancement of polypeptide activity.
[0545] In one embodiment, modulators evaluated in high throughput
screening methods are proteins, often naturally occurring proteins
or fragments of naturally occurring proteins. Thus, e.g., cellular
extracts containing proteins, or random or directed digests of
proteinaceous cellular extracts, are used. In this way, libraries
of proteins are made for screening in the methods of the invention.
Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred.
Particularly useful test compound will be directed to the class of
proteins to which the target belongs, e.g., substrates for enzymes,
or ligands and receptors.
[0546] Use of Soft Agar Growth and Colony Formation to Identify and
Characterize Modulators
[0547] Normal cells require a solid substrate to attach and grow.
When cells are transformed, they lose this phenotype and grow
detached from the substrate. For example, transformed cells can
grow in stirred suspension culture or suspended in semi-solid
media, such as semi-solid or soft agar. The transformed cells, when
transfected with tumor suppressor genes, can regenerate normal
phenotype and once again require a solid substrate to attach to and
grow. Soft agar growth or colony formation in assays are used to
identify modulators of cancer sequences, which when expressed in
host cells, inhibit abnormal cellular proliferation and
transformation. A modulator reduces or eliminates the host cells'
ability to grow suspended in solid or semisolid media, such as
agar.
[0548] Techniques for soft agar growth or colony formation in
suspension assays are described in Freshney, Culture of Animal
Cells a Manual of Basic Technique (3rd ed., 1994). See also, the
methods section of Garkavtsev et al. (1996), supra.
[0549] Evaluation of Contact Inhibition and Growth Density
Limitation to Identify and Characterize Modulators
[0550] Normal cells typically grow in a flat and organized pattern
in cell culture until they touch other cells. When the cells touch
one another, they are contact inhibited and stop growing.
Transformed cells, however, are not contact inhibited and continue
to grow to high densities in disorganized foci. Thus, transformed
cells grow to a higher saturation density than corresponding normal
cells. This is detected morphologically by the formation of a
disoriented monolayer of cells or cells in foci. Alternatively,
labeling index with (.sup.3H)-thymidine at saturation density is
used to measure density limitation of growth, similarly an MTT or
Alamar blue assay will reveal proliferation capacity of cells and
the the ability of modulators to affect same. See Freshney (1994),
supra. Transformed cells, when transfected with tumor suppressor
genes, can regenerate a normal phenotype and become contact
inhibited and would grow to a lower density.
[0551] In this assay, labeling index with .sup.3H)-thymidine at
saturation density is a preferred method of measuring density
limitation of growth. Transformed host cells are transfected with a
cancer-associated sequence and are grown for 24 hours at saturation
density in non-limiting medium conditions. The percentage of cells
labeling with (.sup.3H)-thymidine is determined by incorporated
cpm.
[0552] Contact independent growth is used to identify modulators of
cancer sequences, which had led to abnormal cellular proliferation
and transformation. A modulator reduces or eliminates contact
independent growth, and returns the cells to a normal
phenotype.
[0553] Evaluation of Growth Factor or Serum Dependence to Identify
and Characterize Modulators
[0554] Transformed cells have lower serum dependence than their
normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst.
37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970));
Freshney, supra. This is in part due to release of various growth
factors by the transformed cells. The degree of growth factor or
serum dependence of transformed host cells can be compared with
that of control. For example, growth factor or serum dependence of
a cell is monitored in methods to identify and characterize
compounds that modulate cancer-associated sequences of the
invention.
[0555] Use of Tumor-specific Marker Levels to Identify and
Characterize Modulators
[0556] Tumor cells release an increased amount of certain factors
(hereinafter "tumor specific markers") than their normal
counterparts. For example, plasminogen activator (PA) is released
from human glioma at a higher level than from normal brain cells
(see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and
Potential Interference with Tumor Growth, in Biological Responses
in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor
Angiogenesis Factor (TAF) is released at a higher level in tumor
cells than their normal counterparts. See, e.g., Folkman,
Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is
released from endothelial tumors (Ensoli, B et al).
[0557] Various techniques which measure the release of these
factors are described in Freshney (1994), supra. Also, see, Unkless
et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland &
Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J.
Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor
Vascularization, and Potential Interference with Tumor Growth, in
Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985);
Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor
specific marker levels are monitored in methods to identify and
characterize compounds that modulate cancer-associated sequences of
the invention.
[0558] Invasiveness into Matrigel to Identify and Characterize
Modulators
[0559] The degree of invasiveness into Matrigel or an extracellular
matrix constituent can be used as an assay to identify and
characterize compounds that modulate cancer associated sequences.
Tumor cells exhibit a positive correlation between malignancy and
invasiveness of cells into Matrigel or some other extracellular
matrix constituent. In this assay, tumorigenic cells are typically
used as host cells. Expression of a tumor suppressor gene in these
host cells would decrease invasiveness of the host cells.
Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994),
supra, can be used. Briefly, the level of invasion of host cells is
measured by using filters coated with Matrigel or some other
extracellular matrix constituent. Penetration into the gel, or
through to the distal side of the filter, is rated as invasiveness,
and rated histologically by number of cells and distance moved, or
by prelabeling the cells with .sup.1251 and counting the
radioactivity on the distal side of the filter or bottom of the
dish. See, e.g., Freshney (1984), supra.
[0560] Evaluation of Tumor Growth In Vivo to Identify and
Characterize Modulators
[0561] Effects of cancer-associated sequences on cell growth are
tested in transgenic or immune-suppressed organisms. Transgenic
organisms are prepared in a variety of art-accepted ways. For
example, knock-out transgenic organisms, e.g., mammals such as
mice, are made, in which a cancer gene is disrupted or in which a
cancer gene is inserted. Knock-out transgenic mice are made by
insertion of a marker gene or other heterologous gene into the
endogenous cancer gene site in the mouse genome via homologous
recombination. Such mice can also be made by substituting the
endogenous cancer gene with a mutated version of the cancer gene,
or by mutating the endogenous cancer gene, e.g., by exposure to
carcinogens.
[0562] To prepare transgenic chimeric animals, e.g., mice, a DNA
construct is introduced into the nuclei of embryonic stem cells.
Cells containing the newly engineered genetic lesion are injected
into a host mouse embryo, which is re-implanted into a recipient
female. Some of these embryos develop into chimeric mice that
possess germ cells some of which are derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric
mice can be derived according to U.S. Pat. No. 6,365,797, issued
Apr. 2, 2002; U.S. Pat. No. 6,107,540 issued Aug. 22, 2000; Hogan
et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold
Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, Robertson, ed., IRL Press,
Washington, D.C., (1987).
[0563] Alternatively, various immune-suppressed or immune-deficient
host animals can be used. For example, a genetically athymic "nude"
mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921
(1974)), a SCID mouse, a thymectornized mouse, or an irradiated
mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978);
Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host.
Transplantable tumor cells (typically about 106 cells) injected
into isogenic hosts produce invasive tumors in a high proportion of
cases, while normal cells of similar origin will not. In hosts
which developed invasive tumors, cells expressing cancer-associated
sequences are injected subcutaneously or orthotopically. Mice are
then separated into groups, including control groups and treated
experimental groups) e.g. treated with a modulator). After a
suitable length of time, preferably 4-8 weeks, tumor growth is
measured (e.g., by volume or by its two largest dimensions, or
weight) and compared to the control. Tumors that have statistically
significant reduction (using, e.g., Student's T test) are said to
have inhibited growth.
[0564] In Vitro Assays to Identify and Characterize Modulators
[0565] Assays to identify compounds with modulating activity can be
performed in vitro. For example, a cancer polypeptide is first
contacted with a potential modulator and incubated for a suitable
amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the
cancer polypeptide levels are determined in vitro by measuring the
level of protein or mRNA. The level of protein is measured using
immunoassays such as Western blotting, ELISA and the like with an
antibody that selectively binds to the cancer polypeptide or a
fragment thereof. For measurement of mRNA, amplification, e.g.,
using PCR, LCR, or hybridization assays, e. g., Northern
hybridization, RNAse protection, dot blotting, are preferred. The
level of protein or mRNA is detected using directly or indirectly
labeled detection agents, e.g., fluorescently or radioactively
labeled nucleic acids, radioactively or enzymatically labeled
antibodies, and the like, as described herein.
[0566] Alternatively, a reporter gene system can be devised using a
cancer protein promoter operably linked to a reporter gene such as
luciferase, green fluorescent protein, CAT, or P-gal. The reporter
construct is typically transfected into a cell. After treatment
with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured according to
standard techniques known to those of skill in the art (Davis GF,
supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol.
1998: 9:624).
[0567] As outlined above, in vitro screens are done on individual
genes and gene products. That is, having identified a particular
differentially expressed gene as important in a particular state,
screening of modulators of the expression of the gene or the gene
product itself is performed.
[0568] In one embodiment, screening for modulators of expression of
specific gene(s) is performed. Typically, the expression of only
one or a few genes is evaluated. In another embodiment, screens are
designed to first find compounds that bind to differentially
expressed proteins. These compounds are then evaluated for the
ability to modulate differentially expressed activity. Moreover,
once initial candidate compounds are identified, variants can be
further screened to better evaluate structure activity
relationships.
[0569] Binding Assays to Identify and Characterize Modulators
[0570] In binding assays in accordance with the invention, a
purified or isolated gene product of the invention is generally
used. For example, antibodies are generated to a protein of the
invention, and immunoassays are run to determine the amount and/or
location of protein. Alternatively, cells comprising the cancer
proteins are used in the assays.
[0571] Thus, the methods comprise combining a cancer protein of the
invention and a candidate compound such as a ligand, and
determining the binding of the compound to the cancer protein of
the invention. Preferred embodiments utilize the human cancer
protein; animal models of human disease of can also be developed
and used. Also, other analogous mammalian proteins also can be used
as appreciated by those of skill in the art. Moreover, in some
embodiments variant or derivative cancer proteins are used.
[0572] Generally, the cancer protein of the invention, or the
ligand, is non-diffusibly bound to an insoluble support. The
support can, e.g., be one having isolated sample receiving areas (a
microtiter plate, an array, etc.). The insoluble supports can be
made of any composition to which the compositions can be bound, is
readily separated from soluble material, and is otherwise
compatible with the overall method of screening. The surface of
such supports can be solid or porous and of any convenient
shape.
[0573] Examples of suitable insoluble supports include microtiter
plates, arrays, membranes and beads. These are typically made of
glass, plastic (e.g., polystyrene), polysaccharide, nylon,
nitrocellulose, or Teflon.TM., etc. Microtiter plates and arrays
are especially convenient because a large number of assays can be
carried out simultaneously, using small amounts of reagents and
samples. The particular manner of binding of the composition to the
support is not crucial so long as it is compatible with the
reagents and overall methods of the invention, maintains the
activity of the composition and is nondiffusable. Preferred methods
of binding include the use of antibodies which do not sterically
block either the ligand binding site or activation sequence when
attaching the protein to the support, direct binding to "sticky" or
ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface, etc. Following binding of the protein or
ligand/binding agent to the support, excess unbound material is
removed by washing. The sample receiving areas may then be blocked
through incubation with bovine serum albumin (BSA), casein or other
innocuous protein or other moiety.
[0574] Once a cancer protein of the invention is bound to the
support, and a test compound is added to the assay. Alternatively,
the candidate binding agent is bound to the support and the cancer
protein of the invention is then added. Binding agents include
specific antibodies, non-natural binding agents identified in
screens of chemical libraries, peptide analogs, etc.
[0575] Of particular interest are assays to identify agents that
have a low toxicity for human cells. A wide variety of assays can
be used for this purpose, including proliferation assays, cAMP
assays, labeled in vitro protein-protein binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, functional assays (phosphorylation assays, etc.) and the
like.
[0576] A determination of binding of the test compound (ligand,
binding agent, modulator, etc.) to a cancer protein of the
invention can be done in a number of ways. The test compound can be
labeled, and binding determined directly; e.g., by attaching all or
a portion of the cancer protein of the invention to a solid
support, adding a labeled candidate compound (e.g., a fluorescent
label), washing off excess reagent, and determining whether the
label is present on the solid support. Various blocking and washing
steps can be utilized as appropriate.
[0577] In certain embodiments, only one of the components is
labeled, e.g., a protein of the invention or ligands labeled.
Alternatively, more than one component is labeled with different
labels, e.g., I.sup.125, for the proteins and a fluorophor for the
compound. Proximity reagents, e.g., quenching or energy transfer
reagents are also useful.
[0578] Competitive Binding to Identify and Characterize
Modulators
[0579] In one embodiment, the binding of the "test compound" is
determined by competitive binding assay with a "competitor." The
competitor is a binding moiety that binds to the target molecule
(e.g., a cancer protein of the invention). Competitors include
compounds such as antibodies, peptides, binding partners, ligands,
etc. Under certain circumstances, the competitive binding between
the test compound and the competitor displaces the test compound.
In one embodiment, the test compound is labeled. Either the test
compound, the competitor, or both, is added to the protein for a
time sufficient to allow binding. Incubations are performed at a
temperature that facilitates optimal activity, typically between
four and 40.degree. C. Incubation periods are typically optimized,
e.g., to facilitate rapid high throughput screening; typically
between zero and one hour will be sufficient. Excess reagent is
generally removed or washed away. The second component is then
added, and the presence or absence of the labeled component is
followed, to indicate binding.
[0580] In one embodiment, the competitor is added first, followed
by the test compound. Displacement of the competitor is an
indication that the test compound is binding to the cancer protein
and thus is capable of binding to, and potentially modulating, the
activity of the cancer protein. In this embodiment, either
component can be labeled. Thus, e.g., if the competitor is labeled,
the presence of label in the post-test compound wash solution
indicates displacement by the test compound. Alternatively, if the
test compound is labeled, the presence of the label on the support
indicates displacement.
[0581] In an alternative embodiment, the test compound is added
first, with incubation and washing, followed by the competitor. The
absence of binding by the competitor indicates that the test
compound binds to the cancer protein with higher affinity than the
competitor. Thus, if the test compound is labeled, the presence of
the label on the support, coupled with a lack of competitor
binding, indicates that the test compound binds to and thus
potentially modulates the cancer protein of the invention.
[0582] Accordingly, the competitive binding methods comprise
differential screening to identity agents that are capable of
modulating the activity of the cancer proteins of the invention. In
this embodiment, the methods comprise combining a cancer protein
and a competitor in a first sample. A second sample comprises a
test compound, the cancer protein, and a competitor. The binding of
the competitor is determined for both samples, and a change, or
difference in binding between the two samples indicates the
presence of an agent capable of binding to the cancer protein and
potentially modulating its activity. That is, if the binding of the
competitor is different in the second sample relative to the first
sample, the agent is capable of binding to the cancer protein.
[0583] Alternatively, differential screening is used to identify
drug candidates that bind to the native cancer protein, but cannot
bind to modified cancer proteins. For example the structure of the
cancer protein is modeled and used in rational drug design to
synthesize agents that interact with that site, agents which
generally do not bind to site-modified proteins. Moreover, such
drug candidates that affect the activity of a native cancer protein
are also identified by screening drugs for the ability to either
enhance or reduce the activity of such proteins.
[0584] Positive controls and negative controls can be used in the
assays. Preferably control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples occurs for a time sufficient to allow for
the binding of the agent to the protein. Following incubation,
samples are washed free of non-specifically bound material and the
amount of bound, generally labeled agent determined. For example,
where a radiolabel is employed, the samples can be counted in a
scintillation counter to determine the amount of bound
compound.
[0585] A variety of other reagents can be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc. which are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., can be used. The mixture of components
is added in an order that provides for the requisite binding.
[0586] Use of Polynucleotides to Down-regulate or Inhibit a Protein
of the Invention.
[0587] Polynucleotide modulators of cancer can be introduced into a
cell containing the target nucleotide sequence by formation of a
conjugate with a ligand-binding molecule, as described in WO
91/04753. Suitable ligand-binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell. Alternatively, a polynucleotide
modulator of cancer can be introduced into a cell containing the
target nucleic acid sequence, e.g., by formation of a
polynucleotide-lipid complex, as described in WO 90/10448. It is
understood that the use of antisense molecules or knock out and
knock in models may also be used in screening assays as discussed
above, in addition to methods of treatment.
[0588] Inhibitory and Antisense Nucleotides
[0589] In certain embodiments, the activity of a cancer-associated
protein is down-regulated, or entirely inhibited, by the use of
antisense polynucleotide or inhibitory small nuclear RNA (snRNA),
i.e., a nucleic acid complementary to, and which can preferably
hybridize specifically to, a coding mRNA nucleic acid sequence,
e.g., a cancer protein of the invention, mRNA, or a subsequence
thereof. Binding of the antisense polynucleotide to the mRNA
reduces the translation and/or stability of the mRNA.
[0590] In the context of this invention, antisense polynucleotides
can comprise naturally occurring nucleotides, or synthetic species
formed from naturally occurring subunits or their close homologs.
Antisense polynucleotides may also have altered sugar moieties or
inter-sugar linkages. Exemplary among these are the
phosphorothioate and other sulfur containing species which are
known for use in the art. Analogs are comprised by this invention
so long as they function effectively to hybridize with nucleotides
of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad,
Calif.; Sequitor, Inc., Natick, Mass.
[0591] Such antisense polynucleotides can readily be synthesized
using recombinant means, or can be synthesized in vitro. Equipment
for such synthesis is sold by several vendors, including Applied
Biosystems. The preparation of other oligonucleotides such as
phosphorothioates and alkylated derivatives is also well known to
those of skill in the art.
[0592] Antisense molecules as used herein include antiserse or
sense oligonucleotides. Sense oligonucleotides can, e.g., be
employed to block transcription by binding to the anti-sense
strand. The antisense and sense oligonucleotide comprise a single
stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target mRNA (sense) or DNA (antisense) sequences for
cancer molecules. Antisense or sense oligonucleotides, according to
the present invention, comprise a fragment generally at least about
12 nucleotides, preferably from about 12 to 30 nucleotides. The
ability to derive an antsense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in,
e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van der Krol
et al. (BioTechniques 6:958 (1988)).
[0593] Ribozymes
[0594] In addition to antisense polynucleotides, ribozymes can be
used to target and inhibit transcription of cancer-associated
nucleotide sequences. A ribozyme is an RNA molecule that
catalytically cleaves other RNA molecules. Different kinds of
ribozymes have been described, including group I ribozymes,
hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead
ribozymes (see, e.g., Castanollo et al., Adv. in Pharmacology 25:
289-317 (1994) for a general review of the properties of different
ribozymes).
[0595] The general features of hairpin ribozymes are described,
e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990);
European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678.
Methods of preparing are well known to those of skill in the art
(see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA
90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:3945
(1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699-703
(1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994), and
Yamada et al., Virology 205:121-126 (1994)).
[0596] Use of Modulators in Phenotypic Screening
[0597] In one embodiment, a test compound is administered to a
population of cancer cells, which have an associated cancer
expression profile. By "administration" or "contacting" herein is
meant that the modulator is added to the cells in such a manner as
to allow the modulator to act upon the cell, whether by uptake and
intracellular action, or by action at the cell surface. In some
embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a
peptide) is put into a viral construct such as an adenoviral or
retroviral construct, and added to the cell, such that expression
of the peptide agent is accomplished, e.g., PCT US97/01019.
Regulatable gene therapy systems can also be used. Once the
modulator has been administered to the cells, the cells are washed
if desired and are allowed to incubate under preferably
physiological conditions for some period. The cells are then
harvested and a new gene expression profile is generated. Thus,
e.g., cancer tissue is screened for agents that modulate, e.g.,
induce or suppress, the cancer phenotype. A change in at least one
gene, preferably many, of the expression profile indicates that the
agent has an effect on cancer activity. Similarly, altering a
biological function or a signaling pathway is indicative of
modulator activity. By defining such a signature for the cancer
phenotype, screens for new drugs that alter the phenotype are
devised. With this approach, the drug target need not be known and
need not be represented in the original gene/protein expression
screening platform, nor does the level of transcript for the target
protein need to change. The modulator inhibiting function will
serve as a surrogate marker
[0598] As outlined above, screens are done to assess genes or gene
products. That is, having identified a particular differentially
expressed gene as important in a particular state, screening of
modulators of either the expression of the gene or the gene product
itself is performed.
[0599] Use of Modulators to Affect Peptides of the Invention
[0600] Measurements of cancer polypeptide activity, or of the
cancer phenotype are performed using a variety of assays. For
example, the effects of modulators upon the function of a cancer
polypeptide(s) are measured by examining parameters described
above. A physiological change that affects activity is used to
assess the influence of a test compound on the polypeptides of this
invention. When the functional outcomes are determined using intact
cells or animals, a variety of effects can be assesses such as, in
the case of a cancer associated with solid tumors, tumor growth,
tumor metastasis, neovascularization, hormone release,
transcriptional changes to both known and uncharacterized genetic
markers (e.g., by Northern blots), changes in cell metabolism such
as cell growth or pH changes, and changes in intracellular second
messengers such as cGNIP.
[0601] Methods of Identifying Characterizing Cancer-associated
Sequences
[0602] Expression of various gene sequences is correlated with
cancer. Accordingly, disorders based on mutant or variant cancer
genes are determined. In one embodiment, the invention provides
methods for identifying cells containing variant cancer genes,
e.g., determining the presence of, all or part, the sequence of at
least one endogenous cancer gene in a cell. This is accomplished
using any number of sequencing techniques. The invention comprises
methods of identifying the cancer genotype of an individual, e.g.,
determining all or part of the sequence of at least one gene of the
invention in the individual. This is generally done in at least one
tissue of the individual, e.g., a tissue set forth in Table I, and
may include the evaluation of a number of tissues or different
samples of the same tissue. The method may include comparing the
sequence of the sequenced gene to a known cancer gene, i.e., a
wild-type gene to determine the presence of family members,
homologies, mutations or variants. The sequence of all or part of
the gene can then be compared to the sequence of a known cancer
gene to determine if any differences exist. This is done using any
number of known homology programs, such as BLAST, Bestfit, etc. The
presence of a difference in the sequence between the cancer gene of
the patient and the known cancer gene correlates with a disease
state or a propensity for a disease state, as outlined herein.
[0603] In a preferred embodiment, the cancer genes are used as
probes to determine the number of copies of the cancer gene in the
genome. The cancer genes are used as probes to determine the
chromosomal localization of the cancer genes. Information such as
chromosomal localization finds use in providing a diagnosis or
prognosis in particular when chromosomal abnormalities such as
translocations, and the like are identified in the cancer gene
locus.
[0604] XIV.) Kits/Articles of Manufacture
[0605] 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.
[0606] 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.
[0607] 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.
[0608] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0609] 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.
[0610] 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 251P5G2 and modulating the function of
251P5G2.
[0611] The label can be on or associated with the container. A
label a can be on a container when letters, numbers or other
characters forming the label are molded or etched into the
container itself; a label can be associated with a container when
it is present within a receptacle or carrier that also holds the
container, e.g., as a package insert. The label can indicate that
the composition is used for diagnosing, treating, prophylaxing or
prognosing a condition, such as a neoplasia of a tissue set forth
in Table I. The article of manufacture can further comprise a
second container comprising a pharmaceutically-acceptable buffer,
such as phosphate-buffered saline, Ringer's solution and/ordextrose
solution. It can further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, stirrers, needles, syringes, and/or package inserts with
indications and/or instructions for use.
EXAMPLES
[0612] 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
[0613] SSH-Generated Isolation of cDNA Fragment of the 251P5G2
Gene
[0614] To isolate genes that are over-expressed in prostate cancer
we used the Suppression Subtractive Hybridization (SSH) procedure
using cDNA derived from prostate cancer tissues. The 251P5G2 SSH
cDNA sequence was derived from a prostate cancer metastasis minus
cDNAs derived from a pool of 9 normal tissues. The 251P5G2 cDNA was
identified as highly expressed in the prostate cancer
metastasis.
[0615] Materials and Methods
[0616] Human Tissues:
[0617] The patient cancer and normal tissues were purchased from
different sources such as the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues were purchased from Clontech, Palo Alto,
Calif.
[0618] RNA Isolation:
[0619] Tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA.
Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA
Mini and Midi kits. Total and mRNA were quantified by
spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel
electrophoresis.
[0620] Oligonucleotides:
[0621] The following HPLC purified oligonucleotides were used.
2 DPNCDN (cDNA synthesis primer): 5'TTTTGATCAAGCTT.sub.303- ' (SEQ
ID NO: 41) Adaptor 1:
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 42)
3'GGCCCGTCCTAG5' (SEQ ID NO: 43) Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 44)
3'CGGCTCCTAG5' (SEQ ID NO: 45) PCR primer 1:
5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: 46) Nested primer (NP)1:
5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 47) Nested primer (NP)2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 48)
[0622] Suppression Subtractive Hybridization:
[0623] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in prostate cancer. The SSH reaction utilized cDNA from
prostate cancer and normal tissues.
[0624] The gene 251P5G2 sequence was derived from a prostate cancer
metastasis minus normal tissue cDNA subtraction. The SSH DNA
sequence (FIG. 1) was identified.
[0625] The cDNA derived from of pool of normal tissues was used as
the source of the "driver" cDNA, while the cDNA from prostate
cancer metastasis was used as the source of the "tester" cDNA.
Double stranded cDNAs corresponding to tester and driver cDNAs were
synthesized from 2 .mu.g of poly(A).sup.+ RNA isolated from the
relevant xenograft tissue, as described above, using CLONTECH's
PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN
as primer. First- and second-strand synthesis were carried out as
described in the Kit's user manual protocol (CLONTECH Protocol No.
PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested
with Dpn II for 3 hrs at37.degree. C. Digested cDNA was extracted
with phenol/chloroform (1:1) and ethanol precipitated.
[0626] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from the relevant tissue source (see above) with a
mix of digested cDNAs derived from the nine normal tissues:
stomach, skeletal muscle, lung, brain, liver, kidney, pancreas,
small intestine, and heart.
[0627] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l, 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
160.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.
[0628] The first hybridization was performed by adding 1.5 .mu.l
(600 ng) of driver cDNA to each of two tubes containing 1.5 .mu.l
(20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final
volume of 4 .mu.l, the samples were overlaid with mineral oil,
denatured in an MJ Research thermal cycler at 98.degree. C. for 1.5
minutes, and then were allowed to hybridize for 8 hrs at 68.degree.
C. The two hybridizations were then mixed together with an
additional 1 .mu.l of fresh denatured driver cDNA and were allowed
to hybridize overnight at 68.degree. C. The second hybridization
was then diluted in 200 .mu.l of 20 mM Hepes, pH 8.3, 50 mM NaCl,
0.2 mM EDTA, heated at 70.degree. C. for 7 min. and stored at
-20.degree. C.
[0629] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0630] 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, and
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0631] 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 ul of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0632] 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.
[0633] RT-PCR Expression Analysis:
[0634] First strand cDNAs can be generated from 1 .mu.g of mRNA
with oligo (dT)12-18 priming using the Gibco-BRL Superscript
Preamplification system. The manufacturer's protocol was used which
included an incubation for 50 min at 42.degree. C. with reverse
transcriptase followed by RNAse H treatment at 37.degree. C. for 20
min. After completing the reaction, the volume can be increased to
200 .mu.l with water prior to normalization. First strand cDNAs
from 16 different normal human tissues can be obtained from
Clontech.
[0635] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 49) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 50) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl.sub.2,
50 mM KCl, pH8.3) and 1X Klentaq DNA polymerase (Clontech). Five
.mu.l of the PCR reaction can be removed at 18, 20, and 22 cycles
and used for agarose gel electrophoresis. PCR was performed using
an MJ Research thermal cycler under the following conditions:
Initial denaturation can be at 94.degree. C. for 15 sec, followed
by a 18, 20, and 22 cycles of 94.degree. C. for 15, 65.degree. C.
for 2 min, 72.degree. C. for 5 sec. A final extension at 72.degree.
C. was carried out for 2 min. After agarose gel electrophoresis,
the band intensities of the 283 b.p. .beta.-actin bands from
multiple tissues were compared by visual inspection. Dilution
factors for the first strand cDNAs were calculated to result in
equal .beta.-actin band intensities in all tissues after 22 cycles
of PCR. Three rounds of normalization can be required to achieve
equal band intensities in all tissues after 22 cycles of PCR.
[0636] To determine expression levels of the 251P5G2 gene, 5 .mu.l
of normalized first strand cDNA were analyzed by PCR using 26, and
30 cycles of amplification. Semi-quantitative expression analysis
can be achieved by comparing the PCR products at cycle numbers that
give light band intensities. The primers used for RT-PCR were
designed using the 251P5G2 SSH sequence and are listed below:
3 251P5G2.1 5'-AGTGATTCAAAGAGCTGTGGAGA-3' (SEQ ID NO: 51) 251P5G2.2
5'-GGCCAGAGCGCACTTACCTACC-3' (SEQ ID NO: 52)
[0637] A typical RT-PCR expression analysis is shown in FIG. 14.
First strand cDNA was prepared from vital pool 1 (liver, lung and
kidney), vital pool 2 (pancreas, colon and stomach), prostate
cancer metastasis to lymph node, prostate cancer pool, bladder
cancer pool, and cancer metastasis pool. Normalization was
performed by PCR using primers to actin and GAPDH. Semiquantitative
PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of
amplification. Results show strong expression of 251P5G2 in
prostate cancer metastasis, prostate cancer pool, and cancer
metastasis pool. Expression of 251P5G2 was also detected in bladder
cancer pool, but not in vital pool 1 and vital pool 2.
Example 2
[0638] Isolation of Full Length 251P5G2 Encoding cDNA
[0639] The 251P5G2 SSH cDNA sequence was derived from a
substraction consisting of prostate cancer metastasis to lymph node
minus a mixture of 9 normal tissues: stomach, skeletal muscle,
lung, brain, liver, kidney, pancreas, small intestine and heart.
The SSH cDNA sequence (FIG. 1) was designated 251P5G2.
[0640] The 251P5G2 SSH DNA sequence of 162 bp (FIG. 1) was novel
and did not show homology to any known gene. 251P5G2 v.1 (clone
4.7) of 2157 bp was cloned from prostate cancer cDNA library,
revealing an ORF of 255 amino acids (FIG. 2 and FIG. 3). Other
variants of 251P5G2 were also identified and these are listed in
FIGS. 2 and 3.
[0641] 251P5G2 v.1, v.2, v.3, and v.4 proteins are 255 amino acids
in length and differ from each other by one amino acid as shown in
FIG. 11. 251P5G2 v.5, v.6, v.7, v.8, v.9, and v.10 code for the
same protein as 251P5G2 v.1. 251P5G2 v.12 codes for a protein of
1266 amino acids in length. 251P5G2 v.13 codes for the same protein
as 251P5G2 v.12.
[0642] 251P5G2 v.1 and variants v.2 through v.11 are novel, and did
not show significant homology to known human genes. The 251P5G2 v.1
protein showed homology to the mouse vomeronasal 1 receptor C3, 44%
identity and 60% homology over 234 amino acids (FIG. 4A). 251P5G2
v.12 protein aligns with the protein XM.sub.--063686 at 100%
identity over 1213 amino acids, ranging from position 54 to 1266 of
251P5G2 v.12 protein (FIG. 4B). XM.sub.--063686 protein is a
hypothetical protein predicted by automated computational analysis
using GenomeScan.
Example 3
[0643] Chromosomal Mapping of 251P5G2
[0644] 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 Cornell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0645] 251P5G2 maps to chromosome 15q11.2 using 251P5G2 sequence
and the NCBI BLAST tool located on the World Wide Web at
(.ncbi.nlm.nih.gov/genom-
e/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4
[0646] Expression Analysis of 251P5G2 in Normal Tissues and Patient
Specimens
[0647] Expression analysis by RT-PCR demonstrated that 251P5G2 is
strongly expressed in prostate cancer patient specimens (FIG. 14).
First strand cDNA was prepared from vital pool 1 (liver, lung and
kidney), vital pool 2 (pancreas, colon and stomach), prostate
cancer metastasis to lymph node, prostate cancer pool, bladder
cancer pool, and cancer metastasis pool. Normalization was
performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to 251P5G2, was performed at
26 and 30 cycles of amplification. Results show strong expression
of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and
cancer metastasis pool. Expression of 251P5G2 was also detected in
bladder cancer pool, but not in vital pool 1 and vital pool 2.
[0648] Northern blot analysis of 251P5G2 is a technique known to
those skilled in the art to detect 251P5G2 protein production.
Northern blotting detects relative levels of mRNA expressed from a
251P5G2 gene. Specific mRNA is measured using a nucleic acid
hybridization technique and the signal is detected on an
autoradiogram. The stronger the signal, the more abundant is the
mRNA. For 251P5G2 genes that produce mRNA that contains an open
reading frame flanked by a good Kozak translation initiation site
and a stop codon, in the vast majority of cases the synthesized
mRNA is expressed as a protein.
[0649] The level of expression of the 251P5G2 gene is determined in
various normal tissues and in various tumor tissues and tumor cell
lines using the technique of Northern blotting, which detects
production of messenger RNA. It is well known in the art that the
production of messenger RNA, that encodes the protein, is a
necessary step in the production of the protein itself. Thus,
detection of high levels of messenger RNA by, for example, Northern
blot, is a way of determining that the protein itself is produced.
The Northern blot technique is used as a routine procedure because
it does not require the time delays (as compared to Western
blotting, immunoblotting or immunohistochemistry) involved in
isolating or synthesizing the protein, preparing an immunological
composition of the protein, eliciting a humoral immune response,
harvesting the antibodies, and verifying the specificity
thereof.
[0650] The Kozak consensus sequence for translation initiation
CCACCATGG, where the ATG start codon is noted, is the sequence with
the highest established probability of initiating translation. This
was confirmed by Peri and Pandey Trends in Genetics (2001) 17:
685-687. The conclusion is consistent with the general knowledge in
the art that, with rare exceptions, expression of an mRNA is
predictive of expression of its encoded protein. This is
particularly true for mRNA with an open reading frame and a Kozak
consensus sequence for translation initiation.
[0651] It is understood in the art that the absolute levels of
messenger RNA present and the amounts of protein produced do not
always provide a 1:1 correlation. In those instances where the
Northern blot has shown mRNA to be present, it is almost always
possible to detect the presence of the corresponding protein in the
tissue which provided a positive result in the Northern blot. The
levels of the protein compared to the levels of the mRNA may be
differential, but generally, cells that exhibit detectable mRNA
also exhibit detectable corresponding protein and vice versa. This
is particularly true where the mRNA has an open reading frame and a
good Kozak sequence (See, Peri and Pandey, supra.).
[0652] Occasionally those skilled in the art encounter a rare
occurrence where there is no detectable protein in the presence of
corresponding mRNA. (See, Fu, L., et al., Embo. Journal,
15:4392-4401 (1996)). In many cases, a reported lack of protein
expression is due to technical limitations of the protein detection
assay. These limitations are readily known to those skilled in the
art. These limitations include but are not limited to, available
antibodies that only detect denatured protein and not native
protein present in a cell and unstable proteins with very short
half-life. Short-lived proteins are still functional and have been
previously described to induce tumor formation. (See, e.g.,
Reinstein, et al., Oncogene, 19: 5944-5950). In such situations,
when more sensitive detection techniques are performed and/or other
antibodies are generated, protein expression is detected. When
studies fail to take these principles into account, they are likely
to report artifactually lowered correlations of mRNA to protein.
Outside of these rare exceptions the use of Northern blot analysis
is recognized to those skilled in the art to be predictive and
indicative of the detection of 251P5G2 protein production.
[0653] Extensive northern blot analysis of 251P5G2 in multiple
human normal tissues is shown in FIG. 15. Two multiple tissue
northern blots (Clontech) both with 2 .mu.g of mRNA/lane were
probed with the 251P5G2 SSH sequence. Expression of 251P5G2 was
detected in normal prostate and testis but not in any other normal
tissues tested.
[0654] Expression of 251P5G2 in prostate cancer metastasis patient
specimens and human normal tissues is shown in FIG. 16. RNA was
extracted from two prostate cancer metastasis to lymph node
isolated from two different patients (Met1 and Met2), as well as
from normal bladder (NB), normal kidney (NK), normal lung (NL),
normal breast (NBr), normal ovary (NO), and normal pancreas (NPa).
Northern blot with 10 .mu.g of total RNA/lane was probed with
251P5G2 SSH sequence. Results show strong expression of 251P5G2 in
the prostate cancer metastasis specimens but not in the normal
tissues tested.
[0655] Expression of 251P5G2 was also detected in prostate cancer
patient specimens and prostate cancer xenograft tissues (FIG. 17).
RNA was extracted from prostate cancer xenografts (LAPC4AD,
LAPCA4AI, LAPC-9AD, LAPC-9AI), prostate cancer cell lines (LNCaP
and PC3), normal prostate (N), and prostate cancer patient tumors
(T). Northern blots with 10 .mu.g of total RNA were probed with the
251P5G2 SSH fragment. Results show expression of 251P5G2 in the
LAPC-9AD xenograft and in prostate tumor tissues. Lower level
expression was detected in the other xenograft tissues and LNCaP
cell line but not in PC3. The lower panel represents
ethidium-bromide staining of the gel confirming the quality of the
RNA.
[0656] FIG. 18 shows expression of 251 p5g2 in human normal and
cancer tissues. First strand cDNA was prepared from a panel of 13
normal tissues, prostate cancer pool, bladder cancer pool, kidney
cancer pool, colon cancer pool, lung cancer pool, ovary cancer
pool, breast cancer pool, pancreas cancer pool, 2 different
prostate cancer metastasis specimens to lymph node, and a pool of
prostate cancer LAPC xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD, and
LAPC-9AI). Normalization was performed by PCR using primers to
actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2,
was performed at 26 and 30 cycles of amplification. A standard
curve was generated using plasmid DNA containing 251P5G2 of known
copy number. The experiment was performed in duplicate. Results
show strong expression of 251P5G2 in prostate cancer metastasis,
prostate cancer pool, and cancer metastasis pool. Expression of
251P5G2 was also detected in bladder cancer pool. Amongst normal
tissues, very weak expression was detected in hear, prostate,
skeletal muscle and testis but not in any other normal tissue
tested.
[0657] FIG. 19 shows expression of 251P5G2 in prostate cancer
patient specimens. First strand cDNA was prepared from normal
prostate, prostate cancer cell lines (PC3, DU145, LNCaP, 293T), and
a panel of prostate cancer patient specimens. Normalization was
performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to 251P5G2, was performed at
26 and 30 cycles of amplification. Results show expression of
251P5G2 in 10 out of 19 patient specimens. Very strong expression
was detected in 5 out of the 10 expressing tumors. Expression was
also detected in LNCaP but not in the other cell lines tested nor
in normal prostate.
[0658] Expression of 251P5G2 in bladdercancer patient specimens is
shown in FIG. 20. First strand cDNA was prepared from normal
bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82), and a
panel of bladder cancer patient specimens. Normalization was
performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to 251P5G2, was performed at
26 and 30 cycles of amplification. Results show expression of
251P5G2 in 5 out of 9 patient specimens, but not in the cell lines
tested nor in normal bladder.
[0659] The restricted expression of 251P5G2 in normal tissues and
the expression detected in prostate cancer, prostate cancer
metastasis, and bladder cancer suggest that 251P5G2 is a potential
therapeutic target and a diagnostic marker for human cancers.
Example 5
[0660] Transcript Variants of 251P5G2
[0661] 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.
[0662] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified by full-length cloning experiment,
or by use of full-length transcript and EST sequences. First, all
human ESTs were grouped into clusters which show direct or indirect
identity with each other. Second, ESTs in the same cluster were
further grouped into sub-clusters and assembled into a consensus
sequence. The original gene sequence is compared to the consensus
sequence(s) or other full-length sequences. Each consensus sequence
is a potential splice variant for that gene. Even when a variant is
identified that is not a full-length clone, that portion of the
variant is very useful for antigen generation and for further
cloning of the full-length splice variant, using techniques known
in the art.
[0663] Moreover, computer programs are available in the art that
identify transcript variants based on genomic sequences.
Genomic-based transcript variant identification programs include
FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in
Drosophila genomic DNA," Genome Research. 2000 April;
10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bi-
n/EmptyGrailForm) and Gen Scan (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. Jun. 8, 2001; 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. Nov. 7, 2000;
97(23):12690-3.
[0664] 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. Aug. 17, 1999; 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. Oct. 1, 1997;
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.
Jan. 24, 2001; 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. Aug. 7, 1997; 1353(2): 191-8).
[0665] 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 251P5G2 has a particular expression profile related to cancer.
Alternative transcripts and splice variants of 251P5G2 may also be
involved in cancers in the same or different tissues, thus serving
as tumor-associated markers/antigens.
[0666] The exon composition of the original transcript, designated
as 251P5G2 v.1, is shown in Table LI. Using the full-length gene
and EST sequences, two transcript variants were identified,
designated as 251P5G2 v.12 and v.13. Compared with 251P5G2 v.1,
transcript variant 251P5G2 v.12 has spliced out two fragments from
variant 251P5G2 v.1, as shown in FIG. 12. Theoretically, each
different combination of exons in spatial order, e.g. exons 2 and
3, is a potential splice variant. FIG. 12 shows the schematic
alignment of exons of the two transcript variants. Tables LII (a)
and (b) through LV (a) and (b) are set forth on a variant by
variant basis. LII (a) and (b) shows nucleotide sequence of the
transcript variant. Table LIII (a) and (b) shows the alignment of
the transcript variant with nucleic acid sequence of 251P5G2 v.1.
Table LIV (a) and (b) lays out amino acid translation of the
transcript variant for the identified reading frame orientation.
Table LV (a) and (b) displays alignments of the amino acid sequence
encoded by the splice variant with that of 251P5G2 v.1.
Example 6
[0667] Single Nucleotide Polymorphisms of 251P5G2
[0668] 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).
[0669] 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). Using the
methods described above, ten SNPs were identified in the original
transcript, 251P5G2 v.1, at positions 768 (T/G), 975 (T/A), 1005
(G/A), 1270 (A/G), 1459 (A/G), 1921 (A/G), 540 (GfT), 481 (C/T),
280 (G/A) and 162 (A/T). The transcripts or proteins with
alternative alleles were designated as variants 251P5G2 v.2, v.3,
v.4, v.5, v.6, v.7, v.8, v.9, v.10 and v.11, respectively. FIG. 10
shows the schematic alignment of the SNP variants. FIG. 11 shows
the schematic alignment of protein variants, corresponding to
nucleotide variants. Nucleotide variants that code for the same
amino acid sequence as variant 1 are not shown in FIG. 11. These
alleles of the SNPS, though shown separately here, can occur in
different combinations (haplotypes) and in any one of the
transcript variants (such as 251P5G2 v.12) that contains the
sequence context of the SNPs.
Example 7
[0670] Production of Recombinant 251P5G2 in Prokarvotic Systems
[0671] To express recombinant 251P5G2 and 251P5G2 variants in
prokaryotic cells, the full or partial length 251P5G2 and 251P5G2
variant cDNA sequences are cloned into any one of a variety of
expression vectors known in the art. One or more of the following
regions of 251P5G2 variants are expressed: the full length sequence
presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
contiguous amino acids from 251P5G2, variants, or analogs
thereof.
[0672] A. In Vitro Transcription and Translation Constructs:
[0673] pCRII: To generate 251P5G2 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 251P5G2 cDNA. The PCRII vector has Sp6 and T7 promoters
flanking the insert to drive the transcription of 251P5G2 RNA for
use as probes in RNA in situ hybridization experiments. These
probes are used to analyze the cell and tissue expression of
251P5G2 at the RNA level. Transcribed 251P5G2 RNA representing the
cDNA amino acid coding region of the 251P5G2 gene is used in in
vitro translation systems such as the TnT.TM. Coupled
Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize
251P5G2 protein.
[0674] B. Bacterial Constructs:
[0675] pGEX Constructs: To generate recombinant 251P5G2 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the 251P5G2 cDNA protein coding sequence
are cloned into the pGEX family of GST-fusion vectors (Amersham
Pharmacia Biotech, Piscataway, N.J.). These constructs allow
controlled expression of recombinant 251P5G2 protein sequences with
GST fused at the amino-terminus and a six histidine epitope (6X
His) at the carboxyl-terminus. The GST and 6X 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 6X
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 251P5G2-related protein. The ampicillin resistance
gene and pBR322 origin permits selection and maintenance of the
pGEX plasmids in E. coli.
[0676] pMAL Constructs: To generate, in bacteria, recombinant
251P5G2 proteins that are fused to maltose-binding protein (MBP),
all or parts of the 251P5G2 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 251P5G2 protein sequences with
MBP fused at the amino-terminus and a 6X His epitope tag at the
carboxyl-terminus. The MBP and 6X 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 6X 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
251P5G2. 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.
[0677] pET Constructs: To express 251P5G2 in bacterial cells, all
or parts of the 251P5G2 cDNA protein coding sequence are cloned
into the pET family of vectors (Novagen, Madison, Wis.). These
vectors allow tightly controlled expression of recombinant 251P5G2
protein in bacteria with and without fusion to proteins that
enhance solubility, such as NusA and thioredoxin (Trx), and epitope
tags, such as 6X 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
251P5G2 protein are expressed as amino-terminal fusions to
NusA.
[0678] C. Yeast Constructs:
[0679] pESC Constructs: To express 251P5G2 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 251P5G2 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 251P5G2. In addition, expression in
yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations that are found when expressed
in eukaryotic cells.
[0680] PESP Constructs: To express 251P5G2 in the yeast species
Saccharomyces pombe, all or parts of the 251P5G2 cDNA protein
coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of expression of a 251P5G2
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
[0681] Production of Recombinant 251P5G2 in Higher Eukarvotic
Systems
[0682] A. Mammalian Constructs:
[0683] To express recombinant 251P5G2 in eukaryotic cells, the full
or partial length 251P5G2 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 251P5G2 are expressed in these constructs,
amino acids 1 to 255, 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 251P5G2 v.1 through v.11; amino acids 1
to 1266, 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 251P5G2 v.12 and v.13, variants, or analogs thereof.
[0684] 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-251P5G2 polyclonal serum,
described herein.
[0685] pcDNA4/HisMax Constructs: To express 251P5G2 in mammalian
cells, a 251P5G2 ORF, or portions thereof, of 251P5G2 are cloned
into pcDNA41HisMax Version A (Invitrogen, Carlsbad, Calif.).
Protein expression is driven from the cytomegalovirus (CMV)
promoter and the SP16 translational enhancer. The recombinant
protein has Xpress.TM. and six histidine (6X 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.
[0686] pcDNA3.1/MycHis Constructs: To express 251P5G2 in mammalian
cells, a 251P5G2 ORF, or portions thereof, of 251P5G2 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 6X His epitope fused
to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains
the bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli.
[0687] pcDNA3.1/CT-GFP-TOPO Construct: To express 251P5G2 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, a 251P5G2 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
pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 251P5G2
protein.
[0688] PAPtag: A 251P5G2 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 251P5G2 protein while fusing the IgG.kappa. signal sequence to
the amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgG.kappa. signal sequence is
fused to the amino-terminus of a 251P5G2 protein. The resulting
recombinant 251P5G2 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 251P5G2
proteins. Protein expression is driven from the CMV promoter and
the recombinant proteins also contain myc and 6X 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.
[0689] pTag5: A 251P5G2 ORF, or portions thereof, is cloned into
pTag-5. This vector is similar to pAPtag but without the alkaline
phosphatase fusion. This construct generates 251P5G2 protein with
an amino-terminal IgGK signal sequence and myc and 6X His epitope
tags at the carboxyl-terminus that facilitate detection and
affinity purification. The resulting recombinant 251P5G2 protein is
optimized for secretion into the media of transfected mammalian
cells, and is used as immunogen or ligand to identify proteins such
as ligands or receptors that interact with the 251P5G2 proteins.
Protein expression is driven from the CMV promoter. The Zeocin
resistance gene present in the vector allows for selection of
mammalian cells expressing the protein, and the ampicillin
resistance gene permits selection of the plasmid in E. coli.
[0690] PsecFc: A 251P5G2 ORF, or portions thereof, is also cloned
into psecFc. The psecFc vector was assembled by cloning the human
immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2
(Invitrogen, California). This construct generates an IgG1 Fc
fusion at the carboxyl-terminus of the 251P5G2 proteins, while
fusing the IgGk signal sequence to N-terminus. 251P5G2 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant 251P5G2 proteins are optimized for secretion into the
media of transfected mammalian cells, and can be used as immunogens
or to identify proteins such as ligands or receptors that interact
with 251P5G2 protein. Protein expression is driven from the CMV
promoter. The hygromycin resistance gene present in the vector
allows for selection of mammalian cells that express the
recombinant protein, and the ampicillin resistance gene permits
selection of the plasmid in E. coli.
[0691] pSR.alpha. Constructs: To generate mammalian cell lines that
express 251P5G2 constitutively, 251P5G2 ORF, or portions thereof,
of 251P5G2 were cloned into pSR.alpha. constructs. Amphotropic and
ecotropic retroviruses were generated by transfection of pSR.alpha.
constructs into the 293T-10A1 packaging line or co-transfection of
pSR.alpha. and a helper plasmid (containing deleted packaging
sequences) into the 293 cells, respectively. The retrovirus is used
to infect a variety of mammalian cell lines, resulting in the
integration of the cloned gene, 251P5G2, into the host cell-lines.
Protein expression is driven from a long terminal repeat (LTR). The
Neomycin resistance gene present in the vector allows for selection
of mammalian cells that express the protein, and the ampicillin
resistance gene and ColE1 origin permit selection and maintenance
of the plasmid in E coli. The retroviral vectors can thereafter be
used for infection and generation of various cell lines using, for
example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
[0692] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
251P5G2 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: 53) is added to cloning primer at the 3' end of
the ORF. Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6X His fusion
proteins of the full-length 251P5G2 proteins.
[0693] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 251P5G2. High virus fiter
leading to high level expression of 251P5G2 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. A 251P5G2 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, 251P5G2 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.
[0694] Regulated Expression Systems: To control expression of
251P5G2 in mammalian cells, coding sequences of 251P5G2, or
portions thereof, are cloned into regulated mammalian expression
systems such as the T-Rex System (Invitrogen), the GeneSwitch
System (Invitrogen) and the tightiy-regulated Ecdysone System
(Sratagene). These systems allow the study of the temporal and
concentration dependent effects of recombinant 251P5G2. These
vectors are thereafter used to control expression of 251P5G2 in
various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0695] B. Baculovirus Expression Systems
[0696] To generate recombinant 251P5G2 proteins in a baculovirus
expression system, 251P5G2 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-251P5G2 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.
[0697] Recombinant 251P5G2 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 251P5G2 protein can be detected using anti-251P5G2 or
anti-His-tag antibody. 251P5G2 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 251P5G2.
Example 9
[0698] Antigenicity Profiles and Secondary Structure
[0699] FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B),
FIGS. 8(A & B), and FIGS. 9(A & B) depict graphically five
amino acid profiles of 251P5G2 variants 1 and 12, each assessment
available by accessing the ProtScale website located on the World
Wide Web at (.expasy.ch/cgi-bin/protscale.pl- ) on the ExPasy
molecular biology server.
[0700] 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 251P5G2 protein. Each of the above amino acid
profiles of 251P5G2 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.
[0701] 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.
[0702] 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.
[0703] Antigenic sequences of the 251P5G2 variant proteins
indicated, e.g., by the profiles set forth in FIGS. 5(A & B),
FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), and/or
FIGS. 9(A & B) are used to prepare immunogens, either peptides
or nucleic acids that encode them, to generate therapeutic and
diagnostic anti-251P5G2 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
251P5G2 protein variants 1 and 12 listed in FIGS. 2 and 3. In
particular, peptide immunogens of the invention can comprise, a
peptide region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5;
a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value less than 0.5 in the Hydropathicity profile of FIG. 6; a
peptde region of at least 5 amino acids of FIGS. 2 and 3 in any
whole number increment that includes an amino acid position having
a value greater than 0.5 in the Percent Accessible Residues
profiles of FIG. 7; a peptide region of at least 5 amino acids of
FIGS. 2 and 3 in any whole number increment that includes an amino
acid position having a value greater than 0.5 in the Average
Flexibility profiles on FIG. 8; and, a peptide region of at least 5
amino acids of FIGS. 2 and 3 in any whole number increment that
includes an amino acid position having a value greater than 0.5 in
the Beta-turn profile of FIG. 9. Peptide immunogens of the
invention can also comprise nucleic acids that encode any of the
forgoing.
[0704] 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.
[0705] The secondary structure of 251P5G2 protein variants 1 and
12, namely the predicted presence and location of alpha helices,
extended strands, and random coils, is predicted from the primary
amino acid sequence using the HNN--Hierarchical Neural Network
method (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/). The analysis indicates that
251P5G2 variant 1 is composed of 40.39% alpha helix, 18.82%
extended strand, and 40.78% random coil (FIG. 13A). Variant 12 is
composed of 42.28% alpha helix, 8.33% extended strand, and 49.39%
random coil (FIG. 13B). Analysis for the potential presence of
transmembrane domains in the 251P5G2 variant proteins was carried
out using a variety of transmembrane prediction algorithms accessed
from the ExPasy molecular biology server located on the World Wide
Web at (.expasy.ch/tools/). Shown graphically in FIGS. 13C and 13D
are the results of analysis of variant 1 depicting the presence and
location of 6 transmembrane domains using the TMpred program (FIG.
13C) and 5 transmembrane domains using the TMHMM program (FIG.
13D). Shown graphically in FIGS. 13E and 13F are the results of
analysis of variant 12 depicting the presence and location of 6
transmembrane domains using the TMpred program (FIG. 13E) and 3
transmembrane domains using the TMHMM program (FIG. 13F). The
results of each program, namely the amino acids encoding the
transmembrane domains are summarized in Table VI.
Example 10
[0706] Generation of 251P5G2 Polyclonal Antibodies
[0707] Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. In addition to immunizing with a full
length 251P5G2 protein variant, computer algorithms are employed in
design of immunogens that, based on amino acid sequence analysis
contain characteristics of being antigenic and available for
recognition by the immune system of the immunized host (see the
Example entitled "Antigenicity Profiles and Secondary Structure").
Such regions would be predicted to be hydrophilic, flexible, in
beta-turn conformations, and be exposed on the surface of the
protein (see, e.g., FIGS. 5(A & B), FIGS. 6(A & B), FIGS.
7(A & B), FIGS. 8(A & B), or FIGS. 9(A & B) for amino
acid profiles that indicate such regions of 251P5G2 protein
variants).
[0708] For example, recombinant bacterial fusion proteins or
peptdes containing hydrophilic, flexible, beta-turn regions of
251P5G2 protein variants are used as antigens to generate polyconal
antibodies in New Zealand White rabbits. For example, in 251P5G2
variant 1, such regions include, but are not limited to, amino
acids 28-40, amino acids 65-85, and amino acids 200-222. In
sequence specific for variant 12, such regions include, but are not
limited to, amino acids 236-251, amino acids 540-598, amino acids
832-978, and amino acids 1151-1242 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 65-85 of 251P5G2 variant
1 is conjugated to KLH and used to immunize the rabbit.
Alternatively the immunizing agent may include all or portions of
the 251P5G2 variant proteins, analogs or fusion proteins thereof.
For example, the 251P5G2 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.
[0709] In one embodiment, a GST-fusion protein encoding the whole
cDNA of 251P5G2 variant 1, amino acids 1-255 fused to GST, is
produced and 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
Recombinant 251P5G2 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., Umes, M., Grosmaire, L.,
Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).
[0710] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the section entitled "Production of
Recombinant 251P5G2 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native protein. In one embodiment, amino acids 60-85 of variant 1,
encoding a loop between transmembrane domains, is cloned into the
TagS 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 251P5G2 protein is then used as
immunogen.
[0711] 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).
[0712] 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.
[0713] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with the Tag5-251P5G2
variant 1 protein, the full-length 251P5G2 variant 1 cDNA is cloned
into pCDNA 3.1 myc-his expression vector (Invitrogen, see the
Example entitled "Production of Recombinant 251P5G2 in Eukaryotic
Systems"). After transfection of the constructs into 293T cells,
cell lysates are probed with the anti-251P5G2 serum and with
anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.)
to determine specific reactivity to denatured 251P5G2 protein using
the Western blot technique. In addition, the immune serum is tested
by fluorescence microscopy, flow cytometry and immunoprecipitation
against 293T and other recombinant 251P5G2-expressing cells to
determine specific recognition of native protein. Western blot,
immunoprecipitation, fluorescent microscopy, and flow cytometric
techniques using cells that endogenously express 251P5G2 are also
carried out to test reactivity and specificity.
[0714] Anti-serum from rabbits immunized with 251P5G2 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-251P5G2 variant 1 fusion
protein is first purified by passage over a column of GST protein
covalenily coupled to AffiGel matrix (BioRad, Hercules, Calif.).
The antiserum is then affinity purified by passage over a column
composed of a MBP-251P5G2 fusion protein covalenfy 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
[0715] Generation of 251P5G2 Monoclonal Antibodies (mAbs)
[0716] In one embodiment, therapeutic mAbs to 251P5G2 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
251P5G2 variants, for example those that would disrupt the
interaction with ligands and binding partners. Immunogens for
generation of such mAbs include those designed to encode or contain
the entire 251P5G2 protein variant sequence, regions of the 251P5G2
protein variants predicted to be antigenic from computer analysis
of the amino acid sequence (see, e.g., FIGS. 5(A & B), FIGS.
6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), or FIGS. 9(A
& B), 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 251P5G2 variant, such as 293T-251P5G2
variant 1 or 300.19-251P5G2 variant 1murine Pre-B cells, are used
to immunize mice.
[0717] To generate mAbs to a 251P5G2 variant, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen or 10.sup.7 251P5G2-expressing cells mixed in
complete Freund's adjuvant. Mice are then subsequently immunized IP
every 2-4 weeks with, typically, 10-50 .mu.g of protein immunogen
or 10.sup.7 cells mixed in incomplete Freund's adjuvant.
Alternatively, MPL-TDM adjuvant is used in immunizations. In
addition to the above protein and cell-based immunization
strategies, a DNA-based immunization protocol is employed in which
a mammalian expression vector encoding a 251P5G2 variant sequence
is used to immunize mice by direct injection of the plasmid DNA.
For example, amino acids 60-85 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 251P5G2 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 251P5G2 variant.
[0718] 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).
[0719] In one embodiment for generating 251P5G2 monoclonal
antibodies, a Tag5-251P5G2 variant 1 antigen encoding amino acids
60-85, is expressed and purified from stably transfected 293T
cells. Balb C mice are initially immunized intraperitoneally with
25 .mu.g of the Tag5-251P5G2 variant 1 protein mixed in complete
Freund's adjuvant. Mice are subsequently immunized every two weeks
with 25 .mu.g of the antigen mixed in incomplete Freund's adjuvant
for a total of three immunizations. ELISA using the Tag5 antigen
determines the titer of serum from immunized mice. Reactivity and
specificity of serum to full length 251P5G2 variant 1 protein is
monitored by Western blotting, immunoprecipitation and flow
cytometry using 293T cells transfected with an expression vector
encoding the 251P5G2 variant 1 cDNA (see e.g., the Example entitled
"Production of Recombinant 251P5G2 in Eukaryotic Systems" and FIG.
20. Other recombinant 251P5G2 variant 1-expressing cells or cells
endogenously expressing 251P5G2 variant 1 are also used Mice
showing the strongest reactivity are rested and given a final
injection of Tag5 antigen in PBS and then sacrificed four days
later. The spleens of the sacrificed mice are harvested and fused
to SPO/2 myeloma cells using standard procedures (Harlow and Lane,
1988). Supernatants from HAT selected growth wells are screened by
ELISA, Western blot, immunoprecipitation, fluorescent microscopy,
and flow cytometry to identify 251P5G2 specific antibody-producing
clones.
[0720] In another embodiment, a Tag5 antigen encoding amino acids
800-1266 of variant 12 is produced, purified and used as immunogen
to derive monoclonal antibodies specific to 251P5G2 variant 12.
Hybridoma supernatants are then screened on both 251P5G2 variant 1-
and 251P5G2 variant 12-expressing cells to identify specific
anti-251P5G2 variant 12 monoclonal antibodies.
[0721] The binding affinity of a 251P5G2 monoclonal antibody is
determined using standard technologies. Affinity measurements
quantify the strength of antibody to epitope binding and are used
to help define which 251P5G2 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
[0722] HLA Class I and Class II Binding Assays
[0723] 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.1251-radiolabeled probe peptides as described. Following
incubation, MHG-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 radiolabefed peptdes 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.
[0724] 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.
[0725] Binding assays as outined above may be used to analyze HLA
supenmotif and/or HLA motif-bearing peptides (see Table IV).
Example 13
[0726] Identification of HLA Supermotif and Motif-Bearing CTL
Candidate Epitopes
[0727] 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
supernotif- and motif-bearing epitopes for the inclusion in such a
vaccine composition. Calculation of population coverage is
performed using the strategy described below.
[0728] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0729] The searches performed to identify the motif-bearing peptide
sequences in the Example entitled "Antigenicity Profiles" and
Tables VIII-XXI and XXII-XLIX employ the protein sequence data from
the gene product of 251P5G2 set forth in FIGS. 2 and 3, the
specific search peptides used to generate the tables are listed in
Table VII.
[0730] Computer searches for epitopes bearing HLA Class I or Class
II supermotfs or motifs are performed as follows. All translated
251P5G2 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.
[0731] 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
[0732] where a.sub.ji is a coefficient which represents the effect
of the presence of a given amino acid (j) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial
assumption of this method is that the effects at each position are
essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at position i in the
peptide, it is assumed to contribute a constant amount j.sub.i to
the free energy of binding of the peptide irrespective of the
sequence of the rest of the peptide.
[0733] 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.
[0734] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0735] Protein sequences from 251P5G2 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).
[0736] 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.
[0737] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0738] The 251P5G2 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-reactvity 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.
[0739] Selection of HLA-B7 Supermotif Bearing Epitopes
[0740] The 251P5G2 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.
[0741] Selection of A1 and A24 Motif-Bearing Epitopes
[0742] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 251P5G2 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0743] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs are identified using analogous
methodology.
Example 14
[0744] Confirmation of Immunogenicity
[0745] 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:
[0746] Target Cell Lines for Cellular Screening:
[0747] The .221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the pepide-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
pepfide-specific CTLs to recognize endogenous antigen.
[0748] Primary CTL Induction Cultures:
[0749] 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-essental amino acids,
sodium pyruvate, L-glutamine and penicillin/streptomycin). The
monocytes are purilied 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
supematants. 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 IL4 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.
[0750] Induction of CTL with DC and Peptide: CD8+ T-cells are
isolated by positive selection with Dynal immunomagnetic beads
(Dynabeads.RTM. M450) 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+ T-cells (enough fora 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.
[0751] 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.
[0752] Restimulation of the induction cultures with peptide-pulsed
adherent cells: Seven and fourteen days after the primary
induction, the cells are restimulated with peptide-pulsed adherent
cells. The PBMCs are thawed and washed twice with RPMI and DNAse.
The cells are resuspended at 5.times.10.sup.6 cells/ml and
irradiated at .about.4200 rads. The PBMCs are plated at
2.times.10.sup.6 in 0.5 ml complete medium per well and incubated
for 2 hours at 37.degree. C. The plates are washed twice with RPMI
by tapping the plate gently to remove the nonadherent cells and the
adherent cells pulsed with 10 .mu.g/ml of peptide in the presence
of 3 .mu.g/ml .beta..sub.2 microglobulin in 0.25 ml RPMI/5%AB per
well for 2 hours at 37.degree. C. Peptide solution from each well
is aspirated and the wells are washed once with RPMI. Most of the
media is aspirated from the induction cultures (CD8+ cells) and
brought to 0.5 ml with fresh media. The cells are then transferred
to the wells containing the peptide-pulsed adherent cells. Twenty
four hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant human IL2 is added the
next day and again 2-3 days later at 50 IU/ml (Tsai et al.,
Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days
later, the cultures are assayed for CTL activity in a .sup.51Cr
release assay. In some experiments the cultures are assayed for
peptide-specific recognition in the in situ IFN.sub.Y 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.
[0753] Measurement of CTL Lytic Activity by .sup.51Cr Release
[0754] 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. Peptde-pulsed targets are
prepared by incubating the cells with 10 .mu.g/ml peptide overnight
at 37.degree. C.
[0755] Adherent target cells are removed from culture flasks with
trypsin-EDTA. Target cells are labeled with 200 .mu.Ci of .sup.51Cr
sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37.degree.
C. Labeled target cells are resuspended at 10.sup.6 per ml and
diluted 1:10 with K562 cells at a concentration of
3.3.times.10.sup.6/ml (an NK-sensitive erythroblastoma cell line
used to reduce non-specific lysis). Target cells (100 .mu.l) and
effectors (100 .mu.l) are plated in 96 well round-bottom plates and
incubated for 5 hours at 37.degree. C. At that time, 100 .mu.l of
supernatant are collected from each well and percent lysis is
determined according to the formula:
[(cpm of the test sample-cpm of the spontaneous .sup.51Cr release
sample)/(cpm of the maximal .sup.51Cr release sample-cpm of the
spontaneous .sup.51Cr release sample)].times.100.
[0756] 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.
[0757] In situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-specific and Endogenous Recognition
[0758] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH8.2) overnight
at 4.degree. C. The plates are washed with Ca.sup.2+, Mg.sup.2+
free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours,
after which the CTLs (100 .mu.l/well) and targets (100 .mu.l/well)
are added to each well, leaving empty wells for the standards and
blanks (which received media only). The target cells, either
peptide-pulsed or endogenous targets, are used at a concentration
of 1.times.10.sup.6 cells/ml. The plates are incubated for 48 hours
at 37.degree. C. with 5% CO.sub.2.
[0759] 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.
[0760] CTL Expansion
[0761] 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 sftu IFN.gamma. assay using the same
targets as before the expansion.
[0762] 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 5'10.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 M, sodium pyruvate, 25 mM 2-ME,
L-glutamine and gentamicin.
[0763] Immunogenicity of A2 Supermotif-Bearing Peptides
[0764] 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.
[0765] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 251P5G2. 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.
[0766] Evaluation of A*03/A11 Immunogenicity
[0767] 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.
[0768] Evaluation of B7 Immunogenicity
[0769] 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.
[0770] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also confirmed using similar methodology
Example 15
[0771] Implementation of the Extended Supermotif to Improve the
Binding Capacity of Native Epitopes by Creating Analogs
[0772] 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.
[0773] Analoging at Primary Anchor Residues
[0774] 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.
[0775] 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.
[0776] 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.
[0777] 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).
[0778] 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.
[0779] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0780] 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.
[0781] 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.
[0782] 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).
[0783] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0784] 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.
[0785] Analoging at Secondary Anchor Residues
[0786] 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.
[0787] 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 251P5G2-expressing tumors.
[0788] Other Analoging Strategies
[0789] 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).
[0790] 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
[0791] Identification and Confirmation of 251P5G2-Derived Sequences
with HLA-DR Binding Motifs
[0792] 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.
[0793] Selection of HLA-DR-Supermotif-Bearing Epitopes
[0794] To identify 251P5G2derived, HLA class II HTL epitopes, a
251P5G2 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).
[0795] 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.
[0796] The 251P5G2-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. 251P5G2-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0797] Selection of DR3 Motif Peptides
[0798] 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.
[0799] To efficiently identify peptides that bind DR3, target
251P5G2 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.
[0800] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0801] 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
[0802] Immunogenicity of 251P5G2-Derived HTL Epitones
[0803] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0804] 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 251P5G2-expressing
tumors.
Example 18
[0805] Calculation of Phenotypic Frequencies of HLA-Supertypes in
Various Ethnic Backgrounds to Determine Breadth of Population
Coverage
[0806] 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.
[0807] 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].
[0808] 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).
[0809] 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 Al and A24 are combined with the coverage of the
A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV
(G). An analogous approach can be used to estimate population
coverage achieved with combinations of class II motif-bearing
epitopes.
[0810] 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.
[0811] 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
[0812] CTL Recognition Of Endogenously Processed Antigens After
Priming
[0813] 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.
[0814] 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 251P5G2
expression vectors.
[0815] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
251P5G2 antigen. The choice of transgenic mouse model to be used
for such an analysis depends upon the epitope(s) that are being
evaluated. In addition to HLA-A*0201/K.sup.b transgenic mice,
several other transgenic mouse models including mice with human
A11, which may also be used to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for
HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed, which may be used to evaluate HTL
epitopes.
Example 20
[0816] Activity Of CTL-HTL Coniuqated Epitopes In Transgenic
Mice
[0817] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 251P5G2-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 251P5G2-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.
[0818] 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.
[0819] 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)
[0820] 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.
[0821] 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.
[0822] The results are analyzed to assess the magnitude of the CTL
responses of animals injected with the immunogenic CTU/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
[0823] Selection of CTL and HTL Epitopes for Inclusion in a
251P5G2-Specific Vaccine
[0824] 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.
[0825] 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.
[0826] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 251P5G2 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 251P5G2. For example, if it has been observed
that patients who spontaneously clear 251P5G2-expressing cells
generate an immune response to at least three (3) epitopes from
251P5G2 antigen, then at least three epitopes should be included
for HLA class I. A similar rationale is used to determine HLA class
II epitopes.
[0827] 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/.
[0828] 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.
[0829] 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 251P5G2, 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.
[0830] 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 251P5G2.
Example 22
[0831] Construction of "Minigene" Multi-Epitope DNA Plasmids
[0832] 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.
[0833] 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 251P5G2, are
selected such that multiple supermofifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 251P5G2 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.
[0834] Such a construct may additionally include sequences that
direct the HTL epitopes to the endoplasmic reticulum. For example,
the li protein may be fused to one or more HTL epitopes as
described in the art, wherein the CLIP sequence of the li 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.
[0835] 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.
[0836] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa lg-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disdosed 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.
[0837] 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.
[0838] 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 dones are screened by sequencing.
Example 23
[0839] The Plasmid Construct and the Degree to Which it Induces
Immunogenicity
[0840] 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).
[0841] 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.
[0842] 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.
[0843] 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.
[0844] 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.
[0845] 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.
[0846] 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).
[0847] 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-specitic activity in an alpha, beta and/or
gamma IFN ELISA.
[0848] 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
[0849] Peptide Compositions for Prophylactic Uses
[0850] Vaccine compositions of the present invention can be used to
prevent 251P5G2 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
251P5G2-associated tumor.
[0851] 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 251P5G2-associated disease.
[0852] 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
[0853] Polyepitopic Vaccine Compositions Derived from Native
251P5G2 Sequences
[0854] A native 251P5G2 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, ie., 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.
[0855] The vaccine composition will include, for example, multiple
CTL epitopes from 251P5G2 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.
[0856] 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 251P5G2, 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.
[0857] 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
[0858] Polyepitopic Vaccine Compositions from Multiple Antigens
[0859] The 251P5G2 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
251P5G2 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 251P5G2 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 251P5G2
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
[0860] Use of Peptides to Evaluate an Immune Response
[0861] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 251P5G2. 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.
[0862] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 251P5G2 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising a 251P5G2
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, P2-microglobulin, and peptide are refolded by dilution. The
45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0863] 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 251P5G2 epitope, and thus the
status of exposure to 251P5G2, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
[0864] Use of Peptide Epitopes to Evaluate Recall Responses
[0865] 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 251P5G2-associated disease or who have been
vaccinated with a 251P5G2 vaccine.
[0866] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
251P5G2 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.
[0867] 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.
[0868] 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).
[0869] 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).
[0870] 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.
[0871] 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
effectoritarget (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.
[0872] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 251P5G2 or a 251P5G2 vaccine.
[0873] 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 251P5G2
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 10U/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
[0874] Induction Of Specific CTL Response In Humans
[0875] 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:
[0876] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0877] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0878] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0879] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0880] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0881] 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.
[0882] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0883] 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.
[0884] The vaccine is found to be both safe and efficacious.
Example 30
[0885] Phase II Trials In Patients Expressing 251P5G2
[0886] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 251P5G2. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 251P5G2, 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:
[0887] 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.
[0888] 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 251P5G2.
[0889] 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 251P5G2-associated disease.
Example 31
[0890] Induction of CTL Responses Using a Prime Boost Protocol
[0891] 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.
[0892] For example, the initial immunization may be performed using
an expression vector, such as that constructed in the Example
entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids" in
the form of naked nucleic acid administered IM (or SC or ID) in the
amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to
1000 .mu.g) can also be administered using a gene gun. Following an
incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-10.sup.7 to 5.times.10.sup.9 pfu. An
alternative recombinant virus, such as an MVA, canarypox,
adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient
blood samples are obtained before immunization as well as at
intervals following administration of the initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells
are isolated from fresh heparinized blood by Ficoll-Hypaque density
gradient centrifugation, aliquoted in freezing media and stored
frozen. Samples are assayed for CTL and HTL activity.
[0893] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 251P5G2 is generated.
Example 32
[0894] Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0895] 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
251P5G2 protein from which the epitopes in the vaccine are
derived.
[0896] For example, a cocktail of epitope-comprising peptides is
administered ex vivo to PBMC, or isolated DC therefrom. A
pharmaceutical to facilitate harvesting of DC can be used, such as
Progenipoietin.TM. (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After
pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
[0897] 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.
[0898] 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.
[0899] Ex Vivo Activation of CTL/HTL Responses
[0900] Alternatively, ex vivo CTL or HTL responses to 251P5G2
antigens can be induced by incubating, in tissue culture, the
patients, 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
[0901] An Alternative Method of Identifying and Confirming
Motif-Bearing Peptides
[0902] 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. 251P5G2.
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.
[0903] 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
251P5G2 to isolate peptides corresponding to 251P5G2 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.
[0904] 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
[0905] Complementary Polynucleotides
[0906] Sequences complementary to the 251P5G2-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 251P5G2. 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 251P5G2. 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 251P5G2-encoding
transcript.
Example 35
[0907] Purification of Naturally-occurring or Recombinant 251P5G2
Using 251P5G2-Specific Antibodies
[0908] Naturally occurring or recombinant 251P5G2 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 251P5G2. An immunoaffinity column is constructed by covalently
coupling anti-251P5G2 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 manufacturers instructions.
[0909] Media containing 251P5G2 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 251P5G2 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/251P5G2 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
[0910] Identification of Molecules Which Interact with 251P5G2
[0911] 251P5G2, or biologically active fragments thereof, are
labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
251P5G2, washed, and any wells with labeled 251P5G2 complex are
assayed. Data obtained using different concentrations of 251P5G2
are used to calculate values for the number, affinity, and
association of 251P5G2 with the candidate molecules.
Example 37
[0912] In Vivo Assay for 251P5G2 Tumor Growth Promotion
[0913] The effect of the 251P5G2 protein on tumor cell growth is
evaluated in vivo by evaluating tumor development and growth of
cells expressing or lacking 251P5G2. For example, SCID mice are
injected subcutaneously on each flank with 1.times.10.sup.6 of
either 3T3, prostate or bladder cancer cell lines (e.g. PC3 or J82
cells) containing tkNeo empty vector or 251P5G2. At least two
strategies may be used: (1) Constitutive 251P5G2 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 Jul. 5, 1989), adenovinus (such as
Adenovinus 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 251P5G2-expressing
cells grow at a faster rate and whether tumors produced by
251P5G2-expressing cells demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs).
[0914] Additionally, mice can be implanted with 1.times.10.sup.5 of
the same cells orthotopically to determine if 251P5G2 has an effect
on local growth in the bladder or prostate, and whether 251P5G2
affects the ability of the cells to metastasize, specifically to
lymph nodes and bone (Fu. X. et al, Int J Cancer. 1992, 52:987; Fu.
X. et al, Int J Cancer. 1991, 49:938). The assay is also useful to
determine the 251P5G2 inhibitory effect of candidate therapeutic
compositions, such as for example, 251P5G2 intrabodies, 251P5G2
antisense molecules and ribozymes.
Example 38
[0915] 251P5G2 Monoclonal Antibody-mediated Inhibition of Bladder,
and Prostate Tumors In Vivo
[0916] The significant expression of 251P5G2 in cancer tissues,
together with its restrictive expression in normal tissues makes
251P5G2 a good target for antibody therapy. Similarly, 251P5G2 is a
target for T cell-based immunotherapy. Thus, the therapeutic
efficacy of anti-251P5G2 mAbs in human prostate cancer xenograft
mouse models is evaluated by using recombinant cell lines such as
PC3-251P5G2, and 3T3-251P5G2 (see, e.g., Kaighn, M. E., et al.,
Invest Urol, 1979. 17(1): p. 16-23), as well as human prostate
xenograft models such as LAPC9 (Saffran et al, Proc Natl Acad Sci
USA. 2001, 98:2658). Similarly, anti-251P5G2 mAbs are evaluated in
human bladder cancer xenograft models using recombinant cell lines
such as J82-251P5G2.
[0917] Antibody efficacy on tumor growth and metastasis formation
is studied, e.g., in a mouse orthotopic bladder cancer xenograft
model, and a mouse prostate cancer xenograft model. The antibodies
can be unconjugated, as discussed in this Example, or can be
conjugated to a therapeutic modality, as appreciated in the art.
Anti-251P5G2 mAbs inhibit formation of prostate and bladder
xenografts. Anti-251P5G2 mAbs also retard the growth of established
orthotopic tumors and prolonged survival of tumor-bearing mice.
These results indicate the utility of anti-251P5G2 mAbs in the
treatment of local and advanced stages of prostate and bladder
cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or on
the World Wide Web at (.pnas.org/cgi/doi/10.1073/pnas.-
051624698).
[0918] Administration of the anti-251P5G2 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 251P5G2
as an attractive target for immunotherapy and demonstrate the
therapeutic potential of anti-251P5G2 mAbs for the treatment of
local and metastatic cancer. This example demonstrates that
unconjugated 251P5G2 monoclonal antibodies are effective to inhibit
the growth of human bladder and prostate tumor xenografts grown in
SCID mice; accordingly a combination of such efficacious monoclonal
antibodies is also effective.
[0919] Tumor Inhibition Using Multiple Unconjugated 251P5G2
mAbs
[0920] Materials and Methods
[0921] 251P5G2 Monoclonal Antibodies:
[0922] Monoclonal antibodies are raised against 251P5G2 as
described in the Example entitled "Generation of 251P5G2 Monoclonal
Antibodies (mAbs)." The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation for their capacity to
bind 251P5G2. Epitope mapping data for the anti-251P5G2 mAbs, as
determined by ELISA and Western analysis, recognize epitopes on the
251P5G2 protein. Immunohistochemical analysis of prostate cancer
tissues and cells with these antibodies is performed.
[0923] 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 SCABER, J82, A498, 769P, CaOv1 or PA1 tumor xenografts.
[0924] Cell Lines
[0925] The bladder and prostate carcinoma cell lines, J82 and PC3
as well as the fibroblast line NIH 3T3 (American Type Culture
Collection) are maintained in media supplemented with L-glutamine
and 10% FBS.
[0926] A J82-251P5G2, PC3-251P5G2 and 3T3-251P5G2 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.
[0927] Xenograft Mouse Models.
[0928] 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.p. antibody injections
are started on the same day as tumor-cell injections. As a control,
mice are injected with either purified mouse IgG (ICN) or PBS; or a
purified monoclonal antibody that recognizes an irrelevant antigen
not expressed in human cells. Tumor sizes are determined by caliper
measurements, and the tumor volume is calculated as:
Length.times.Width.times.Height. Mice with s.c. tumors greater than
1.5 cm in diameter are sacrificed.
[0929] Orthotopic injections are performed under anesthesia by
using ketamine/xylazine. For bladder orthotopic studies, an
incision is made through the abdomen to expose the bladder, and
tumor cells (5.times.10.sup.5) mixed with Matrigel are injected
into the bladder wall in a 10-.mu.l volume. To monitor tumor
growth, mice are palpated and blood is collected on a weekly basis
to measure BTA levels. For prostate orthopotic models, an incision
is made through the abdominal muscles to expose the bladder and
seminal vesicles, which then are delivered through the incision to
expose the dorsal prostate. Tumor cells e.g. LAPC-9 cells
(5.times.10.sup.5) mixed with Matrigel are injected into the
prostate in a 10-.mu.l volume (Yoshida Y et al, Anticancer Res.
1998, 18:327; Ahn et al, Tumour Biol. 2001, 22:146). To monitor
tumor growth, blood is collected on a weekly basis measuring PSA
levels. The mice are segregated into groups for the appropriate
treatments, with anti-251P5G2 or control mAbs being injected
i.p.
[0930] Anti-251P5G2 mAbs Inhibit Growth of 251P5G2-Expressing
Xenograft-Cancer Tumors
[0931] The effect of anti-251P5G2 mAbs on tumor formation is tested
on the growth and progression of bladder, and prostate cancer
xenografts using J82-251P5G2, and PC3-251P5G2 orthotopic models. As
compared with the s.c. tumor model, the orthotopic model, which
requires injection of tumor cells directly in the mouse bladder,
and 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; Fu,
X, et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell
Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic
model more representative of human disease progression and allowed
us to follow the therapeutic effect of mAbs on clinically relevant
end points.
[0932] Accordingly, tumor cells are injected into the mouse
bladder, or prostate, and 2 days later, the mice are segregated
into two groups and treated with either: a) 200-500 .mu.g, of
anti-251P5G2 Ab, or b) PBS three times per week for two to five
weeks.
[0933] 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 bladder
cancer, anti-STEAP-1 for prostate cancer models (Lin S et al,
Cancer Detect Prev. 2001; 25:202; Saffran, D., et al., PNAS
supra).
[0934] Mice bearing established orthotopic tumors are administered
1000 .mu.g injections of either anti-251P5G2 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.
[0935] These studies demonstrate a broad anti-tumor efficacy of
anti-251P5G2 antibodies on initiation and progression of prostate
and kidney cancer in xenograft mouse models. Anti-251P5G2
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-251P5G2 mAbs demonstrate a dramatic
inhibitory effect on the spread of local bladder and prostate tumor
to distal sites, even in the presence of a large tumor burden.
Thus, anti-251P5G2 mAbs are efficacious on major clinically
relevant end points (tumor growth), prolongation of survival, and
health.
Example 39
[0936] Therapeutic and Diagnostic use of Anti-251P5G2 Antibodies in
Humans
[0937] Anti-251P5G2 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-251P5G2 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 251P5G2 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-251P5G2 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0938] As determined by flow cytometry, anti-251P5G2 mAb
specifically binds to carcinoma cells. Thus, anti-251P5G2
antibodies are used in diagnostic whole body imaging applications,
such as radioimmunosdintigraphy 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 251P5G2. Shedding or release of an extracellular
domain of 251P5G2 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 251P5G2 by
anti-251P5G2 antibodies in serum and/or urine samples from suspect
patients.
[0939] Anti-251P5G2 antibodies that specifically bind 251P5G2 are
used in therapeutic applications for the treatment of cancers that
express 251P5G2. Anti-251P5G2 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-251P5G2 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 "251P5G2 Monoclonal Antibody-mediated Inhibition
of Bladder and Lung Tumors In Vivo"). Either conjugated and
unconjugated anti-251P5G2 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
[0940] Human Clinical Trials for the Treatment and Diagnosis of
Human Carcinomas through Use of Human Anti-251P5G2 Antibodies In
vivo
[0941] Antibodies are used in accordance with the present invention
which recognize an epitope on 251P5G2, and are used in the
treatment of certain tumors such as those listed in Table I. Based
upon a number of factors, including 251P5G2 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.
[0942] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-251P5G2 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-251P5G2
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-251P5G2 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).
[0943] II.) Monotherapy: In connection with the use of the
anti-251P5G2 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.
[0944] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-251P5G2 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 251P5G2. In connection with the use of the anti-251P5G2
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)-251P5G2 antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses 251P5G2 (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.
[0945] Dose and Route of Administration
[0946] 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-251P5G2
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-251P5G2 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-251P5G2 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-251P5G2 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.
[0947] Three distinct delivery approaches are useful for delivery
of anti-251P5G2 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.
[0948] Clinical Development Plan (CDP)
[0949] Overview: The CDP follows and develops treatments of
anti-251P5G2 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-251P5G2 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
251P5G2 expression levels in their tumors as determined by
biopsy.
[0950] 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 251P5G2. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-251P5G2 antibodies are
found to be safe upon human administration.
Example 41
[0951] Human Clinical Trial Adiunctive Therapy with Human
Anti-251P5G2 Antibody and Chemotherapeutic Agent
[0952] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-251P5G2 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-251P5G2 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-251P5G2 antibody
with dosage of antibody escalating from approximately about 25
mg/m.sup.2to about 275 mg/m.sup.2 over the course of the treatment
in accordance with the following schedule:
4 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175
225 275 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2 mg/m.sup.2
mg/m.sup.2 Chemotherapy + + + + + + (standard dose)
[0953] 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 251P5G2. 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.
[0954] The anti-251P5G2 antibodies are demonstrated to be safe and
elfacious, Phase II trials confirm the efficacy and refine optimum
dosing.
Example 42
[0955] Human Clinical Trial: Monotherapy with Human Anti-251P5G2
Antibody
[0956] Anti-251P5G2 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-251P5G2
antibodies.
Example 43
[0957] Human Clinical Trial: Diagnostic Imaging with Anti-251P5G2
Antibody
[0958] Once again, as the adjunctive therapy discussed above is
safe within the safety criteria discussed above, a human clinical
trial is conducted concerning the use of anti-251P5G2 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
[0959] Homology Comparison of 251P5G2 to Known Sequences
[0960] The 251P5G2 protein of FIG. 3 has 255 amino acids with
calculated molecular weight of 29.3 kDa, and pl of 9.4. Three
variants of 251P5G2 have been identified, 251P5G2 v.1, v.2 and v 3,
which differ from variant 1 by 1 amino acid each at aa positions 16
and 95 respectively. The 251P5G2 protein exhibits homology to a
previously cloned murine gene, namely vomeronasal 1 receptor, C3
(gi 20821692), and shows 44% identity and 60% homology to that gene
over the length of the protein (FIG. 4). 251P5G2 is a
multi-transmembrane protein, predicted to carry 5 or 6
transmembrane domains. Bioinformatic analysis indicates that the
251P5G2 protein may localize to the endoplasmic reticulum or
peroxisome (see Table L). However, based on it topology and
similarity to vomeronasal receptor suggest that 251P5G2 may also
localize to the plasma membrane. Motif analysis revealed the
presence of a vomeronasal organ pheromone receptor family
signature, a rhodopsin-like GPCR superfamily signature, and
iodothyronine deiodinase motif (see Table VI).
[0961] The vomeronasal receptors share sequence homology to other
families of G protein-coupled receptors, and are distantly related
to the T2R bitter taste receptors and rhodopsin-like GPCRs.
G-protein coupled receptors are seven-transmembrane receptors that
exhibit an extracellular amino-terminus, three extracellular loops,
three intracellular loops and an intracellular carboxyl terminus.
G-protein coupled receptors are stimulated by a variety of stimuli,
including polypeptide hormones, neurotransmitters, chemokines and
phospholipids (Civelli O et al, Trends Neurosci. 2001, 24:230;
Vrecl M et al Mol Endocrinol. 1998, 12:1818). Ligand binding
traditionally occurs between the first and second extracellular
loops of the GPCR. Upon ligand binding GPCRs transduce signals
across the cell surface membrane by associating with trimeric G
proteins. Vomeronasal receptors are expressed in the apical regions
of the vomeranasal organs, often in neurons expressing Gi2 subunits
of G proteins. Vomeronasal receptors are activated by pheromones
and signal by activating the ERK-MAPK and inositol trisphosphate
pathways (Dudley C A et al, Brain Res. 2001, 915:32; Wekesa K S et
al, Endocrinology. 1997, 138:3497). Although the function of
vomeronasal receptors is not well understood, they appear to be
associated with motility, cell proliferation and programmed cell
death (Riccardi D. Cell Calcium. 1999, 26:77) all of which have a
direct effect on tumor growth and progression. This is further
supported by recent studies associating GPCRs with cellular
transformation. In particular, KSHV G protein-coupled receptor was
found to transform NIH 3T3 cells in vitro and induces multifocal
KS-like lesions in KSHV-GPCR-transgenic mice (Schwarz M, Murphy P
M. J Immunol 2001, 167:505). KSHV-GPCR was capable of producing its
effect on endothelial cells and fibroblasts by activating defined
signaling pathways, including the AKT survival pathway (Montaner S
et al, Cancer Res 2001, 61:2641). In addition, KSHV-GPCR induced
the activation of mitogenic pathways such as AP-1 and NFkB,
resulting in the expression of pro-inflammatory genes (Schwarz M,
Murphy P M. J Immunol 2001, 167:505).
[0962] Accordingly, when 251P5G2 functions as a regulator of tumor
formation, cell proliferation, invasion or cell signaling, 251P5G2
is used for therapeutic, diagnostic, prognostic and/or preventative
purposes.
Example 45
[0963] Identification and Confirmation of Potential Signal
Transduction Pathways
[0964] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J Neurochem. 2001; 76:217-223). In particular, GPCRs
have been reported to activate MAK cascades as well as G proteins,
and been associated with the EGFR pathway in epithelial cells
(Naor, Z., et al, Trends Endocrinol Metab. 2000, 11:91; Vacate
Fetal, Cancer Res. 2000, 60:5310; Della Rocca G. J., et al, J Biol
Chem. 1999, 274:13978). In addition, GPCRs transmit their signals
by activating the protein kinase A or the phospholipase C pathways,
generating inositol 1,4,5-trisphosphate (IP3) and diacyl-glycerol
(DAG) (Breer, 1993, Ciba Found Symp 179: 97; Bruch, 1996, Comp
Biochem Physiol B Biochem Mol Biol 113:451).
[0965] Using immunoprecipitation and Western blotting techniques,
proteins are identified that associate with 251P5G2 and mediate
signaling events. Several pathways known to play a role in cancer
biology can be regulated by 251P5G2, including phospholipid
pathways such as P13K, AKT, etc, adhesion and migration pathways,
including FAK, Rho, Rac-1, etc, as well as mitogenic/survival
cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J
Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003; J. Cell Biol.
1997, 138:913). Using Western blotting and other techniques, the
ability of 251P5G2 to regulate these pathways is confirmed. Cells
expressing or lacking 251P5G2 are either left untreated or
stimulated with cytokines, androgen and anti-integrin antibodies.
Cell lysates are analyzed using anti-phospho-specific antibodies
(Cell Signaling, Santa Cruz Biotechnology) in order to detect
phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and
other signaling molecules.
[0966] To confirm that 251P5G2 directly or indirectly activates
known signal transduction pathways in cells, luciferase (luc) based
transcriptional reporter assays are carried out in cells expressing
individual genes. These transcriptional reporters contain
consensus-binding sites for known transcription factors that lie
downstream of well-characterized signal transduction pathways. The
reporters and examples of these associated transcription factors,
signal transduction pathways, and activation stimuli are listed
below.
[0967] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0968] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0969] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0970] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0971] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0972] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0973] 7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion
[0974] 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.
[0975] Signaling pathways activated by 251P5G2 are mapped and used
for the identification and validation of therapeutic targets. When
251P5G2 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 46
[0976] 251P5G2 Functions as a GPCR Inhibitors
[0977] Sequence and homology analysis of 251P5G2 indicated that
251P5G2 is a member of the pheromone GPCR family. Vomeronasal
receptors are known to regulate biological-responses by activating
PLC. In order to confirm that 251P5G2 functions as a GPCR and
mediates the activation PLC, phosphorylation of PLC is investigated
in PC3 and PC3-251P5G2 cells. Control PC3 and PC3-1251P5G2 cells
are grown in a low concentration of fetal bovine serum (FBS) and
stimulated with 10% FBS, pheromones and LPA. PLC phosphorylation is
investigated by western blotting.
[0978] GPCRs are activated by ligand binding to the extracellular
loops, resulting in the binding of trimeric G proteins to GPCR and
their activation. Using this information, several therapeutic and
small molecule strategies are utilized to inhibit GPCR activation
or downstream signaling events.
[0979] One strategy inhibits receptor and ligand binding. Recent
studies using several types of GPCRs, have demonstrated the
effectiveness of this strategy (Fawzi A B, et al. 2001, Mol.
Pharmacol., 59:30). Using a compound named SCH-202676, they
inhibited agonist and antagonist binding to GPCRs by allosterically
hindering ligand-GPCR interaction. Using this and even more
specific allosteric (small molecule) inhibitors, signal
transduction through 251P5G2 can be inhibited, thereby providing
therapeutic, prognostic, diagnostic and/or prophylactic
benefit.
[0980] A second approach is to inhibit G alpha subunit activation.
Activation of GPCRs results in the exchange of GTP for GDP on the G
alpha subunit of the trimeric G protein. Inhibition of Ga
activation prevents the activation of downstream signaling cascades
and therefore biological effects of GPCR. One molecule used to
inhibit GDP exchange on G.alpha. subunits is Suranim (Freissmuth M
et al, 1996, Mol. Pharmacol, 49:602). Since suranim functions as a
universal G.alpha. inhibitor, it prevents the activation of most
G.alpha. subunits.
[0981] A third approach is to inhibit G.alpha. subunit association
with GPCR. In order for trimeric G proteins to be activated
following GPCR/ligand interaction, it is necessary for them to
associate with their corresponding GPCR. Mutational analysis has
mapped the interaction of G.alpha. to the first and third
intracellular loops of GPCRs (Heller R at al. 1996, Biochem.
Biophys. Res. Commun). Several studies have used synthetic (small
molecule) peptides corresponding to the intracellular sequence of
loops 1 and 3 as inhibitors (Mukherjee, S., et al. 1999, J. Biol.
Chem.). Using such short peptides that serve as receptor mimics,
they are used to compete for binding of Ga subunits to 251P5G2 and
thereby provide therapeutic, prognostic, diagnostic and/or
prophylactic benefit.
[0982] Thus, compounds and small molecules designed to inhibit
251P5G2 function and downstream signaling events are used for
therapeutic diagnostic, prognostic and/or preventative
purposes.
Example 47
[0983] Regulation of Transcription
[0984] The cell surface localization of 251P5G2 and its similarity
to GPCRs indicate that it is effectively used as a modulator of the
transcriptional regulation of eukaryotic genes. Regulation of gene
expression is confirmed, e.g., by studying gene expression in cells
expressing or lacking 251P5G2. For this purpose, two types of
experiments are performed.
[0985] In the first set of experiments, RNA from parental and
251P5G2-expressing cells are extracted and hybridized to
commercially available gene arrays (Clontech) (Smid-Koopman E et
al. Br J Cancer. 2000. 83:246). Resting cells as well as cells
treated with FBS, pheromones, or growth factors are compared.
Differentially expressed genes are identified in accordance with
procedures known in the art. The differentially expressed genes are
then mapped to biological pathways (Chen K et al. Thyroid. 2001.
11:41.).
[0986] 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.
[0987] Thus, 251P5G2 plays a role in gene regulation, and it is
used as a target for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 48
[0988] Involvement in Tumor Progression
[0989] The 251P5G2 gene can contribute to the growth of cancer
cells. The role of 251P5G2 in tumor growth is confirmed in a
variety of primary and transfected cell lines including prostate,
and bladder cell lines, as well as NIH 3T3 cells engineered to
stably express 251P5G2. Parental cells lacking 251P5G2 and cells
expressing 251P5G2 are evaluated for cell growth using a
well-documented proliferation assay (Fraser S P, et al., Prostate
2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs.
1996, 7:288). The effect of 251P5G2 can also observed on cell cycle
progression. Control and 251P5G2-expressing cells are grown in low
serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells
are analyzed for BrdU and propidium iodide incorporation by FACS
analysis.
[0990] To confirm the role of 251P5G2 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH-3T3 cells lacking 251P5G2 are compared to NIH-3T3
cells expressing 251P5G2, using a soft agar assay under stringent
and more permissive conditions (Song Z. et al. Cancer Res. 2000;
60:6730).
[0991] To confirm the role of 251P5G2 in invasion and metastasis of
cancer cells, a well-established assay is used. A non-limiting
example is the use of an assay which provides a basement membrane
or an analog thereof used to detect whether cells are invasive
(e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer
Res. 1999; 59:6010)). Control cells, including prostate, and
bladder cell lines lacking 251P5G2 are compared to cells expressing
251P5G2. Cells are loaded with the fluorescent dye, calcein, and
plated in the top well of a support structure coated with a
basement membrane analog (e.g. the Transwell insert) and used in
the assay. Invasion is determined by fluorescence of cells in the
lower chamber relative to the fluorescence of the entire cell
population.
[0992] 251P5G2 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 251P5G2 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 251P5G2, including normal and tumor prostate,
colon and lung cells. Engineered and parental cells are treated
with various chemotherapeutic agents, such as etoposide, flutamide,
etc, and protein synthesis inhibitors, such as cycloheximide. Cells
are stained with annexin V-FITC and cell death is measured by FACS
analysis. The modulation of cell death by 251P5G2 can play a
critical role in regulating tumor progression and tumor load.
[0993] When 251P5G2 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 49
[0994] Involvement in Angiogenesis
[0995] 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,
251P5G2 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 251P5G2 in angiogenesis,
enhancement or inhibition, is confirmed.
[0996] For example, endothelial cells engineered to express 251P5G2
are evaluated using tube formation and proliferation assays. The
effect of 251P5G2 is also confirmed in animal models in vivo. For
example, cells either expressing or lacking 251P5G2 are implanted
subcutaneously in immunocompromised mice. Endothelial cell
migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. 251P5G2 affects angiogenesis, and
it is used as a target for diagnostic, prognostic, preventative
and/or therapeutic purposes.
Example 50
[0997] Involvement in Protein-Protein Interactions
[0998] Several GPCRs have been shown to interact with other
proteins, thereby regulating signal transduction, gene
transcription, transformation and cell adhesion (Sexton P M et al,
Cell Signal. 2001, 13:73; Turner C E, J Cell Sci. 2000, 23:4139).
Using immunoprecipitation techniques as well as two yeast hybrid
systems, proteins are identified that associate with 251P5G2.
Immunoprecipitates from cells expressing 251P5G2 and cells lacking
251P5G2 are compared for specific protein-protein associations.
[0999] Studies are performed to confirm the extent of association
of 251P5G2 with effector molecules, such as nuclear proteins,
transcription factors, kinases, phsophates etc. Studies comparing
251P5G2 positive and 251P5G2 negative cells as well as studies
comparing unstimulated/resting cells and cells treated with
epithelial cell activators, such as cytokines, growth factors and
anti-integrin Ab reveal unique interactions.
[1000] 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 251P5G2-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
colorimetric reporter activity. Specific association with effector
molecules and transcription factors directs one of skill to the
mode of action of 251P5G2, 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 251P5G2.
[1001] Thus it is found that 251P5G2 associates with proteins and
small molecules. Accordingly, 251P5G2 and these proteins and small
molecules are used for diagnostic, prognostic, preventative and/or
therapeutic purposes.
Example 51
[1002] Involvement in Adhesion
[1003] Cell adhesion plays a critical role in tissue colonization
and metastasis. The presence of link motif in 251P5G2 is indicative
of its role in cell adhesion. To confirm that 251P5G2 plays a role
in cell adhesion, control cells lacking 251P5G2 are compared to
cells expressing 251P5G2, using techniques previously described
(see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and
Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one
embodiment, cells labeled with a fluorescent indicator, such as
calcein, are incubated on tissue culture wells coated with media
alone or with matrix proteins. Adherent cells are detected by
fluorimetric analysis and percent adhesion is calculated. This
experimental system can be used to identify proteins, antibodies
and/or small molecules that modulate cell adhesion to extracellular
matrix and cell-cell interaction. Since cell adhesion plays a
critical role in tumor growth, progression, and, colonization, the
gene involved in this process can serves as a diagnostic,
preventative and therapeutic modality.
[1004] 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.
[1005] 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.
5TABLE I Tissues that Express 251P5G2: a. Malignant Tissues
Prostate Bladder
[1006]
6TABLE 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
[1007]
7TABLE 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
[1008] TABLE IV:
[1009] HLA Class I/II Motifs/Supermotifs
8TABLE IV A HLA Class I Supermotifs/Motifs POSITION POSITION
POSITION 3 (Primary C Terminus 2 (Primary Anchor) Anchor) (Primary
Anchor) SUPERMOTIFS 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
[1010] 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.
9TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, I, L A, V,
I, L, P, C, S, T A, V, I, L, C, S, T, M, Y
[1011]
10TABLE IV (C) HLA Class II Motifs MOTIFS 1.degree. anchor 1 2 3 4
5 1.degree. anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH
MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM
deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL
M IV deleterious C G GRD N G DR3 MOTIFS 1.degree. anchor 1 2 3
1.degree. anchor 4 5 1.degree. anchor 6 Motif a preferred LIVMFY D
Motif b preferred LIVMFAY DNQEST KRH DR Supermotif MFLIVWY
VMSTACPLI Italicized residues indicate less preferred or
"tolerated" residues
[1012]
11TABLE IV (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
[1013]
12TABLE IV (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
VTLMISAGNCDF KRYH dele- DEP 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 YFW YFW P 1.degree. Anchor ferred AVTMSLI LIVM 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 (LMFWY)} AIV
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)} dele- AGP G G te- rious POSITION: 9 or C- C- 1 2 3 4 5
6 7 8 terminus terminus A1 pre- GFYW 1.degree. Anchor DEA YFW P
DEQN YFW 1.degree. Anchor 9-mer ferred STM Y dele- DE RHKLIVMP A G
A te- rious A1 pre- GRHK ASTCLIVM 1.degree. Anchor GSTC A LIVM DE
1.degree. Anchor 9-mer ferred DEAS STC Y dele- A RHKDEP DE PQN RHK
PG GP te- YFW rious dele- AGP G G te- rious B51 Pre- LIVMFWY
1.degree. Anchor FWY STC FWY G FWY 1.degree. Anchor ferred P
{overscore (LIVFWYAM)} dele- AGPDERHKSTC DE G DEQN GDE te- rious
B5301 pre- LIVMFWY 1.degree. Anchor FWY STC FWY LIVM FWY 1.degree.
Anchor ferred P FWY {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
[1014]
13TABLE IV (F) Summary of HLA-supertypes Overall phenotypic
frequencies of HLA-supertypes in different ethnic populations
Specificity Phenotypic frequency Supertype Position 2 C-Terminus
Caucasian N.A. Black Japanese Chinese Hispanic Average B7 P
AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3 AILMVST RK 37.5 42.1 45.8
52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 A24
YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D)
FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1
21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4
B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY
(LIV) 10.0 25.1 1.6 9.0 5.9 10.3
[1015]
14TABLE IV (G) Calculated population coverage afforded by different
HLA-supertype combinations HLA-supertypes Phenotypic frequency
Caucasian N.A Blacks Japanese Chinese Hispanic Average A2, A3 and
B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, A24, B44 99.5 98.1
100.0 99.5 99.4 99.3 and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2, A3,
B7, A24 B44, A1, B27, B62, and B 58 Motifs indicate the residues
defining supertype specificites. The motifs incorporate residues
determined on the basis of published data to be recognized by
multiple alleles within the supertype. Residues within brackets are
additional residues also predicted to be tolerated by multiple
alleles within the supertype.
[1016]
15TABLE V Frequently Occurring Motifs avrg. % Name identity
Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type
Nucleic acid-binding protein functions as tanscription 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
Ubiquinone/plastoquinone proton translocation across the (complex
I), various chains membrane 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 Aspartyl
or acid proteases, centered on protease a catalytic aspartyl
residue Collagen 42% Collagen triple helix repeat extracellular
structural proteins involved (20 copies) in 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 (rhodopsin family)
regions, with the N-terminus located extracellularly while the
C-terminus is cytoplasmic. Signal through G proteins
[1017]
16TABLE VI Motifs and Post-translational Modifications of 251P5G2
N-glycosylation site 145-148 NVTQ (SEQ ID NO: 54) N-myristoylation
site 168-173 GLFFTL (SEQ ID NO: 55) Leucine zipper pattern 31-52
LRPERTYLPVCHVALIHMVVLL (SEQ ID NO: 56)
[1018]
17TABLE VII Search Peptides 251P5G2 Variant 1, original sequence,
nonamers, decamers. MPFISKINLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV
CHVALIHMVV LLTMVFLSPQ (SEQ ID NO: 57) LFESLNFQND FKYEASFYLR
RVIRVLSTCT TCLLGMLQVV NISPSISWLV RFKWKSTIFT FHLFSWSLSF PVSSSLIFYT
VASSNVTQJM LHVSKYCSLF PTNSIIRGLF FTLSLFRDVF LKQIMLFSSV YMMTLTQELQ
EILVPSQPQP LPKDLCRGKS HQHILLPVSF SVGMYKMDFI ISTSSTLPWA YDRGV
251P5G2 Variant 2 nonamers VLA SQPTLCSFFS ASSP. (SEQ ID NO: 58)
decamers LVLA SQPTLCSFFS ASSPF (SEQ ID NO: 59) 251P5G2 Variant 3
nonamers FYLR RVIRDLSICT TCL. (SEQ ID NO: 60) decamers SFYLR
RVIRDLSICT TCLL. (SEQ ID NO: 61) 251P5G2 Variant 4 nonamers SICT
TCLLDMLQVV NIS. (SEQ ID NO: 62) decamers LSICT TCLLDMLQVV NISP.
(SEQ ID NO: 63) 251P5G2 Variant 12 251P5G2 Variant 12a Nonamers
ISPSISWLIMLFSSVY. (SEQ ID NO: 64) Decamers NISPSTSWLIMLFSSVYM. (SEQ
ID NO: 65) 15-mers MLQVVNISPSISWLIMLFSSVYMMTLIQ. (SEQ ID NO: 66)
251P5G2 Variant 12b Nonamers. SHQHILLPTQATFAAATGLWAALT-
TVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTTLTGHSA (SEQ ID NO: 67)
LSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLGWERVVQRRLEVPRPQAAPATSATPS-
RDPSPPCHQRR DAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLTHVRCAQ-
GLEAASANLPGAPGRSSSCALRYRSGPSV SSAPSPAEPPAHQRLLFLPRAPQAVSGP-
QEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEILKFSEKE
TGGGILGLELPATAARLSGLNSIMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSECYGHSFLTMK-
ET STKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAWWGKVPR-
KDLIVMLRDTDMNKRDKQKR TALHLASANGNSEVVQLLLDRRCQLNVLDNKKRTALI-
KAVQCQEDECVLMLLEHGADGNIQDEYGNTALHYAIYN
EDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCGSASIVN-
LL LEQNVDVSSQDLSGQTAREYAVSSHHHVICELLSDY1EKQMLKISSENSNPVTTI-
LNIKLPLKVEEEIKKGGSNP VGLPENLTNGASAGNGDDGLIPQRKSRKPENQQFPDT-
ENEEYHSDEQNDTQKQLSEEQNTGISQDEILTNKQKQI
EVAEKEMNSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQNQLRENKILEEIESVKEKLLKTIQLNEE-
AL TKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFSLRQLGL-
AQHAQASVQQLCYKWGHTEK TEQQAQEQGAALRSQIGDPCGVPLSEGGTAAGDQGPG-
THLPPREPPASPGTPSLVRLASGAPAAALPPPTGKNGR
SPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSNRETHQAFRDKDDLPFFKTQQSPRHTK-
DL GQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSLPHFHVS-
AGGVCPTTLGSNREIT Decamers: KSHQHILLPTQATFAAATGLWAAL-
TTVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTTLTGHS (SEQ ID NO: 68)
ALSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLCWERVVQRRLEVPRPQAAPATSATP-
SRDPSPPCHQR RDAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLTHVRCA-
QGLEAASANLPGAPGRSSSCCARYRSGPS VSSAPSPAEPPAHQRLLFLPPAPQAVSG-
PQEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEILKFSEK
ETGGGILGLELPATAARLSGLNSIMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSEGYCHSFLIM-
KE TSTKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAWWGKVP-
RKDLIVMLRDTDMNKRDKQK RTALHLASANGNSEVVQLLLDRRCQLNVLDNKKRTAL-
TKAVQCQEDECVLMLLEHGADGNIQDEYGNTALHYAIY
NEDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCGSASIV-
NL LLEQNVDVSSQDLSGQTAREYAVSSHHHVICELLSDYKEKQMLKISSENSNPVIT-
ILNIKLPLKVEEEIKKHGSN PVGLPENLThGASAGNGDDGLIPQRKSRKPENQQFPD-
TENEEYHSDEQNDTQKQLSEEQNTGISQDEILTNKQKQ
IEVAEKEMNSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQHQLRENKILEETESVKEKLLKTIQLNE-
EA LTKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFSLRQLG-
LAQHAQASVQQLCYKWGHTE KTEQQAQEQGAALRSQTGDPGGVPLSEGGTAAGDQGP-
GTHLPPREPRASPGTPSLVRLASGARAAALPPPTGKNG
RSPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSNRETHQAFRDKDDLPFFKTQQSPRHT-
KD LGQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSLPHFHV-
SAGGVGPTTLGSNREIT 15-mers. DLCRGKSHQHILLPTQATFAAATG-
LWAALTTVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTT (SEQ ID NO: 69)
LTGHSALSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLGWERVVQRRLEVPRPQAAPA-
TSATPSRDPSP PCHQRRDAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLT-
HVRCAQGLEAASANLPGAPGRSSSCALRY RSGPSVSSAPSPAEPPAHQRLLFLPRAP-
QAVSGPQEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEIL
KFSEKETGGGILGLELPATAARLSGLNSTMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSEGYGH-
SF LIMKETSTKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAW-
WGKVPRKDLIVMLRDTDMNK RDKQKRTALHLASANGNSEVVQLLLDRRCQLNVLDNK-
KRTALIKAVQCQEDECVLMLLEHGADGHIQDEYGNTAL
HYAIYNEDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCG-
SA SIVNLLLEQNDVSSQDLSGQTAREYAVSSHHIIVICELLSDYKEKQMLKISSENS-
NPVITILNIKLPLKVEEEIK KHGSNPVGLPENLTHGASAGNGDDGLIPQRKSRKPEN-
QQFPDTENEEYHSDEQNDTQKQLSEEQNTGISQDEILT
HKQKQIEVAEKEMMSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQNQLREHKILEEIESVKEKLLKT-
IQ LNEEALTKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFS-
LRQLGLAQHAQASVQQLCYK WGHTEKTEQQAQEQGAALRSQIGDPGGVPLSEGGTAA-
GDQGPGTHLPPREPRASPGTPSLVRLASGARAAALPPP
TGKNGRSPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSHRETHQAFRDKDDLPFFKTQQ-
SP RHTKDLGQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSL-
PHFHVSAGGVGPTTLGSNRE IT
[1019]
18TABLE VIII V1-A1-9mers: 251P5G2 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 10 ASQPTLFSF 7.500
245 STLPWAYDR 5.000 205 PSQPQPLPK 1.500 228 VSFSVGMYK 1.500 209
QPLPKDLCR 1.250 72 KYEASFYLR 0.900 243 TSSTLPWAY 0.750 163
NSIIRGLFF 0.750 21 ASSPFLLFL 0.750 196 IQELQEILV 0.675 65 LNFQNDFKY
0.625 48 MVVLLTMVF 0.500 28 FLDLRPERT 0.500 148 QINLHVSKY 0.500 56
FLSPQLFES 0.500 122 HLFSWSLSF 0.500 231 SVGMYKMDF 0.500 224
ILLPVSFSV 0.500 183 QIMLFSSVY 0.500 20 SASSPFLLF 0.500 199
LQEILVPSQ 0.270 131 PVSSSLIFY 0.250 236 KMDFIISTS 0.250 116
STIFTFHLF 0.250 61 LFESLNFQN 0.225 105 SISWLVRFK 0.200 64 SLNFQNDFK
0.200 128 LSFPVSSSL 0.150 63 ESLNFQNDF 0.150 90 TTCLLGMLQ 0.125 68
QNDFKYEAS 0.125 103 SPSISWLVR 0.125 130 FPVSSSLIF 0.125 32
RPERTYLPV 0.113 54 MVFLSPQLF 0.100 168 GLFFTLSLF 0.100 172
TLSLFRDVF 0.100 174 SLFRDVFLK 0.100 101 NISPSISWL 0.100 158
SLFPINSII 0.100 8 VLASQPTLF 0.100 115 KSTIFTFHL 0.075 132 VSSSLIFYT
0.075 187 FSSVYMMTL 0.075 19 FSASSPFLL 0.075 241 ISTSSTLPW 0.075
143 SSNVTQINL 0.075 147 TQINLHVSK 0.060 40 VCHVALIHM 0.050 42
HVALIHMVV 0.050 117 TIFTFHLFS 0.050 9 LASQPTLFS 0.050 156 YCSLFPINS
0.050 242 STSSTLPWA 0.050 189 SVYMMTLIQ 0.050 88 ICTTCLLGM 0.050 87
SICTTCLLG 0.050 145 NVTQINLHV 0.050 227 PVSFSVGMY 0.050 178
DVFLKQIML 0.050 91 TCLLGMLQV 0.050 45 LIHMVVLLT 0.050 50 VLLTMVFLS
0.050 104 PSISWLVRF 0.030 173 LSLFRDVFL 0.030 133 SSSLIFYTV 0.030
126 WSLSFPVSS 0.030 159 LFPINSIIR 0.025 35 RTYLPVCHV 0.025 52
LTMVFLSPQ 0.025 73 YEASFYLRR 0.025 146 VTQINLHVS 0.025 162
INSIIRGLF 0.025 207 QPQPLPKDL 0.025 176 FRDVFLKQI 0.025 119
FTFHLFSWS 0.025 89 CTTCLLGML 0.025 139 YTVASSNVT 0.025 169
LFFTLSLFR 0.025 171 FTLSLFRDV 0.025 7 LVLASQPTL 0.020 140 TVASSNVTQ
0.020 136 LIFYTVASS 0.020 185 MLFSSVYMM 0.020 43 VALIHMVVL 0.020 93
LLGMLQVVN 0.020 150 NLHVSKYCS 0.020 202 ILVPSQPQP 0.020 98
QVVNISPSI 0.020 37 YLPVCHVAL 0.020 44 ALIHMVVLL 0.020 135 SLIFYTVAS
0.020 49 VVLLTMVFL 0.020 198 ELQEILVPS 0.020 188 SSVYMMTLI 0.015 86
LSICTTCLL 0.015 142 ASSNVTQIN 0.015 102 ISPSISWLV 0.015 57
LSPQLFESL 0.015 157 CSLFPINSI 0.015 V2-A1-9mers: 251P5G2 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. 3
ASQPTLCSF 1.500 2 LASQPTLCS 0.050 8 LCSFFSASS 0.020 4 SQPTLCSFF
0.015 5 QPTLCSFFS 0.013 6 PTLCSFFSA 0.013 7 TLCSFFSAS 0.010 1
VLASQPTLC 0.010 9 CSFFSASSP 0.002 V3-A1-9mers: 251P5G2 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. 7 IRDLSICTT 0.025 9
DLSICTTCL 0.010 6 VIRDLSICT 0.005 4 RRVIRDLSI 0.003 5 RVIRDLSIC
0.001 8 RDLSICTTC 0.001 2 YLRRVIRDL 0.000 3 LRRVIRDLS 0.000 1
FYLRRVIRD 0.000 V4-A1-9mers: 251P5G2 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. 7 LLDMLQVVN 1.000 4 TTCLLDMLQ 0.125
5 TCLLDMLQV 0.050 2 ICTTCLLDM 0.050 3 CTTCLLDML 0.025 6 CLLDMLQVV
0.010 9 DMLQVVNIS 0.005 1 SICTTCLLD 0.005 8 LDMLQVVNI 0.001
V12A-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25;
each start position is specified, the length of peptide is 9 amino
acids, and the end position for each peptide is the start position
plus eight. 8 LIMLFSSVY 0.500 4 SISWLIMLF 0.500 1 ISPSISWLI 0.015 2
SPSISWLIM 0.013 7 WLIMLFSSV 0.010 3 PSISWLIML 0.008 5 ISWLIMLFS
0.008 6 SWLIMLFSS 0.003 V12B-A1-9mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. 31 RADPVTWRK 100.000 404
FMEPRYHVR 45.000 758 NSELSLSHK 27.000 439 DTDMNKRDK 25.000 524
YNEDKLMAK 22.500 629 ICELLSDYK 18.000 803 ILEEIESVK 18.000 361
LSEGYGHSF 13.500 538 GADIESKNK 10.000 231 PAEPPAHQR 9.000 461
NSEVVQLLL 6.750 709 FPDTENEEY 6.250 179 GCPPSRNSY 5.000 711
DTENEEYHS 4.500 326 IKEFEELVK 4.500 211 RSSSCALRY 3.750 55
LSFPGTAAR 3.000 905 AQEQGAALR 2.700 865 AQEQEVAGF 2.700 645
SSENSNPVI 2.700 924 LSEGGTAAG 2.700 916 IGDPGGVPL 2.500 845
QASVQQLCY 2.500 1031 DKDDLPFFK 2.500 885 QASVQQLCY 2.500 516
NTALHYAIY 2.500 860 KTEQQAQEQ 2.250 38 RKEPAVLPC 2.250 900
KTEQQAQEQ 2.250 964 AALPPPTGK 2.000 5 ILLPTQATF 2.000 1010
DVSPAMRLK 2.000 197 GLEAASANL 1.800 122 RLEVPRPQA 1.800 738
SQDEILTNK 1.500 401 DSAFMEPRY 1.500 729 LSEEQNTGI 1.350 100
ESEQSATPA 1.350 430 RKDLIVMLR 1.250 413 REDLDKLHR 1.250 1070
HTDTPPHRN 1.250 396 WGDYDDSAF 1.250 819 QLNEEALTK 1.000 43
VLPCCNLEK 1.000 341 KVIQCVFAK 1.000 694 GLIPQRKSR 1.000 540
DIESKNKCG 0.900 719 SDEQNDTQK 0.900 663 KVEEEIKKH 0.900 1022
NRETHQAFR 0.900 786 RLELDETKH 0.900 501 LLEHGADGN 0.900 821
NEEALTKTK 0.900 749 QIEVAEKEM 0.900 806 EIESVKEKL 0.900 600
LLEQNVDVS 0.900 307 GLELPATAA 0.900 1018 KSDSNRETH 0.750 612
LSGQTAREY 0.750 281 LSYPLVLRH 0.750 718 HSDEQNDTQ 0.750 996
FSSGSFLGR 0.750 633 LSDYKEKQM 0.750 867 EQEVAGFSL 0.675 560
KQEVVKFLI 0.675 690 NGDDGLIPQ 0.625 82 RALPGSLPA 0.500 23 LTTVSNPSR
0.500 691 GDDGLIPQR 0.500 1108 GVGPTTLGS 0.500 548 GLTPLLLGV 0.500
139 SRDPSPPCH 0.500 1094 DTTTSLPHF 0.500 1078 NADTPPHRH 0.500 604
NVDVSSQDL 0.500 983 VCDSSGWIL 0.500
[1020]
19TABLE IX Pos 1234567890 Score V1-A1-10mers: 251P5G2 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. 28 FLDLRPERTY 25.000 158
SLFPINSIIR 5.000 64 SLNFQNDFKY 2.500 68 QNDFKYEASF 2.500 72
KYEASFYLRR 2.250 173 LSLFRDVFLK 1.500 10 ASQPTLFSFF 1.500 242
STSSTLPWAY 1.250 146 VTQINLHVSK 1.000 230 FSVGMYKMDF 0.750 19
FSASSPFLLF 0.750 102 ISPSISWLVR 0.750 130 FPVSSSLIFY 0.625 168
GLFFTLSLFR 0.500 178 DVFLKQIMLF 0.500 9 LASQPTLFSF 0.500 244
SSTLPWAYDR 0.300 75 ASFYLRRVIR 0.300 63 ESLNFQNDFK 0.300 171
FTLSLFRDVF 0.250 245 STLPWAYDRG 0.250 236 KMDFIISTSS 0.250 204
VPSQPQPLPK 0.250 47 HMVVLLTMVF 0.250 26 LLFLDLRPER 0.200 128
LSFPVSSSLI 0.150 115 KSTIFTFHLF 0.150 16 FSFFSASSPF 0.150 90
TTCLLGMLQV 0.125 116 STIFTFHLFS 0.125 162 INSIIRGLFF 0.125 89
CTTCLLGMLQ 0.125 58 SPQLFESLNF 0.125 226 LPVSFSVGMY 0.125 56
FLSPQLFESL 0.100 224 ILLPVSFSVG 0.100 7 LVLASQPTLF 0.100 202
ILVPSQPQPL 0.100 101 NISPSISWLV 0.100 227 PVSFSVGMYK 0.100 219
KSHQHILLPV 0.075 188 SSVYMMTLIQ 0.075 163 NSIIRGLFFT 0.075 22
SSPFLLFLDL 0.075 21 ASSPFLLFLD 0.075 142 ASSNVTQINL 0.075 182
KQIMLFSSVY 0.075 147 TQINLHVSKY 0.075 86 LSICTTCLLG 0.075 196
IQELQEILVP 0.068 53 TMVFLSPQLF 0.050 185 MLFSSVYMMT 0.050 87
SICTTCLLGM 0.050 49 VVLLTMVFLS 0.050 105 SISWLVRFKW 0.050 60
QLFESLNFQN 0.050 117 TIFTFHLFSW 0.050 8 VLASQPTLFS 0.050 240
IISTSSTLPW 0.050 45 LIHMVVLLTM 0.050 52 LTMVFLSPQL 0.050 103
SPSISWLVRF 0.050 44 ALIHMVVLLT 0.050 99 VVNISPSISW 0.050 195
LIQELQEILV 0.050 139 YTVASSNVTQ 0.050 223 HILLPVSFSV 0.050 20
SASSPFLLFL 0.050 32 RPERTYLPVC 0.045 104 PSISWLVRFK 0.030 106
ISWLVRFKWK 0.030 221 HQHILLPVSF 0.030 241 ISTSSTLPWA 0.030 132
VSSSLIFYTV 0.030 228 VSFSVGMYKM 0.030 134 SSLIFYTVAS 0.030 167
RGLFFTLSLF 0.025 23 SPFLLFLDLR 0.025 129 SFPVSSSLIF 0.025 209
QPLPKDLCRG 0.025 207 QPQPLPKDLC 0.025 193 MTLIQELQEI 0.025 119
FTFHLFSWSL 0.025 35 RTYLPVCHVA 0.025 176 FRDVFLKQIM 0.025 121
FHLFSWSLSF 0.025 14 TLFSFFSASS 0.020 42 HVALIHMVVL 0.020 198
ELQEILVPSQ 0.020 6 KLVLASQPTL 0.020 93 LLGMLQVVNI 0.020 135
SLIFYTVASS 0.020 225 LLPVSFSVGM 0.020 43 VALIHMVVLL 0.020 92
CLLGMLQVVN 0.020 48 MVVLLTMVFL 0.020 37 YLPVCHVALI 0.020 183
QIMLFSSVYM 0.020 172 TLSLFRDVFL 0.020 57 LSPQLFESLN 0.015
V2-A1-10mers: 251P5G2 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. 4 ASQPTLCSFF 1.500 10 CSFFSASSPF 0.150 3 LASQPTLCSF
0.100 2 VLASQPTLCS 0.050 8 TLCSFFSASS 0.020 6 QPTLCSFFSA 0.013 1
LVLASQPTLC 0.010 5 SQPTLCSFFS 0.007 7 PTLCSFFSAS 0.003 9 LCSFFSASSP
0.001 V3-A1-10mers: 251P5G2 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. 8 IRDLSICTTC 0.025 10 DLSICTTCLL 0.010 6
RVIRDLSICT 0.005 7 VIRDLSICTT 0.001 9 RDLSICTTCL 0.001 5 RRVIRDLSIC
0.001 4 LRRVIRDLSI 0.000 1 SFYLRRVIRD 0.000 3 YLRRVIRDLS 0.000 2
FYLRRVIRDL 0.000 V4-A1-10mers: 251P5G2 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. 8 LLDMLQVVNI 1.000 5 TTCLLDMLQV 0.125
4 CTTCLLDMLQ 0.125 2 SICTTCLLDM 0.050 7 CLLDMLQVVN 0.020 3
ICTTCLLDML 0.010 6 TCLLDMLQVV 0.010 1 LSICTTCLLD 0.007 10
DMLQVVNISP 0.003 9 LDMLQVVNIS 0.001 V12A-A1-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 8
WLIMLFSSVY 0.500 1 NISPSISWLI 0.100 4 PSISWLIMLF 0.075 2 ISPSISWLIM
0.075 5 SISWLIMLFS 0.050 9 LIMLFSSVYM 0.020 3 SPSISWLIML 0.013 6
ISWLIMLFSS 0.008 7 SWLIMLFSSV 0.001 V12B-A1-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 405
FMEPRYHVRR 90.000 759 NSELSLSHKK 27.000 506 GADGNIQDEY 25.000 308
GLELPATAAR 18.000 719 HSDEQNDTQK 15.000 561 KQEVVKFLIK 13.500 43
AVLPCCNLEK 10.000 56 LSFPGTAARK 6.000 821 LNEEALTKTK 4.500 291
IPEILKFSEK 4.500 49 NLEKGSWLSF 4.500 101 ESEQSATPAG 2.700 646
SSENSNPVIT 2.700 529 KLMAKALLLY 2.500 691 NGDDGLIPQR 2.500 917
IGDPGGVPLS 2.500 275 LIQCIPNLSY 2.500 413 RREDLDKLHR 2.250 901
KTEQQAQEQG 2.250 695 GLIPQRKSRK 2.000 83 RALPGSLPAF 2.000 601
LLEQNVDVSS 1.800 557 VHEQKQEVVK 1.800 776 NSMLREEIAK 1.500 362
LSEGYGHSFL 1.350 868 EQEVAGFSLR 1.350 730 LSEEQNTGIS 1.350 1071
HTDTPPHRNA 1.250 357 WDDFCLSEGY 1.250 440 DTDMNKRDKQ 1.250 179
AGCPPSRNSY 1.250 180 GCPPSRNSYR 1.000 787 RLELDETKHQ 0.900 502
LLEHGADGNI 0.900 805 LEEIESVKEK 0.900 740 QDEILTNKQK 0.900 664
KVEEEIKKHG 0.900 807 EIESVKEKLL 0.900 232 PAEPPAHQRL 0.900 39
RKEPAVLPCC 0.900 750 QIEVAEKEMN 0.900 541 DIESKNKCGL 0.900 123
RLEVPRPQAA 0.900 391 KSNVGTWGDY 0.750 948 ASPGTPSLVR 0.750 610
SQDLSGQTAR 0.750 649 NSNPVITILN 0.750 634 LSDYKEKQML 0.750 1019
KSDSNRETHQ 0.750 462 NSEVVQLLLD 0.675 906 AQEQGAALRS 0.675 493
CQEDECVLML 0.675 628 HVICELLSDY 0.500 990 WILPVPTFSS 0.500 105
SATPAGAFLL 0.500 605 NVDVSSQDLS 0.500 32 RADPVTWRKE 0.500 789
ELDETKHQNQ 0.500 572 KANLNALDRY 0.500 1011 DVSPAMRLKS 0.500 1040
KTQQSPRHTK 0.500 612 DLSGQTAREY 0.500 88 SLPAFADLPR 0.500 692
GDDGLIPQRK 0.500 478 VLDNKKRTAL 0.500 1073 DTPPHRNADT 0.500 1079
NADTPPHRHT 0.500 539 GADIESKNKC 0.500 704 KPENQQFPDT 0.450 525
YNEDKLMAKA 0.450 342 KVIQCVFAKK 0.400 655 TILNIKLPLK 0.400 845
AQASVQQLCY 0.375 885 AQASVQQLCY 0.375 1098 TSLPHFH 0.300 673
GSNPVGLPEN 0.300 738 ISQDEILTNK 0.300 258 PSEEALGVGS 0.270 925
LSEGGTAAGD 0.270 397 WGDYDDSAFM 0.250 967 LPPPTGKNGR 0.250 1089
TTLPHRDTTT 0.250 1081 DTPPHRHTTT 0.250 984 VCDSSGWILP 0.250 516
GNTALHYAIY 0.250 142 DPSPPCHQRR 0.250 938 GTHLPPREPR 0.250 400
YDDSAFMEPR 0.250 353 NVDKWDDFCL 0.250 140 SRDPSPPCHQ 0.250 469
LLDRRCQLNV 0.250 712 DTENEEYHSD 0.225 679 LPENLTNGAS 0.225 299
EKETGGGILG 0.225 329 EFEELVKLHS 0.225 373 MKETSTKISG 0.225 861
KTEQQAQEQE 0.225 779 LREEIAKLRL 0.225 858 HTEKTEQQAQ 0.225 731
SEEQNTGISQ 0.225
[1021]
20TABLE X Pos 123456789 Score V1-A2-9mers: 251P5G2 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. 224 ILLPVSFSV 2537.396
191 YMMTLIQEL 603.960 92 CLLGMLQVV 242.674 49 VVLLTMVFL 148.730 184
IMLFSSVYM 124.127 124 FSWSLSFPV 100.540 37 YLPVCHVAL 98.267 164
SIIRGLFFT 75.179 185 MLFSSVYMM 71.872 233 GMYKMDFII 57.337 71
FKYEASFYL 57.122 44 ALIHMVVLL 49.134 182 KQIMLFSSV 46.894 101
NISPSISWL 37.157 85 VLSICTTCL 36.316 67 FQNDFKYEA 36.099 6
KLVLASQPT 26.082 194 TLIQELQEI 23.995 158 SLFPINSII 15.827 53
TMVFLSPQL 15.428 239 FIISTSSTL 13.512 7 LVLASQPTL 11.757 115
KSTIFTFHL 10.757 108 WLVRFKWKS 9.770 35 RTYLPVCHV 7.110 28
FLDLRPERT 6.719 21 ASSPFLLFL 6.703 235 YKMDFIIST 6.312 50 VLLTMVFLS
6.253 171 FTLSLFRDV 6.248 145 NVTQINLHV 6.086 132 VSSSLIFYT 6.067
102 ISPSISWLV 5.789 173 LSLFRDVFL 4.824 56 FLSPQLFES 4.573 165
IIRGLFFTL 4.182 195 LIQELQEIL 4.113 45 LIHMVVLLT 4.006 47 HMVVLLTMV
3.928 91 TCLLGMLQV 3.864 19 FSASSPFLL 3.720 203 LVPSQPQPL 3.178 60
QLFESLNFQ 2.860 232 VGMYKMDFI 2.655 112 FKWKSTIFT 2.173 14
TLFSFFSAS 1.991 167 RGLFFTLSL 1.961 43 VALIHMVVL 1.760 84 RVLSICTTC
1.608 187 FSSVYMMTL 1.475 78 YLRRVIRVL 1.409 247 LPWAYDRGV 1.281
149 INLHVSKYC 1.122 94 LGMLQVVNI 0.985 23 SPFLLFLDL 0.980 98
QVVNISPSI 0.913 128 LSFPVSSSL 0.877 242 STSSTLPWA 0.873 133
SSSLIFYTV 0.863 117 TIFTFHLFS 0.792 196 IQELQEILV 0.767 174
SLFRDVFLK 0.736 30 DLRPERTYL 0.670 168 GLFFTLSLF 0.634 154
SKYCSLFPI 0.619 157 CSLFPINSI 0.580 141 VASSNVTQI 0.567 178
DVFLKQIML 0.519 120 TFHLFSWSL 0.423 81 RVIRVLSIC 0.410 77 FYLRRVIRV
0.378 18 FFSASSPFL 0.375 1 MPFISKLVL 0.360 134 SSLIFYTVA 0.280 38
LPVCHVALI 0.266 25 FLLFLDLRP 0.254 86 LSICTTCLL 0.237 57 LSPQLFESL
0.221 226 LPVSFSVGM 0.209 139 YTVASSNVT 0.195 95 GMLQVVNIS 0.188
119 FTFHLFSWS 0.184 236 KMDFIISTS 0.173 127 SLSFPVSSS 0.171 188
SSVYMMTLI 0.157 180 FLKQIMLFS 0.152 136 LIFYTVASS 0.148 109
LVRFKWKST 0.143 207 QPQPLPKDL 0.139 143 SSNVTQINL 0.139 82
VIRVLSICT 0.132 88 ICTTCLLGM 0.127 40 VCHVALIHM 0.127 8 VLASQPTLF
0.127 220 SHQHILLPV 0.111 41 CHVALIHMV 0.111 3 FISKLVLAS 0.108 225
LLPVSFSVG 0.099 89 CTTCLLGML 0.089 42 HVALIHMVV 0.085 V2-A2-9mers:
251P5G2 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.
1 VLASQPTLC 8.446 7 TLCSFFSAS 0.538 6 PTLCSFFSA 0.062 4 SQPTLCSFF
0.042 5 QPTLCSFFS 0.016 8 LCSFFSASS 0.003 2 LASQPTLCS 0.002 3
ASQPTLCSF 0.001 9 CSFFSASSP 0.000 V3-A2-9mers: 251P5G2 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. 2 YLRRVIRDL 3.435 9
DLSICTTCL 1.602 6 VIRDLSICT 0.543 5 RVIRDLSIC 0.410 8 RDLSICTTC
0.026 7 IRDLSICTT 0.002 4 RRVIRDLSI 0.001 1 FYLRRVIRD 0.000 3
LRRVIRDLS 0.000 V4-A2-9mers: 251P5G2 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. 6 CLLDMLQVV 994.963 5 TCLLDMLQV
3.864 3 CTTCLLDML 0.334 8 LDMLQVVNI 0.210 2 ICTTCLLDM 0.127 7
LLDMLQVVN 0.021 9 DMLQVVNIS 0.014 1 SICTTCLLD 0.002 4 TTCLLDMLQ
0.000 V12A-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start
position plus eight. 7 WLIMLFSSV 607.884 1 ISPSISWLI 0.868 8
LIMLFSSVY 0.100 5 ISWLIMLFS 0.087 4 SISWLIMLF 0.024 2 SPSISWLIM
0.023 3 PSISWLIML 0.007 6 SWLIMLFSS 0.001 V12B-A2-9mers: 251P5G2
Each peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 599
LLLEQNVDV 1793.677 18 GLWAALTTV 1327.748 802 KILEEIESV 572.255 467
LLLDRRCQL 550.915 242 FLPRAPQAV 319.939 528 KLMAKALLL 276.643 334
KLHSLSHKV 243.432 548 GLTPLLLGV 159.970 566 FLIKKKANL 98.267 111
FLLGWERVV 97.070 266 SLSVFQLHL 81.177 261 ALGVGSLSV 69.552 159
GLTRAFQVV 54.181 327 KEFEELVKL 50.726 6 LLPTQATFA 46.451 270
FQLHLIQCI 41.407 533 ALLLYGADI 38.601 369 FLIMKETST 34.279 498
VLMLLEHGA 31.249 305 ILGLELPAT 29.137 777 MLREEIAKL 26.027 555
GVHEQKQEV 24.952 54 WLSFPGTAA 22.853 345 CVFAKKKNV 22.517 1098
SLPHFHVSA 18.878 742 ILTNKQKQI 17.736 35 VTWRKEPAV 13.630 285
LVLRHIPEI 13.206 850 QLCYKWNHT 12.668 47 CNLEKGSWL 11.635 337
SLSHKVIQC 11.426 654 TILNIKLPL 10.868 418 KLHRAAWWG 10.759 476
NVLDNKKRT 9.892 727 KQLSEEQNT 9.784 158 QGLTRAFQV 9.743 427
KVPRKDLIV 8.733 164 FQVVHLAPT 7.994 1089 TLPHRDTTT 7.452 614
GQTAREYAV 7.052 117 RVVQRRLEV 6.086 770 DLLRENSML 5.928 490
VQCQEDECV 5.874 1096 TTSLPHFHV 5.603 378 KISGLIQEM 5.499 755
KEMNSELSL 5.379 652 VITILNIKL 4.993 953 SLVRLASGA 4.968 75
ALSLSSSRA 4.968 818 IQLNEEALT 4.752 620 YAVSSHHHV 4.444 385
EMGSGKSNV 3.767 324 MQIKEFEEL 3.428 796 NQLRENKIL 3.286 644
ISSENSNPV 3.165 1050 LGQDDRAGV 3.165 264 VGSLSVFQL 3.162 277
CIPNLSYPL 2.937 9 TQATFAAAT 2.871 597 VNLLLEQNV 2.856 434 IVMLRDTDM
2.734 982 SVCDSSGWI 2.676 325 QIKEFEELV 2.555 304 GILGLELPA 2.527
362 SEGYGHSFL 2.285 784 KLRLELDET 2.234 813 KLLKTIQLN 2.220 522
AIYNEDKLM 2.186 316 RLSGLNSIM 2.037 500 MLLEHGADG 1.922 514
YGNTALHYA 1.887 836 RQLGLAQHA 1.864 876 RQLGLAQHA 1.864 256
QPSEEALGV 1.861 371 IMKETSTKI 1.838 338 LSHKVIQCV 1.775 13
FAAATGLWA 1.746 105 ATPAGAFLL 1.721 273 HLIQCIPNL 1.671 890
QLCYKWGHT 1.647 840 LAQHAQASV 1.642 880 LAQHAQASV 1.642 104
SATPAGAFL 1.632 309 ELPATAARL 1.602 493 QEDECVLML 1.567 559
QKQEVVKFL 1.539 1113 TLGSNREIT 1.497 817 TIQLNEEAL 1.439 510
IQDEYGNTA 1.404 560 KQEVVKFLI 1.374 197 GLEAASANL 1.367 460
GNSEVVQLL 1.315 904 QAQEQGAAL 1.216 1007 PMFDVSPAM 1.197 14
AAATGLWAA 1.190 278 IPNLSYPLV 1.158 586 ILAVCCGSA 1.098 68
TTLTGHSAL 1.098 11 ATFAAATGL 1.098 184 RNSYRLTHV 1.044
[1022]
21TABLE XI Pos 123456789 Score V1-A2-10mers: 251P5G2 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. 119 FTFHLFSWSL 143.920 172
TLSLFRDVFL 117.493 37 YLPVCHVALI 110.379 56 FLSPQLFESL 91.487 6
KLVLASQPTL 74.768 101 NISPSISWLV 71.726 185 MLFSSVYMMT 70.310 195
LIQELQEILV 66.657 108 WLVRFKWKST 58.275 184 IMLFSSVYMM 51.908 93
LLGMLQVVNI 40.792 48 MVVLLTMVFL 40.197 85 VLSICTTCLL 36.316 202
ILVPSQPQPL 36.316 164 SIIRGLFFTL 32.369 225 LLPVSFSVGM 32.093 150
NLHVSKYCSL 32.044 127 SLSFPVSSSL 21.362 44 ALIHMVVLLT 17.140 20
SASSPFLLFL 14.262 246 TLPWAYDRGV 13.910 183 QIMLFSSVYM 13.901 223
HILLPVSFSV 6.978 84 RVLSICTTCL 6.916 60 QLFESLNFQN 6.557 231
SVGMYKMDFI 5.658 43 VALIHMVVLL 4.292 194 TLIQELQEIL 4.292 148
QINLHVSKYC 3.757 219 KSHQHILLPV 3.655 163 NSIIRGLFFT 3.569 114
WKSTIFTFHL 3.008 100 VNISPSISWL 2.999 73 YEASFYLRRV 2.862 45
LIHMVVLLTM 2.671 90 TTCLLGMLQV 2.222 206 SQPQPLPKDL 2.166 140
TVASSNVTQI 2.100 193 MTLIQELQEI 2.096 52 LTMVFLSPQL 1.866 97
LQVVNISPSI 1.798 40 VCHVALIHMV 1.775 91 TCLLGMLQVV 1.584 87
SICTTCLLGM 1.571 132 VSSSLIFYTV 1.466 131 PVSSSLIFYT 1.052 14
TLFSFFSASS 1.048 29 LDLRPERTYL 1.026 232 VGMYKMDFII 1.019 11
SQPTLFSFFS 0.916 187 FSSVYMMTLI 0.721 156 YCSLFPINSI 0.721 46
IHMVVLLTMV 0.699 241 ISTSSTLPWA 0.697 8 VLASQPTLFS 0.697 81
RVIRVLSICT 0.652 49 VVLLTMVFLS 0.547 117 TIFTFHLFSW 0.506 123
LFSWSLSFPV 0.476 1 MPFISKLVLA 0.469 228 VSFSVGMYKM 0.469 144
SNVTQINLHV 0.454 64 SLNFQNDFKY 0.432 128 LSFPVSSSLI 0.428 18
FFSASSPFLL 0.396 224 ILLPVSFSVG 0.365 12 QPTLFSFFSA 0.357 76
SFYLRRVIRV 0.355 181 LKQIMLFSSV 0.312 82 VIRVLSICTT 0.304 168
GLFFTLSLFR 0.303 17 SFFSASSPFL 0.302 160 FPINSIIRGL 0.295 96
MLQVVNISPS 0.291 22 SSPFLLFLDL 0.265 137 IFYTVASSNV 0.263 51
LLTMVFLSPQ 0.221 50 VLLTMVFLSP 0.178 135 SLIFYTVASS 0.171 180
FLKQIMLFSS 0.160 142 ASSNVTQINL 0.139 233 GMYKMDFIIS 0.134 109
LVRFKWKSTI 0.118 25 FLLFLDLRPE 0.117 92 CLLGMLQVVN 0.113 174
SLFRDVFLKQ 0.105 67 FQNDFKYEAS 0.105 157 CSLFPINSII 0.103 31
LRPERTYLPV 0.101 26 LLFLDLRPER 0.094 35 RTYLPVCHVA 0.091 191
YMMTLIQELQ 0.090 170 FFTLSLFRDV 0.084 133 SSSLIFYTVA 0.076 236
KMDFIISTSS 0.075 88 ICTTCLLGML 0.071 27 LFLDLRPERT 0.065 237
MDFIISTSST 0.065 136 LIFYTVASSN 0.064 42 HVALIHMVVL 0.060
V2-A2-10mers: 251P5G2 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. 1 LVLASQPTLC 2.734 6 QPTLCSFFSA 0.357 8 TLCSFFSASS 0.283
5 SQPTLCSFFS 0.241 2 VLASQPTLCS 0.127 3 LASQPTLCSF 0.004 4
ASQPTLCSFF 0.003 10 CSFFSASSPF 0.002 7 PTLCSFFSAS 0.001 9
LCSFFSASSP 0.000 V3-A2-10mers: 251P5G2 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. 10 DLSICTTCLL 1.602 7 VIRDLSICTT
1.248 6 RVIRDLSICT 0.652 9 RDLSICTTCL 0.110 2 FYLRRVIRDL 0.023 3
YLRRVIRDLS 0.013 5 RRVIRDLSIC 0.001 8 IRDLSICTTC 0.000 4 LRRVIRDLSI
0.000 1 SFYLRRVIRD 0.000 V4-A2-10mers: 251P5G2 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. 8 LLDMLQVVNI 16.317 5
TTCLLDMLQV 2.222 6 TCLLDMLQVV 1.584 2 SICTTCLLDM 1.571 7 CLLDMLQVVN
0.463 3 ICTTCLLDML 0.267 10 DMLQVVNISP 0.003 9 LDMLQVVNIS 0.001 4
CTTCLLDMLQ 0.000 1 LSICTTCLLD 0.000 V12A-A2-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 9
LIMLFSSVYM 23.632 1 NISPSISWLI 10.759 8 WLIMLFSSVY 0.534 3
SPSISWLIML 0.321 5 SISWLIMLFS 0.130 6 ISWLIMLFSS 0.092 7 SWLIMLFSSV
0.068 2 ISPSISWLIM 0.038 4 PSISWLIMLF 0.000 V12B-A2-10mers: 251P5G2
Each peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 599
NLLLEQNVDV 257.342 6 ILLPTQATFA 171.868 338 SLSHKVIQCV 159.970 777
SMLREEIAKL 131.296 656 ILNIKLPLKV 118.238 467 QLLLDRRCQL 79.041 820
QLNEEALTKT 70.272 880 GLAQHAQASV 69.552 840 GLAQHAQASV 69.552 7
LLPTQATFAA 48.984 469 LLDRRCQLNV 47.295 729 QLSEEQNTGI 42.774 158
AQGLTRAFQV 40.900 633 LLSDYKEKQM 34.627 644 KISSENSNPV 33.472 241
LLFLPRAPQA 31.249 84 ALPGSLPAFA 27.324 264 GVGSLSVFQL 24.935 324
IMQIKEFEEL 24.419 267 SLSVFQLHLI 23.995 983 SVCDSSGWIL 23.566 325
MQIKEFEELV 22.322 70 TLTGHSALSL 21.362 678 GLPENLTNGA 20.369 660
KLPLKVEEEI 17.892 335 KLHSLSHKVI 14.971 478 VLDNKKRTAL 14.526 278
CIPNLSYPLV 14.345 1059 VLAPKCRPGT 12.668 916 QIGDPGGVPL 12.043 763
SLSHKKEEDL 10.468 597 IVNLLLEQNV 10.346 867 QEQEVAGFSL 9.878 371
LIMKETSTKI 9.023 305 GILGLELPAT 8.720 577 ALDRYGRTAL 8.545 306
ILGLELPATA 8.446 749 KQIEVAEKEM 7.228 511 IQDEYGNTAL 6.039 1096
TTTSLPHFHV 5.603 14 FAAATGLWAA 5.475 217 ALRYRSGPSV 5.286 1050
DLGQDDRAGV 5.216 556 GVHEQKQEVV 5.013 385 QEMGSGKSNV 5.004 878
QLGLAQHAQA 4.968 957 RLASGARAAA 4.968 838 QLGLAQHAQA 4.968 850
QQLCYKWNHT 4.752 353 NVDKWDDFCL 4.337 76 ALSLSSSRAL 4.272 18
TGLWAALTTV 3.864 548 CGLTPLLLGV 3.864 269 SVFQLHLIQC 3.699 403
SAFMEPRYHV 3.574 300 KETGGGILGL 3.344 374 KETSTKISGL 3.344 830
KVAGFSLRQL 3.009 490 AVQCQEDECV 2.982 110 GAFLLGWERV 2.977 510
NIQDEYGNTA 2.801 309 LELPATAARL 2.613 589 AVCCGSASIV 2.495 286
LVLRHIPEIL 2.362 165 FQVVHLAPTA 2.317 529 KLMAKALLLY 2.220 523
AIYNEDKLMA 2.186 904 QQAQEQGAAL 2.166 1042 QQSPRHTKDL 2.166 197
QGLEAASANL 2.115 266 GSLSVFQLHL 1.961 468 LLLDRRCQLN 1.922 533
KALLLYGADI 1.876 105 SATPAGAFLL 1.721 36 VTWRKEPAVL 1.716 744
LTNKQKQIEV 1.642 456 LASANGNSEV 1.642 498 CVLMLLEHGA 1.608 494
QEDECVLMLL 1.567 491 VQCQEDECVL 1.510 428 KVPRKDLIVM 1.435 862
TEQQAQEQEV 1.352 477 NVLDNKKRTA 1.319 159 QGLTRAFQVV 1.309 493
CQEDECVLML 1.307 994 VPTFSSGSFL 1.304 352 KNVDKWDDFC 1.254 363
SEGYGHSFLI 1.177 434 LIVMLRDTDM 1.161 530 LMAKALLLYG 1.157 818
TIQLNEEALT 1.025 990 WILPVPTFSS 1.011 947 RASPGTPSLV 0.966 501
MLLEHGADGN 0.942 600 LLLEQNVDVS 0.888 155 CLRAQGLTRA 0.868 307
LGLELPATAA 0.836 1052 GQDDRAGVLA 0.826 1003 LGRRCPMFDV 0.783 555
LGVHEQKQEV 0.772
[1023]
22TABLE XII Pos 123456789 Score V1-A3-9mers: 251P5G2 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. 174 SLFRDVFLK 900.000 122
HLFSWSLSF 60.000 168 GLFFTLSLF 45.000 233 GMYKMDFII 27.000 64
SLNFQNDFK 20.000 185 MLFSSVYMM 9.000 158 SLFPINSII 6.750 172
TLSLFRDVF 6.000 245 STLPWAYDR 4.050 44 ALIHMVVLL 2.700 224
ILLPVSFSV 2.025 8 VLASQPTLF 2.000 183 QIMLFSSVY 1.800 14 TLFSFFSAS
1.800 228 VSFSVGMYK 1.500 191 YMMTLIQEL 1.350 194 TLIQELQEI 1.350
148 QINLHVSKY 1.200 231 SVGMYKMDF 1.200 54 MVFLSPQLF 1.000 147
TQINLHVSK 0.900 53 TMVFLSPQL 0.900 165 IIRGLFFTL 0.810 95 GMLQVVNIS
0.810 92 CLLGMLQVV 0.675 152 HVSKYCSLF 0.600 37 YLPVCHVAL 0.600 105
SISWLVRFK 0.600 85 VLSICTTCL 0.600 48 MVVLLTMVF 0.600 108 WLVRFKWKS
0.540 50 VLLTMVFLS 0.540 47 HMVVLLTMV 0.450 116 STIFTFHLF 0.450 6
KLVLASQPT 0.450 184 IMLFSSVYM 0.300 56 FLSPQLFES 0.270 236
KMDFIISTS 0.270 30 DLRPERTYL 0.270 60 QLFESLNFQ 0.225 35 RTYLPVCHV
0.225 178 DVFLKQIML 0.180 23 SPFLLFLDL 0.180 20 SASSPFLLF 0.180 180
FLKQIMLFS 0.180 135 SLIFYTVAS 0.180 127 SLSFPVSSS 0.180 51
LLTMVFLSP 0.180 11 SQPTLFSFF 0.180 209 QPLPKDLCR 0.180 78 YLRRVIRVL
0.135 101 NISPSISWL 0.135 49 VVLLTMVFL 0.135 98 QVVNISPSI 0.135 117
TIFTFHLFS 0.120 131 PVSSSLIFY 0.120 65 LNFQNDFKY 0.120 150
NLHVSKYCS 0.120 72 KYEASFYLR 0.108 28 FLDLRPERT 0.100 239 FIISTSSTL
0.090 7 LVLASQPTL 0.090 195 LIQELQEIL 0.090 45 LIHMVVLLT 0.090 115
KSTIFTFHL 0.081 182 KQIMLFSSV 0.081 103 SPSISWLVR 0.080 73
YEASFYLRR 0.072 164 SIIRGLFFT 0.068 81 RVIRVLSIC 0.068 10 ASQPTLFSF
0.068 42 HVALIHMVV 0.060 145 NVTQINLHV 0.060 1 MPFISKLVL 0.060 136
LIFYTVASS 0.060 130 FPVSSSLIF 0.060 225 LLPVSFSVG 0.060 243
TSSTLPWAY 0.060 25 FLLFLDLRP 0.060 96 MLQVVNISP 0.060 203 LVPSQPQPL
0.060 67 FQNDFKYEA 0.054 107 SWLVRFKWK 0.045 84 RVLSICTTC 0.045 202
ILVPSQPQP 0.045 198 ELQEILVPS 0.041 21 ASSPFLLFL 0.041 169
LFFTLSLFR 0.040 227 PVSFSVGMY 0.036 128 LSFPVSSSL 0.034 205
PSQPQPLPK 0.030 119 FTFHLFSWS 0.030 192 MMTLIQELQ 0.030 163
NSIIRGLFF 0.030 69 NDFKYEASF 0.030 113 KWKSTIFTF 0.027 223
HILLPVSFS 0.027 187 FSSVYMMTL 0.027 38 LPVCHVALI 0.027 3 FISKLVLAS
0.024 V2-A3-9mers: 251P5G2 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. 7 TLCSFFSAS 0.360 1 VLASQPTLC 0.200 4
SQPTLCSFF 0.060 3 ASQPTLCSF 0.022 6 PTLCSFFSA 0.013 8 LCSFFSASS
0.001 5 QPTLCSFFS 0.001 2 LASQPTLCS 0.001 9 CSFFSASSP 0.001
V3-A3-9mers: 251P5G2 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. 9 DLSICTTCL 0.180 2 YLRRVIRDL 0.135 5 RVIRDLSIC 0.045 6
VIRDLSICT 0.020 4 RRVIRDLSI 0.002 8 RDLSICTTC 0.000 1 FYLRRVIRD
0.000 7 IRDLSICTT 0.000 3 LRRVIRDLS 0.000 V4-A3-9mers: 251P5G2 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. 6
CLLDMLQVV 0.450 9 DMLQVVNIS 0.081 3 CTTCLLDML 0.045 7 LLDMLQVVN
0.020 5 TCLLDMLQV 0.009 2 ICTTCLLDM 0.006 1 SICTTCLLD 0.004 8
LDMLQVVNI 0.003 4 TTCLLDMLQ 0.002 V12A-A3-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 8
LIMLFSSVY 1.800 7 WLIMLFSSV 0.900 4 SISWLIMLF 0.600 1 ISPSISWLI
0.013 5 ISWLIMLFS 0.005 3 PSISWLIML 0.004 2 SPSISWLIM 0.004 6
SWLIMLFSS 0.000 V12B-A3-9mers: 251P5G2 Each peptide is a portion of
SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 319 GLNSIMQIK 135.000 341 KVIQCVFAK
81.000 552 LLLGVHEQK 67.500 819 QLNEEALTK 60.000 776 SMLREEIAK
60.000 803 ILEEIESVK 45.000 436 MLRDTDMNK 40.000 43 VLPCCNLEK
40.000 655 ILNIKLPLK 30.000 474 QLNVLDNKK 20.000 694 GLIPQRKSR
13.500 771 LLRENSMLR 12.000 529 LMAKALLLY 12.000 342 VIQCVFAKK
9.000 280 NLSYPLVLR 9.000 562 EVVKFLIKK 8.100 154 CLRAQGLTR 8.000
404 FMEPRYHVR 6.000 528 KLMAKALLL 5.400 5 ILLPTQATF 4.500 631
ELLSDYKEK 4.500 83 ALPGSLPAF 4.500 18 GLWAALTTV 4.500 410 HVRREDLDK
4.000 266 SLSVFQLHL 3.600 441 DMNKRDKQK 3.000 382 LIQEMGSGK 3.000
370 LIMKETSTK 3.000 533 ALLLYGADI 2.700 548 GLTPLLLGV 2.700 473
CQLNVLDNK 2.025 738 SQDEILTNK 2.025 695 LIPQRKSRK 2.000 1001
FLGRRCPMF 2.000 661 PLKVEEEIK 2.000 159 GLTRAFQVV 1.800 197
GLEAASANL 1.800 109 GAFLLGWER 1.800 563 VVKFLIKKK 1.500 273
HLIQCIPNL 1.350 31 RADPVTWRK 1.350 777 MLREEIAKL 1.350 337
SLSHKVIQC 1.200 307 GLELPATAA 0.900 467 LLLDRRCQL 0.900 286
VLRHIPEIL 0.900 566 FLIKKKANL 0.900 1040 TQQSPRHTK 0.900 628
VICELLSDY 0.900 371 IMKETSTKI 0.900 343 IQCVFAKKK 0.900 237
HQRLLFLPR 0.720 651 PVITILNIK 0.675 964 AALPPPTGK 0.675 535
LLYGADIES 0.600 1098 SLPHFHVSA 0.600 794 HQNQLRENK 0.600 464
VVQLLLDRR 0.600 334 KLHSLSHKV 0.600 463 EVVQLLLDR 0.540 561
QEVVKFLIK 0.540 599 LLLEQNVDV 0.450 784 KLRLELDET 0.450 423
AWWGKVPRK 0.450 261 ALGVGSLSV 0.400 275 IQCIPNLSY 0.360 939
HLPPREPRA 0.300 825 LTKTKVAGF 0.300 498 VLMLLEHGA 0.300 538
GADIESKNK 0.300 316 RLSGLNSIM 0.300 54 WLSFPGTAA 0.300 742
ILTNKQKQI 0.300 122 RLEVPRPQA 0.300 953 SLVRLASGA 0.300 304
GILGLELPA 0.270 770 DLLRENSML 0.270 827 KTKVAGFSL 0.270 654
TILNIKLPL 0.270 560 KQEVVKFLI 0.243 571 KANLNALDR 0.240 886
ASVQQLCYK 0.225 846 ASVQQLCYK 0.225 802 KILEEIESV 0.203 422
AAWWGKVPR 0.200 516 NTALHYAIY 0.200 75 ALSLSSSRA 0.200 23 LTTVSNPSR
0.200 786 RLELDETKH 0.200 242 FLPRAPQAV 0.200 347 FAKKKNVDK 0.200 6
LLPTQATFA 0.200 629 ICELLSDYK 0.200 365 YGHSFLIMK 0.180 418
KLHRAAWWG 0.180 990 ILPVPTFSS 0.180 309 ELPATAARL 0.180 659
KLPLKVEEE 0.180 935 GPGTHLPPR 0.180 179 GCPPSRNSY 0.180
[1024]
23TABLE XIII Pos 1234567890 Score V1-A3-10mers: 251P5G2 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. 168
GLFFTLSLFR 120.000 158 SLFPINSIIR 60.000 26 LLFLDLRPER 20.000 64
SLNFQNDFKY 12.000 47 HMVVLLTMVF 6.000 233 GMYKMDFIIS 3.600 53
TMVFLSPQLF 3.000 184 IMLFSSVYMM 2.700 56 FLSPQLFESL 2.700 6
KLVLASQPTL 2.700 37 YLPVCHVALI 1.800 93 LLGMLQVVNI 1.800 182
KQIMLFSSVY 1.620 185 MLFSSVYMMT 1.500 44 ALIHMVVLLT 1.350 119
FTFHLFSWSL 1.350 202 ILVPSQPQPL 1.350 173 LSLFRDVFLK 1.350 146
VTQINLHVSK 1.000 178 DVFLKQIMLF 0.900 127 SLSFPVSSSL 0.900 194
TLIQELQEIL 0.900 23 SPFLLFLDLR 0.900 174 SLFRDVFLKQ 0.900 164
SIIRGLFFTL 0.810 106 ISWLVRFKWK 0.750 85 VLSICTTCLL 0.600 242
STSSTLPWAY 0.600 28 FLDLRPERTY 0.600 150 NLHVSKYCSL 0.600 225
LLPVSFSVGM 0.600 172 TLSLFRDVFL 0.600 227 PVSFSVGMYK 0.600 14
TLFSFFSASS 0.600 147 TQINLHVSKY 0.540 117 TIFTFHLFSW 0.450 171
FTLSLFRDVF 0.450 60 QLFESLNFQN 0.450 204 VPSQPQPLPK 0.400 7
LVLASQPTLF 0.300 95 GMLQVVNISP 0.270 50 VLLTMVFLSP 0.270 71
FKYEASFYLR 0.270 210 PLPKDLCRGK 0.200 236 KMDFIISTSS 0.180 135
SLIFYTVASS 0.180 180 FLKQIMLFSS 0.180 244 SSTLPWAYDR 0.180 140
TVASSNVTQI 0.180 109 LVRFKWKSTI 0.180 130 FPVSSSLIFY 0.180 122
HLFSWSLSFP 0.150 223 HILLPVSFSV 0.135 224 ILLPVSFSVG 0.135 230
FSVGMYKMDF 0.135 48 MVVLLTMVFL 0.135 101 NISPSISWLV 0.135 8
VLASQPTLFS 0.120 75 ASFYLRRVIR 0.100 115 KSTIFTFHLF 0.090 9
LASQPTLFSF 0.090 231 SVGMYKMDFI 0.090 19 FSASSPFLLF 0.090 84
RVLSICTTCL 0.090 42 HVALIHMVVL 0.090 103 SPSISWLVRF 0.090 51
LLTMVFLSPQ 0.090 105 SISWLVRFKW 0.090 45 LIHMVVLLTM 0.090 35
RTYLPVCHVA 0.075 108 WLVRFKWKST 0.075 72 KYEASFYLRR 0.072 193
MTLIQELQEI 0.068 195 LIQELQEILV 0.060 96 MLQVVNISPS 0.060 87
SICTTCLLGM 0.060 V2-A3-10mers: 251P5G2 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. 2 VLASQPTLCS 0.120 8 TLCSFFSASS 0.120
10 CSFFSASSPF 0.050 1 LVLASQPTLC 0.030 3 LASQPTLCSF 0.030 6
QPTLCSFFSA 0.018 4 ASQPTLCSFF 0.015 5 SQPTLCSFFS 0.004 7 PTLCSFFSAS
0.003 9 LCSFFSASSP 0.000 V3-A3-10mers: 251P5G2 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. 10 DLSICTTCLL 0.180 3
YLRRVIRDLS 0.060 6 RVIRDLSICT 0.030 7 VIRDLSICTT 0.015 4 LRRVIRDLSI
0.001 9 RDLSICTTCL 0.001 1 SFYLRRVIRD 0.001 5 RRVIRDLSIC 0.000 8
IRDLSICTTC 0.000 2 FYLRRVIRDL 0.000 V4-A3-10mers: 251P5G2 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. 8
LLDMLQVVNI 1.800 2 SICTTCLLDM 0.060 5 TTCLLDMLQV 0.030 7 CLLDMLQVVN
0.030 10 DMLQVVNISP 0.027 3 ICTTCLLDML 0.009 6 TCLLDMLQVV 0.005 4
CTTCLLDMLQ 0.002 1 LSICTTCLLD 0.000 9 LDMLQVVNIS 0.000
V12A-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. 8 WLIMLFSSVY 18.000 1 NISPSISWLI 0.405 3 SPSISWLIML
0.054 9 LIMLFSSVYM 0.030 5 SISWLIMLFS 0.018 4 PSISWLIMLF 0.005 6
ISWLIMLFSS 0.005 2 ISPSISWLIM 0.002 7 SWLIMLFSSV 0.001
V12B-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine 536 LLYGADIESK 225.000 419 KLHRAAWWGK 180.000 695
GLIPQRKSRK 135.000 382 GLIQEMGSGK 90.000 785 KLRLELDETK 60.000 436
VMLRDTDMNK 60.000 287 VLRHIPEILK 60.000 529 KLMAKALLLY 54.000 342
KVIQCVFAKK 40.500 370 FLIMKETSTK 30.000 803 KILEEIESVK 20.250 405
FMEPRYHVRR 18.000 113 LLGWERVVQR 12.000 308 GLELPATAAR 12.000 574
NLNALDRYGR 12.000 561 KQEWKFLIK 10.800 43 AVLPCCNLEK 9.000 361
CLSEGYGHSF 9.000 88 SLPAFADLPR 8.000 825 ALTKTKVAGF 6.000 326
QIKEFEELVK 6.000 437 MLRDTDMNKR 6.000 747 KQKQIEVAEK 5.400 1040
KTQQSPRHTK 4.500 655 TILNIKLPLK 4.500 552 PLLLGVHEQK 4.500 778
MLREEIAKLR 4.500 49 NLEKGSWLSF 4.000 55 WLSFPGTAAR 4.000 662
PLKVEEEIKK 4.000 1015 AMRLKSDSNR 4.000 23 ALTTVSNPSR 4.000 771
DLLRENSMLR 3.600 629 VICELLSDYK 3.000 262 ALGVGSLSVF 3.000 423
AAWWGKVPRK 3.000 333 LVKLHSLSHK 3.000 343 VIQCVFAKKK 3.000 660
KLPLKVEEEI 2.700 1008 PMFDVSPAMR 2.000 475 QLNVLDNKKR 2.000 954
SLVRLASGAR 1.800 324 IMQIKEFEEL 1.800 70 TLTGHSALSL 1.800 828
KTKVAGFSLR 1.800 160 GLTRAFQVVH 1.800 819 IQLNEEALTk 1.800 264
GVGSLSVFQL 1.620 777 SMLREEIAKL 1.350 275 LIQCIPNLSY 1.200 281
NLSYPLVLRH 1.200 442 DMNKRDKQKR 1.200 852 LCYKWNHTEK 1.000 892
LCYKWGHTEK 1.000 241 LLFLPRAPQA 1.000 729 QLSEEQNTGI 0.900 678
GLPENLTNGA 0.900 938 GTHLPPREPR 0.900 628 HVICELLSDY 0.900 335
KLHSLSHKVI 0.900 267 SLSVFQLHLI 0.900 474 CQLNVLDNKK 0.900 467
QLLLDRRCQL 0.900 562 QEVVKFLIKK 0.810 56 LSFPGTAARK 0.750 563
EVVKFLIKKK 0.675 651 NPVITILNIK 0.675 395 GTWGDYDDSA 0.675 840
GLAQHAQASV 0.600 291 IPEILKFSEK 0.600 7 LLPTQATFAA 0.600 478
VLDNKKRTAL 0.600 880 GLAQHAQASV 0.600 19 GLWAALTTVS 0.600 577
ALDRYGRTAL 0.600 763 SLSHKKEEDL 0.600 365 GYGHSFLIMK 0.540 473
RCQLNVLDNK 0.450 6 ILLPTQATFA 0.450 338 SLSHKVIQCV 0.450 549
GLTPLLLGVH 0.405 469 LLDRRCQLNV 0.400 656 ILNIKLPLKV 0.400 119
VVQRRLEVPR 0.400 520 LHYAIYNEDK 0.300 84 ALPGSLPAFA 0.300 875
SLRQLGLAQH 0.300 155 CLRAQGLTRA 0.300 776 NSMLREEIAK 0.300 5
HILLPTQATF 0.300 661 LPLKVEEEIK 0.300 964 AAALPPPTGK 0.300 886
QASVQQLCYK 0.300 599 NLLLEQNVDV 0.300 846 QASVQQLCYK 0.300 835
SLRQLGLAQH 0.300 269 SVFQLHLIQC 0.300 341 HKVIQCVFAK 0.270 506
GADGNIQDEY 0.270 464 EVVQLLLDRR 0.270
[1025]
24TABLE XIV Pos 123456789 Score V1-A11-9mers: 251P5G2 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. 174 SLFRDVFLK 2.400 147
TQINLHVSK 0.900 245 STLPWAYDR 0.600 72 KYEASFYLR 0.480 64 SLNFQNDFK
0.400 169 LFFTLSLFR 0.160 209 QPLPKDLCR 0.120 159 LFPINSIIR 0.080
103 SPSISWLVR 0.080 76 SFYLRRVIR 0.080 228 VSFSVGMYK 0.080 233
GMYKMDFII 0.072 35 RTYLPVCHV 0.060 27 LFLDLRPER 0.060 48 MVVLLTMVF
0.060 231 SVGMYKMDF 0.040 54 MVFLSPQLF 0.040 42 HVALIHMVV 0.040 105
SISWLVRFK 0.040 145 NVTQINLHV 0.040 98 QVVNISPSI 0.030 49 VVLLTMVFL
0.030 7 LVLASQPTL 0.030 182 KQIMLFSSV 0.027 178 DVFLKQIML 0.024 168
GLFFTLSLF 0.024 73 YEASFYLRR 0.024 203 LVPSQPQPL 0.020 152
HVSKYCSLF 0.020 211 LPKDLCRGK 0.020 224 ILLPVSFSV 0.018 122
HLFSWSLSF 0.016 185 MLFSSVYMM 0.016 107 SWLVRFKWK 0.015 116
STIFTFHLF 0.015 165 IIRGLFFTL 0.012 77 FYLRRVIRV 0.012 67 FQNDFKYEA
0.012 196 IQELQEILV 0.012 32 RPERTYLPV 0.012 242 STSSTLPWA 0.010 89
CTTCLLGML 0.010 81 RVIRLVSIC 0.009 84 RVLSICTTC 0.009 23 SPFLLFLDL
0.008 158 SLFPINSII 0.008 189 SVYMMTLIQ 0.008 183 QIMLFSSVY 0.008 1
MPFISKLVL 0.008 191 YMMTLIQEL 0.008 24 PFLLFLDLR 0.006 92 CLLGMLQVV
0.006 44 ALIHMVVLL 0.006 184 IMLFSSVYM 0.006 118 IFTFHLFSW 0.006 36
TYLPVCHVA 0.006 194 TLIQELQEI 0.006 91 TCLLGMLQV 0.006 239
FIISTSSTL 0.006 111 RFKWKSTIF 0.006 11 SQPTLFSFF 0.006 47 HMVVLLTMV
0.006 179 VFLKQIMLF 0.006 130 FPVSSSLIF 0.006 53 TMVFLSPQL 0.006 13
PTLFSFFSA 0.005 205 PSQPQPLPK 0.004 148 QINLHVSKY 0.004 40
VCHVALIHM 0.004 39 PVCHVALIH 0.004 172 TLSLFRDVF 0.004 131
PVSSSLIFY 0.004 229 SFSVGMYKM 0.004 120 TFHLFSWSL 0.004 37
YLPVCHVAL 0.004 88 ICTTCLLGM 0.004 8 VLASQPTLF 0.004 195 LIQELQEIL
0.004 138 FYTVASSNV 0.004 85 VLSICTTCL 0.004 17 SFFSASSPF 0.004 20
SASSPFLLF 0.004 101 NISPSISWL 0.004 226 LPVSFSVGM 0.003 38
LPVCHVALI 0.003 43 VALIHMVVL 0.003 193 MTLIQELQE 0.003 65 LNFQNDFKY
0.002 99 VVNISPSIS 0.002 52 LTMVFLSPQ 0.002 90 TTCLLGMLQ 0.002 119
FTFHLFSWS 0.002 141 VASSNVTQI 0.002 215 LCRGKSHQH 0.002 140
TVASSNVTQ 0.002 18 FFSASSPFL 0.002 227 PVSFSVGMY 0.002 129
SFPVSSSLI 0.002 80 RRVIRVLSI 0.002 59 PQLFESLNF 0.002 V2-A11-9mers:
251P5G2 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.
4 SQPTLCSFF 0.006 6 PTLCSFFSA 0.005 5 QPTLCSFFS 0.001 7 TLCSFFSAS
0.000 1 VLASQPTLC 0.000 2 LASQPTLCS 0.000 8 LCSFFSASS 0.000 3
ASQPTLCSF 0.000 9 CSFFSASSP 0.000 V3-A11-9mers: 251P5G2 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. 5
RVIRDLSIC 0.009 4 RRVIRDLSI 0.002 1 FYLRRVIRD 0.001 9 DLSICTTCL
0.001 6 VIRDLSICT 0.001 2 YLRRVIRDL 0.000 8 RDLSICTTC 0.000 7
IRDLSICTT 0.000 3 LRRVIRDLS 0.000 V4-A11-9mers: 251P5G2 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. 3
CTTCLLDML 0.010 5 TCLLDMLQV 0.006 6 CLLDMLQVV 0.006 2 ICTTCLLDM
0.004 4 TTCLLDMLQ 0.002 1 SICTTCLLD 0.001 7 LLDMLQVVN 0.000 8
LDMLQVVNI 0.000 9 DMLQVVNIS 0.000 V12A-A11-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 8
LIMLFSSVY 0.008 4 SISWLIMLF 0.008 7 WLIMLFSSV 0.006 2 SPSISWLIM
0.004 1 ISPSISWLI 0.000 6 SWLIMLFSS 0.000 5 ISWLIMLFS 0.000 3
PSISWLIML 0.000 V12B-A11-9mers: 251P5G2 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. 341 KVIQCVFAK 27.000 410 HVRREDLDK
4.000 562 EVVKFLIKK 1.800 31 RADPVTWRK 1.200 776 SMLREEIAK 1.200
319 GLNSIMQIK 1.200 563 VVKFLIKKK 1.000 473 CQLNVLDNK 0.900 370
LIMKETSTK 0.800 436 MLRDTDMNK 0.800 819 QLNEEALTK 0.800 43
VLPCCNLEK 0.800 738 SQDEILTNK 0.600 794 HQNQLRENK 0.600 1040
TQQSPRHTK 0.600 552 LLLGVHEQK 0.600 109 GAFLLGWER 0.480 803
ILEEIESVK 0.400 852 CYKWNHTEK 0.400 474 QLNVLDNKK 0.400 464
VVQLLLDRR 0.400 954 LVRLASGAR 0.400 382 LIQEMGSGK 0.400 695
LIPQRKSRK 0.400 342 VIQCVFAKK 0.400 520 HYAIYNEDK 0.400 655
ILNIKLPLK 0.400 892 CYKWGHTEK 0.400 536 LYGADIESK 0.400 463
EVVQLLLDR 0.360 651 PVITILNIK 0.300 343 IQCVFAKKK 0.300 964
AALPPPTGK 0.300 538 GADIESKNK 0.300 237 HQRLLFLPR 0.240 571
KANLNALDR 0.240 635 DYKEKQMLK 0.240 23 LTTVSNPSR 0.200 347
FAKKKNVDK 0.200 56 SFPGTAARK 0.200 629 ICELLSDYK 0.200 117
RVVQRRLEV 0.180 694 GLIPQRKSR 0.180 561 QEVVKFLIK 0.180 154
CLRAQGLTR 0.160 771 LLRENSMLR 0.160 935 GPGTHLPPR 0.120 119
VQRRLEVPR 0.120 1110 GPTTLGSNR 0.120 905 AQEQGAALR 0.120 427
KVPRKDLIV 0.120 827 KTKVAGFSL 0.090 631 ELLSDYKEK 0.090 759
SELSLSHKK 0.090 422 AAWWGKVPR 0.080 404 FMEPRYHVR 0.080 948
SPGTPSLVR 0.080 88 LPAFADLPR 0.080 280 NLSYPLVLR 0.080 524
YNEDKLMAK 0.080 413 REDLDKLHR 0.072 662 LKVEEEIKK 0.060 971
GKNGRSPTK 0.060 1010 DVSPAMRLK 0.060 441 DMNKRDKQK 0.060 74
SALSLSSSR 0.060 555 GVHEQKQEV 0.060 557 HEQKQEVVK 0.060 791
ETKHQNQLR 0.060 560 KQEVVKFLI 0.054 528 KLMAKALLL 0.048 816
KTIQLNEEA 0.045 887 SVQQLCYKW 0.040 423 AWWGKVPRK 0.040 332
LVKLHSLSH 0.040 326 IKEFEELVK 0.040 1008 MFDVSPAMR 0.040 480
NKKRTALIK 0.040 180 CPPSRNSYR 0.040 847 SVQQLCYKW 0.040 287
LRHIPEILK 0.040 1037 FFKTQQSPR 0.040 434 IVMLRDTDM 0.040 419
LHRAAWWGK 0.040 661 PLKVEEEIK 0.040 365 YGHSFLIMK 0.040 614
GQTAREYAV 0.036 447 KQKRTALHL 0.036 304 GILGLELPA 0.036 785
LRLELDETK 0.030 886 ASVQQLCYK 0.030 1024 ETHQAFRDK 0.030 439
DTDMNKRDK 0.030 333 VKLHSLSHK 0.030 846 ASVQQLCYK 0.030 1096
TTSLPHFHV 0.030 105 ATPAGAFLL 0.030 663 KVEEEIKKH 0.030 821
NEEALTKTK 0.030 450 RTALHLASA 0.030
[1026]
25TABLE XV Pos 1234567890 Score V1-A11-10mers: 251P5G2 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. 146 VTQINLHVSK 1.000 168
GLFFTLSLFR 0.960 72 KYEASFYLRR 0.480 204 VPSQPQPLPK 0.400 227
PVSFSVGMYK 0.400 158 SLFPINSIIR 0.320 26 LLFLDLRPER 0.160 173
LSLFRDVFLK 0.090 84 RVLSICTTCL 0.090 23 SPFLLFLDLR 0.080 35
RTYLPVCHVA 0.060 99 VVNISPSISW 0.040 119 FTFHLFSWSL 0.040 7
LVLASQPTLF 0.030 48 MVVLLTMVFL 0.030 182 KQIMLFSSVY 0.027 208
PQPLPKDLCR 0.024 117 TIFTFHLFSW 0.024 178 DVFLKQIMLF 0.024 109
LVRFKWKSTI 0.020 140 TVASSNVTQI 0.020 231 SVGMYKMDFI 0.020 242
STSSTLPWAY 0.020 90 TTCLLGMLQV 0.020 42 HVALIHMVVL 0.020 106
ISWLVRFKWK 0.020 52 LTMVFLSPQL 0.020 164 SIIRGLFFTL 0.018 6
KLVLASQPTL 0.018 81 RVIRVLSICT 0.018 223 HILLPVSFSV 0.018 71
FKYEASFYLR 0.016 171 FTLSLFRDVF 0.015 193 MTLIQELQEI 0.015 47
HMVVLLTMVF 0.012 64 SLNFQNDFKY 0.012 184 IMLFSSVYMM 0.012 105
SISWLVRFKW 0.012 63 ESLNFQNDFK 0.009 49 VVLLTMVFLS 0.009 97
LQVVNISPSI 0.009 147 TQINLHVSKY 0.009 190 VYMMTLIQEL 0.008 87
SICTTCLLGM 0.008 240 IISTSSTLPW 0.008 183 QIMLFSSVYM 0.008 101
NISPSISWLV 0.008 244 SSTLPWAYDR 0.008 54 MVFLSPQLFE 0.008 45
LIHMVVLLTM 0.008 189 SVYMMTLIQE 0.008 75 ASFYLRRVIR 0.008 102
ISPSISWLVR 0.008 195 LIQELQEILV 0.008 76 SFYLRRVIRV 0.008 202
ILVPSQPQPL 0.006 9 LASQPTLFSF 0.006 53 TMVFLSPQLF 0.006 130
FPVSSSLIFY 0.006 36 TYLPVCHVAL 0.006 221 HQHILLPVSF 0.006 194
TLIQELQEIL 0.006 38 LPVCHVALIH 0.006 123 LFSWSLSFPV 0.006 18
FFSASSPFLL 0.006 12 QPTLFSFFSA 0.006 233 GMYKMDFIIS 0.005 210
PLPKDLCRGK 0.004 1 MPFISKLVLA 0.004 66 NFQNDFKYEA 0.004 172
TLSLFRDVFL 0.004 152 HVSKYCSLFP 0.004 129 SPFVSSSLIF 0.004 56
FLSPQLFESL 0.004 93 LLGMLQVVNI 0.004 17 SFFSASSPFL 0.004 37
YLPVCHVALI 0.004 39 PVCHVALIHM 0.004 20 SASSPFLLFL 0.004 85
VLSICTTCLL 0.004 127 SLSFPVSSSL 0.004 58 SPQLFESLNF 0.004 225
LLPVSFSVGM 0.004 150 NLHVSKYCSL 0.004 186 LFSSVYMMTL 0.004 137
IFYTVASSNV 0.004 95 GMLQVVNISP 0.004 116 STIFTFHLFS 0.003 226
LPVSFSVGMY 0.003 43 VALIHMVVLL 0.003 91 TCLLGMLQVV 0.003 206
SQPQPLPKDL 0.003 98 QVVNISPSIS 0.003 60 QLFESLNFQN 0.002 155
KYCSLFPINS 0.002 156 YCSLFPINSI 0.002 88 ICTTCLLGML 0.002 40
VCHVALIHMV 0.002 215 LCRGKSHQHI 0.002 203 LVPSQPQPLP 0.002
V2-A11-10mers: 251P5G2 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. 6 QPTLCSFFSA 0.006 1 LVLASQPTLC 0.003 3 LASQPTLCSF 0.002
5 SQPTLCSFFS 0.002 2 VLASQPTLCS 0.001 10 CSFFSASSPF 0.000 8
TLCSFFSASS 0.000 9 LCSFFSASSP 0.000 4 ASQPTLCSFF 0.000 7 PTLCSFFSAS
0.000 V3-A11-10mers: 251P5G2 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. 6 RVIRDLSICT 0.018 10 DLSICTTCLL 0.001 9
RDLSICTTCL 0.001 1 SFYLRRVIRD 0.001 2 FYLRRVIRDL 0.001 7 VIRDLSICTT
0.000 4 LRRVIRDLSI 0.000 3 YLRRVIRDLS 0.000 5 RRVIRDLSIC 0.000 8
IRDLSICTTC 0.000 V4-A11-10mers: 251P5G2 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. 5 TTCLLDMLQV 0.020 2 SICTTCLLDM 0.008
8 LLDMLQVVNI 0.004 6 TCLLDMLQVV 0.003 3 ICTTCLLDML 0.002 4
CTTCLLDMLQ 0.002 7 CLLDMLQVVN 0.001 10 DMLQVVNISP 0.000 1
LSICTTCLLD 0.000 9 LDMLQVVNIS 0.000 V12A-A11-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 9
LIMLFSSVYM 0.008 1 NISPSISWLI 0.008 8 WLIMLFSSVY 0.006 3 SPSISWLIML
0.004 5 SISWLIMLFS 0.001 2 ISPSISWLIM 0.000 7 SWLIMLFSSV 0.000 6
ISWLIMLFSS 0.000 4 PSISWLIMLF 0.000 V12B-A11-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 342
KVIQCVFAKK 9.000 43 AVLPCCNLEK 6.000 561 KQEVVKFLIK 3.600 1040
KTQQSPRHTK 3.000 365 GYGHSFLIMK 2.400 419 KLHRAAWWGK 2.400 333
LVKLHSLSHK 2.000 819 IQLNEEALTK 1.800 695 GLIPQRKSRK 1.800 803
KILEEIESVK 1.800 747 KQKQIEVAEK 1.800 382 GLIQEMGSGK 1.800 785
KLRLELDETK 1.200 436 VMLRDTDMNK 1.200 474 CQLNVLDNKK 0.900 287
VLRHIPEILK 0.800 524 IYNEDKLMAK 0.800 536 LLYGADIESK 0.800 326
QIKEFEELVK 0.800 828 KTKVAGFSLR 0.600 655 TILNIKLPLK 0.600 370
FLIMKETSTK 0.600 473 RCQLNVLDNK 0.600 938 GTHLPPREPR 0.600 563
EVVKFLIKKK 0.450 852 LCYKWNHTEK 0.400 119 VVQRRLEVPR 0.400 629
VICELLSDYK 0.400 423 AAWWGKVPRK 0.400 892 LCYKWGHTEK 0.400 661
LPLKVEEEIK 0.300 651 NPVITILNIK 0.300 308 GLELPATAAR 0.240 964
AAALPPPTGK 0.200 291 IPEILKFSEK 0.200 1047 HTKDLGQDDR 0.200 343
VIQCVFAKKK 0.200 347 VFAKKKNVDK 0.200 886 QASVQQLCYK 0.200 846
QASVQQLCYK 0.200 1031 RDKDDLPFFK 0.180 464 EVVQLLLDRR 0.180 264
GVGSLSVFQL 0.180 562 QEVVKFLIKK 0.180 88 SLPAFADLPR 0.160 574
NLNALDRYGR 0.160 154 ACLRAQGLTR 0.120 610 SQDLSGWTAR 0.120 422
RAAWWGKVPR 0.120 954 SLVRLASGAR 0.120 148 HQRRDAACLR 0.120 180
GCPPSRNSYR 0.120 428 KVPRKDLIVM 0.120 808 IESVKEKLLK 0.120 341
HKVIQCVFAK 0.090 635 SDYKEKQMLK 0.080 405 FMEPRYHVRR 0.080 23
ALTTVSNPSR 0.080 55 WLSFPGTAAR 0.080 404 AFMEPRYHVR 0.080 662
PLKVEEEIKK 0.080 1015 AMRLKSDSNR 0.080 113 LLGWERVVQR 0.080 996
TFSSGSFLGR 0.080 437 MLRDTDMNKR 0.080 776 NSMLREEIAK 0.080 771
DLLRENSMLR 0.072 794 KHQNQLRENK 0.060 973 KNGRSPTKQK 0.060 692
GDDGLIPQRK 0.060 410 YHVRREDLDK 0.060 552 PLLLGVHEQK 0.060 1056
RAGVLAPKCR 0.060 353 NVDKWDDFCL 0.060 395 GTWGDYDDSA 0.060 556
GVHEQKQEVV 0.060
[1027]
26TABLE XVI Pos 123456789 Score V1-A24-9mers: 251P5G2 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. 120 TFHLFSWSL 20.000 111
RFKWKSTIF 20.000 18 FFSASSPFL 20.000 179 VFLKQIMLF 15.000 155
KYCSLFPIN 14.400 36 TYLPVCHVA 12.600 167 RGLFFTLSL 12.000 17
SFFSASSPF 10.000 191 YMMTLIQEL 9.504 207 QPQPLPKDL 8.640 57
LSPQLFESL 8.640 195 LIQELQEIL 8.6401 115 KSTIFTFHL 8.000 217
RGKSHQHIL 8.000 77 FYLRRVIRV 7.500 129 SFPVSSSLI 7.500 203
LVPSQPQPL 7.200 53 TMVFLSPQL 7.200 128 LSFPVSSSL 6.720 37 YLPVCHVAL
6.000 44 ALIHMVVLL 6.000 49 VVLLTMVFL 6.000 86 LSICTTCLL 6.000 173
LSLFRDVFL 6.000 7 LVLASQPTL 6.000 239 FIISTSSTL 6.000 143 SSNVTQINL
6.000 234 MYKMDFIIS 6.000 43 VALIHMVVL 6.000 165 IIRGLFFTL 5.760 23
SPFLLFLDL 5.760 78 YLRRVIRVL 5.600 138 FYTVASSNV 5.000 30 DLRPERTYL
4.800 89 CTTCLLGML 4.800 101 NISPSISWL 4.800 21 ASSPFLLFL 4.800 11
SQPTLFSFF 4.320 113 KWKSTIFTF 4.000 19 FSASSPFLL 4.000 85 VLSICTTCL
4.000 187 FSSVYMMTL 4.000 178 DVFLKQIML 4.000 1 MPFISKLVL 4.000 116
STIFTFHLF 3.600 63 ESLNFQNDF 3.600 10 ASQPTLFSF 3.600 48 MVVLLTMVF
3.600 163 NSIIRGLFF 3.000 130 FPVSSSLIF 3.000 162 INSIIRGLF 2.800
229 SFSVGMYKM 2.750 20 SASSPFLLF 2.400 54 MVFLSPQLF 2.400 98
QVVNISPSI 2.100 168 GLFFTLSLF 2.000 172 TLSLFRDVF 2.000 231
SVGMYKMDF 2.000 8 VLASQPTLF 2.000 122 HLFSWSLSF 2.000 152 HVSKYGSLF
2.000 194 TLIQELQEI 1.980 72 KYEASFYLR 1.800 157 CSLFPINSI 1.800
158 SLFPINSII 1.680 188 SSVYMMTLI 1.500 94 LGMLQVVNI 1.500 232
VGMYKMDFI 1.500 38 LPVCHVALI 1.500 75 ASFYLRRVI 1.200 141 VASSNVTQI
1.000 233 GMYKMDFII 1.000 61 LFESLNFQN 0.900 161 PINSIIRGL 0.840
238 DFIISTSST 0.750 184 IMLFSSVYM 0.750 190 VYMMTLIQE 0.750 226
LPVSFSVGM 0.750 137 IFYTVASSN 0.700 186 LFSSVYMMT 0.700 151
LHVSKYCSL 0.600 70 DFKYEASFY 0.500 185 MLFSSVYMM 0.500 88 ICTTCLLGM
0.500 40 VCHVALIHM 0.500 118 IFTFHLFSW 0.500 15 LFSFFSASS 0.500 71
FKYEASFYL 0.480 182 KQIMLFSSV 0.432 184 RVLSICTTC 0.420 81
RVIRVLSIC 0.420 218 GKSHQHILL 0.400 222 QHILLPVSF 0.360 6 KLVLASQPT
0.360 59 PQLFESLNF 0.300 80 RRVIRVLSI 0.300 32 RPERTYLPV 0.300 104
PSISWLVRF 0.300 236 KMDFIISTS 0.280 95 GMLQVVNIS 0.252
V2-A24-9mers: 251P5G2 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. 3 ASQPTLCSF 3.600 4 SQPTLCSFF 3.600 7 TLCSFFSAS 0.120 8
LCSFFSASS 0.100 1 VLASQPTLC 0.100 5 QPTLCSFFS 0.100 2 LASQPTLCS
0.100 6 PTLCSFFSA 0.018 9 CSFFSASSP 0.010 V3-A24-9mers: 251P5G2
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. 2
YLRRVIRDL 5.600 9 DLSICTTCL 4.000 1 FYLRRVIRD 0.750 5 RVIRDLSIC
0.300 4 RRVIRDLSI 0.300 6 VIRDLSICT 0.144 8 RDLSICTIC 0.042 3
LRRVIRDLS 0.014 7 IRDLSICIT 0.010 V4-A24-9mers: 251P5G2 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. 3
CTTCLLDML 4.800 2 ICTTCLLDM 0.500 9 DMLQVVNIS 0.252 6 CLLDMLQVV
0.216 8 LDMLQVVNI 0.150 5 TCLLDMLQV 0.150 7 LLDMLQVVN 0.120 4
TTCLLDMLQ 0.012 1 SICTTCLLD 0.010 V12A-A24-9mers: 251P5G2 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. 4
SISWLIMLF 2.400 1 ISPSISWLI 2.100 3 PSISWLIML 0.600 2 SPSISWLIM
0.500 7 WLIMLFSSV 0.216 6 SWLIMLFSS 0.150 8 LIMLFSSVY 0.150 5
ISWLIMLFS 0.140 V12B-A24-9mers: 251P5G2 Each peptide is a portion
of SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 579 RYGRTALIL 400.000 408 RYHVRREDL
400.000 282 SYPLVLRHI 105.000 364 GYGHSFLIM 30.000 872 GFSLRQLGL
20.000 832 GFSLRQLGL 20.000 528 KLMAKALLL 12.000 648 NSNPVITIL
10.080 218 RYRSGPSVS 10.000 946 RASPGTPSL 9.600 806 EIESVKEKL 9.240
523 IYNEDKLMA 9.000 86 GSLPAFADL 8.640 654 TILNIKLPL 8.400 461
NSEVVQLLL 8.400 460 GNSEVVQLL 8.064 447 KQKRTALHL 8.000 544
KNKCGLTPL 8.000 827 KTKVAGFSL 8.000 546 KCGLTPLLL 8.000 324
MQIKEFEEL 7,920 491 QCQEDECVL 7.200 883 HAQASVQQL 7.200 904
QAQEQGAAL 7.200 351 KNVDKWDDF 7.200 674 NPVGLPENL 7.200 277
CIPNLSYPL 7.200 843 HAQASVQQL 7.200 467 LLLDRRCQL 7.200 156
RAQGLTRAF 7.200 47 CNLEKGSWL 7.200 721 EQNDTQKQL 7.200 592
GSASIVNLL 6.720 521 YAIYNEDKL 6.600 566 FLIKKKANL 6.000 309
ELPATAARL 6.000 796 NQLRENKIL 6.000 735 TGISQDEIL 6.000 105
ATPAGAFLL 6.000 273 HLIQCIPNL 6.000 68 TTLTGHSAL 6.000 76 LSLSSSRAL
6.000 233 EPPAHQRLL 6.000 867 EQEVAGFSL 6.000 459 NGNSEVVQL 6.000
770 DLLRENSML 6.000 817 TIQLNEEAL 6.000 1042 QSPRHTKDL 6.000 197
GLEAASANL 6.000 286 VLRHIPEIL 5.600 593 SASIVNLLL 5.600 652
VITILNIKL 5.280 781 EIAKLRLEL 5.280 777 MLREEIAKL 5.280 302
GGGILGLEL 5.280 186 SYRLTHVRC 5.000 513 EYGNTALHY 5.000 916
IGDPGGVPL 4.800 80 SSRALPGSL 4.800 1051 GQDDRAGVL 4.800 152
AACLRAQGL 4.800 15 AATGLWAAL 4.800 104 SATPAGAFL 4.800 604
NVDVSSQDL 4.800 560 KQEVVKFLI 4.200 36 TWRKEPAVL 4.000 425
WGKVPRKDL 4.000 1106 AGGVGPTTL 4.000 958 ASGARAAAL 4.000 870
VAGFSLRQL 4.000 687 SAGNGDDGL 4.000 312 ATAARLSGL 4.000 266
SLSVFQLHL 4.000 147 HQRRDAACL 4.000 983 VCDSSGWIL 4.000 830
VAGFSLRQL 4.000 1060 APKCRPGTL 4.000 264 VGSLSVFQL 4.000 11
ATFAAATGL 4.000 763 LSHKKEEDL 4.000 591 CGSASIVNL 4.000 374
ETSTKISGL 4.000 300 ETGGGILGL 4.000 70 LTGHSALSL 4.000 5 ILLPTQATF
3.600 321 NSIMQIKEF 3.300 262 LGVGSLSVF 3.000 361 LSEGYGHSF 3.000
865 AQEQEVAGF 3.000 988 GWILPVPTF 3.000 83 ALPGSLPAF 3.000 1021
SNRETHQAF 2.880 660 LPLKVEEEI 2.310 558 EQKQEVVKF 2.200 270
FQLHLIQCI 2.160 825 LTKTKVAGF 2.000 396 WGDYDDSAF 2.000 1001
FLGRRCPMF 2.000
[1028]
27TABLE XVII Pos 1234567890 Score V1-A-24-10mers: 251P5G2 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. 190
VYMMTLIQEL 475.200 77 FYLRRVIRVL 420.000 36 TYLPVCHVAL 360.000 238
DFIISTSSTL 30.000 17 SFFSASSPFL 20.000 186 LFSSVYMMTL 20.000 18
FFSASSPFLL 20.000 70 DFKYEASFYL 20.000 129 SFPVSSSLIF 15.000 84
RVLSICTTCL 12.00 6 KLVLASQPTL 12.00 155 KYCSLFPINS 10.00 202
ILVPSQPQPL 8.640 22 SSPFLLFLDL 8.640 164 SIIRGLFFTL 8.640 160
FPINSIIRGL 8.400 217 RGKSHQHILL 8.000 206 SQPSPLPKDL 7.200 194
TLIQELQEIL 7.200 52 LTMVFLSPQL 7.200 56 FLSPQLFESL 6.912 43
VALIHMVVLL 6.000 48 MVVLLTMVFL 6.000 167 RGLFFTLSLF 6.000 138
FYTVASSNVT 6.000 175 LFRDVFLKQI 6.000 100 VNISPSISWL 6.000 127
SLSFPVSSSL 5.600 10 ASQPTLFSFF 6.184 234 MYKMDFIIST 5.000 88
ICTTCLLGML 4.800 115 KSTIFTFHLF 4.800 85 VLSICTTCLL 4.000 20
SASSPFLLFL 4.000 172 TLSLFRDVFL 4.000 142 ASSNVTQINL 4.000 42
HVALIHMVVL 4.000 150 NLHVSKYCSL 4.000 119 FTFHLFSWSL 4.000 47
HMVVLLTMVF 3.600 53 TMVFLSPQLF 3.600 58 SPQLFESLNF 3.0001 230
FSVGMYKMDF 3.000 7 LVLASQPTLF 3.000 171 FTLSLFRDVF 3.000 19
FSASSPFLLF 2.400 221 HQHILLPVSF 2.400 97 LQVVNISPSI 2.100 157
CSLFPINSII 2.100 68 QNDFKYEASF 2.000 178 DVFLKQIMLF 2.000 9
LASQPTLFSF 2.000 162 INSIIRGLFF 2.000 103 SPSISWLVRF 2.000 16
FSFFSASSPF 2.000 193 MTLIQELQEI 1.980 37 YLPVCHVALI 1.500 72
KYEASFYLRR 1.500 232 VGMYKMDFII 1.500 177 RDVFLKQIML 1.200 156
YCSLFPINSI 1.200 215 LCRGKSHQHI 1.200 128 LSFPVSSSLI 1.200 74
EASFYLRRVI 1.200 179 VFLKQIMLFS 1.050 93 LLGMLQVVNI 1.000 153
VSKYCSLFPI 1.000 140 TVASSNVTQI 1.000 187 FSSVYMMTLI 1.000 231
SVGMYKMDFI 1.000 111 RFKWKSTIFT 1.000 109 LVRFKWKSTI 1.000 27
LFLDLRPERT 0.900 55 VFLSPQLFES 0.825 66 NFQNDFKYEA 0.825 184
IMLFSSVYMM 0.750 183 QIMLFSSVYM 0.750 225 LLPVSFSVGM 0.750 118
IFTFHLFSWS 0.720 170 FFTLSLFRDV 0.720 45 LIHMVVLLTM 0.700 123
LFSWSLSFPV 0.600 29 LDLRPERTYL 0.600 228 VSFSVGMYKM 0.550 137
IFYTVASSNV 0.500 120 TFHLFSWSLS 0.500 76 SFYLRRVIRV 0.500 87
SICTTCLLGM 0.500 161 PINSIIRGLF 0.420 166 IRGLFFTLSL 0.400 216
CRGKSHQHIL 0.400 114 WKSTIFTFHL 0.400 81 RVIRVLSICT 0.360 182
KQIMLFSSVY 0.300 32 RPRTYLPVC 0.300 151 LHVSKYCSLF 0.300 121
FHLFSWSLSF 0.300 219 KSHQHILLPV 0.280 35 RTYLPVCHVA 0.280 236
KMDFIISTSS 0.280 V2-A24-10mers: 251P5G2 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 4 ASQPTLCSFF 4.320 3 LASQPTLCSF 2.000
10 CSFFSASSPF 2.000 1 LVLASQPTLC 0.150 5 SQPTLCSFFS 0.150 6
QPTLCSFFSA 0.120 8 TLCSFFSASS 0.100 2 VLASQPTLCS 0.100 7 PTLCSFFSAS
0.018 9 LCSFFSASSP 0.010 V3-A24-10mers: 251P5G2 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 2 FYLRRVIRDL 420.000 10
DLSICTTCLL 4.000 9 RDLSICTTCL 1.200 6 RVIRDLSICT 0.360 3 YLRRVIRDLS
0.140 7 VIRDLSICTT 0.120 4 LRRVIRDLSI 0.100 1 SFYLRRVIRD 0.050 5
RRVIRDLSIC 0.030 8 IRDLSICTTC 0.014 V4-A24-10mers: 251P5G2 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. 3
ICTTCLLDML 4.800 8 LLDMLQVVNI 1.000 2 SICTTCLLDM 0.500 7 CLLDMLQVVN
0.216 6 TCLLDMLQVV 0.180 5 TTCLLDMLQV 0.100 9 LDMLQVVNIS 0.025 10
DMLQVVNISP 0.021 1 LSICTTCLLD 0.015 4 CTTCLLDMLQ 0.012
V12A-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO:
25; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. 3 SPSISWLIML 4.000 1 NISPSISWLI 1.680 9
LIMLFSSVYM 0.750 2 ISPSISWLIM 0.750 4 PSISWLIMLF 0.360 7 SWLIMLFSSV
0.216 8 WLIMLFSSVY 0.150 5 SISWLIMLFS 0.140 6 ISWLIMLFSS 0.100
V12B-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO:
25; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. 521 HYAIYNEDKL 220.000 636 DYKEKQMLKI 66.000
566 KFLIKKKANL 60.000 1009 MFDVSPAMRL 20.000 1001 SFLGRRCPMF 15.000
1029 AFRDKDDLPF 12.000 817 KTIQLNEEAL 12.000 270 VFQLHLIQCI 10.800
460 NGNSEVVQLL 10.080 219 RYRSGPSVSS 10.000 580 RYGRTALILA 10.000
545 KNKCGLTPLL 9.600 604 QNVDVSSQDL 8.640 277 QCIPNLSYPL 8.640 286
LVLRHIPEIL 8.400 115 GWERVVQRRL 8.400 654 ITILNIKLPL 8.400 830
KVAGFSLRQL 8.000 324 IMQIKEFEEL 7.920 537 LYGADIESKN 7.700 493
CQEDECVLML 7.200 83 RALPGSLPAF 7.200 674 SNPVGLPENL 7.200 197
QGLEAASANL 7.200 1051 LGQDDRAGVL 7.200 461 GNSEVVQLLL 6.720 592
CGSASIVNLL 6.720 777 SMLREEIAKL 6.600 753 VAEKEMNSEL 6.600 624
SSHHHVICEL 6.160 1082 TPPHRHTTTL 6.000 807 EIESVKEKLL 6.000 717
EYHSDEQNDT 6.000 541 DIESKNKCGL 6.000 1060 LAPKCRPGTL 6.000 362
LSEGYGHSFL 6.000 266 GSLSVFQLHL 6.000 796 QNQLRENKIL 6.000 279
IPNLSYPLVL 6.000 181 CPPSRNSYRL 6.000 467 QLLLDRRCQL 6.000 47
CCNLEKGSWL 6.000 559 EQKQEVVKFL 5.600 648 ENSNPVITIL 5.600 593
GSASIVNLLL 5.600 853 CYKWNHTEKT 5.500 893 CYKWGHTEKT 5.500 302
TGGGILGLEL 5.280 187 SYRLTHVRCA 5.000 514 EYGNTALHYA 5.000 620
EYAVSSHHHV 5.000 429 VPRKDLIVML 4.800 15 AAATGLWAAL 4.800 701
KSRKPENQQF 4.800 80 SSSRALPGSL 4.800 983 SVCDSSGWIL 4.800 916
QIGDPGGVPL 4.800 1042 QQSPRHTKDL 4.800 152 DAACLRAQGL 4.800 634
LSDYKEKQML 4.800 511 IQDEYGNTAL 4.800 105 SATPAGAFLL 4.800 190
LTHVRCAQGL 4.800 660 KLPLKVEEEI 4.620 411 HVRREDLDKL 4.400 904
QQAQEQGAAL 4.000 425 WWGKVPRKDL 4.000 687 ASAGNGDDGL 4.000 491
VQCQEDECVL 4.000 1091 LPHRDTTTSL 4.000 994 VPTFSSGSFL 4.000 932
AGDQGPGTHL 4.000 11 QATFAAATGL 4.000 459 ANGNSEVVQL 4.000 949
SPGTPSLVRL 4.000 763 SLSHKKEEDL 4.000 353 NVDKWDDFCL 4.000 41
EPAVLPCCNL 4.000 764 LSHKKEEDLL 4.000 62 AARKEFSTTL 4.000 161
LTRAFQVVHL 4.000 870 EVAGFSLRQL 4.000 209 APGRSSSCAL 4.000 478
VLDNKKRTAL 4.000 1027 HQAFRDKDDL 4.000 832 AGFSLRQLGL 4.000 1106
SAGGVGPTTL 4.000 70 TLTGHSALSL 4.000 68 STTLTGHSAL 4.000 735
NTGISQDEIL 4.000 958 LASGARAAAL 4.000 36 VTWRKEPAVL 4.000 872
AGFSLRQLGL 4.000 591 CCGSASIVNL 4.000 577 ALDRYGRTAL 4.000 76
ALSLSSSRAL 4.000 104 QSATPAGAFL 4.000 264 GVGSLSVFQL 4.000 865
QAQEQEVAGF 3.600 1021 DSNRETHQAF 3.600
[1029]
28TABLE XVIII Pos 123456789 Score V1-B7-9mers: 251P5G2 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. 207 QPQPLPKDL 80.00 23
SPFLLFLDL 80.00 1 MPFISKLVL 80.00 30 DLRPERTYL 60.00 78 YLRRVIRVL
40.00 165 IIRGLFFTL 40.00 178 DVFLKQIML 20.00 203 LVPSQPQPL 20.00 7
LVLASQPTL 20.00 49 VVLLTMVFL 20.00 226 LPVSFSVGM 20.00 191
YMMTLIQEL 12.00 44 ALIHMVVLL 12.00 21 ASSPFLLFL 12.00 43 VALIHMVVL
12.00 38 LPVCHVALI 8.000 109 LVRFKWKST 5.000 195 LIQELQEIL 4.000 53
TMVFLSPQL 4.000 217 RGKSHQHIL 4.000 57 LSPQLFESL 4.000 86 LSICTTCLL
4.000 173 LSLFRDVFL 4.000 239 FIISTSSTL 4.000 115 KSTIFTFHL 4.000
85 VLSICTTCL 4.000 101 NISPSISWL 4.000 128 LSFPVSSSL 4.000 37
YLPVCHVAL 4.000 247 LPWAYDRGV 4.000 143 SSNVTQINL 4.000 19
FSASSPFLL 4.000 89 CTTCLLGML 4.000 167 RGLFFTLSL 4.000 187
FSSVYMMTL 4.000 98 QVVNISPSI 2.000 75 ASFYLRRVI 1.800 141 VASSNVTQI
1.200 232 VGMYKMDFI 1.200 32 RPERTYLPV 1.200 94 LGMLQVVNI 1.200 185
MLFSSVYMM 1.000 82 VIRVLSICT 1.000 88 ICTTCLLGM 1.000 40 VCHVALIHM
1.000 184 IMLFSSVYM 1.000 145 NVTQINLHV 1.000 42 HVALIHMVV 1.000
157 CSLFPINSI 0.600 74 EASFYLRRV 0.600 84 RVLSICTTC 0.500 81
RVIRVLSIC 0.500 12 QPTLFSFFS 0.400 151 LHVSKYCSL 0.400 18 FFSASSPFL
0.400 161 PINSIIRGL 0.400 58 SPQLFESLN 0.400 233 GMYKMDFII 0.400
120 TFHLFSWSL 0.400 194 TLIQELQEI 0.400 188 SSVYMMTLI 0.400 130
FPVSSSLIF 0.400 71 FKYEASFYL 0.400 158 SLFPINSII 0.400 218
GKSHQHILL 0.400 46 IHMVVLLTM 0.300 204 VPSQPQPLP 0.300 35 RTYLPVCHV
0.300 124 FSWSLSFPV 0.200 182 KQIMLFSSV 0.200 91 TCLLGMLQV 0.200
102 ISPSISWLV 0.200 171 FTLSLFRDV 0.200 133 SSSLIFYTV 0.200 103
SPSISWLVR 0.200 47 HMVVLLTMV 0.200 160 FPINSIIRG 0.200 209
QPLPKDLCR 0.200 211 LPKDLCRGK 0.200 224 ILLPVSFSV 0.200 92
CLLGMLQVV 0.200 231 SVGMYKMDF 0.100 6 KLVLASQPT 0.100 139 YTVASSNVT
0.100 229 SFSVGMYKM 0.100 45 LIHMVVLLT 0.100 99 VVNISPSIS 0.100 215
LCRGKSHQH 0.100 242 STSSTLPWA 0.100 149 INLHVSKYC 0.100 152
HVSKYCSLF 0.100 164 SIIRGLFFT 0.100 177 RDVFLKQIM 0.100 67
FQNDFKYEA 0.100 54 MVFLSPQLF 0.100 132 VSSSLIFYT 0.100 48 MVVLLTMVF
0.100 134 SSLIFYTVA 0.100 9 LASQPTLFS 0.090 20 SASSPFLLF 0.090
V2-B7-9mers: 251P5G2 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. 5 QPTLCSFFS 0.400 1 VLASQPTLC 0.100 2 LASQPTLCS 0.090 3
ASQPTLCSF 0.060 7 TLCSFFSAS 0.020 8 LCSFFSASS 0.020 4 SQPTLCSFF
0.020 6 PTLCSFFSA 0.010 9 CSFFSASSP 0.010 V3-B7-9mers: 251P5G2 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. 2
YLRRVIRDL 40.00 9 DLSICTTCL 4.000 6 VIRDLSICT 1.000 5 RVIRDLSIC
0.500 4 RRVIRDLSI 0.040 3 LRRVIRDLS 0.030 8 RDLSICTTC 0.010 7
IRDLSICTT 0.003 1 FYLRRVIRD 0.001 V4-B7-9mers: 251P5G2 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 3
CTTCLLDML 4.000 2 ICTTCLLDM 1.000 6 CLLDMLQVV 0.200 5 TCLLDMLQV
0.200 8 LDMLQVVNI 0.120 9 DMLQVVNIS 0.020 4 TTCLLDMLQ 0.010 1
SICTTCLLD 0.010 7 LLDMLQVVN 0.006 V12A-B7-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 2
SPSISWLIM 20.000 1 ISPSISWLI 0.400 3 PSISWLIML 0.400 7 WLIMLFSSV
0.200 8 LIMLFSSVY 0.060 4 SISWLIMLF 0.020 5 ISWLIMLFS 0.020 6
SWLIMLFSS 0.002 V12B-B7-9mers: 251P5G2 Each peptide is a portion of
SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 1060 APKCRPGTL 240.000 428 VPRKDLIVM
200.000 233 EPPAHQRLL 80.000 674 NPVGLPENL 80.000 286 VLRHIPEIL
40.0 147 HQRRDAACL 40.000 777 MLREEIAKL 40.000 152 AACLRAQGL 36.000
15 AATGLWAAL 36.000 125 VPRPQAAPA 20.000 434 IVMLRDTDM 15.000 104
SATPAGAFL 12.000 830 VAGFSLRQL 12.000 958 ASGARAAAL 12.000 946
RASPGTPSL 12.000 687 SAGNGDDGL 12.000 312 ATAARLSGL 12.000 521
YAIYNEDKL 12.000 1106 AGGVGPTTL 12.000 883 HAQASVQQL 12.000 11
ATFAAATGL 12.000 870 VAGFSLRQL 12.000 843 HAQASVQQL 12.000 528
KLMAKALLL 12.000 904 QAQEQGAAL 12.000 593 SASIVNLLL 12.000 1027
QAFRDKDDL 12.00 105 ATPAGAFLL 12.000 61 AARKEFSTT 9.000 170
APTAPDGGA 9.000 425 WGKVPRKDL 9.000 1082 PPHRHTTTL 8.000 660
LPLKVEEEI 8.000 181 PPSRNSYRL 8.000 650 NPVITILNI 8.000 208
APGRSSSCA 6.000 621 AVSSHHHVI 6.000 604 NVDVSSQDL 6.000 781
EIAKLRLEL 6.000 1006 CPMFDVSPA 6.000 228 APSPAEPPA 6.000 467
LLLDRRCQL 6.000 588 AVCCGSASI 6.000 577 LDRYGRTAL 6.000 243
LPRAPQAVS 6.000 591 CGSASIVNL 4.000 76 LSLSSSRAL 4.000 817
TIQLNEEAL 4.000 309 ELPATAARL 4.000 544 KNKCGLTPL 4.000 491
QCQEDECVL 4.000 86 GSLPAFADL 4.000 735 TGISQDEIL 4.000 1042
QSPRHTKDL 4.000 592 GSASIVNLL 4.000 278 IPNLSYPLV 4.000 266
SLSVFQLHL 4.000 648 NSNPVITIL 4.000 36 TWRKEPAVL 4.000 460
GNSEVVQLL 4.000 256 QPSEEALGV 4.000 374 ETSTKISGL 4.000 566
FLIKKKANL 4.000 277 CIPNLSYPL 4.000 411 VRREDLDKL 4.000 302
GGGILGLEL 4.000 47 CNLEKGSWL 4.000 796 NQLRENKIL 4.000 264
VGSLSVFQL 4.000 447 KQKRTALHL 4.000 300 ETGGGILGL 4.000 324
MQIKEFEEL 4.000 827 KTKVAGFSL 4.000 68 TTLTGHSAL 4.000 654
TILNIKLPL 4.000 546 KCGLTPLLL 4.000 652 VITILNIKL 4.000 115
WERVVQRRL 4.000 763 LSHKKEEDL 4.000 721 EQNDTQKQL 4.000 770
DLLRENSML 4.000 459 NGNSEVVQL 4.000 209 PGRSSSCAL 4.000 273
HLIQCIPNL 4.000 470 DRRCQLNVL 4.000 70 LTGHSALSL 4.000 522
AIYNEDKLM 3.000 84 LPGSLPAFA 2.000 7 LPTQATFAA 2.000 285 LVLRHIPEI
2.000 944 EPRASPGTP 2.000 1073 TPPHRNADT 2.000 28 NPSRADPVT 2.000
1043 SPRHTKDLG 2.000 976 SPTKQKSVC 2.000 922 VPLSEGGTA 2.000 94
LPRSCPESE 2.000 1081 TPPHRHTTT 2.000 982 SVCDSSGWI 2.000
[1030]
29TABLE XIX Pos 1234567890 Score V1-B7-10mers: 251P5G2 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. 160 FPINSIIRGL 80.000 48
MVVLLTMVFL 20.000 42 HVALIHMVVL 20.000 84 RVLSICTTCL 20.000 109
LVRFKWKSTI 20.000 142 ASSNVTQINL 12.000 20 SASSPFLLFL 12.000 43
VALIHMVVLL 12.000 52 LTMVFLSPQL 12.000 88 ICTTCLLGML 4.000 172
TLSLFRDVFL 4.000 119 FTFHLFSWSL 4.000 127 SLSFPVSSSL 4.000 85
VLSICTTCLL 4.000 22 SSPFLLFLDL 4.000 217 RGKSHQHILL 4.000 202
ILVPSQPQPL 4.000 100 VNISPSISWL 4.000 194 TLIQELQEIL 4.000 206
SQPQPLPKDL 4.000 6 KLVLASQPTL 4.000 150 NLHVSKYCSL 4.000 56
FLSPQLFESL 4.000 164 SIIRGLFFTL 4.000 215 LCRGKSHQHI 4.000 207
QPQPLPKDLC 3.000 183 QIMLFSSVYM 3.000 140 TVASSNVTQI 2.000 1
MPFISKLVLA 2.000 12 QPTLFSFFSA 2.000 231 SVGMYKMDFI 2.000 74
EASFYLRRVI 1.800 190 VYMMTLIQEL 1.200 232 VGMYKMDFII 1.200 184
IMLFSSVYMM 1.000 82 VIRVLSICTT 1.000 225 LLPVSFSVGM 1.000 87
SICTTCLLGM 1.000 45 LIHMVVLLTM 1.000 228 VSFSVGMYKM 1.000 29
LDLRPERTYL 0.600 156 YCSLFPINSI 0.600 32 RPERTYLPVC 0.600 211
LPKDLCRGKS 0.600 39 PVCHVALIHM 0.500 81 RVIRVLSICT 0.500 36
TYLPVCHVAL 0.400 70 DFKYEASFYL 0.400 18 FFSASSPFLL 0.400 97
LQVVNISPSI 0.400 186 LFSSVYMMTL 0.400 128 LSFPVSSSLI 0.400 77
FYLRRVIRVL 0.400 103 SPSISWLVRF 0.400 187 FSSVYMMTLI 0.400 193
MTLIQELQEI 0.400 238 DFIISTSSTL 0.400 175 LFRDVFLKQI 0.400 17
SFFSASSPFL 0.400 79 LRRVIRVLSI 0.400 216 CRGKSHQHIL 0.400 177
RDVFLKQIML 0.400 226 LPVSFSVGMY 0.400 166 IRGLFFTLSL 0.400 37
YLPVCHVALI 0.400 93 LLGMLQVVNI 0.400 153 VSKYCSLFPI 0.400 157
CSLFPINSII 0.400 114 WKSTIFTFHL 0.400 130 FPVSSSLIFY 0.400 58
SPQLFESLNF 0.400 44 ALIHMVVLLT 0.300 78 YLRRVIRVLS 0.300 91
TCLLGMLQVV 0.200 195 LIQELQEILV 0.200 90 TTCLLGMLQV 0.200 101
NISPSISWLV 0.200 219 KSHQHILLPV 0.200 40 VCHVALIHMV 0.200 209
QPLPKDLCRG 0.200 132 VSSSLIFYTV 0.200 144 SNVTQINLHV 0.200 165
IIRGLFFTLS 0.200 38 LPVCHVALIH 0.200 246 TLPWAYDRGV 0.200 204
VPSQPQPLPK 0.200 23 SPFLLFLDLR 0.200 223 HILLPVSFSV 0.200 99
VVNISPSISW 0.150 163 NSIIRGLFFT 0.100 98 QVVNISPSIS 0.100 241
ISTSSTLPWA 0.100 178 DVFLKQIMLF 0.100 35 RTYLPVCHVA 0.100 133
SSSLIFYTVA 0.100 49 VVLLTMVFLS 0.100 148 QINLHVSKYC 0.100 30
DLRPERTYLP 0.100 185 MLFSSVYMMT 0.100 7 LVLASQPTLF 0.100
V2-B7-10mers: 251P5G2 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. 6 QPTLCSFFSA 2.000 1 LVLASQPTLC 0.500 3 LASQPTLCSF 0.060
4 ASQPTLCSFF 0.060 2 VLASQPTLCS 0.030 10 CSFFSASSPF 0.020 8
TLCSFFSASS 0.020 5 SQPTLCSFFS 0.020 9 LCSFFSASSP 0.010 7 PTLCSFFSAS
0.002 V3-B7-10mers: 251P5G2 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. 10 DLSICTTCLL 4.000 7 VIRDLSICTT 1.000 6
RVIRDLSICT 0.500 2 FYLRRVIRDL 0.400 9 RDLSICTTCL 0.400 4 LRRVIRDLSI
0.400 3 YLRRVIRDLS 0.300 5 RRVIRDLSIC 0.010 8 IRDLSICTTC 0.003 1
SFYLRRVIRD 0.001 V4-B7-10mers: 251P5G2 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. 3 ICTTCLLDML 4.000 2 SICTTCLLDM 1.000
5 TTCLLDMLQV 0.200 6 TCLLDMLQVV 0.200 8 LLDMLQVVNI 0.120 7
CLLDMLQVVN 0.020 4 CTTCLLDMLQ 0.010 10 DMLQVVNISP 0.010 1
LSICTTCLLD 0.010 9 LDMLQVVNIS 0.006 V12A-B7-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 3
SPSISWLIML 80.000 9 LIMLFSSVYM 3.000 2 ISPSISWLIM 1.000 1
NISPSISWLI 0.400 6 ISWLIMLFSS 0.020 7 SWLIMLFSSV 0.020 5 SISWLIMLFS
0.020 8 WLIMLFSSVY 0.020 4 PSISWLIMLF 0.002 V12B-B7-10mers: 251P5G2
Each peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 429
VPRKDLIVML 800.000 62 AARKEFSTTL 360.000 209 APGRSSSCAL 240.000 411
HVRREDLDKL 200.000 41 EPAVLPCCNL 120.000 994 VPTFSSGSFL 80.000 279
IPNLSYPLVL 80.000 1091 LPHRDTTTSL 80.000 1082 TPPHRHTTTL 80.000 949
SPGTPSLVRL 80.000 181 CPPSRNSYRL 80.000 1007 CPMFDVSPAM 60.000 161
LTRAFQVVHL 40.000 315 AARLSGLNSI 36.000 15 AAATGLWAAL 36.000 983
SVCDSSGWIL 20.000 830 KVAGFSLRQL 20.000 126 VPRPQAAPAT 20.000 264
GVGSLSVFQL 20.000 286 LVLRHIPEIL 20.000 870 EVAGFSLRQL 20.000 1060
LAPKCRPGTL 12.000 958 LASGARAAAL 12.000 105 SATPAGAFLL 12.000 152
DAACLRAQGL 12.000 872 AGFSLRQLGL 12.000 76 ALSLSSSRAL 12.000 459
ANGNSEVVQL 12.000 1106 SAGGVGPTTL 12.000 832 AGFSLRQLGL 12.000 11
QATFAAATGL 12.000 687 ASAGNGDDGL 12.000 1061 APKCRPGTLC 9.000 235
PPAHQRLLFL 8.000 217 ALRYRSGPSV 6.000 945 EPRASPGTPS 6.000 353
NVDKWDDFCL 6.000 467 QLLLDRRCQL 6.000 577 ALDRYGRTAL 5.400 932
AGDQGPGTHL 5.400 955 LVRLASGARA 5.000 192 HVRCAQGLEA 5.000 428
KVPRKDLIVM 5.000 796 QNQLRENKIL 4.000 266 GSLSVFQLHL 4.000 559
EQKQEVVKFL 4.000 1027 HQAFRDKDDL 4.000 545 KNKCGLTPLL 4.000 916
QIGDPGGVPL 4.000 624 SSHHHVICEL 4.000 593 GSASIVNLLL 4.000 197
QGLEAASANL 4.000 654 ITILNIKLPL 4.000 70 TLTGHSALSL 4.000 68
STTLTGHSAL 4.000 1051 LGQDDRAGVL 4.000 460 NGNSEVVQLL 4.000 904
QQAQEQGAAL 4.000 777 SMLREEIAKL 4.000 104 QSATPAGAFL 4.000 763
SLSHKKEEDL 4.000 302 TGGGILGLEL 4.000 277 QCIPNLSYPL 4.000 591
CCGSASIVNL 4.000 190 LTHVRCAQGL 4.000 461 GNSEVVQLLL 4.000 470
LDRRCQLNVL 4.000 47 CCNLEKGSWL 4.000 604 QNVDVSSQDL 4.000 36
VTWRKEPAVL 4.000 817 KTIQLNEEAL 4.000 674 SNPVGLPENL 4.000 491
VQCQEDECVL 4.000 324 IMQIKEFEEL 4.000 592 CGSASIVNLL 4.000 80
SSSRALPGSL 4.000 1042 QQSPRHTKDL 4.000 648 ENSNPVITIL 4.000 735
NTGISQDEIL 4.000 764 LSHKKEEDLL 4.000 753 VAEKEMNSEL 3.600 253
GPQEQPSEEA 3.000 1003 LGRRCPMFDV 3.000 490 AVQCQEDECV 3.000 522
YAIYNEDKLM 3.000 675 NPVGLPENLT 3.000 95 LPRSCPESEQ 3.000 407
EPRYHVRRED 3.000 589 AVCCGSASIV 3.000 144 SPPCHQRRDA 3.000 128
RPQAAPATSA 3.000 138 TPSRDPSPPC 3.000 244 LPRAPQAVSG 2.000 8
LPTQATFAAA 2.000 652 PVITILNIKL 2.000 1065 RPGTLCHTDT 2.000 34
DPVTWRKEPA 2.000 923 VPLSEGGTAA 2.000 581 YGRTALILAV 2.000 1044
SPRHTKDLGQ 2.000
[1031]
30TABLE XX Pos 123456789 Score V1-B35-9mers: 251P5G2 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. 226 LPVSFSVGM 40.000 1
MPFISKLVL 20.000 207 QPQPLPKDL 20.000 23 SPFLLFLDL 20.000 130
FPVSSSLIF 20.000 115 KSTIFTFHL 10.000 243 TSSTLPWAY 10.000 38
LPVCHVALI 8.000 217 RGKSHQHIL 6.000 187 FSSVYMMTL 5.000 21
ASSPFLLFL 5.000 19 FSASSPFLL 5.000 10 ASQPTLFSF 5.000 173 LSLFRDVFL
5.000 86 LSICTTCLL 5.000 128 LSFPVSSSL 5.000 143 SSNVTQINL 5.000 63
ESLNFQNDF 5.000 163 NSIIRGLFF 5.000 57 LSPQLFESL 5.000 30 DLRPERTYL
4.500 247 LPWAYDRGV 4.000 20 SASSPFLLF 3.000 165 IIRGLFFTL 3.000 43
VALIHMVVL 3.000 78 YLRRVIRVL 3.000 106 ISWLVRFKW 2.500 241
ISTSSTLPW 2.500 32 RPERTYLPV 2.400 184 IMLFSSVYM 2.000 12 QPTLFSFFS
2.000 157 CSLFPINSI 2.000 188 SSVYMMTLI 2.000 183 QIMLFSSVY 2.000
185 MLFSSVYMM 2.000 65 LNFQNDFKY 2.000 167 RGLFFTLSL 2.000 195
LIQELQEIL 2.000 58 SPQLFESLN 2.000 40 VCHVALIHM 2.000 75 ASFYLRRVI
2.000 148 QINLHVSKY 2.000 88 ICTTCLLGM 2.000 141 VASSNVTQI 1.200
211 LPKDLCRGK 1.200 116 STIFTFHLF 1.000 53 TMVFLSPQL 1.000 37
YLPVCHVAL 1.000 124 FSWSLSFPV 1.000 7 LVLASQPTL 1.000 49 VVLLTMVFL
1.000 85 VLSICTTCL 1.000 44 ALIHMVVLL 1.000 203 LVPSQPQPL 1.000 178
DVFLKQIML 1.000 239 FIISTSSTL 1.000 133 SSSLIFYTV 1.000 8 VLASQPTLF
1.000 101 NISPSISWL 1.000 162 INSIIRGLF 1.000 172 TLSLFRDVF 1.000
11 SQPTLFSFF 1.000 191 YMMTLIQEL 1.000 54 MVFLSPQLF 1.000 168
GLFFTLSLF 1.000 231 SVGMYKMDF 1.000 48 MVVLLTMVF 1.000 89 CTTCLLGML
1.000 102 ISPSISWLV 1.000 152 HVSKYCSLF 1.000 122 HLFSWSLSF 1.000
70 DFKYEASFY 0.900 194 TLIQELQEI 0.600 111 RFKWKSTIF 0.600 74
EASFYLRRV 0.600 113 KWKSTIFTF 0.600 142 ASSNVTQIN 0.500 134
SSLIFYTVA 0.500 132 VSSSLIFYT 0.500 100 VNISPSISW 0.500 104
PSISWLVRF 0.500 126 WSLSFPVSS 0.500 98 QVVNISPSI 0.400 233
GMYKMDFII 0.400 182 KQIMLFSSV 0.400 35 RTYLPVCHV 0.400 177
RDVFLKQIM 0.400 158 SLFPINSII 0.400 94 LGMLQVVNI 0.400 232
VGMYKMDFI 0.400 82 VIRVLSICT 0.300 9 LASQPTLFS 0.300 180 FLKQIMLFS
0.300 109 LVRFKWKST 0.300 145 NVTQINLHV 0.200 171 FTLSLFRDV 0.200
29 LDLRPERTY 0.200 131 PVSSSLIFY 0.200 160 FPINSIIRG 0.200 209
QPLPKDLCR 0.200 V2-B35-9mers: 251P5G2 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. 3 ASQPTLCSF 5.000 5 QPTLCSFFS 2.000
4 SQPTLCSFF 1.000 2 LASQPTLCS 0.300 8 LCSFFSASS 0.100 7 TLCSFFSAS
0.100 1 VLASQPTLC 0.100 9 CSFFSASSP 0.050 6 PTLCSFFSA 0.010
V3-B35-9mers: 251P5G2 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. 2 YLRRVIRDL 3.000 9 DLSICTTCL 1.000 6 VIRDLSICT 0.600 5
RVIRDLSIC 0.300 4 RRVIRDLSI 0.080 3 LRRVIRDLS 0.030 8 RDLSICTTC
0.020 7 IRDLSICTT 0.003 1 FYLRRVIRD 0.001 V4-B35-9mers: 251P5G2
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. 2
ICTTCLLDM 2.000 3 CTTCLLDML 1.000 6 CLLDMLQVV 0.400 5 TCLLDMLQV
0.300 9 DMLQVVNIS 0.100 8 LDMLQVVNI 0.040 7 LLDMLQVVN 0.030 4
TTCLLDMLQ 0.010 1 SICTTCLLD 0.010 V12A-B35-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 2
SPSISWLIM 40.000 8 LIMLFSSVY 2.000 1 ISPSISWLI 2.000 4 SISWLIMLF
1.000 3 PSISWLIML 0.500 5 ISWLIMLFS 0.500 7 WLIMLFSSV 0.200 6
SWLIMLFSS 0.010 V12B-B35-9mers: 251P5G2 Each peptide is a portion
of SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 428 VPRKDLIVM 180.000 1060 APKCRPGTL
60.000 674 NPVGLPENL 20.000 993 VPTFSSGSF 20.000 233 EPPAHQRLL
20.000 211 RSSSCALRY 20.000 709 FPDTENEEY 18.000 80 SSRALPGSL
15.000 256 QPSEEALGV 12.000 612 LSGQTAREY 10.000 401 DSAFMEPRY
10.000 777 MLREEIAKL 9.000 650 NPVITILNI 8.000 660 LPLKVEEEI 8.000
981 KSVCDSSGW 7.500 946 RASPGTPSL 6.000 544 KNKCGLTPL 6.000 125
VPRPQAAPA 6.000 885 QASVQQLCY 6.000 447 KQKRTALHL 6.000 1021
SNRETHQAF 6.000 243 LPRAPQAVS 6.000 845 QASVQQLCY 6.000 156
RAQGLTRAF 6.000 827 KTKVAGFSL 6.000 904 QAQEQGAAL 6.000 1042
QSPRHTKDL 5.000 321 NSIMQIKEF 5.000 958 ASGARAAAL 5.000 103
QSATPAGAF 5.000 75 LSLSSSRAL 5.000 648 NSNPVITIL 5.000 86 GSLPAFADL
5.000 763 LSHKKEEDL 5.000 592 GSASIVNLL 5.000 1027 QAFRDKDDL 4.500
558 EQKQEVVKF 4.500 147 HQRRDAACL 4.500 628 VICELLSDY 4.000 316
RLSGLNSIM 4.000 278 IPNLSYPLV 4.000 351 KNVDKWDDF 4.000 127
RPQAAPATS 4.000 378 KISGLIQEM 4.000 687 SAGNGDDGL 3.000 491
QCQEDECVL 3.000 1006 CPMFDVSPA 3.000 522 AIYNEDKLM 3.000 870
VAGFSLRQL 3.000 1090 LPHRDTTTS 3.000 593 SASIVNLLL 3.000 830
VAGFSLRQL 3.000 521 YAIYNEDKL 3.000 633 LSDYKEKQM 3.000 922
VPLSEGGTA 3.000 825 LTKTKVAGF 3.000 15 AATGLWAAL 3.000 286
VLRHIPEIL 3.000 104 SATPAGAFL 3.000 940 LPPREPRAS 3.000 152
AACLRAQGL 3.000 883 HAQASVQQL 3.000 425 WGKVPRKDL 3.000 843
HAQASVQQL 3.000 371 IMKETSTKI 2.400 546 KCGLTPLLL 2.000 375
TSTKISGLI 2.000 228 APSPAEPPA 2.000 170 APTAPDGGA 2.000 275
IQCIPNLSY 2.000 208 APGRSSSCA 2.000 179 GCPPSRNSY 2.000 1035
LPFFKTQQS 2.000 391 SNVGTWGDY 2.000 1081 TPPHRHTTT 2.000 951
TPSLVRLAS 2.000 7 LPTQATFAA 2.000 84 LPGSLPAFA 2.000 234 PPAHQRLLF
2.000 516 NTALHYAIY 2.000 644 ISSENSNPV 2.000 434 IVMLRDTDM 2.000
40 EPAVLPCCN 2.000 267 LSVFQLHLI 2.000 1082 PPHRHTTTL 2.000 976
SPTKQKSVC 2.000 310 LPATAARLS 2.000 131 APATSATPS 2.000 28
NPSRADPVT 2.000 572 ALNLALDRY 2.000 460 GNSEVVQLL 2.000 467
LLLDRRCQL 2.000 1073 TPPHRNADT 2.000 181 PPSRNSYRL 2.000 205
LPGAPGRSS 2.000 529 LMAKALLLY 2.000 528 KLMAKALLL 2.000 721
EQNDTQKQL 2.000 47 CNLEKGSWL 2.000 222 GPSVSSAPS 2.000
[1032]
31TABLE XXI Pos 1234567890 Score V1-B35-10mers: 251P5G2 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. 226
LPVSFSVGMY 40.000 130 FPVSSSLIFY 40.000 58 SPQLFESLNF 30.000 103
SPSISWLVRF 20.000 160 FPINSIIRGL 20.000 211 LPKDLCRGKS 12.000 228
VSFSVGMYKM 10.000 115 KSTIFTFHLF 10.000 217 RGKSHQHILL 6.000 153
VSKYCSLFPI 6.000 230 FSVGMYKMDF 5.000 19 FSASSPFLLF 5.000 16
FSFFSASSPF 5.000 22 SSPFLLFLDL 5.000 142 ASSNVTQINL 5.000 10
ASQPTLFSFF 5.000 182 KQIMLFSSVY 4.000 43 VALIHMVVLL 3.000 9
LASQPTLFSF 3.000 20 SASSPFLLFL 3.000 167 RGLFFTLSLF 2.000 184
IMLFSSVYMM 2.000 147 TQINLHVSKY 2.000 12 QPTLFSFFSA 2.000 128
LSFPVSSSLI 2.000 187 FSSVYMMTLI 2.000 87 SICTTCLLGM 2.000 84
RVLSICTTCL 2.000 207 QPQPLPKDLC 2.000 1 MPFISKLVLA 2.000 157
CSLFPINSII 2.000 6 KLVLASQPTL 2.000 242 STSSTLPWAY 2.000 183
QIMLFSSVYM 2.000 225 LLPVSFSVGM 2.000 219 KSHQHILLPV 2.000 45
LIHMVVLLTM 2.000 64 SLNFQNDFKY 2.000 32 RPERTYLPVC 1.200 109
LVRFKWKSTI 1.200 74 EASFYLRRVI 1.200 215 LCRGKSHQHI 1.200 171
FTLSLFRDVF 1.000 202 ILVPSQPQPL 1.000 221 HQHILLPVSF 1.000 132
VSSSLIFYTV 1.000 164 SIIRGLFFTL 1.000 178 DVFLKQIMLF 1.000 7
LVLASQPTLF 1.000 42 HVALIHMVVL 1.000 119 FTFHLFSWSL 1.000 150
NLHVSKYCSL 1.000 172 TLSLFRDVFL 1.000 88 ICTTCLLGML 1.000 58
MVVLLTMVFL 1.000 100 VNISPSISWL 1.000 47 HMVVLLTMVF 1.000 194
TLIQELQEIL 1.000 206 SQPQPLPKDL 1.000 162 INSIIRGLFF 1.000 127
SLSFPVSSSL 1.000 56 FLSPQLFESL 1.000 53 TMVFLSPQLF 1.000 85
VLSICTTCLL 1.000 52 LTMVFLSPQL 1.000 193 MTLIQELQEI 0.600 28
FLDLRPERTY 0.600 105 SISWLVRFKW 0.500 241 ISTSSTLPWA 0.500 124
FSWSLSFPVS 0.500 240 IISTSSTLPW 0.500 57 LSPQLFESLN 0.500 163
NSIIRGLFFT 0.500 99 VVNISPSISW 0.500 134 SSLIFYTVAS 0.500 117
TIFTFHLFSW 0.500 133 SSSLIFYTVA 0.500 126 WSLSFPVSSS 0.500 97
LQVVNISPSI 0.400 231 SVGMYKMDFI 0.400 93 LLGMLQVVNI 0.400 232
VGMYKMDFII 0.400 195 LIQELQEILV 0.400 140 TVASSNVTQI 0.400 37
YLPVCHVALI 0.400 156 YCSLFPINSI 0.400 69 NDFKYEASFY 0.300 68
QNDFKYEASF 0.300 180 FLKQIMLFSS 0.300 165 IIRGLFFTLS 0.300 82
VIRVLSICTT 0.300 78 YLRRVIRVLS 0.300 141 VASSNVTQIN 0.300 209
QPLPKDLCRG 0.300 70 DFKYEASFYL 0.300 175 LFRDVFLKQI 0.240 177
RDVFLKQIML 0.200 144 SNVTQINLHV 0.200 38 LPVCHVALIH 0.200 39
PVCHVALIHM 0.200 V2-B35-10mers: 251P5G2 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. 4 ASQPTLCSFF 5.000 10 CSFFSASSPF
5.000 3 LASQPTLCSF 3.000 6 QPTLCSFFSA 2.000 8 TLCSFFSASS 0.100 1
LVLASQPTLC 0.100 5 SQPTLCSFFS 0.100 2 VLASQPTLCS 0.100 7 PTLCSFFSAS
0.010 9 LCSFFSASSP 0.010 V3-B35-10mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 7; each start position is specified, length
of peptide is 10 amino acids, and the end position for each peptide
is the start position plus nine. 10 DLSICTTCLL 1.000 7 VIRDLSIGTT
0.600 3 YLRRVIRDLS 0.300 9 RDLSICTTCL 0.200 6 RVIRDLSICT 0.200 4
LRRVIRDLSI 0.120 2 FYLRRVIRDL 0.100 5 RRVIRDLSIC 0.030 8 IRDLSICTTC
0.003 1 SFYLRRVIRD 0.001 V4-B35-10mers: 251P5G2 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. 2 SICTTCLLDM 2.000 3
ICTTCLLDML 1.000 5 TTCLLDMLQV 0300 6 TCLLDMLQVV 0.200 7 CLLDMLQVVN
0.200 8 LLDMLQVVNI 0.120 1 LSICTTCLLD 0.050 4 CTTCLLDMLQ 0.010 10
DMLQVVNISP 0.010 9 LDMLQVVNIS 0.010 V12A-B35-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 3
SPSISWLIML 20.000 2 ISPSISWLIM 10.000 8 WLIMLFSSVY 2.000 9
LIMLFSSVYM 2.000 6 ISWLIMLFSS 0.500 4 PSISWLIMLF 0.500 1 NISPSISWLI
0.400 5 SISWLIMLFS 0.100 7 SWLIMLFSSV 0.020 V12B-B35-10mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
429 VPRKDLIVML 60.000 701 KSRKPENQQF 45.000 1007 CPMFDVSPAM 40.000
994 VPTFSSGSFL 20.000 234 EPPAHQRLLF 20.000 279 IPNLSYPLVL 20.000
181 CPPSRNSYRL 20.000 209 APGRSSSCAL 20.000 58 FPGTAARKEF 20.000
949 SPGTPSLVRL 20.000 1082 TPPHRHTTTL 20.000 391 KSNVGTWGDY 20.000
41 EPAVLPCCNL 20.000 1091 LPHRDTTTSL 20.000 29 NPSRADPVTW 15.000
572 KANLNALDRY 12.000 1000 GSFLGRRCPM 10.000 107 TPAGAFLLGW 10.000
62 AARKEFSTTL 9.000 522 YAIYNEDKLM 9.000 865 QAQEQEVAGF 9.000 749
KQIEVAEKEM 8.000 764 LSHKKEEDLL 7.500 428 KVPRKDLIVM 6.000 545
KNKCGLTPLL 6.000 945 EPRASPGTPS 6.000 492 QCQEDECVLM 6.000 83
RALPGSLPAF 6.000 1061 APKCRPGTLC 6.000 125 VPRPQAAPAT 6.000 1021
DSNRETHQAF 5.000 339 LSHKVIQCVF 5.000 624 SSHHHVICEL 5.000 266
GSLSVFQLHL 5.000 104 QSATPAGAFL 5.000 687 ASAGNGDDGL 5.000 80
SSSRALPGSL 5.000 593 GSASIVNLLL 5.000 348 FAKKKNVDKW 4.500 411
HVRREDLDKL 4.500 982 KSVCDSSGWI 4.000 128 RPQAAPATSA 4.000 1065
RPGTLCHTDT 4.000 633 LLSDYKEKQM 4.000 529 KLMAKALLLY 4.000 253
GPQEQPSEEA 4.000 645 ISSENSNPVI 4.000 315 AARLSGLNSI 3.600 11
QATFAAATGL 3.000 559 EQKQEVVKFL 3.000 1106 LAPKCRPGTL 3.000 914
RSQIGDPGGV 3.000 152 DAACLRAQGL 3.000 958 LASGARAAAL 3.000 89
LPAFADLPRS 3.000 15 AAATGLWAAL 3.000 105 SATPAGAFLL 3.000 847
ASVQQLCYKW 2.500 388 GSGKSNVGTW 2.500 887 ASVQQLCYKW 2.500 533
KALLLYGADI 2.400 810 SVKEKLLKTI 2.400 634 LSDYKEKQML 2.250 179
AGCPPSRNSY 2.000 275 LIQCIPNLSY 2.000 138 TPSRDPSPPC 2.000 817
KTIQLNEEAL 2.000 318 LSGLNSIMQI 2.000 364 EGYGHSFLIM 2.000 675
NPVGLPENLT 2.000 983 SVCDSSGWIL 2.000 1051 LGQDDRAGVL 2.000 144
SPPCHQRRDA 2.000 916 QIGDPGGVPL 2.000 361 CLSEGYGHSF 2.000 45
LPCCNLEKGS 2.000 34 DPVTWRKEPA 2.000 885 AWASVQQLCY 2.000 8
LPTQATFAAA 2.000 516 GNTALHYAIY 2.000 461 GNSEVVQLLL 2.000 923
VPLSEGGTAA 2.000 830 KVAGFSLRQL 2.000 612 DLSGQTAREY 2.000 628
HVICELLSDY 2.000 206 LPGAPGRSSS 2.000 282 LSYPLVLRHI 2.000 845
AQASVQQLCY 2.000 197 QGLEAASANL 2.000 604 QNVDVSSQDL 2.000 434
LIVMLRDTDM 2.000 235 PPAHQRLLFL 2.000 1013 SPAMRLKSDS 2.000 992
LPVPTFSSGS 2.000 688 SAGNGDDGLI 1.800 506 GADGNIQDEY 1.800 324
IMQIKEFEEL 1.500 777 SMLREEIAKL 1.500
[1033] Tables XXI-XLIX:
32TABLE XXII Pos 123456789 Score V1A-A1-9mers: 251P5G2 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. 131 PVSSSLIFY 19 243
TSSTLPWAY 19 148 QINLHVSKY 18 196 IQELQEILV 18 65 LNFQNDFKY 17 227
PVSFSVGMY 17 29 LDLRPERTY 16 32 RPERTYLPV 16 183 QIMLFSSVY 16 205
PSQPQPLPK 16 21 ASSPFLLFL 15 70 DFKYEASFY 15 72 KYEASFYLR 15 20
SASSPFLLF 14 193 MTLIQELQE 14 116 STIFTFHLF 13 175 LFRDVFLKQ 13 212
PKDLCRGKS 13 28 FLDLRPFRT 12 51 LLTMVFLSP 12 219 KSHQHILLP 12 245
STLPWAYDR 12 23 SPFLLFLDL 11 45 LIHMVVLLT 11 61 LFESLNFQN 11 153
VSKYCSLFP 11 163 NSIIRGLFF 11 176 FRDVFLKQI 11 199 LQEILVESQ 11 68
QNDFKYEAS 10 87 SICTTCLLG 10 119 FTFHLFSWS 10 128 LSFPVSSSL 10 143
SSNVTQINL 10 171 FTLSLFRDV 10 209 QPLPKDLCR 10 236 KMDFIISTS 10 241
ISTSSTLPW 10 10 ASQPTLFSF 9 13 PTLFSFFSA 9 90 TTCLLGMLQ 9 103
SPSISWLVR 9 106 ISWLVRFKW 9 133 SSSLIFYTV 9 139 YTVASSNVT 9
V2A-HLA-A1-9mers: 251P5G2 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. 3 ASQPTLCSF 9 6 PTLCSFFSA 9 2 LASQPTLCS 7 1
VLASQPTLC 6 7 TLCSFFSAS 4 9 CSFFSASSP 4 V3A-HLA-A1-9mers: 251P5G2
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. 7
IRDLSICTT 10 1 FYLRRVIRD 6 4 RRVIRDLSI 6 2 YLRRVIRDL 5 6 VIRDLSICT
5 V4A-HLA-A1-9mers: 251P5G2 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. 7 LLDMLQVVN 12 1 SICTTCLLD 10 4 TTCLLDMLQ 8 3
CTTCLLDML 7 2 ICTTCLLDM 6 5 TCLLDMLQV 6 V12A-HLA-A1-9mers: 251P5G2
Each peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 8
LIMLFSSVY 16 5 ISWLIMLFS 10 3 PSISWLIML 9 2 SPSISWLIM 8
[1034]
33TABLE XXIII Pos 123456789 Score V1A-HLA-A0201-9mers: 251P5G2 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. 44
ALIHMVVLL 30 92 CLLGMLQVV 28 191 YMMTLIQEL 28 224 8LLPVSFSV 28 78
YLRRVIRVL 26 194 TLIQELQEI 26 37 YLPVCHVAL 25 165 IIRGLFFTL 25 101
NISPSISWL 24 47 HMVVLLTMV 23 85 VLSICTTCL 23 158 SLFPINSII 23 195
LIQELQEIL 23 239 FIISTSSTL 23 164 SIIRGLFFT 22 7 LVLASQPTL 21 30
DLRPERTYL 21 161 PINSIIRGL 21 21 ASSPFLLFL 20 35 RTYLPVCHV 20 185
MLFSSVYMM 20 43 VALIHMVVL 19 45 LIHMVVLLT 19 49 VVLLTMVFL 19 53
TMVFLSPQL 19 56 FLSPQLFES 19 135 SLIFYTVAS 19 60 QLFESLNFQ 18 89
CTTCLLGML 18 127 SLSFPVSSS 18 136 LIFYTVASS 18 171 FTLSLFRDV 18 233
GMYKMDFII 18 26 LLFLDLRPE 17 50 VLLTMVFLS 17 77 FYLRRVIRV 17 94
LGMLQVVNI 17 128 LSFPVSSSL 17 141 VASSNVTQI 17 157 CSLFPINSI 17 167
RGLFFTLSL 17 174 SLFRDVFLK 17 184 IMLFSSVYM 17 220 SHQHILLPV 17 3
FISKLVLAS 16 38 LPVCHVALI 16 41 CHVALIHMV 16 42 HVALIHMVV 16 86
LSICTTCLL 16 95 GMLQVVNIS 16 182 KQIMLFSSV 16 203 LVPSQPQPL 16 6
KLVLASQPT 15 23 SPFLLFLDL 15 28 FLDLRPERT 15 57 LSPQLFESL 15 74
EASFYLRRV 15 80 RRVIRVLSI 15 91 TCLLGMLQV 15 93 EASFYLRRV 15 80
RRVIRVLSI 15 91 TCLLGMLQV 15 93 LLGMLQVVN 15 98 QVVNISPSI 15 105
SISWLVRFK 15 133 SSSLIFYTV 15 148 QINLHVSKY 15 151 LHVSKYCSL 15 154
SKYCSLFPI 15 168 GLFFTLSLF 15 173 LSLFRDVFL 15 178 DVFLKQIML 15 198
ELQEILVPS 15 202 ILVPSQPQP 15 223 HILLPVSFS 15 235 YKMDFIIST 15 242
STSSTLPWA 15 247 LPWAYDRGV 15 25 FLLFLDLRP 14 46 IHMVVLLTM 14 51
LLTMVFLSP 14 71 FKYEASFYL 14 82 VIRVLSICT 14 122 HLFSWSLSF 14 145
NVTQINLHV 14 187 FSSVYMMTL 14 236 KMDFIISTS 14 V2A-HLA-A0201-9mers:
251P5G2 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.
1 VLASQPTLC 13 7 TLCSFFSAS 12 2 LASQPTLCS 10 3 ASQPTLCSF 10 6
PTLCSFFSA 9 8 LCSFFSASS 6 V3A-HLA-A0201-9mers: 251P5G2 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. 2 YLRRVIRDL 26 9
DLSICTTCL 21 6 VIRDLSICT 15 7 IRDLSICTT 12 V4A-HLA-A0201-9mers:
251P5G2 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.
6 CLLDMLQVV 27 3 CTTCLLDML 18 8 LDMLQVVNI 17 7 LLDMLQVVN 15 9
DMLQVVNIS 14 5 TCLLDMLQV 13 V12A-HLA-A0201-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 7
WLIMLFSSV 25 4 SISWLIMLF 15 3 PSISWLIML 13 8 LIMLFSSVY 12
V12B-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start
position plus eight. 777 MLREEIAKL 30 548 GLTPLLLGV 29 802
KILEEIESV 29 18 GLWAALTTV 28 599 LLLEQNVDV 28 261 ALGVGSLSV 27 273
HLIQCIPNL 27 566 FLIKKKANL 26 111 FLLGWERVV 25 334 KLHSLSHKV 25 467
LLLDRRCQL 25 533 ALLLYGADI 25 266 SLSVFQLHL 24 528 KLMAKALLL 24 285
LVLRHIPEI 23 312 ATAARLSGL 23 946 RASPGTPSL 23 197 GLEAASANL 22 242
FLPRAPQAV 22 305 ILGLELPAT 22 327 KEFEELVKL 22 378 KISGLIQEM 22 15
AATGLWAAL 21 286 VLRHIPEIL 21 300 ETGGGILGL 21 315 ARLSGLNSI 21 371
IMKETSTKI 21 652 VITILNIKL 21 654 TILNIKLPL 21 770 DLLRENSML 21 781
EIAKLRLEL 21 784 KLRLELDET 21 1098 SLPHFHVSA 21 5 ILLPTQATF 20 83
ALPGSLPAF 20 159 GLTRAFQVV 20 277 CIPNLSYPL 20 325 QIKEFEELV 20 369
FLIMKETST 20 586 ILAVCCGSA 20 588 AVOCOSASI 20 840 LAQHAQASV 20 880
LAQHAQASV 20 14 AAATGLWAA 19 35 VTWRKEPAV 19 68 TTLTGHSAL 19 70
LTGHSALSL 19 104 SATPAGAFL 19 304 GILGLELPA 19 309 ELPATAARL 19 411
VRREDLDKL 19 432 DLIVMLRDT 19 457 SANGNSEVV 19 498 VLMLLEHGA 19 521
YAIYNEDKL 19 555 GVHEQKQEV 19 576 ALDRYGRTA 19 655 ILNIKLPLK 19 687
SAGNGDDGL 19 742 ILTNKQKQI 19 809 SVKEKLLKT 19 817 TIQLNEEAL 19 904
QAQEQGAAL 19 956 RLASGARAA 19 965 ALPPPTGKN 19 4 HILLPTQAT 18 11
ATFAAATGL 18 105 ATPAGAFLL 18 450 RTALHLASA 18 456 ASANGNSEV 18 477
VLDNKKRTA 18 485 ALIKAVQCQ 18 551 PLLLGVHEQ 18 584 ALILAVCCG 18 592
GSASIVNLL 18 595 SIVNLLLEQ 18 624 SHHHVICEL 18 656 LNIKLPLKV 18 659
KLPLKVEEE 18 830 VAGFSLRQL 18 843 HAQASVQQL 18 870 VAGFSLRQL 18 883
HAQASVQQL 18 916 IGDPGGVPL 18 953 SLVRLASGA 18 1112 TTLGSNREI 18 86
GSLPAFADL 17 117 RVVQRRLEV 17 152 AACLRAQGL 17 161 TRAFQVVHL 17 259
EEALGVGSL 17 316 RLSGLNSIM 17 330 EELVKLHSL 17 337 SLSHKVIQC 17 374
ETSTKISGL 17 385 EMGSGKSNV 17 403 AFMEPRYHV 17 427 KVPRKDLIV 17 459
NGNSEVVQL 17 460 GNSEVVQLL 17 493 QEDECVLML 17 529 LMAKALLLY 17 535
LLYGADIES 17 581 GRTALILAV 17 593 SASIVNLLL 17 620 YAVSSHHHV 17 648
NSNPVITIL 17 677 GLPENLTNG 17 813 KLLKTIQLN 17 914 SQIGDPGGV 17 939
HLPPREPRA 17 990 ILPVPTFSS 17 6 LLPTQATFA 16 62 ARKEFSTTL 16 75
ALSLSSSRA 16 110 AFLLGWERV 16 200 AASANLPGA 16 217 LRYRSGPSV 16 235
PAHQRLLFL 16 240 LLFLPRAPQ 16 270 FQLHLIQGI 16 280 NLSYPLVLR 16 307
GLELPATAA 16 318 SGLNSIMQI 16 338 LSHKVIQCV 16 500 MLLEHGADG 16 518
ALHYAIYNE 16 544 KNKCGLTPL 16 545 NKCGLTPLL 16 552 LLLGVHEQK 16 569
KKKANLNAL 16 591 CGSASIVNL 16 628 VICELLSDY 16 632 LLSDYKEKQ 16 694
GLIPQRKSR 16 806 EIESVKEKL 16 819 QLNEEALTK 16 827 KTKVAGFSL 16 911
ALRSQIGDP 16 923 PLSEGGTAA 16 985 DSSGWILPV 16 1050 LGQDDRAGV 16
1058 VLAPKCRPG 16 1089 TLPHRDTTT 16 1106 AGGVGPTTL 16 47 CNLEKGSWL
15 54 WLSFPGTAA 15 76 LSLSSSRAL 15 80 SSRALPGSL 15 82 RALPGSLPA 15
122 RLEVPRPQA 15 129 QAAPATSAT 15 168 HLAPTAPDG 15 264 VGSLSVFQL 15
293 ILKFSEKET 15 302 GGGILGLEL 15 306 LGLELRATA 15 345 CVFAKKKNV 15
370 LIMKETSTK 15 381 GLIQEMGSG 15 415 DLDKLHRPA 15 474 QLNVLDNKK 15
478 LDNKKRTAL 15 482 KRTALIKAV 15 531 AKALLLYGA 15 534 LLLYGADIE 15
559 QKQEVVKFL 15 600 LLEQNVDVS 15 611 DLSGQTARE 15 621 AVSSHHHVI 15
644 ISSENSNPV 15 728 QLSEEQNTG 15 753 AEKEMNSEL 15 755 KEMNSELSL 15
762 SLSHKKEED 15 799 RENKILEEI 15 834 SLRQLGLAQ 15 850 QLCYKWNHT 15
874 SLRQLGLAQ 15 957 LASGARAAA 15 958 ASGARAAAL 15 982 SVCDSSGWI 15
1027 QAFRDKDDL 15 1096 TTSLPHFHV 15 1104 VSAGGVGPT 15 1105
SAGGVGPTT 15 43 VLPCCNLEK 14 77 SLSSSRALP 14 112 LLGWERVVQ 14 155
LRAQGLTRA 14 162 RAFQVVHLA 14 232 AEPPAHQRL 14 256 QPSEEALGV 14 267
LSVFQLHLI 14 278 IPNLSYPLV 14 282 SYPLVLRHI 14 324 MQIKEFEEL 14 360
CLSEGYGHS 14 420 HRAAWWGKV 14 429 PRKDLIVML 14 452 ALHLASANG 14 470
DRRCQLNVL 14 501 LLEHGADGN 14 522 AIYNEDKLM 14 553 LLGVHEQKQ 14 577
LDRYGRTAL 14 578 DRYGRTALI 14 597 VNLLLEQNV 14 604 NVDVSSQDL 14 660
LPLKVEEEI 14 668 IKKHGSNPV 14 670 KHGSNPVGL 14 734 NTGISQDEI 14 771
LLRENSMLR 14 810 VKEKLLKTI 14 816 KTIQLNEEA 14 824 ALTKTKVAG 14 839
GLAQHAQAS 14 879 GLAQHAWAS 14 890 QLCYKWGHT 14 932 GDQGPGTHL 14 947
ASPGTPSLV 14 949 PGTPSLVRL 14 950 GTPSLVRLA 14 1088 TTLPHRDTT 14
1113 TLGSNREIT 14
[1035]
34TABLE XXIV Pos 123456789 score V1A-HLA-A0202-9mers: 251P5G2
Noresultsfound. V2A-HLA-A0202-9mers: 251P5G2 Noresultsfound.
V3A-HLA-A0202-9mers: 251P5G2 Noresultsfound. V4A-HLA-A0202-9mers:
251P5G2 Noresultsfound. V12A-HLA-A0202-9mers: 251P5G2 No results
found. V12B-HLA-A0202-9mers: 251P5G2 Noresultsfound.
[1036]
35TABLE XXV Pos 123456789 Score V1A-HLA-A0203-9mers: 251P5G2
Noresultsfound. V2A-HLA-A0203-9mers: 251P5G2 Noresultsfound.
V3A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V4A-HLA-A0203-9mers:
251P5G2 Noresultsfound. V12A-HLA-A0203-9mers: 251P5G2
Noresultsfound. V12B-HLA-A0203-9mers: 251P5G2 Noresultsfound.
[1037]
36TABLE XXVI Pos 123456789 Score V1A-HLA-A3-9mers: 251P5G2 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. 81
RVIRVLSIC 24 174 SLFRDVFLK 24 183 QIMLFSSVY 24 39 PVCHVALIH 21 64
SLNFQNDFK 21 78 YLRRVIRVL 21 84 RVLSICTTC 21 44 ALIHMVVLL 20 48
MVVLLTMVF 20 147 TQINLHVSK 20 148 QINLHVSKY 20 172 TLSLFRDVF 20 224
ILLPVSFSV 20 105 SISWLVRFK 19 122 HLFSWSLSF 19 135 SLIFYTVAS 19 140
TVASSNVTQ 19 165 IIRGLFFTL 19 7 LVLASQPTL 18 30 DLRPERTYL 18 92
CLLGMLQVV 18 93 LLGMLQVVN 18 189 SVYMMTLIQ 18 202 ILVPSQPQP 18 214
DLCRGKSHQ 18 227 PVSFSVGMY 18 239 FIISTSSTL 18 8 VLASQPTLF 17 42
HVALIHMVV 17 49 VVLLTMVFL 17 76 SFYLRRVIR 17 152 HVSKYCSLF 17 158
SLFPINSII 17 164 SIIRGLFFT 17 168 GLFFTLSLF 17 205 PSQPQPLPK 17 222
QHILLPVSF 17 225 LLPVSFSVG 17 51 LLTMVFLSP 16 54 MVFLSPQLF 16 107
SWLVRFKWK 16 109 LVRFKWKST 16 127 SLSFPVSSS 16 131 PVSSSLIFY 16 163
NSIIRGLFF 16 213 KDLCRGKSH 16 231 SVGMYKMDF 16 25 FLLFLDLRP 15 29
LDLRPERTY 15 37 YLPVCHVAL 15 98 QVVNISPSI 15 99 VVNISPSIS 15 101
NISPSISWL 15 137 IFYTVASSN 15 198 ELQEILVPS 15 209 QPLPKDLCR 15 228
VSFSVGMYK 15 6 KLVLASQPT 14 14 TLFSFFSAS 14 28 FLDLRPERT 14 180
FLKQIMLFS 14 200 QEILVPSQP 14 223 HILLPVSFS 14 56 FLSPQLFES 13 60
QLFESLNFQ 13 70 DFKYEASFY 13 96 MLQVVNISP 13 103 SPSISWLVR 13 108
WLVRFKWKS 13 136 LIFYTVASS 13 145 NVTQINLHV 13 178 DVFLKQIML 13 194
TLIQELQEI 13 215 LCRGKSHQH 13 245 STLPWAYDR 13 3 FISKLVLAS 12 5
SKLVLASQP 12 10 ASQPTLFSF 12 45 LIHMVVLLT 12 50 VLLTMVFLS 12 80
RRVIRVLSI 12 85 VLSICTTCL 12 87 SICTTCLLG 12 113 KWKSTIFTF 12 211
LPKDLCRGK 12 26 LLFLDLRPE 11 43 VALIHMVVL 11 82 VIRVLSICT 11 91
TCLLGMLQV 11 104 PSISWLVRF 11 111 RFKWKSTIF 11 117 TIFTFHLFS 11 167
RGLFFTLSL 11 182 KQIMLFSSV 11 185 MLFSSVYMM 11 197 QELQEILVP 11 203
LVPSQPQPL 11 240 IISTSSTLP 11 V2A-HLA-A3-9mers: 251P5G2 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. 7
TLCSFFSAS 14 1 VLASQPTLC 13 3 ASQPTLCSF 12 4 SQPTLCSFF 8 9
CSFFSASSP 8 V3A-HLA-A3-9mers: 251P5G2 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. 5 RVIRDLSIC 23 2 YLRRVIRDL 17 4
RRVIRDLSI 12 6 VIRDLSICT 12 9 DLSICTTCL 12 8 RDLSICTTC 11
V4A-HLA-V3-9mers: 251P5G2 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. 7 LLDMLQVVN 18 6 CLLDMLQVV 17 1 SICTTCLLD 12 5
TCLLDMLQV 9 V12A-HLA-A3-9mers: 251P5G2 Each peptide is a portion of
SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 9 LIMLFSSVY 22 8 WLIMLFSSV 18 5
SISWLIMLF 14 V12B-HLA-A3-9mers: 251P5G2 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 each peptide is the
start position plus eight. 819 QLNEEALTK 33 803 ILEEIESVK 30 154
CLRAQGLTR 29 410 HVRREDLDK 29 5 ILLPTQATF 28 552 LLLGVHEQK 28 341
KVIQCVFAK 27 370 LIMKETSTK 25 382 LIQEMGSGK 25 436 MLRDTDMNK 25 655
ILNIKLPLK 24 43 VLPCCNLEK 23 576 ALDRYGRTA 23 651 PVITILNIK 23 694
GLIPQRKSR 23 695 LIPQRKSRK 23 1010 DVSPAMRLK 23 342 VIQCVFAKK 22
474 QLNVLDNKK 22 528 KLMAKALLL 22 533 ALLLYGADI 22 631 ELLSDYKEK 22
332 LVKLHSLSH 21 563 VVKFLIKKK 21 661 PLKVEEEIK 21 834 SLRQLGLAQ 21
874 SLRQLGLAQ 21 956 RLASGARAA 21 1103 HVSAGGVGP 21 83 ALPGSLPAF 20
117 RVVQRRLEV 20 261 ALGVGSLSV 20 319 GLNSIMQIK 20 418 KLHRAAWWG 20
427 KVPRKDLIV 20 480 NKKRTALIK 20 562 EVVKFLIKK 20 588 AVCCGSASI 20
681 NLTNGASAG 20 786 RLELDETKH 20 953 SLVRLASGA 20 954 LVRLASGAR 20
964 AALPPPTGK 20 1001 FLGRRCPMF 20 56 SFPGTAARK 19 112 LLGWERVVQ 19
118 VVQRRLEVP 19 165 QVVHLAPTA 19 216 ALRYRSGPS 19 316 RLSGLNSIM 19
326 IKEFEELVK 19 457 LLLDRRCQL 19 500 MLLEHGADG 19 770 DLLRENSML 19
771 LLRENSMLR 19 829 KVAGFSLRQ 19 915 QIGDPGGVP 19 31 RADPVTWRK 18
42 AVLPCCNLE 18 111 FLLGWERVV 18 122 RLEVPRPQA 18 239 RLLFLPRAP 18
249 AVSGPQEQP 18 280 NLSYPLVLR 18 291 PEILKFSEK 18 309 ELPATAARL 18
557 HEQKQEVVK 18 618 REYAVSSHH 18 627 HVICELLSD 18 628 VICELLSDY 18
663 KVEEEIKKH 18 824 ALTKTKVAG 18 923 PLSEGGTAA 18 971 GKNGRSPTK 18
1062 KCRPGTLCH 18 18 GLWAALTTV 17 75 ALSLSSSRA 17 124 EVPRPQAAP 17
159 GLTRAFQVV 17 160 LTRAFQVVH 17 168 HLAPTAPDG 17 188 RLTHVRCAQ 17
203 ANLPGAPGR 17 218 RYRSGPSVS 17 288 RHIPEILKF 17 369 FLIMDETST 17
452 ALHLASANG 17 463 EVVQLLLDR 17 534 LLLYGADIE 17 566 FLIKKKANL 17
584 ALILAVCCG 17 598 NLLLEQNVD 17 599 LLLEQNVDV 17 621 AVSSHHHVI 17
728 QLSEEQNTG 17 740 DEILTNKQK 17 742 ILTNKQKQI 17 776 SMLREEIAK 17
777 MLREEIAKL 17 784 KLRLELDET 17 785 LRLELDETK 17 809 SVKEKLLKT 17
911 ALRSQIGDP 17 965 ALPPPTGKN 17 1054 DRAGVLAPK 17 1089 TLPHRDTTT
17 1098 SLPHFHVSA 17 87 SLPAFADLP 16 211 RSSSCALRY 16 307 GLELPATAA
16 347 FAKKKNVDK 16 381 GLIQEMGSG 16 423 AWWGKVPRK 16 434 IVMLRDTDM
16 466 QLLLDRRCQ 16 477 VLDNKKRTA 16 485 ALIKAVQCQ 16 522 AIYNEKDLM
16 535 LLYGADIES 16 548 GLTPLLLGV 16 561 QEVVKFLIK 16 586 ILAVCCGSA
16 596 IVNLLLEQN 16 747 QKQIEVAEK 16 797 QLRENKILE 16 802 KILEEIESV
16 821 NEEALTKTK 16 869 EVAGFSLRQ 16 973 NGRSPTKQK 16 989 WILPVPTFS
16 1108 GVGPTTLGS 16
[1038]
37TABLE XXVII Pos 123456789 score V1A-HLA-A26-9mers: 251P5G2 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. 178
DVFLKQIML 28 101 NISPSISWL 26 116 STIFTFHLF 26 131 PVSSSLIFY 26 148
QINLHVSKY 26 227 PVSFSVGMY 26 165 IIRGLFFTL 25 168 GLFFTLSLF 25 185
MLFSSVYMM 25 30 DLRPERTYL 24 54 MVFLSPQLF 24 70 DFKYEASFY 24 89
CTTCLLGML 24 161 PINSIIRGL 24 44 ALIHMVVLL 23 49 VVLLTMVFL 23 179
VFLKQIMLF 23 48 MVVLLTMVF 22 122 HLFSWSLSF 22 152 HVSKYCSLF 22 203
LVPSQPQPL 22 239 FIISTSSTL 22 8 VLASQPTLF 21 183 QIMLFSSVY 21 195
LIQELQEIL 21 198 ELQEILVPS 21 231 SVGMYKMDF 21 7 LVLASQPTL 20 17
SFFSASSPF 20 78 YLRRVIRVL 20 37 YLPVCHVAL 19 104 PSISWLVRF 19 229
SFSVGMYKM 19 111 RFKWKSTIF 18 119 FTFHLFSWS 18 136 LIFYTVASS 18 172
TLSLFRDVF 18 3 FISKLVLAS 17 10 ASQPTLFSF 17 11 SWPTLFSFF 17 14
TLFSFFSAS 17 60 QLFESLNFQ 17 81 RVIRVLSIC 17 194 TLIQELEQI 17 201
EILVPSQPQ 17 238 DFIISTSST 17 13 PTLFSFFSA 16 18 FFSASSPFL 16 56
FLSPQLFES 16 57 LSPQLFESL 16 63 ESLNFQNDF 16 85 VLSICTTCL 16 113
KWKSTIFTF 16 164 SIIRGLFFT 16 171 FTLSLFRDV 16 214 DLCRGKSHQ 16 242
STSSTLPWA 16 2 PFISKLVLA 15 20 SASSPFLLF 15 23 SPFLLFLDL 15 35
TRYLPVCHV 15 52 LTMVFLSPQ 15 69 NDFKYEASF 15 92 CLLGMLQVV 15 105
SISWLVRFK 15 120 TFHLFSWSL 15 151 LHVSKYCSL 15 191 YMMTLIQEL 15 210
PLPKDLCRG 15 222 QHILLPVSF 15 225 LLPVSFSVG 15 21 ASSPFLLFL 14 26
LLFLDLRPE 14 45 LIHMVVLLT 14 65 LNFQNDFKY 14 127 SLSFPVSSS 14 128
LSFPVSSSL 14 140 TVASSNVTQ 14 146 VTQINLHVS 14 174 SLFRDVFLK 14 180
FLKQIMLFS 14 246 TLPWAYDRG 14 40 VCHVALIHM 13 43 VALIHMVVL 13 51
LLTMVFLSP 13 88 ICTTCLLGM 13 158 SLFPINSII 13 186 LFSSVYMMT 13 187
FSSVYMMTL 13 202 ILVPSQPQP 13 V2A-HLA-A26-9mers: 251P5G2 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. 3
ASQPTLCSF 17 6 PTLCSFFSA 16 7 TLCSFFSAS 15 4 SQPTLCSFF 13 1
VLASQPTLC 11 V3A-HLA-A269mers: 251P5G2 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. 9 DLSICTTCL 22 2 YLRRVIRDL 20 5
RVIRDLSIC 17 6 VIRDLSICT 12 V12A-HLA-A26-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 5
SISWLIMLF 26 9 LIMLFSSVY 21 8 WLIMLFSSV 17 4 PSISWLIML 16 2
ISPSISWLI 2 V12B-HLA-A26-9mers: 251P5G2 Each peptide is a portion
of SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 1094 DTTTSLPHF 33 300 ETGGGILGL 31
374 ETSTKISGL 31 83 ALPGSLPAF 28 628 VICELLSDY 28 781 EIAKLRLEL 28
825 LTKTKVAGF 28 378 KISGLIQEM 27 516 NTALHYAIY 26 806 EIESVKEKL 26
273 HLIQCIPNL 25 309 ELPATAARL 25 312 ATAARLSGL 25 558 EQKQEVVKF 24
724 DTQKQLSEE 24 770 DLLRENSML 24 777 MLREEIAKL 24 994 PTFSSGSFL 23
463 EVVQLLLDR 22 562 EVVKFLIKK 22 566 FLIKKKANL 22 751 EVAEKEMNS 22
1010 DVSPAMRLK 22 5 ILLPTQATF 21 11 ATFAAATGL 21 604 NVDVSSQDL 21
1024 ETHQAFRDK 21 68 TTLTGHSAL 20 70 LTGHSALSL 20 259 EEALGVGSL 20
277 CIPNLSYPL 20 316 RLSGLNSIM 20 330 EELVKLHSL 20 357 DDFCLSEGY 20
432 DLIVMLRDT 20 631 ELLSDYKEK 20 652 VITILNIKL 20 667 EIKKHGSNP 20
827 KTKVAGFSL 20 869 EVAGFSLRQ 20 1001 FLGRRCPMF 20 1072 DTPPHRNAD
20 105 ATPAGAFLL 19 124 EVPRPQAAP 19 288 RHIPEILKF 19 328 EFEELVKLH
19 401 DSAFMEPRY 19 470 DRRCQLNVL 19 494 EDECVLMLL 19 606 DVSSQDLSG
19 611 DLSGQTARE 19 749 QIEVAEKEM 19 791 ETKHQNQLR 19 809 SVKEKLLKT
19 817 TIQLNEEAL 19 1080 DTPPHRHTT 19 93 DLPRSCPES 18 197 GLEAASANL
18 262 LGVGSLSVF 18 292 EILKFSEKE 18 321 NSIMQIKEF 18 327 KEFEELVKL
18 341 LVIQCVFAK 18 415 DLDKLHRAA 18 434 IVMLRDTDM 18 467 LLLDRRCQL
18 522 AIYNEDKLM 18 540 DIESKNKCG 18 654 TILNIKLPL 18 741 EILTNKQKQ
18 788 ELDETKHQN 18 65 EFSTTLTGH 17 266 SLSVFQLHL 17 289 HIPEILKFS
17 331 ELVKLHSLS 17 429 PRKDLIVML 17 439 DTDMNKRDK 17 450 RTALHLASA
17 513 EYGNTALHY 17 528 KLMAKALLL 17 563 VVKFLIKKK 17 627 HVICELLSD
17 663 KVEEEIKKH 17 711 DTENEEYHS 17 802 KILEEIESV 17 865 AQEQEVAGF
17 949 PGTPSLVRL 17 950 GTPSLVRLA 17 988 GWILPVPTF 17 1030
RDKDDLPFF 17 8 PTQATFAAA 16 161 TRAFQVVHL 16 285 LVLRHIPEI 16 286
VLRHIPEIL 16 324 MQIKEFEEL 16 342 VIQCVFAKK 16 381 GLIQEMGSG 16 394
GTWGDYDDS 16 464 VVQLLLDRR 16 485 ALIKAVQCQ 16 493 QEDECVLML 16 497
CVLMLLEHG 16 509 NIQDEYGNT 16 526 EDKLMAKAL 16 529 LMAKALLLY 16 548
GLTPLLLGV 16 549 LTPLLLGVH 16 582 RTALILAVC 16 595 SIVNLLLEQ 16 596
IVNLLLEQN 16 615 QTAREYAVS 16 651 PVITILNIK 16 653 ITILNIKLP 16 677
GLPENLTNG 16 721 EQNDTQKQL 16 760 ELSLSHKKE 16 790 DETKHQNQL 16 805
EEIESVKEK 16 812 EKLLKTIQL 16 1000 SFLGRRCPM 16 1034 DLPFFKTQQ 16
1049 DLGQDDRAG 16
[1039]
38TABLE XXVIII Pos 123456789 score V1A-HLA-B0702-9mers: 251P5G2
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. 1
MPFISKLVL 23 23 SPFLLFLDL 23 207 QPQPLPKDL 21 32 RPERTYLPV 20 21
ASSPFLLFL 18 38 LPVCHVALI 18 130 FPVSSSLIF 18 226 LPVSFSVGM 18 247
LPWAYDRGV 17 165 IIRGLFFTL 16 204 VPSQPQPLP 16 18 FFSASSPFL 15 30
DLRPERTYL 15 44 ALIHMVVLL 15 103 SPSISWLVR 15 20 SASSPFLLF 14 85
VLSICTTCL 14 167 RGLFFTLSL 14 37 YLPVCHVAL 13 43 VALIHMVVL 13 49
VVLLTMVFL 13 78 YLRRVIRVL 13 101 NISPSISWL 13 173 LSLFRDVFL 13 209
QPLPKDLCR 13 7 LVLASQPTL 12 115 KSTIFTFHL 12 187 FSSVYMMTL 12 218
GKSHQHILL 12 12 QPTLFSFFS 11 19 FSASSPFLL 11 35 RTYLPVCHV 11 53
TMVFLSPQL 11 57 LSPQLFESL 11 86 LSICTTCLL 11 128 LSFPVSSSL 11 191
YMMTLIQEL 11 203 LVPSQPQPL 11 217 RGKSHQHIL 11 V2A-HLA-B0702-9mers:
251P5G2 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.
5 QPTLCSFFS 11 3 ASQPTLCSF 9 2 LASQPTLCS 8 4 SQPTLCSFF 7 6
PTLCSFFSA 7 V3A-HLA-B0702-9mers: 251P5G2 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. 9 DLSICTTCL 14 2 YLRRVIRDL 12 4
RRVIRDLSI 9 6 VIRDLSICT 8 7 IRDLSICTT 8 V4A-B0702-9mers: 251P5G2
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. 3
CTTCLLDML 10 8 LDMLQVVNI 10 2 ICTTCLLDM 9 5 TCLLDMLQV 8 6 CLLDMLQVV
7 7 LLDMLQVVN 4 V12A-B0702-9mers: 251P5G2 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. 3 SPSISWLIM 20 4 PSISWLIML 10
V12B-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO:
25; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. 228 APSPAEPPA 23 170 APTAPDGGA 22 181
PPSRNSYRL 22 233 EPPAHQRLL 22 674 NPVGLPENL 22 1060 APKCRPGTL 22
1082 PPHRHTTTL 22 125 VPRPQAAPA 21 28 NPSRADPVTI 20 234 PPAHQRLLFJ
20 256 QPSEEALGV 20 428 VPRKDLIVM 20 84 LPGSLPAFA 19 208 APGRSSSCA
19 1006 CPMFDVSPA 19 7 LPTQATFAA 18 243 LPRAPQAVS 18 278 IPNLSYPLV
18 650 NPVITILNI 18 98 CPESEQSAT 17 144 PPCHQRRDA 17 660 LPLKVEEEI
17 678 LPENLTNGA 17 916 IGDPGGVPL 17 922 VPLSEGGTA 17 932 GDQGPGTHL
17 941 PPREPRASP 17 948 SPGTPSLVR 17 1073 TPPHRNADT 17 1081
TPPHRHTTT 17 15 AATGLWAAL 16 106 TPAGAFLLG 16 141 DPSPPCHQR 16 205
LPGAPGRSS 16 300 ETGGGILGL 16 781 EIAKLRLEL 16 946 RASPGTPSL 16 951
ASGARAAAL 16 958 ASGARAAAL 16 993 VPTFSSGSF 16 36 TWRKEPAVL 15 131
APATSATPS 15 173 APDGGAGCP 15 230 SPAEPPAHQ 15 528 KLMAKALLL 15 577
LDRYGRTAL 15 591 CGSASIVNL 15 918 DPGGVPLSE 15 11 ATFAAATGL 14 54
WLSFPGTAA 14 88 LPAFADLPR 14 104 SATPAGAFL 14 161 TRAFQVVHL 14 222
GSPVSSAPS 14 246 APQAVSGPQ 14 266 SLSVFQLHL 14 302 GGGILGLEL 14 312
ATAARLSGL 14 406 EPRYHVRRE 14 425 WGKVPRKDL 14 445 RDKQKRTAL 14 478
LDNKKRTAL 14 493 QEDECVLML 14 541 IESKNKCGL 14 545 NKCGLTPLL 14 546
KCGLTPLLL 14 579 RYGRTALIL 14 593 SASIVNLLL 14 670 KHGSNPVGL 14 755
KEMNSELSL 14 832 GFSLRQLGL 14 872 GFSLRQLGL 14 944 EPRASPGTP 14
1091 PHRDTTTSL 14 1106 AGGVGPTTL 14 41 PAVLPCCNL 13 62 ARKEFSTTL 13
70 LTGHSALSL 13 80 SSRALPGSL 13 86 GSLPAFADL 13 105 ATPAGAFLL 13
127 RPQAAPATS 13 137 TPSRDPSPP 13 147 HQRRDAACL 13 209 PGRSSSCAL 13
232 AEPPAHQRL 13 235 PAHQRLLFL 13 259 EEALGVGSL 13 264 VGSLSVFQL 13
279 PNLSYPLVL 13 309 ELPATAARL 13 327 KEFEELVKL 13 374 ETSTKISGL 13
403 AFMEPRYHV 13 447 KQKRTALHL 13 459 NGNSEVVQL 13 460 GNSEVVQLL 13
470 DRRCQLNVL 13 544 KNKCGLTPL 13 550 TPLLLGVHE 13 569 KKKANLNAL 13
647 ENSNPVITI 13 654 TILNIKLPL 13 777 MLREEIAKL 13 779 REEIAKLRL 13
807 IESVKEKLL 13 904 QAQEQGAAL 13 935 GPGTHLPPR 13 949 PGTPSLVRL 13
957 LASGARAAA 13 985 DSSGWILPV 13 1009 FDVSPAMRL 13 40 EPAVLPCCN 12
61 AARKEFSTT 12 83 ALPGSLPAF 12 94 LPRSCPESE 12 115 WERVVQRRL 12
122 RLEVPRPQA 12 128 PQAAPATSA 12 152 AACLRAQGL 12 197 GLEAASANL 12
200 AASANLPGA 12 254 QEQPSEEAL 12 286 VLRHIPEIL 12 298 EKETGGGIL 12
324 MQIKEFEEL 12 362 SEGYGHSFL 12 408 RYHVRREDL 12 411 VRREDLDKL 12
429 PRKDLIVML 12 461 NSEVVQLLL 12 511 QDEYGNTAL 12 526 EDKLMAKAL 12
559 QKQEVVKFL 12 566 FLIKKKANL 12 592 GSASIVNLL 12 648 NSNPVITIL 12
703 KPENQQFPD 12 753 AEKEMNSEL 12 812 EKLLKTIQL 12 827 KTKVAGFSL 12
843 HAQASVQQL 12 867 EQEVAGFSL 12 883 HAQASVQQL 12 966 LPPPTGKNG 12
968 PPTGKNGRS 12 976 SPTKQKSVC 12 983 VCDSSGWIL 12 991 LPVPTFSSG 12
994 PTFSSGSFL 12 1032 KDDLPFFKT 12 1043 SPRHTKDLG 12 1051 GQDDRAGVL
12 1064 RPGTLCHTD 12 1079 ADTPPHRHT 12 1090 LPHRDTTTS 12 1099
LPHFHVSAG 12 4 HILLPTQAT 11 13 FAAATGLWA 11 33 DPVTWRKEP 11 47
CNLEKGSWL 11 57 FPGTAARKE 11 68 TTLTGHSAL 11 76 LSLSSSRAL 11 82
RALPGSLPA 11 102 EQSATPAGA 11 103 QSATPAGAF 11 149 RRDAACLRA 11 156
RAQOLTRAF 11 261 ALGVGSLSV 11 273 HLIQCIPNL 11 277 CIPNLSYPL 11 283
YPLVLRHIP 11 290 IPEILKFSE 11 304 GILGLELPA 11 307 GLELPATAA 11 310
LPATAARLS 11 316 RLSGLNSIM 11 330 EELVKLHSL 11 378 KISGLIQEM 11 467
LLLDRRCQL 11 491 QCQEDECVL 11 494 EDEGVLMLL 11 527 DKLMAKALL 11 604
NVDVSSQDL 11 621 AVSSHHHVI 11 634 SDYKEKQML 11 687 SAGNGDDGL 11 696
IPQRKSRKP 11 709 FPDTENEEY 11 721 EQNDTQKQL 11 763 LSHKKEEDL 11 764
SHKKEEDLL 11 790 DETKHQNQL 11 806 EIESVKEKL 11 817 TIQLNEEAL 11 830
VAGFSLRQL 11 870 VAGFSLRQL 11 923 PLSEGGTAA 11 930 AAGDQGPGT 11 940
LPPREPRAS 11 962 RAAALPPPT 11 988 GWILPVPTF 11 1003 GRRCPMFDV 11
1012 SPAMRLKSD 11 1029 FRDKDDLPF 11 1035 LPFFKTQQS 11 1042
QSPRHTKDL 11 1096 TTSLPHFHV 11 1104 VSAGGVGPT 11 1105 SAGGVGPIT 11
1110 GPTTLGSNR 11
[1040]
39TABLE XXIX Pos 123456789 score V1A-HLA-B08-9mers: 251P5G2 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. 111
RFKWKSTIF 26 78 YLRRVIRVL 23 30 DLRPERTYL 22 165 IIRGLFFTL 21 178
DVFLKQIML 21 151 LHVSKYCSL 20 217 RGKSHQHIL 20 23 SPFLLFLDL 19 37
YLPVCHVAL 19 173 LSLFRDVFL 19 44 ALIHMVVLL 18 232 VGMYKMDFI 18 85
VLSICTTCL 17 109 LVRFKWKST 17 113 KWKSTIFTF 17 180 FLKQIMLFS 17 207
QPQPLPKDL 17 1 MPFISKLVL 16 28 FLDLRPERT 16 43 VALIHMVVL 16 195
LIQELQEIL 16 209 QPLPKDLCR 16 211 LPKDLCRGK 16 215 LCRGKSHQH 16 80
RRVIRVLSI 15 101 NISPSISWL 15 158 SLFPINSII 15 161 PINSIIRGL 15 239
FIISTSSTL 15 163 NSIIRGLFF 14 8 VLASQPTLF 13 38 LPVCHVALI 13 49
VVLLTMVFL 13 71 FKYEASFYL 13 122 HLFSWSLSF 13 130 FPVSSSLIF 13 143
SSNVTQINL 13 168 GLFFTLSLF 13 172 TLSLFRDVF 13 194 TLIQELQEI 13 4
ISKLVLASQ 12 20 SASSPFLLF 12 76 SFYLRRVIR 12 82 VIRVLSICT 12 107
SWLVRFKWK 12 141 VASSNVTQI 12 153 VSDYCSLFP 12 187 FSSVYMMTL 12 191
YMMTLIQEL 12 V2A-HLA-B08-9mers: 251P5G2 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. 4 SQPTLCSFF 8 1 VLASQPTLC 7 5
QPTLCSFFS 7 7 TLCSFFSAS 7 3 ASQPTLCSF 6 2 LASQPTLCS 4
V3A-HLA-B08-9mers: 251P5G2 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. 2 YLRRVIRDL 23 9 DLSICTTCL 16 4 RRVIRDLSI 14 6
VIRDLSICT 11 V4A-HLA-B08-9mers: 251P5G2 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. 3 CTTCLLDML 10 8 LDMLQVVNI 9 7
LLDMLQVVN 7 1 SICTTCLLD 6 6 CLLDMLQVV 6 V12A-HLA-B08-9mers: 251P5G2
Each peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 5
SISWLIMLF 13 4 PSISWLIML 10 3 SPSISWLIM 8 2 ISPSISWLI 7 8 WLIMLFSSV
6 V12B-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start
position plus eight. 1060 APKCRPGTL 34 445 RDKQKRTAL 31 347
FAKKKNVDK 27 566 FLIKKKANL 27 825 LTKTKVAGF 27 528 KLMAKALLL 26 526
EDKLMAKAL 25 777 MLREEIAKL 25 467 LLLDRRCQL 24 479 DNKKRTALI 24 567
LIKKKANLN 24 742 ILTNKQKQI 24 753 AEKEMNSEL 24 782 IAKLRLELD 24 809
SVKEKLLKT 24 152 AACLRAQGL 23 286 VLRHIPEIL 23 330 EELVKLHSL 23 374
ETSTKISGL 23 807 IESVKEKLL 23 812 EKLLKTIQL 23 47 CNLEKGSWL 22 62
ARKEFSTTL 22 235 PAHQRLLFL 22 425 WGKVPRKDL 22 429 PRKDLIVML 22 542
ESKNKCGLT 22 635 DYKEKQMLK 22 764 SHKKEEDLL 22 216 ALRYRSGPS 21 353
VDKWDDFCL 21 478 LDNKKRTAL 21 569 KKKANLNAL 21 744 TNKQKQIEV 21 827
KTKVAGFSL 21 977 PTKQKSVCD 21 1001 FLGRRCPMF 21 297 SEKETGGGI 20
447 KQKRTALHL 20 544 KNKCGLTPL 20 558 EQKQEVVKF 20 763 LSHKKEEDL 20
832 GFSLRQLGL 20 872 GFSLRQLGL 20 266 SLSVFQLHL 19 293 ILKFSEKET 19
337 SLSHKVIQC 19 339 SHKVIQCVF 19 371 IMKETSTKI 19 408 RYHVRREDL 19
411 VRREDLDKL 19 659 KLPLKVEEE 19 698 QRKSRKPEN 19 701 SRKPENQQF 19
762 SLSHKKEED 19 958 ASGARAAAL 19 1051 GQDDRAGVL 19 80 SSRALPGSL 18
233 EPPAHQRLL 18 273 HLIQCIPNL 18 309 ELPATAARL 18 312 ATAARLSGL 18
577 LDRYGRTAL 18 115 WERVVQRRL 17 147 HQRRDAACL 17 197 GLEAASANL 17
209 PGRSSSCAL 17 369 FLIMKETST 17 443 NKRDKQKRT 17 477 VLDNKKRTA 17
593 SASIVNLLL 17 655 ILNIKLPLK 17 661 PLKVEEEIK 17 696 IPQRKSRKP 17
770 DLLRENSML 17 781 EIAKLRLEL 17 823 EALTKTKVA 17 850 QLCYKWNHT 17
890 QLCYKWGHT 17 904 QAQEQGAAL 17 968 PPTGKNGRS 17 968 PPTGKNGRS 17
1012 SPAMRLKSD 17 1014 AMRLKSDSN 17 1021 SNRETHQAF 17 1058
VLAPKCRPG 17 36 TWRKEPAVL 16 49 LEKGSWLSF 16 104 SATPAGAFL 16 181
PPSRNSYRL 16 325 QIKEFEELV 16 351 KNVDKWDDF 16 406 EPRYHVRRE 16 470
DRRCQLNVL 16 540 DIESKNKCG 16 641 MLKISSENS 16 652 VITILNIKL 16 657
NIKLPLKVE 16 667 EIKKHGSNP 16 674 NPVGLPENL 16 687 SAGNGDDGL 16 806
EIESVKEKL 16 814 LLKTIQLNE 16 817 TIQLNEEAL 16 843 HAQASVQQL 16 883
HAQASVQQL 16 1016 RLKSDSNRE 16 1028 AFRDKDDLP 16 1030 RDKDDLPFF 16
1035 LPFFKTQQS 16 1082 PPHRHTTTL 16 1091 PHRDTTTSL 16
[1041]
40TABLE XXX Pos 123456789 score V1A-HLA-B1510-9mers: 251P5G2 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. 46
IHMVVLLTM 20 151 LHVSKYCSL 20 222 QHILLPVSF 19 78 YLRRVIRVL 16 37
YLPVCHVAL 15 43 VALIHMVVL 15 101 NISPSISWL 14 165 IIRGLFFTL 14 187
FSSVYMMTL 14 1 MPFISKLVL 13 21 ASSPFLLFL 13 30 DLRPERTYL 13 44
ALIHMVVLL 13 49 VVLLTMVFL 13 161 PINSIIRGL 13 191 YMMTLIQEL 13 207
QPQPLPKDL 13 7 LVLASQPTL 12 18 FFSASSPFL 12 19 FSASSPFLL 12 53
TMVFLSPQL 12 128 LSFPVSSSL 12 173 LSLFRDVFL 12 218 GKSHQHILL 12 41
CHVALIHMV 11 57 LSPQLFESL 11 71 FKYEASFYL 11 85 VLSICTTCL 11 120
TFHLFSWSL 11 143 SSNVTQINL 11 172 TLSLFRDVF 11 195 LIQELQEIL 11 203
LVPSQPQPL 11 239 FIISTSSTL 11 23 SPFLLFLDL 10 86 LSICTTCLL 10 89
CTTCLLGML 10 104 PSISWLVRF 10 115 KSTIFTFHL 10 121 FHLFSWSLS 10 162
INSIIRGLF 10 167 RGLFFTLSL 10 178 DVFLKQIML 10 184 IMLFSSVYM 10 217
RGKSHQHIL 10 220 SHQHILLPV 10 229 SFSVGMYKM 10 V2A-B1510-9mers:
251P5G2 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.
3 ASQPTLCSF 8 4 SQPTLCSFF 6 V3A-HLA-B1510-9mers: 251P5G2 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. 2
YLRRVIRDL 14 9 DLSICTTCL 11 V4A-HLA-B1510-9mers: 251P5G2 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. 3
CTTCLLDML 10 2 ICTTCLLDM 8 7 LLDMLQVVN 4 V12A-HLA-B1510-9mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 9 amino acids, and
the end position for each peptide is the start position plus eight.
1 ISPSISWL 16 4 PSISWLIML 10 5 SISWLIMLF 8 3 SPSISWLIM 7
V12B-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start
position plus eight. 670 KHGSNPVGL 24 624 SHHHVICEL 22 190
THVRCAQGL 21 625 HHHVICELL 21 764 SHKKEEDLL 21 1091 PHRDTTTSL 21
288 RHIPEILKF 19 339 SHKVIQCVF 19 916 IGDPGGVPL 17 541 IESKNKCGL 16
949 PGTPSLVRL 16 36 TWRKEPAVL 15 115 WERVVQRRL 15 161 TRAFQVVHL 15
781 EIAKLRLEL 15 938 THLPPREPR 15 946 RASPGTPSL 15 181 PPSRNSYRL 14
232 AEPPAHQRL 14 259 EEALGVGSL 14 300 ETGGGILGL 14 429 PRKDLIVML 14
460 GNSEVVQLL 14 556 VHEQKQEVV 14 591 CGSASIVNL 14 779 REEIAKLRL 14
807 IESVKEKLL 14 842 QHAQASVQQ 14 882 QHAQASVQQ 14 1051 GQDDRAGVL
14 1106 AGGVGPTTL 14 68 TTLTGHSAL 13 76 LSLSSSRAL 13 86 GSLPAFADL
13 233 EPPAHQRLL 13 254 QEQPSEEAL 13 273 HLIQCIPNL 13 279 PNLSYPLVL
13 298 EKETGGGIL 13 309 ELPATAARL 13 327 KEFEELVKL 13 335 LHSLSHKVI
13 366 GHSFLIMKE 13 374 ETSTKISGL 13 425 WGKVPRKDL 13 445 RDKQKRTAL
13 459 NGNSEVVQL 13 478 LDNKKRTAL 13 491 QCQEDECVL 13 511 QDEYGNTAL
13 577 LDRYGRTAL 13 592 GSASIVNLL 13 648 NSNPVITIL 13 717 YHSDEQNDT
13 793 KHQNQLREN 13 806 EIESVKEKL 13 817 TIQLNEEAL 13 867 EQEVAGFSL
13 932 GDQGPGTHL 13 1009 FDVSPAMRL 13 1069 CHTDTPPHR 13 15
AATGLWAAL 12 62 ARKEFSTTL 12 72 GHSALSLSS 12 104 SATPAGAFL 12 197
GLEAASANL 12 264 VGSLSVFQL 12 266 SLSVFQLHL 12 302 GGGILGLEL 12 324
MQIKEFEEL 12 330 EELVKLHSL 12 408 RYHVRREDL 12 461 NSEVVQLLL 12 467
LLLDRRCQL 12 470 DRRCQLNVL 12 493 QEDECVLML 12 494 EDECVLMLL 12 521
YAIYNEDKL 12 526 EDKLMAKAL 12 545 NKCGLTPLL 12 559 QKQEVVKFL 12 566
FLIKKKANL 12 569 KKKANLNAL 12 634 SDYKEKQML 12 654 TILNIKLPL 12 674
NPVGLPENL 12 721 EQNDTQKQL 12 753 AEKEMNSEL 12 777 MLREEIAKL 12 830
VAGFSLRQL 12 832 GFSLRQLGL 12 870 VAGFSLRQL 12 872 GFSLRQLGL 12 896
GHTEKTEQQ 12 904 QAQEQGAAL 12 1025 THQAFRDKD 12 1060 APKCRPGTL 12
1102 FHVSAGGVG 12 3 QHILLPTQA 11 5 ILLPTQATF 11 41 PAVLPCCNL 11 47
CNLEKGSWL 11 80 SSRALPGSL 11 105 ATPAGAFLL 11 146 CHQRRDAAC 11 167
VHLAPTAPD 11 209 PGRSSSCAL 11 235 PAHQRLLFL 11 286 VLRHIPEIL 11 312
ATAARLSGL 11 362 SEGYGHSFL 11 409 YHVRREDLD 11 411 VRREDLDKL 11 503
EHGADGNIQ 11 519 LHYAIYNED 11 527 DKLMAKALL 11 544 KNKCGLTPL 11 546
KCGLTPLLL 11 558 EQKQEVVKF 11 593 SASIVNLLL 11 604 NVDVSSQDL 11 687
SAGNGDDGL 11 735 TGISQDEIL 11 763 LSHKKEEDL 11 790 DETKHQNQL 11 812
EKLLKTIQL 11 827 KTKVAGFSL 11 843 HAQASVQQL 11 856 NHTEKTEQQ 11 883
HAQASVQQL 11 958 ASGARAAAL 11 988 GWILPVPTF 11 1027 QAFRDKDDL 11
1045 RHTKDLGQD 11 1082 PPHRHTTTL 11 1085 RHTTTLPHR 11
[1042]
41TABLE XXXI Pos 123456789 score V1A-HLA-B2705-9mers: 251P5G2 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. 80
RRVIRVLSI 24 110 VRFKWKSTI 24 34 ERTYLPVCH 20 216 CRGKSHQHI 19 83
IRVLSICTT 18 111 RFKWKSTIF 18 128 LSFPVSSSL 18 167 RGLFFTLSL 18 168
GLFFTLSLF 18 176 FRDVFLKQI 18 178 DVFLKQIML 18 179 VFLKQIMLF 18 1
MPFISKLVL 17 7 LVLASQPTL 17 59 PQLFESLNF 17 69 NDFKYEASF 17 23
SPFLLFLDL 16 65 LNFQNDFKY 16 101 NISPSISWL 16 104 PSISWLVRF 16 113
KWKSTIFTF 16 122 HLFSWSLSF 16 169 LFFTLSLFR 16 209 QPLPKDLCR 16 217
RGKSHQHIL 16 222 QHILLPVSF 16 17 SFFSASSPF 15 27 LFLDLRPER 15 44
ALIHMVVLL 15 48 MVVLLTMVF 15 53 TMVFLSPQL 15 54 MVFLSPQLF 15 63
ESLNFQNDF 15 78 YLRRVIRVL 15 143 SSNVTQINL 15 147 TQINLHVSK 15 165
IIRGLFFTL 15 177 RDVFLKQIM 15 191 YMMTLIQEL 15 228 VSFSVGMYK 15 239
FIISTSSTL 15 245 STLPWAYDR 15 10 ASQPTLFSF 14 21 ASSPFLLFL 14 24
PFLLFLDLR 14 30 DLRPERTYL 14 43 VALIHMVVL 14 49 VVLLTMVFL 14 57
LSPQLFESL 14 71 FKYEASFYL 14 72 KYEASFYLR 14 76 SFYLRRVIR 14 120
TFHLFSWSL 14 130 FPVSSSLIF 14 157 CSLFPINSI 14 161 PINSIIRGL 14 166
IRGLFFTLS 14 173 LSLFRDVFL 14 174 SLFRDVFLK 14 184 IMLFSSVYM 14 185
MLFSSVYMM 14 195 LIQELQEIL 14 218 GKSHQHILL 14 233 GMYKMDFII 14 18
FFSASSPFL 13 46 IHMVVLLTM 13 73 YEASFYLRR 13 103 SPSISWLVR 13 144
SNVTQINLH 13 148 QINLHVSKY 13 151 LHVSKYCSL 13 152 HVSKYCSLF 13 158
SLFPINSII 13 163 NSIIRGLFF 13 194 TLIQELQEI 13 213 KDLCRGKSH 13 215
LCRGKSHQH 13 229 SFSVGMYKM 13 8 VLASQPTLF 12 11 SQPTLFSFF 12 20
SASSPFLLF 12 29 LDLRPERTY 12 31 LRPERTYLP 12 75 ASFYLRRVI 12 79
LRRVIRVLS 12 85 VLSICTTCL 12 86 LSICTTCLL 12 89 CTTCLLGML 12 105
SISWLVRFK 12 107 SWLVRFKWK 12 115 KSTIFTFHL 12 116 STIFTFHLF 12 159
LFPINSIIR 12 162 INSIIRGLF 12 172 TLSLFRDVF 12 183 QIMLFSSVY 12 187
FSSVYMMTL 12 205 PSQPQPLPK 12 207 QPQPLPKDL 12 231 SVGMYKMDF 12 19
FSASSPFLL 11 37 YLPVCHVAL 11 39 PVCHVALIH 11 64 SLNFQNDFK 11 70
DFKYEASFY 11 88 ICTTCLLGM 11 94 LGMLQVVNI 11 98 QVVNISPSI 11 114
WKSTIFTFH 11 131 PVSSSLIFY 11 211 LPKDLCRGK 11 226 LPVSFSVGM 11
V2A-B2705-9mers: 251P5G2 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. 3 ASQPTLCSF 14 4 SQPTLCSFF 12 9 CSFFSASSP 7
V3A-HLA-B2705-9mers: 251P5G2 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. 4 RRVIRDLSI 24 7 IRDLSICTT 17 2 YLRRVIRDL 14 9
DLSICTTCL 12 3 LRRVIRDLS 11 V4A-HLA-B2705-9mers: 251P5G2 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. 3
CTTCLLDML 12 2 ICTTCLLDM 11 8 LDMLQVVNI 11 9 DMLQVVNIS 7 5
TCLLDMLQV 5 7 LLDMLQVVN 5 V12A-HLA-B2705-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 5
SISWLIMLF 15 4 PSISWLIML 14 9 LIMLFSSVY 12 2 ISPSISWLI 10 3
SPSISWLIM 10 V12B-HLA-B2705-9mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. 1015 MRLKSDSNR 29 62
ARKEFSTTL 26 785 LRLELDETK 26 210 GRSSSCALR 25 429 PRKDLIVML 25
1054 DRAGVLAPK 25 30 SRADPVTWR 24 288 RHIPEILKF 24 315 ARLSGLNSI 24
411 VRREDLDKL 24 412 RREDLDKLH 24 701 SRKPENQQF 24 835 LRQLGLAQH 24
875 LRQLGLAQH 24 1029 FRDKDDLPF 24 437 LRDTDMNKR 23 470 DRRCQLNVL
23 778 LREEIAKLR 23 1022 NRETHQAFR 23 161 TRAFQVVHL 22 287
LRHIPEILK 22 617 AREYAVSSH 22 139 SRDPSPPCH 21 148 QRRDAACLR 21 327
KEFEELVKL 21 578 DRYGRTALI 21 691 GDDGLIPQR 21 109 GAFLLGWER 20 183
SRNSYRLTH 20 273 HLIQCIPNL 20 988 GWILPVPTF 20 31 RADPVTWRK 19 155
LRAQGLTRA 19 445 RDKQKRTAL 19 471 RRCQLNVLD 19 566 FLIKKKANL 19 946
RASPGTPSL 19 1076 HRNADTPPH 19 1084 HRHTTTLPH 19 5 ILLPTQATF 18 121
RRLEVPRPQ 18 203 ANLPGAPGR 18 300 ETGGGILGL 18 423 AWWGKVPRK 18 430
RKDLIVMLR 18 581 GRTALILAV 18 610 QDLSGQTAR 18 777 MLREEIAKL 18 779
REEIAKLRL 18 932 GDQGPGTHL 18 971 GKNGRSPTK 18 11 ATFAAATGL 17 55
LSFPGTAAR 17 86 GSLPAFADL 17 114 GWERVVQRR 17 149 RRDAACLRA 17 156
RAQGLTRAF 17 176 GGAGCPPSR 17 197 GLEAASANL 17 238 QRLLFLPRA 17 262
LGVGSLSVF 17 316 RLSGLNSIM 17 341 KVIQCVFAK 17 378 KISGLIQEM 17 413
REDLDKLHR 17 538 GADIESKNK 17 544 KNKCGLTPL 17 558 EQKQEVVKF 17 562
EVVKFLIKK 17 591 CGSASIVNL 17 618 REYAVSSHH 17 648 NSNPVITIL 17 663
KVEEEIKKH 17 694 GLIPQRKSR 17 738 SQDEILTNK 17 786 RLELDETKH 17 832
GFSLRQLGL 17 872 GFSLRQLGL 17 905 AQEQGAALR 17 964 AALPPPTGK 17 974
GRSPTKQKS 17 1110 GPTTLGSNR 17 56 SFPGTAARK 16 74 SALSLSSSR 16 83
ALPGSLPAF 16 120 QRRLEVPRP 16 211 RSSSCALRY 16 219 YRSGPSVSS 16 281
LSYPLVLRH 16 291 PEILKFSEK 16 302 GGGILGLEL 16 321 NSIMQIKEF 16 324
MQIKEFEEL 16 444 KRDKQKRTA 16 460 GNSEVVQLL 16 463 EVVQLLLDR 16 473
CQLNVLDNK 16 482 KRTALIKAV 16 552 LLLGVHEQK 16 563 VVKFLIKKK 16 579
RYGRTALIL 16 592 GSASIVNLL 16 634 SDYKEKQML 16 662 LKVEEEIKK 16 674
NPVGLPENL 16 695 LIPQRKSRK 16 719 SDEQNDTQK 16 759 SELSLSHKK 16 790
DETKHQNQL 16 798 LRENKILEE 16 805 EEIESVKEK 16 808 ESVKEKLLK 16 812
EKLLKTIQL 16 865 AQEQEVAGF 16 949 PGTPSLVRL 16 955 VRLASGARA 16
1004 RRCPMFDVS 16 1009 FDVSPAMRL 16 1030 RDKDDLPFF 16 1085
RHTTTLPHR 16 1106 AGGVGPTTL 16 15 AATGLWAAL 15 36 TWRKEPAVL 15 41
PAVLPCCNL 15 47 CNLEKGSWL 15 49 LEKGSWLSF 15 68 TTLTGHSAL 15 115
WERVVQRRL 15 179 GCPPSRNSY 15 217 LRYRSGPSV 15 259 EEALGVGSL 15 330
EELVKLHSL 15 333 VKLHSLSHK 15 347 FAKKKNVDK 15 370 LIMKETSTK 15 374
ETSTKISGL 15 397 GDYDDSAFM 15 422 AAWWGKVPR 15 464 VVQLLLDRR 15 474
QLNVLDNKK 15 475 LNVLDNKKR 15 478 LDNKKRTAL 15 511 QDEYGNTAL 15 528
KLMAKALLL 15 571 KANLNALDR 15 624 SHHHVICEL 15 635 DYKEKQMLK 15 651
PVITILNIK 15 652 VITILNIKL 15 654 TILNIKLPL 15 740 DEILTNKQK 15 753
AEKEMNSEL 15 755 KEMNSELSL 15 770 DLLRENSML 15 781 EIAKLRLEL 15 794
HQNQLRENK 15 799 RENKILEEI 15 803 ILEEIESVK 15 819 QLNEEALTK 15 846
ASVQQLCYK 15 886 ASVQQLCYK 15 892 CYKWGHTEK 15 916 IGDPGGVPL 15 935
GPGTHLPPR 15 994 PTFSSGSFL 15 1047 TKDLGQDDR 15 1051 GQDDRAGVL 15
1077 RNADTPPHR 15 23 LTTVSNPSR 14 37 WRKEPAVLP 14 70 LTGHSALSL 14
76 LSLSSSRAL 14 105 ATPAGAFLL 14 113 LGWERVVQR 14 126 PRPQAAPAT 14
181 PPSRNSYRL 14 185 NSYRLTHVR 14 231 PAEPPAHQR 14 232 AEPPAHQRL 14
235 PAHQRLLFL 14 244 PRAPQAVSG 14 264 VGSLSVFQL 14 265 GSLSVFQLH 14
279 PNLSYPLVL 14 308 LELPATAAR 14 309 ELPATAARL 14 318 SGLNSIMQI 14
319 GLNSIMQIK 14 326 IKEFEELVK 14 339 SHKVIQCVF 14 342 VIQCVFAKK 14
343 IQCVFAKKK 14 351 KNVDKWDDF 14 407 PRYHVRRED 14 408 RYHVRREDL 14
436 MLRDTDMNK 14 441 DMNKRDKQK 14 447 KQKRTALHL 14 449 KRTALHLAS 14
459 NGNSEVVQL 14 461 NSEVVQLLL 14 521 YAIYNEDKL 14 527 DKLMAKALL 14
536 LYGADIESK 14 541 IESKNKCGL 14 545 NKCGLTPLL 14 546 KCGLTPLLL 14
557 HEQKQEVVK 14 569 KKKANLNAL 14 572 ANLNALDRY 14 577 LDRYGRTAL 14
629 ICELLSDYK 14 655 ILNIKLPLK 14 692 DDGLIPQRK 14 747 QKQIEVAEK 14
758 NSELSLSHK 14 765 HKKEEDLLR 14 772 LRENSMLRE 14 776 SMLREEIAK 14
796 NQLRENKIL 14 806 EIESVKEKL 14 828 TKVAGFSLR 14 849 QQLCYKWNH 14
868 QEVAGFSLR 14 931 AGDQGPGTH 14 938 THLPPREPR 14 948 SPGTPSLVR 14
967 PPPTGKNGR 14 996 FSSGSFLGR 14 1003 GRRCPMFDV 14 1007 PMFDVSPAM
14 1027 QAFRDKDDL 14 1038 FKTQQSPRH 14 1062 KCRPGTLCH 14 1093
RDTTTSLPH 14
[1043]
42TABLE XXXII Pos 123456789 score V1A-HLA-B2709-9mers: 251P5G2 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. 80
RRVIRVLSI 25 110 VRFKWKSTI 20 176 FRDVFLKQI 19 216 CRGKSHQHI 18 167
RGLFFTLSL 17 35 RTYLPVCHV 15 217 RGKSHQHIL 15 44 ALIHMVVLL 14 1
MPFISKLVL 13 7 LVLASQPTL 13 21 ASSPFLLFL 13 23 SPFLLFLDL 13 32
RPERTYLPV 13 43 VALIHMVVL 13 49 VVLLTMVFL 13 53 TMVFLSPQL 13 115
KSTIFTFHL 13 128 LSFPVSSSL 13 168 GLFFTLSLF 13 173 LSLFRDVFL 13 177
RDVFLKQIM 13 185 MLFSSVYMM 13 218 GKSHQHILL 13 233 GMYKMDFII 13 34
ERTYLPVCH 12 59 PQLFESLNF 12 71 FKYEASFYL 12 77 FYLRRVIRV 12 79
LRRVIRVLS 12 83 IRVLSICTT 12 91 TCLLGMLQV 12 104 PSISWLVRF 12 111
RFKWKSTIF 12 122 HLFSWSLSF 12 151 LHVSKYCSL 12 158 SLFPINSII 12 161
PINSIIRGL 12 178 DVFLKQIML 12 184 IMLFSSVYM 12 224 ILLPVSFSV 12 239
FIISTSSTL 12 V2A-B2709-9mers: 251P5G2 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. 3 ASQPTLCSF 10 4 SQPTLCSFF 8
V3A-B2709-9mers: 251P5G2 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. 4 RRVIRDLSI 24 3 LRRVIRDLS 11 7 IRDLSICTT 11
V4A-HLA-B2709-9mers: 251P5G2 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. 5 TCLLDMLQV 13 2 ICTTCLLDM 11 3 CTTCLLDML 11 8
LDMLQVVNI 11 6 CLLDMLQVV 10 V12A-HLA-B2709-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 4
PSISWLIML 12 2 ISPSISWLI 11 3 SPSISWLIM 9 8 WLIMLFSSV 9 5 SISWLIMLF
8 V12B-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO:
25; each start position is specified, the length of peptide is 9
amino acids, and the end position for each peptide is the start
position plus eight. 581 GRTALILAV 23 161 TRAFQVVHL 22 315
ARLSGLNSI 22 62 ARKEFSTTL 21 217 LRYRSGPSV 21 411 VRREDLDKL 21 429
PRKDLIVML 21 482 KRTALIKAV 21 1003 GRRCPMFDV 21 470 DRRCQLNVL 20
578 DRYGRTALI 20 701 SRKPENQQF 20 1029 FRDKDDLPF 20 420 HRAAWWGKV
18 86 GSLPAFADL 16 121 RRLEVPRPQ 16 149 RRDAACLRA 16 579 RYGRTALIL
16 592 GSASIVNLL 16 779 REEIAKLRL 16 946 RASPGTPSL 16 288 RHIPEILKF
15 327 KEFEELVKL 15 471 RRCQLNVLD 15 1004 RRCPMFDVS 15 11 ATFAAATGL
14 117 RVVQRRLEV 14 197 GLEAASANL 14 210 GRSSSCALR 14 238 QRLLFLPRA
14 279 PNLSYPLVL 14 302 GGGILGLEL 14 397 GDYDDSAFM 14 408 RYHVRREDL
14 412 RREDLDKLH 14 445 RDKQKRTAL 14 447 KQKRTALHL 14 449 KRTALHLAS
14 459 NGNSEVVQL 14 460 GNSEVVQLL 14 528 KLMAKALLL 14 548 GLTPLLLGV
14 654 TILNIKLPL 14 670 KHGSNPVGL 14 832 GFSLRQLGL 14 872 GFSLRQLGL
14 949 PGTPSLVRL 14 974 GRSPTKQKS 14 988 GWILPVPTF 14 1009
FDVSPAMRL 14 1030 RDKDDLPFF 14 1051 GQDDRAGVL 14 76 LSLSSSRAL 13
120 QRRLEVPRP 13 187 YRLTHVRCA 13 232 AEPPAHQRL 13 244 PRAPQAVSG 13
273 HLIQCIPNL 13 426 GKVPRKDLI 13 467 LLLDRRCQL 13 515 GNTALHYAI 13
546 KCGLTPLLL 13 614 GQTAREYAV 13 755 KEMNSELSL 13 799 RENKILEEI 13
812 EKLLKTIQL 13 916 IGDPGGVPL 13 932 GDQGPGTHL 13 955 VRLASGARA 13
994 PTFSSGSFL 13 1015 MRLKSDSNR 13 1027 QAFRDKDDL 13 5 ILLPTQATF 12
15 AATGLWAAL 12 37 WRKEPAVLP 12 41 PAVLPCCNL 12 47 CNLEKGSWL 12 68
TTLTGHSAL 12 70 LTGHSALSL 12 104 SATPAGAFL 12 105 ATPAGAFLL 12 110
AFLLGWERV 12 126 PRPQAAPAT 12 139 SRDPSPPCH 12 147 HQRRDAACL 12 152
AACLRAQGL 12 156 RAQGLTRAF 12 158 QGLTRAFQV 12 159 GLTRAFQVV 12 181
PPSRNSYRL 12 183 SRNSYRLTH 12 184 RNSYRLTHV 12 190 THVRCAQGL 12 264
VGSLSVFQL 12 309 ELPATAARL 12 316 RLSGLNSIM 12 330 EELVKLHSL 12 364
GYGHSFLIM 12 407 PRYHVRRED 12 444 KRDKQKRTA 12 493 QEDECVLML 12 527
DKLMAKALL 12 544 KNKCGLTPL 12 566 FLIKKKANL 12 569 KKKANLNAL 12 591
CGSASIVNL 12 617 AREYAVSSH 12 634 SDYKEKQML 12 674 NPVGLPENL 12 698
QRKSRKPEN 12 735 TGISQDEIL 12 770 DLLRENSML 12 772 LRENSMLRE 12 778
LREEIAKLR 12 785 LRLELDETK 12 790 DETKHQNQL 12 796 NQLRENKIL 12 802
KILEEIESV 12 827 KTKVAGFSL 12 843 HAQASVQQL 12 883 HAQASVQQL 12 942
PREPRASPG 12 958 ASGARAAAL 12 961 ARAAALPPP 12 975 RSPTKQKSV 12
1076 HRNADTPPH 12 18 GLWAALTTV 11 30 SRADPVTWR 11 115 WERVVQRRL 11
116 ERVVQRRLE 11 148 QRRDAACLR 11 155 LRAQGLTRA 11 192 VRCAQGLEA 11
209 PGRSSSCAL 11 219 YRSGPSVSS 11 235 PAHQRLLFL 11 254 QEQPSEEAL 11
259 EEALGVGSL 11 266 SLSVFQLHL 11 277 CIPNLSYPL 11 285 LVLRHIPEI 11
286 VLRHIPEIL 11 300 ETGGGILGL 11 312 ATAARLSGL 11 318 SGLNSIMQI 11
324 MQIKEFEEL 11 334 KLHSLSHKV 11 345 CVFAKKKNV 11 351 KNVDKWDDF 11
353 VDKWDDFCL 11 427 KVPRKDLIV 11 437 LRDTDMNKR 11 461 NSEVVQLLL 11
491 QCQEDECVL 11 521 YAIYNEDKL 11 522 AIYNEDKLM 11 526 EDKLMAKAL 11
533 ALLLYGADI 11 541 IESKNKCGL 11 545 NKCGLTPLL 11 555 GVHEQKQEV 11
559 QKQEVVKFL 11 593 SASIVNLLL 11 597 VNLLLEQNV 11 599 LLLEQNVDV 11
625 HHHVICELL 11 648 NSNPVITIL 11 650 NPVITILNI 11 652 VITILNIKL 11
721 EQNDTQKQL 11 742 ILTNKQKQI 11 753 AEKEMNSEL 11 781 EIAKLRLEL 11
798 LRENKILEE 11 806 EIESVKEKL 11 807 IESVKEKLL 11 830 VAGFSLRQL 11
835 LRQLGLAQH 11 870 VAGFSLRQL 11 875 LRQLGLAQH 11 912 LRSQIGDPG 11
945 PRASPGTPS 11 983 VCDSSGWIL 11 1007 PMFDVSPAM 11 1044 PRHTKDLGQ
11 1060 APKCRPGTL 11 1084 HRHTTTLPH 11 1106 AGGVGPTTL 11
[1044]
43TABLE XXXIII Pos 123456789 score V1A-HLA-B4402-9mers: 251P5G2
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. 21
ASSPFLLFL 19 44 ALIHMVVLL 19 101 NISPSISWL 18 10 ASQPTLFSF 17 116
STIFTFHLF 17 23 SPFLLFLDL 16 75 ASFYLRRVI 16 78 YLRRVIRVL 16 163
NSIIRGLFF 16 197 QELQEILVP 16 200 QEILVPSQP 16 207 QPQPLPKDL 16 222
QHILLPVSF 16 20 SASSPFLLF 15 54 MVFLSPQLF 15 63 ESLNFQNDF 15 86
LSICTTCLL 15 104 PSISWLVRF 15 113 KWKSTIFTF 15 128 LSFPVSSSL 15 158
SLFPINSII 15 161 PINSIIRGL 15 179 VFLKQIMLF 15 191 YMMTLIQEL 15 243
TSSTLPWAY 15 1 MPFISKLVL 14 11 SQPTLFSFF 14 29 LDLRPERTY 14 30
DLRPERTYL 14 37 YLPVCHVAL 14 62 FESLNFQND 14 65 LNFQNDFKY 14 100
VNISPSISW 14 162 INSIIRGLF 14 168 GLFFTLSLF 14 172 TLSLFRDVF 14 178
DVFLKQIML 14 239 FIISTSSTL 14 17 SFFSASSPF 13 33 PERTYLPVC 13 43
VALIHMVVL 13 48 MVVLLTMVF 13 49 VVLLTMVFL 13 69 NDFKYEASF 13 106
ISWLVRFKW 13 122 HLFSWSLSF 13 131 PVSSSLIFY 13 148 QINLHVSKY 13 157
CSLFPINSI 13 165 IIRGLFFTL 13 167 RGLFFTLSL 13 173 LSLFRDVFL 13 176
FRDVFLKQI 13 183 QIMLFSSVY 13 218 GKSHQHILL 13 7 LVLASQPTL 12 8
VLASQPTLF 12 19 FSASSPFLL 12 57 LSPQLFESL 12 59 PQLFESLNF 12 85
VLSICTTCL 12 94 LGMLQVVNI 12 115 KSTIFTFHL 12 141 VASSNVTQI 12 143
SSNVTQINL 12 152 HVSKYCSLF 12 187 FSSVYMMTL 12 194 TLIQELQEI 12 203
LVPSQPQPL 12 227 PVSFSVGMY 12 241 ISTSSTLPW 12 18 FFSASSPFL 11 53
TMVFLSPQL 11 70 DFKYEASFY 11 73 YEASFYLRR 11 80 RRVIRVLSI 11 89
CTTCLLGML 11 110 VRFKWKSTI 11 118 IFTFHLFSW 11 120 TFHLFSWSL 11 129
SFPVSSSLI 11 130 FPVSSSLIF 11 217 RGKSHQHIL 11 231 SVGMYKMDF 11 38
LPVCHVALI 10 71 FKYEASFYL 10 98 QVVNISPSI 10 111 RFKWKSTIF 10 151
LHVSKYCSL 10 154 SKYCSLFPI 10 188 SSVYMMTLI 10 195 LIQELQEIL 10 232
VGMYKMDFI 9 V2A-B4402-9mers: 251P5G2 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. 3 ASQPTLCSF 17 4 SQPTLCSFF 13
V3A-HLA-B4402-9mers: 251P5G2 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. 2 YLRRVIRDL 15 9 DLSICTTCL 12 4 RRVIRDLSI 10
V4A-HLA-B4402-9mers: 251P5G2 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. 8 LDMLQVVNI 12 3 CTTCLLDML 11 1 SICTTCLLD 4 5
TCLLDMLQV 4 6 CLLDMLQVV 4 V12A-HLA-B4402-9mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 9 amino acids, and the end
position for each peptide is the start position plus eight. 1
ISPSISWL 16 5 SISWLIMLF 16 4 PSISWLIML 15 9 LIMLFSSVY 13 2
ISPSISWLI 11 V12B-HLA-B4402-9mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 9 amino acids, and the end position for each
peptide is the start position plus eight. 232 AEPPAHQRL 27 327
KEFEELVKL 26 254 QEQPSEEAL 25 330 EELVKLHSL 25 259 EEALGVGSL 24 755
KEMNSELSL 24 807 IESVKEKLL 24 493 QEDECVLML 23 753 AEKEMNSEL 23 49
LEKGSWLSF 22 541 IESKNKCGL 22 790 DETKHQNQL 22 115 WERVVQRRL 21 288
RHIPEILKF 21 362 SEGYGHSFL 21 779 REEIAKLRL 21 799 RENKILEEI 20 297
SEKETGGGI 19 321 NSIMQIKEF 19 502 LEHGADGNI 19 83 ALPGSLPAF 18 526
EDKLMAKAL 18 780 EEIAKLRLE 18 805 EEIESVKEK 18 123 LEVPRPQAA 17 300
ETGGGILGL 17 308 LELPATAAR 17 374 ETSTKISGL 17 648 NSNPVITIL 17 740
DEILTNKQK 17 812 EKLLKTIQL 17 958 ASGARAAAL 17 11 ATFAAATGL 16 15
AATGLWAAL 16 64 KEFSTTLTG 16 105 ATPAGAFLL 16 152 AACLRAQGL 16 179
GCPPSRNSY 16 273 HLIQCIPNL 16 348 AKKKNVDKW 16 569 KKKANLNAL 16 572
ANLNALDRY 16 721 EQNDTQKQL 16 988 GWILPVPTF 16 1060 APKCRPGTL 16 5
ILLPTQATF 15 86 GSLPAFADL 15 156 RAQGLTRAF 15 233 EPPAHQRLL 15 291
PEILKFSEK 15 312 ATAARLSGL 15 318 SGLNSIMQI 15 429 PRKDLIVML 15 467
LLLDRRCQL 15 506 ADGNIQDEY 15 513 EYGNTALHY 15 521 YAIYNEDKL 15 528
KLMAKALLL 15 546 KCGLTPLLL 15 558 EQKQEVVKF 15 591 CGSASIVNL 15 647
ENSNPVITI 15 666 EEIKKHGSN 15 701 SRKPENQQF 15 768 EEDLLRENS 15 787
LELDETKHQ 15 796 NQLRENKIL 15 821 NEEALTKTK 15 916 IGDPGGVPL 15 946
RASPGTPSL 15 949 PGTPSLVRL 15 1042 QSPRHTKDL 15 1051 GQDDRAGVL 15
1106 AGGVGPTTL 15 62 ARKEFSTTL 14 68 TTLTGHSAL 14 76 LSLSSSRAL 14
101 SEQSATPAG 14 103 QSATPAGAF 14 104 SATPAGAFL 14 264 VGSLSVFQL 14
279 PNLSYPLVL 14 299 KETGGGILG 14 309 ELPATAARL 14 315 ARLSGLNSI 14
324 MQIKEFEEL 14 384 QEMGSGKSN 14 388 SGKSNVGTW 14 416 LDKLHRAAW 14
447 KQKRTALHL 14 459 NGNSEVVQL 14 462 SEVVQLLLD 14 494 EDECVLMLL 14
512 DEYGNTALH 14 545 NKCGLTPLL 14 592 GSASIVNLL 14 593 SASIVNLLL 14
621 AVSSHHHVI 14 624 SHHHVICEL 14 637 KEKQMLKIS 14 646 SENSNPVIT 14
654 TILNIKLPL 14 670 KHGSNPVGL 14 731 EEQNTGISQ 14 759 SELSLSHKK 14
767 KEEDLLREN 14 777 MLREEIAKL 14 781 EIAKLRLEL 14 817 TIQLNEEAL 14
822 EEALTKTKV 14 832 GFSLRQLGL 14 865 AQEQEVAGF 14 872 GFSLRQLGL 14
29 PSRADPVTW 13 39 KEPAVLPCC 13 58 PGTAARKEF 13 181 PPSRNSYRL 13
198 LEAASANLP 13 211 RSSSCALRY 13 234 PPAHQRLLF 13 235 PAHQRLLFL 13
262 LGVGSLSVF 13 266 SLSVFQLHL 13 282 SYPLVLRHI 13 285 LVLRHIPEI 13
298 EKETGGGIL 13 335 LHSLSHKVI 13 339 SHKVIQCVF 13 396 WGDYDDSAF 13
405 MEPRYHVRR 13 413 REDLDKLHR 13 417 DKLHRAAWW 13 425 WGKVPRKDL 13
426 GKVPRKDLI 13 445 RDKQKRTAL 13 460 GNSEVVQLL 13 461 NSEVVQLLL 13
470 DRRCQLNVL 13 478 LDNKKRTAL 13 525 NEDKLMAKA 13 529 LMAKALLLY 13
533 ALLLYGADI 13 559 QKQEVVKFL 13 566 FLIKKKANL 13 577 LDRYGRTAL 13
579 RYGRTALIL 13 630 CELLSDYKE 13 650 NPVITILNI 13 664 VEEEIKKHG 13
665 EEEIKKHGS 13 674 NPVGLPENL 13 679 PENLTNGAS 13 688 AGNGDDGLI 13
715 EEYHSDEQN 13 720 DEQNDTQKQ 13 730 SEEQNTGIS 13 735 TGISQDEIL 13
806 EIESVKEKL 13 830 VAGFSLRQL 13 858 TEKTEQQAQ 13 870 VAGFSLRQL 13
898 TEKTEQQAQ 13 906 QEQGAALRS 13 925 SEGGTAAGD 13 1021 SNRETHQAF
13 1027 QAFRDKDDL 13 1082 PPHRHTTTL 13
[1045]
44TABLE XXXIV Pos 123456789 score V1A-HLA-B5101-9mers: 251P5G2 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. 38
LPVCHVALI 24 141 VASSNVTQI 24 247 LPWAYDRGV 24 1 MPFISKLVL 23 43
VALIHMVVL 23 74 EASFYLRRV 22 94 LGMLQVVNI 21 32 RPERTYLPV 20 23
SPFLLFLDL 19 207 QPQPLPKDL 18 232 VGMYKMDFI 18 92 CLLGMLQVV 16 154
SKYCSLFPI 16 233 GMYKMDFII 16 35 RTYLPVCHV 15 75 ASFYLRRVI 15 77
FYLRRVIRV 15 78 YLRRVIRVL 15 103 SPSISWLVR 15 110 VRFKWKSTI 15 160
FPINSIIRG 15 167 RGLFFTLSL 15 7 LVLASQPTL 14 157 CSLFPINSI 14 209
QPLPKDLCR 14 211 LPKDLCRGK 14 224 ILLPVSFSV 14 9 LASQPTLFS 13 71
FKYEASFYL 13 80 RRVIRVLSI 13 130 FPVSSSLIF 13 171 FTLSLFRDV 13 176
FRDVFLKQI 13 178 DVFLKQIML 13 216 CRGKSHQHI 13 226 LPVSFSVGM 13 20
SASSPFLLF 12 91 TCLLGMLQV 12 129 SFPVSSSLI 12 145 NVTQINLHV 12 158
SLFPINSII 12 165 IIRGLFFTL 12 188 SSVYMMTLI 12 194 TLIQELQEI 12 217
RGKSHQHIL 12 30 DLRPERTYL 11 42 HVALIHMVV 11 49 VVLLTMVFL 11 102
ISPSISWLV 11 128 LSFPVSSSL 11 133 SSSLIFYTV 11 173 LSLFRDVFL 11 196
IQELQEILV 11 204 VPSQPQPLP 11 V2A-B5101-9mers: 251P5G2 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. 2 LASQPTLCS 13 5
QPTLCSFFS 10 V3A-HLA-B5101-9mers 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. 2 YLRRVIRDL 12 4 RRVIRDLSI 11 9
DLSICTTCL 11 1 FYLRRVIRD 7 7 IRDLSICTT 6 V4A-HLA-B5101-9mers:
251P5G2 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.
6 CLLDMLQVV 16 8 LDMLQVVNI 15 5 TCLLDMLQV 12 9 DMLQVVNIS 11 3
CTTCLLDML 7 V12A-HLA-B5101-9mers: 251P5G2 Each peptide is a portion
of SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 2 ISPSISWLI 13 3 SPSISWLIM 12 8
WLIMLFSSV 9 4 PSISWLIML 8 9 LIMLFSSVY 5 6 ISWLIMLFS 4 7 SWLIMLFSS 4
5 SISWLIMLF 1 V12B-B5101-9mers: 251P5G2 Each peptide is a portion
of SEQ ID NO: 25; each start position is specified, the length of
peptide is 9 amino acids, and the end position for each peptide is
the start position plus eight. 660 LPLKVEEEI 27 650 NPVITILNI 24
256 QPSEEALGV 22 457 SANGNSEVV 22 620 YAVSSHHHV 22 278 IPNLSYPLV 21
363 EGYGHSFLI 21 318 SGLNSIMQI 20 521 YAIYNEDKL 20 593 SASIVNLLL 20
830 VAGFSLRQL 20 840 LAQHAQASV 20 843 HAQASVQQL 20 870 VAGFSLRQL 20
880 LAQHAQASV 20 883 HAQASVQQL 20 233 EPPAHQRLL 19 578 DRYGRTALI 19
674 NPVGLPENL 19 918 DPGGVPLSE 19 1060 APKCRPGTL 19 1082 PPHRHTTTL
19 158 QGLTRAFQV 18 235 PAHQRLLFL 18 371 IMKETSTKI 18 823 EALTKTKVA
18 904 QAQEQGAAL 18 15 AATGLWAAL 17 152 AACLRAQGL 17 181 PPSRNSYRL
17 335 LHSLSHKVI 17 428 VPRKDLIVM 17 688 AGNGDDGLI 17 908 QGAALRSQI
17 1027 QAFRDKDDL 17 1050 LGQDDRAGV 17 1112 TTLGSNREI 17 21
AALTTVSNP 16 33 DPVTWRKEP 16 41 PAVLPCCNL 16 57 FPGTAARKE 16 104
SATPAGAFL 16 111 FLLGWERVV 16 243 LPRAPQAVS 16 264 VGSLSVFQL 16 282
SYPLVLRHI 16 459 NGNSEVVQL 16 484 TALIKAVQC 16 517 TALHYAIYN 16 591
CGSASIVNL 16 599 LLLEQNVDV 16 616 TAREYAVSS 16 636 YKEKQMLKI 16 687
SAGNGDDGL 16 696 IPQRKSRKP 16 729 LSEEQNTGI 16 810 VKEKLLKTI 16 946
RASPGTPSL 16 949 PGTPSLVRL 16 1106 AGGVGPTTL 16 44 LPCCNLEKG 15 130
AAPATSATP 15 162 RAFQVVHLA 15 169 LAPTAPDGG 15 217 LRYRSGPSV 15 260
EALGVGSLS 15 285 LVLRHIPEI 15 306 LGLELPATA 15 310 LPATAARLS 15 347
FAKKKNVDK 15 470 DRRCQLNVL 15 479 DNKKRTALI 15 550 TPLLLGVHE 15 575
NALDRYGRT 15 583 TALILAVCC 15 645 SSENSNPVI 15 647 ENSNPVITI 15 656
LNIKLPLKV 15 693 DGLIPQRKS 15 916 IGDPGGVPL 15 922 VPLSEGGTA 15 940
LPPREPRAS 15 944 EPRASPGTP 15 948 SPGTPSLVR 15 966 LPPPTGKNG 15 985
DSSGWILPV 15 1081 TPPHRHTTT 15 1090 LPHRDTTTS 15 1105 SAGGVGPTT 15
13 FAAATGLWA 14 18 GLWAALTTV 14 28 NPSRADPVT 14 61 AARKEFSTT 14 89
PAFADLPRS 14 106 TPAGAFLLG 14 113 LGWERVVQR 14 127 RPQAAPATS 14 131
APATSATPS 14 141 DPSPPCHQR 14 151 DAACLRAQG 14 205 LPGAPGRSS 14 230
SPAEPPAHQ 14 262 LGVGSLSVF 14 270 FQLHLIQCI 14 279 PNLSYPLVL 14 283
YPLVLRHIP 14 315 ARLSGLNSI 14 338 LSHKVIQCV 14 406 EPRYHVRRE 14 421
RAAWWGKVP 14 451 TALHLASAN 14 455 LASANGNSE 14 530 MAKALLLYG 14 742
ILTNKQKQI 14 910 AALRSQIGD 14 951 TPSLVRLAS 14 964 AALPPPTGK 14 968
PPTGKNGRS 14 976 SPTKQKSVC 14 991 LPVPTFSSG 14 1035 LPFFKTQQS 14
1059 LAPKCRPGT 14 1078 NADTPPHRH 14 1099 LPHFHVSAG 14 10 QATFAAATG
13 17 TGLWAALTT 13 31 RADPVTWRK 13 82 RALPGSLPA 13 84 LPGSLPAFA 13
88 LPAFADLPR 13 94 LPRSCPESE 13 107 PAGAFLLGW 13 143 SPPCHQRRD 13
172 TAPDGGAGC 13 209 PGRSSSCAL 13 215 CALRYRSGP 13 245 RAPQAVSGP 13
246 APQAVSGPQ 13 252 GPQEQPSEE 13 267 LSVFQLHLI 13 297 SEKETGGGI 13
302 GGGILGLEL 13 327 KEFEELVKL 13 386 MGSGKSNVG 13 402 SAFMEPRYH 13
425 WGKVPRKDL 13 502 LEHGADGNI 13 533 ALLLYGADI 13 556 VHEQKQEVV 13
559 QKQEVVKFL 13 621 AVSSHHHVI 13 644 ISSENSNPV 13 676 VGLPENLTN 13
678 LPENLTNGA 13 709 FPDTENEEY 13 735 TGISQDEIL 13 752 VAEKEMNSE 13
770 DLLRENSML 13 782 IAKLRLELD 13 795 QNQLRENKI 13 802 KILEEIESV 13
864 QAQEQEVAG 13 957 LASGARAAA 13 960 GARAAALPP 13 963 AAALPPPTG 13
967 PPPTGKNGR 13 1055 RAGVLAPKC 13 1064 RPGTLCHTD 13 1073 TPPHRNADT
13 1074 PPHRNADTP 13
[1046]
45TABLE XXXV Pos 1234567890 score V1A-HLA-A0201-10mers: 251P5G2
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 start position plus nine. 37
YLPVCHVALI 26 93 LLGMLQVVNI 26 56 FLSPQLFESL 25 6 KLVLASQPTL 24 164
SIIRGLFFTL 24 202 ILVPSQPQPL 24 43 VALIHMVVLL 23 44 ALIHMVVLLT 23
127 SLSFPVSSSL 23 150 NLHVSKYCSL 22 172 TLSLFRDVFL 22 194
TLIQELQEIL 22 195 LIQELQEILV 22 223 HILLPVSFSV 22 20 SASSPFLLFL 21
45 LIHMVVLLTM 21 85 VLSICTTCLL 21 135 SLIFYTVASS 21 246 TLPWAYDRGV
21 87 SICTTCLLGM 20 101 NISPSISWLV 20 184 IMLFSSVYMM 20 193
MTLIQELQEI 20 82 VIRVLSICTT 19 225 LLPVSFSVGM 19 42 HVALIHMVVL 18
52 LTMVFLSPQL 18 73 YEASFYLRRV 18 119 FTFHLFSWSL 18 140 TVASSNVTQI
18 160 FPINSIIRGL 18 190 VYMMTLIQEL 18 3 FISKLVLASQ 17 36
TYLPVCHVAL 17 46 IHMVVLLTMV 17 76 SFYLRRVIRV 17 77 FYLRRVIRVL 17 79
LRRVIRVLSI 17 100 VNISPSISWL 17 156 YCSLFPINSI 17 25 FLLFLDLRPE 16
26 LLFLDLRPER 16 40 VCHVALIHMV 16 48 MVVLLTMVFL 16 50 VLLTMVFLSP 16
84 RVLSICTTCL 16 91 TCLLGMLQVV 16 92 CLLGMLQVVN 16 174 SLFRDVFLKQ
16 180 FLKQIMLFSS 16 198 ELQEILVPSQ 16 219 KSHQHILLPV 16 224
ILLPVSFSVG 16 14 TLFSFFSASS 15 31 LRPERTYLPV 15 51 LLTMVFLSPQ 15 88
ICTTCLLGML 15 90 TTCLLGMLQV 15 108 WLVRFKWKST 15 109 LVRFKWKSTI 15
117 TIFTFHLFSW 15 122 HLFSWSLSFP 15 158 SLFPINSIIR 15 166
IRGLFFTLSL 15 183 QIMLFSSVYM 15 185 MLFSSVYMMT 15 8 VLASQPTLFS 14
17 SFFSASSPFL 14 29 LDLRPERTYL 14 64 SLNFQNDFKY 14 78 YLRRVIRVLS 14
96 MLQVVNISPS 14 132 VSSSLIFYTV 14 175 LFRDVFLKQI 14 181 LKQIMLFSSV
14 186 LFSSVYMMTL 14 231 SVGMYKMDFI 14 28 FLDLRPERTY 13 60
QLFESLNFQN 13 95 GMLQVVNISP 13 97 LQVVNISPSI 13 128 LSFPVSSSLI 13
137 IFYTVASSNV 13 144 SNVTQINLHV 13 238 DFIISTSSTL 13 9 LASQPTLFSF
12 22 SSPFLLFLDL 12 30 DLRPERTYLP 12 105 SISWLVRFKW 12 123
LFSWSLSFPV 12 136 LIFYTVASSN 12 142 ASSNVTQINL 12 165 IIRGLFFTLS 12
168 GLFFTLSLFR 12 215 LCRGKSHQHI 12 V2A-A0201-10mers: 251P5G2 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. 2
VLASQPTLCS 14 8 TLCSFFSASS 14 3 LASQPTLCSF 12 1 LVLASQPTLC 8
V3A-A0201-10mers: 251P5G2 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. 7 VIRDLSICTT 20 10 DLSICTTCLL 19 2 FYLRRVIRDL
17 4 LRRVIRDLSI 13 3 YLRRVIRDLS 12 9 RDLSICTTCL 12 6 RVIRDLSICT 10
V4A-A0201-10mers: 251P5G2 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. 8 LLDMLQVVNI 26 2 SICTTCLLDM 19 6 TCLLDMLQVV 16
3 ICTTCLLDML 15 5 TTCLLDMLQV 15 7 CLLDMLQVVN 15
V12A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. 2 NISPSISWLI 18 10 LIMLFSSVYM 17 4 SPSISWLIML
16 8 SWLIMLFSSV 16 9 WLIMLFSSVY 12 6 SISWLIMLFS 11 7 ISWLIMLFSS 9
V12B-HLA-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start
position plus nine. 777 SMLREEIAKL 30 840 GLAQHAQASV 27 880
GLAQHAQASV 27 338 SLSHKVIQCV 26 656 ILNIKLPLKV 26 267 SLSVFQLHLI 25
70 TLTGHSALSL 24 217 ALRYRSGPSV 24 599 NLLLEQNVDV 24 76 ALSLSSSRAL
23 305 GILGLELPAT 23 469 LLDRRCQLNV 23 577 ALDRYGRTAL 23 820
QLNEEALTKT 23 830 KVAGFSLRQL 23 161 LTRAFQVVHL 22 285 PLVLRHIPEI 22
324 IMQIKEFEEL 22 467 QLLLDRRCQL 22 478 VLDNKKRTAL 22 763
SLSHKKEEDL 22 1050 DLGQDDRAGV 22 15 AAATGLWAAL 21 315 AARLSGLNSI 21
371 LIMKETSTKI 21 536 LLYGADIESK 21 660 KLPLKVEEEI 21 729
QLSEEQNTGI 21 916 QIGDPGGVPL 21 958 LASGARAAAL 21 1059 VLAPKCRPGT
21 6 ILLPTQATFA 20 84 ALPGSLPAFA 20 306 ILGLELPATA 20 411
HVRREDLDKL 20 600 LLLEQNVDVS 20 644 KISSENSNPV 20 810 SVKEKLLKTI 20
1106 SAGGVGPTTL 20 264 GVGSLSVFQL 19 273 LHLIQCIPNL 19 278
CIPNLSYPLV 19 502 LLEHGADGNI 19 654 ITILNIKLPL 19 678 GLPENLTNGA 19
744 LTNKQKQIEV 19 798 QLRENKILEE 19 870 EVAGFSLRQL 19 957
RLASGARAAA 19 18 TGLWAALTTV 18 36 VTWRKEPAVL 18 62 AARKEFSTTL 18
113 LLGWERVVQR 18 155 CLRAQGLTRA 18 241 LLFLPRAPQA 18 261
EALGVGSLSV 18 286 LVLRHIPEIL 18 300 KETGGGILGL 18 309 LELPATAARL 18
327 IKEFEELVKL 18 335 KLHSLSHKVI 18 403 SAFMEPRYHV 18 456
LASANGNSEV 18 459 ANGNSEVVQL 18 548 CGLTPLLLGV 18 553 LLLGVHEQKQ 18
581 YGRTALILAV 18 586 LILAVCCGSA 18 588 LAVCCGSASI 18 624
SSHHHVICEL 18 802 NKILEEIESV 18 815 LLKTIQLNEE 18 817 KTIQLNEEAL 18
825 ALTKTKVAGF 18 835 SLRQLGLAQH 18 875 SLRQLGLAQH 18 949
SPGTPSLVRL 18 1060 LAPKCRPGTL 18 7 LLPTQATFAA 17 44 VLPCCNLEKG 17
105 SATPAGAFLL 17 112 FLLGWERVVQ 17 184 SRNSYRLTHV 17 190
LTHVRCAQGL 17 242 LFLPRAPQAV 17 259 SEEALGVGSL 17 282 LSYPLVLRHI 17
330 FEELVKLHSL 17 429 VPRKDLIVML 17 470 LDRRCQLNVL 17 529
KLMAKALLLY 17 530 LMAKALLLYG 17 533 KALLLYGADI 17 541 DIESKNKCGL 17
544 SKNKCGLTPL 17 556 GVHEQKQEVV 17 569 IKKKANLNAL 17 597
IVNLLLEQNV 17 647 SENSNPVITI 17 687 ASAGNGDDGL 17 804 ILEEIESVKE 17
983 SVCDSSGWIL 17 1099 SLPHFHVSAG 17 14 FAAATGLWAA 16 68 STTLTGHSAL
16 110 GAFLLGWERV 16 152 DAACLRAQGL 16 197 QGLEAASANL 16 262
ALGVGSLSVF 16 334 VKLHSLSHKV 16 490 AVQCQEDECV 16 494 QEDECVLMLL 16
510 NIQDEYGNTA 16 523 AIYNEDKLMA 16 549 GLTPLLLGVH 16 589
AVCCGSASIV 16 591 CCGSASIVNL 16 633 LLSDYKEKQM 16 670 KKHGSNPVGL 16
737 GISQDEILTN 16 753 VAEKEMNSEL 16 838 QLGLAQHAQA 16 843
QHAQASVQQL 16 878 QLGLAQHAQA 16 883 QHAQASVQQL 16 947 RASPGTPSLV 16
169 HLAPTAPDGG 15 277 QCIPNLSYPL 15 302 TGGGILGLEL 15 312
PATAARLSGL 15 361 CLSEGYGHSF 15 370 FLIMKETSTK 15 374 KETSTKISGL 15
455 HLASANGNSE 15 460 NGNSEVVQLL 15 468 LLLDRRCQLN 15 482
KKRTALIKAV 15 493 CQEDECVLML 15 501 MLLEHGADGN 15 511 IQDEYGNTAL 15
535 LLLYGADIES 15 566 KFLIKKKANL 15 585 ALILAVCCGS 15 587
ILAVCCGSAS 15 592 CGSASIVNLL 15 601 LLEQNVDVSS 15 621 YAVSSHHHVI 15
650 SNPVITILNI 15 652 PVITILNIKL 15 655 TILNIKLPLK 15 668
EIKKHGSNPV 15 695 GLIPQRKSRK 15 772 LLRENSMLRE 15 799 LRENKILEEI 15
832 AGFSLRQLGL 15 872 AGFSLRQLGL 15 904 QQAQEQGAAL 15 985
CDSSGWILPV 15 1017 RLKSDSNRET 15 5 HILLPTQATF 14 23 ALTTVSNPSR 14
27 VSNPSRADPV 14 80 SSSRALPGSL 14 83 RALPGSLPAF 14 104 QSATPAGAFL
14 111 AFLLGWERVV 14 123 RLEVPRPQAA 14 195 CAQGLEAASA 14 205
NLPGAPGRSS 14 220 YRSGPSVSSA 14 233 AEPPAHQRLL 14 235 PPAHQRLLFL 14
266 GSLSVFQLHL 14 279 IPNLSYPLVL 14 281 NLSYPLVLRH 14 318
LSGLNSIMQI 14 320 GLNSIMQIKE 14 353 NVDKWDDFCL 14 382 GLIQEMGSGK 14
383 LIQEMGSGKS 14 395 GTWGDYDDSA 14 420 LHRAAWWGKV 14 427
GKVPRKDLIV 14 434 LIVMLRDTDM 14 437 MLRDTDMNKR 14 453 ALHLASANGN 14
457 ASANGNSEVV 14 461 GNSEVVQLLL 14 486 ALIKAVQCQE 14 500
LMLLEHGADG 14 519 ALHYAIYNED 14 521 HYAIYNEDKL 14 534 ALLLYGADIE 14
555 LGVHEQKQEV 14 567 FLIKKKANLN 14 568 LIKKKANLNA 14 593
GSASIVNLLL 14 596 SIVNLLLEQN 14 614 SGQTAREYAV 14 688 SAGNGDDGLI 14
735 NTGISQDEIL 14 742 EILTNKQKQI 14 779 LREEIAKLRL 14 784
AKLRLELDET 14 814 KLLKTIQLNE 14 818 TIQLNEEALT 14 867 QEQEVAGFSL 14
930 TAAGDQGPGT 14 975 GRSPTKQKSV 14 990 WILPVPTFSS 14 991
ILPVPTFSSG 14 1089 TTLPHRDTTT 14 1091 LPHRDTTTSL 14
[1047]
46TABLE XXXVI Pos 123456789 score V1A-HLA-A0202-10mers: 251P5G2
Noresultsfound. V2A-HLA-A0202-10mers: 251P5G2 Noresultsfound.
V3A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V4A-HLA-A0202-10mers:
251P5G2 Noresultsfound. V12A-HLA-A0202-10mers: 251P5G2
Noresultsfound. V12B-HLA-A0202-10mers: 251P5G2 Noresultsfound.
[1048]
47TABLE XXXVII Pos 123456789 score V1A-HLA-A0203-10mers: 251P5G2
Noresultsfound. V2A-HLA-A0203-10mers: 251P5G2 Noresultsfound.
V3A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V4A-HLA-A0203-10mers:
251P5G2 Noresultsfound. V12A-HLA-A0203-10mers: 251P5G2
Noresultsfound. V12B-A0203-10mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. 8 LPTQATFAAA 27 957
RLASGARAAA 27 7 LLPTQATFAA 19 14 FAAATGLWAA 19 54 SWLSFPGTAA 19 123
RLEVPRPQAA 19 145 PPCHQRRDAA 19 193 VRCAQGLEAA 19 307 LGLELPATAA 19
415 EDLDKLHRAA 19 903 EQQAQEQGAA 19 923 VPLSEGGTAA 19 956
VRLASGARAA 19 84 ALPGSLPAFA 18 102 SEQSATPAGA 18 125 EVPRPQAAPA 18
195 CAQGLEAASA 18 306 ILGLELPATA 18 450 KRTALHLASA 18 525
YNEDKLMAKA 18 680 PENLTNGASA 18 838 QLGLAQHAQA 18 878 QLGLAQHAQA 18
955 LVRLASGARA 18 9 PTQATFAAAT 17 15 AAATGLWAAL 17 55 WLSFPGTAAR 17
124 LEVPRPQAAP 17 146 PCHQRRDAAC 17 194 RCAQGLEAAS 17 308
GLELPATAAR 17 416 DLDKLHRAAW 17 904 QQAQEQGAAL 17 924 PLSEGGTAAG 17
958 LASGARAAAL 17
[1049]
48TABLE XXXVIII Pos 1234567890 score V1A-HLA-A3-10mers: 251P5G2
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. 224
ILLPVSFSVG 24 227 PVSFSVGMYK 24 92 CLLGMLQVVN 22 28 FLDLRPERTY 21
78 YLRRVIRVLS 21 7 LVLASQPTLF 21 42 HVALIHMVVL 20 81 RVIRVLSICT 20
109 LVRFKWKSTI 20 210 PLPKDLCRGK 20 214 DLCRGKSHQH 20 44 ALIHMVVLLT
19 135 SLIFYTVASS 19 164 SIIRGLFFTL 19 182 KQIMLFSSVY 19 6
KLVLASQPTL 18 158 SLFPINSIIR 18 189 SVYMMTLIQE 18 50 VLLTMVFLSP 17
64 SLNFQNDFKY 17 84 RVLSICTTCL 17 98 QVVNISPSIS 17 146 VTQINLHVSK
17 168 GLFFTLSLFR 17 174 SLFRDVFLKQ 17 26 LLFLDLRPER 16 30
DLRPERTYLP 16 60 QLFESLNFQN 16 127 SLSFPVSSSL 16 136 LIFYTVASSN 16
165 IIRGLFFTLS 16 178 DVFLKQIMLF 16 202 ILVPSQPQPL 16 14 TLFSFFSASS
15 45 LIHMVVLLTM 15 48 MVVLLTMVFL 15 56 FLSPQLFESL 15 82 VIRVLSICTT
15 108 WINRFKWKST 15 140 TVASSNVTQI 15 172 TLSLFRDVFL 15 183
QIMLFSSVYM 15 8 VLASQPTLFS 14 54 MVFLSPQLFE 14 93 LLGMLQVVNI 14 106
ISWLVRFKWK 14 145 NVTQINLHVS 14 161 PINSIIRGLF 14 204 VPSQPQPLPK 14
225 LLPVSFSVGM 14 3 FISKLVLASQ 13 4 ISKLVASQP 13 10 ASQPTLFSFF 13
35 RTYLPVCHVA 13 37 YLPVCHVALI 13 49 VVLLTMVFLS 13 51 LLTMVFLSPQ 13
63 ESLNFQNDFK 13 96 MLQVVNISPS 13 99 VVNISPSISW 13 102 ISPSISWLVR
13 104 PSISWLVRFK 13 122 HLFSWSLSFP 13 147 TQINLHVSKY 13 152
HVSKYCSLFP 13 162 INSIIRGLFF 13 167 RGLFFTLSLF 13 180 FLKQIMLFSS 13
194 TLIQELQEIL 13 198 ELQEILVPSQ 13 201 EILVPSQPQP 13 223
HILLPVSFSV 13 240 IISTSSTLPW 13 25 FLLFLDLRPE 12 33 PERTYLPVCH 12
69 NDFKYEASFY 12 72 KYEASFYLRR 12 75 ASFYLRRVIR 12 87 SICTTCLLGM 12
150 NLHVSKYCSL 12 173 LSLFRDVFLK 12 185 MLFSSVYMMT 12 221
HQHILLPVSF 12 239 FIISTSSTLP 12 32 RPERTYLPVC 11 68 QNDFKYEASF 11
85 VLSICTTCLL 11 101 NISPSISWLV 11 121 FHLFSWSLSF 11 148 QINLHVSKYC
11 171 FTLSLFRDVF 11 231 SVGMYKMDFI 11 238 DFIISTSSTL 11
V2A-HLA-A3-10mers: 251P5G2 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. 1 LVLASQPTLC 16 8 TLCSFFSASS 15 2 VLASQPTLCS 14
4 ASQPTICSFF 13 9 LCSFFSASSP 8 10 CSFFSASSPF 7 V3A-HLA-A3-10mers:
251P5G2 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.
6 RVIRDLSICT 20 3 YLRRVIRDLS 17 7 VIRDLSICTT 16 10 DLSICTTCLL 11 4
LRRVIRDLSI 8 5 RRVIRDLSIC 8 8 IRDLSICTTC 8 V4A-HLA-A3-10mers:
251P5G2 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.
7 CLLDMLQVVN 21 8 LLDMLQVVNI 14 2 SICTTCLLDM 12 V12A-HLA-A3-10mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
9 WLIMLFSSVY 26 10 LIMLFSSVYM 13 6 SISWLIMLFS 12
V12B-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO:
25; each start position is specified, the length of peptide is 10
amino acids, and the end position for each peptide is the start
position plus nine. 43 AVLPCCNLEK 31 785 KLRLELDETK 31 803
KILEEIESVK 31 370 FLIMKETSTK 30 326 QIKEFEELVK 29 342 KVIQCVFAKK 29
382 GLIQEMGSGK 29 695 GLIPQRKSRK 29 536 LLYGADIESK 28 419
KLHRAAWWGK 26 552 PLLLGVHEQK 26 160 GLTRAFQVVH 25 333 LVKLHSLSHK 25
819 IQLNEEALTK 25 262 ALGVGSLSVF 24 529 KLMAKALLLY 24 835
SLRQLGLAQH 24 875 SLRQLGLAQH 24 112 FLLGWERVVQ 23 287 VLRHIPEILK 23
5 HILLPTQATF 22 154 ACLRAQGLTR 22 549 GLTPLLLGVH 22 628 HVICELLSDY
22 6 ILLPTQATFA 21 113 LLGWERVVQR 21 119 VVQRRLEVPR 21 217
ALRYRSGPSV 21 306 ILGLELPATA 21 343 VIQCVFAKKK 21 612 DLSGQTAREY 21
655 TILNIKLPLK 21 662 PLKVEEEIKK 21 778 MLREEIAKLR 21 70 TLTGHSALSL
20 88 SLPAFADLPR 20 189 RLTHVRCAQG 20 240 RLLFLPRAPQ 20 243
FLPRAPQAVS 20 361 CLSEGYGHSF 20 428 KVPRKDLIVM 20 523 AIYNEDKLMA 20
563 EVVKFLIKKK 20 587 ILAVCCGSAS 20 771 DLLRENSMLR 20 825
ALTKTKVAGF 20 954 SLVRLASGAR 20 955 LVRLASGARA 20 957 RLASGARAAA 20
23 ALTTVSNPSR 19 49 NLEKGSWLSF 19 56 LSFPGTAARK 19 84 ALPGSLPAFA 19
125 EVPRPQAAPA 19 192 HVRCAQGLEA 19 275 LIQCIPNLSY 19 308
GLELPATAAR 19 332 ELVKLHSLSH 19 467 QLLLDRRCQL 19 486 ALIKAVQCQE 19
534 ALLLYGADIE 19 577 ALDRYGRTAL 19 589 AVCCGSASIV 19 629
VICELLSDYK 19 798 QLRENKILEE 19 830 KVAGFSLRQL 19 838 QLGLAQHAQA 19
878 QLGLAQHAQA 19 891 QLCYKWGHTE 19 912 ALRSQIGDPG 19 922
GVPLSEGGTA 19 966 ALPPPTGKNG 19 973 KNGRSPTKQK 19 55 WLSFPGTAAR 18
118 RVVQRRLEVP 18 433 DLIVMLRDTD 18 437 MLRDTDMNKR 18 513
DEYGNTALHY 18 557 VHEQKQEVVK 18 676 PVGLPENLTN 18 814 LKKLTIQLNE 18
916 QIGDPGGVPL 18 1040 KTQQSPRHTK 18 1054 DDRAGVLAPK 18 19
GLWAALTTVS 17 78 SLSSSRALPG 17 205 NLPGAPGRSS 17 241 LLFLPRAPQA 17
281 NLSYPLVLRH 17 294 ILKFSEKETG 17 317 RLSGLNSIMQ 17 335
KLHSLSHKVI 17 453 ALHLASANGN 17 477 NVLDNKKRTA 17 480 DNKKRTALIK 17
574 NLNALDRYGR 17 585 ALILAVCCGS 17 599 NLLLEQNVDV 17 600
LLLEQNVDVS 17 622 AVSSHHHVIC 17 656 ILNIKLPLKV 17 747 KQKQIEVAEK 17
772 LLRENSMLRE 17 804 ILEEIESVKE 17 810 SVKEKLLKTI 17 964
AAALPPPTGK 17 991 ILPVPTFSSG 17 993 PVPTFSSGSF 17 1011 DVSPAMRLKS
17 1031 RDKDDLPFFK 17 94 DLPRSCPESE 16 123 RLEVPRPQAA 16 155
CLRAQGLTRA 16 183 PSRNSYRLTH 16 286 LVLRHIPEIL 16 291 IPEILKFSEK 16
346 CVFAKKKNVD 16 410 YHVRREDLDK 16 411 HVRREDLDKL 16 436
VMLRDTDMNK 16 455 HLASANGNSE 16 469 LLDRRCQLNV 16 475 QLNVLDNKKR 16
484 RTALIKAVQC 16 501 MLLEHGADGN 16 510 NIQDEYGNTA 16 520
LHYAIYNEDK 16 561 KQEVVKFLIK 16 567 FLIKKKANLN 16 601 LLEQNVDVSS 16
635 SDYKEKQMLK 16 740 QDEILTNKQK 16 840 GLAQHAQASV 16 851
QLCYKWNHTE 16 870 EVAGFSLRQL 16 880 GLAQHAQASV 16 948 ASPGTPSLVR 16
971 TGKNGRSPTK 16 1109 GVGPTTLGSN 16 26 TVSNPSRADP 15 76 ALSLSSSRAL
15 166 QVVHLAPTAP 15 169 HLAPTAPDGG 15 179 AGCPPSRNSY 15 198
GLEAASANLP 15 218 LRYRSGPSVS 15 250 AVSGPQEQPS 15 310 ELPATAARLS 15
347 VFAKKKNVDK 15 416 DLDKLHRAAW 15 439 RDTDMNKRDK 15 473
RCQLNVLDNK 15 524 IYNEDKLMAK 15 556 GVHEQKQEVV 15 571 KKANLNALDR 15
586 LILAVCCGSA 15 616 QTAREYAVSS 15 719 HSDEQNDTQK 15 737
GISQDEILTN 15 787 RLELDETKHQ 15 794 KHQNQLRENK 15 808 IESVKEKLLK 15
821 LNEEALTKTK 15 852 LCYKWNHTEK 15 892 LCYKWGHTEK 15 924
PLSEGGTAAG 15 940 HLPPREPRAS 15 983 SVCDSSGWIL 15 990 WILPVPTFSS 15
1002 FLGRRCPMFD 15 1017 RLKSDSNRET 15 1059 VLAPKCRPGT 15 1090
TLPHRDTTTS 15 1099 SLPHFHVSAG 15
[1050]
49TABLE XXXIX Pos 1234567890 score V1A-HLA-A26-10mers: 251P502 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. 178
DVFLKQIMLF 33 42 HVALIHMVVL 24 164 SIIRGLFFTL 24 39 PVCHVALIHM 23
48 MVVLLTMVFL 23 56 FLSPQLFESL 23 87 SICTTCLLGM 23 238 DFIISTSSTL
23 45 LIHMVVLLTM 22 198 ELQEILVPSQ 22 242 STSSTLPWAY 22 70
DFKYEASFYL 21 119 FTFHLFSWSL 21 150 NLHVSKYCSL 21 161 PINSIIRGLF 21
171 FTLSLFRDVF 21 7 LVLASQPTLF 20 52 LTMVFLSPQL 20 185 LFSSVYMMTL
20 28 FLDLRPERTY 19 64 SLNFQNDFKY 19 84 RVLSICTTCL 19 183
QIMLFSSVYM 19 194 TLIQELQEIL 19 202 ILVPSQPQPL 19 225 LLPVSFSVGM 19
17 SFFSASSPFL 18 127 SLSFPVSSSL 18 147 TQINLHVSKY 18 201 EILVPSQPQP
18 3 FISKLVLASQ 17 6 KLVLASQPTL 17 10 ASQPTLFSFF 17 117 TIFTFHLFSW
17 129 SFPVSSSLIF 17 140 TVASSNVTQI 17 172 TLSLFRDVFL 17 185
MLFSSVYMMT 17 245 STLPWAYDRG 17 9 LASQPTLFSF 16 13 PTLFSFFSAS 16 19
FSASSPFLLF 16 30 DLRPERTYLP 16 85 VLSICTTCLL 16 100 VNISPSISWL 16
103 SPSISWLVRF 16 122 HLFSWSLSFP 16 135 SLIFYTVASS 16 165
IIRGLFFTLS 16 193 MTLIQELQQEI 16 226 LPVSFSVGMY 16 228 VSFSVGMYKM
16 V2A-A26-10mers: 251P5G2 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. 3 LASQPTLCSF 16 7 PTLCSFFSAS 16 4 ASQPTLCSFF 13
10 CSFFSASSPF 13 8 TLCSFFSASS 11 1 LVLASQPTLC 10 2 VLASQPTLCS 10
V3A-A26-HLA-10mers: 251P5G2 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. 10 DLSICTTCLL 22 7 VIRDLSICTT 15 2 FYLRRVIRDL
12 6 RVIRDLSICT 12 1 SFYLRRVIRD 11 V4A-HLA-A26-10mers: 251P5G2 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. 2
SICTTCLLDM 22 5 TTCLLDMLQV 15 8 LLDMLQVVNI 15 3 ICTTCLLDML 13 4
CTTCLLDMLQ 11 7 CLLDMLQVVN 10 V12A-HLA-1A26-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 9
WLIMLFSSVY 21 10 LIMLFSSVYM 19 5 PSISWLIMLF 18 6 SISWLIMLFS 14 4
SPSISWLIML 13 2 NISPSISWLI 12 3 ISPSISWLIM 10 V12B-HLA-A26-10mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 10 amino acids, and
the end position for each peptide is the start position plus nine.
870 EVAGFSLRQL 32 612 DLSGQTAREY 27 628 HVICELLSDY 27 541
DIESKNKCGL 26 830 KVAGFSLRQL 26 807 EIESVKEKLL 25 825 ALTKTKVAGF 25
161 LTRAFQVVHL 24 264 GVGSLSVFQL 24 752 EVAEKEMNSE 24 262
ALGVGSLSVF 23 411 HVRREDLDKL 23 428 KVPRKDLIVM 23 607 DVSSQDLSGQ 23
301 ETGGGILGLE 22 464 EVVQLLLDRR 22 529 KLMAKALLLY 22 563
EVVKFLIKKK 22 782 EIAKLRLELD 22 817 KTIQLNEEAL 22 993 PVPTFSSGSF 22
5 HILLPTQATF 21 275 LIQCIPNLSY 21 361 CLSEGYGHSF 21 652 PVITILNIKL
21 712 DTENEEYHSD 21 49 NLEKGSWLSF 20 68 STTLTGHSAL 20 190
LTHVRCAQGL 20 353 NVDKWDDFCL 20 364 EGYGHSFLIM 20 559 EQKQEVVKFL 20
654 ITILNIKLPL 20 916 QIGDPGGVPL 20 1001 SFLGRRCPMF 20 1073
DTPPHRNADT 20 36 VTWRKEPAVL 19 70 TLTGHSALSL 19 342 KVIQCVFAKK 19
513 DEYGNTALHY 19 709 QFPDTENEEY 19 789 ELDETKHQNQ 19 806
EEIESVKEKL 19 983 SVCDSSGWIL 19 1011 DVSPAMRLKS 19 1081 DTPPHRHTTT
19 83 RALPGSLPAF 18 125 EVPRPQAAPA 18 269 SVFQLHLIQC 18 286
LVLRHIPEIL 18 293 EILKFSEKET 18 305 GILGLELPAT 18 401 DDSAFMEPRY 18
416 DLDKLHRAAW 18 434 LIVMLRDTDM 18 440 DTDMNKRDKQ 18 478
VLDNKKRTAL 18 577 ALDRYGRTAL 18 633 LLSDYKEKQM 18 648 ENSNPVITIL 18
668 EIKKHGSNPV 18 725 DTQKQLSEEQ 18 735 NTGISQDEIL 18 792
ETKHQNQLRE 18 865 QAQEQEVAGF 18 1021 DSNRETHQAF 18 1025 ETHQAFRDKD
18 76 ALSLSSSRAL 17 103 EQSATPAGAF 17 152 DAACLRAQGL 17 234
EPPAHQRLLF 17 333 LVKLHSLSHK 17 378 TKISGLIQEM 17 467 QLLLDRRCQL 17
558 HEQKQEVVKF 17 742 EILTNKQKQI 17 781 EEIAKLRLEL 17 810
SVKEKLLKTI 17 1009 MFDVSPAMRL 17 1029 AFRDKDDLPF 17 1030 FRDKDDLPFF
17 1035 DLPFFKTQQS 17 1050 DLGQDDRAGV 17 1109 GVGPTTLGSN 17 41
EPAVLPCCNL 16 94 DLPRSCPESE 16 278 CIPNLSYPLV 16 332 ELVKLHSLSH 16
375 ETSTKISGLI 16 377 STKISGLIQE 16 433 DLIVMLRDTD 16 447
DKQKRTALHL 16 494 QEDECVLMLL 16 527 EDKLMAKALL 16 566 KFLIKKKANL 16
579 DRYGRTALIL 16 632 ELLSDYKEKQ 16 763 SLSHKKEEDL 16 769
EEDLLRENSM 16 770 EDLLRENSML 16 828 KTKVAGFSLR 16 843 QHAQASVQQL 16
883 QHAQASVQQL 16 978 PTKQKSVCDS 16 1094 RDTTTSLPHF 16 1095
DTTTSLPHFH 16 71 LTGHSALSLS 15 106 ATPAGAFLLG 15 118 RVVQRRLEVP 15
172 PTAPDGGAGC 15 288 LRHIPEILKF 15 300 KETGGGILGL 15 310
ELPATAARLS 15 321 LNSIMQIKEF 15 329 EFEELVKLHS 15 374 KETSTKISGL 15
429 VPRKDLIVML 15 516 GNTALHYAIY 15 536 LLYGADIESK 15 569
IKKKANLNAL 15 572 KANLNALDRY 15 591 CCGSASIVNL 15 596 SIVNLLLEQN 15
655 TILNIKLPLK 15 721 DEQNDTQKQL 15 737 GISQDEILTN 15 755
EKEMNSELSL 15 761 ELSLSHKKEE 15 771 DLLRENSMLR 15 772 LLRENSMLRE 15
798 QLRENKILEE 15 820 QLNEEALTKT 15 835 SLRQLGLAQH 15 848
SVQQLCYKWN 15 875 SLRQLGLAQH 15 888 SVQQLCYKWG 15 988 SGWILPVPTF 15
991 ILPVPTFSSG 15 1099 SLPHFHVSAG 15
[1051]
50TABLE XL Pos 1234567890 score V1A-HLA-B0702-10mers: 251P5G2 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. 103
SPSISWLVRF 20 160 FPINSIIRGL 20 1 MPFISKLVLA 18 58 SPQLFESLNF 18 12
QPTLFSFFSA 17 20 SASSPFLLFL 15 166 IRGLFFTLSL 15 172 TLSLFRDVFL 15
204 VPSQPQPLPK 15 36 TYLPVGHVAL 14 42 HVALIHMVVL 14 56 FLSPQLFESL
14 142 ASSNVTQINL 14 18 FFSASSPFLL 13 22 SSPFLLFLDL 13 32
RPERTYLPVC 13 43 VALIHMVVLL 13 48 MVVLLTMVFL 13 84 RVLSIGTTCL 13 85
VLSICTTCLL 13 114 WKSTIFTFHL 13 202 ILVPSQPQPL 13 6 KLVLASQPTL 12
17 SFFSASSPFL 12 29 LDLRPERTYL 12 38 LPVCHVALIH 12 52 LTMVFLSPQL 12
127 SLSFPVSSSL 12 162 INSIIRGLFF 12 186 LFSSVYMMTL 12 216
CRGKSHQHIL 12 10 ASQPTLFSFF 11 33 ALIHMVVLLT 11 74 EASFYLRRVI 11 77
FYLRRVIRVL 11 79 LRRVIRVLSI 11 188 ICTTCLLGML 11 130 FPVSSSLIFY 11
164 SIIRGLFFTL 11 177 RDVFLKQIML 11 190 VYMMTLIQEL 11 207
QPQPLPKDLC 11 209 QPLPKDLCRG 11 211 LPKDLCRGKS 11 217 RGKSHQHILL 11
226 LPVSFSVGMY 11 19 FSASSPFLLF 10 23 SPFLLFLDLR 10 70 DFKYEASFYL
10 93 LLGMLQVVNI 10 100 VNISPSISWL 10 119 FTFHLFSWSL 10 133
SSSLIFYTVA 10 150 NLHVSKYCSL 10 194 TLIQELQEIL 10 206 SQPQPLPKDL 10
215 LCRGKSHQHI 10 219 KSHQHILLPV 10 238 DFIISTSSTL 10 31 LRPERTYLPV
9 46 IHMVVLLTMV 9 101 NISPSISWLV 9 111 RFKWKSTIFT 9 123 LFSWSLSFPV
9 132 VSSSLIFYTV 9 140 TVASSNVTQI 9 183 QIMLFSSVYM 9 187 FSSVYMMTLI
9 V2A-B0702-10mers: 251P5G2 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. 6 QPTLCSFFSA 17 4 ASQPTLCSFF 10 3 LASQPTLCSF 8
V3A-B0702-10mers: 251P5G2 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. 9 RDLSICTTGL 13 10 DLSICTTCLL 13 2 FYLRRVIRDL
10 4 LRRVIRDLSI 10 7 VIRDLSICTT 8 6 RVIRDLSICT 7
V4A-HLA-B0702-10mers: 251P5G2 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. 3 ICTTCLLDML 11 8 LLDMLQVVNI 10 2 SICTTCLLDM 8
5 TTGLLDMLQV 8 6 TCLLOMLQVV 7 V12A-HLA-B0702-10mers: 251P5G2 Each
peptide is a portion of SEQ ID NO: 25; each start position is
specified, the length of peptide is 10 amino acids, and the end
position for each peptide is the start position plus nine. 4
SPSISWLIML 22 2 NISPSISWLI 9 3 ISPSISWLIM 9 10 LIMLFSSVYM 9 8
SWLIMLFSSV 7 5 PSISWLIMLF 6 6 SISWLIMLFS 5 7 ISWLIMLFSS 1 9
WLIMLFSSVY 1 V12B-HLA-B0702-10mers: 251P5G2 Each peptide is a
portion of SEQ ID NO: 25; each start position is specified, the
length of peptide is 10 amino acids, and the end position for each
peptide is the start position plus nine. 429 VPRKDLIVML 25 209
APGRSSSCAL 24 235 PPAHQRLLFL 24 279 IPNLSYPLVL 24 949 SPGTPSLVRL 23
41 EPAVLPCCNL 22 1082 TPPHRHTTTL 22 1091 LPHRDTTTSL 22 126
VPRPQAAPAT 21 234 EPPAHQRLLF 21 994 VPTFSSGSFL 21 181 CPPSRNSYRL 20
8 LPTQATFAAA 19 128 RPQAAPATSA 19 704 KPENQQFPDT 19 1007 CPMFDVSPAM
19 1065 RPGTLCHTDT 19 58 FPGTAARKEF 18 145 PPCHQRRDAA 18 182
PPSRNSYRLT 18 675 NPVGLPENLT 18 923 VPLSEGGTAA 18 34 DPVTWRKEPA 17
144 SPPCHQRRDA 17 229 APSPAEPPAH 17 62 AARKEFSTTL 16 161 LTRAFQVVHL
16 174 APDGGAGCPP 16 253 GPQEQPSEEA 16 445 KRDKQKRTAL 16 459
ANGNSEVVQL 16 577 ALDRYGRTAL 16 945 EPRASPGTPS 16 958 LASGARAAAL 16
15 AAATGLWAAL 15 76 ALSLSSSRAL 15 107 TPAGAFLLGW 15 132 APATSATPSR
15 244 LPRAPQAVSG 15 300 KETGGGILGL 15 302 TGGGILGLEL 15 670
KKHGSNPVGL 15 687 ASAGNGDDGL 15 781 EEIAKLRLEL 15 29 NPSRADPVTW 14
70 TLTGHSALSL 14 86 PGSLPAFADL 14 138 TPSRDPSPPC 14 171 APTAPDGGAG
14 247 APQAVSGPQE 14 311 LPATAARLSG 14 407 EPRYHVRRED 14 461
GNSEVVQLLL 14 478 VLDNKKRTAL 14 511 IQDEYGNTAL 14 546 NKCGLTPLLL 14
579 DRYGRTALIL 14 592 CGSASIVNLL 14 648 ENSNPVITIL 14 654
ITILNIKLPL 14 830 KVAGFSLRQL 14 832 AGFSLRQLGL 14 870 EVAGFSLRQL 14
872 AGFSLRQLGL 14 916 QIGDPGGVPL 14 932 AGDQGPGTHL 14 1042
SPRHTKDLGQ 14 1106 SAGGVGPTTL 14 104 QSATPAGAFL 13 142 DPSPPCHQRR
13 206 LPGAPGRSSS 13 223 GPSVSSAPSP 13 233 AEPPAHQRLL 13 257
QPSEEALGVG 13 327 IKEFEELVKL 13 362 LSEGYGHSFL 13 425 WWGKVPRKDL 13
447 DKQKRTALHL 13 470 LDRRCQLNVL 13 493 CQEDECVLML 13 526
NEDKLMAKAL 13 544 SKNKCGLTPL 13 545 KNKCGLTPLL 13 559 EQKQEVVKFL 13
569 IKKKANLNAL 13 591 CCGSASIVNL 13 593 GSASIVNLLL 13 697
IPQRKSRKPE 13 755 EKEMNSELSL 13 904 QQAQEQGAAL 13 919 DPGGVPLSEG 13
942 PPREPRASPG 13 946 PRASPGTPSL 13 952 TPSLVRLASG 13 977
SPTKQKSVCD 13 1029 AFRDKDDLPF 13 1061 APKCRPGTLC 13 36 VTWRKEPAVL
12 80 SSSRALPGSL 12 84 ALPGSLPAFA 12 85 LPGSLPAFAD 12 89 LPAFADLPRS
12 95 LPRSCPESEQ 12 259 SEEALGVGSL 12 264 GVGSLSVFQL 12 266
GSLSVFQLHL 12 277 QCIPNLSYPL 12 291 IPEILKFSEK 12 309 LELPATAARL 12
324 IMQIKEFEEL 12 353 NVDKWDDFCL 12 364 EGYGHSFLIM 12 387
MGSGKSNVGT 12 411 HVRREDLDKL 12 527 EDKLMAKALL 12 528 DKLMAKALLL 12
566 KFLIKKKANL 12 763 SLSHKKEEDL 12 779 LREEIAKLRL 12 812
KEKLLKTIQL 12 817 KTIQLNEEAL 12 843 QHAQASVQQL 12 883 QHAQASVQQL 12
936 GPGTHLPPRE 12 941 LPPREPRASP 12 962 ARAAALPPPT 12 969
PPTGKNGRSP 12 1075 PPHRNADTPP 12 1083 PPHRHTTTLP 12
[1052]
51TABLE XLI Pos 123456789 score V1A-HLA-B08-10mers: 251P5G2
Noresultsfound. V2A-HLA-B08-10mers: 251P5G2 Noresultsfound.
V3A-HLA-B08-10mers: 251P5G2 Noresultsfound. V4A-HLA-B08-10mers:
251P5G2 Noresultsfound. V12A-HLA-B08-10mers: 251P5G2
Noresultsfound. V12B-HLA-B08-10mers: 251P5G2 Noresultsfound.
[1053]
52TABLE XLII Pos 123456789 score V1A-HLA-B1510-10mers: 251P5G2
Noresultsfound. V2A-HLA-B1510-10mers: 251P5G2 Noresultsfound.
V3A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V4A-HLA-B1510-10mers:
251P5G2 Noresultsfound. V12A-HLA-B1510-10mers: 251P5G2
Noresultsfound. V12B-HLA-B1510-10mers: 251P5G2 Noresultsfound.
[1054]
53TABLE XLIII Pos 123456789 score V1A-HLA-B2705-10mers: 251P5G2
Noresultsfound. V2A-HLA-B2705-10mers: 251P5G2 Noresultsfound.
V3A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V4A-HLA-B2705-10mers:
251P5G2 Noresultsfound. V12A-HLA-B2705-10mers: 251P5G2
Noresultsfound. V12B-HLA-B2705-10mers: 251P5G2 Noresultsfound.
[1055]
54TABLE XLIV Pos 123456789 score V1A-HLA-B2709-10mers: 251P5G2
Noresultsfound. V2A-HLA-B2709-10mers: 251P5G2 Noresultsfound.
V3A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V4A-HLA-B2709-10mers:
251P5G2 Noresultsfound. V12A-HLA-B2709-10mers: 251P5G2
Noresultsfound. V12B-HLA-B2709-10mers: 251P5G2 Noresultsfound.
[1056]
55TABLE XLV Pos 1234567890 score V1A-HLA-B4402-10mers: 251P5G2 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. 62
FESLNFQNDF 23 160 FPINSIIRGL 18 10 ASQPTLFSFF 17 36 TYLPVCHVAL 17
77 FYLRRVIRVL 17 164 SIIRGLFFTL 17 178 DVFLKQIMLF 17 100 VNISPSISWL
16 142 ASSNVTQINL 16 147 TQINLHVSKY 16 56 FLSPQLFESL 15 182
KQIMLFSSVY 15 206 SQPQPLPKDL 15 242 STSSTLPWAY 15 20 SASSPFLLFL 14
22 SSPFLLFLDL 14 28 FLDLRPERTY 14 43 VALIHMVVLL 14 69 NDFKYEASFY 14
74 EASFYLRRVI 14 103 SPSISWLVRF 14 105 SISWLVRFKW 14 112 FKWKSTIFTF
14 115 KSTIFTFHLF 14 117 TIFTFHLFSW 14 128 LSFPVSSSLI 14 156
YCSLFPINSI 14 190 VYMMTLIQEL 14 200 QEILVPSQPQ 14 202 ILVPSQPQPL 14
238 DFIISTSSTL 14 240 IISTSSTLPW 14 6 KLVLASQPTL 13 7 LVLASQPTLF 13
17 SFFSASSPFL 13 18 FFSASSPFLL 13 19 FSASSPFLLF 13 29 LDLRPERTYL 13
33 PERTYLPVCH 13 42 HVALIHMVVL 13 58 SPQLFESLNF 13 85 VLSICTTCLL 13
114 WKSTIFTFHL 13 129 SFPVSSSLIF 13 166 IRGLFFTLSL 13 167
RGLFFTLSLF 13 171 FTLSLFRDVF 13 172 TLSLFRDVFL 13 175 LFRDVFLKQI 13
194 TLIQELQEIL 13 197 QELQEILVPS 13 9 LASQPTLFSF 12 16 FSFFSASSPF
12 47 HMVVLLTMVF 12 48 MVVLLTMVFL 12 52 LTMVFLSPQL 12 53 TMVFLSPQLF
12 64 SLNFQNDFKY 12 73 YEASFYLRRV 12 84 RVLSICTTCL 12 88 ICTTCLLGML
12 110 VRFKWKSTIF 12 119 FTFHLFSWSL 12 121 FHLFSWSLSF 12 127
SLSFPVSSSL 12 130 FPVSSSLIFY 12 161 PINSIIRGLF 12 162 INSIIRGLFF 12
186 LFSSVYMMTL 12 217 RGKSHQHILL 12 221 HQHILLPVSF 12 37 YLPVCHVALI
11 68 QNDFKYEASF 11 99 VVNISPSISW 11 150 NLHVSKYCSL 11 151
LHVSKYCSLF 11 177 RDVFLKQIML 11 216 CRGKSHQHIL 11 226 LPVSFSVGMY 11
230 FSVGMYKMDF 11 V2A-B4402-10mers: 251P5G2 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. 4 ASQPTLCSFF 16 3
LASQPTLCSF 12 10 CSFFSASSPF 12 V3A-B4402-10mers: 251P5G2 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. 2
FYLRRVIRDL 16 10 DLSICTTCLL 13 9 RDLSICTTCL 12 4 LRRVIRDLSI 9
V4A-HLA-B4402-10mers: 251P5G2 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. 3 ICTTCLLDML 12 8 LLDMLQVVNI 11 1 LSICTTCLLD 5
V12A-HLA-B4402-10mers Each peptide is a portion of SEQ ID NO: 25;
each start position is specified, the length of peptide is 10 amino
acids, and the end position for each peptide is the start position
plus nine. 5 PSISWLIMLF 17 2 NISPSISWLI 14 4 SPSISWLIML 14 9
WLIMLFSSVY 14 V12B-HLA-B4402-10mers Each peptide is a portion of
SEQ ID NO: 25; each start position is specified, the length of
peptide is 10 amino acids, and the end position for each peptide is
the start position plus nine. 233 AEPPAHQRLL 29 526 NEDKLMAKAL 27
781 EEIAKLRLEL 27 300 KETGGGILGL 26 806 EEIESVKEKL 26 309
LELPATAARL 25 812 KEKLLKTIQL 25 374 KETSTKISGL 24 721 DEQNDTQKQL 24
330 FEELVKLHSL 23 494 QEDECVLMLL 23 513 DEYGNTALHY 23 558 HEQKQEWKF
23 259 SEEALGVGSL 22 647 SENSNPVITI 22 298 SEKETGGGIL 21 363
SEGYGHSFLI 20 867 QEQEVAGFSL 20 774 RENSMLREEI 19 76 ALSLSSSRAL 18
179 AGCPPSRNSY 18 577 ALDRYGRTAL 18 12 ATFAAATGLW 17 83 RALPGSLPAF
17 328 KEFEELVKLH 17 459 ANGNSEVVQL 17 648 ENSNPVITIL 17 732
EEQNTGISQD 17 777 SMLREEIAKL 17 823 EEALTKTKVA 17 1042 QQSPRHTKDL
17 15 AAATGLWAAL 16 29 NPSRADPVTW 16 103 EQSATPAGAF 16 105
SATPAGAFLL 16 209 APGRSSSCAL 16 234 EPPAHQRLLF 16 277 QCIPNLSYPL 16
321 LNSIMQIKEF 16 652 PVITILNIKL 16 817 KTIQLNEEAL 16 832
AGFSLRQLGL 16 870 EVAGFSLRQL 16 872 AGFSLRQLGL 16 124 LEVPRPQAAP 15
262 ALGVGSLSVF 15 288 LRHIPEILKF 15 292 PEILKFSEKE 15 445
KRDKQKRTAL 15 478 VLDNKKRTAL 15 529 KLMAKALLLY 15 546 NKCGLTPLLL 15
559 EQKQEVVKFL 15 654 ITILNIKLPL 15 667 EEIKKHGSNP 15 741
DEILTNKQKQ 15 742 EILTNKQKQI 15 756 KEMNSELSLS 15 760 SELSLSHKKE 15
769 EEDLLRENSM 15 807 EIESVKEKLL 15 830 KVAGFSLRQL 15 958
LASGARAAAL 15 1029 AFRDKDDLPF 15 58 FPGTAARKEF 14 62 AARKEFSTTL 14
65 KEFSTTLTGH 14 68 STTLTGHSAL 14 80 SSSRALPGSL 14 156 LRAQGLTRAF
14 255 QEQPSEEALG 14 260 EEALGVGSLS 14 273 LHLIQCIPNL 14 279
IPNLSYPLVL 14 315 AARLSGLNSI 14 331 EELVKLHSLS 14 406 MEPRYHVRRE 14
414 REDLDKLHRA 14 416 DLDKLHRAAW 14 429 VPRKDLIVML 14 467
QLLLDRRCQL 14 470 LDRRGQLNVL 14 527 EDKLMAKALL 14 528 DKLMAKALLL 14
569 IKKKANLNAL 14 591 CCGSASIVNL 14 592 CGSASIVNLL 14 612
DLSGQTAREY 14 624 SSHHHVICEL 14 628 HVICELLSDY 14 631 CELLSDYKEK 14
650 SNPVITILNI 14 670 KKHGSNPVGL 14 687 ASAGNGDDGL 14 754
AEKEMNSELS 14 770 EDLLRENSML 14 788 LELDETKHQN 14 825 ALTKTKVAGF 14
845 AQASVQQLCY 14 847 ASVQQLCYKW 14 885 AQASVQQLCY 14 887
ASVQQLCYKW 14 932 AGDQGPGTHL 14 944 REPRASPGTP 14 949 SPGTPSLVRL 14
1106 SAGGVGPTTL 14
[1057]
56TABLE XLVI Pos 123456789 score V1A-HLA-B5101-10mers: 251P5G2
Noresultsfound. V2A-HLA-B5101-10mers: 251P5G2 Noresultsfound.
V3A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V4A-HLA-B5101-10mers:
251P5G2 Noresultsfound. V12A-HLA-B5101-10mers: 251P5G2
Noresultsfound. V12B-HLA-B5101-10mers: 251P5G2 Noresultsfound.
[1058]
57TABLE XLVII Pos 123456789012345 score V12A-HLA-DRB10101-15mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 15 amino acids, and
the end position for each peptide is the start position plus
fourteen. 1 MLQVVNISPSISWL 33 2 MLQVVNISPSISWL 29 11
SISWLIMLFSSVYMM 27 9 SPSISWLIMLFSSVY 24 12 ISWLIMLFSSVYMMT 23 13
SWLIMLFSSVYMMTL 23 5 VVNISPSISWLIMLF 22 3 LQVVNISPSISWLIM 18 14
WLIMLFSSVYMMTLI 17 10 PSISWLIMLFSSVYM 15 6 VNISPSISWLIMLFS 14
V12B-HLA-DRB10101-15mers: 251P5G2 Each peptide is a portion of SEQ
ID NO: 25; each start position is specified, the length of peptide
is 15 amino acids, and the end position for each peptide is the
start position plus fourteen. 169 AFQVVHLAPTAPDGG 32 40
PVTWRKEPAVLPCGN 30 386 SGLIQEMGSGKSNVG 30 644 EKQMLKISSENSNPV 30
656 NPVITILNIKLPLKV 29 10 HILLPTQATFAAATG 28 567 QEVVKFLIKKKANLN 28
993 SGWILPVPTFSSGSF 28 57 KGSWLSFPGTAARKE 27 428 AAWWGKVPRKDLIVM 27
437 KDLIVMLRDTDMNKR 27 501 DECVLMLLEHGADGN 27 529 IYNEDKLMAKALLLY
27 659 ITILNIKLPLKVEEE 27 958 PSLVRLASGARAAAL 27 6 KSHQHILLPTQATFA
26 16 QATFAAATGLWAALT 26 84 LSSSRALPGSLPAFA 26 94 LPAFADLPRSCPESE
26 155 RRDAACLRAQGLTRA 26 158 AACLRAQGLTRAFQV 26 195
LTHVRCAQGLEAASA 26 220 SCALRYRSGPSVSSA 26 222 ALRYRSGPSVSSAPS 26
332 IKEFEELVKLHSLSH 26 374 SFLIMKETSTKISGL 26 600 ASIVNLLLEQNVDVS
26 671 EEEIKKHGSNPVGLP 26 925 PGGVPLSEGGTAAGD 26 992
SSGWILPVPTFSSGS 26 1012 CPMFDVSPAMRLKSD 26 9 QHILLPTQATFAAAT 25 70
KEFSTTLTGHSALSL 25 73 STTLTGHSALSLSSS 25 118 LLGWERVVQRRLEVP 25 198
VRCAQGLEAASANLP 25 208 SANLPGAPGRSSSGA 25 244 QRLLFLPRAPQAVSG 25
286 NLSYPLVLRHIPEIL 25 310 GILGLELPATAARLS 25 313 GLELPATAARLSGLN
25 335 FEELVKLHSLSHKVI 25 338 LVKLHSLSHKVIQCV 25 536
MAKALLLYGADIESK 25 830 ALTKTKVAGFSLRQL 25 919 RSQIGDPGGVPLSEG 25 23
TGLWAALTFVSNPSR 24 69 RKEFSTTLTGHSALS 24 79 HSALSLSSSRALPGS 24 128
RLEVPRPQAAPATSA 24 201 AQGLEAASANLPGAP 24 245 RLLFLPRAPQAVSGP 24
267 ALGVGSLSVFQLHLI 24 281 IQCIPNLSYPLVLRH 24 317 PATAARLSGLNSIMQ
24 346 HKVIQCVFAKKKNVD 24 364 DFCLSEGYGHSFLIM 24 375
FLIMKETSTKISGLI 24 513 DGNIQDEYGNTALHY 24 590 ALILAVCGGSASIVN 24
592 ILAVCCGSASIVNLL 24 612 DVSSQDLSGQTAREY 24 681 PVGLPENLTNGASAG
24 699 DGLIPQRKSRKPENQ 24 815 SVKEKLLKTIQLNEE 24 915
GAALRSQIGDPGGVP 24 940 QGPGTHLPPREPRAS 24 943 GTHLPPREPRASPGT 24
986 QKSVCDSSGWILPVP 24 1104 SLPHFHVSAGGVGPT 24 22 ATGLWAALTTVSNPS
23 103 SCPESEQSATPAGAF 23 309 GGILGLELPATAARL 23 493
IKAVQCQEDECVLML 23 615 SQDLSGQTAREYAVS 23 655 SNPVITILNIKLPLK 23
810 LEEIESVKEKLLKTI 23 957 TPSLVRLASGARAAA 23 1038 KDDLPFFKTQQSPRH
23 1041 LPFFKIQQSPRHTKD 23 1093 TITLPHRDTTTSLPH 23 78
GHSALSLSSSRALPG 22 87 SRALPGSLPAFADLP 22 126 QRRLEVPRPQAAPAT 22 190
RNSYRLTHVRCAQGL 22 277 QLHLIQCIPNLSYPL 22 289 YPLVLRHIPEILKFS 22
305 KETGGGILGLELPAT 22 320 AARLSGLNSIMQIKE 22 362 WDDFCLSEGYGHSFL
22 524 ALHYAIYNEDKLMAK 22 549 SKNKCGLTPLLLGVH 22 559
LLGVHEQKQEVVKFL 22 589 TALILAVCCGSASIV 22 603 VNLLLEQNVDVSSQD 22
632 HHVICELLSDYKEKQ 22 647 MLKISSENSNPVITI 22 807 NKILEEIESVKEKLL
22 842 RQLGLAQHAQASVQQ 22 870 QAQEQEVAGFSLRQL 22 882
RQLGLAQHAQASVQQ 22 996 ILPVPTFSSGSFLGR 22 1109 HVSAGGVGPTTLGSN 22
14 PTQATFAAATGLWAA 21 264 SEEALGVGSLSVFQL 21 569 VVKFLIKKKANLNAL 21
607 LEQNVDVSSQDLSGQ 21 757 EVAEKEMNSELSLSH 21 804 LRENKILEEIESVKE
21 828 EEALTKTKVAGFSLR 21 60 WLSFPGTAARKEFST 20 114 AGAFLLGWERVVQRR
20 225 YRSGPSVSSAPSPAE 20 256 VSGPQEQPSEEALGV 20 300
LKFSEKETGGGILGL 20 306 ETGGGILGLELPATA 20 327 NSIMQIKEFEELVKL 20
372 GHSFLIMKETSTKIS 20 639 LSDYKEKQMLKISSE 20 733 KQLSEEQNTGISQDE
20 1011 RCPMFDVSPAMRLKS 20 1032 HQAFRDKDDLPFFKT 20 1106
PHFHVSAGGVGPTTL 20 65 GTAARKEFSTILTGH 19 123 RVVQRRLEVPRPQAA 19 241
PAHQRLLFLPRAPQA 19 259 PQEQPSEEALGVGSL 19 273 LSVFQLHLIQCIPNL 19
299 ILKFSEKETGGGILG 19 414 RYHVRREDLDKLHRA 19 456 RTALHLASANGNSEV
19 478 RCQLNVLDNKKRTAL 19 504 VLMLLEHGADGNIQD 19 534
KLMAKALLLYGADIE 19 571 KFLIKKKANLNALDR 19 577 KANLNALDRYGRTAL 19
588 RTALILAVCCGSASI 19 732 QKQLSEEQNTGISQD 19 781 NSMLREEIAKLRLEL
19 836 VAGFSLRQLGLAQHA 19 838 GFSLRQLGLAQHAQA 19 876
VAGFSLRQLGLAQHA 19 878 GFSLRQLGLAQHAQA 19 917 ALRSQIGDPGGVPLS 19
948 PREPRASPGTPSLVR 19 991 DSSGWILPVPTFSSG 19 1002 FSSGSFLGRRCPMFD
19 50 LPCCNLEKGSWLSFP 18 52 CGNLEKGSWLSFPGT 18 108 EQSATPAGAFLLGWE
18 113 PAGAFLLGWERVVQR 18 193 YRLTHVRCAQGLEAA 18 261
EQPSEEALGVGSLSV 18 288 SYPLVLRHIPEILKF 18 301 KFSEKETGGGILGLE 18
324 SGLNSIMQIKEFEEL 18 356 KKNVDKWDDFCLSEG 18 385 ISGLIQEMGSGKSNV
18 399 VGTWGDYDDSAFMEP 18 402 WGDYDDSAFMEPRYH 18 419
REDLDKLHRAAWWGK 18 427 RAAWWGKVPRKDLIV 18 450 KRDKQKRTALHLASA 18
468 SEVVQLLLDRRCQLN 18 502 ECVLMLLEHGADGNI 18 517 QDEYGNTALHYAIYN
18 537 AKALLLYGADIESKN 18 544 GADIESKNKCGLTPL 18 561
GVHEQKQEVVKFLIK 18 570 VKFLIKKKANLNALD 18 580 LNALDRYGRTALILA 18
583 LDRYGRTALILAVCC 18 623 AREYAVSSHHHVICE 18 670 VEEEIKKHGSNPVGL
18 683 GLPENLTNGASAGNG 18 780 ENSMLREEIAKLRLE 18 785
REEIAKLRLELDETK 18 798 TKHQNQLRENKILEE 18 813 IESVKEKLLKTIQLN 18
819 KLLKTIQLNEEALTK 18 825 QLNEEALTKTKVAGF 18 835 KVAGFSLRQLGLAQH
18 856 QLCYKWNHTEKTEQQ 18 875 EVAGFSLRQLGLAQH 18 896
QLCYKWGHTEKTEQQ 18 926 GGVPLSEGGTAAGDQ 18 956 GTPSLVRLASGARAA 18
959 SLVRLASGARAAALP 18 972 LPPPTGKNGRSPTKQ 18 997 LPVPTFSSGSFLGRR
18 1004 SGSFLGRRCPMFDVS 18 1053 TKDLGQDDRAGVLAP 18 1105
LPHFHVSAGGVGPTT 18 8 HQHILLPTQATFAAA 17 19 FAAATGLWAALTTVS 17 30
TTVSNPSRADPVTWR 17 38 ADPVTWRKEPAVLPC 17 112 TPAGAFLLGWERWQ 17 121
WERVVQRRLEVPRPQ 17 131 VPRPQAAPATSATPS 17 134 PQAAPATSATPSRDP 17
161 LRAQGLTRAFQVVHL 17 168 RAFQVVHLAPTAPDG 17 176 APTAPDGGAGCPPSR
17 243 HQRLLFLPRAPQAVS 17 262 QPSEEALGVGSLSVF 17 263
PSEEALGVGSLSVFQ 17 276 FQLHLIQCIPNLSYP 17 285 PNLSYPLVLRHIPEI 17
308 GGGILGLELPATAAR 17 379 KETSTKISGLIQEMG 17 383 TKISGLIQEMGSGKS
17 424 KLHRPAWWGKVPRKD 17 448 MNKRDKQKRTALHLA 17 453
KQKRTALHLASANGN 17 467 NSEVVQLLLDRRCQL 17 470 VVQLLLDRRCQLNVL 17
480 QLNVLDNKKRTALIK 17 481 LNVLDNKKRTALIKA 17 482 NVLDNKKRTALIKAV
17 503 CVLMLLEHGADGNIQ 17 531 NEDKLMAKALLLYGA 17 546
DIESKNKCGLTPLLL 17 552 KCGLTPLLLGVHEQK 17 553 CGLTPLLLGVHEQKQ 17
579 NLNALDRYGRTALIL 17 599 SASIVNLLLEQNVDV 17 602 IVNLLLEQNVDVSSQ
17 636 CELLSDYKEKQMLKI 17 640 SDYKEKQMLKISSEN 17 663
NIKLPLKVEEEIKKH 17 685 PENLTNGASAGNGDD 17 692 ASAGNGDDGLIPQRK 17
696 NGDDGLIPQRKSRKP 17 745 QDEILTNKQKQIEVA 17 747 EILTNKQKQIEVAEK
17 772 KKEEDLLRENSMLRE 17 778 LRENSMLREEIAKLR 17 790
KLRLELDETKHQNQL 17 829 EALTKTKVAGFSLRQ 17 833 KTKVAGFSLRQLGLA 17
839 FSLRQLGLAQHAQAS 17 841 LRQLGLAQHAQASVQ 17 851 QASVQQLCYKWNHTE
17 868 EQQAQEQEVAGFSLR 17 873 EQEVAGFSLRQLGLA 17 879
FSLRQLGLAQHAQAS 17 881 LRQLGLAQHAQASVQ 17 891 QASVQQLCYKWGHTE 17
935 TAAGDQGPGTHLPPR 17 969 AAALPPPTGKNGRSP 17 995 WILPVPTFSSGSFLG
17 1014 MFDVSPAMRLKSDSN 17 1099 RDTTTSLPHFHVSAG 17 1111
SAGGVGPTTLGSNRE 17 29 LTTVSNPSRADPVTW 16 44 RKEPAVLPCCNLEKG 16 58
GSWLSFPGTAARKEF 16 76 LTGHSALSLSSSRAL 16 81 ALSLSSSRALPGSLP 16 85
SSSRALPGSLPAFAD 16 88 RALPGSLPAFADLPR 16 90 LPGSLPAFADLPRSC 16 105
PESEQSATPAGAFLL 16 106 ESEQSATPAGAFLLG 16 107 SEQSATPAGAFLLGW 16
129 LEVPRPQAAPATSAT 16 150 PPCHQRRDAACLRAQ 16 167 TRAFQVVHLAPTAPD
16 172 VVHLAPTAPDGGAGC 16 177 PTAPDGGAGCPPSRN 16 205
EAASANLPGAPGRSS 16 226 RSGPSVSSAPSPAEP 16 230 SVSSAPSPAEPPAHQ 16
242 AHQRLLFLPRAPQAV 16 246 LLFLPRAPQAVSGPQ 16 248 FLPRAPQAVSGPQEQ
16 253 PQAVSGPQEQPSEEA 16 265 EEALGVGSLSVFQLH 16 270
VGSLSVFQLHLIQCI 16 272 SLSVFQLHLIQCIPN 16 274 SVFQLHLIQCIPNLS 16
282 QCIPNLSYPLVLRHI 16 290 PLVLRHIPEILKFSE 16 312 LGLELPATAARLSGL
16 323 LSGLNSIMQIKEFEE 16 341 LHSLSHKVIQCVFAK 16 342
HSLSHKVIQCVFAKK 16 373 HSFLIMKETSTKISG 16 382 STKISGLIQEMGSGK 16
431 WGKVPRKDLIVMLRD 16 458 ALHLASANGNSEVVQ 16 472 QLLLDRRCQLNVLDN
16 505 LMLLEHGADGNIQDE 16 526 HYAIYNEDKLMAKAL 16 530
YNEDKLMAKALLLYG 16 538 KALLLYGADIESKNK 16 574 IKKKANLNALDRYGR 16
581 NALDRYGRTALILAV 16 582 ALORYORTALILAVO 16 591 LILAVCCGSASIVNL
16 604 NLLLEQNVDVSSQDL 16 608 EQNVDVSSQDLSGQT 16 610
NVDVSSQDLSGQTAR 16 619 SGQTAREYAVSSHHH 16 668 LKVEEEIKKHGSNPV 16
673 EIKKHGSNPVGLPEN 16 682 VGLPENLTNGASAGN 16 766 ELSLSHKKEEDLLRE
16 771 HKKEEDLLRENSMLR 16 820 LLKTIQLNEEALTKT 16 826
LNEEALTKTKVAGFS 16 843 QLGLAQHAQASVQQL 16 869 QQAQEQEVAGFSLRQ 16
883 QLGLAQHAQASVQQL 16 894 VQQLCYKWGHTEKTE 16 907 TEQQAQEQGAALRSQ
16 909 QQAQEQGAALRSQIG 16 918 LRSQIGDPGGVPLSE 16 954
SPGTPSLVRLASGAR 16 960 LVRLASGARPAALPP 16 961 VRLASGARAAALPPP 16
962 RLASGARAAALPPPT 16 963 LASGARAAALPPPTG 16 1054 KDLGQDDRAGVLAPK
16 1055 DLGQDDRAGVLAPKC 16 1063 GVLAPKCRPGTLCHT 16 1069
CRPGTLCHTDTPPHR 16 1077 TDTPPHRNADTPPHR 16 1095 TLPHRDTTTSLPHFH 16
7 SHQHILLPTQATFAA 15 26 WAALTTVSNPSRADP 15 27 AALTTVSNPSRADPV 15 39
DPVTWRKEPAVLPCC 15 51 PCCNLEKGSWLSFPG 15 56 EKGSWLSFPGTAARK 15 99
DLPRSCPESEQSATP 15 120 GWERVVQRRLEVPRP 15 137 APATSATPSRDPSPP 15
159 ACLRAQGLTRAFQVV 15 165 GLTRAFQVVHLAPTA 15 170 FQVVHLAPTAPDGGA
15 181 DGGAGCPPSRNSYRL 15 192 SYRLTHVRGAQGLEA 15 196
THVRCAQGLEAASAN 15 221 CALRYRSGPSVSSAP 15 227 SGPSVSSAPSPAEPP 15
275 VFQLHLIQCIPNLSY 15 311 ILGLELPATAARLSG 15 333 KEFEELVKLHSLSHK
15 337 ELVKLHSLSHKVIQC 15 343 SLSHKVIQCVFAKKK 15 371
YGHSFLIMKETSTKI 15 393 GSGKSNVGTWGDYDD 15 405 YDDSAFMEPRYHVRR 15
438 DLIVMLRDTDMNKRD 15 487 KKRTALIKAVQCQED 15 490 TALIKAVQGQEDECV
15 548 ESKNKCGLTPLLLGV 15 556 TPLLLGVHEQKQEVV 15 586
YGRTALILAVCGGSA 15 641 DYKEKQMLKISSENS 15 737 EEQNTGISQDEILTN 15
752 KQKQIEVAEKEMNSE 15 760 EKEMNSELSLSHKKE 15 777 LLRENSMLREEIAKL
15 818 EKLLKTIQLNEEALT 15 863 HTEKTEQQAQEQEVA 15 866
KTEQQAQEQEVAGFS 15 903 HTEKTEQQAQEQGAA 15 906 KTEQQAQEQGAALRS 15
908 EQQAQEQGAALRSQI 15 927 GVPLSEGGTAAGDQG 15 936 AAGDQGPGTHLPPRE
15 949 REPRASPGTPSLVRL 15 980 GRSPTKQKSVCDSSG 15 985
KQKSVCDSSGWILPV 15 1010 RRCPMFDVSPAMRLK 15 1023 LKSDSNRETHQAFRD 15
1045 KTQQSPRHTKDLGQD 15 1058 QDDRAGVLAPKCRPG 15 1059
DDRAGVLAPKCRPGT 15 1085 ADTPPHRHTTTLPHR 15 1086 DTPPHRHTTTLPHRD 15
1094 TTLPHRDTTTSLPHF 15
[1059]
58TABLE XLVIII Pos 123456789012345 score V12A-HLA-DRBI0301-15mers:
251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start
position is specified, the length of peptide is 15 amino acids, and
the end position for each peptide is the start position plus
fourteen. 1 MLQVVNISPSISWL 22 12 ISWLIMLFSSVYMMT 20 13
SWLIMLFSSVYMMTL 13 14 WLIMLFSSVYMMTLI 13 2 MLQVVNISPSISWLI 12 5
VVNISPSISWLIMLF 12 8 ISPSISWLIMLFSSV 12 3 LQVVNISPSISWLIM 11 4
QVVNISPSISWLIML 11 9 SPSISWLIMLFSSVY 11 15 LIMLFSSVYMMTLIQ 11
V12B-HLA-DRB1-15meres: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
15 amino acids, and the end position for each peptide is the start
position plus fourteen. 470 VVQLLLDRRCQLNVL 37 635 ICELLSDYKEKQMLK
30 1020 AMRLKSDSNRETHQA 29 8 HQHILLPTQATFAAA 28 569 VVKFLIKKKANLNAL
27 655 SNPVITILNIKLPLK 27 810 LEEIESVKEKLLKTI 27 1053
TKDLGQDDRAGVLAP 27 278 LHLIQCIPNLSYPLV 26 481 LNVLDNKKRTALIKA 26
580 LNALDRYGRTALILA 26 544 GADIESKNKCGLTPL 25 766 ELSLSHKKEEDLLRE
25 724 HSDEQNDTQKQLSEE 24 439 LIVMLRDTDMNKRDK 23 712
NQQFPDTENEEYHSD 23 780 ENSMLREEIAKLRLE 23 740 NTGISQDEILTNKQK 22
790 KLRLELDETKHQNQL 22 73 STTLTGHSALSLSSS 21 79 HSALSLSSSRALPGS 21
432 GKVPRKDLIVMLRDT 21 828 EEALTKTKVAGFSLR 21 46 EPAVLPCCNLEKGSW 20
52 CCNLEKGSWLSFPGT 20 115 GAFLLGWERVVQRRL 20 243 HQRLLFLPRAPQAVS 20
267 ALGVGSLSVFQLHLI 20 289 YPLVLRHIPEILKFS 20 329 IMQIKEFEELVKLHS
20 414 RYHVRREDLDKLHRA 20 501 DECVLMLLEHGADGN 20 532
EDKLMAKALLLYGAD 20 556 TPLLLGVHEQKQEVV 20 600 ASIVNLLLEQNVDVS 20
602 IVNLLLEQNVDVSSQ 20 631 HHHVICELLSDYKEK 20 661 ILNIKLPLKVEEEIK
20 817 KEKLLKTIQLNEEAL 20 821 LKTIQLNEEALTKTK 20 919
RSQIGDPGGVPLSEG 20 38 ADPVTWRKEPAVLPC 19 265 EEALGVGSLSVFQLH 19 290
PLVLRHIPEILKFSE 19 296 IPEILKFSEKETGGG 19 327 NSIMQIKEFEELVKL 19
354 AKKKNVDKWDDFCLS 19 364 DFCLSEGYGHSFLIM 19 480 QLNVLDNKKRTALIK
19 571 KFLIKKKANLNALDR 19 645 KQMLKISSENSNPVI 19 665
KLPLKVEEEIKKHGS 19 679 SNPVGLPENLTNGAS 19 720 NEEYHSDEQNDTQKQ 19
745 QDEILTNKQKQIEVA 19 806 ENKILEEIESVKEKL 19 833 KTKVAGFSLRQLGLA
19 873 EQEVAGFSLRQLGLA 19 960 LVRLASGARAAALPP 19 986
QKSVCDSSGWILPVP 19 996 ILPVPTFSSGSFLGR 19 1014 MFDVSPAMRLKSDSN 19
26 WAALTTVSNPSRADP 18 29 LTTVSNPSRADPVTW 18 122 ERVVQRRLEVPRPQA 18
163 AQGLTRAFQVVHLAP 18 246 LLFLPRAPQAVSGPQ 18 269 GVGSLSVFQLHLIQC
18 293 LRHIPEILKFSEKET 18 297 PEILKFSEKETGGGI 18 323
LSGLNSIMQIKEFEE 18 349 IQCVFAKKKNVDKWD 18 356 KKNVDKWDDFCLSEG 18
419 REDLDKLHRAAWWGK 18 436 RKDLIVMLRDTDMNK 18 445 DTDMNKRDKQKRTAL
18 446 TDMNKRDKQKRTALH 18 472 QLLLDRRCQLNVLDN 18 479
CQLNVLDNKKRTALI 18 530 YNEDKLMAKALLLYG 18 559 LLGVHEQKQEVVKFL 18
567 QEVVKFLIKKKANLN 18 608 EQNVDVSSQDLSGQT 18 615 SQDLSGQTAREYAVS
18 698 DDGLIPQRKSRKPEN 18 711 ENQQFPDTENEEYHS 18 746
DEILTNKQKQIEVAE 18 773 KEEDLLRENSMLREE 18 1004 SGSFLGRRCPMFDVS 18
1032 HQAFRDKDDLPFFKT 18 1033 QAFRDKDDLPFFKTQ 18 1054
KDLGQDDRAGVLAPK 18 1094 TTLPHRDTTTSLPHF 18
[1060]
59TABLE XLIX Pos 123456789012345 score V12A-HLA-DR1-0410-15mers:
251P5G2 Noresultsfound. V12B-DR-0401-15mers: 251P5G2 Each peptide
is a portion of SEQ ID NO: 25; each start position is specified,
the length of peptide is 15 amino acids, and the end position for
each peptide is the start position plus fourteen. 23
TGLWAALTTVSNPSR 28 118 LLGWERVVQRRLEVP 28 428 AAWWGKVPRKDLIVM 28
720 NEEYHSDEQNDTQKQ 28 22 ATGLWAALTTVSNPS 26 243 HQRLLFLPRAPQAVS 26
246 LLFLPRAPQAVSGPQ 26 320 AARLSGLNSIMQIKE 26 338 LVKLHSLSHKVIQCV
26 374 SFLIMKETSTKISGL 26 470 VVQLLLDRRCQLNVL 26 602
IVNLLLEQNVDVSSQ 26 632 HHVICELLSDYKEKQ 26 644 EKQMLKISSENSNPV 26
647 MLKISSENSNPVITI 26 655 SNPVITILNIKLPLK 26 732 QKQLSEEQNTGISQD
26 790 KLRLELDETKHQNQL 26 841 LRQLGLAQHAQASVQ 26 881
LRQLGLAQHAQASVQ 26 993 SGWILPVPTFSSGSF 26 1014 MFDVSPAMRLKSDSN 26
1020 AMRLKSDSNRETHQA 26 1038 KDDLPFFKTQQSPRH 26 1053
TKDLGQDDRAGVLAP 26 16 QATFAAATGLWAALT 22 57 KGSWLSFPGTAARKE 22 69
RKEFSTTLTGHSALS 22 94 LPAFADLPRSCPESE 22 167 TRAFQVVHLAPTAPD 22 222
ALRYRSGPSVSSAPS 22 286 NLSYPLVLRHIPEIL 22 332 IKEFEELVKLHSLSH 22
527 YAIYNEDKLMAKALL 22 540 LLLYGADIESKNKCG 22 623 AREYAVSSHHHVICE
22 856 QLCYKWNHTEKTEQQ 22 896 QLCYKWGHTEKTEQQ 22 10 HILLPTQATFAAATG
20 26 WAALTTVSNPSRADP 20 46 EPAVLPCCNLEKGSW 20 79 HSALSLSSSRALPGS
20 115 GAFLLGWERVVQRRL 20 163 AQGLTRAFQVVHLAP 20 170
FQVVHLAPTAPDGGA 20 195 LTHVRCAQGLEAASA 20 208 SANLPGAPGRSSSCA 20
228 GPSVSSAPSPAEPPA 20 267 ALGVGSLSVFQLHLI 20 270 VGSLSVFQLHLIQCI
20 275 VFQLHLIQCIPNLSY 20 278 LHLIQCIPNLSYPLV 20 281
IQCIPNLSYPLVLRH 20 290 PLVLRHIPEILKFSE 20 296 IPEILKFSEKETGGG 20
308 GGGILGLELPATAAR 20 309 GGILGLELPATAARL 20 323 LSGLNSIMQIKEFEE
20 329 IMQIKEFEELVKLHS 20 335 FEELVKLHSLSHKVI 20 346
HKVIQCVFAKKKNVD 20 375 FLIMKETSTKISGLI 20 382 STKISGLIQEMGSGK 20
385 ISGLIQEMGSGKSNV 20 386 SGLIQEMGSGKSNVG 20 414 RYHVRREDLDKLHRA
20 419 REDLDKLHRAAWWGK 20 422 LDKLHRAAWWGKVPR 20 437
KDLIVMLRDTDMNKR 20 439 LIVMLRDTDMNKRDK 20 456 RTALHLASANGNSEV 20
478 RCQLNVLDNKKRTAL 20 489 RTALIKAVQCQEDEC 20 501 DECVLMLLEHGADGN
20 502 ECVLMLLEHGADGNI 20 513 DGNIQDEYGNTALHY 20 526
HYAIYNEDKLMAKAL 20 539 ALLLYGADIESKNKC 20 555 LTPLLLGVHEQKQEV 20
556 TPLLLGVHEQKQEVV 20 559 LLGVHEQKQEVVKFL 20 566 KQEVVKFLIKKKANL
20 567 QEVVKFLIKKKANLN 20 577 KANLNALDRYGRTAL 20 580
LNALDRYGRTALILA 20 588 RTALILAVCCGSASI 20 589 TALILAVCCGSASIV 20
599 SASIVNLLLEQNVDV 20 600 ASIVNLLLEQNVDVS 20 608 EQNVDVSSQDLSGQT
20 635 ICELLSDYKEKQMLK 20 658 VITILNIKLPLKVEE 20 665
KLPLKVEEEIKKHGS 20 671 EEEIKKHGSNPVGLP 20 679 SNPVGLPENLTNGAS 20
681 PVGLPENLTNGASAG 20 685 PENLTNGASAGNGDD 20 740 NTGISQDEILTNKQK
20 745 QDEILTNKQKQIEVA 20 753 QKQIEVAEKEMNSEL 20 760
EKEMNSELSLSHKKE 20 774 EEDLLRENSMLREEI 20 780 ENSMLREEIAKLRLE 20
788 IAKLRLELDETKHQN 20 792 RLELDETKHQNQLRE 20 806 ENKILEEIESVKEKL
20 807 NKILEEIESVKEKLL 20 810 LEEIESVKEKLLKTI 20 823
TIQLNEEALTKTKVA 20 833 KTKVAGFSLRQLGLA 20 843 QLGLAQHAQASVQQL 20
851 QASVQQLCYKWNHTE 20 873 EQEVAGFSLRQLGLA 20 883 QLGLAQHAQASVQQL
20 957 TPSLVRLASGARAAA 20 958 PSLVRLASGARAAAL 20 960
LVRLASGARAAALPP 20 996 ILPVPTFSSGSFLGR 20 7 SHQHILLPTQATFAA 18 65
GTAARKEFSTTLTGH 18 66 TAARKEFSTTLTGHS 18 75 TLTGHSALSLSSSRA 18 78
GHSALSLSSSRALPG 18 125 VQRRLEVPRPQAAPA 18 160 CLRAQGLTRAFQVVH 18
200 CAQGLEAASANLPGA 18 216 GRSSSCALRYRSGPS 18 225 YRSGPSVSSAPSPAE
18 236 SPAEPPAHQRLLFLP 18 249 LPRAPQAVSGPQEQP 18 264
SEEALGVGSLSVFQL 18 269 GVGSLSVFQLHLIQC 18 400 GTWGDYDDSAFMEPR 18
406 DDSAFMEPRYHVRRE 18 411 MEPRYHVRREDLDKL 18 446 TDMNKRDKQKRTALH
18 452 DKQKRTALHLASANG 18 464 ANGNSEVVQLLLDRR 18 469
EVVQLLLDRRCQLNV 18 518 DEYGNTALHYAIYNE 18 523 TALHYAIYNEDKLMA 18
541 LLYGADIESKNKCGL 18 596 CCGSASIVNLLLEQN 18 607 LEQNVDVSSQDLSGQ
18 611 VDVSSQDLSGQTARE 18 638 LLSDYKEKQMLKISS 18 652
SENSNPVITILNIKL 18 682 VGLPENLTNGASAGN 18 696 NGDDGLIPQRKSRKP 18
721 EEYHSDEQNDTQKQL 18 724 HSDEQNDTQKQLSEE 18 737 EEQNTGISQDEILTN
18 742 GISQDEILTNKQKQI 18 743 ISQDEILTNKQKQIE 18 756
IEVAEKEMNSELSLS 18 771 HKKEEDLLRENSMLR 18 815 SVKEKLLKTIQLNEE 18
824 IQLNEEALTKTKVAG 18 835 KVAGFSLRQLGLAQH 18 840 SLRQLGLAQHAQASV
18 859 YKWNHTEKTEQQAQE 18 875 EVAGFSLRQLGLAQH 18 880
SLRQLGLAQHAQASV 18 899 YKWGHTEKTEQQAQE 18 911 AQEQGAALRSQIGDP 18
954 SPGTPSLVRLASGAR 18 985 KQKSVCDSSGWILPV 18 1010 RRCPMFDVSPAMRLK
18 1017 VSPAMRLKSDSNRET 18 1068 KCRPGTLCHTDTPPH 18 1090
HRHTTTLPHRDTTTS 18 1094 TTLPHRDTTTSLPHF 18 1099 RDTTTSLPHFHVSAG 18
350 QCVFAKKKNVDKWDD 17 40 PVTWRKEPAVLPCCN 16 114 AGAFLLGWERVVQRR 16
190 RNSYRLTHVRCAQGL 16 273 LSVFQLHLIQCIPNL 16 359 VDKWDDFCLSEGYGH
16 368 SEGYGHSFLIMKETS 16 372 GHSFLIMKETSTKIS 16 517
QDEYGNTALHYAIYN 16 524 ALHYAIYNEDKLMAK 16 583 LDRYGRTALILAVCC 16
712 NQQFPDTENEEYHSD 16 858 CYKWNHTEKTEQQAQ 16 898 CYKWGHTEKTEQQAQ
16 992 SSGWILPVPTFSSGS 16 1012 CPMFDVSPAMRLKSD 16 1040
DLPFFKTQQSPRHTK 16 1041 LPFFKTQQSPRHTKD 16 373 HSFLIMKETSTKISG 15
438 DLIVMLRDTDMNKRD 15 472 QLLLDRRCQLNVLDN 15 481 LNVLDNKKRTALIKA
15 571 KFLIKKKANLNALDR 15 1062 AGVLAPKCRPGTLCH 15 1093
TTTLPHRDTTTSLPH 15 8 HQHILLPTQATFAAA 14 9 QHILLPTQATFAAAT 14 29
LTTVSNPSRADPVTW 14 58 GSWLSFPGTAARKEF 14 73 STTLTGHSALSLSSS 14 87
SRALPGSLPAFADLP 14 91 PGSLPAFADLPRSCP 14 116 AFLLGWERVVQRRLE 14 126
QRRLEVPRPQAAPAT 14 128 RLEVPRPQAAPATSA 14 158 AACLRAQGLTRAFQV 14
169 AFQVVHLAPTAPDGG 14 192 SYRLTHVRCAQGLEA 14 201 AQGLEAASANLPGAP
14 244 QRLLFLPRAPQAVSG 14 253 PQAVSGPQEQPSEEA 14 272
SLSVFQLHLIQCIPN 14 277 QLHLIQCIPNLSYPL 14 289 YPLVLRHIPEILKFS 14
293 LRHIPEILKFSEKET 14 311 ILGLEPATAARLSG 14 313 GLELPATAARLSGLN 14
326 LNSIMQIKEFEELVK 14 336 EELVKLHSLSHKVIQ 14 345 SHKVIQCVFAKKKNV
14 356 KKNVDKWDDFCLSEG 14 389 IQEMGSGKSNVGTWG 14 436
RKDLIVMLRDTDMNK 14 458 ALHLASANGNSEVVQ 14 467 NSEVVQLLLDRRCQL 14
468 SEVVQLLLDRRCQLN 14 480 QLNVLDNKKRTALIK 14 490 TALIKAVQCQEDECV
14 493 IKAVQCQEDECVLML 14 503 CVLMLLEHGADGNIQ 14 504
VLMLLEHGADGNIQD 14 505 LMLLEHGADGNIQDE 14 522 NTALHYAIYNEDKLM 14
533 DKLMAKALLLYGADI 14 538 KALLLYGADIESKNK 14 552 KCGLTPLLLGVHEQK
14 557 PLLLGVHEQKQEVVK 14 590 ALILAVCCGSASIVN 14 592
ILAVCCGSASIVNLL 14 603 VNLLLEQNVDVSSQD 14 604 NLLLEQNVDVSSQDL 14
610 NVDVSSQDLSGQTAR 14 625 EYAVSSHHHVICELL 14 631 HHHVICELLSDYKEK
14 636 CELLSDYKEKQMKLI 14 645 KQMLKISSENSNPVI 14 656
NPVITILNIKLPLKV 14 667 PLKVEEEIKKHGSNP 14 698 DDGLIPQRKSRKPEN 14
781 NSMLREEIAKLRLEL 14 785 REEIAKLRLELDETK 14 817 KEKLLKTIQLNEEAL
14 818 EKLLKTIQLNEEALT 14 821 LKTIQLNEEALTKTK 14 838
GFSLRQLGLAQHAQA 14 878 GFSLRQLGLAQHAQA 14 891 QASVQQLCYKWGHTE 14
919 RSQIGDPGGVPLSEG 14 925 PGGVPLSEGGTAAGD 14 927 GVPLSEGGTAAGDQG
14 986 QKSVCDSSGWILPVP 14 1011 RCPMFDVSPAMRLKS 14 1071
PGTLCHTDTPPHRNA 14 1102 TTSLPHFHVSAGGVG 14 1107 HFHVSAGGVGPTTLG 14
1112 AGGVGPTTLGSNREI 14 V12A-HLA-DRB1-15mers: 251P5G2 Each peptide
is a portion of SEQ ID NO: 25; each start position is specified,
the length of peptide is 15 amino acids, and the end position for
each peptide is the start position plus fourteen. 1 MLQVVNISPSISWL
21 2 MLQVVNISPSISWLI 19 9 SPSISWLIMLFSSVY 18 11 SISWLIMLFSSVYMM 16
12 ISWLIMLFSSVYMMT 13 10 PSISWLIMLFSSVYM 12 15 LIMLFSSVYMMTLIQ 9
V12B-DRB1-1101-15mers: 251P5G2 Each peptide is a portion of SEQ ID
NO: 25; each start position is specified, the length of peptide is
15 amino acids, and the end position for each peptide is the start
position plus fourteen. 332 IKEFEELVKLHSLSH 32 286 NLSYPLVLRHIPEIL
31 94 LPAFADLPRSCPESE 30 567 QEVVKFLIKKKANLN 27 428 AAWWGKVPRKDLIVM
24 166 LTRAFQVVHLAPTAP 23 954 SPGTPSLVRLASGAR 22 118
LLGWERVVQRRLEVP 21 338 LVKLHSLSHKVIQCV 21 371 YGHSFLIMKETSTKI 21
436 RKDLIVMLRDTDMNK 21 480 QLNVLDNKKRTALIK 21 656 NPVITILNIKLPLKV
21 1002 FSSGSFLGRRCPMFD 21 1014 MFDVSPAMRLKSDSN 21 29
LTTVSNPSRADPVTW 20 125 VQRRLEVPRPQAAPA 20 192 SYRLTHVRCAQGLEA 20
243 HQRLLFLPRAPQAVS 20 293 LRHIPEILKFSEKET 20 297 PEILKFSEKETGGGI
20 419 REDLDKLHRAAWWGK 20 502 ECVLMLLEHGADGNI 20 526
HYAIYNEDKLMAKAL 20 577 KANLNALDRYGRTAL 20 641 DYKEKQMLKISSENS 20
667 PLKVEEEIKKHGSNP 20 668 LKVEEEIKKHGSNPV 20 771 HKKEEDLLRENSMLR
20 841 LRQLGLAQHAQASVQ 20 881 LRQLGLAQHAQASVQ 20 960
LVRLASGARAAALPP 20 386 SGLIQEMGSGKSNVG 19 623 AREYAVSSHHHVICE 19
169 AFQVVHLAPTAPDGG 18 382 STKISGLIQEMGSGK 18 501 DECVLMLLEHGADGN
18 569 VVKFLIKKKANLNAL 18 589 TALILAVCCGSASIV 18 644
EKQMLKISSENSNPV 18 858 CYKWNHTEKTEQQAQ 18 891 QASVQQLCYKWGHTE 18
898 CYKWGHTEKTEQQAQ 18 992 SSGWILPVPTFSSGS 18 993 SGWILPVPTFSSGSF
18 1105 LPHFHVSAGGVGPTT 18 40 PVTWRKEPAVLPCCN 17 119 LGWERWQRRLEVPR
17 167 TRAFQVVHLAPTAPD 17 190 RNSYRLTHVRCAQGL 17 442
MLRDTDMNKRDKQKR 17 486 NKKRTALIKAVQCQE 17 563 HEQKQEVVKFLIKKK 17
583 LDRYGRTALILAVCC 17 746 DEILTNKQKQIEVAE 17 836 VAGFSLRQLGLAQHA
17 876 VAGFSLRQLGLAQHA 17 1090 HRHTTTLPHRDTTTS 17 16
QATFAAATGLWAALT 16 23 TGLWAALTTVSNPSR 16 57 KGSWLSFPGTAARKE 16 62
SFPGTAARKEFSTTL 16 69 RKEFSTTLTGHSALS 16 189 SRNSYRLTHVRCAQG 16 216
GRSSSCALRYRSGPS 16 222 ALRYRSGPSVSSAPS 16 299 ILKFSEKETGGGILG 16
334 EFEELVKLHSLSHKV 16 349 IQCVFAKKKNVDKWD 16 359 VDKWDDFCLSEGYGH
16 372 GHSFLIMKETSTKIS 16 402 WGDYDDSAFMEPRYH 16 408
SAFMEPRYHVRREDL 16 517 QDEYGNTALHYAIYN 16 555 LTPLLLGVHEQKQEV 16
698 DDGLIPQRKSRKPEN 16 782 SMLREEIAKLRLELD 16 1060 DRAGVLAPKCRPGTL
16 1099 RDTTTSLPHFHVSAG 16 36 SRADPVTWRKEPAVL 15 80 SALSLSSSRALPGSL
15 115 GAFLLGWERVVQRRL 15 147 DPSPPCHQRRDAACL 15 287
LSYPLVLRHIPEILK 15 418 RREDLDKLHRAAWWG 15 445 DTDMNKRDKQKRTAL 15
447 DMNKRDKQKRTALHL 15 468 SEVVQLLLDRRCQLN 15 469 EVVQLLLDRRCQLNV
15 530 YNEDKLMAKALLLYG 15 552 KCGLTPLLLGVHEQK 15 568
EVVKFLIKKKANLNA 15 625 EYAVSSHHHVICELL 15 635 ICELLSDYKEKQMLK 15
725 SDEQNDTQKQLSEEQ 15 753 QKQIEVAEKEMNSEL 15 763 MNSELSLSHKKEEDL
15 814 ESVKEKLLKTIQLNE 15 825 QLNEEALTKTKVAGF 15 834
TKVAGFSLRQLGLAQ 15 874 QEVAGFSLRQLGLAQ 15 940 QGPGTHLPPREPRAS 15
977 GKNGRSPTKQKSVCD 15 1046 TQQSPRHTKDLGQDD 15 1074 LCHTDTPPHRNADTP
15
[1061]
60TABLE L Protein Properties of 251P5G2 Bioinformatic Program URL
Outcome ORF ORF finder 722-1489 Protein length 255aa Transmembrane
TM Pred http://www.ch.embnet.org/ 6TM, N-terminal outside, aa 7-29,
region 43-62, 82-100, 118-138, 158-176, 180-199 HMMTop
http://www.enzim.hu/hmmtop/ 6TM, aa 7-29, 38-58, 85-109, 118-142,
169-193, 224-243 Sosui http://www.genome.ad.jp/SOSui/ 5TM, aa 6-28,
39-61, 86-108, 119-141, 166-188 TMHMM
http://www.cbs.dtu.dk/services/TMHMM 5TM, N terminal inside, aa
7-29, 44-62, 83-102, 117-139, 159-181 Signal Peptide Signal P
http://www.cbs.dtu.dk/services/SignalP/ cleavage between aa 129-130
pl pl/MW tool http://www.expasy.ch/tools/ pl 9.4 Molecular weight
pl/MW tool http://www.expasy.ch/tools/ 29.3 kD Localization PSORT
http://psort.nibb.ac.jp/ microbody (peroxisome) 74.8%,
mitochondrial inner membrane 71.4%, plasma membrane 65.0%,
mitochondrial intermembrane 30.4% PSORT II http://psort.nibb.ac.jp/
44.4%: endoplasmic reticulum, 22.2%: mitochondrial, 22.2%: Golgi,
11.1%: nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/
Vomeronasal organ pheromone receptor family, V1R Prints
http://www.biochem.ucl.ac.uk/ Rhodopsin-like GPCR superfamily
signature Blocks http://www.blocks.fhcrc.org/ lodothyronine
deiodinase aa 2-29
[1062]
61TABLE LI Exon compositions of 251P5G2 v.1 Exon number Start End 1
1 2156
[1063]
62TABLE LII(a). Nucleotide sequence of transcript variant 251P5G2
v.12 (SEQ ID NO: 70) gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccagyt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 aatgcctttc atttctaagc tggtattggc atctcagcca
acacttttct ccttcttttc 780 tgcgtcttct ccttttctgc tttttctgga
tctcaggcca gagcgcactt acctaccagt 840 ctgtcatgtg gccctcatcc
acatggtggt ccttctcacc atggtgttct tgtctccaca 900 gctctttgaa
tcactgaatt ttcagaatga cttcaaatat gaggcatctt tctacctgag 960
gagggtgatc agggtcctct ccatttgtac cacctgcctc ctggacatgc tgcaggtcgt
1020 caacatcagc cccagcattt cctggttgat aatgctgttc tcaagtgtct
acatgatgac 1080 tctcattcag gaactacagg agatcctggt accttcacag
ccccagcctc tacctaagga 1140 tctttgcaga ggcaagagcc atcagcacat
cctgctgccg actcaagcaa cttttgctgc 1200 agcaactgga ctatgggctg
cactaaccac cgtatcaaat ccaagcagag cagatcctgt 1260 gacctggaga
aaggagccgg ctgtccttcc ctgctgtaac ctagagaaag gaagctggct 1320
gtccttccct ggcacagctg cacgcaagga attttccacc acgctcaccg ggcacagcgc
1380 gctgagcctc tccagttcgc gggccctccc cggctcgctc ccggctttcg
cagacctccc 1440 ccgctcctgc cctgagtCCg agcagagcgC aacgccagcc
ggcgcCttCC tcctgggctg 1500 ggagcgagtg gtgcagcggc ggctcgaagt
cccccggcct caagcagccc ccgcgactag 1560 cgcgacaccc tcgcgggatc
cgagtccacc ctgccaccag cgccgggacg ccgcgtgcct 1620 cagagcccaa
gggctgaccc gggccttcca ggtggtccat ctcgctccta cggctcccga 1680
cggtggcgct gggtgtcccc catcccgcaa ttcctaccgg ctgacccatg tgcgctgcgc
1740 ccaggggctg gaggctgcca gcgccaacct tcccggcgct ccggggcgga
gcagctcctg 1800 cgccctgcgc taccgcagcg gcccttcagt cagctccgcg
ccgtcccccg cagagccccc 1860 ggcgcaccag cgcctgcttt tccttccccg
agcgcctcaa gcagtctctg ggccgcagga 1920 acagccctct gaagaggcgc
ttggtgtagg aagcctctca gttttccagt tacacctaat 1980 acagtgtatt
ccaaatctaa gttacccact agtacttcgg cacattccag aaattctgaa 2040
attttctgaa aaggaaactg gtggtggaat tctaggctta gaattaccag cgacagctgc
2100 tcgcctctca ggattaaaca gcataatgca aatcaaagag tttgaagaat
tggtaaaact 2160 tcacagcttg tcacacaaag tcattcagtg tgtgtttgca
aagaaaaaaa atgtagacaa 2220 atgggatgac ttttgtctta gtgagggtta
tggacattca ttcttaataa tgaaagaaac 2280 gtcgactaaa atatcaggtt
taattcagga gatggggagc ggcaagagca acgtgggcac 2340 ttggggagac
tacgacgaca gcgccttcat ggagccgagg taccacgtcc gtcgagaaga 2400
tctggacaag ctccacagag ctgcctggtg gggtaaagtc cccagaaagg atctcatcgt
2460 catgctcagg gacactgaca tgaacaagag ggacaagcaa aagaggactg
ctctacattt 2520 ggcctctgcc aatggaaatt cagaagtagt acaactcctg
ctggacagac gatgtcaact 2580 taacgtcctt gacaacaaaa aaaggacagc
tctgataaag gccgtacaat gccaggaaga 2640 tgaatgtgtg ttaatgttgc
tggaacatgg cgctgatgga aatattcaag atgagtatgg 2700 aaataccgct
ctacactatg ctatctacaa tgaagataaa ttaatggcca aagcactgct 2760
cttatatggt gctgatattg aatcaaaaaa caagtgtggc ctcacaccac ttttgcttgg
2820 cgtacatgaa caaaaacagg aagtggtgaa atttttaatc aagaaaaaag
ctaatttaaa 2880 tgcacttgat agatatggaa gaactgccct catacttgct
gtatgttgtg gatcagcaag 2940 tatagtcaat cttctacttg agcaaaatgt
tgatgtatct tctcaagatc tatctggaca 3000 gacggccaga gagtatgctg
tttctagtca tcatcatgta atttgtgaat tactttctga 3060 ctataaagaa
aaacagatgc taaaaatctc ttctgaaaac agcaatccag tgataaccat 3120
ccttaatatc aaacttccac tcaaggttga agaagaaata aagaagcatg gaagtaatcc
3180 tgtgggatta ccagaaaacc tgactaatgg tgccagtgct ggcaatggtg
atgatggatt 3240 aattccacaa aggaagagca gaaaacctga aaatcagcaa
tttcctgaca ctgagaatga 3300 agagtatcac agtgacgaac aaaatgatac
ccagaaacaa ctttctgaag aacagaacac 3360 tggaatatca caagatgaga
ttctgactaa taaacaaaag cagatagaag tggctgaaaa 3420 ggaaatgaat
tctgagcttt ctcttagtca taagaaagaa gaagatctct tgcgtgaaaa 3480
cagcatgttg cgggaagaaa ttgccaagct aagactggaa ctagatgaaa caaaacatca
3540 gaaccagcta agggaaaata aaattttgga ggaaattgaa agtgtaaaag
aaaaacttct 3600 aaagactata caactgaatg aagaagcatt aacgaaaacc
aaggtggctg gtttctcttt 3660 gcgccagctt ggccttgccc agcatgcaca
agcctcagtg caacagctgt gctacaaatg 3720 gaaccacaca gagaaaacag
agcagcaggc tcaggagcag gaggtggctg gtttctcttt 3780 ycgccagctt
ggccttgccc agcatgcaca agcctcagta caacaactgt gctacaaatg 3840
gggccacaca gagaaaacag agcagcaggc tcaggagcag ggagctgcgc tgaggtccca
3900 gataggcgac cctggcgggg tgcccctgag cgaagggggg acagcagcag
gagaccaggg 3960 tccagggacc cacctcccac cgagggaacc tcgagcctcc
cctggcaccc ctagcttggt 4020 ccgcctggcc tccggagccc gagctgctgc
gcttccccca cccacaggga aaaacggccg 4080 atctccaacc aaacagaaat
ctgtgtgtga ctcctctggt tggatactgc cagtccccac 4140 attttcttcc
gggagttttc ttggcagaag gtgcccaatg tttgatgttt cgccagccat 4200
gaggctgaaa agtgacagca atagagaaac acatcagyct ttccgcgaca aagatgacct
4260 tcccttcttc aaaactcagc aatctccacg gcacacaaag gacttaggac
aagatgaccg 4320 agctggagtg ctcgccccaa aatgcaggcc cggaacactc
tgccacacgg acacaccacc 4380 acacagaaat geggacacac caccacacag
acacaccacc acyctyccac acagagacac 4440 caccacatcg ttgccacact
ttcatgtgtc agctggcggt gtgggcccca cgactctggg 4500 ctctaataga
gaaattactt ag 4522
[1064]
63TABLE LIII(a) Nucleotide seguence alignment of 251P5G2 v.1 (SEQ
ID NO: 71) and 251P5G2 v.12 (SEQ ID NO: 72) Score = 2009 bits
(1045), Expect = 0.0 Identites = 1047/1048(99%) Strand = Plus/Plus
251P5G2v.1: 1
gttttttttttttttttttttttttttttttattttaagggattcgtttaataggacttg 60
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 1
gttttttttttttttttttttttttttttttattttaagggattcgtttaataggacttg 60
251P5G2v.1: 61 tggtaagtggaataatgccatgcaaaggtccccatgtctaaccaccaggtt-
ctaggcatg 120 .vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v12: 61
tggtaagtggaataatyccatgcaaaggtccccatgtctaaccaccaggttctagg- catg 120
251P5G2v.1: 121 tattatggtatatgagaaatgggaattcaggct-
gcagatgaaatcaaggttgataaccag 180 .vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline. 251P5G2v.12: 121
tattatggtatatgagaaatgggaattcaggctgca- gatgaaatcaaggttgataaccag 180
251P5G2v.1: 181
ctgactctaaaacaaaaacattaacttgaattacagatttgggcctaatgtaattataag 240
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 181
ctgactctaaaacaaaaacattaacttgaattacagatttgggcctaatgtaattataag 240
251P5G2v.1: 241 cattcttaaaagtgaaagaaataataagagaaactgagtgctgtgatgtg-
agtcagttaa 300 .vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 241
cattcttaaaagtgaaagaaataataagagaaactgagtgctgtgatgtgagt- cagttaa 300
251P5G2v.1: 301 acttttttttcaactttttctttaggtgat-
tattttcccttaacataaaatttactttag 360 .vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline. 251P5G2v.12: 301
acttttttttcaactttttctttaggtgattat- tttcccttaacataaaatttactttag 360
251P5G2v.1: 361
ctcaactatacaaacatgtgagttattgttatgtaaccatcactcttcattaagaaatgc 420
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 361
ctcaactatacaaacatgtgagttattgttatgtaaccatcactcttcattaagaaatgc 420
251P5G2v.1: 421 tttgtaaaaagtgagccagtttttcatatacattcttcaaaatacattct-
caacattata 480 .vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 421
tttgtaaaaagtgagccagtttttcatatacattcttcaaaatacattctcaa- cattata 480
251P5G2v.1: 481 catcaaattatatatacatacatgcacaca-
tacactatatatatcaaggatttatatgag 540 .vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline. 251P5G2v.12: 481
catcaaattatatatacatacatgcacacatac- actatatatatcaaggatttatatgag 540
251P5G2v.1: 541
aggattaattaagaaaaaaattagtggaataaaaataatgtttatgataattttggccat 600
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 541
aggattaattaagaaaaaaattagtggaataaaaataatgtttatgataattttggccat 600
251P5G2v.1: 601 agaatatataatacagatgatgtgaagtacaaaatgttttttatacttca-
tattttgatg 660 .vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 601
agaatatataatacagatgatgtgaagtacaaaatgttttttatacttcatat- tttgatg 660
251P5G2v.1: 661 tacaaagtatgtttgtctttgtaattcaga-
tgattactttgcacttgtgttcccatgaaa 720 .vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline. 251P5G2v.12: 661
tacaaagtatgtttgtctttgtaattcagatga- ttactttgcacttgtgttcccatgaaa 720
251P5G2v.1: 721
aatgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttc 780
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 721
aatgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttc 780
251P5G2v.1: 781 tgcgtcttctccttttctgctttttctggatctcaggccagagcgcactt-
acctaccagt 840 .vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 781
tgcgtcttctcottttctgctttttctggatctcaggccagagcgcacttacc- taccagt 840
251P5G2v.1: 841 ctgtcatgtggccctcatccacatggtggt-
ccttctcaccatggtgttcttgtctccaca 900 .vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline. 251P5G2v.12: 841
ctgtcatgtggccctcatccacatggtggtcct- tctcaccatggtgttcttgtctccaca 900
251P5G2v.1: 901
gctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgag 960
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 901
gctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgag 960
251P5G2v.1: 961 gagggtgatcagggtcctctccatttgtaccacctgcctcctgggcatgc-
tgcaggtcgt 1020 .vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 961
gagggtgatcagggtcctctccatttgtaccacctgcctcctggacatgctg- caggtcgt 1020
251P5G2v.1: 1021 caacatcagccccagcatttcctggtt- g 1048
.vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
251P5G2v.12: 1021 caacatcagccccagcatttcctggttg 1048 Score = 254
bits (132), Expect = 3e-64 Identities 132/132(100%) Strand =
Plus/Plus 251P5G2v.1: 1271 ataatgctgttctcaagtgtctacatgat-
gactctcattcaggaactacaggagatcctg 1330 .vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline. 251P5G2v.12: 1049
ataatgctgttctcaagtgtctacatgat- gactctcattcaggaactacaggagatcctg 1108
251P5G2v.1: 1331
gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 1390
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline. 251P5G2v.12: 1109
gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 1168
251P5G2v.1: 1391 atcctgctgccg 1402
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline. 251P5G2v.12: 1169
atcctgctgccg 1180
[1065]
64TABLE LIV(a) Peptide sequences of protein coded by 251P5G2 v.12
(SEQ ID NO: 73) MPFISKLVLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV
CHVALIHMVV LLTMVFLSPQ 60 LFESLNFQND FKYEASFYLR RVTRVLSICT
TCLLDMLQVV NTSPSISWLI MLFSSVYMMT 120 LIQELQEILV PSQPQPLPKD
LCRGKSHQHI LLPTQATFAA ATGLWAALTT VSNPSRADPV 180 TWRKEPAVLP
CCNLEKGSWL SFPGTAARKE FSTTLTGHSA LSLSSSRALP GSLPAFADLP 240
RSCPESEQSA TPAGAFLLGW ERVVQRRLEV PRPQAAPATS ATPSRDPSPP CHQRRDAACL
300 RAQGLTRAFQ VVHLAPTAPD GGAGCPPSRN SYRLTHVRCA QGLEAASANL
PGAPGRSSSC 360 ALRYRSGPSV SSAPSPAEPP AHQRLLFLPR APQAVSGPQE
QPSEEALGVG SLSVFQLHLI 420 QCIPNLSYPL VLRHIPEILK FSEKETGGGI
LGLELPATAA RLSGLNSIMQ IKEFEELVKL 480 HSLSHKVIQC VFAKKKNVDK
WDDFCLSEGY GHSFLIMKET STKISGLIQE MGSGKSNVGT 540 WGDYDDSAFM
EPRYHVRRED LDKLHRAAWW GKVPRKDLIV MLRDTDMNKR DKQKRTALHL 600
ASANGNSEVV QLLLDRRCQL NVLDNKKRTA LIKAVOCQED ECVLMLLEHG ADGNIQDEYG
660 NTALHYAIYN EDKLMAKALL LYGADIESKN KCGLTPLLLG VHEQKQEVVK
FLIKKKANLN 720 ALDRYGRTAL ILAVCCGSAS IVNLLLEQNV DVSSQDLSGQ
TAREYAVSSH HHVICELLSD 780 YKEKQMLKIS SENSNPVITI LNIKLPLKVE
EEIKKHGSNP VGLPENLTNG ASAGNGDDGL 840 IPQRKSRKPE MQQFPDTENE
EYHSDEQNDT QKQLSEEQNT GISQDEILTN KQKQIEVAEK 900 EMNSELSLSH
KKEEDLLREH SMLREEIAKL RLELDETKHQ NQLRENKILE EIESVKEKLL 960
KTIQLNEEAL TKTKVAGFSL RQLGLAQHAQ ASVQQLCYKW NHTEKTEQQA QEQEVAGFSL
1020 RQLGLAQHAQ ASVQQLCYKW GHTEKTEQQA QEQGAALRSQ IGDPGGVPLS
EGGTAAGDQG 1080 PGTHLPPREP RASPGTPSLV RLASGARAAA LPPPTGKNGR
SPTKQKSVCD SSGWILPVPT 1140 FSSGSFLGRR CPMFDVSPAM RLKSDSNRET
HQAFRDKDDL PFFKTQQSPR HTKDLGQDDR 1200 AGVLAPKCRP GTLCHTDTPP
HRNADTPPHR HTTTLPHRDT TTSLPHFHVS AGGVGPTTLG 1260 SNREIT 1266
[1066]
65TABLE LV(a) Amino acid seguence alignment of 121P1F1 v.1 (SEQ ID
NO: 74) and 251P5G2 v.12 (SEQ ID NO: 75) Score = 269 bits (688),
Expect = 2e-71 Identities = 152/227 (66%), Positives = 152/227
(66%), Gaps = 74/227 (32%) 251P5G2v.1: 1
MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIH- MVVLLTMVFLSPQ 60
MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIH- MVVLLTMVFLSPQ
251P5G2v.12: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYL-
PVCHVALIHMVVLLTMVFLSPQ 60 251P5G2v.1: 61
LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLCMLQVVNISPSISWLVRFKWKSTIFT 120
LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLL MLQVVNISPSISWL 251P5G2v.12: 61
LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLDMLQVVNISPSISWL----------- 109
251P5G2v.1: 121 FHLFSWSLSFPVSSSLIFYTVASSNVTQINLHVSKYCSLFPINSIIRGLF-
FTLSLFRDVF 180 251P5G2v.12: 109 ---------------------------
---------------------------------- 109 251P5G2v.1: 181
LKQIMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 227
IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 251P5G2v.12: 110
---IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 153
[1067]
66TABLE LII(b) Nucleotide seguence of transcript variant
251P5G2v.13 (SEQ ID NO: 76) atgcctttca tttctaagct ggtattggca
tctcagccaa cacttttctc cttcttttct 60 gcgtcttctc cttttctgct
ttttctggat ctcaggccag agcgcactta cctaccagtc 120 tgtcatgtgg
ccctcatcca catggtggtc cttctcacca tggtgttctt gtctccacag 180
ctctttgaat cactgaattt tcagaatgac ttcaaataty aggcatcttt ctacctgagg
240 agggtgatca gggtcctctc catttgtacc acctgcctcc tggacatgct
ycaggtcgtc 300 aacatcagcc ccagcatttc ctggttgata atgctgttct
caagtgtcta catgatgact 360 ctcattcagg aactacagga gatcctggta
ccttcacagc cccagcctct acctaaggat 420 dtttgcagag gcaagagcca
tcagcacatc ctgctgccga ctcaagcaac ttttgctgca 480 gcaactggac
tatgggctgc actaaccacc gtatcaaatc caagcagagc agatcctgtg 540
acctggagaa aggagccggc tgtccttccc tgctgtaacc tagagaaagg aagctggctg
600 tccttccctg gcacagctgc acgcaaggaa ttttccacca cgctcaccgy
gcacaycgcg 660 ctgagcctct ccagttcgcg ggccctcccc ggctcgctcc
cggctttcgc agacctcccc 720 cgctcctgcc ctgagtccga gcagagcgca
acyccagecy gcgccttcct cctyggctgg 780 yagcgagtgg tgcagcggcg
gctcgaagtc ccccggcctc aagcagcccc cgcgactagc 840 gcgacaccct
cgcgggatcc gagtccaccc tgccaccagc gccgggacgc cgcgtgcctc 900
agagcccaag ggctgacccg ggccttccag gtggtccatc tcgctcctac ggctcccgac
960 ggtggcgctg ggtgtccccc atcccgcaat tcctaccggc tgacccatgt
gcgctgcgcc 1020 caggggctgg aggctgccag cgccaacctt cccggcgctc
cggggcggag cagctcctgc 1080 gccctgcgct accgcagcgg cccttcagtc
agctccgcgc cgtcccccgc agagcccccg 1140 gcgcaccagc gcctgctttt
ccttccccga gcgcctcaag cagtctctgg gccgcaggaa 1200 cagccctctg
aagaggcgct tggtgtagga agcctctcag ttttccagtt acacctaata 1260
cagtgtattc caaatctaag ttacccacta 4tacttcggc acattccaga aattctgaaa
1320 ttttctgaaa agyaaactgg tgytggaatt ctaggcttag aattaccagc
gacagctgct 1380 cgcctctcag gattaaacag cataatgcaa atcaaagagt
ttgaagaatt ggtaaaactt 1440 cacagcttgt cacacaaagt cattcagtgt
gtgtttgcaa agaaaaaaaa tgtagacaaa 1500 tgggat9act tttgtcttag
tgagggttat ggacattcat tcttaataat gaaagaaacg 1560 tcgactaaaa
tatcaggttt aattcaggag atggggagcg gcaagagcaa cgtgggcact 1620
tggggagact acyacgacag cyccttcatg gagccgaggt accacgtccg tcgagaagat
1680 ctggacaagc tccacagagc tgcctggtgg ggtaaagtcc ccagaaagga
tctcatcgtc 1740 atgctcaggg acactyacat gaacaagagg gacaagcaaa
agaggactgc tctacatttg 1800 gcctctgcca atggaaattc agaagtagta
caactcctgc tggacagacg atgtcaactt 1860 aacgtccttg acaacaaaaa
aaggacagct ctgataaagg ccgtacaatg ccaggaagat 1920 gaatgtgtgt
taatgttgct ggaacatggc gctyatggaa atattcaaga tgagtatgga 1980
aataccgctc tacactatgc tatctacaat gaagataaat taatggccaa agcactgctc
2040 ttatatggtg ctgatattga atcaaaaaac aagtytggcc toacaccact
tttgcttggc 2100 gtacatgaac aaaaacagga agtggtgaaa tttttaatca
agaaaaaagc taatttaaat 2160 gcacttgata gatatggaag aactgccctc
atacttgctg tatgttgtgg atcagcaagt 2220 atagtcaatc ttctacttga
gcaaaatgtt gatgtatctt ctcaagatct atctggacag 2280 acggccagag
agtatgctgt ttctagtcat catcatgtaa tttgtgaatt actttctgac 2340
tataaagaaa aacagatgct aaaaatctct tctgaaaaca gcaatccagt gataaccatc
2400 cttaatatca aacttccact caaggttgaa gaagaaataa agaagcatgg
aagtaatcct 2460 gtgggattac cagaaaacct gactaatggt gccagtgctg
gcaatggtga tgatggatta 2520 attccacaaa ggaagagcag aaaacctgaa
aatcagcaat ttcctgacac tgagaatgaa 2580 gagtatcaca gtgacgaaca
aaatgatacc cagaaacaac tttctgaaga acagaacact 2640 ggaatatcac
aagatgagat tctgactaat aaacaaaagc agatagaagt ggctgaaaag 2700
gaaatgaatt ctgagctttc tcttagtcat aagaaagaag aagatctctt gcgtgaaaac
2760 agcatgttgc gggaagaaat tgccaagcta agactggaac tagatgaaac
aaaacatcag 2820 aaccagctaa gggaaaataa aattttggag gaaattgaaa
gtgtaaaaga aaaacttcta 2880 aagactatac aactgaatga agaagcatta
acgaaaacca aggtggctgg tttctctttg 2940 cgccagcttg gccttgccca
gcatgcacaa gcctcagtgc aacagctgtg ctacaaatgg 3000 aaccacacag
agaaaacaga gcagcaggct caggagcagg aggtggctgg tttctctttg 3060
cgccagcttg gccttgccca gcatgcacaa gcctcagtac aacaactgtg ctacaaatgg
3120 ggccacacag agaaaacaga gcagcaggct caggagcagg gagct9cgct
gaggtcccag 3180 ataggcgacC ctggcggggt gcccctgagc gaagggggga
cagcagcagg agaccagggt 3240 ccaggyacCC acctcccacC gagggaacct
cgagcctccc ctggcacccc tagcttggtc 3300 cgcctggcct ccggagcccg
agctgctgcg cttcccccac ccacagggaa aaacggccga 3360 tctccaacca
aacagaaatc tgtgtgtgac tcctctggtt ggatactgcc agtccccaca 3420
ttttcttccg ggagttttct tggcagaayg tgcccaatgt ttgatgtttc gccagccatg
3480 aggctgaaaa gtgacagcaa tagagaaaca catcaggctt tccgcgacaa
agatgacctt 3540 cccttcttca aaactcayca atctccacgg cacacaaagg
acttaggaca agatgaccga 3600 gctggagtgc tcgccccaaa atgcaggccc
ggaacactct gccacacgga cacaccacca 3660 cacagaaatg cggacacacc
accacacaga cacaccacca cgctgccaca cagagacacc 3720 accacatcgt
tgccacactt tcatgtgtca gctggcggtg tgggccccac gactctgggc 3780
tctaatagag aaattactta g 3801
[1068]
67TABLE LIII(b) Nucleotide sequence alignment of 251P5G2 v.1 (SEQ
ID NO: 77) and 251P5G2 v.13 (SEQ ID NO: 78) Score = 623 bits (324),
Expect = e-175 Identities = 326/327 (99%) Strand = Plus/Plus Query:
722 atgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttct
781
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
Sbjct: 1 atgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttt-
tct 60 Query: 782 gcgtcttctccttttctgctttttctggatctcaggccag-
agcgcacttacctaccagtc 841 .vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline. Sbjct: 61
ggtcttctccttttctgctttttc- tggatctcaggccagagcgcacttacctaccagtc 120
Query: 842
tgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccacag 901
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
Sbjct: 121 tgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctc-
cacag 180 Query: 902 ctctttgaatcactgaattttcagaatgacttcaaat-
atgaggcatctttctacctgagg 961 .vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline. Sbjct: 181
ctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgagg 240
Query: 962 agggtgatcagggtcctctccatttgtaccacctycctcctgggcatgctgcagg-
tcgtc 1021 .vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline. Sbjct: 241
agggtgatcagggtcctctccatttgtaccacctgcc- tcctggacatgctgcaggtcgtc 300
Query: 1022 aacatcagccccagcatttcctggttg 1048
.vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline. Sbjct: 301 aacatcagccccagcatttcctggttg 327 Score = 254 bits
(132), Expect = 3e-64 Identities = 132/132(100%) Strand Plus/Plus
Query: 1271 ataatgctgttctcaagtgtctacatgatgactc-
tcattcaggaactacaggagatcctg 1330 .vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline. Sbjct: 328
ataatgctgttctcaagtgtctacatgatyactctcattcaggaactacaggagatcctg 387
Query: 1331 gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccat-
cagcac 1390 .vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline. Sbjct: 388
gtaccttcacagccccagcctctacc- taaggatctttgcagaggcaagagccatcagcac 447
Query: 1391 atcctgctgccg 1402
.vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
Sbjct: 448 atcctgctgccg 459
[1069]
68TABLE LIV(b) Peptide sequences of protein coded by 251P5G2 v.13
(SEQ ID NO: 79) MPFISKLVLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV
CHVALIHMVV LLTMVFLSPQ 60 LFESLNFQND FKYEASFYLR RVIRVLSICT
TCLLDMLQVV NISPSISWLI MLFSSVYMMT 120 LIQELQEILV PSQPQPLPKD
LCRGKSHQHI LLPTQATFAA ATGLWAALTT VSNPSPADPV 180 TWRKEPAVLP
CCNLEKGSWL SFPGTAARKE FSTTLTGHSA LSLSSSRALP GSLPAFADLP 240
RSCPESEQSA TPAGAFLLGW ERVVQRRLEV PRPQAAPATS ATPSRDPSPP CHQRRDAACL
300 RAQGLTRAFQ VVHLAPTAPD GGAGCPPSRN SYRLTHVRCA QGLEAASANL
PGAPGRSSSC 360 ALRYRSGPSV SSAPSPAEPP AHQRLLFLPR APQAVSGPQE
QPSEEALGVG SLSVFQLHLI 420 QCIPNLSYPL VLRHIPEILK FSEKETGGGI
LGLELPATAA RLSGLNSIMQ IKEFEELVKL 480 HSLSHKVIQC VFAKKKNVDK
WDDFCLSEGY GHSFLIMKET STKISGLIQE MGSGKSNVGT 540 WGDYDDSAFM
EPRYHVRRED LDKLHRAAWW GKVPRKDLIV MLRDTDMNKR DKQKRTALHL 600
ASANGNSEVV QLLLDRRCQL NVLDNKKRTA LIKAVQCQED ECVLMLLEHG ADGNIQDEYG
660 NTALHYAIYN EDKLMAKALL LYGADIESKN KCGLTPLLLG VHEQKQEVVK
FLIKKKANLN 720 ALDRYGRTAL ILAVCCGSAS IVNLLLEQNV DVSSQDLSGQ
TAREYAVSSH HHVICELLSD 780 YKEKQMLKIS SEMSNPVITI LNIKLPLKVE
EEIKKHGSNP VGLPENLTNG ASAGNGDDGL 840 IPQRKSRKPE NQQFPDTENE
EYHSDEQNDT QKQLSEEQNT GISQDEILTN KQKQIEVAEK 900 EMNSELSLSH
KKEEDLLREN SMLREEIAKL RLELDETKHQ NQLRENKILE EIESVKEKLL 960
KTIQLNEEAL TKTKVAGFSL RQLGLAQHAQ ASVQQLCYKW NHTEKTEQQA QEQEVAGFSL
1020 RQLGLAQHAQ ASVQQLCYKW GHTEKTEQQA QEQGAALRSQ IGDPGGVPLS
EGGTAAGDQG 1080 PGTHLPPREP RASPGTPSLV RLASGARAAA LPPPTGKNGR
SPTKQKSVCD SSGWILPVPT 1140 FSSGSFLGRR CPMFDVSPAM RLKSDSNRET
HQAFRDKDDL PFFKTQQSPR HTKDLGQDDR 1200 AGVLAPKCRP GTLCHTDTPP
HRNADTPPHR HTTTLPHRDT TTSLPHFHVS AGGVGPTTLG 1260 SNREIT 1266
[1070]
69TABLE LV(b) Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID
NO: 80) and 251P5G2 v.13 (SEQ ID NO: 81) Score = 269 bits (688),
Expect = 2e-71 Identities = 152/227 (66%), Positives = 152/227
(66%), Gaps = 74/227 (32%) 251P5G2v.1: 1
MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMV- FLSPQ 60
MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMV- FLSPQ
251P5G2v.13: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALI-
HMVVLLTMVFLSPQ 60 251P5G2v.1: 61 LFESLNFQNDFKYEASFYLRRVIRV-
LSICTTCLLGMLQVVNISPSISWLVRFKWKSTIFT 120 LFESLNFQNDFKYEASFYLRRVIRV-
LSICTTCLL MLQVVNISPSISWL 251P5G2v.13: 61
LFESLNFQNDFKYEASFYLRRVIRVL- SICTTCLLDMLQVVNISPSISWL----------- 109
251P5G2v. 1: 121
FHLFSWSLSFPVSSSLIFYTVASSNVTQINLHVSKYCSLFPINSIIRGLFFTLSLFRDVF 180
251P5G2v.13: 109 --------------------------------------------------
----------- 109 251P5G2v.1: 181 LKQIMLFSSVYMMTLIQELQEILVPS-
QPQPLPKDLCRGKSHQHILLP 227 IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGK-
SHQHILLP 251P5G2v.13: 110
---IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSH- QHILLP 153
[1071]
Sequence CWU 1
1
83 1 162 DNA Homo sapians 1 gatcaccctc ctcaggtaga aagatgcctc
atatttgaag tcattctgaa aattcagtga 60 ttcaaagagc tgtggagaca
agaacaccat ggtgagaagg accaccatgt ggataagggc 120 cacatgacag
actggtaggt aagtgcgctc tggcctgaga tc 162 2 2156 DNA Homo sapians CDS
(722)...(1489) 2 gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccaggt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca tct cag
cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct ttt ctg
ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta cca gtc
tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc acc atg
gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat ttt cag
aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg
atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg ggc atg 1009 Arg
Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90
95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg gtg agg ttt
1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg
Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc ttc cat ttg ttc tca
tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe
Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt agt agc ctg atc ttt
tac act gtg gct tct tcc 1153 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg acc cag atc aat ttg
cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn Val Thr Gln Ile Asn
Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 cca ata aac
tcc ata atc aga gga ctg ttt ttc act ctg tca tta ttc 1249 Pro Ile
Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175
aga gat gtt ttt ctt aaa caa ata atg ctg ttc tca agt gtc tac atg
1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr
Met 180 185 190 atg act ctc att cag gaa cta cag gag atc ctg gta cct
tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val
Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat ctt tgc aga ggc
aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg
Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg
gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu Pro Val Ser Phe
Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 atc tca acc
tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag 1489 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val * 245 250 255
aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag atctgataaa
1549 agggtaatca atgtgatgta aactataaga caaatgttta aaaggttaat
tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc attatagaga
ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg taggataaaa
gtagtaggag attgcttctt gaagatttaa 1729 aattatattg agtgtaatta
tttgcattaa aataatttta aatgttttga atagcaagta 1789 ttgatataat
taaactttcg aataacttag tgctttgcct ttattcctaa tgtttatatg 1849
gaagcatgtg gtcaatgttt gatgcattac agctctgagc ggtccttctg tattaggtgg
1909 tcatcattta tatacttctc cataaaagat taaggacctg gaaatgtaag
atacatgaag 1969 aaaatctaag tggagaggct gtttgtggtt aagtgataac
agtgttgtaa gcgatgcatg 2029 aggtaggtgt tcagtgcata tcctctgcat
tttattaata aacactgtaa aatttagaag 2089 aaaattgttt caccaaatgc
acataaaact aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc 2156 3 255
PRT Homo sapians 3 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val
Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95
Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100
105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser
Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val
Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys
Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys
Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220
Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225
230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly
Val 245 250 255 4 2156 DNA Homo sapians CDS (722)...(1489) 4
gttttttttt tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg
60 tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccaggt
tctaggcatg 120 tattatggta tatgagaaat gggaattcag gctgcagatg
aaatcaaggt tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa
ttacagattt gggcctaatg taattataag 240 cattcttaaa agtgaaagaa
ataataagag aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt
caactttttc tttaggtgat tattttccct taacataaaa tttactttag 360
ctcaactata caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc
420 tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct
caacattata 480 catcaaatta tatatacata catgcacaca tacactatat
atatcaagga tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat
aaaaataatg tttatgataa ttttggccat 600 agaatatata atacagatga
tgtgaagtac aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat
gtttgtcttt gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 a atg
cct ttc att tct aag ctg gta ttg gca tct cag cca aca ctt tgc 769 Met
Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Cys 1 5 10
15 tcc ttc ttt tct gcg tct tct cct ttt ctg ctt ttt ctg gat ctc agg
817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg
20 25 30 cca gag cgc act tac cta cca gtc tgt cat gtg gcc ctc atc
cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile
His Met 35 40 45 gtg gtc ctt ctc acc atg gtg ttc ttg tct cca cag
ctc ttt gaa tca 913 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln
Leu Phe Glu Ser 50 55 60 ctg aat ttt cag aat gac ttc aaa tat gag
gca tct ttc tac ctg agg 961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu
Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg atc agg gtc ctc tcc att
tgt acc acc tgc ctc ctg ggc atg 1009 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 ctg cag gtc gtc aac
atc agc ccc agc att tcc tgg ttg gtg agg ttt 1057 Leu Gln Val Val
Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg
aaa tcc aca att ttt acc ttc cat ttg ttc tca tgg tct ctc 1105 Lys
Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120
125 agt ttt cct gtt agt agt agc ctg atc ttt tac act gtg gct tct tcc
1153 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser
Ser 130 135 140 aat gtg acc cag atc aat ttg cat gtc agt aaa tac tgt
tca ctt ttc 1201 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr
Cys Ser Leu Phe 145 150 155 160 cca ata aac tcc ata atc aga gga ctg
ttt ttc act ctg tca tta ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 aga gat gtt ttt ctt aaa
caa ata atg ctg ttc tca agt gtc tac atg 1297 Arg Asp Val Phe Leu
Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 atg act ctc
att cag gaa cta cag gag atc ctg gta cct tca cag ccc 1345 Met Thr
Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205
cag cct cta cct aag gat ctt tgc aga ggc aag agc cat cag cac atc
1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg gtg ggc atg tac aag atg
gac ttc atc 1441 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys
Met Asp Phe Ile 225 230 235 240 atc tca acc tcc tca aca ttg cca tgg
gca tat gac cgt ggt gtc tag 1489 Ile Ser Thr Ser Ser Thr Leu Pro
Trp Ala Tyr Asp Arg Gly Val * 245 250 255 aggctagtgg gcagtgtcta
taccattgtc aggtttttgg tgctactgag atctgataaa 1549 agggtaatca
atgtgatgta aactataaga caaatgttta aaaggttaat tgtatgaatc 1609
ctgtcatgag ttaaattatt cagagtgttc attatagaga ataatccaaa gttaaaataa
1669 ttggataatt tatttgtatg taggataaaa gtagtaggag attgcttctt
gaagatttaa 1729 aattatattg agtgtaatta tttgcattaa aataatttta
aatgttttga atagcaagta 1789 ttgatataat taaactttcg aataacttag
tgctttgcct ttattcctaa tgtttatatg 1849 gaagcatgtg gtcaatgttt
gatgcattac agctctgagc ggtccttctg tattaggtgg 1909 tcatcattta
tatacttctc cataaaagat taaggacctg gaaatgtaag atacatgaag 1969
aaaatctaag tggagaggct gtttgtggtt aagtgataac agtgttgtaa gcgatgcatg
2029 aggtaggtgt tcagtgcata tcctctgcat tttattaata aacactgtaa
aatttagaag 2089 aaaattgttt caccaaatgc acataaaact aataaaatag
agtggatttt gatatgtccc 2149 tcgtgcc 2156 5 255 PRT Homo sapians 5
Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Cys 1 5
10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu
Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu
Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro
Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr
Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln Val Val Asn
Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys
Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser
Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135
140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe
145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu
Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu
Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu
Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser
Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 6
2156 DNA Homo sapians CDS (722)...(1489) 6 gttttttttt tttttttttt
tttttttttt tattttaagg gattcgttta ataggacttg 60 tggtaagtgg
aataatgcca tgcaaaggtc cccatgtcta accaccaggt tctaggcatg 120
tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt tgataaccag
180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt gggcctaatg
taattataag 240 cattcttaaa agtgaaagaa ataataagag aaactgagtg
ctgtgatgtg agtcagttaa 300 actttttttt caactttttc tttaggtgat
tattttccct taacataaaa tttactttag 360 ctcaactata caaacatgtg
agttattgtt atgtaaccat cactcttcat taagaaatgc 420 tttgtaaaaa
gtgagccagt ttttcatata cattcttcaa aatacattct caacattata 480
catcaaatta tatatacata catgcacaca tacactatat atatcaagga tttatatgag
540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg tttatgataa
ttttggccat 600 agaatatata atacagatga tgtgaagtac aaaatgtttt
ttatacttca tattttgatg 660 tacaaagtat gtttgtcttt gtaattcaga
tgattacttt gcacttgtgt tcccatgaaa 720 a atg cct ttc att tct aag ctg
gta ttg gca tct cag cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu
Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg
tct tct cct ttt ctg ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala
Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc
act tac cta cca gtc tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg
Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 gtg
gtc ctt ctc acc atg gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val
Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55
60 ctg aat ttt cag aat gac ttc aaa tat gag gca tct ttc tac ctg agg
961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg
65 70 75 80 agg gtg atc agg gac ctc tcc att tgt acc acc tgc ctc ctg
ggc atg 1009 Arg Val Ile Arg Asp Leu Ser Ile Cys Thr Thr Cys Leu
Leu Gly Met 85 90 95 ctg cag gtc gtc aac atc agc ccc agc att tcc
tgg ttg gtg agg ttt 1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile
Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc
ttc cat ttg ttc tca tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe
Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt
agt agc ctg atc ttt tac act gtg gct tct tcc 1153 Ser Phe Pro Val
Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg
acc cag atc aat ttg cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn
Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150
155 160 cca ata aac tcc ata atc aga gga ctg ttt ttc act ctg tca tta
ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser
Leu Phe 165 170 175 aga gat gtt ttt ctt aaa caa ata atg ctg ttc tca
agt gtc tac atg 1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 atg act ctc att cag gaa cta cag gag
atc ctg gta cct tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln
Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat
ctt tgc aga ggc aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys
Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg
gtg agt ttc tcg gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu
Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235
240 atc tca acc tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag
1489 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val *
245 250 255 aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag
atctgataaa 1549 agggtaatca atgtgatgta aactataaga caaatgttta
aaaggttaat tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc
attatagaga ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg
taggataaaa gtagtaggag attgcttctt gaagatttaa 1729 aattatattg
agtgtaatta tttgcattaa aataatttta aatgttttga atagcaagta 1789
ttgatataat taaactttcg
aataacttag tgctttgcct ttattcctaa tgtttatatg 1849 gaagcatgtg
gtcaatgttt gatgcattac agctctgagc ggtccttctg tattaggtgg 1909
tcatcattta tatacttctc cataaaagat taaggacctg gaaatgtaag atacatgaag
1969 aaaatctaag tggagaggct gtttgtggtt aagtgataac agtgttgtaa
gcgatgcatg 2029 aggtaggtgt tcagtgcata tcctctgcat tttattaata
aacactgtaa aatttagaag 2089 aaaattgttt caccaaatgc acataaaact
aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc 2156 7 255 PRT Homo
sapians 7 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr
Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe
Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His
Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe
Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp
Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg
Asp Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln
Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110
Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115
120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser
Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys
Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe
Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile
Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu
Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro
Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu
Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235
240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245
250 255 8 2156 DNA Homo sapians CDS (722)...(1489) 8 gttttttttt
tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg 60
tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccaggt tctaggcatg
120 tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt
tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt
gggcctaatg taattataag 240 cattcttaaa agtgaaagaa ataataagag
aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt caactttttc
tttaggtgat tattttccct taacataaaa tttactttag 360 ctcaactata
caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc 420
tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct caacattata
480 catcaaatta tatatacata catgcacaca tacactatat atatcaagga
tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg
tttatgataa ttttggccat 600 agaatatata atacagatga tgtgaagtac
aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat gtttgtcttt
gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 a atg cct ttc att
tct aag ctg gta ttg gca tct cag cca aca ctt ttc 769 Met Pro Phe Ile
Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc
ttt tct gcg tct tct cct ttt ctg ctt ttt ctg gat ctc agg 817 Ser Phe
Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30
cca gag cgc act tac cta cca gtc tgt cat gtg gcc ctc atc cac atg 865
Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35
40 45 gtg gtc ctt ctc acc atg gtg ttc ttg tct cca cag ctc ttt gaa
tca 913 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu
Ser 50 55 60 ctg aat ttt cag aat gac ttc aaa tat gag gca tct ttc
tac ctg agg 961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe
Tyr Leu Arg 65 70 75 80 agg gtg atc agg gtc ctc tcc att tgt acc acc
tgc ctc ctg gac atg 1009 Arg Val Ile Arg Val Leu Ser Ile Cys Thr
Thr Cys Leu Leu Asp Met 85 90 95 ctg cag gtc gtc aac atc agc ccc
agc att tcc tgg ttg gtg agg ttt 1057 Leu Gln Val Val Asn Ile Ser
Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg aaa tcc aca
att ttt acc ttc cat ttg ttc tca tgg tct ctc 1105 Lys Trp Lys Ser
Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 agt ttt
cct gtt agt agt agc ctg atc ttt tac act gtg gct tct tcc 1153 Ser
Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135
140 aat gtg acc cag atc aat ttg cat gtc agt aaa tac tgt tca ctt ttc
1201 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu
Phe 145 150 155 160 cca ata aac tcc ata atc aga gga ctg ttt ttc act
ctg tca tta ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe
Thr Leu Ser Leu Phe 165 170 175 aga gat gtt ttt ctt aaa caa ata atg
ctg ttc tca agt gtc tac atg 1297 Arg Asp Val Phe Leu Lys Gln Ile
Met Leu Phe Ser Ser Val Tyr Met 180 185 190 atg act ctc att cag gaa
cta cag gag atc ctg gta cct tca cag ccc 1345 Met Thr Leu Ile Gln
Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 cag cct cta
cct aag gat ctt tgc aga ggc aag agc cat cag cac atc 1393 Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220
ctg ctg ccg gtg agt ttc tcg gtg ggc atg tac aag atg gac ttc atc
1441 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe
Ile 225 230 235 240 atc tca acc tcc tca aca ttg cca tgg gca tat gac
cgt ggt gtc tag 1489 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr
Asp Arg Gly Val * 245 250 255 aggctagtgg gcagtgtcta taccattgtc
aggtttttgg tgctactgag atctgataaa 1549 agggtaatca atgtgatgta
aactataaga caaatgttta aaaggttaat tgtatgaatc 1609 ctgtcatgag
ttaaattatt cagagtgttc attatagaga ataatccaaa gttaaaataa 1669
ttggataatt tatttgtatg taggataaaa gtagtaggag attgcttctt gaagatttaa
1729 aattatattg agtgtaatta tttgcattaa aataatttta aatgttttga
atagcaagta 1789 ttgatataat taaactttcg aataacttag tgctttgcct
ttattcctaa tgtttatatg 1849 gaagcatgtg gtcaatgttt gatgcattac
agctctgagc ggtccttctg tattaggtgg 1909 tcatcattta tatacttctc
cataaaagat taaggacctg gaaatgtaag atacatgaag 1969 aaaatctaag
tggagaggct gtttgtggtt aagtgataac agtgttgtaa gcgatgcatg 2029
aggtaggtgt tcagtgcata tcctctgcat tttattaata aacactgtaa aatttagaag
2089 aaaattgttt caccaaatgc acataaaact aataaaatag agtggatttt
gatatgtccc 2149 tcgtgcc 2156 9 255 PRT Homo sapians 9 Met Pro Phe
Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser
Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25
30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met
35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe
Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser
Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr
Thr Cys Leu Leu Asp Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro
Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile
Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val
Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val
Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155
160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe
165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val
Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val
Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly
Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val
Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser
Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 10 2156 DNA
Homo sapians CDS (722)...(1489) 10 gttttttttt tttttttttt tttttttttt
tattttaagg gattcgttta ataggacttg 60 tggtaagtgg aataatgcca
tgcaaaggtc cccatgtcta accaccaggt tctaggcatg 120 tattatggta
tatgagaaat gggaattcag gctgcagatg aaatcaaggt tgataaccag 180
ctgactctaa aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag
240 cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg
agtcagttaa 300 actttttttt caactttttc tttaggtgat tattttccct
taacataaaa tttactttag 360 ctcaactata caaacatgtg agttattgtt
atgtaaccat cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt
ttttcatata cattcttcaa aatacattct caacattata 480 catcaaatta
tatatacata catgcacaca tacactatat atatcaagga tttatatgag 540
aggattaatt aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat
600 agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca
tattttgatg 660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt
gcacttgtgt tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca
tct cag cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala
Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct
ttt ctg ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro
Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta
cca gtc tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu
Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc
acc atg gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu
Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat
ttt cag aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn
Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80
agg gtg atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg ggc atg
1009 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly
Met 85 90 95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg
gtg agg ttt 1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp
Leu Val Arg Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc ttc cat
ttg ttc tca tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe Thr Phe
His Leu Phe Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt agt agc
ctg atc ttt tac act gtg gct tct tcc 1153 Ser Phe Pro Val Ser Ser
Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg acc cag
atc aat ttg cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn Val Thr
Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160
cca ata aac tcc ata atc aga gga ctg ttt ttc act ctg tca tta ttc
1249 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu
Phe 165 170 175 aga gat gtt ttt ctt aaa cag ata atg ctg ttc tca agt
gtc tac atg 1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser
Ser Val Tyr Met 180 185 190 atg act ctc att cag gaa cta cag gag atc
ctg gta cct tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln Glu
Ile Leu Val Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat ctt
tgc aga ggc aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys Asp
Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg gtg
agt ttc tcg gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu Pro
Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240
atc tca acc tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag
1489 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val *
245 250 255 aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag
atctgataaa 1549 agggtaatca atgtgatgta aactataaga caaatgttta
aaaggttaat tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc
attatagaga ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg
taggataaaa gtagtaggag attgcttctt gaagatttaa 1729 aattatattg
agtgtaatta tttgcattaa aataatttta aatgttttga atagcaagta 1789
ttgatataat taaactttcg aataacttag tgctttgcct ttattcctaa tgtttatatg
1849 gaagcatgtg gtcaatgttt gatgcattac agctctgagc ggtccttctg
tattaggtgg 1909 tcatcattta tatacttctc cataaaagat taaggacctg
gaaatgtaag atacatgaag 1969 aaaatctaag tggagaggct gtttgtggtt
aagtgataac agtgttgtaa gcgatgcatg 2029 aggtaggtgt tcagtgcata
tcctctgcat tttattaata aacactgtaa aatttagaag 2089 aaaattgttt
caccaaatgc acataaaact aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc
2156 11 255 PRT Homo sapians 11 Met Pro Phe Ile Ser Lys Leu Val Leu
Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser
Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr
Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu
Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu
Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70
75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly
Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu
Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu
Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu
His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser
Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp
Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190
Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195
200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met
Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala
Tyr Asp Arg Gly Val 245 250 255 12 2156 DNA Homo sapians CDS
(722)...(1489) 12 gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccaggt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca tct cag
cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct ttt ctg
ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta cca gtc
tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc acc atg
gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat ttt cag
aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg
atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg ggc atg 1009 Arg
Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90
95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg gtg agg ttt
1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg
Phe
100 105 110 aaa tgg aaa tcc aca att ttt acc ttc cat ttg ttc tca tgg
tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser
Trp Ser Leu 115 120 125 agt ttt cct gtt agt agt agc ctg atc ttt tac
act gtg gct tct tcc 1153 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe
Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg acc cag atc aat ttg cat
gtc agt aaa tac tgt tca ctt ttc 1201 Asn Val Thr Gln Ile Asn Leu
His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 cca ata aac tcc
ata atc aga gga ctg ttt ttc act ctg tca tta ttc 1249 Pro Ile Asn
Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 aga
gat gtt ttt ctt aaa caa ata atg ctg ttc tca agt gtc tac atg 1297
Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180
185 190 atg act ctc att cag gaa cta cag gag atc ctg gta cct tca cag
ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser
Gln Pro 195 200 205 cag cct cta cct aag gat ctt tgc aga ggc aag agc
cat cag cac atc 1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys
Ser His Gln His Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg gtg ggc
atg tac aag atg gac ttc atc 1441 Leu Leu Pro Val Ser Phe Ser Val
Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 13 255 PRT Homo
sapians 13 14 2156 DNA Homo sapians CDS (722)...(1489) 14
gttttttttt tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg
60 tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccaggt
tctaggcatg 120 tattatggta tatgagaaat gggaattcag gctgcagatg
aaatcaaggt tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa
ttacagattt gggcctaatg taattataag 240 cattcttaaa agtgaaagaa
ataataagag aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt
caactttttc tttaggtgat tattttccct taacataaaa tttactttag 360
ctcaactata caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc
420 tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct
caacattata 480 catcaaatta tatatacata catgcacaca tacactatat
atatcaagga tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat
aaaaataatg tttatgataa ttttggccat 600 agaatatata atacagatga
tgtgaagtac aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat
gtttgtcttt gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 a atg
cct ttc att tct aag ctg gta ttg gca tct cag cca aca ctt ttc 769 Met
Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10
15 tcc ttc ttt tct gcg tct tct cct ttt ctg ctt ttt ctg gat ctc agg
817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg
20 25 30 cca gag cgc act tac cta cca gtc tgt cat gtg gcc ctc atc
cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile
His Met 35 40 45 gtg gtc ctt ctc acc atg gtg ttc ttg tct cca cag
ctc ttt gaa tca 913 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln
Leu Phe Glu Ser 50 55 60 ctg aat ttt cag aat gac ttc aaa tat gag
gca tct ttc tac ctg agg 961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu
Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg atc agg gtc ctc tcc att
tgt acc acc tgc ctc ctg ggc atg 1009 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 ctg cag gtc gtc aac
atc agc ccc agc att tcc tgg ttg gtg agg ttt 1057 Leu Gln Val Val
Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg
aaa tcc aca att ttt acc ttc cat ttg ttc tca tgg tct ctc 1105 Lys
Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120
125 agt ttt cct gtt agt agt agc ctg atc ttt tac act gtg gct tct tcc
1153 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser
Ser 130 135 140 aat gtg acc cag atc aat ttg cat gtc agt aaa tac tgt
tca ctt ttc 1201 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr
Cys Ser Leu Phe 145 150 155 160 cca ata aac tcc ata atc aga gga ctg
ttt ttc act ctg tca tta ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 aga gat gtt ttt ctt aaa
caa ata atg ctg ttc tca agt gtc tac atg 1297 Arg Asp Val Phe Leu
Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 atg act ctc
att cag gaa cta cag gag atc ctg gta cct tca cag ccc 1345 Met Thr
Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205
cag cct cta cct aag gat ctt tgc aga ggc aag agc cat cag cac atc
1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg gtg ggc atg tac aag atg
gac ttc atc 1441 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys
Met Asp Phe Ile 225 230 235 240 atc tca acc tcc tca aca ttg cca tgg
gca tat gac cgt ggt gtc tag 1489 Ile Ser Thr Ser Ser Thr Leu Pro
Trp Ala Tyr Asp Arg Gly Val * 245 250 255 aggctagtgg gcagtgtcta
taccattgtc aggtttttgg tgctactgag atctgataaa 1549 agggtaatca
atgtgatgta aactataaga caaatgttta aaaggttaat tgtatgaatc 1609
ctgtcatgag ttaaattatt cagagtgttc attatagaga ataatccaaa gttaaaataa
1669 ttggataatt tatttgtatg taggataaaa gtagtaggag attgcttctt
gaagatttaa 1729 aattatattg agtgtaatta tttgcattaa aataatttta
aatgttttga atagcaagta 1789 ttgatataat taaactttcg aataacttag
tgctttgcct ttattcctaa tgtttatatg 1849 gaagcatgtg gtcaatgttt
gatgcattac agctctgagc ggtccttctg tattaggtgg 1909 tcatcattta
tgtacttctc cataaaagat taaggacctg gaaatgtaag atacatgaag 1969
aaaatctaag tggagaggct gtttgtggtt aagtgataac agtgttgtaa gcgatgcatg
2029 aggtaggtgt tcagtgcata tcctctgcat tttattaata aacactgtaa
aatttagaag 2089 aaaattgttt caccaaatgc acataaaact aataaaatag
agtggatttt gatatgtccc 2149 tcgtgcc 2156 15 255 PRT Homo sapians 15
Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5
10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu
Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu
Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro
Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr
Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln Val Val Asn
Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys
Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser
Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135
140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe
145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu
Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu
Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu
Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser
Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 16
2156 DNA Homo sapians CDS (722)...(1489) 16 gttttttttt tttttttttt
tttttttttt tattttaagg gattcgttta ataggacttg 60 tggtaagtgg
aataatgcca tgcaaaggtc cccatgtcta accaccaggt tctaggcatg 120
tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt tgataaccag
180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt gggcctaatg
taattataag 240 cattcttaaa agtgaaagaa ataataagag aaactgagtg
ctgtgatgtg agtcagttaa 300 actttttttt caactttttc tttaggtgat
tattttccct taacataaaa tttactttag 360 ctcaactata caaacatgtg
agttattgtt atgtaaccat cactcttcat taagaaatgc 420 tttgtaaaaa
gtgagccagt ttttcatata cattcttcaa aatacattct caacattata 480
catcaaatta tatatacata catgcacaca tacactatat atatcaagga tttatatgat
540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg tttatgataa
ttttggccat 600 agaatatata atacagatga tgtgaagtac aaaatgtttt
ttatacttca tattttgatg 660 tacaaagtat gtttgtcttt gtaattcaga
tgattacttt gcacttgtgt tcccatgaaa 720 a atg cct ttc att tct aag ctg
gta ttg gca tct cag cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu
Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg
tct tct cct ttt ctg ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala
Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc
act tac cta cca gtc tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg
Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 gtg
gtc ctt ctc acc atg gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val
Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55
60 ctg aat ttt cag aat gac ttc aaa tat gag gca tct ttc tac ctg agg
961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg
65 70 75 80 agg gtg atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg
ggc atg 1009 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu
Leu Gly Met 85 90 95 ctg cag gtc gtc aac atc agc ccc agc att tcc
tgg ttg gtg agg ttt 1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile
Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc
ttc cat ttg ttc tca tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe
Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt
agt agc ctg atc ttt tac act gtg gct tct tcc 1153 Ser Phe Pro Val
Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg
acc cag atc aat ttg cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn
Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150
155 160 cca ata aac tcc ata atc aga gga ctg ttt ttc act ctg tca tta
ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser
Leu Phe 165 170 175 aga gat gtt ttt ctt aaa caa ata atg ctg ttc tca
agt gtc tac atg 1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 atg act ctc att cag gaa cta cag gag
atc ctg gta cct tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln
Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat
ctt tgc aga ggc aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys
Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg
gtg agt ttc tcg gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu
Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235
240 atc tca acc tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag
1489 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val *
245 250 255 aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag
atctgataaa 1549 agggtaatca atgtgatgta aactataaga caaatgttta
aaaggttaat tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc
attatagaga ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg
taggataaaa gtagtaggag attgcttctt gaagatttaa 1729 aattatattg
agtgtaatta tttgcattaa aataatttta aatgttttga atagcaagta 1789
ttgatataat taaactttcg aataacttag tgctttgcct ttattcctaa tgtttatatg
1849 gaagcatgtg gtcaatgttt gatgcattac agctctgagc ggtccttctg
tattaggtgg 1909 tcatcattta tatacttctc cataaaagat taaggacctg
gaaatgtaag atacatgaag 1969 aaaatctaag tggagaggct gtttgtggtt
aagtgataac agtgttgtaa gcgatgcatg 2029 aggtaggtgt tcagtgcata
tcctctgcat tttattaata aacactgtaa aatttagaag 2089 aaaattgttt
caccaaatgc acataaaact aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc
2156 17 255 PRT Homo sapians 17 Met Pro Phe Ile Ser Lys Leu Val Leu
Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser
Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr
Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu
Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu
Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70
75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly
Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu
Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu
Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu
His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser
Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp
Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190
Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195
200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met
Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala
Tyr Asp Arg Gly Val 245 250 255 18 2156 DNA Homo sapians CDS
(722)...(1489) 18 gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccaggt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 tatcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca tct cag
cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct ttt ctg
ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta cca gtc
tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc acc atg
gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat ttt cag
aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg
atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg ggc atg 1009 Arg
Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90
95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg gtg agg ttt
1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg
Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc ttc cat ttg ttc tca
tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe
Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt agt agc ctg atc ttt
tac act gtg gct tct tcc 1153 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg acc cag atc aat ttg
cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn Val Thr Gln Ile Asn
Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 cca ata aac
tcc ata atc aga gga ctg ttt ttc act ctg tca tta ttc 1249 Pro Ile
Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165
170 175 aga gat gtt ttt ctt aaa caa ata atg ctg ttc tca agt gtc tac
atg 1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val
Tyr Met 180 185 190 atg act ctc att cag gaa cta cag gag atc ctg gta
cct tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu
Val Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat ctt tgc aga
ggc aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys Asp Leu Cys
Arg Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg gtg agt ttc
tcg gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu Pro Val Ser
Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 atc tca
acc tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag 1489 Ile
Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val * 245 250
255 aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag
atctgataaa 1549 agggtaatca atgtgatgta aactataaga caaatgttta
aaaggttaat tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc
attatagaga ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg
taggataaaa gtagtaggag attgcttctt gaagatttaa 1729 aattatattg
agtgtaatta tttgcattaa aataatttta aatgttttga atagcaagta 1789
ttgatataat taaactttcg aataacttag tgctttgcct ttattcctaa tgtttatatg
1849 gaagcatgtg gtcaatgttt gatgcattac agctctgagc ggtccttctg
tattaggtgg 1909 tcatcattta tatacttctc cataaaagat taaggacctg
gaaatgtaag atacatgaag 1969 aaaatctaag tggagaggct gtttgtggtt
aagtgataac agtgttgtaa gcgatgcatg 2029 aggtaggtgt tcagtgcata
tcctctgcat tttattaata aacactgtaa aatttagaag 2089 aaaattgttt
caccaaatgc acataaaact aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc
2156 19 255 PRT Homo sapians 19 Met Pro Phe Ile Ser Lys Leu Val Leu
Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser
Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr
Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu
Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu
Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70
75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly
Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu
Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu
Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu
His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser
Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp
Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190
Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195
200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met
Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala
Tyr Asp Arg Gly Val 245 250 255 20 2156 DNA Homo sapians CDS
(722)...(1489) 20 gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccaggt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagta ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca tct cag
cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct ttt ctg
ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta cca gtc
tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc acc atg
gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat ttt cag
aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg
atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg ggc atg 1009 Arg
Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90
95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg gtg agg ttt
1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg
Phe 100 105 110 aaa tgg aaa tcc aca att ttt acc ttc cat ttg ttc tca
tgg tct ctc 1105 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe
Ser Trp Ser Leu 115 120 125 agt ttt cct gtt agt agt agc ctg atc ttt
tac act gtg gct tct tcc 1153 Ser Phe Pro Val Ser Ser Ser Leu Ile
Phe Tyr Thr Val Ala Ser Ser 130 135 140 aat gtg acc cag atc aat ttg
cat gtc agt aaa tac tgt tca ctt ttc 1201 Asn Val Thr Gln Ile Asn
Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 cca ata aac
tcc ata atc aga gga ctg ttt ttc act ctg tca tta ttc 1249 Pro Ile
Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175
aga gat gtt ttt ctt aaa caa ata atg ctg ttc tca agt gtc tac atg
1297 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr
Met 180 185 190 atg act ctc att cag gaa cta cag gag atc ctg gta cct
tca cag ccc 1345 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val
Pro Ser Gln Pro 195 200 205 cag cct cta cct aag gat ctt tgc aga ggc
aag agc cat cag cac atc 1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg
Gly Lys Ser His Gln His Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg
gtg ggc atg tac aag atg gac ttc atc 1441 Leu Leu Pro Val Ser Phe
Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 atc tca acc
tcc tca aca ttg cca tgg gca tat gac cgt ggt gtc tag 1489 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val * 245 250 255
aggctagtgg gcagtgtcta taccattgtc aggtttttgg tgctactgag atctgataaa
1549 agggtaatca atgtgatgta aactataaga caaatgttta aaaggttaat
tgtatgaatc 1609 ctgtcatgag ttaaattatt cagagtgttc attatagaga
ataatccaaa gttaaaataa 1669 ttggataatt tatttgtatg taggataaaa
gtagtaggag attgcttctt gaagatttaa 1729 aattatattg agtgtaatta
tttgcattaa aataatttta aatgttttga atagcaagta 1789 ttgatataat
taaactttcg aataacttag tgctttgcct ttattcctaa tgtttatatg 1849
gaagcatgtg gtcaatgttt gatgcattac agctctgagc ggtccttctg tattaggtgg
1909 tcatcattta tatacttctc cataaaagat taaggacctg gaaatgtaag
atacatgaag 1969 aaaatctaag tggagaggct gtttgtggtt aagtgataac
agtgttgtaa gcgatgcatg 2029 aggtaggtgt tcagtgcata tcctctgcat
tttattaata aacactgtaa aatttagaag 2089 aaaattgttt caccaaatgc
acataaaact aataaaatag agtggatttt gatatgtccc 2149 tcgtgcc 2156 21
255 PRT Homo sapians 21 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser
Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe
Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro
Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr
Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe
Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg
Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90
95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe
100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp
Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr
Val Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser
Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg
Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu
Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu
Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln
Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215
220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile
225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg
Gly Val 245 250 255 22 2156 DNA Homo sapians CDS (722)...(1489) 22
gttttttttt tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg
60 tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccaggt
tctaggcatg 120 tattatggta tatgagaaat gggaattcag gctgcagatg
atatcaaggt tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa
ttacagattt gggcctaatg taattataag 240 cattcttaaa agtgaaagaa
ataataagag aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt
caactttttc tttaggtgat tattttccct taacataaaa tttactttag 360
ctcaactata caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc
420 tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct
caacattata 480 catcaaatta tatatacata catgcacaca tacactatat
atatcaagga tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat
aaaaataatg tttatgataa ttttggccat 600 agaatatata atacagatga
tgtgaagtac aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat
gtttgtcttt gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 a atg
cct ttc att tct aag ctg gta ttg gca tct cag cca aca ctt ttc 769 Met
Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10
15 tcc ttc ttt tct gcg tct tct cct ttt ctg ctt ttt ctg gat ctc agg
817 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg
20 25 30 cca gag cgc act tac cta cca gtc tgt cat gtg gcc ctc atc
cac atg 865 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile
His Met 35 40 45 gtg gtc ctt ctc acc atg gtg ttc ttg tct cca cag
ctc ttt gaa tca 913 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln
Leu Phe Glu Ser 50 55 60 ctg aat ttt cag aat gac ttc aaa tat gag
gca tct ttc tac ctg agg 961 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu
Ala Ser Phe Tyr Leu Arg 65 70 75 80 agg gtg atc agg gtc ctc tcc att
tgt acc acc tgc ctc ctg ggc atg 1009 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 ctg cag gtc gtc aac
atc agc ccc agc att tcc tgg ttg gtg agg ttt 1057 Leu Gln Val Val
Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 aaa tgg
aaa tcc aca att ttt acc ttc cat ttg ttc tca tgg tct ctc 1105 Lys
Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120
125 agt ttt cct gtt agt agt agc ctg atc ttt tac act gtg gct tct tcc
1153 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser
Ser 130 135 140 aat gtg acc cag atc aat ttg cat gtc agt aaa tac tgt
tca ctt ttc 1201 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr
Cys Ser Leu Phe 145 150 155 160 cca ata aac tcc ata atc aga gga ctg
ttt ttc act ctg tca tta ttc 1249 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 aga gat gtt ttt ctt aaa
caa ata atg ctg ttc tca agt gtc tac atg 1297 Arg Asp Val Phe Leu
Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 atg act ctc
att cag gaa cta cag gag atc ctg gta cct tca cag ccc 1345 Met Thr
Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205
cag cct cta cct aag gat ctt tgc aga ggc aag agc cat cag cac atc
1393 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His
Ile 210 215 220 ctg ctg ccg gtg agt ttc tcg gtg ggc atg tac aag atg
gac ttc atc 1441 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys
Met Asp Phe Ile 225 230 235 240 atc tca acc tcc tca aca ttg cca tgg
gca tat gac cgt ggt gtc tag 1489 Ile Ser Thr Ser Ser Thr Leu Pro
Trp Ala Tyr Asp Arg Gly Val * 245 250 255 aggctagtgg gcagtgtcta
taccattgtc aggtttttgg tgctactgag atctgataaa 1549 agggtaatca
atgtgatgta aactataaga caaatgttta aaaggttaat tgtatgaatc 1609
ctgtcatgag ttaaattatt cagagtgttc attatagaga ataatccaaa gttaaaataa
1669 ttggataatt tatttgtatg taggataaaa gtagtaggag attgcttctt
gaagatttaa 1729 aattatattg agtgtaatta tttgcattaa aataatttta
aatgttttga atagcaagta 1789 ttgatataat taaactttcg aataacttag
tgctttgcct ttattcctaa tgtttatatg 1849 gaagcatgtg gtcaatgttt
gatgcattac agctctgagc ggtccttctg tattaggtgg 1909 tcatcattta
tatacttctc cataaaagat taaggacctg gaaatgtaag atacatgaag 1969
aaaatctaag tggagaggct gtttgtggtt aagtgataac agtgttgtaa gcgatgcatg
2029 aggtaggtgt tcagtgcata tcctctgcat tttattaata aacactgtaa
aatttagaag 2089 aaaattgttt caccaaatgc acataaaact aataaaatag
agtggatttt gatatgtccc 2149 tcgtgcc 2156 23 255 PRT Homo sapians 23
Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5
10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu
Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu
Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro
Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr
Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln Val Val Asn
Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys
Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser
Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135
140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe
145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu
Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu
Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu
Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser
Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 24
4522 DNA Homo sapians CDS (722)...(4522) 24 gttttttttt tttttttttt
tttttttttt tattttaagg gattcgttta ataggacttg 60 tggtaagtgg
aataatgcca tgcaaaggtc cccatgtcta accaccaggt tctaggcatg 120
tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt tgataaccag
180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt gggcctaatg
taattataag 240 cattcttaaa agtgaaagaa ataataagag aaactgagtg
ctgtgatgtg agtcagttaa 300 actttttttt caactttttc tttaggtgat
tattttccct taacataaaa tttactttag 360 ctcaactata caaacatgtg
agttattgtt atgtaaccat cactcttcat taagaaatgc 420 tttgtaaaaa
gtgagccagt ttttcatata cattcttcaa aatacattct caacattata 480
catcaaatta tatatacata catgcacaca tacactatat atatcaagga tttatatgag
540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg tttatgataa
ttttggccat 600 agaatatata atacagatga tgtgaagtac aaaatgtttt
ttatacttca
tattttgatg 660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt
gcacttgtgt tcccatgaaa 720 a atg cct ttc att tct aag ctg gta ttg gca
tct cag cca aca ctt ttc 769 Met Pro Phe Ile Ser Lys Leu Val Leu Ala
Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc ttt tct gcg tct tct cct
ttt ctg ctt ttt ctg gat ctc agg 817 Ser Phe Phe Ser Ala Ser Ser Pro
Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 cca gag cgc act tac cta
cca gtc tgt cat gtg gcc ctc atc cac atg 865 Pro Glu Arg Thr Tyr Leu
Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 gtg gtc ctt ctc
acc atg gtg ttc ttg tct cca cag ctc ttt gaa tca 913 Val Val Leu Leu
Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 ctg aat
ttt cag aat gac ttc aaa tat gag gca tct ttc tac ctg agg 961 Leu Asn
Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80
agg gtg atc agg gtc ctc tcc att tgt acc acc tgc ctc ctg gac atg
1009 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Asp
Met 85 90 95 ctg cag gtc gtc aac atc agc ccc agc att tcc tgg ttg
ata atg ctg 1057 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp
Leu Ile Met Leu 100 105 110 ttc tca agt gtc tac atg atg act ctc att
cag gaa cta cag gag atc 1105 Phe Ser Ser Val Tyr Met Met Thr Leu
Ile Gln Glu Leu Gln Glu Ile 115 120 125 ctg gta cct tca cag ccc cag
cct cta cct aag gat ctt tgc aga ggc 1153 Leu Val Pro Ser Gln Pro
Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly 130 135 140 aag agc cat cag
cac atc ctg ctg ccg act caa gca act ttt gct gca 1201 Lys Ser His
Gln His Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala 145 150 155 160
gca act gga cta tgg gct gca cta acc acc gta tca aat cca agc aga
1249 Ala Thr Gly Leu Trp Ala Ala Leu Thr Thr Val Ser Asn Pro Ser
Arg 165 170 175 gca gat cct gtg acc tgg aga aag gag ccg gct gtc ctt
ccc tgc tgt 1297 Ala Asp Pro Val Thr Trp Arg Lys Glu Pro Ala Val
Leu Pro Cys Cys 180 185 190 aac cta gag aaa gga agc tgg ctg tcc ttc
cct ggc aca gct gca cgc 1345 Asn Leu Glu Lys Gly Ser Trp Leu Ser
Phe Pro Gly Thr Ala Ala Arg 195 200 205 aag gaa ttt tcc acc acg ctc
acc ggg cac agc gcg ctg agc ctc tcc 1393 Lys Glu Phe Ser Thr Thr
Leu Thr Gly His Ser Ala Leu Ser Leu Ser 210 215 220 agt tcg cgg gcc
ctc ccc ggc tcg ctc ccg gct ttc gca gac ctc ccc 1441 Ser Ser Arg
Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu Pro 225 230 235 240
cgc tcc tgc cct gag tcc gag cag agc gca acg cca gcc ggc gcc ttc
1489 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro Ala Gly Ala
Phe 245 250 255 ctc ctg ggc tgg gag cga gtg gtg cag cgg cgg ctc gaa
gtc ccc cgg 1537 Leu Leu Gly Trp Glu Arg Val Val Gln Arg Arg Leu
Glu Val Pro Arg 260 265 270 cct caa gca gcc ccc gcg act agc gcg aca
ccc tcg cgg gat ccg agt 1585 Pro Gln Ala Ala Pro Ala Thr Ser Ala
Thr Pro Ser Arg Asp Pro Ser 275 280 285 cca ccc tgc cac cag cgc cgg
gac gcc gcg tgc ctc aga gcc caa ggg 1633 Pro Pro Cys His Gln Arg
Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290 295 300 ctg acc cgg gcc
ttc cag gtg gtc cat ctc gct cct acg gct ccc gac 1681 Leu Thr Arg
Ala Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 305 310 315 320
ggt ggc gct ggg tgt ccc cca tcc cgc aat tcc tac cgg ctg acc cat
1729 Gly Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr
His 325 330 335 gtg cgc tgc gcc cag ggg ctg gag gct gcc agc gcc aac
ctt ccc ggc 1777 Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala
Asn Leu Pro Gly 340 345 350 gct ccg ggg cgg agc agc tcc tgc gcc ctg
cgc tac cgc agc ggc cct 1825 Ala Pro Gly Arg Ser Ser Ser Cys Ala
Leu Arg Tyr Arg Ser Gly Pro 355 360 365 tca gtc agc tcc gcg ccg tcc
ccc gca gag ccc ccg gcg cac cag cgc 1873 Ser Val Ser Ser Ala Pro
Ser Pro Ala Glu Pro Pro Ala His Gln Arg 370 375 380 ctg ctt ttc ctt
ccc cga gcg cct caa gca gtc tct ggg ccg cag gaa 1921 Leu Leu Phe
Leu Pro Arg Ala Pro Gln Ala Val Ser Gly Pro Gln Glu 385 390 395 400
cag ccc tct gaa gag gcg ctt ggt gta gga agc ctc tca gtt ttc cag
1969 Gln Pro Ser Glu Glu Ala Leu Gly Val Gly Ser Leu Ser Val Phe
Gln 405 410 415 tta cac cta ata cag tgt att cca aat cta agt tac cca
cta gta ctt 2017 Leu His Leu Ile Gln Cys Ile Pro Asn Leu Ser Tyr
Pro Leu Val Leu 420 425 430 cgg cac att cca gaa att ctg aaa ttt tct
gaa aag gaa act ggt ggt 2065 Arg His Ile Pro Glu Ile Leu Lys Phe
Ser Glu Lys Glu Thr Gly Gly 435 440 445 gga att cta ggc tta gaa tta
cca gcg aca gct gct cgc ctc tca gga 2113 Gly Ile Leu Gly Leu Glu
Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455 460 tta aac agc ata
atg caa atc aaa gag ttt gaa gaa ttg gta aaa ctt 2161 Leu Asn Ser
Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu 465 470 475 480
cac agc ttg tca cac aaa gtc att cag tgt gtg ttt gca aag aaa aaa
2209 His Ser Leu Ser His Lys Val Ile Gln Cys Val Phe Ala Lys Lys
Lys 485 490 495 aat gta gac aaa tgg gat gac ttt tgt ctt agt gag ggt
tat gga cat 2257 Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu
Gly Tyr Gly His 500 505 510 tca ttc tta ata atg aaa gaa acg tcg act
aaa ata tca ggt tta att 2305 Ser Phe Leu Ile Met Lys Glu Thr Ser
Thr Lys Ile Ser Gly Leu Ile 515 520 525 cag gag atg ggg agc ggc aag
agc aac gtg ggc act tgg gga gac tac 2353 Gln Glu Met Gly Ser Gly
Lys Ser Asn Val Gly Thr Trp Gly Asp Tyr 530 535 540 gac gac agc gcc
ttc atg gag ccg agg tac cac gtc cgt cga gaa gat 2401 Asp Asp Ser
Ala Phe Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp 545 550 555 560
ctg gac aag ctc cac aga gct gcc tgg tgg ggt aaa gtc ccc aga aag
2449 Leu Asp Lys Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg
Lys 565 570 575 gat ctc atc gtc atg ctc agg gac act gac atg aac aag
agg gac aag 2497 Asp Leu Ile Val Met Leu Arg Asp Thr Asp Met Asn
Lys Arg Asp Lys 580 585 590 caa aag agg act gct cta cat ttg gcc tct
gcc aat gga aat tca gaa 2545 Gln Lys Arg Thr Ala Leu His Leu Ala
Ser Ala Asn Gly Asn Ser Glu 595 600 605 gta gta caa ctc ctg ctg gac
aga cga tgt caa ctt aac gtc ctt gac 2593 Val Val Gln Leu Leu Leu
Asp Arg Arg Cys Gln Leu Asn Val Leu Asp 610 615 620 aac aaa aaa agg
aca gct ctg ata aag gcc gta caa tgc cag gaa gat 2641 Asn Lys Lys
Arg Thr Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp 625 630 635 640
gaa tgt gtg tta atg ttg ctg gaa cat ggc gct gat gga aat att caa
2689 Glu Cys Val Leu Met Leu Leu Glu His Gly Ala Asp Gly Asn Ile
Gln 645 650 655 gat gag tat gga aat acc gct cta cac tat gct atc tac
aat gaa gat 2737 Asp Glu Tyr Gly Asn Thr Ala Leu His Tyr Ala Ile
Tyr Asn Glu Asp 660 665 670 aaa tta atg gcc aaa gca ctg ctc tta tat
ggt gct gat att gaa tca 2785 Lys Leu Met Ala Lys Ala Leu Leu Leu
Tyr Gly Ala Asp Ile Glu Ser 675 680 685 aaa aac aag tgt ggc ctc aca
cca ctt ttg ctt ggc gta cat gaa caa 2833 Lys Asn Lys Cys Gly Leu
Thr Pro Leu Leu Leu Gly Val His Glu Gln 690 695 700 aaa cag gaa gtg
gtg aaa ttt tta atc aag aaa aaa gct aat tta aat 2881 Lys Gln Glu
Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 705 710 715 720
gca ctt gat aga tat gga aga act gcc ctc ata ctt gct gta tgt tgt
2929 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys
Cys 725 730 735 gga tca gca agt ata gtc aat ctt cta ctt gag caa aat
gtt gat gta 2977 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln
Asn Val Asp Val 740 745 750 tct tct caa gat cta tct gga cag acg gcc
aga gag tat gct gtt tct 3025 Ser Ser Gln Asp Leu Ser Gly Gln Thr
Ala Arg Glu Tyr Ala Val Ser 755 760 765 agt cat cat cat gta att tgt
gaa tta ctt tct gac tat aaa gaa aaa 3073 Ser His His His Val Ile
Cys Glu Leu Leu Ser Asp Tyr Lys Glu Lys 770 775 780 cag atg cta aaa
atc tct tct gaa aac agc aat cca gtg ata acc atc 3121 Gln Met Leu
Lys Ile Ser Ser Glu Asn Ser Asn Pro Val Ile Thr Ile 785 790 795 800
25 1266 PRT Homo sapians 25 Met Pro Phe Ile Ser Lys Leu Val Leu Ala
Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro
Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu
Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu
Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn
Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80
Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Asp Met 85
90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Ile Met
Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu Ile Gln Glu Leu
Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln Pro Leu Pro Lys
Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His Ile Leu Leu Pro
Thr Gln Ala Thr Phe Ala Ala 145 150 155 160 Ala Thr Gly Leu Trp Ala
Ala Leu Thr Thr Val Ser Asn Pro Ser Arg 165 170 175 Ala Asp Pro Val
Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys 180 185 190 Asn Leu
Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg 195 200 205
Lys Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala Leu Ser Leu Ser 210
215 220 Ser Ser Arg Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu
Pro 225 230 235 240 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro
Ala Gly Ala Phe 245 250 255 Leu Leu Gly Trp Glu Arg Val Val Gln Arg
Arg Leu Glu Val Pro Arg 260 265 270 Pro Gln Ala Ala Pro Ala Thr Ser
Ala Thr Pro Ser Arg Asp Pro Ser 275 280 285 Pro Pro Cys His Gln Arg
Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290 295 300 Leu Thr Arg Ala
Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 305 310 315 320 Gly
Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His 325 330
335 Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly
340 345 350 Ala Pro Gly Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser
Gly Pro 355 360 365 Ser Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro
Ala His Gln Arg 370 375 380 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala
Val Ser Gly Pro Gln Glu 385 390 395 400 Gln Pro Ser Glu Glu Ala Leu
Gly Val Gly Ser Leu Ser Val Phe Gln 405 410 415 Leu His Leu Ile Gln
Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu 420 425 430 Arg His Ile
Pro Glu Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly 435 440 445 Gly
Ile Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455
460 Leu Asn Ser Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu
465 470 475 480 His Ser Leu Ser His Lys Val Ile Gln Cys Val Phe Ala
Lys Lys Lys 485 490 495 Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser
Glu Gly Tyr Gly His 500 505 510 Ser Phe Leu Ile Met Lys Glu Thr Ser
Thr Lys Ile Ser Gly Leu Ile 515 520 525 Gln Glu Met Gly Ser Gly Lys
Ser Asn Val Gly Thr Trp Gly Asp Tyr 530 535 540 Asp Asp Ser Ala Phe
Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp 545 550 555 560 Leu Asp
Lys Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys 565 570 575
Asp Leu Ile Val Met Leu Arg Asp Thr Asp Met Asn Lys Arg Asp Lys 580
585 590 Gln Lys Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser
Glu 595 600 605 Val Val Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn
Val Leu Asp 610 615 620 Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val
Gln Cys Gln Glu Asp 625 630 635 640 Glu Cys Val Leu Met Leu Leu Glu
His Gly Ala Asp Gly Asn Ile Gln 645 650 655 Asp Glu Tyr Gly Asn Thr
Ala Leu His Tyr Ala Ile Tyr Asn Glu Asp 660 665 670 Lys Leu Met Ala
Lys Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser 675 680 685 Lys Asn
Lys Cys Gly Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln 690 695 700
Lys Gln Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 705
710 715 720 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val
Cys Cys 725 730 735 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln
Asn Val Asp Val 740 745 750 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala
Arg Glu Tyr Ala Val Ser 755 760 765 Ser His His His Val Ile Cys Glu
Leu Leu Ser Asp Tyr Lys Glu Lys 770 775 780 Gln Met Leu Lys Ile Ser
Ser Glu Asn Ser Asn Pro Val Ile Thr Ile 785 790 795 800 Leu Asn Ile
Lys Leu Pro Leu Lys Val Glu Glu Glu Ile Lys Lys His 805 810 815 Gly
Ser Asn Pro Val Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser 820 825
830 Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys
835 840 845 Pro Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr
His Ser 850 855 860 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu
Glu Gln Asn Thr 865 870 875 880 Gly Ile Ser Gln Asp Glu Ile Leu Thr
Asn Lys Gln Lys Gln Ile Glu 885 890 895 Val Ala Glu Lys Glu Met Asn
Ser Glu Leu Ser Leu Ser His Lys Lys 900 905 910 Glu Glu Asp Leu Leu
Arg Glu Asn Ser Met Leu Arg Glu Glu Ile Ala 915 920 925 Lys Leu Arg
Leu Glu Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg 930 935 940 Glu
Asn Lys Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu 945 950
955 960 Lys Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val
Ala 965 970 975 Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala
Gln Ala Ser 980 985 990 Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr
Glu Lys Thr Glu Gln 995 1000 1005 Gln Ala Gln Glu Gln Glu Val Ala
Gly Phe Ser Leu Arg Gln Leu Gly 1010 1015 1020 Leu Ala Gln His Ala
Gln Ala Ser Val Gln Gln Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly
His Thr Glu Lys Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045
1050 1055 Leu Arg Ser Gln Ile Gly Asp Pro Gly Gly Val Pro Leu Ser
Glu Gly 1060 1065 1070 Gly Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr
His Leu Pro Pro Arg 1075 1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr
Pro Ser Leu Val Arg Leu Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala
Ala Leu Pro Pro Pro Thr Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser
Pro Thr Lys Gln Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu 1125
1130 1135 Pro Val Pro Thr Phe Ser Ser Gly Ser Phe Leu
Gly Arg Arg Cys Pro 1140 1145 1150 Met Phe Asp Val Ser Pro Ala Met
Arg Leu Lys Ser Asp Ser Asn Arg 1155 1160 1165 Glu Thr His Gln Ala
Phe Arg Asp Lys Asp Asp Leu Pro Phe Phe Lys 1170 1175 1180 Thr Gln
Gln Ser Pro Arg His Thr Lys Asp Leu Gly Gln Asp Asp Arg 1185 1190
1195 1200 Ala Gly Val Leu Ala Pro Lys Cys Arg Pro Gly Thr Leu Cys
His Thr 1205 1210 1215 Asp Thr Pro Pro His Arg Asn Ala Asp Thr Pro
Pro His Arg His Thr 1220 1225 1230 Thr Thr Leu Pro His Arg Asp Thr
Thr Thr Ser Leu Pro His Phe His 1235 1240 1245 Val Ser Ala Gly Gly
Val Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu 1250 1255 1260 Ile Thr
1265 26 3801 DNA Homo sapians CDS (1)...(3801) 26 atg cct ttc att
tct aag ctg gta ttg gca tct cag cca aca ctt ttc 48 Met Pro Phe Ile
Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 tcc ttc
ttt tct gcg tct tct cct ttt ctg ctt ttt ctg gat ctc agg 96 Ser Phe
Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30
cca gag cgc act tac cta cca gtc tgt cat gtg gcc ctc atc cac atg 144
Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35
40 45 gtg gtc ctt ctc acc atg gtg ttc ttg tct cca cag ctc ttt gaa
tca 192 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu
Ser 50 55 60 ctg aat ttt cag aat gac ttc aaa tat gag gca tct ttc
tac ctg agg 240 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe
Tyr Leu Arg 65 70 75 80 agg gtg atc agg gtc ctc tcc att tgt acc acc
tgc ctc ctg gac atg 288 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr
Cys Leu Leu Asp Met 85 90 95 ctg cag gtc gtc aac atc agc ccc agc
att tcc tgg ttg ata atg ctg 336 Leu Gln Val Val Asn Ile Ser Pro Ser
Ile Ser Trp Leu Ile Met Leu 100 105 110 ttc tca agt gtc tac atg atg
act ctc att cag gaa cta cag gag atc 384 Phe Ser Ser Val Tyr Met Met
Thr Leu Ile Gln Glu Leu Gln Glu Ile 115 120 125 ctg gta cct tca cag
ccc cag cct cta cct aag gat ctt tgc aga ggc 432 Leu Val Pro Ser Gln
Pro Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly 130 135 140 aag agc cat
cag cac atc ctg ctg ccg act caa gca act ttt gct gca 480 Lys Ser His
Gln His Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala 145 150 155 160
gca act gga cta tgg gct gca cta acc acc gta tca aat cca agc aga 528
Ala Thr Gly Leu Trp Ala Ala Leu Thr Thr Val Ser Asn Pro Ser Arg 165
170 175 gca gat cct gtg acc tgg aga aag gag ccg gct gtc ctt ccc tgc
tgt 576 Ala Asp Pro Val Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys
Cys 180 185 190 aac cta gag aaa gga agc tgg ctg tcc ttc cct ggc aca
gct gca cgc 624 Asn Leu Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr
Ala Ala Arg 195 200 205 aag gaa ttt tcc acc acg ctc acc ggg cac agc
gcg ctg agc ctc tcc 672 Lys Glu Phe Ser Thr Thr Leu Thr Gly His Ser
Ala Leu Ser Leu Ser 210 215 220 agt tcg cgg gcc ctc ccc ggc tcg ctc
ccg gct ttc gca gac ctc ccc 720 Ser Ser Arg Ala Leu Pro Gly Ser Leu
Pro Ala Phe Ala Asp Leu Pro 225 230 235 240 cgc tcc tgc cct gag tcc
gag cag agc gca acg cca gcc ggc gcc ttc 768 Arg Ser Cys Pro Glu Ser
Glu Gln Ser Ala Thr Pro Ala Gly Ala Phe 245 250 255 ctc ctg ggc tgg
gag cga gtg gtg cag cgg cgg ctc gaa gtc ccc cgg 816 Leu Leu Gly Trp
Glu Arg Val Val Gln Arg Arg Leu Glu Val Pro Arg 260 265 270 cct caa
gca gcc ccc gcg act agc gcg aca ccc tcg cgg gat ccg agt 864 Pro Gln
Ala Ala Pro Ala Thr Ser Ala Thr Pro Ser Arg Asp Pro Ser 275 280 285
cca ccc tgc cac cag cgc cgg gac gcc gcg tgc ctc aga gcc caa ggg 912
Pro Pro Cys His Gln Arg Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290
295 300 ctg acc cgg gcc ttc cag gtg gtc cat ctc gct cct acg gct ccc
gac 960 Leu Thr Arg Ala Phe Gln Val Val His Leu Ala Pro Thr Ala Pro
Asp 305 310 315 320 ggt ggc gct ggg tgt ccc cca tcc cgc aat tcc tac
cgg ctg acc cat 1008 Gly Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser
Tyr Arg Leu Thr His 325 330 335 gtg cgc tgc gcc cag ggg ctg gag gct
gcc agc gcc aac ctt ccc ggc 1056 Val Arg Cys Ala Gln Gly Leu Glu
Ala Ala Ser Ala Asn Leu Pro Gly 340 345 350 gct ccg ggg cgg agc agc
tcc tgc gcc ctg cgc tac cgc agc ggc cct 1104 Ala Pro Gly Arg Ser
Ser Ser Cys Ala Leu Arg Tyr Arg Ser Gly Pro 355 360 365 tca gtc agc
tcc gcg ccg tcc ccc gca gag ccc ccg gcg cac cag cgc 1152 Ser Val
Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro Ala His Gln Arg 370 375 380
ctg ctt ttc ctt ccc cga gcg cct caa gca gtc tct ggg ccg cag gaa
1200 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala Val Ser Gly Pro Gln
Glu 385 390 395 400 cag ccc tct gaa gag gcg ctt ggt gta gga agc ctc
tca gtt ttc cag 1248 Gln Pro Ser Glu Glu Ala Leu Gly Val Gly Ser
Leu Ser Val Phe Gln 405 410 415 tta cac cta ata cag tgt att cca aat
cta agt tac cca cta gta ctt 1296 Leu His Leu Ile Gln Cys Ile Pro
Asn Leu Ser Tyr Pro Leu Val Leu 420 425 430 cgg cac att cca gaa att
ctg aaa ttt tct gaa aag gaa act ggt ggt 1344 Arg His Ile Pro Glu
Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly 435 440 445 gga att cta
ggc tta gaa tta cca gcg aca gct gct cgc ctc tca gga 1392 Gly Ile
Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455 460
tta aac agc ata atg caa atc aaa gag ttt gaa gaa ttg gta aaa ctt
1440 Leu Asn Ser Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys
Leu 465 470 475 480 cac agc ttg tca cac aaa gtc att cag tgt gtg ttt
gca aag aaa aaa 1488 His Ser Leu Ser His Lys Val Ile Gln Cys Val
Phe Ala Lys Lys Lys 485 490 495 aat gta gac aaa tgg gat gac ttt tgt
ctt agt gag ggt tat gga cat 1536 Asn Val Asp Lys Trp Asp Asp Phe
Cys Leu Ser Glu Gly Tyr Gly His 500 505 510 tca ttc tta ata atg aaa
gaa acg tcg act aaa ata tca ggt tta att 1584 Ser Phe Leu Ile Met
Lys Glu Thr Ser Thr Lys Ile Ser Gly Leu Ile 515 520 525 cag gag atg
ggg agc ggc aag agc aac gtg ggc act tgg gga gac tac 1632 Gln Glu
Met Gly Ser Gly Lys Ser Asn Val Gly Thr Trp Gly Asp Tyr 530 535 540
gac gac agc gcc ttc atg gag ccg agg tac cac gtc cgt cga gaa gat
1680 Asp Asp Ser Ala Phe Met Glu Pro Arg Tyr His Val Arg Arg Glu
Asp 545 550 555 560 ctg gac aag ctc cac aga gct gcc tgg tgg ggt aaa
gtc ccc aga aag 1728 Leu Asp Lys Leu His Arg Ala Ala Trp Trp Gly
Lys Val Pro Arg Lys 565 570 575 gat ctc atc gtc atg ctc agg gac act
gac atg aac aag agg gac aag 1776 Asp Leu Ile Val Met Leu Arg Asp
Thr Asp Met Asn Lys Arg Asp Lys 580 585 590 caa aag agg act gct cta
cat ttg gcc tct gcc aat gga aat tca gaa 1824 Gln Lys Arg Thr Ala
Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu 595 600 605 gta gta caa
ctc ctg ctg gac aga cga tgt caa ctt aac gtc ctt gac 1872 Val Val
Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp 610 615 620
aac aaa aaa agg aca gct ctg ata aag gcc gta caa tgc cag gaa gat
1920 Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val Gln Cys Gln Glu
Asp 625 630 635 640 gaa tgt gtg tta atg ttg ctg gaa cat ggc gct gat
gga aat att caa 1968 Glu Cys Val Leu Met Leu Leu Glu His Gly Ala
Asp Gly Asn Ile Gln 645 650 655 gat gag tat gga aat acc gct cta cac
tat gct atc tac aat gaa gat 2016 Asp Glu Tyr Gly Asn Thr Ala Leu
His Tyr Ala Ile Tyr Asn Glu Asp 660 665 670 aaa tta atg gcc aaa gca
ctg ctc tta tat ggt gct gat att gaa tca 2064 Lys Leu Met Ala Lys
Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser 675 680 685 aaa aac aag
tgt ggc ctc aca cca ctt ttg ctt ggc gta cat gaa caa 2112 Lys Asn
Lys Cys Gly Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln 690 695 700
aaa cag gaa gtg gtg aaa ttt tta atc aag aaa aaa gct aat tta aat
2160 Lys Gln Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu
Asn 705 710 715 720 gca ctt gat aga tat gga aga act gcc ctc ata ctt
gct gta tgt tgt 2208 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile
Leu Ala Val Cys Cys 725 730 735 gga tca gca agt ata gtc aat ctt cta
ctt gag caa aat gtt gat gta 2256 Gly Ser Ala Ser Ile Val Asn Leu
Leu Leu Glu Gln Asn Val Asp Val 740 745 750 tct tct caa gat cta tct
gga cag acg gcc aga gag tat gct gtt tct 2304 Ser Ser Gln Asp Leu
Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser 755 760 765 agt cat cat
cat gta att tgt gaa tta ctt tct gac tat aaa gaa aaa 2352 Ser His
His His Val Ile Cys Glu Leu Leu Ser Asp Tyr Lys Glu Lys 770 775 780
cag atg cta aaa atc tct tct gaa aac agc aat cca gtg ata acc atc
2400 Gln Met Leu Lys Ile Ser Ser Glu Asn Ser Asn Pro Val Ile Thr
Ile 785 790 795 800 ctt aat atc aaa ctt cca ctc aag gtt gaa gaa gaa
ata aag aag cat 2448 Leu Asn Ile Lys Leu Pro Leu Lys Val Glu Glu
Glu Ile Lys Lys His 805 810 815 gga agt aat cct gtg gga tta cca gaa
aac ctg act aat ggt gcc agt 2496 Gly Ser Asn Pro Val Gly Leu Pro
Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830 gct ggc aat ggt gat gat
gga tta att cca caa agg aag agc aga aaa 2544 Ala Gly Asn Gly Asp
Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835 840 845 cct gaa aat
cag caa ttt cct gac act gag aat gaa gag tat cac agt 2592 Pro Glu
Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His Ser 850 855 860
gac gaa caa aat gat acc cag aaa caa ctt tct gaa gaa cag aac act
2640 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu Gln Asn
Thr 865 870 875 880 gga ata tca caa gat gag att ctg act aat aaa caa
aag cag ata gaa 2688 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys
Gln Lys Gln Ile Glu 885 890 895 gtg gct gaa aag gaa atg aat tct gag
ctt tct ctt agt cat aag aaa 2736 Val Ala Glu Lys Glu Met Asn Ser
Glu Leu Ser Leu Ser His Lys Lys 900 905 910 gaa gaa gat ctc ttg cgt
gaa aac agc atg ttg cgg gaa gaa att gcc 2784 Glu Glu Asp Leu Leu
Arg Glu Asn Ser Met Leu Arg Glu Glu Ile Ala 915 920 925 aag cta aga
ctg gaa cta gat gaa aca aaa cat cag aac cag cta agg 2832 Lys Leu
Arg Leu Glu Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg 930 935 940
gaa aat aaa att ttg gag gaa att gaa agt gta aaa gaa aaa ctt cta
2880 Glu Asn Lys Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu
Leu 945 950 955 960 aag act ata caa ctg aat gaa gaa gca tta acg aaa
acc aag gtg gct 2928 Lys Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr
Lys Thr Lys Val Ala 965 970 975 ggt ttc tct ttg cgc cag ctt ggc ctt
gcc cag cat gca caa gcc tca 2976 Gly Phe Ser Leu Arg Gln Leu Gly
Leu Ala Gln His Ala Gln Ala Ser 980 985 990 gtg caa cag ctg tgc tac
aaa tgg aac cac aca gag aaa aca gag cag 3024 Val Gln Gln Leu Cys
Tyr Lys Trp Asn His Thr Glu Lys Thr Glu Gln 995 1000 1005 cag gct
cag gag cag gag gtg gct ggt ttc tct ttg cgc cag ctt ggc 3072 Gln
Ala Gln Glu Gln Glu Val Ala Gly Phe Ser Leu Arg Gln Leu Gly 1010
1015 1020 ctt gcc cag cat gca caa gcc tca gta caa caa ctg tgc tac
aaa tgg 3120 Leu Ala Gln His Ala Gln Ala Ser Val Gln Gln Leu Cys
Tyr Lys Trp 1025 1030 1035 1040 ggc cac aca gag aaa aca gag cag cag
gct cag gag cag gga gct gcg 3168 Gly His Thr Glu Lys Thr Glu Gln
Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050 1055 ctg agg tcc cag ata
ggc gac cct ggc ggg gtg ccc ctg agc gaa ggg 3216 Leu Arg Ser Gln
Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly 1060 1065 1070 ggg
aca gca gca gga gac cag ggt cca ggg acc cac ctc cca ccg agg 3264
Gly Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro Arg
1075 1080 1085 gaa cct cga gcc tcc cct ggc acc cct agc ttg gtc cgc
ctg gcc tcc 3312 Glu Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val
Arg Leu Ala Ser 1090 1095 1100 gga gcc cga gct gct gcg ctt ccc cca
ccc aca ggg aaa aac ggc cga 3360 Gly Ala Arg Ala Ala Ala Leu Pro
Pro Pro Thr Gly Lys Asn Gly Arg 1105 1110 1115 1120 tct cca acc aaa
cag aaa tct gtg tgt gac tcc tct ggt tgg ata ctg 3408 Ser Pro Thr
Lys Gln Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130 1135
cca gtc ccc aca ttt tct tcc ggg agt ttt ctt ggc aga agg tgc cca
3456 Pro Val Pro Thr Phe Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys
Pro 1140 1145 1150 atg ttt gat gtt tcg cca gcc atg agg ctg aaa agt
gac agc aat aga 3504 Met Phe Asp Val Ser Pro Ala Met Arg Leu Lys
Ser Asp Ser Asn Arg 1155 1160 1165 gaa aca cat cag gct ttc cgc gac
aaa gat gac ctt ccc ttc ttc aaa 3552 Glu Thr His Gln Ala Phe Arg
Asp Lys Asp Asp Leu Pro Phe Phe Lys 1170 1175 1180 act cag caa tct
cca cgg cac aca aag gac tta gga caa gat gac cga 3600 Thr Gln Gln
Ser Pro Arg His Thr Lys Asp Leu Gly Gln Asp Asp Arg 1185 1190 1195
1200 gct gga gtg ctc gcc cca aaa tgc agg ccc gga aca ctc tgc cac
acg 3648 Ala Gly Val Leu Ala Pro Lys Cys Arg Pro Gly Thr Leu Cys
His Thr 1205 1210 1215 gac aca cca cca cac aga aat gcg gac aca cca
cca cac aga cac acc 3696 Asp Thr Pro Pro His Arg Asn Ala Asp Thr
Pro Pro His Arg His Thr 1220 1225 1230 acc acg ctg cca cac aga gac
acc acc aca tcg ttg cca cac ttt cat 3744 Thr Thr Leu Pro His Arg
Asp Thr Thr Thr Ser Leu Pro His Phe His 1235 1240 1245 gtg tca gct
ggc ggt gtg ggc ccc acg act ctg ggc tct aat aga gaa 3792 Val Ser
Ala Gly Gly Val Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu 1250 1255
1260 att act tag 3801 Ile Thr * 1265 27 1266 PRT Homo sapians 27
Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5
10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu
Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu
Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro
Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr
Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Asp Met 85 90 95 Leu Gln Val Val Asn
Ile Ser Pro Ser Ile Ser Trp Leu Ile Met Leu 100 105 110 Phe Ser Ser
Val Tyr Met Met Thr Leu Ile Gln Glu Leu Gln Glu Ile 115 120 125 Leu
Val Pro Ser Gln Pro Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly 130 135
140 Lys Ser His Gln His Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala
145 150 155 160 Ala Thr Gly Leu Trp Ala Ala Leu Thr Thr Val Ser Asn
Pro Ser Arg 165 170 175 Ala Asp Pro Val Thr Trp Arg Lys Glu Pro Ala
Val Leu Pro Cys Cys 180 185 190 Asn Leu Glu Lys Gly Ser Trp Leu Ser
Phe Pro Gly Thr Ala Ala Arg 195 200 205 Lys Glu Phe Ser Thr Thr Leu
Thr Gly His Ser Ala Leu Ser Leu Ser 210 215 220 Ser Ser Arg Ala Leu
Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu Pro 225 230 235 240 Arg Ser
Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro Ala Gly Ala Phe 245 250 255
Leu Leu Gly Trp Glu Arg Val Val Gln Arg Arg Leu Glu Val Pro Arg
260 265 270 Pro Gln Ala Ala Pro Ala Thr Ser Ala Thr Pro Ser Arg Asp
Pro Ser 275 280 285 Pro Pro Cys His Gln Arg Arg Asp Ala Ala Cys Leu
Arg Ala Gln Gly 290 295 300 Leu Thr Arg Ala Phe Gln Val Val His Leu
Ala Pro Thr Ala Pro Asp 305 310 315 320 Gly Gly Ala Gly Cys Pro Pro
Ser Arg Asn Ser Tyr Arg Leu Thr His 325 330 335 Val Arg Cys Ala Gln
Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly 340 345 350 Ala Pro Gly
Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser Gly Pro 355 360 365 Ser
Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro Ala His Gln Arg 370 375
380 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala Val Ser Gly Pro Gln Glu
385 390 395 400 Gln Pro Ser Glu Glu Ala Leu Gly Val Gly Ser Leu Ser
Val Phe Gln 405 410 415 Leu His Leu Ile Gln Cys Ile Pro Asn Leu Ser
Tyr Pro Leu Val Leu 420 425 430 Arg His Ile Pro Glu Ile Leu Lys Phe
Ser Glu Lys Glu Thr Gly Gly 435 440 445 Gly Ile Leu Gly Leu Glu Leu
Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455 460 Leu Asn Ser Ile Met
Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu 465 470 475 480 His Ser
Leu Ser His Lys Val Ile Gln Cys Val Phe Ala Lys Lys Lys 485 490 495
Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu Gly Tyr Gly His 500
505 510 Ser Phe Leu Ile Met Lys Glu Thr Ser Thr Lys Ile Ser Gly Leu
Ile 515 520 525 Gln Glu Met Gly Ser Gly Lys Ser Asn Val Gly Thr Trp
Gly Asp Tyr 530 535 540 Asp Asp Ser Ala Phe Met Glu Pro Arg Tyr His
Val Arg Arg Glu Asp 545 550 555 560 Leu Asp Lys Leu His Arg Ala Ala
Trp Trp Gly Lys Val Pro Arg Lys 565 570 575 Asp Leu Ile Val Met Leu
Arg Asp Thr Asp Met Asn Lys Arg Asp Lys 580 585 590 Gln Lys Arg Thr
Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu 595 600 605 Val Val
Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp 610 615 620
Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp 625
630 635 640 Glu Cys Val Leu Met Leu Leu Glu His Gly Ala Asp Gly Asn
Ile Gln 645 650 655 Asp Glu Tyr Gly Asn Thr Ala Leu His Tyr Ala Ile
Tyr Asn Glu Asp 660 665 670 Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr
Gly Ala Asp Ile Glu Ser 675 680 685 Lys Asn Lys Cys Gly Leu Thr Pro
Leu Leu Leu Gly Val His Glu Gln 690 695 700 Lys Gln Glu Val Val Lys
Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 705 710 715 720 Ala Leu Asp
Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys 725 730 735 Gly
Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn Val Asp Val 740 745
750 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser
755 760 765 Ser His His His Val Ile Cys Glu Leu Leu Ser Asp Tyr Lys
Glu Lys 770 775 780 Gln Met Leu Lys Ile Ser Ser Glu Asn Ser Asn Pro
Val Ile Thr Ile 785 790 795 800 Leu Asn Ile Lys Leu Pro Leu Lys Val
Glu Glu Glu Ile Lys Lys His 805 810 815 Gly Ser Asn Pro Val Gly Leu
Pro Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830 Ala Gly Asn Gly Asp
Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835 840 845 Pro Glu Asn
Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His Ser 850 855 860 Asp
Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu Gln Asn Thr 865 870
875 880 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys Gln Lys Gln Ile
Glu 885 890 895 Val Ala Glu Lys Glu Met Asn Ser Glu Leu Ser Leu Ser
His Lys Lys 900 905 910 Glu Glu Asp Leu Leu Arg Glu Asn Ser Met Leu
Arg Glu Glu Ile Ala 915 920 925 Lys Leu Arg Leu Glu Leu Asp Glu Thr
Lys His Gln Asn Gln Leu Arg 930 935 940 Glu Asn Lys Ile Leu Glu Glu
Ile Glu Ser Val Lys Glu Lys Leu Leu 945 950 955 960 Lys Thr Ile Gln
Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala 965 970 975 Gly Phe
Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln Ala Ser 980 985 990
Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu Lys Thr Glu Gln 995
1000 1005 Gln Ala Gln Glu Gln Glu Val Ala Gly Phe Ser Leu Arg Gln
Leu Gly 1010 1015 1020 Leu Ala Gln His Ala Gln Ala Ser Val Gln Gln
Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly His Thr Glu Lys Thr Glu
Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050 1055 Leu Arg Ser Gln
Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly 1060 1065 1070 Gly
Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro Arg 1075
1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val Arg Leu
Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala Ala Leu Pro Pro Pro Thr
Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser Pro Thr Lys Gln Lys Ser
Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130 1135 Pro Val Pro Thr
Phe Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys Pro 1140 1145 1150 Met
Phe Asp Val Ser Pro Ala Met Arg Leu Lys Ser Asp Ser Asn Arg 1155
1160 1165 Glu Thr His Gln Ala Phe Arg Asp Lys Asp Asp Leu Pro Phe
Phe Lys 1170 1175 1180 Thr Gln Gln Ser Pro Arg His Thr Lys Asp Leu
Gly Gln Asp Asp Arg 1185 1190 1195 1200 Ala Gly Val Leu Ala Pro Lys
Cys Arg Pro Gly Thr Leu Cys His Thr 1205 1210 1215 Asp Thr Pro Pro
His Arg Asn Ala Asp Thr Pro Pro His Arg His Thr 1220 1225 1230 Thr
Thr Leu Pro His Arg Asp Thr Thr Thr Ser Leu Pro His Phe His 1235
1240 1245 Val Ser Ala Gly Gly Val Gly Pro Thr Thr Leu Gly Ser Asn
Arg Glu 1250 1255 1260 Ile Thr 1265 28 255 PRT Homo sapians 28 Met
Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10
15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg
20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile
His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln
Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu
Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile
Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln Val Val Asn Ile
Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys Ser
Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser Phe
Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140
Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145
150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser
Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser
Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile
Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu Cys
Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser Phe
Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser Thr
Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 29 255
PRT Homo sapians 29 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Cys 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val
Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95
Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100
105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser
Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val
Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys
Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys
Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220
Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225
230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly
Val 245 250 255 30 255 PRT Homo sapians 30 Met Pro Phe Ile Ser Lys
Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser
Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu
Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45
Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50
55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu
Arg 65 70 75 80 Arg Val Ile Arg Asp Leu Ser Ile Cys Thr Thr Cys Leu
Leu Gly Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser
Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe
His Leu Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser
Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile
Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile
Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175
Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180
185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln
Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His
Gln His Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr
Lys Met Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro
Trp Ala Tyr Asp Arg Gly Val 245 250 255 31 255 PRT Homo sapians 31
Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5
10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu
Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu
Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro
Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr
Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser
Ile Cys Thr Thr Cys Leu Leu Asp Met 85 90 95 Leu Gln Val Val Asn
Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys
Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser
Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135
140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe
145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu
Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe
Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu
Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu
Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu Leu Pro Val Ser
Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser
Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 32
1266 PRT Homo sapians 32 Met Pro Phe Ile Ser Lys Leu Val Leu Ala
Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro
Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu
Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu
Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn
Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80
Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Asp Met 85
90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Ile Met
Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu Ile Gln Glu Leu
Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln Pro Leu Pro Lys
Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His Ile Leu Leu Pro
Thr Gln Ala Thr Phe Ala Ala 145 150 155 160 Ala Thr Gly Leu Trp Ala
Ala Leu Thr Thr Val Ser Asn Pro Ser Arg 165 170 175 Ala Asp Pro Val
Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys 180 185 190 Asn Leu
Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg 195 200 205
Lys Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala Leu Ser Leu Ser 210
215 220 Ser Ser Arg Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu
Pro 225 230 235 240 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro
Ala Gly Ala Phe 245 250 255 Leu Leu Gly Trp Glu Arg Val Val Gln Arg
Arg Leu Glu Val Pro Arg 260 265 270 Pro Gln Ala Ala Pro Ala Thr Ser
Ala Thr Pro Ser Arg Asp Pro Ser 275 280 285 Pro Pro Cys His Gln Arg
Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290 295 300 Leu Thr Arg Ala
Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 305 310 315 320 Gly
Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His 325 330
335 Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly
340 345 350 Ala Pro Gly Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser
Gly Pro 355 360 365 Ser Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro
Ala His Gln Arg 370 375 380 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala
Val Ser Gly Pro Gln Glu 385 390 395 400 Gln Pro Ser Glu Glu Ala Leu
Gly Val Gly Ser Leu Ser Val
Phe Gln 405 410 415 Leu His Leu Ile Gln Cys Ile Pro Asn Leu Ser Tyr
Pro Leu Val Leu 420 425 430 Arg His Ile Pro Glu Ile Leu Lys Phe Ser
Glu Lys Glu Thr Gly Gly 435 440 445 Gly Ile Leu Gly Leu Glu Leu Pro
Ala Thr Ala Ala Arg Leu Ser Gly 450 455 460 Leu Asn Ser Ile Met Gln
Ile Lys Glu Phe Glu Glu Leu Val Lys Leu 465 470 475 480 His Ser Leu
Ser His Lys Val Ile Gln Cys Val Phe Ala Lys Lys Lys 485 490 495 Asn
Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu Gly Tyr Gly His 500 505
510 Ser Phe Leu Ile Met Lys Glu Thr Ser Thr Lys Ile Ser Gly Leu Ile
515 520 525 Gln Glu Met Gly Ser Gly Lys Ser Asn Val Gly Thr Trp Gly
Asp Tyr 530 535 540 Asp Asp Ser Ala Phe Met Glu Pro Arg Tyr His Val
Arg Arg Glu Asp 545 550 555 560 Leu Asp Lys Leu His Arg Ala Ala Trp
Trp Gly Lys Val Pro Arg Lys 565 570 575 Asp Leu Ile Val Met Leu Arg
Asp Thr Asp Met Asn Lys Arg Asp Lys 580 585 590 Gln Lys Arg Thr Ala
Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu 595 600 605 Val Val Gln
Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp 610 615 620 Asn
Lys Lys Arg Thr Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp 625 630
635 640 Glu Cys Val Leu Met Leu Leu Glu His Gly Ala Asp Gly Asn Ile
Gln 645 650 655 Asp Glu Tyr Gly Asn Thr Ala Leu His Tyr Ala Ile Tyr
Asn Glu Asp 660 665 670 Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr Gly
Ala Asp Ile Glu Ser 675 680 685 Lys Asn Lys Cys Gly Leu Thr Pro Leu
Leu Leu Gly Val His Glu Gln 690 695 700 Lys Gln Glu Val Val Lys Phe
Leu Ile Lys Lys Lys Ala Asn Leu Asn 705 710 715 720 Ala Leu Asp Arg
Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys 725 730 735 Gly Ser
Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn Val Asp Val 740 745 750
Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser 755
760 765 Ser His His His Val Ile Cys Glu Leu Leu Ser Asp Tyr Lys Glu
Lys 770 775 780 Gln Met Leu Lys Ile Ser Ser Glu Asn Ser Asn Pro Val
Ile Thr Ile 785 790 795 800 Leu Asn Ile Lys Leu Pro Leu Lys Val Glu
Glu Glu Ile Lys Lys His 805 810 815 Gly Ser Asn Pro Val Gly Leu Pro
Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830 Ala Gly Asn Gly Asp Asp
Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835 840 845 Pro Glu Asn Gln
Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His Ser 850 855 860 Asp Glu
Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu Gln Asn Thr 865 870 875
880 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys Gln Lys Gln Ile Glu
885 890 895 Val Ala Glu Lys Glu Met Asn Ser Glu Leu Ser Leu Ser His
Lys Lys 900 905 910 Glu Glu Asp Leu Leu Arg Glu Asn Ser Met Leu Arg
Glu Glu Ile Ala 915 920 925 Lys Leu Arg Leu Glu Leu Asp Glu Thr Lys
His Gln Asn Gln Leu Arg 930 935 940 Glu Asn Lys Ile Leu Glu Glu Ile
Glu Ser Val Lys Glu Lys Leu Leu 945 950 955 960 Lys Thr Ile Gln Leu
Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala 965 970 975 Gly Phe Ser
Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln Ala Ser 980 985 990 Val
Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu Lys Thr Glu Gln 995
1000 1005 Gln Ala Gln Glu Gln Glu Val Ala Gly Phe Ser Leu Arg Gln
Leu Gly 1010 1015 1020 Leu Ala Gln His Ala Gln Ala Ser Val Gln Gln
Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly His Thr Glu Lys Thr Glu
Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050 1055 Leu Arg Ser Gln
Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly 1060 1065 1070 Gly
Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro Arg 1075
1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val Arg Leu
Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala Ala Leu Pro Pro Pro Thr
Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser Pro Thr Lys Gln Lys Ser
Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130 1135 Pro Val Pro Thr
Phe Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys Pro 1140 1145 1150 Met
Phe Asp Val Ser Pro Ala Met Arg Leu Lys Ser Asp Ser Asn Arg 1155
1160 1165 Glu Thr His Gln Ala Phe Arg Asp Lys Asp Asp Leu Pro Phe
Phe Lys 1170 1175 1180 Thr Gln Gln Ser Pro Arg His Thr Lys Asp Leu
Gly Gln Asp Asp Arg 1185 1190 1195 1200 Ala Gly Val Leu Ala Pro Lys
Cys Arg Pro Gly Thr Leu Cys His Thr 1205 1210 1215 Asp Thr Pro Pro
His Arg Asn Ala Asp Thr Pro Pro His Arg His Thr 1220 1225 1230 Thr
Thr Leu Pro His Arg Asp Thr Thr Thr Ser Leu Pro His Phe His 1235
1240 1245 Val Ser Ala Gly Gly Val Gly Pro Thr Thr Leu Gly Ser Asn
Arg Glu 1250 1255 1260 Ile Thr 1265 33 228 PRT Homo sapians 33 Phe
Leu Leu Phe Leu Asp Leu Arg Pro Glu Arg Thr Tyr Leu Pro Val 1 5 10
15 Cys His Val Ala Leu Ile His Met Val Val Leu Leu Thr Met Val Phe
20 25 30 Leu Ser Pro Gln Leu Phe Glu Ser Leu Asn Phe Gln Asn Asp
Phe Lys 35 40 45 Tyr Glu Ala Ser Phe Tyr Leu Arg Arg Val Ile Arg
Val Leu Ser Ile 50 55 60 Cys Thr Thr Cys Leu Leu Gly Met Leu Gln
Val Val Asn Ile Ser Pro 65 70 75 80 Ser Ile Ser Trp Leu Val Arg Phe
Lys Trp Lys Ser Thr Ile Phe Thr 85 90 95 Phe His Leu Phe Ser Trp
Ser Leu Ser Phe Pro Val Ser Ser Ser Leu 100 105 110 Ile Phe Tyr Thr
Val Ala Ser Ser Asn Val Thr Gln Ile Asn Leu His 115 120 125 Val Ser
Lys Tyr Cys Ser Leu Phe Pro Ile Asn Ser Ile Ile Arg Gly 130 135 140
Leu Phe Phe Thr Leu Ser Leu Phe Arg Asp Val Phe Leu Lys Gln Ile 145
150 155 160 Met Leu Phe Ser Ser Val Tyr Met Met Thr Leu Ile Gln Glu
Leu Gln 165 170 175 Glu Ile Leu Val Pro Ser Gln Pro Gln Pro Leu Pro
Lys Asp Leu Cys 180 185 190 Arg Gly Lys Ser His Gln His Ile Leu Leu
Pro Val Ser Phe Ser Val 195 200 205 Gly Met Tyr Lys Met Asp Phe Ile
Ile Ser Thr Ser Ser Thr Leu Pro 210 215 220 Trp Ala Tyr Asp 225 34
234 PRT Homo sapians 34 Phe Tyr Ile Phe Ile Ile Leu Gly His Arg Pro
Lys Pro Met Asp Leu 1 5 10 15 Ile Ser Cys Gln Gln Thr Phe Ile His
Ile Met Leu Phe Phe Thr Ala 20 25 30 Gly Asp Ile Leu His Thr Asp
Ile Phe Glu Ser Met Asn Ile Glu Asn 35 40 45 Asp Phe Lys Cys Lys
Thr Thr Phe Tyr Ile Cys Arg Val Met Arg Gly 50 55 60 Leu Ser Ile
Cys Thr Thr Cys Leu Leu Ser Val Phe Gln Ala Val Thr 65 70 75 80 Ile
Ser Pro Asn Thr Ser Leu Leu Ala Lys Phe Lys His Lys Leu Lys 85 90
95 Lys Tyr Thr Ile Asn Ala Phe Phe Tyr Ile Trp Ser Phe Asn Leu Ser
100 105 110 Phe Ser Ser Asn Leu Ile Phe Tyr Val Gly Ala Tyr Thr Asn
Val Ser 115 120 125 Glu Thr Asn Gln Met Lys Val Thr Lys Tyr Cys Ser
Leu Phe Pro Met 130 135 140 Asn Tyr Ile Ile Arg Gly Leu Ile Leu Thr
Val Thr Thr Ser Arg Asp 145 150 155 160 Val Phe Leu Val Gly Val Met
Leu Ile Thr Ser Thr Tyr Met Val Ile 165 170 175 Ile Leu Phe Arg His
Gln Arg Gln Cys Lys His Leu His Ser Ile Arg 180 185 190 His Leu Arg
Ala Ser Pro Glu Lys Lys Ala Thr Gln Thr Ile Leu Leu 195 200 205 Leu
Val Val Phe Phe Val Val Met Tyr Trp Val Asp Phe Ile Ile Ser 210 215
220 Ser Thr Ser Val Leu Leu Trp Met Tyr Asp 225 230 35 1213 PRT
Homo sapians 35 Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser Leu Asn
Phe Gln Asn 1 5 10 15 Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg
Arg Val Ile Arg Val 20 25 30 Leu Ser Ile Cys Thr Thr Cys Leu Leu
Asp Met Leu Gln Val Val Asn 35 40 45 Ile Ser Pro Ser Ile Ser Trp
Leu Ile Met Leu Phe Ser Ser Val Tyr 50 55 60 Met Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln 65 70 75 80 Pro Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His 85 90 95 Ile
Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala Ala Thr Gly Leu Trp 100 105
110 Ala Ala Leu Thr Thr Val Ser Asn Pro Ser Arg Ala Asp Pro Val Thr
115 120 125 Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys Asn Leu Glu
Lys Gly 130 135 140 Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg Lys
Glu Phe Ser Thr 145 150 155 160 Thr Leu Thr Gly His Ser Ala Leu Ser
Leu Ser Ser Ser Arg Ala Leu 165 170 175 Pro Gly Ser Leu Pro Ala Phe
Ala Asp Leu Pro Arg Ser Cys Pro Glu 180 185 190 Ser Glu Gln Ser Ala
Thr Pro Ala Gly Ala Phe Leu Leu Gly Trp Glu 195 200 205 Arg Val Val
Gln Arg Arg Leu Glu Val Pro Arg Pro Gln Ala Ala Pro 210 215 220 Ala
Thr Ser Ala Thr Pro Ser Arg Asp Pro Ser Pro Pro Cys His Gln 225 230
235 240 Arg Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly Leu Thr Arg Ala
Phe 245 250 255 Gln Val Val His Leu Ala Pro Thr Ala Pro Asp Gly Gly
Ala Gly Cys 260 265 270 Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His
Val Arg Cys Ala Gln 275 280 285 Gly Leu Glu Ala Ala Ser Ala Asn Leu
Pro Gly Ala Pro Gly Arg Ser 290 295 300 Ser Ser Cys Ala Leu Arg Tyr
Arg Ser Gly Pro Ser Val Ser Ser Ala 305 310 315 320 Pro Ser Pro Ala
Glu Pro Pro Ala His Gln Arg Leu Leu Phe Leu Pro 325 330 335 Arg Ala
Pro Gln Ala Val Ser Gly Pro Gln Glu Gln Pro Ser Glu Glu 340 345 350
Ala Leu Gly Val Gly Ser Leu Ser Val Phe Gln Leu His Leu Ile Gln 355
360 365 Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu Arg His Ile Pro
Glu 370 375 380 Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly Gly Ile
Leu Gly Leu 385 390 395 400 Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser
Gly Leu Asn Ser Ile Met 405 410 415 Gln Ile Lys Glu Phe Glu Glu Leu
Val Lys Leu His Ser Leu Ser His 420 425 430 Lys Val Ile Gln Cys Val
Phe Ala Lys Lys Lys Asn Val Asp Lys Trp 435 440 445 Asp Asp Phe Cys
Leu Ser Glu Gly Tyr Gly His Ser Phe Leu Ile Met 450 455 460 Lys Glu
Thr Ser Thr Lys Ile Ser Gly Leu Ile Gln Glu Met Gly Ser 465 470 475
480 Gly Lys Ser Asn Val Gly Thr Trp Gly Asp Tyr Asp Asp Ser Ala Phe
485 490 495 Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp Leu Asp Lys
Leu His 500 505 510 Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys Asp
Leu Ile Val Met 515 520 525 Leu Arg Asp Thr Asp Met Asn Lys Arg Asp
Lys Gln Lys Arg Thr Ala 530 535 540 Leu His Leu Ala Ser Ala Asn Gly
Asn Ser Glu Val Val Gln Leu Leu 545 550 555 560 Leu Asp Arg Arg Cys
Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr 565 570 575 Ala Leu Ile
Lys Ala Val Gln Cys Gln Glu Asp Glu Cys Val Leu Met 580 585 590 Leu
Leu Glu His Gly Ala Asp Gly Asn Ile Gln Asp Glu Tyr Gly Asn 595 600
605 Thr Ala Leu His Tyr Ala Ile Tyr Asn Glu Asp Lys Leu Met Ala Lys
610 615 620 Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser Lys Asn Lys
Cys Gly 625 630 635 640 Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln
Lys Gln Glu Val Val 645 650 655 Lys Phe Leu Ile Lys Lys Lys Ala Asn
Leu Asn Ala Leu Asp Arg Tyr 660 665 670 Gly Arg Thr Ala Leu Ile Leu
Ala Val Cys Cys Gly Ser Ala Ser Ile 675 680 685 Val Asn Leu Leu Leu
Glu Gln Asn Val Asp Val Ser Ser Gln Asp Leu 690 695 700 Ser Gly Gln
Thr Ala Arg Glu Tyr Ala Val Ser Ser His His His Val 705 710 715 720
Ile Cys Glu Leu Leu Ser Asp Tyr Lys Glu Lys Gln Met Leu Lys Ile 725
730 735 Ser Ser Glu Asn Ser Asn Pro Val Ile Thr Ile Leu Asn Ile Lys
Leu 740 745 750 Pro Leu Lys Val Glu Glu Glu Ile Lys Lys His Gly Ser
Asn Pro Val 755 760 765 Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser
Ala Gly Asn Gly Asp 770 775 780 Asp Gly Leu Ile Pro Gln Arg Lys Ser
Arg Lys Pro Glu Asn Gln Gln 785 790 795 800 Phe Pro Asp Thr Glu Asn
Glu Glu Tyr His Ser Asp Glu Gln Asn Asp 805 810 815 Thr Gln Lys Gln
Leu Ser Glu Glu Gln Asn Thr Gly Ile Ser Gln Asp 820 825 830 Glu Ile
Leu Thr Asn Lys Gln Lys Gln Ile Glu Val Ala Glu Lys Glu 835 840 845
Met Asn Ser Glu Leu Ser Leu Ser His Lys Lys Glu Glu Asp Leu Leu 850
855 860 Arg Glu Asn Ser Met Leu Arg Glu Glu Ile Ala Lys Leu Arg Leu
Glu 865 870 875 880 Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg Glu
Asn Lys Ile Leu 885 890 895 Glu Glu Ile Glu Ser Val Lys Glu Lys Leu
Leu Lys Thr Ile Gln Leu 900 905 910 Asn Glu Glu Ala Leu Thr Lys Thr
Lys Val Ala Gly Phe Ser Leu Arg 915 920 925 Gln Leu Gly Leu Ala Gln
His Ala Gln Ala Ser Val Gln Gln Leu Cys 930 935 940 Tyr Lys Trp Asn
His Thr Glu Lys Thr Glu Gln Gln Ala Gln Glu Gln 945 950 955 960 Glu
Val Ala Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala 965 970
975 Gln Ala Ser Val Gln Gln Leu Cys Tyr Lys Trp Gly His Thr Glu Lys
980 985 990 Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala Leu Arg Ser
Gln Ile 995 1000 1005 Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly
Gly Thr Ala Ala Gly 1010 1015 1020 Asp Gln Gly Pro Gly Thr His Leu
Pro Pro Arg Glu Pro Arg Ala Ser 1025 1030 1035 1040 Pro Gly Thr Pro
Ser Leu Val Arg Leu Ala Ser Gly Ala Arg Ala Ala 1045 1050 1055 Ala
Leu Pro Pro Pro Thr Gly Lys Asn Gly Arg Ser Pro Thr Lys Gln 1060
1065 1070 Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu Pro Val Pro
Thr Phe 1075 1080 1085 Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys Pro
Met Phe Asp Val Ser 1090 1095 1100 Pro Ala Met Arg Leu Lys Ser Asp
Ser Asn Arg Glu Thr His Gln Ala 1105 1110 1115
1120 Phe Arg Asp Lys Asp Asp Leu Pro Phe Phe Lys Thr Gln Gln Ser
Pro 1125 1130 1135 Arg His Thr Lys Asp Leu Gly Gln Asp Asp Arg Ala
Gly Val Leu Ala 1140 1145 1150 Pro Lys Cys Arg Pro Gly Thr Leu Cys
His Thr Asp Thr Pro Pro His 1155 1160 1165 Arg Asn Ala Asp Thr Pro
Pro His Arg His Thr Thr Thr Leu Pro His 1170 1175 1180 Arg Asp Thr
Thr Thr Ser Leu Pro His Phe His Val Ser Ala Gly Gly 1185 1190 1195
1200 Val Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu Ile Thr 1205 1210
36 1213 PRT Homo sapians 36 Met Val Phe Leu Ser Pro Gln Leu Phe Glu
Ser Leu Asn Phe Gln Asn 1 5 10 15 Asp Phe Lys Tyr Glu Ala Ser Phe
Tyr Leu Arg Arg Val Ile Arg Val 20 25 30 Leu Ser Ile Cys Thr Thr
Cys Leu Leu Asp Met Leu Gln Val Val Asn 35 40 45 Ile Ser Pro Ser
Ile Ser Trp Leu Ile Met Leu Phe Ser Ser Val Tyr 50 55 60 Met Met
Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln 65 70 75 80
Pro Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His 85
90 95 Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala Ala Thr Gly Leu
Trp 100 105 110 Ala Ala Leu Thr Thr Val Ser Asn Pro Ser Arg Ala Asp
Pro Val Thr 115 120 125 Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys
Asn Leu Glu Lys Gly 130 135 140 Ser Trp Leu Ser Phe Pro Gly Thr Ala
Ala Arg Lys Glu Phe Ser Thr 145 150 155 160 Thr Leu Thr Gly His Ser
Ala Leu Ser Leu Ser Ser Ser Arg Ala Leu 165 170 175 Pro Gly Ser Leu
Pro Ala Phe Ala Asp Leu Pro Arg Ser Cys Pro Glu 180 185 190 Ser Glu
Gln Ser Ala Thr Pro Ala Gly Ala Phe Leu Leu Gly Trp Glu 195 200 205
Arg Val Val Gln Arg Arg Leu Glu Val Pro Arg Pro Gln Ala Ala Pro 210
215 220 Ala Thr Ser Ala Thr Pro Ser Arg Asp Pro Ser Pro Pro Cys His
Gln 225 230 235 240 Arg Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly Leu
Thr Arg Ala Phe 245 250 255 Gln Val Val His Leu Ala Pro Thr Ala Pro
Asp Gly Gly Ala Gly Cys 260 265 270 Pro Pro Ser Arg Asn Ser Tyr Arg
Leu Thr His Val Arg Cys Ala Gln 275 280 285 Gly Leu Glu Ala Ala Ser
Ala Asn Leu Pro Gly Ala Pro Gly Arg Ser 290 295 300 Ser Ser Cys Ala
Leu Arg Tyr Arg Ser Gly Pro Ser Val Ser Ser Ala 305 310 315 320 Pro
Ser Pro Ala Glu Pro Pro Ala His Gln Arg Leu Leu Phe Leu Pro 325 330
335 Arg Ala Pro Gln Ala Val Ser Gly Pro Gln Glu Gln Pro Ser Glu Glu
340 345 350 Ala Leu Gly Val Gly Ser Leu Ser Val Phe Gln Leu His Leu
Ile Gln 355 360 365 Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu Arg
His Ile Pro Glu 370 375 380 Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly
Gly Gly Ile Leu Gly Leu 385 390 395 400 Glu Leu Pro Ala Thr Ala Ala
Arg Leu Ser Gly Leu Asn Ser Ile Met 405 410 415 Gln Ile Lys Glu Phe
Glu Glu Leu Val Lys Leu His Ser Leu Ser His 420 425 430 Lys Val Ile
Gln Cys Val Phe Ala Lys Lys Lys Asn Val Asp Lys Trp 435 440 445 Asp
Asp Phe Cys Leu Ser Glu Gly Tyr Gly His Ser Phe Leu Ile Met 450 455
460 Lys Glu Thr Ser Thr Lys Ile Ser Gly Leu Ile Gln Glu Met Gly Ser
465 470 475 480 Gly Lys Ser Asn Val Gly Thr Trp Gly Asp Tyr Asp Asp
Ser Ala Phe 485 490 495 Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp
Leu Asp Lys Leu His 500 505 510 Arg Ala Ala Trp Trp Gly Lys Val Pro
Arg Lys Asp Leu Ile Val Met 515 520 525 Leu Arg Asp Thr Asp Met Asn
Lys Arg Asp Lys Gln Lys Arg Thr Ala 530 535 540 Leu His Leu Ala Ser
Ala Asn Gly Asn Ser Glu Val Val Gln Leu Leu 545 550 555 560 Leu Asp
Arg Arg Cys Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr 565 570 575
Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp Glu Cys Val Leu Met 580
585 590 Leu Leu Glu His Gly Ala Asp Gly Asn Ile Gln Asp Glu Tyr Gly
Asn 595 600 605 Thr Ala Leu His Tyr Ala Ile Tyr Asn Glu Asp Lys Leu
Met Ala Lys 610 615 620 Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser
Lys Asn Lys Cys Gly 625 630 635 640 Leu Thr Pro Leu Leu Leu Gly Val
His Glu Gln Lys Gln Glu Val Val 645 650 655 Lys Phe Leu Ile Lys Lys
Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr 660 665 670 Gly Arg Thr Ala
Leu Ile Leu Ala Val Cys Cys Gly Ser Ala Ser Ile 675 680 685 Val Asn
Leu Leu Leu Glu Gln Asn Val Asp Val Ser Ser Gln Asp Leu 690 695 700
Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser Ser His His His Val 705
710 715 720 Ile Cys Glu Leu Leu Ser Asp Tyr Lys Glu Lys Gln Met Leu
Lys Ile 725 730 735 Ser Ser Glu Asn Ser Asn Pro Val Ile Thr Ile Leu
Asn Ile Lys Leu 740 745 750 Pro Leu Lys Val Glu Glu Glu Ile Lys Lys
His Gly Ser Asn Pro Val 755 760 765 Gly Leu Pro Glu Asn Leu Thr Asn
Gly Ala Ser Ala Gly Asn Gly Asp 770 775 780 Asp Gly Leu Ile Pro Gln
Arg Lys Ser Arg Lys Pro Glu Asn Gln Gln 785 790 795 800 Phe Pro Asp
Thr Glu Asn Glu Glu Tyr His Ser Asp Glu Gln Asn Asp 805 810 815 Thr
Gln Lys Gln Leu Ser Glu Glu Gln Asn Thr Gly Ile Ser Gln Asp 820 825
830 Glu Ile Leu Thr Asn Lys Gln Lys Gln Ile Glu Val Ala Glu Lys Glu
835 840 845 Met Asn Ser Glu Leu Ser Leu Ser His Lys Lys Glu Glu Asp
Leu Leu 850 855 860 Arg Glu Asn Ser Met Leu Arg Glu Glu Ile Ala Lys
Leu Arg Leu Glu 865 870 875 880 Leu Asp Glu Thr Lys His Gln Asn Gln
Leu Arg Glu Asn Lys Ile Leu 885 890 895 Glu Glu Ile Glu Ser Val Lys
Glu Lys Leu Leu Lys Thr Ile Gln Leu 900 905 910 Asn Glu Glu Ala Leu
Thr Lys Thr Lys Val Ala Gly Phe Ser Leu Arg 915 920 925 Gln Leu Gly
Leu Ala Gln His Ala Gln Ala Ser Val Gln Gln Leu Cys 930 935 940 Tyr
Lys Trp Asn His Thr Glu Lys Thr Glu Gln Gln Ala Gln Glu Gln 945 950
955 960 Glu Val Ala Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His
Ala 965 970 975 Gln Ala Ser Val Gln Gln Leu Cys Tyr Lys Trp Gly His
Thr Glu Lys 980 985 990 Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala
Leu Arg Ser Gln Ile 995 1000 1005 Gly Asp Pro Gly Gly Val Pro Leu
Ser Glu Gly Gly Thr Ala Ala Gly 1010 1015 1020 Asp Gln Gly Pro Gly
Thr His Leu Pro Pro Arg Glu Pro Arg Ala Ser 1025 1030 1035 1040 Pro
Gly Thr Pro Ser Leu Val Arg Leu Ala Ser Gly Ala Arg Ala Ala 1045
1050 1055 Ala Leu Pro Pro Pro Thr Gly Lys Asn Gly Arg Ser Pro Thr
Lys Gln 1060 1065 1070 Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu
Pro Val Pro Thr Phe 1075 1080 1085 Ser Ser Gly Ser Phe Leu Gly Arg
Arg Cys Pro Met Phe Asp Val Ser 1090 1095 1100 Pro Ala Met Arg Leu
Lys Ser Asp Ser Asn Arg Glu Thr His Gln Ala 1105 1110 1115 1120 Phe
Arg Asp Lys Asp Asp Leu Pro Phe Phe Lys Thr Gln Gln Ser Pro 1125
1130 1135 Arg His Thr Lys Asp Leu Gly Gln Asp Asp Arg Ala Gly Val
Leu Ala 1140 1145 1150 Pro Lys Cys Arg Pro Gly Thr Leu Cys His Thr
Asp Thr Pro Pro His 1155 1160 1165 Arg Asn Ala Asp Thr Pro Pro His
Arg His Thr Thr Thr Leu Pro His 1170 1175 1180 Arg Asp Thr Thr Thr
Ser Leu Pro His Phe His Val Ser Ala Gly Gly 1185 1190 1195 1200 Val
Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu Ile Thr 1205 1210 37 14 PRT
Tetanus toxoid 37 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile
Thr Glu 1 5 10 38 21 PRT Plasmodium falciparum 38 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 39 16 PRT Strepcococcus 39 Gly Ala Val Asp Ser Ile
Leu Gly Gly Val Ala Thr Tyr Gly Ala Ala 1 5 10 15 40 13 PRT
Artificial Sequence Pan DR-binding epitope 40 Xaa Lys Xaa Val Ala
Ala Trp Thr Leu Lys Ala Ala Xaa 1 5 10 41 14 DNA Artificial
Sequence Primer 41 ttttgatcaa gctt 14 42 42 DNA Artificial Sequence
Primer 42 ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag 42 43 12
DNA Artificial Sequence Primer 43 gatcctgccc gg 12 44 40 DNA
Artificial Sequence Primer 44 gtaatacgac tcactatagg gcagcgtggt
cgcggccgag 40 45 10 DNA Artificial Sequence Primer 45 gatcctcggc 10
46 22 DNA Artificial Sequence Primer 46 ctaatacgac tcactatagg gc 22
47 22 DNA Artificial Sequence Primer 47 tcgagcggcc gcccgggcag ga 22
48 20 DNA Artificial Sequence Primer 48 agcgtggtcg cggccgagga 20 49
25 DNA Artificial Sequence Primer 49 atatcgccgc gctcgtcgtc gacaa 25
50 26 DNA Artificial Sequence Primer 50 agccacacgc agctcattgt
agaagg 26 51 23 DNA Artificial Sequence RT-PCR Primer 51 agtgattcaa
agagctgtgg aga 23 52 22 DNA Artificial Sequence RT-PCR Primer 52
ggccagagcg cacttaccta cc 22 53 24 DNA Artificial Sequence Epitope
Tag 53 gattacaagg atgacgacga taag 24 54 4 PRT Homo sapians 54 Asn
Val Thr Gln 1 55 6 PRT Homo sapians 55 Gly Leu Phe Phe Thr Leu 1 5
56 22 PRT Homo sapians 56 Leu Arg Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile 1 5 10 15 His Met Val Val Leu Leu 20 57 255
PRT Homo sapians 57 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln
Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu
Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val
Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met
Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln
Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val
Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95
Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100
105 110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser
Leu 115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val
Ala Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys
Tyr Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly
Leu Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys
Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220
Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met Asp Phe Ile 225
230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr Asp Arg Gly
Val 245 250 255 58 17 PRT Homo sapians 58 Val Leu Ala Ser Gln Pro
Thr Leu Cys Ser Phe Phe Ser Ala Ser Ser 1 5 10 15 Pro 59 19 PRT
Homo sapians 59 Leu Val Leu Ala Ser Gln Pro Thr Leu Cys Ser Phe Phe
Ser Ala Ser 1 5 10 15 Ser Pro Phe 60 17 PRT Homo sapians 60 Phe Tyr
Leu Arg Arg Val Ile Arg Asp Leu Ser Ile Cys Thr Thr Cys 1 5 10 15
Leu 61 19 PRT Homo sapians 61 Ser Phe Tyr Leu Arg Arg Val Ile Arg
Asp Leu Ser Ile Cys Thr Thr 1 5 10 15 Cys Leu Leu 62 17 PRT Homo
sapians 62 Ser Ile Cys Thr Thr Cys Leu Leu Asp Met Leu Gln Val Val
Asn Ile 1 5 10 15 Ser 63 19 PRT Homo sapians 63 Leu Ser Ile Cys Thr
Thr Cys Leu Leu Asp Met Leu Gln Val Val Asn 1 5 10 15 Ile Ser Pro
64 16 PRT Homo sapians 64 Ile Ser Pro Ser Ile Ser Trp Leu Ile Met
Leu Phe Ser Ser Val Tyr 1 5 10 15 65 18 PRT Homo sapians 65 Asn Ile
Ser Pro Ser Ile Ser Trp Leu Ile Met Leu Phe Ser Ser Val 1 5 10 15
Tyr Met 66 28 PRT Homo sapians 66 Met Leu Gln Val Val Asn Ile Ser
Pro Ser Ile Ser Trp Leu Ile Met 1 5 10 15 Leu Phe Ser Ser Val Tyr
Met Met Thr Leu Ile Gln 20 25 67 1121 PRT Homo sapians 67 Ser His
Gln His Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala Ala 1 5 10 15
Thr Gly Leu Trp Ala Ala Leu Thr Thr Val Ser Asn Pro Ser Arg Ala 20
25 30 Asp Pro Val Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys
Asn 35 40 45 Leu Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala
Ala Arg Lys 50 55 60 Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala
Leu Ser Leu Ser Ser 65 70 75 80 Ser Arg Ala Leu Pro Gly Ser Leu Pro
Ala Phe Ala Asp Leu Pro Arg 85 90 95 Ser Cys Pro Glu Ser Glu Gln
Ser Ala Thr Pro Ala Gly Ala Phe Leu 100 105 110 Leu Gly Trp Glu Arg
Val Val Gln Arg Arg Leu Glu Val Pro Arg Pro 115 120 125 Gln Ala Ala
Pro Ala Thr Ser Ala Thr Pro Ser Arg Asp Pro Ser Pro 130 135 140 Pro
Cys His Gln Arg Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly Leu 145 150
155 160 Thr Arg Ala Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp
Gly 165 170 175 Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu
Thr His Val 180 185 190 Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala
Asn Leu Pro Gly Ala 195 200 205 Pro Gly Arg Ser Ser Ser Cys Ala Leu
Arg Tyr Arg Ser Gly Pro Ser 210 215 220 Val Ser Ser Ala Pro Ser Pro
Ala Glu Pro Pro Ala His Gln Arg Leu 225 230 235 240 Leu Phe Leu Pro
Arg Ala Pro Gln Ala Val Ser Gly Pro Gln Glu Gln 245 250 255 Pro Ser
Glu Glu Ala Leu Gly Val Gly Ser Leu Ser Val Phe Gln Leu 260 265 270
His Leu Ile Gln Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu Arg 275
280 285 His Ile Pro Glu Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly
Gly 290 295 300 Ile Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu
Ser Gly Leu 305 310 315 320 Asn Ser Ile Met Gln Ile Lys Glu Phe Glu
Glu Leu Val Lys Leu His 325 330 335 Ser Leu Ser His Lys Val Ile Gln
Cys Val Phe Ala Lys Lys Lys
Asn 340 345 350 Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu Gly Tyr
Gly His Ser 355 360 365 Phe Leu Ile Met Lys Glu Thr Ser Thr Lys Ile
Ser Gly Leu Ile Gln 370 375 380 Glu Met Gly Ser Gly Lys Ser Asn Val
Gly Thr Trp Gly Asp Tyr Asp 385 390 395 400 Asp Ser Ala Phe Met Glu
Pro Arg Tyr His Val Arg Arg Glu Asp Leu 405 410 415 Asp Lys Leu His
Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys Asp 420 425 430 Leu Ile
Val Met Leu Arg Asp Thr Asp Met Asn Lys Arg Asp Lys Gln 435 440 445
Lys Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu Val 450
455 460 Val Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp
Asn 465 470 475 480 Lys Lys Arg Thr Ala Leu Ile Lys Ala Val Gln Cys
Gln Glu Asp Glu 485 490 495 Cys Val Leu Met Leu Leu Glu His Gly Ala
Asp Gly Asn Ile Gln Asp 500 505 510 Glu Tyr Gly Asn Thr Ala Leu His
Tyr Ala Ile Tyr Asn Glu Asp Lys 515 520 525 Leu Met Ala Lys Ala Leu
Leu Leu Tyr Gly Ala Asp Ile Glu Ser Lys 530 535 540 Asn Lys Cys Gly
Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln Lys 545 550 555 560 Gln
Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn Ala 565 570
575 Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys Gly
580 585 590 Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn Val Asp
Val Ser 595 600 605 Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg Glu Tyr
Ala Val Ser Ser 610 615 620 His His His Val Ile Cys Glu Leu Leu Ser
Asp Tyr Lys Glu Lys Gln 625 630 635 640 Met Leu Lys Ile Ser Ser Glu
Asn Ser Asn Pro Val Ile Thr Ile Leu 645 650 655 Asn Ile Lys Leu Pro
Leu Lys Val Glu Glu Glu Ile Lys Lys His Gly 660 665 670 Ser Asn Pro
Val Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser Ala 675 680 685 Gly
Asn Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys Pro 690 695
700 Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His Ser Asp
705 710 715 720 Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu Gln
Asn Thr Gly 725 730 735 Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys Gln
Lys Gln Ile Glu Val 740 745 750 Ala Glu Lys Glu Met Asn Ser Glu Leu
Ser Leu Ser His Lys Lys Glu 755 760 765 Glu Asp Leu Leu Arg Glu Asn
Ser Met Leu Arg Glu Glu Ile Ala Lys 770 775 780 Leu Arg Leu Glu Leu
Asp Glu Thr Lys His Gln Asn Gln Leu Arg Glu 785 790 795 800 Asn Lys
Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu Lys 805 810 815
Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala Gly 820
825 830 Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln Ala Ser
Val 835 840 845 Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu Lys Thr
Glu Gln Gln 850 855 860 Ala Gln Glu Gln Glu Val Ala Gly Phe Ser Leu
Arg Gln Leu Gly Leu 865 870 875 880 Ala Gln His Ala Gln Ala Ser Val
Gln Gln Leu Cys Tyr Lys Trp Gly 885 890 895 His Thr Glu Lys Thr Glu
Gln Gln Ala Gln Glu Gln Gly Ala Ala Leu 900 905 910 Arg Ser Gln Ile
Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly Gly 915 920 925 Thr Ala
Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro Arg Glu 930 935 940
Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val Arg Leu Ala Ser Gly 945
950 955 960 Ala Arg Ala Ala Ala Leu Pro Pro Pro Thr Gly Lys Asn Gly
Arg Ser 965 970 975 Pro Thr Lys Gln Lys Ser Val Cys Asp Ser Ser Gly
Trp Ile Leu Pro 980 985 990 Val Pro Thr Phe Ser Ser Gly Ser Phe Leu
Gly Arg Arg Cys Pro Met 995 1000 1005 Phe Asp Val Ser Pro Ala Met
Arg Leu Lys Ser Asp Ser Asn Arg Glu 1010 1015 1020 Thr His Gln Ala
Phe Arg Asp Lys Asp Asp Leu Pro Phe Phe Lys Thr 1025 1030 1035 1040
Gln Gln Ser Pro Arg His Thr Lys Asp Leu Gly Gln Asp Asp Arg Ala
1045 1050 1055 Gly Val Leu Ala Pro Lys Cys Arg Pro Gly Thr Leu Cys
His Thr Asp 1060 1065 1070 Thr Pro Pro His Arg Asn Ala Asp Thr Pro
Pro His Arg His Thr Thr 1075 1080 1085 Thr Leu Pro His Arg Asp Thr
Thr Thr Ser Leu Pro His Phe His Val 1090 1095 1100 Ser Ala Gly Gly
Val Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu Ile 1105 1110 1115 1120
Thr 68 1122 PRT Homo sapians 68 Lys Ser His Gln His Ile Leu Leu Pro
Thr Gln Ala Thr Phe Ala Ala 1 5 10 15 Ala Thr Gly Leu Trp Ala Ala
Leu Thr Thr Val Ser Asn Pro Ser Arg 20 25 30 Ala Asp Pro Val Thr
Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys 35 40 45 Asn Leu Glu
Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg 50 55 60 Lys
Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala Leu Ser Leu Ser 65 70
75 80 Ser Ser Arg Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu
Pro 85 90 95 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro Ala
Gly Ala Phe 100 105 110 Leu Leu Gly Trp Glu Arg Val Val Gln Arg Arg
Leu Glu Val Pro Arg 115 120 125 Pro Gln Ala Ala Pro Ala Thr Ser Ala
Thr Pro Ser Arg Asp Pro Ser 130 135 140 Pro Pro Cys His Gln Arg Arg
Asp Ala Ala Cys Leu Arg Ala Gln Gly 145 150 155 160 Leu Thr Arg Ala
Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 165 170 175 Gly Gly
Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His 180 185 190
Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly 195
200 205 Ala Pro Gly Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser Gly
Pro 210 215 220 Ser Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro Ala
His Gln Arg 225 230 235 240 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala
Val Ser Gly Pro Gln Glu 245 250 255 Gln Pro Ser Glu Glu Ala Leu Gly
Val Gly Ser Leu Ser Val Phe Gln 260 265 270 Leu His Leu Ile Gln Cys
Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu 275 280 285 Arg His Ile Pro
Glu Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly 290 295 300 Gly Ile
Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 305 310 315
320 Leu Asn Ser Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu
325 330 335 His Ser Leu Ser His Lys Val Ile Gln Cys Val Phe Ala Lys
Lys Lys 340 345 350 Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu
Gly Tyr Gly His 355 360 365 Ser Phe Leu Ile Met Lys Glu Thr Ser Thr
Lys Ile Ser Gly Leu Ile 370 375 380 Gln Glu Met Gly Ser Gly Lys Ser
Asn Val Gly Thr Trp Gly Asp Tyr 385 390 395 400 Asp Asp Ser Ala Phe
Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp 405 410 415 Leu Asp Lys
Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys 420 425 430 Asp
Leu Ile Val Met Leu Arg Asp Thr Asp Met Asn Lys Arg Asp Lys 435 440
445 Gln Lys Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu
450 455 460 Val Val Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val
Leu Asp 465 470 475 480 Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val
Gln Cys Gln Glu Asp 485 490 495 Glu Cys Val Leu Met Leu Leu Glu His
Gly Ala Asp Gly Asn Ile Gln 500 505 510 Asp Glu Tyr Gly Asn Thr Ala
Leu His Tyr Ala Ile Tyr Asn Glu Asp 515 520 525 Lys Leu Met Ala Lys
Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser 530 535 540 Lys Asn Lys
Cys Gly Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln 545 550 555 560
Lys Gln Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 565
570 575 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys
Cys 580 585 590 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn
Val Asp Val 595 600 605 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg
Glu Tyr Ala Val Ser 610 615 620 Ser His His His Val Ile Cys Glu Leu
Leu Ser Asp Tyr Lys Glu Lys 625 630 635 640 Gln Met Leu Lys Ile Ser
Ser Glu Asn Ser Asn Pro Val Ile Thr Ile 645 650 655 Leu Asn Ile Lys
Leu Pro Leu Lys Val Glu Glu Glu Ile Lys Lys His 660 665 670 Gly Ser
Asn Pro Val Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser 675 680 685
Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 690
695 700 Pro Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His
Ser 705 710 715 720 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu
Glu Gln Asn Thr 725 730 735 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn
Lys Gln Lys Gln Ile Glu 740 745 750 Val Ala Glu Lys Glu Met Asn Ser
Glu Leu Ser Leu Ser His Lys Lys 755 760 765 Glu Glu Asp Leu Leu Arg
Glu Asn Ser Met Leu Arg Glu Glu Ile Ala 770 775 780 Lys Leu Arg Leu
Glu Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg 785 790 795 800 Glu
Asn Lys Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu 805 810
815 Lys Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala
820 825 830 Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln
Ala Ser 835 840 845 Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu
Lys Thr Glu Gln 850 855 860 Gln Ala Gln Glu Gln Glu Val Ala Gly Phe
Ser Leu Arg Gln Leu Gly 865 870 875 880 Leu Ala Gln His Ala Gln Ala
Ser Val Gln Gln Leu Cys Tyr Lys Trp 885 890 895 Gly His Thr Glu Lys
Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala 900 905 910 Leu Arg Ser
Gln Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly 915 920 925 Gly
Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro Arg 930 935
940 Glu Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val Arg Leu Ala Ser
945 950 955 960 Gly Ala Arg Ala Ala Ala Leu Pro Pro Pro Thr Gly Lys
Asn Gly Arg 965 970 975 Ser Pro Thr Lys Gln Lys Ser Val Cys Asp Ser
Ser Gly Trp Ile Leu 980 985 990 Pro Val Pro Thr Phe Ser Ser Gly Ser
Phe Leu Gly Arg Arg Cys Pro 995 1000 1005 Met Phe Asp Val Ser Pro
Ala Met Arg Leu Lys Ser Asp Ser Asn Arg 1010 1015 1020 Glu Thr His
Gln Ala Phe Arg Asp Lys Asp Asp Leu Pro Phe Phe Lys 1025 1030 1035
1040 Thr Gln Gln Ser Pro Arg His Thr Lys Asp Leu Gly Gln Asp Asp
Arg 1045 1050 1055 Ala Gly Val Leu Ala Pro Lys Cys Arg Pro Gly Thr
Leu Cys His Thr 1060 1065 1070 Asp Thr Pro Pro His Arg Asn Ala Asp
Thr Pro Pro His Arg His Thr 1075 1080 1085 Thr Thr Leu Pro His Arg
Asp Thr Thr Thr Ser Leu Pro His Phe His 1090 1095 1100 Val Ser Ala
Gly Gly Val Gly Pro Thr Thr Leu Gly Ser Asn Arg Glu 1105 1110 1115
1120 Ile Thr 69 1127 PRT Homo sapians 69 Asp Leu Cys Arg Gly Lys
Ser His Gln His Ile Leu Leu Pro Thr Gln 1 5 10 15 Ala Thr Phe Ala
Ala Ala Thr Gly Leu Trp Ala Ala Leu Thr Thr Val 20 25 30 Ser Asn
Pro Ser Arg Ala Asp Pro Val Thr Trp Arg Lys Glu Pro Ala 35 40 45
Val Leu Pro Cys Cys Asn Leu Glu Lys Gly Ser Trp Leu Ser Phe Pro 50
55 60 Gly Thr Ala Ala Arg Lys Glu Phe Ser Thr Thr Leu Thr Gly His
Ser 65 70 75 80 Ala Leu Ser Leu Ser Ser Ser Arg Ala Leu Pro Gly Ser
Leu Pro Ala 85 90 95 Phe Ala Asp Leu Pro Arg Ser Cys Pro Glu Ser
Glu Gln Ser Ala Thr 100 105 110 Pro Ala Gly Ala Phe Leu Leu Gly Trp
Glu Arg Val Val Gln Arg Arg 115 120 125 Leu Glu Val Pro Arg Pro Gln
Ala Ala Pro Ala Thr Ser Ala Thr Pro 130 135 140 Ser Arg Asp Pro Ser
Pro Pro Cys His Gln Arg Arg Asp Ala Ala Cys 145 150 155 160 Leu Arg
Ala Gln Gly Leu Thr Arg Ala Phe Gln Val Val His Leu Ala 165 170 175
Pro Thr Ala Pro Asp Gly Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser 180
185 190 Tyr Arg Leu Thr His Val Arg Cys Ala Gln Gly Leu Glu Ala Ala
Ser 195 200 205 Ala Asn Leu Pro Gly Ala Pro Gly Arg Ser Ser Ser Cys
Ala Leu Arg 210 215 220 Tyr Arg Ser Gly Pro Ser Val Ser Ser Ala Pro
Ser Pro Ala Glu Pro 225 230 235 240 Pro Ala His Gln Arg Leu Leu Phe
Leu Pro Arg Ala Pro Gln Ala Val 245 250 255 Ser Gly Pro Gln Glu Gln
Pro Ser Glu Glu Ala Leu Gly Val Gly Ser 260 265 270 Leu Ser Val Phe
Gln Leu His Leu Ile Gln Cys Ile Pro Asn Leu Ser 275 280 285 Tyr Pro
Leu Val Leu Arg His Ile Pro Glu Ile Leu Lys Phe Ser Glu 290 295 300
Lys Glu Thr Gly Gly Gly Ile Leu Gly Leu Glu Leu Pro Ala Thr Ala 305
310 315 320 Ala Arg Leu Ser Gly Leu Asn Ser Ile Met Gln Ile Lys Glu
Phe Glu 325 330 335 Glu Leu Val Lys Leu His Ser Leu Ser His Lys Val
Ile Gln Cys Val 340 345 350 Phe Ala Lys Lys Lys Asn Val Asp Lys Trp
Asp Asp Phe Cys Leu Ser 355 360 365 Glu Gly Tyr Gly His Ser Phe Leu
Ile Met Lys Glu Thr Ser Thr Lys 370 375 380 Ile Ser Gly Leu Ile Gln
Glu Met Gly Ser Gly Lys Ser Asn Val Gly 385 390 395 400 Thr Trp Gly
Asp Tyr Asp Asp Ser Ala Phe Met Glu Pro Arg Tyr His 405 410 415 Val
Arg Arg Glu Asp Leu Asp Lys Leu His Arg Ala Ala Trp Trp Gly 420 425
430 Lys Val Pro Arg Lys Asp Leu Ile Val Met Leu Arg Asp Thr Asp Met
435 440 445 Asn Lys Arg Asp Lys Gln Lys Arg Thr Ala Leu His Leu Ala
Ser Ala 450 455 460 Asn Gly Asn Ser Glu Val Val Gln Leu Leu Leu Asp
Arg Arg Cys Gln 465 470 475 480 Leu Asn Val Leu Asp Asn Lys Lys Arg
Thr Ala Leu Ile Lys Ala Val 485 490 495 Gln Cys Gln Glu Asp Glu Cys
Val Leu Met Leu Leu Glu His Gly Ala 500 505 510 Asp Gly Asn Ile Gln
Asp Glu Tyr Gly Asn Thr Ala Leu His Tyr Ala 515 520 525 Ile Tyr Asn
Glu Asp Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr Gly 530 535
540 Ala Asp Ile Glu Ser Lys Asn Lys Cys Gly Leu Thr Pro Leu Leu Leu
545 550 555 560 Gly Val His Glu Gln Lys Gln Glu Val Val Lys Phe Leu
Ile Lys Lys 565 570 575 Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr Gly
Arg Thr Ala Leu Ile 580 585 590 Leu Ala Val Cys Cys Gly Ser Ala Ser
Ile Val Asn Leu Leu Leu Glu 595 600 605 Gln Asn Val Asp Val Ser Ser
Gln Asp Leu Ser Gly Gln Thr Ala Arg 610 615 620 Glu Tyr Ala Val Ser
Ser His His His Val Ile Cys Glu Leu Leu Ser 625 630 635 640 Asp Tyr
Lys Glu Lys Gln Met Leu Lys Ile Ser Ser Glu Asn Ser Asn 645 650 655
Pro Val Ile Thr Ile Leu Asn Ile Lys Leu Pro Leu Lys Val Glu Glu 660
665 670 Glu Ile Lys Lys His Gly Ser Asn Pro Val Gly Leu Pro Glu Asn
Leu 675 680 685 Thr Asn Gly Ala Ser Ala Gly Asn Gly Asp Asp Gly Leu
Ile Pro Gln 690 695 700 Arg Lys Ser Arg Lys Pro Glu Asn Gln Gln Phe
Pro Asp Thr Glu Asn 705 710 715 720 Glu Glu Tyr His Ser Asp Glu Gln
Asn Asp Thr Gln Lys Gln Leu Ser 725 730 735 Glu Glu Gln Asn Thr Gly
Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys 740 745 750 Gln Lys Gln Ile
Glu Val Ala Glu Lys Glu Met Asn Ser Glu Leu Ser 755 760 765 Leu Ser
His Lys Lys Glu Glu Asp Leu Leu Arg Glu Asn Ser Met Leu 770 775 780
Arg Glu Glu Ile Ala Lys Leu Arg Leu Glu Leu Asp Glu Thr Lys His 785
790 795 800 Gln Asn Gln Leu Arg Glu Asn Lys Ile Leu Glu Glu Ile Glu
Ser Val 805 810 815 Lys Glu Lys Leu Leu Lys Thr Ile Gln Leu Asn Glu
Glu Ala Leu Thr 820 825 830 Lys Thr Lys Val Ala Gly Phe Ser Leu Arg
Gln Leu Gly Leu Ala Gln 835 840 845 His Ala Gln Ala Ser Val Gln Gln
Leu Cys Tyr Lys Trp Asn His Thr 850 855 860 Glu Lys Thr Glu Gln Gln
Ala Gln Glu Gln Glu Val Ala Gly Phe Ser 865 870 875 880 Leu Arg Gln
Leu Gly Leu Ala Gln His Ala Gln Ala Ser Val Gln Gln 885 890 895 Leu
Cys Tyr Lys Trp Gly His Thr Glu Lys Thr Glu Gln Gln Ala Gln 900 905
910 Glu Gln Gly Ala Ala Leu Arg Ser Gln Ile Gly Asp Pro Gly Gly Val
915 920 925 Pro Leu Ser Glu Gly Gly Thr Ala Ala Gly Asp Gln Gly Pro
Gly Thr 930 935 940 His Leu Pro Pro Arg Glu Pro Arg Ala Ser Pro Gly
Thr Pro Ser Leu 945 950 955 960 Val Arg Leu Ala Ser Gly Ala Arg Ala
Ala Ala Leu Pro Pro Pro Thr 965 970 975 Gly Lys Asn Gly Arg Ser Pro
Thr Lys Gln Lys Ser Val Cys Asp Ser 980 985 990 Ser Gly Trp Ile Leu
Pro Val Pro Thr Phe Ser Ser Gly Ser Phe Leu 995 1000 1005 Gly Arg
Arg Cys Pro Met Phe Asp Val Ser Pro Ala Met Arg Leu Lys 1010 1015
1020 Ser Asp Ser Asn Arg Glu Thr His Gln Ala Phe Arg Asp Lys Asp
Asp 1025 1030 1035 1040 Leu Pro Phe Phe Lys Thr Gln Gln Ser Pro Arg
His Thr Lys Asp Leu 1045 1050 1055 Gly Gln Asp Asp Arg Ala Gly Val
Leu Ala Pro Lys Cys Arg Pro Gly 1060 1065 1070 Thr Leu Cys His Thr
Asp Thr Pro Pro His Arg Asn Ala Asp Thr Pro 1075 1080 1085 Pro His
Arg His Thr Thr Thr Leu Pro His Arg Asp Thr Thr Thr Ser 1090 1095
1100 Leu Pro His Phe His Val Ser Ala Gly Gly Val Gly Pro Thr Thr
Leu 1105 1110 1115 1120 Gly Ser Asn Arg Glu Ile Thr 1125 70 4522
DNA Homo sapians 70 gttttttttt tttttttttt tttttttttt tattttaagg
gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc
cccatgtcta accaccaggt tctaggcatg 120 tattatggta tatgagaaat
gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa
aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240
cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa
300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa
tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat
cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata
cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata
catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt
aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600
agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg
660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt
tcccatgaaa 720 aatgcctttc atttctaagc tggtattggc atctcagcca
acacttttct ccttcttttc 780 tgcgtcttct ccttttctgc tttttctgga
tctcaggcca gagcgcactt acctaccagt 840 ctgtcatgtg gccctcatcc
acatggtggt ccttctcacc atggtgttct tgtctccaca 900 gctctttgaa
tcactgaatt ttcagaatga cttcaaatat gaggcatctt tctacctgag 960
gagggtgatc agggtcctct ccatttgtac cacctgcctc ctggacatgc tgcaggtcgt
1020 caacatcagc cccagcattt cctggttgat aatgctgttc tcaagtgtct
acatgatgac 1080 tctcattcag gaactacagg agatcctggt accttcacag
ccccagcctc tacctaagga 1140 tctttgcaga ggcaagagcc atcagcacat
cctgctgccg actcaagcaa cttttgctgc 1200 agcaactgga ctatgggctg
cactaaccac cgtatcaaat ccaagcagag cagatcctgt 1260 gacctggaga
aaggagccgg ctgtccttcc ctgctgtaac ctagagaaag gaagctggct 1320
gtccttccct ggcacagctg cacgcaagga attttccacc acgctcaccg ggcacagcgc
1380 gctgagcctc tccagttcgc gggccctccc cggctcgctc ccggctttcg
cagacctccc 1440 ccgctcctgc cctgagtccg agcagagcgc aacgccagcc
ggcgccttcc tcctgggctg 1500 ggagcgagtg gtgcagcggc ggctcgaagt
cccccggcct caagcagccc ccgcgactag 1560 cgcgacaccc tcgcgggatc
cgagtccacc ctgccaccag cgccgggacg ccgcgtgcct 1620 cagagcccaa
gggctgaccc gggccttcca ggtggtccat ctcgctccta cggctcccga 1680
cggtggcgct gggtgtcccc catcccgcaa ttcctaccgg ctgacccatg tgcgctgcgc
1740 ccaggggctg gaggctgcca gcgccaacct tcccggcgct ccggggcgga
gcagctcctg 1800 cgccctgcgc taccgcagcg gcccttcagt cagctccgcg
ccgtcccccg cagagccccc 1860 ggcgcaccag cgcctgcttt tccttccccg
agcgcctcaa gcagtctctg ggccgcagga 1920 acagccctct gaagaggcgc
ttggtgtagg aagcctctca gttttccagt tacacctaat 1980 acagtgtatt
ccaaatctaa gttacccact agtacttcgg cacattccag aaattctgaa 2040
attttctgaa aaggaaactg gtggtggaat tctaggctta gaattaccag cgacagctgc
2100 tcgcctctca ggattaaaca gcataatgca aatcaaagag tttgaagaat
tggtaaaact 2160 tcacagcttg tcacacaaag tcattcagtg tgtgtttgca
aagaaaaaaa atgtagacaa 2220 atgggatgac ttttgtctta gtgagggtta
tggacattca ttcttaataa tgaaagaaac 2280 gtcgactaaa atatcaggtt
taattcagga gatggggagc ggcaagagca acgtgggcac 2340 ttggggagac
tacgacgaca gcgccttcat ggagccgagg taccacgtcc gtcgagaaga 2400
tctggacaag ctccacagag ctgcctggtg gggtaaagtc cccagaaagg atctcatcgt
2460 catgctcagg gacactgaca tgaacaagag ggacaagcaa aagaggactg
ctctacattt 2520 ggcctctgcc aatggaaatt cagaagtagt acaactcctg
ctggacagac gatgtcaact 2580 taacgtcctt gacaacaaaa aaaggacagc
tctgataaag gccgtacaat gccaggaaga 2640 tgaatgtgtg ttaatgttgc
tggaacatgg cgctgatgga aatattcaag atgagtatgg 2700 aaataccgct
ctacactatg ctatctacaa tgaagataaa ttaatggcca aagcactgct 2760
cttatatggt gctgatattg aatcaaaaaa caagtgtggc ctcacaccac ttttgcttgg
2820 cgtacatgaa caaaaacagg aagtggtgaa atttttaatc aagaaaaaag
ctaatttaaa 2880 tgcacttgat agatatggaa gaactgccct catacttgct
gtatgttgtg gatcagcaag 2940 tatagtcaat cttctacttg agcaaaatgt
tgatgtatct tctcaagatc tatctggaca 3000 gacggccaga gagtatgctg
tttctagtca tcatcatgta atttgtgaat tactttctga 3060 ctataaagaa
aaacagatgc taaaaatctc ttctgaaaac agcaatccag tgataaccat 3120
ccttaatatc aaacttccac tcaaggttga agaagaaata aagaagcatg gaagtaatcc
3180 tgtgggatta ccagaaaacc tgactaatgg tgccagtgct ggcaatggtg
atgatggatt 3240 aattccacaa aggaagagca gaaaacctga aaatcagcaa
tttcctgaca ctgagaatga 3300 agagtatcac agtgacgaac aaaatgatac
ccagaaacaa ctttctgaag aacagaacac 3360 tggaatatca caagatgaga
ttctgactaa taaacaaaag cagatagaag tggctgaaaa 3420 ggaaatgaat
tctgagcttt ctcttagtca taagaaagaa gaagatctct tgcgtgaaaa 3480
cagcatgttg cgggaagaaa ttgccaagct aagactggaa ctagatgaaa caaaacatca
3540 gaaccagcta agggaaaata aaattttgga ggaaattgaa agtgtaaaag
aaaaacttct 3600 aaagactata caactgaatg aagaagcatt aacgaaaacc
aaggtggctg gtttctcttt 3660 gcgccagctt ggccttgccc agcatgcaca
agcctcagtg caacagctgt gctacaaatg 3720 gaaccacaca gagaaaacag
agcagcaggc tcaggagcag gaggtggctg gtttctcttt 3780 gcgccagctt
ggccttgccc agcatgcaca agcctcagta caacaactgt gctacaaatg 3840
gggccacaca gagaaaacag agcagcaggc tcaggagcag ggagctgcgc tgaggtccca
3900 gataggcgac cctggcgggg tgcccctgag cgaagggggg acagcagcag
gagaccaggg 3960 tccagggacc cacctcccac cgagggaacc tcgagcctcc
cctggcaccc ctagcttggt 4020 ccgcctggcc tccggagccc gagctgctgc
gcttccccca cccacaggga aaaacggccg 4080 atctccaacc aaacagaaat
ctgtgtgtga ctcctctggt tggatactgc cagtccccac 4140 attttcttcc
gggagttttc ttggcagaag gtgcccaatg tttgatgttt cgccagccat 4200
gaggctgaaa agtgacagca atagagaaac acatcaggct ttccgcgaca aagatgacct
4260 tcccttcttc aaaactcagc aatctccacg gcacacaaag gacttaggac
aagatgaccg 4320 agctggagtg ctcgccccaa aatgcaggcc cggaacactc
tgccacacgg acacaccacc 4380 acacagaaat gcggacacac caccacacag
acacaccacc acgctgccac acagagacac 4440 caccacatcg ttgccacact
ttcatgtgtc agctggcggt gtgggcccca cgactctggg 4500 ctctaataga
gaaattactt ag 4522 71 1180 DNA Homo sapians 71 gttttttttt
tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg 60
tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccaggt tctaggcatg
120 tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt
tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt
gggcctaatg taattataag 240 cattcttaaa agtgaaagaa ataataagag
aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt caactttttc
tttaggtgat tattttccct taacataaaa tttactttag 360 ctcaactata
caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc 420
tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct caacattata
480 catcaaatta tatatacata catgcacaca tacactatat atatcaagga
tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg
tttatgataa ttttggccat 600 agaatatata atacagatga tgtgaagtac
aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat gtttgtcttt
gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 aatgcctttc
atttctaagc tggtattggc atctcagcca acacttttct ccttcttttc 780
tgcgtcttct ccttttctgc tttttctgga tctcaggcca gagcgcactt acctaccagt
840 ctgtcatgtg gccctcatcc acatggtggt ccttctcacc atggtgttct
tgtctccaca 900 gctctttgaa tcactgaatt ttcagaatga cttcaaatat
gaggcatctt tctacctgag 960 gagggtgatc agggtcctct ccatttgtac
cacctgcctc ctgggcatgc tgcaggtcgt 1020 caacatcagc cccagcattt
cctggttgat aatgctgttc tcaagtgtct acatgatgac 1080 tctcattcag
gaactacagg agatcctggt accttcacag ccccagcctc tacctaagga 1140
tctttgcaga ggcaagagcc atcagcacat cctgctgccg 1180 72 1180 DNA Homo
sapians 72 gttttttttt tttttttttt tttttttttt tattttaagg gattcgttta
ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta
accaccaggt tctaggcatg 120 tattatggta tatgagaaat gggaattcag
gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa aacaaaaaca
ttaacttgaa ttacagattt gggcctaatg taattataag 240 cattcttaaa
agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa 300
actttttttt caactttttc tttaggtgat tattttccct taacataaaa tttactttag
360 ctcaactata caaacatgtg agttattgtt atgtaaccat cactcttcat
taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata cattcttcaa
aatacattct caacattata 480 catcaaatta tatatacata catgcacaca
tacactatat atatcaagga tttatatgag 540 aggattaatt aagaaaaaaa
ttagtggaat aaaaataatg tttatgataa ttttggccat 600 agaatatata
atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg 660
tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt tcccatgaaa
720 aatgcctttc atttctaagc tggtattggc atctcagcca acacttttct
ccttcttttc 780 tgcgtcttct ccttttctgc tttttctgga tctcaggcca
gagcgcactt acctaccagt 840 ctgtcatgtg gccctcatcc acatggtggt
ccttctcacc atggtgttct tgtctccaca 900 gctctttgaa tcactgaatt
ttcagaatga cttcaaatat gaggcatctt tctacctgag 960 gagggtgatc
agggtcctct ccatttgtac cacctgcctc ctggacatgc tgcaggtcgt 1020
caacatcagc cccagcattt cctggttgat aatgctgttc tcaagtgtct acatgatgac
1080 tctcattcag gaactacagg agatcctggt accttcacag ccccagcctc
tacctaagga 1140 tctttgcaga ggcaagagcc atcagcacat cctgctgccg 1180 73
1266 PRT Homo sapians 73 Met Pro Phe Ile Ser Lys Leu Val Leu Ala
Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro
Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu
Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu
Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn
Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80
Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Asp Met 85
90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Ile Met
Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu Ile Gln Glu Leu
Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln Pro Leu Pro Lys
Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His Ile Leu Leu Pro
Thr Gln Ala Thr Phe Ala Ala 145 150 155 160 Ala Thr Gly Leu Trp Ala
Ala Leu Thr Thr Val Ser Asn Pro Ser Arg 165 170 175 Ala Asp Pro Val
Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys 180 185 190 Asn Leu
Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg 195 200 205
Lys Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala Leu Ser Leu Ser 210
215 220 Ser Ser Arg Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu
Pro 225 230 235 240 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro
Ala Gly Ala Phe 245 250 255 Leu Leu Gly Trp Glu Arg Val Val Gln Arg
Arg Leu Glu Val Pro Arg 260 265 270 Pro Gln Ala Ala Pro Ala Thr Ser
Ala Thr Pro Ser Arg Asp Pro Ser 275 280 285 Pro Pro Cys His Gln Arg
Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290 295 300 Leu Thr Arg Ala
Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 305 310 315 320 Gly
Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His 325 330
335 Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly
340 345 350 Ala Pro Gly Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser
Gly Pro 355 360 365 Ser Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro
Ala His Gln Arg 370 375 380 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala
Val Ser Gly Pro Gln Glu 385 390 395 400 Gln Pro Ser Glu Glu Ala Leu
Gly Val Gly Ser Leu Ser Val Phe Gln 405 410 415 Leu His Leu Ile Gln
Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu 420 425 430 Arg His Ile
Pro Glu Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly 435 440 445 Gly
Ile Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455
460 Leu Asn Ser Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu
465 470 475 480 His Ser Leu Ser His Lys Val Ile Gln Cys Val Phe Ala
Lys Lys Lys 485 490 495 Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser
Glu Gly Tyr Gly His 500 505 510 Ser Phe Leu Ile Met Lys Glu Thr Ser
Thr Lys Ile Ser Gly Leu Ile 515 520 525 Gln Glu Met Gly Ser Gly Lys
Ser Asn Val Gly Thr Trp Gly Asp Tyr 530 535 540 Asp Asp Ser Ala Phe
Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp 545 550 555 560 Leu Asp
Lys Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys 565 570 575
Asp Leu Ile Val Met Leu Arg Asp Thr Asp Met Asn Lys Arg Asp Lys 580
585 590 Gln Lys Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser
Glu 595 600 605 Val Val Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn
Val Leu Asp 610 615 620 Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val
Gln Cys Gln Glu Asp 625 630 635 640 Glu Cys Val Leu Met Leu Leu Glu
His Gly Ala Asp Gly Asn Ile Gln 645 650 655 Asp Glu Tyr Gly Asn Thr
Ala Leu His Tyr Ala Ile Tyr Asn Glu Asp 660 665 670 Lys Leu Met Ala
Lys Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser 675 680 685 Lys Asn
Lys Cys Gly Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln 690 695 700
Lys Gln Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 705
710
715 720 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys
Cys 725 730 735 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn
Val Asp Val 740 745 750 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg
Glu Tyr Ala Val Ser 755 760 765 Ser His His His Val Ile Cys Glu Leu
Leu Ser Asp Tyr Lys Glu Lys 770 775 780 Gln Met Leu Lys Ile Ser Ser
Glu Asn Ser Asn Pro Val Ile Thr Ile 785 790 795 800 Leu Asn Ile Lys
Leu Pro Leu Lys Val Glu Glu Glu Ile Lys Lys His 805 810 815 Gly Ser
Asn Pro Val Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830
Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835
840 845 Pro Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His
Ser 850 855 860 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu
Gln Asn Thr 865 870 875 880 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn
Lys Gln Lys Gln Ile Glu 885 890 895 Val Ala Glu Lys Glu Met Asn Ser
Glu Leu Ser Leu Ser His Lys Lys 900 905 910 Glu Glu Asp Leu Leu Arg
Glu Asn Ser Met Leu Arg Glu Glu Ile Ala 915 920 925 Lys Leu Arg Leu
Glu Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg 930 935 940 Glu Asn
Lys Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu 945 950 955
960 Lys Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala
965 970 975 Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln
Ala Ser 980 985 990 Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu
Lys Thr Glu Gln 995 1000 1005 Gln Ala Gln Glu Gln Glu Val Ala Gly
Phe Ser Leu Arg Gln Leu Gly 1010 1015 1020 Leu Ala Gln His Ala Gln
Ala Ser Val Gln Gln Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly His
Thr Glu Lys Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050
1055 Leu Arg Ser Gln Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu
Gly 1060 1065 1070 Gly Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His
Leu Pro Pro Arg 1075 1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr Pro
Ser Leu Val Arg Leu Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala Ala
Leu Pro Pro Pro Thr Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser Pro
Thr Lys Gln Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130
1135 Pro Val Pro Thr Phe Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys
Pro 1140 1145 1150 Met Phe Asp Val Ser Pro Ala Met Arg Leu Lys Ser
Asp Ser Asn Arg 1155 1160 1165 Glu Thr His Gln Ala Phe Arg Asp Lys
Asp Asp Leu Pro Phe Phe Lys 1170 1175 1180 Thr Gln Gln Ser Pro Arg
His Thr Lys Asp Leu Gly Gln Asp Asp Arg 1185 1190 1195 1200 Ala Gly
Val Leu Ala Pro Lys Cys Arg Pro Gly Thr Leu Cys His Thr 1205 1210
1215 Asp Thr Pro Pro His Arg Asn Ala Asp Thr Pro Pro His Arg His
Thr 1220 1225 1230 Thr Thr Leu Pro His Arg Asp Thr Thr Thr Ser Leu
Pro His Phe His 1235 1240 1245 Val Ser Ala Gly Gly Val Gly Pro Thr
Thr Leu Gly Ser Asn Arg Glu 1250 1255 1260 Ile Thr 1265 74 227 PRT
Homo sapians 74 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro
Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu
Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys
His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val
Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn
Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile
Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu
Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105
110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu
115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala
Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr
Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu
Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln
Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln
Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu
Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu
Leu Pro 225 75 153 PRT Homo sapians 75 Met Pro Phe Ile Ser Lys Leu
Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala
Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg
Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val
Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55
60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg
65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu
Asp Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp
Leu Ile Met Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His Ile
Leu Leu Pro 145 150 76 3801 DNA Homo sapians 76 atgcctttca
tttctaagct ggtattggca tctcagccaa cacttttctc cttcttttct 60
gcgtcttctc cttttctgct ttttctggat ctcaggccag agcgcactta cctaccagtc
120 tgtcatgtgg ccctcatcca catggtggtc cttctcacca tggtgttctt
gtctccacag 180 ctctttgaat cactgaattt tcagaatgac ttcaaatatg
aggcatcttt ctacctgagg 240 agggtgatca gggtcctctc catttgtacc
acctgcctcc tggacatgct gcaggtcgtc 300 aacatcagcc ccagcatttc
ctggttgata atgctgttct caagtgtcta catgatgact 360 ctcattcagg
aactacagga gatcctggta ccttcacagc cccagcctct acctaaggat 420
ctttgcagag gcaagagcca tcagcacatc ctgctgccga ctcaagcaac ttttgctgca
480 gcaactggac tatgggctgc actaaccacc gtatcaaatc caagcagagc
agatcctgtg 540 acctggagaa aggagccggc tgtccttccc tgctgtaacc
tagagaaagg aagctggctg 600 tccttccctg gcacagctgc acgcaaggaa
ttttccacca cgctcaccgg gcacagcgcg 660 ctgagcctct ccagttcgcg
ggccctcccc ggctcgctcc cggctttcgc agacctcccc 720 cgctcctgcc
ctgagtccga gcagagcgca acgccagccg gcgccttcct cctgggctgg 780
gagcgagtgg tgcagcggcg gctcgaagtc ccccggcctc aagcagcccc cgcgactagc
840 gcgacaccct cgcgggatcc gagtccaccc tgccaccagc gccgggacgc
cgcgtgcctc 900 agagcccaag ggctgacccg ggccttccag gtggtccatc
tcgctcctac ggctcccgac 960 ggtggcgctg ggtgtccccc atcccgcaat
tcctaccggc tgacccatgt gcgctgcgcc 1020 caggggctgg aggctgccag
cgccaacctt cccggcgctc cggggcggag cagctcctgc 1080 gccctgcgct
accgcagcgg cccttcagtc agctccgcgc cgtcccccgc agagcccccg 1140
gcgcaccagc gcctgctttt ccttccccga gcgcctcaag cagtctctgg gccgcaggaa
1200 cagccctctg aagaggcgct tggtgtagga agcctctcag ttttccagtt
acacctaata 1260 cagtgtattc caaatctaag ttacccacta gtacttcggc
acattccaga aattctgaaa 1320 ttttctgaaa aggaaactgg tggtggaatt
ctaggcttag aattaccagc gacagctgct 1380 cgcctctcag gattaaacag
cataatgcaa atcaaagagt ttgaagaatt ggtaaaactt 1440 cacagcttgt
cacacaaagt cattcagtgt gtgtttgcaa agaaaaaaaa tgtagacaaa 1500
tgggatgact tttgtcttag tgagggttat ggacattcat tcttaataat gaaagaaacg
1560 tcgactaaaa tatcaggttt aattcaggag atggggagcg gcaagagcaa
cgtgggcact 1620 tggggagact acgacgacag cgccttcatg gagccgaggt
accacgtccg tcgagaagat 1680 ctggacaagc tccacagagc tgcctggtgg
ggtaaagtcc ccagaaagga tctcatcgtc 1740 atgctcaggg acactgacat
gaacaagagg gacaagcaaa agaggactgc tctacatttg 1800 gcctctgcca
atggaaattc agaagtagta caactcctgc tggacagacg atgtcaactt 1860
aacgtccttg acaacaaaaa aaggacagct ctgataaagg ccgtacaatg ccaggaagat
1920 gaatgtgtgt taatgttgct ggaacatggc gctgatggaa atattcaaga
tgagtatgga 1980 aataccgctc tacactatgc tatctacaat gaagataaat
taatggccaa agcactgctc 2040 ttatatggtg ctgatattga atcaaaaaac
aagtgtggcc tcacaccact tttgcttggc 2100 gtacatgaac aaaaacagga
agtggtgaaa tttttaatca agaaaaaagc taatttaaat 2160 gcacttgata
gatatggaag aactgccctc atacttgctg tatgttgtgg atcagcaagt 2220
atagtcaatc ttctacttga gcaaaatgtt gatgtatctt ctcaagatct atctggacag
2280 acggccagag agtatgctgt ttctagtcat catcatgtaa tttgtgaatt
actttctgac 2340 tataaagaaa aacagatgct aaaaatctct tctgaaaaca
gcaatccagt gataaccatc 2400 cttaatatca aacttccact caaggttgaa
gaagaaataa agaagcatgg aagtaatcct 2460 gtgggattac cagaaaacct
gactaatggt gccagtgctg gcaatggtga tgatggatta 2520 attccacaaa
ggaagagcag aaaacctgaa aatcagcaat ttcctgacac tgagaatgaa 2580
gagtatcaca gtgacgaaca aaatgatacc cagaaacaac tttctgaaga acagaacact
2640 ggaatatcac aagatgagat tctgactaat aaacaaaagc agatagaagt
ggctgaaaag 2700 gaaatgaatt ctgagctttc tcttagtcat aagaaagaag
aagatctctt gcgtgaaaac 2760 agcatgttgc gggaagaaat tgccaagcta
agactggaac tagatgaaac aaaacatcag 2820 aaccagctaa gggaaaataa
aattttggag gaaattgaaa gtgtaaaaga aaaacttcta 2880 aagactatac
aactgaatga agaagcatta acgaaaacca aggtggctgg tttctctttg 2940
cgccagcttg gccttgccca gcatgcacaa gcctcagtgc aacagctgtg ctacaaatgg
3000 aaccacacag agaaaacaga gcagcaggct caggagcagg aggtggctgg
tttctctttg 3060 cgccagcttg gccttgccca gcatgcacaa gcctcagtac
aacaactgtg ctacaaatgg 3120 ggccacacag agaaaacaga gcagcaggct
caggagcagg gagctgcgct gaggtcccag 3180 ataggcgacc ctggcggggt
gcccctgagc gaagggggga cagcagcagg agaccagggt 3240 ccagggaccc
acctcccacc gagggaacct cgagcctccc ctggcacccc tagcttggtc 3300
cgcctggcct ccggagcccg agctgctgcg cttcccccac ccacagggaa aaacggccga
3360 tctccaacca aacagaaatc tgtgtgtgac tcctctggtt ggatactgcc
agtccccaca 3420 ttttcttccg ggagttttct tggcagaagg tgcccaatgt
ttgatgtttc gccagccatg 3480 aggctgaaaa gtgacagcaa tagagaaaca
catcaggctt tccgcgacaa agatgacctt 3540 cccttcttca aaactcagca
atctccacgg cacacaaagg acttaggaca agatgaccga 3600 gctggagtgc
tcgccccaaa atgcaggccc ggaacactct gccacacgga cacaccacca 3660
cacagaaatg cggacacacc accacacaga cacaccacca cgctgccaca cagagacacc
3720 accacatcgt tgccacactt tcatgtgtca gctggcggtg tgggccccac
gactctgggc 3780 tctaatagag aaattactta g 3801 77 459 DNA Homo
sapians 77 atgcctttca tttctaagct ggtattggca tctcagccaa cacttttctc
cttcttttct 60 gcgtcttctc cttttctgct ttttctggat ctcaggccag
agcgcactta cctaccagtc 120 tgtcatgtgg ccctcatcca catggtggtc
cttctcacca tggtgttctt gtctccacag 180 ctctttgaat cactgaattt
tcagaatgac ttcaaatatg aggcatcttt ctacctgagg 240 agggtgatca
gggtcctctc catttgtacc acctgcctcc tgggcatgct gcaggtcgtc 300
aacatcagcc ccagcatttc ctggttgata atgctgttct caagtgtcta catgatgact
360 ctcattcagg aactacagga gatcctggta ccttcacagc cccagcctct
acctaaggat 420 ctttgcagag gcaagagcca tcagcacatc ctgctgccg 459 78
459 DNA Homo sapians 78 atgcctttca tttctaagct ggtattggca tctcagccaa
cacttttctc cttcttttct 60 gcgtcttctc cttttctgct ttttctggat
ctcaggccag agcgcactta cctaccagtc 120 tgtcatgtgg ccctcatcca
catggtggtc cttctcacca tggtgttctt gtctccacag 180 ctctttgaat
cactgaattt tcagaatgac ttcaaatatg aggcatcttt ctacctgagg 240
agggtgatca gggtcctctc catttgtacc acctgcctcc tggacatgct gcaggtcgtc
300 aacatcagcc ccagcatttc ctggttgata atgctgttct caagtgtcta
catgatgact 360 ctcattcagg aactacagga gatcctggta ccttcacagc
cccagcctct acctaaggat 420 ctttgcagag gcaagagcca tcagcacatc
ctgctgccg 459 79 1266 PRT Homo sapians 79 Met Pro Phe Ile Ser Lys
Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser
Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu
Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45
Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50
55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu
Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu
Leu Asp Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser
Trp Leu Ile Met Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu
Ile Gln Glu Leu Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln
Pro Leu Pro Lys Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His
Ile Leu Leu Pro Thr Gln Ala Thr Phe Ala Ala 145 150 155 160 Ala Thr
Gly Leu Trp Ala Ala Leu Thr Thr Val Ser Asn Pro Ser Arg 165 170 175
Ala Asp Pro Val Thr Trp Arg Lys Glu Pro Ala Val Leu Pro Cys Cys 180
185 190 Asn Leu Glu Lys Gly Ser Trp Leu Ser Phe Pro Gly Thr Ala Ala
Arg 195 200 205 Lys Glu Phe Ser Thr Thr Leu Thr Gly His Ser Ala Leu
Ser Leu Ser 210 215 220 Ser Ser Arg Ala Leu Pro Gly Ser Leu Pro Ala
Phe Ala Asp Leu Pro 225 230 235 240 Arg Ser Cys Pro Glu Ser Glu Gln
Ser Ala Thr Pro Ala Gly Ala Phe 245 250 255 Leu Leu Gly Trp Glu Arg
Val Val Gln Arg Arg Leu Glu Val Pro Arg 260 265 270 Pro Gln Ala Ala
Pro Ala Thr Ser Ala Thr Pro Ser Arg Asp Pro Ser 275 280 285 Pro Pro
Cys His Gln Arg Arg Asp Ala Ala Cys Leu Arg Ala Gln Gly 290 295 300
Leu Thr Arg Ala Phe Gln Val Val His Leu Ala Pro Thr Ala Pro Asp 305
310 315 320 Gly Gly Ala Gly Cys Pro Pro Ser Arg Asn Ser Tyr Arg Leu
Thr His 325 330 335 Val Arg Cys Ala Gln Gly Leu Glu Ala Ala Ser Ala
Asn Leu Pro Gly 340 345 350 Ala Pro Gly Arg Ser Ser Ser Cys Ala Leu
Arg Tyr Arg Ser Gly Pro 355 360 365 Ser Val Ser Ser Ala Pro Ser Pro
Ala Glu Pro Pro Ala His Gln Arg 370 375 380 Leu Leu Phe Leu Pro Arg
Ala Pro Gln Ala Val Ser Gly Pro Gln Glu 385 390 395 400 Gln Pro Ser
Glu Glu Ala Leu Gly Val Gly Ser Leu Ser Val Phe Gln 405 410 415 Leu
His Leu Ile Gln Cys Ile Pro Asn Leu Ser Tyr Pro Leu Val Leu 420 425
430 Arg His Ile Pro Glu Ile Leu Lys Phe Ser Glu Lys Glu Thr Gly Gly
435 440 445 Gly Ile Leu Gly Leu Glu Leu Pro Ala Thr Ala Ala Arg Leu
Ser Gly 450 455 460 Leu Asn Ser Ile Met Gln Ile Lys Glu Phe Glu Glu
Leu Val Lys Leu 465 470 475 480 His Ser Leu Ser His Lys Val Ile Gln
Cys Val Phe Ala Lys Lys Lys 485 490 495 Asn Val Asp Lys Trp Asp Asp
Phe Cys Leu Ser Glu Gly Tyr Gly His 500 505 510 Ser Phe Leu Ile Met
Lys Glu Thr Ser Thr Lys Ile Ser Gly Leu Ile 515 520 525 Gln Glu Met
Gly Ser Gly Lys Ser Asn Val Gly Thr Trp Gly Asp Tyr 530 535 540 Asp
Asp Ser Ala Phe Met Glu Pro Arg Tyr His Val Arg Arg Glu Asp 545 550
555 560 Leu Asp Lys Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg
Lys 565 570 575 Asp Leu Ile Val Met Leu Arg Asp Thr Asp Met Asn Lys
Arg Asp Lys 580 585 590 Gln Lys Arg Thr Ala Leu His Leu Ala Ser Ala
Asn Gly Asn Ser Glu 595 600 605 Val Val Gln Leu Leu Leu Asp Arg Arg
Cys Gln Leu Asn Val Leu Asp 610 615 620 Asn Lys Lys Arg Thr Ala Leu
Ile Lys Ala Val Gln Cys Gln Glu Asp 625 630 635 640 Glu Cys Val Leu
Met Leu Leu Glu His Gly Ala Asp Gly Asn Ile Gln 645 650 655 Asp Glu
Tyr Gly Asn Thr Ala Leu His Tyr Ala Ile Tyr Asn Glu Asp 660 665 670
Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser 675
680 685 Lys Asn Lys Cys Gly Leu Thr Pro Leu Leu Leu Gly Val His Glu
Gln 690 695 700 Lys Gln Glu Val Val Lys Phe Leu Ile Lys Lys Lys Ala
Asn Leu Asn 705 710
715 720 Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys
Cys 725 730 735 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn
Val Asp Val 740 745 750 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg
Glu Tyr Ala Val Ser 755 760 765 Ser His His His Val Ile Cys Glu Leu
Leu Ser Asp Tyr Lys Glu Lys 770 775 780 Gln Met Leu Lys Ile Ser Ser
Glu Asn Ser Asn Pro Val Ile Thr Ile 785 790 795 800 Leu Asn Ile Lys
Leu Pro Leu Lys Val Glu Glu Glu Ile Lys Lys His 805 810 815 Gly Ser
Asn Pro Val Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830
Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835
840 845 Pro Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His
Ser 850 855 860 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu
Gln Asn Thr 865 870 875 880 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn
Lys Gln Lys Gln Ile Glu 885 890 895 Val Ala Glu Lys Glu Met Asn Ser
Glu Leu Ser Leu Ser His Lys Lys 900 905 910 Glu Glu Asp Leu Leu Arg
Glu Asn Ser Met Leu Arg Glu Glu Ile Ala 915 920 925 Lys Leu Arg Leu
Glu Leu Asp Glu Thr Lys His Gln Asn Gln Leu Arg 930 935 940 Glu Asn
Lys Ile Leu Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu 945 950 955
960 Lys Thr Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala
965 970 975 Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln
Ala Ser 980 985 990 Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu
Lys Thr Glu Gln 995 1000 1005 Gln Ala Gln Glu Gln Glu Val Ala Gly
Phe Ser Leu Arg Gln Leu Gly 1010 1015 1020 Leu Ala Gln His Ala Gln
Ala Ser Val Gln Gln Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly His
Thr Glu Lys Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050
1055 Leu Arg Ser Gln Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu
Gly 1060 1065 1070 Gly Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His
Leu Pro Pro Arg 1075 1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr Pro
Ser Leu Val Arg Leu Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala Ala
Leu Pro Pro Pro Thr Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser Pro
Thr Lys Gln Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130
1135 Pro Val Pro Thr Phe Ser Ser Gly Ser Phe Leu Gly Arg Arg Cys
Pro 1140 1145 1150 Met Phe Asp Val Ser Pro Ala Met Arg Leu Lys Ser
Asp Ser Asn Arg 1155 1160 1165 Glu Thr His Gln Ala Phe Arg Asp Lys
Asp Asp Leu Pro Phe Phe Lys 1170 1175 1180 Thr Gln Gln Ser Pro Arg
His Thr Lys Asp Leu Gly Gln Asp Asp Arg 1185 1190 1195 1200 Ala Gly
Val Leu Ala Pro Lys Cys Arg Pro Gly Thr Leu Cys His Thr 1205 1210
1215 Asp Thr Pro Pro His Arg Asn Ala Asp Thr Pro Pro His Arg His
Thr 1220 1225 1230 Thr Thr Leu Pro His Arg Asp Thr Thr Thr Ser Leu
Pro His Phe His 1235 1240 1245 Val Ser Ala Gly Gly Val Gly Pro Thr
Thr Leu Gly Ser Asn Arg Glu 1250 1255 1260 Ile Thr 1265 80 227 PRT
Homo sapians 80 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro
Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu
Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys
His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val
Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn
Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile
Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu
Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105
110 Lys Trp Lys Ser Thr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu
115 120 125 Ser Phe Pro Val Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala
Ser Ser 130 135 140 Asn Val Thr Gln Ile Asn Leu His Val Ser Lys Tyr
Cys Ser Leu Phe 145 150 155 160 Pro Ile Asn Ser Ile Ile Arg Gly Leu
Phe Phe Thr Leu Ser Leu Phe 165 170 175 Arg Asp Val Phe Leu Lys Gln
Ile Met Leu Phe Ser Ser Val Tyr Met 180 185 190 Met Thr Leu Ile Gln
Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro 195 200 205 Gln Pro Leu
Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln His Ile 210 215 220 Leu
Leu Pro 225 81 153 PRT Homo sapians 81 Met Pro Phe Ile Ser Lys Leu
Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala
Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg
Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val
Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55
60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg
65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys Leu Leu
Asp Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile Ser Trp
Leu Ile Met Leu 100 105 110 Phe Ser Ser Val Tyr Met Met Thr Leu Ile
Gln Glu Leu Gln Glu Ile 115 120 125 Leu Val Pro Ser Gln Pro Gln Pro
Leu Pro Lys Asp Leu Cys Arg Gly 130 135 140 Lys Ser His Gln His Ile
Leu Leu Pro 145 150 82 255 PRT Homo sapians 82 Met Pro Phe Ile Ser
Lys Leu Val Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe
Ser Ala Ser Ser Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro
Glu Arg Thr Tyr Leu Pro Val Cys His Val Ala Leu Ile His Met 35 40
45 Val Val Leu Leu Thr Met Val Phe Leu Ser Pro Gln Leu Phe Glu Ser
50 55 60 Leu Asn Phe Gln Asn Asp Phe Lys Tyr Glu Ala Ser Phe Tyr
Leu Arg 65 70 75 80 Arg Val Ile Arg Val Leu Ser Ile Cys Thr Thr Cys
Leu Leu Gly Met 85 90 95 Leu Gln Val Val Asn Ile Ser Pro Ser Ile
Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys Ser Thr Ile Phe Thr
Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser Phe Pro Val Ser Ser
Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn Val Thr Gln
Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160 Pro
Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170
175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met
180 185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser
Gln Pro 195 200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser
His Gln His Ile 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met
Tyr Lys Met Asp Phe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu
Pro Trp Ala Tyr Asp Arg Gly Val 245 250 255 83 1140 PRT Homo
sapians 83 Met Pro Phe Ile Ser Lys Leu Val Leu Ala Ser Gln Pro Thr
Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala Ser Ser Pro Phe Leu Leu Phe
Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr Leu Pro Val Cys His
Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr Met Val Phe
Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn Asp
Phe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg
Val Leu Ser Ile Cys Thr Thr Cys Leu Leu Asp Met 85 90 95 Leu Gln
Val Val Asn Ile Ser Pro Ser Ile Ser Trp Leu Ile Met Leu 100 105 110
Phe Ser Ser Val Tyr Met Met Thr Leu Ile Gln Glu Leu Gln Glu Ile 115
120 125 Leu Val Pro Ser Gln Pro Gln Pro Leu Pro Lys Asp Leu Cys Arg
Gly 130 135 140 Lys Ser His Gln His Ile Leu Leu Pro Thr Gln Ala Thr
Phe Ala Ala 145 150 155 160 Ala Thr Gly Leu Trp Ala Ala Leu Thr Thr
Val Ser Asn Pro Ser Arg 165 170 175 Ala Asp Pro Val Thr Trp Arg Lys
Glu Pro Ala Val Leu Pro Cys Cys 180 185 190 Asn Leu Glu Lys Gly Ser
Trp Leu Ser Phe Pro Gly Thr Ala Ala Arg 195 200 205 Lys Glu Phe Ser
Thr Thr Leu Thr Gly His Ser Ala Leu Ser Leu Ser 210 215 220 Ser Ser
Arg Ala Leu Pro Gly Ser Leu Pro Ala Phe Ala Asp Leu Pro 225 230 235
240 Arg Ser Cys Pro Glu Ser Glu Gln Ser Ala Thr Pro Ala Gly Ala Phe
245 250 255 Leu Leu Gly Trp Glu Arg Val Val Gln Arg Arg Leu Glu Val
Pro Arg 260 265 270 Pro Gln Ala Ala Pro Ala Thr Ser Ala Thr Pro Ser
Arg Asp Pro Ser 275 280 285 Pro Pro Cys His Gln Arg Arg Asp Ala Ala
Cys Leu Arg Ala Gln Gly 290 295 300 Leu Thr Arg Ala Phe Gln Val Val
His Leu Ala Pro Thr Ala Pro Asp 305 310 315 320 Gly Gly Ala Gly Cys
Pro Pro Ser Arg Asn Ser Tyr Arg Leu Thr His 325 330 335 Val Arg Cys
Ala Gln Gly Leu Glu Ala Ala Ser Ala Asn Leu Pro Gly 340 345 350 Ala
Pro Gly Arg Ser Ser Ser Cys Ala Leu Arg Tyr Arg Ser Gly Pro 355 360
365 Ser Val Ser Ser Ala Pro Ser Pro Ala Glu Pro Pro Ala His Gln Arg
370 375 380 Leu Leu Phe Leu Pro Arg Ala Pro Gln Ala Val Ser Gly Pro
Gln Glu 385 390 395 400 Gln Pro Ser Glu Glu Ala Leu Gly Val Gly Ser
Leu Ser Val Phe Gln 405 410 415 Leu His Leu Ile Gln Cys Ile Pro Asn
Leu Ser Tyr Pro Leu Val Leu 420 425 430 Arg His Ile Pro Glu Ile Leu
Lys Phe Ser Glu Lys Glu Thr Gly Gly 435 440 445 Gly Ile Leu Gly Leu
Glu Leu Pro Ala Thr Ala Ala Arg Leu Ser Gly 450 455 460 Leu Asn Ser
Ile Met Gln Ile Lys Glu Phe Glu Glu Leu Val Lys Leu 465 470 475 480
His Ser Leu Ser His Lys Val Ile Gln Cys Val Phe Ala Lys Lys Lys 485
490 495 Asn Val Asp Lys Trp Asp Asp Phe Cys Leu Ser Glu Gly Tyr Gly
His 500 505 510 Ser Phe Leu Ile Met Lys Glu Thr Ser Thr Lys Ile Ser
Gly Leu Ile 515 520 525 Gln Glu Met Gly Ser Gly Lys Ser Asn Val Gly
Thr Trp Gly Asp Tyr 530 535 540 Asp Asp Ser Ala Phe Met Glu Pro Arg
Tyr His Val Arg Arg Glu Asp 545 550 555 560 Leu Asp Lys Leu His Arg
Ala Ala Trp Trp Gly Lys Val Pro Arg Lys 565 570 575 Asp Leu Ile Val
Met Leu Arg Asp Thr Asp Met Asn Lys Arg Asp Lys 580 585 590 Gln Lys
Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu 595 600 605
Val Val Gln Leu Leu Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp 610
615 620 Asn Lys Lys Arg Thr Ala Leu Ile Lys Ala Val Gln Cys Gln Glu
Asp 625 630 635 640 Glu Cys Val Leu Met Leu Leu Glu His Gly Ala Asp
Gly Asn Ile Gln 645 650 655 Asp Glu Tyr Gly Asn Thr Ala Leu His Tyr
Ala Ile Tyr Asn Glu Asp 660 665 670 Lys Leu Met Ala Lys Ala Leu Leu
Leu Tyr Gly Ala Asp Ile Glu Ser 675 680 685 Lys Asn Lys Cys Gly Leu
Thr Pro Leu Leu Leu Gly Val His Glu Gln 690 695 700 Lys Gln Glu Val
Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn 705 710 715 720 Ala
Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys 725 730
735 Gly Ser Ala Ser Ile Val Asn Leu Leu Leu Glu Gln Asn Val Asp Val
740 745 750 Ser Ser Gln Asp Leu Ser Gly Gln Thr Ala Arg Glu Tyr Ala
Val Ser 755 760 765 Ser His His His Val Ile Cys Glu Leu Leu Ser Asp
Tyr Lys Glu Lys 770 775 780 Gln Met Leu Lys Ile Ser Ser Glu Asn Ser
Asn Pro Val Ile Thr Ile 785 790 795 800 Leu Asn Ile Lys Leu Pro Leu
Lys Val Glu Glu Glu Ile Lys Lys His 805 810 815 Gly Ser Asn Pro Val
Gly Leu Pro Glu Asn Leu Thr Asn Gly Ala Ser 820 825 830 Ala Gly Asn
Gly Asp Asp Gly Leu Ile Pro Gln Arg Lys Ser Arg Lys 835 840 845 Pro
Glu Asn Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His Ser 850 855
860 Asp Glu Gln Asn Asp Thr Gln Lys Gln Leu Ser Glu Glu Gln Asn Thr
865 870 875 880 Gly Ile Ser Gln Asp Glu Ile Leu Thr Asn Lys Gln Lys
Gln Ile Glu 885 890 895 Val Ala Glu Lys Glu Met Asn Ser Glu Leu Ser
Leu Ser His Lys Lys 900 905 910 Glu Glu Asp Leu Leu Arg Glu Asn Ser
Met Leu Arg Glu Glu Ile Ala 915 920 925 Lys Leu Arg Leu Glu Leu Asp
Glu Thr Lys His Gln Asn Gln Leu Arg 930 935 940 Glu Asn Lys Ile Leu
Glu Glu Ile Glu Ser Val Lys Glu Lys Leu Leu 945 950 955 960 Lys Thr
Ile Gln Leu Asn Glu Glu Ala Leu Thr Lys Thr Lys Val Ala 965 970 975
Gly Phe Ser Leu Arg Gln Leu Gly Leu Ala Gln His Ala Gln Ala Ser 980
985 990 Val Gln Gln Leu Cys Tyr Lys Trp Asn His Thr Glu Lys Thr Glu
Gln 995 1000 1005 Gln Ala Gln Glu Gln Glu Val Ala Gly Phe Ser Leu
Arg Gln Leu Gly 1010 1015 1020 Leu Ala Gln His Ala Gln Ala Ser Val
Gln Gln Leu Cys Tyr Lys Trp 1025 1030 1035 1040 Gly His Thr Glu Lys
Thr Glu Gln Gln Ala Gln Glu Gln Gly Ala Ala 1045 1050 1055 Leu Arg
Ser Gln Ile Gly Asp Pro Gly Gly Val Pro Leu Ser Glu Gly 1060 1065
1070 Gly Thr Ala Ala Gly Asp Gln Gly Pro Gly Thr His Leu Pro Pro
Arg 1075 1080 1085 Glu Pro Arg Ala Ser Pro Gly Thr Pro Ser Leu Val
Arg Leu Ala Ser 1090 1095 1100 Gly Ala Arg Ala Ala Ala Leu Pro Pro
Pro Thr Gly Lys Asn Gly Arg 1105 1110 1115 1120 Ser Pro Thr Lys Gln
Lys Ser Val Cys Asp Ser Ser Gly Trp Ile Leu 1125 1130 1135 Pro Val
Pro Thr 1140
* * * * *
References